Italian Purine Club

Cari amici “purinergici”, questa newsletter nasce con l’intenzione di aggiornare i nostri soci sulle recenti pubblicazioni relative al mondo delle purine.

La riuscita di questa iniziativa si basa essenzialmente sulla generosa disponibilità mostrata da diversi colleghi (che speriamo possano diventare un numero sempre maggiore) che hanno deciso di dedicare un po' del loro tempo ad indagare in lungo e in largo la recente letteratura scientifica al fine di scovare qualche articolo di pregio da portare alla nostra attenzione, distillandone i contenuti e fornendo una loro opinione critica sull’elaborato. 

Inviate il vostro Literature Highlight al curatore di questa sezione Dr. Luca Antonioli lucaant[at]gmail.com

F. Pedata, E. Adinolfi, L. Antonioli, D. Dal Ben, P. Di Iorio, M. Fumagalli, F. Trettel

 

 

Giancarlo Pepeu (1930 - 2021)

 

Ci ha lasciato domenica 21 novembre scorso all’età di 91 anni Giancarlo Pepeu , Professore Emerito di Farmacologia dell’Università di Firenze. Il professor Pepeu nato a Milano nel 1930, ha poi conseguito presso l’Ateneo fiorentino la laurea in Medicina e Chirurgia nel 1954. Subito ha iniziato la sua attività di ricerca presso l’ateneo Fiorentino nell’Istituto di Farmacologia allora diretto dal Professor Mario Aiiazzi-Mancini proseguendo i suoi studi dal 1958 come “Postdoctoral Fellow” presso il Department of Pharmacology della Yale University. Rientrato in Italia è stato dal 1961 al 1968 assistente a Sassari, aiuto a Pisa, divenuto ordinario di farmacologia nel 1968 a Cagliari è rientrato a Firenze nel 1974 dove ha insegnato Farmacologia a Medicina fino al 2005. Nell’Ateneo Fiorentino è stato prorettore alla ricerca e ai rapporti internazionali ed è stato Direttore del Dipartimento di Farmacologia oltre a svolgere vari incarichi organizzativi. Dagli anni 50 ha partecipato attivamente alla vita della Società Italiana di Farmacologia dove prima Consigliere è stato poi Presidente dal 1995 al 1999. Ha svolto una vita di ricerca appassionata e dedicata al mondo delle Neurofarmacologia, disciplina nascente, dove i suoi interessi si sono focalizzati sul sistema colinergico cerebrale con ricerche di base e rivolte allo studio in particolare della demenza di Alzheimer dove è divenuto un punto di riferimento a livello internazionale. Ha ricevuto molti riconoscimenti da parte del mondo scientifico internazionale fra cui il “Life Time Achievement Award of Alzheimer’s Association”, è stato eletto “Honorary fellow della British Pharmacological Society”, ha ricevuto il Copernicus Medal della Polish Academy of Sciences e il titolo di “Honorary Doctor of Sciences” dell’Università dell’Arizona. Il suo interesse primariamente colinergico non gli ha impedito di seguire e stimolare anche altri campi di ricerca come quello del sistema purinergico a livello cerebrale con studi dediti alla comprensione di meccanismi di base e implicazione in patologie come lo stroke cerebrale. E’ stato infatti un promotore di una prima riunione di ricercatori interessati a questo ambito di ricerca a Firenze nel 1985 con l’obiettivo di costituire un “Purine Club Italiano”. La sua prima pubblicazione scientifica del 1955 è stata seguita da oltre 300 pubblicazioni su riviste internazionali e contributi su libri di cui è stato editore. Con il suo interesse, dedizione e passione è stato un catalizzatore per molti studenti che lo hanno seguito con entusiasmo nella vita scientifica formando un laboratorio attivo nel campo della Neurofarmacologia. Ha seguito con partecipe attenzione la carriera scientifica e accademica dei suoi allievi spesso anche fuori dai confini nazionali costituendo sempre un punto di riferimento fondamentale. Ha sempre promosso collaborazioni scientifiche trasferendo agli allievi l’importanza della internazionalità del mondo della ricerca. Professore attento alla didattica e al trasferimento di rigorose informazioni ha formato numerosi studenti nel campo della Farmacologia. Le sue numerose qualità, includendo la sua
cultura e la sua apertura di mente, ne fanno certo una persona che ha influito in modo importante nel
mondo scientifico- accademico ma anche nell’ambito personale di chi lo ha conosciuto.

Prof. Giancarlo Pepeu

Laurea ad Honorem a Jeoffrey Burstock da parte dell’Università di Ferrara (3-4 Giugno 2009)

 

 

Intervista al Prof. Giancarlo Pepeu

Firenze, 22 novembre 2017

 

tratta da  Felicita Pedata all’indirizzo WEB: 

http://www.sonsci.com/wp-content/uploads/2019/09/Pepeu_intervista-.pdf

 

Professor Pepeu, buonasera. La ringrazio per aver accettato di partecipare al nostro progetto. Vorrei iniziare questa intervista chiedendole qualcosa sui primi anni della sua formazione e su cosa l’ha spinta allo studio del cervello.

La mia formazione è cominciata qui a Firenze. Io sono in realtà di famiglia triestina e vivevo a Trieste. Sono venuto a Firenze perché i miei genitori hanno deciso che bisognava che io sciacquassi i panni in Arno [ride]. Sono venuto a Firenze nel 1948 e mi sono laureato nel 1954. Sono entrato in Farmacologia nel 1952 per preparare la tesi e ho lavorato, in quel periodo, con il professor Aiazzi Mancini, fondatore della Scuola farmacologica fiorentina. L’argomento della tesi è stato lo studio di un insetticida, il Parathion, che veniva utilizzato per combattere la mosca dell’ulivo. Visto adesso lo studio sembra un po’ assurdo perché cercava di dimostrare qual era il massimo contenuto di Parathion contenuto nell’olio di oliva per uso alimentare che non era tossico nel ratto. [ride]. Oggi sembra impensabile perché non è ammesso alcun insetticida nell’olio di oliva. Poiché il Parathion è un inibitore delle colinesterasi, questa ricerca mi ha obbligato a misurare le colinesterasi nel cervello dei ratti per dimostrare che l’olio contenente un certo numero di parti per milione, non ricordo quante, di Parathion non era tossico nel ratto., Certamente con gli occhi di oggi questa ricerca era totalmente inadeguata. E infatti il Parathion oggi per le coltivazioni di ulivo non si usa più. Finita la tesi, sono rimasto a Firenze perché mi è stato offerto immediatamente un posto, che adesso diremmo di post-doc, ma allora si chiamava pomposamente “assistente straordinario” con uno stipendio del tutto inadeguato per vivere, ma per fortuna mio padre, medico, mi aveva detto che se ero bravo era disposto a sostenermi finanziariamente per alcuni anni dopo la laurea. Dopo quattro anni sono riuscito ad andare in America. I rapporti con l’estero erano molto limitati a quell’epoca, non c’era quella rete che si è creata dopo e che ci ha permesso di mandare all’estero i nostri allievi con facilità. Ho scritto tante lettere in giro e un bel giorno è arrivata una risposta dalla Yale University dove esisteva una borsa di studio istituita dalla famiglia Toscanini per onorare la scomparsa di una ballerina, Fornaroli Toscanini, ed era dedicata a ricercatori italiani che volessero andare a Yale. Mi fu offerta, andai a Yale, dove trovai Nicola Giarman, che si occupava di sistema nervoso centrale. Il resto del dipartimento si occupava prevalentemente di chemioterapia dei tumori. Arrivato con un inglese piuttosto approssimato, ci furono le interviste, mi chiesero cosa volessi fare, con quell’atteggiamento di apertura con quale non ti impongono nulla di forza ma ti orientano, [il professor Giarman] mi propose di lavorare su tre possibili argomenti: la neonata serotonina, la noradrenalina, e l’acetilcolina. Mi disse che nessuno se ne occupava e feci una rapida valutazione. Di serotonina se ne occupava lui, personalmente. Mi ero già reso conto che sulla noradrenalina c’era già qualcuno in laboratorio che ci lavorava. Così gli dissi che mi interessava occuparmi di acetilcolina. Inoltre qualcosa sapevo già dalla mia tesi di laurea. Detto, fatto: e da allora per tutto il resto della mia vita mi sono occupato, non solo, ma prevalentemente del sistema colinergico. Non è stata una scelta, una vocazione, ma una serie di casualità come spesso succede nella vita. Da allora mi sono occupato di tanti aspetti del sistema colinergico e continuo ancora adesso.

Prima di soffermarci un po’ sul periodo a Yale vorrei chiederle una cosa: lei prima ha citato il professor Aiazzi Mancini e la scuola di Firenze. Cos’è stata la scuola di Firenze negli anni della sua formazione per quanto riguarda la farmacologia?

È stata una scuola che per alcuni anni è stata vivacissima. Non tutti si occupava di sistema nervoso centrale, anzi, prevalentemente si occupavano di cardiofarmacologia, poi c’era il gruppo di chemioterapia. Era molto articolata. E lo è ancora, anche adesso, anche se si è un po’ ridotta come si è ridotta tutta l’università. Tra i miei colleghi che si sono occupati di sistema nervoso centrale c’è stato Sandro Guidotti, che è andato in America, ha lavorato ed è ancora attivo alla University of Illinois di Chicago lavorando inizialmente come braccio destro di Erminio (Mimmo) Costa, di cui avrà sentito parlare perché ha formato molta gente in Italia. Un altro era Flavio Moroni che è andato in pensione dopo di me e si è occupato prevalentemente di trasmissioni aminoacidergica, di glutammato. Il direttore, Aiazzi Mancini è andato in pensione poco dopo che mi sono laureato, nel 1957 e il suo posto fu preso, per incarico, dal suo allievo Alberto Giotti. Tornai dagli USA, dove mi avevano offerto di restare, nel dicembre del 1960 perché Alberto Giotti mi aveva offerto di seguirlo come aiuto nella sede nella quale lui sarebbe andato dopo aver vinto il concorso. Il che significò che nella primavera del 1961 mi ritrovai assistente all’Università di Sassari e il passaggio da Yale a Sassari fu un trauma non da poco [sorride].

Quali furono le differenze, se c’erano, nei modi di portare avanti la ricerca che lei notò venendo dall’Italia e andando negli States?

Sa, era tutto diverso perché la prima cosa che colpiva era la ricchezza di quel momento. Uno esprimeva un desiderio e otteneva l’apparecchio di cui aveva bisogno. Poi c’era un forte senso responsabilizzazione e autonomia. Cioè io mi sono ritrovato a passare da un ambiente in cui, mi trovavo benissimo, ma gli esperimenti mi venivano indicati quasi quotidianamente tra “fai così e fai colà” ad una situazione in cui mi veniva dato il problema e tu cercavi la soluzione. Vai in giro, chiedi consiglio, dimmi hai bisogno di qualcosa, però mi porti i risultati tra una settimana e mi dici a che punto sei. Questo ovviamente è un modo di fare che ti motiva in una maniera tremenda perché sei tu che devi far tutto e decidere tutto. Questo è stato per me, se poi è capitato a tutti non so, a me è capitato ed è stato decisivo per la mia formazione ed è stato poi il metodo che io ho usato con il mio largo gruppo di collaboratori, con i quali ho sempre seguito la stessa tecnica: il problema è il tuo, vieni a chiedermi consiglio, ma le cose te le risolvi da te.

Chi ricorda dei suoi colleghi di lavoro in America?

Quello che era il mio capo in quel momento era appunto Nicola Giarman, che allora era professore associato, poi divenuto ordinario o full professor, che purtroppo è morto in un incidente automobilistico poco prima di trasferirsi a Harvard. Era un uomo che nonostante il cognome era di discendenza italiane, suo nonno veniva dalla Puglia e come molti italiani aveva cambiato il nome durante la guerra. Delle altre persone che si occupavano di sistema nervoso centrale e con le quali ho collaborato c’era uno psichiatra che è diventato rapidamente molto famoso: Daniel (Danny) Freedman. È stato uno dei più importanti psichiatri americani di formazione psicoanalitica che però aveva anche interesse per la parte biologica delle malattie mentali e ho pubblicato con lui, un paio di lavori. Era un uomo di straordinaria intelligenza. [sfoglia pagine e mostra un volume]. Gli altri molti amici scomparsi erano gente brava ma non hanno lasciato una traccia importante, salvo Bill Prusoff nel campo degli antivirali.

Su cosa avete lavorato lei e il professor Freedman?

Il primo lavoro che mi viene in mente subito, non si sorprenda di lavori ne ho fatti tanti, sono passati sessanta anni [ride], è stato su una sostanza che in quel momento era appena entrata nella ricerca e in terapia: il Dimetilaminoetanolo. C’era l’idea, che ho smentito, che questa sostanza aumentasse i livelli di acetilcolina e per questa via, in qualche maniera avrebbe potuto influenzare i processi cognitivi. Come vede dopo sessanta anni siamo sempre allo stesso punto.

Questo testimonia però il fatto che ci fosse un’attenzione nei confronti dell’incontro tra psichiatria, psicologia cognitiva e farmacologia.

Sì, assolutamente.

Grazie professore, torniamo invece in Italia e alla proposta di Alberto Giotti che poi la condusse al Sassari nel 1961. Cosa arrivò questa offerta e cosa ha significato per lei andare a Sassari?

Arrivò una lettera che purtroppo non mi riesce di ritrovare tra le tante carte che ho conservato, anzi che mia madre ha conservato [ride]. Nella primavera del 1960 mi fu offerto di restare in America da Giarman e dal direttore del dipartimento Arlnold Welch, un chemioterapista. Addirittura mi fu offerto di restare lì per essere il responsabile di una unità di neurofarmacologia. Io accettai, devo dire, però avevo usufruito di una borsa di studio Fulbright per coprire i costi di viaggio: il biglietto per il viaggio, in nave, fu pagato in questo modo. Per questo avevo l’obbligo di ritornare in Italia. L’ufficio legale di Yale chiese agli organi predisposti se si poteva ottenere un’esenzione ma fu risposto che dovevo in tutti i modi rientrare in Italia per almeno un anno. Simultaneamente, nel giro di poche settimane, arrivò la lettera di Giotti in cui mi chiedeva se fossi disponibile a seguirlo nella sede in cui sarebbe stato chiamato, dato che aveva vinto da poco il concorso. Io mi trovavo con la porta americana chiusa e comunque per tenerla aperta avrei dovuto ricorrere a tutta una serie di manovre, il che non mi andava e la speranza di riuscire era debole. L’alternativa era venire in Italia, col posto offerto la carriera era quasi assicurata e con la speranza, che allora avevo, che il Paese sarebbe migliorato e avrebbe investito nell’Università e nella ricerca. Inoltre avevo i genitori anziani - che non hanno interferito nelle mie decisioni, ma mi faceva piacere di essere più vicino a loro. E così sono rientrato a Natale ’61 per poi andare due mesi dopo a Sassari. Dove non c’era niente. Devo dire che nel periodo di Sassari con Giotti e con quei pochi soldi che riusciva a raccogliere abbiamo costruito un piccolo Istituto ma io non ho una produzione scientifica fatta a Sassari. Tuttavia nella seconda metà del 1962 tornai in America per sei mesi per completare ricerche lasciate in sospeso. Giotti fu generoso. Partii alla fine della sessione di esami per tornare in autunno.

Dove andò?

Sempre a Yale. Furono mesi molto produttivi perché ormai ero entrato nel meccanismo e c’erano molti lavori da finire.

L’Istituto di Sassari era dedicato alla neuro-farmacologia?

No, l’Istituto di Sassari era l’istituto di una piccola Università, lo è anche adesso in fondo, con un carico di didattico pesante. Dovevamo tenere i corsi di farmacologia a medicina e farmacia ma non veterinaria. A Sassari diventai assistente ordinario, passo decisivo della carriera universitaria e presi la libera docenza in Farmacologia e Farmacognosia e imparai a insegnare, a fare lezione. Questo perché Giotti veniva spesso nel Continente e quindi toccava a me, con un paio di giovani locali. Tutto era molto limitato dai pochi soldi disponibili. Come Giotti faceva notare, il bilancio dell’Istituto era molto più piccolo di quello che aveva una normale famiglia [ride].

Quando ci fu un punto di svolta?

Il punto di svolta fu quando Giotti fu chiamato a Pisa. A Pisa trovai una situazione del tutto diversa. Prima di tutto per un anno andai a lavorare da Moruzzi. Poi a Pisa fui in grado di ricominciare a dosare l’acetilcolina, con il metodo biologico che avevo usato a Yale, e sviluppai la tecnica delle coppette corticali per studiare la liberazione di acetilcolina dalla corteccia cerebrale. Pubblicai insieme ad Adolfo Pazzagli, poi diventato professore di psichiatra a Firenze, un lavoro sui rapporti tra amnesia indotta da scopolamina e i livelli cerebrali di acetilcolina. Anche se l’ipotesi conteneva un errore di fondo, fu un lavoro molto citato in quelli anni. Fu Giotti che in quel periodo disse “senti vai a lavorare da Moruzzi”. In fondo la neurofarmacologia italiana non aveva ancora una sua precisa identità. Le neuroscienze erano parte della fisiologia nella quale operavano scienziati di valore internazionale quali Moruzzi e Infantellina. Avere lavorato con Moruzzi era come aver ottenuto un timbro di garanzia. Così sono andato da Moruzzi, nel cui laboratorio mi sono occupato di tutt’altro che l’acetilcolina. Venni messo a lavorare con Marchiavafa sull’elettrofisiologia del sistema ottico. Fu l’unico mio periodo come elettrofisiologo. Registravamo l’attività dei neuroni del tetto ottico del gatto in risposta a stimoli visivi. Il contatto con Moruzzi fu interessante e utile. Veniva a vedere come procedevano gli esperimenti e il suo rigoroso modo di ragionare era affascinante. Gli proposi di provare dei farmaci e la reazione fu “come si può pensare di immettere qualcosa di cui non si conosce niente in un sistema che è ancora ignoto?” [ride]. Devo dire che fu un’esperienza molto utile per me sotto tanti punti di vista. Conobbi diverse persone poi diventarono ricercatori importanti come Berlucchi, Rizzolati, Strata. Poi non ebbi occasioni per collaborare con loro ma è rimasto un rapporto di reciproca stima e conoscenza che mi ha sempre fatto piacere.

Nonostante la battuta di Moruzzi questo testimonia l’interazione tra campi diversi di studio del sistema nervoso centrale?

Sì, che poi l’atteggiamento negativo di Moruzzi non era del tutto vero perché quando le cose non andavano bene, si iniettava un po’ di anfetamina per tirare su il gatto e si proseguiva [ride].

Nel frattempo al suo lavoro con Moruzzi alternava l’attività con Giotti a Pisa.

Sì, andavo al mattino all’Istituto, facevo lezione e stavo con il mio embrionale gruppo. Poi andavo da Moruzzi, lavoravo lì diverse ore e poi nel tardo pomeriggio ritornavo in farmacologia. Erano anni in cui lavoravo praticamente senza limiti.

Ci puo’ parlare di questo periodo pisano, di che cosa era l’ambiente della farmacologia e della neurofarmacologia a Pisa negli anni Sessanta?

Il Dipartimento di Farmacologia era piccolo. Quando siamo venuti via, Giotti lavorava con alcuni collaboratori su problemi di cardiofarmacologia e io lavoravo sul sistema nervoso centrale, ovviamente. Pubblicai diversi lavori e tra l’altro venni in contatto con Giovanni Cassano allora studente, diventato poi un famoso psichiatra. Iniziai a collaborare con un amico che diventò professore di neurologia a Firenze, Luigi Amaducci. Lo avevo conosciuto in una circostanza divertente. Chi vinceva una borsa Fulbright, nel mio caso la borsa per il viaggio, doveva seguire un training program, indovini dove? A Napoli, provi a immaginare quanta America poteva esserci. Per una settimana andammo in un palazzo seicentesco affittato dalla Fulbright dove ci insegnavano come si viveva in America. Ridicolissimo. Lì però ho conosciuto diverse persone, oltre Luigi Amaducci, allora assistente a Modena. Ci siamo ritrovati in America, lui era a Harvard, io a Yale ed eravamo sufficientemente vicini per incontrarci. A Harvard avevo alcuni amici venuti da Firenze e si formò un gruppettino e si consolidò l’amicizia. Quando Amaducci tornò in Italia, le vicende accademiche lo portarono a Firenze e al mio ritorno dall’America condivisi con lui un appartamentino che fu il mio pied à terre fiorentino quando ero a Sassari. Cominciò una collaborazione che si materializzò prima a Pisa e poi divenne intensa dopo il mio ritorno definivo a Firenze. Come già raccontato, a Pisa conobbi Cassano, che da studente venne da me un giorno portandomi un lavoro che descriveva una nuova tecnica, sviluppata in Svezia, che allora sembrava rivoluzionaria ed è ancora usata solo in qualche industria farmaceutica, per visualizzare la distribuzione dei farmaci nell’organismo. Veniva iniettato in vena in un topo un farmaco marcato con un isotopo radioattivo, per esempio la nicotina marcata con trizio. Poi il topo veniva congelato in un tempo brevissimo a – 80 °C, tagliato a fette sottilissime con un grosso microtomo congelatore e le fettine erano poste a contatto con carta fotosensibile sulla quale compariva la mappa della distribuzione della nicotina in tutto il corpo compreso il cervello. Cassano venne da me con questo lavoro appena pubblicato da ricercatori svedesi e mi disse “io vorrei fare questo”. Gli risposi che era molto bello ma che io non ero in grado di comprare l’attrezzatura. “Lei certamente può permettersi di andare in Svezia”, gli dissi. Era figlio di Cataldo Cassano, clinico medico a Pisa e senatore. Andò in Svezia, ci rimase per due anni, il primo anno a proprie spese e l’anno dopo pagato, e ritornò con tutta l’attrezzatura e mise su la tecnica. Pubblicò diversi lavori di cui un paio anche insieme a me. Uno, pubblicato sugli Archives Internationales de Pharmacodynamie sulla distribuzione del glucosio nel cervello di gatto, di cui mi ero totalmente dimenticato perché non fu un lavoro glorioso e i risultati furono abbastanza ovvi ma, con mia sorpresa, è stato citato pochi giorni fa. Gli Archives era una rivista fondata dal premio Nobel belga Heymans, che è vissuta fino a quando è nato lo “European Journal Pharmacology”, sua diretta continuazione.

In quegli anni i suoi temi di ricerca continuarono a essere collegati all’acetilcolina. Ricorda scoperte o laboratori?

Una delle cose più importanti è stato il lavoro con Pazzagli sul rapporto tra scopolamina, modifica dei livelli di acetilcolina e amnesia che mi portò per la prima volta a studiare i meccanismi della memoria. Mi ero accorto che se addestravo a lungo il ratto nel labirinto l’effetto amnesico di scopolamina scompariva perché evidentemente cambiava il tipo di memoria nella quale il ricordo del percorso del labirinto era immagazzinato. A Pisa pubblicai anche un lavoro su Science sulle differenze nei livelli di acetilcolina nella corteccia cerebrale nel sonno e nella veglia. I lavori di elettrofisiologia li ho persi di vista , ne pubblicai due con il gruppo di Moruzzi ma non so se abbiano avuto un minimo di risonanza o meno. Da Pisa tornai a Firenze nel 1965 e ci rimasi per tre anni come aiuto di Giotti, in qualità di assistente ordinario con titolo di aiuto.

A Firenze l’ambiente com’era? Ricorda qualche collega particolare?

L’ambiente era ancora abbastanza provinciale ma cominciava ad aprirsi. Paolo Procacci, iniziava a lavorare sul dolore e mi coinvolse nella fondazione dell’Associazione Italiana Studio Dolore e nell’organizzazione del First World Congress on Pain. Amaducci inseriva la neurologia fiorentina nel circuito internazionale. In clinica c’erano Francesco Antonini, il fondatore della geriatria italiana e Federico Sicuteri con le sue ipotesi sulla patogenesi della emicrania ma mi lasciava molto perplesso, specie nel suo modo di fare. Era un uomo intelligente e brillante ma forse un po’ troppo immaginifico dal punto di vista scientifico. Poi nel 1968 vinsi il concorso e divenni professore ordinario di farmacologia nella facoltà di Farmacia di Cagliari.

Prima di aprire la parentesi di Cagliari vorrei domandare cosa pensa dello stato della neurofarmacologia negli anni Sessanta. Già allora, in particolare, erano evidenti le applicazioni pratiche per aspetti legati alla cognizione e al comportamento. Nel corso della sua carriera in questo decennio, la maggior parte dei finanziamenti per la ricerca provenivano dal pubblico o cominciavano già ad affacciarsi dei privati?

In quegli anni lì e anche negli anni cagliaritani i finanziamenti venivano dal Ministero e dal CNR ed erano accettabili come quantità. Non che si nuotasse nell’oro, però i fondi che Giotti riusciva a passarmi erano sufficienti. Non c’era quella situazione di ansia e angoscia che hanno adesso i miei ex-collaboratori, che non riescono più a trovare soldi sufficienti. A Cagliari i fondi in pratica li ricevevo tutti dall’Università e dal CNR, non avevo ancora rapporti con l’industria. Nel momento in cui l’università si rendeva conto che il docente stava a Cagliari e non era un docente “aeronautico”, come venivano chiamati quelli che facevano avanti e indietro, ma che prendeva casa e stava a Cagliari, aiutava molto. I funzionari dell’Università avevano una rete di rapporti romani. Se andavo da loro dicendo che avevo bisogno di comprare una centrifuga mi veniva suggerito di andare a Roma, al Ministero della Pubblica Istruzione dove c’era un “cugino” che mi poteva aiutare. E io andavo al Ministero, davo la mancia al portiere che mi accompagnava dal “cugino” che mi ascoltava con cortesia, non mi prometteva niente, mi raccomandava di portare i suoi saluti al cugino di Cagliari e dopo un mese arrivava una assegnazione speciale di fondi per l’acquisto della centrifuga. Credo che siano abitudini scomparse.

Veniamo al ’68, periodo del concorso da ordinario e dell’arrivo a Cagliari.

Mi ritrovai a Cagliari di nuovo senza laboratorio e senza collaboratori. Per un breve periodo potei disporre di un laboratorio a Firenze e di un paio di collaboratori con i quali continuai lo studio degli effetti di psicofarmaci sulla liberazione di acetilcolina, andando a Firenze il week end e lunedì. Rimasi a Cagliari sei anni e in questo tempo riuscii ad attrezzare un Istituto di 9 locali compresa un’aula e formai un gruppo di una decina di collaboratori fra assistenti, borsisti e tecnici con i quali pubblicai un discreto numero di lavori. Una parte delle attrezzature potei poi portarle a Firenze perché erano apparecchi comprati con i fondi del CNR. Portai anche i fondi del CNR che erano abbastanza sostanziosi per ricominciare a lavorare a Firenze quando vi ritornai nel 1974. A Cagliari ripresi a studiare i meccanismi colinergici cerebrali sia misurando i cambiamenti del contenuto di acetilcolina in varie regioni del cervello sia studiando la liberazione di acetilcolina dalla corteccia cerebrale con la tecnica della coppetta. Questa utile tecnica consisteva nell’aprire la teca cranica, rimuovere la dura meninge e applicare un piccolo cilindro di plastica sulla corteccia del gatto o del ratto. Si formava così una coppetta nella quale veniva messa una soluzione di Ringer in cui diffondeva l’acetilcolina dai tessuti sottostanti. Questa tecnica è stata utilizzata fino alla introduzione della microdialisi e ha fornito dei dati importanti. Ho pubblicato lavori che hanno avuto una buona diffusione per i dati interessanti che contenevano. Gli anni trascorsi a Cagliari furono importanti per la mia formazione accademica e potei inserire diversi giovani nella ricerca. Quando arrivai a Cagliari, sulla cattedra di farmacologia di medicina c’era il prof Rodolfo Paoletti, di scuola milanese. Eravamo amici ma non collaborammo perché si occupava di metabolismo lipidico. Dopo qualche tempo Ghighi Gessa tornò dagli Stati Uniti e cominciò la crescita della scuola farmacologica cagliaritana che allora si occupava soprattutto di dopamina. Diventai membro della British Pharmacology Society, la società inglese di farmacologia, e partecipai frequentemente alle sue riunioni in Inghilterra che mi erano molto utili dal punto di vista culturale e per l’inserimento internazionale.

In quegli anni quali erano i trend internazionali nell’ambito della ricerca farmacologica?

Era il momento della dopamina e degli antipsicotici, fondamentalmente. C’era anche la serotonina, ma l’interesse era soprattutto per la dopamina. Cominciavano a diffondersi gli antidepressivi ed era il momento degli inibitori delle monoaminossidasi. Mentre ero a Cagliari organizzai con Gessa un convegno in onore del prof. Herman Blaschko di Oxford, scopritore proprio di questi enzimi. Era un periodo molto divertente dal punto di vista culturale, stimolante, con tutti questi nuovi farmaci. Tentai di lavorare anche sui rapporti tra acetilcolina e serotonina, ed ero interessato soprattutto alla corteccia cerebrale. Per cui in realtà quello che facevo suscitava poco interesse perché l’interesse era tutto era concentrato sullo striato. Tuttavia dimostrai, senza troppo successo come pubblicazioni, che esisteva un rapporto tra le due sostanze a livello corticale, cosa che adesso sappiamo nel dettaglio più fine. Furono sei anni pesanti, dal punto psicologico, perché non volevo restare a Cagliari e quindi avevo l’idea che prima o poi sarei andato via. Poi fu richiamato a Firenze da Giotti perché in quel momento c’erano 1200 studenti iscritti al IV anno e i corsi andavano divisi. Era un momento cruciale per l’Università e rientrai a Firenze.

Un’ultima domanda sul periodo cagliaritano. Gessa non era ancora rientrato, ma ricorda qualcuno tra i suoi allievi?

Di Chiara lo ricordo. Era studente e molto vivace. Dovevano ancora formare il gruppo, non esisteva. Gessa aveva fatto dei bei lavori in America e aveva una rete importante di rapporti internazionali, ma non c’era ancora una scuola, che poi è nata subito dopo.

Il ritorno a Firenze. Finalmente ce la fece.

Tornai a Firenze e comprai casa [ride]. Cominciai a costruire il mio gruppo. A quel punto avevo anche lì la mia completa autonomia: Giotti era il direttore e non è che accettasse molto questa mia indipendenza, ma dopotutto si occupava di ben altro. Cominciò quel periodo fortunatissimo per le neuroscienze italiane che fu l’esistenza di Fidia e del suo competitore Sigma-Tau. Questo per la quantità di denaro che Fidia investiva nella ricerca per studiare i suoi prodotti che, a prescindere dalla maggiore o minore efficacia e utilità clinica, erano interessanti dal punto di vista scientifico. Avendo quei fondi potevo fare anche altre cose, to perché i fondi che ottenevo da Fidia non erano vincolati solo per lo studio dei suoi prodotti ma potevo usarli per studiare il sistema colinergico. Furono anni d’oro che mi permisero di pagare borse di studio, di mandare in America alcuni studenti per brevi soggiorni, ad esempio partecipare ai Neuroscience meetings. Io stesso andavo con i miei studenti in America a questi grandi eventi scientifici che cominciavano ad affermarsi. C’era poi tutto un altro versante finanziato da Sigma-Tau ma non ero inserito. C’erano anche i soldi del CNR ottenibili con relativa facilità, insomma sono stati anni molto floridi. Andavo in America molto di frequente c invitato a congressi e nella stagione dello Neuroscience per quasi un mese in quanto, prima e dopo il meeting tenevo numerosi seminari in diverse università e industrie. Ricordo che in un mese arrivai a fare almeno sei seminari sul sistema colinergico. Mettemmo a punto la tecnica della microdialisi per studiare la liberazione di acetilcolina anche da strutture cerebrali profonde in ratti liberi di muoversi. Pubblicai tanto in quel periodo e costruì il gruppo che poi è rimasto unito anche dopo il mio pensionamento. Fra l’altro dimenticavo i miei rapporti con Andrea Nistri, professore alla SISSA, mio studente proprio nel periodo in cui sono andato a Cagliari. Con lui i ho avuto molte interazioni al punto che lo considero il mio primo e migliore tra gli allievi: è molto conosciuto, vada a trovarlo perché è bravo.

Su cosa lavoravate insieme con Nistri?

Abbiamo pubblicato un lavoro sull’effetto dell’anfetamina sui gatti, in particolare sul sistema colinergico e sui rapporti con la dopamina. Nistri iniziò subito a chiedermi di lavorare sul midollo spinale, l’argomento principale delle sue ricerche per tutta la sua vita. Lo finanziai, e pubblicò un paio di lavori sul sistema colinergico nel midollo spinale di rana. Lui insistette e io lo lasciai fare, nonostante mi interessasse poco il midollo spinale. Poi con me lavorò Felicita Pedata, attualmente ordinario di farmacologia nella Scuola di Scienze Biologiche a Firenze. A un certo punto nello studiare i meccanismi di regolazione della liberazione dell’acetilcolina saltò fuori la adenosina, che a livello presinaptico modula la liberazione dell’acetilcolina. Da allora passai a lei il tema della adenosina e in tutti questi anni lei ha lavorato costantemente per un po’ di tempo insieme a me, nel senso che discutevamo gli esperimenti e i lavori venivano scritti insieme, poi acquistò totale autonomia. Adesso è una delle esperte di purine in Italia ed è attualmente presidente dell’Italian Purine Club, società che si occupa dello studio dell’adenosina e delle altre purine. A Firenze il rapporto con Amaducci divenne più stretto e venni trascinato nel campo dell’Alzheimer nell’ambito del progetto finalizzato del CNR sull’invecchiamento. Da allora mi sono interessato alla malattia di Alzheimer e cominciammo a lavorare su ratti vecchi nei quali dimostrammo una ipofunzione colinergica. Il rapporto tra Alzheimer e sistema colinergico è particolare e ben noto. Fui il primo a distruggere il nucleo basale e studiare gli effetti sul comportamento, seppure in maniera impropria perché distruggevo il nucleo basale mediante elettrocoagulazione causando danni un po’ estesi. Però i dati che pubblicai sugli effetti sulla distruzione del nucleo basale sono stati forse i primi lavori nel campo e non sono stati smentiti da nessuno. Non erano dati puliti ma davano informazioni sui rapporti fra sistema cognitivo e sistema colinergico. Facemmo e pubblicammo numerosi lavori sulla fosfatidilserina. I lavori sulla fosfatidilserina continuano a essere citati perché questa sostanza, assunta per bocca è venduta nelle farmacie come nutraceutico. Credo che per bocca non faccia nulla. Però nel ratto vecchio, somministrata per via parenterale, i suoi effetti di miglioramento del sistema colinergico sono sorprendenti. Sono stati anni in cui ho prodotto bene tant’è vero che, con mia sorpresa, secondo Research Gate ho attualmente superato le 11.000 citazioni.

Qual era lo stato dell’arte dello studio su questa malattia?

Nasceva proprio in quel momento. Quando iniziai si parlava di demenza senile e arteriosclerosi. Lo studio dell’Alzheimer l’ho visto svilupparsi proprio in quegli anni lì.

C’era interesse da parte delle aziende e del settore pubblico o c’era una ricerca spinta da una sola delle parti?

All’inizio la ricerca sull’invecchiamento fu sviluppata dalle aziende private, ma poi fu lanciato un grande progetto sull’invecchiamento da parte del CNR. Voleva indirizzare la ricerca del Paese verso questo problema coinvolgendo neurologi, geriatri e farmacologi. Fu un progetto, mi sembra quinquennale molto intelligente, condotto bene, con abbastanza soldi, era utile ed era un’occasione per ritrovarsi e discutere insieme. Fu una delle cose più utili fatte dal CNR. Si chiamava “Progetto finalizzato invecchiamento”. Venni in contatto con diversa gente che occupava posizioni importanti in Italia. In quel periodo ero influenzato fortemente dal punto di vista culturale da Francesco Antonini, forse questo nome dice qualcosa, il fondatore della geriatria italiana. Ho insegnato anche farmacologia nella Scuola di specializzazione di Geriatria da quando ero a Cagliari fino a che le scuole erano qualcosa di decente. Poi un bel giorno, nessuno mi disse qualcosa, morì, come la Scuola di Psichiatria. Insegnai anche in quella Scuola, ma improvvisamente gli specialisti non vennero più perché non c’era tempo per la didattica e dovevano stare tutti in corsia.

Mi diceva che queste erano foto della Scuola di Farmacologia.

Qui era il 1957, Aiazzi Mancini lascia, va in pensione, e Giotti viene ufficialmente nominato suo successore anche se poi la cattedra a Firenze l’ha avuta dopo qualche anno. Questi sono allievi. Eccomi qui, travestito da americano insieme a tutto il gruppo di farmacologi a Yale. Questo nel momento in cui siamo venuti via da Pisa, questa è mia moglie che avevo da poco conosciuto e che aveva iniziato a lavorare con me.

Firenze e l’inizio degli studi sull’Alzheimer. Qual è il suo coinvolgimento sulla prima fase di studio sull’Alzheimer in Italia?

In quegli anni il mio rapporto con lo studio di questa malattia aveva due aspetti: il rapporto con la clinica, con la geriatria, nella quale non facevo ricerca pur essendo al corrente di cosa si facesse; e l’altro aspetto, gli inibitori delle colinesterasi, di cui mi sono occupato dal primo giorno in cui sono entrati in uso per il trattamento dell’Alzheimer. Mi sono occupato della malattia attraverso la Scuola di Geriatria e la Clinica neurologica con Amaducci per quanto riguarda gli aspetti clinici, ma senza far ricerca, o solo ricerca marginale. Ho un paio di lavori con Amaducci, uno sul piano statistico-diagnostico. Il secondo piano è sugli animali da esperimento, con i quali ho cominciato a lavorare sui farmaci inibitori delle colinesterasi che per un breve periodo hanno rappresentato una speranza, con quella illusoria ipotesi per cui il sistema colinergico stava all’Alzheimer come il sistema dopaminergico stava al Parkinson. In quel periodo ho collaborato con la Bayer, ho avuto alcuni contratti perchè stava cercando di sviluppare un nuovo inibitore delle colinesterasi per cui ho fatto più studi sul suo effetto sulla liberazione dell’acetilcolina dalla corteccia cerebrale e dall’ippocampo. Avevo in mano la tecnica della micro-dialisi e si sono rivolte a me diverse industrie con contratti ben precisi perché conveniva loro pagare il lavoro fatto da me e dai miei collaboratori anziché mettere su la stessa tecnica nei loro laboratori. Questo ovviamente mi permetteva di farmi pagare bene e di reinvestire quei soldi in borse di studio, viaggi con gli studenti. Per cui in tutto questo tempo, dagli Ottanta, fino al pensionamento nel 2005, non ebbi problemi finanziari e riuscii sempre a mantenere i diversi gruppi dando loro autonomia. Lavoravamo molto su animali vecchi. Facevamo lesioni del nucleo basale con varie tecniche sempre più perfezionate, poi sono riuscito a procurarmi uno dei tanti ceppi di topolini transgenici che sviluppano una patologia simile all’Alzheimer. Comprarli allora era molto costoso, ma un amico canadese mi regalò due coppie di topolini transgenici con cui poi è stata fatta una colonia con cui continuano a lavorare studiosi come la prof.ssa Fiorella Casamenti, che è stata una mia collaboratrice per molti anni. In questo periodo c’è tutto lo spettro della ricerca sui vari modelli di Alzheimer e sui farmaci da usare per contrastarlo. Non posso dire che ho scoperto cose sensazionali, certamente, perché non è un campo in cui si arriva in fondo studiando solo il sistema colinergico e io non sono un neuropatologo per cui gli aspetti morfologici o patogenetici non li posso studiare. Tuttavia mi sono divertito in quegli anni. E poi il numero delle citazioni dei miei lavori di quelli anni è alto, gli articoli sono citati frequentemente e ciò dimostra che erano e sono ancora utili. Con questo arriviamo al 2002 quando andai fuori ruolo e smisi di fare lezione ma, dato che avevo la possibilità, sono rimasto fino al 2005 quando sono andato definitivamente in pensione. Poi, sono stato nominato professore emerito e continuo ad andare in dipartimento, una volta alla settimana, e a pubblicare qualche lavoro. Questa estate ho pubblicato una review sul sistema colinergico e le malattie neuro- degenerative su “Brain Research”.

Oltre alle pubblicazioni che continua a sfornare ha anche lasciato una scuola a Firenze.

Sì, ma nessuno segue più la linea colinergica, che è morta con me. C’è Renato Corradetti che si occupa di serotonina ed elettrofisiologia. Felicita Pedata si occupa di adenosina e della numerosa popolazione di recettori dell’ adenosina e delle purine in generale, e del loro coinvolgimento nell’ictus. Fiorella Casamenti è rimasta legata ai problemi della malattia di Alzheimer e adesso si occupa di una sostanza derivata dall’olio di oliva, oleoeuropeina, come mi sembra si chiami, che ha effetti meravigliosi nei topolini transgenici. Credo stiano iniziando un piccolo trial clinico con i limitati fondi disponibili, perché le sperimentazioni cliniche su larga scala costano cifre iperboliche. Ha ottenuto addirittura un finanziamento dalla Regione Toscana su questo progetto. Poi c’è Mariagrazia Giovannini che si occupa in parte di Alzheimer, ma in soprattutto dei rapporti fra neuroni e glia. Quello che mi fa effetto è vedere come in laboratorio ci sia adesso tanta istochimica che ha raggiunto dei livelli inaspettati di specificità e utilità con l’immuno-istochimica e con la possibilità di quantificare le immagini. Ricordo quanta fatica ho fatto a suo tempo per visualizzare i neuroni colinergici e mettere in evidenza la colinoacetiltransferasi. Ce n’è voluto prima di metterla su perché eravamo del tutto sprovveduti in quel campo lì…

Una retrospettiva. Cosa ha visto cambiare nella farmacologia nel corso della sua carriera e in particolare all’interno del panorama italiano?

Prima di tutto devo dire che forse sono uno degli ultimi che ha visto un ospedale psichiatrico prima dell’arrivo degli antipsicotici. L’ho detto spesso ai miei studenti. Durante l’estate tornavo dai miei genitori a Trieste. Mio padre, medico, non voleva che restassi in casa a non far niente e mi affidava a uno dei suoi amici e così frequentai diversi reparti. Nell’estate del 1953 andai per diverse settimane all’ospedale psichiatrico della città. Non era ancora quello di Basaglia ma era considerato una struttura moderna, a piccoli padiglioni separati. In quell’estate non erano ancora arrivati gli antipsicotici, perché la sintesi della clorpromazina era del 1951 e l’entrata in commercio del 1954. Non c’erano farmaci ma c’era l’ elettroshock. E ne ho visti fare ogni giorno. La mattina era come una catena di montaggio. C’erano i barbiturici. A parte queste due terapie, il manicomio era come quello descritto da Mario Tobino nel suo libro “Le libere donne di Magliano” del 1953. Quindi ho visto questa enorme trasformazione. Nei libri di farmacologia del 1952 non c’era niente e poi con l’arrivo degli antipsicotici è nata la neurofarmacologia. Le neuroscienze erano allora dominio della fisiologia, come le ho detto prima. I suoi temi principali erano l’epilessia, il sonno con le ricerche di Moruzzi . I a parte i barbiturici, non c’erano farmaci e quindi nessuno li studiava; tutto è cominciato proprio alla metà degli anni Cinquanta. Il mio primo lavoro fatto a Yale su farmaci e acetilcolina cerebrale, guardato con occhi odierni, è grossolano e primitivo. Tuttavia c’erano in letteratura solo tre lavori sull’effetto dei farmaci sui livelli di acetilcolina nel cervello prima del nostro. Pubblicato sul British Journal of Pharmacology ha avuto centinaia di citazioni. Quindi tutto è nato in quel momento e sono subito nati gruppi brillantissimi. Pensi al gruppo della scuola cagliaritana, che è basata sull’intelligenza dei suoi collaboratori ma che deve molto all’esperienza americana in questo campo. Come diceva lei, siamo figli ben riusciti della neurofarmacologia e delle neuroscienze americane [ride]. Quello che mi dispiace è che in questo momento ho l’impressione che ci sia un disfacimento: in primis perché ci sono pochi soldi; secondo, perché ci sono stati molti insuccessi, guardi al campo dell’Alzheimer abbandonato dalle multinazionali farmaceutiche perché gli enormi investimenti fatti non hanno dato i risultati attesi. Il tutto in attesa che qualcuna delle start-up o dei piccoli gruppi venga fuori con un’idea geniale e brillante. Va comunque ribadito che in questo momento la carenza di fondi per la ricerca in Italia è drammatica.

Prima raccontava di un certo momento della sua carriera in cui i fondi venivano dal settore pubblico. Poi l’ingresso dell’industria farmaceutica. Oggi col paradigma traslazionale assistiamo a un’inversione di tendenza?

Sì, ma le industrie non investono più nelle neuroscienze perché non ne vedono più la vera utilità.

E questo non rischia di generare un circolo vizioso per cui ci sono sempre meno soldi per la ricerca?

Lo si sta creando e c’è meno gente. I giovani più bravi vanno via. L’ultima delle mie allieve più brave, che si è laureata con me in Scienze biologiche nel 2002-2003, è capitata nel momento nel quale mi fu offerto un piccolo contratto da un’industria straniera per studiare l’effetto di un nuovo “cognition enhancer” sul sistema colinergico.. Così chiesi alla Giovannini a chi si poteva affidare l’esecuzione del progetto, sotto la mia guida. Mi disse che c’era questa ragazza appena laureata, Susanna Rosi, molto brava e intelligente. Così chiamo questa ragazza dicendogli che gli offrivo un anno di stipendio fino al mio pensionamento, dopo il quale non potevo offrire nulla. Capiva benissimo che era solo per un anno ma lei accettò. Condusse il progetto, pubblicammo un lavoro, niente di straordinario, ma si rispondeva alla domanda postaci dimostrando che il farmaco stimolava il sistema colinergico. Proprio allo scadere del contratto di questa ragazza, uno dei miei amici americani mi disse che aveva un posto libero da affidare a qualcuno in gamba. Chiamo la ragazza, le ribadisco che non potevo fare nulla per lei dopo il contratto ma le offrivo la possibilità di andare negli Stati Uniti. Lei mi dice che è fidanzata, ci deve pensare …[ride]. Tre giorni dopo mi informa che accetta: “e il fidanzato?”, chiedo, “l’ho lasciato”, dice in lacrime. Era il 2004. Questa ragazza oggi è full-professor di neuroscienze alla University of California a San Francisco con un laboratorio di 10 collaboratori e grants dal NIH, dalla NASA e da fondazioni private. Abbiamo perso una ricercatrice con doti intellettuali e capacità non comuni che rimpiange di non aver avuto possibilità di carriera in Italia.

Durante la sua carriera lei ha attraversato non solo la neurofarmacologia ma, dall’incontro con Moruzzi alle varie collaborazioni con neurologi, clinici e neuro- psicologi, come dicevo ci rendiamo conto che esiste una specie di eccezione italiana per quanto riguarda le neuroscienze. Può dare un suo giudizio su quello che sono state le neuroscienze nel XX secolo ed eventualmente una raccomandazione per il futuro delle neuroscienze in Italia.

Le neuroscienze italiane si sono sviluppate perché c’erano solide basi culturali, una forte fisiologia attiva nel campo del sistema nervoso centrale e una buona neurologia con interessi sperimentali. Non nascevano nel vuoto, si sono formate da solide radici. Poi, con lo sviluppo dei nuovi farmaci, è comparsa la neurofarmacologia, il che è stato di estrema utilità anche per le neuroscienze non farmacologiche perché la neurofarmacologia ha fornito strumenti di indagine importanti e attratto finanziamenti. È stato un innesto importante. Personaggi come Gessa nascono farmacologi e diventano neuroscienziati. Io stesso non so se definirmi farmacologo o neuroscienziato. Il mio timore è che tutto questo muoia perché non ha finanziamenti adeguati, che sono diventati irraggiungibili. Anche l’iter burocratico per l’uso di animali è diventato complesso e quasi impossibile. Non dico di usare i gatti, per carità, ma anche l’uso di topolini transgenici richiede un iter burocratico assurdo dietro il quale c’è la chiara volontà di ostacolare la ricerca sugli animali. Noto nei miei colleghi stanchezza e frustrazione. Il risultato è che la produzione scientifica in questo campo si sta riducendo notevolmente. Lo vedo anche adesso, mentre sto scrivendo un lavoro in collaborazione con un amico italiano che è stato tanto in America e che ora ha la mia età ed è attivo a Ginevra, Ezio Giacobini, non so se l’ha sentito nominare. Ezio mi ha proposto un mese e mezzo fa di lavorare a uno studio, una rassegna, sulla gender influence sulla therapy of Alzheimer with cholinesterase inhibitors; io ho allargato il tema a gender influence on the cholinergic system and Alzheimer therapy. Sto scrivendo la parte pre-clinica e lui fa quella clinica. Mi rendo conto che la letteratura scientifica italiana ormai è scarsa, purtroppo. Io ovviamente non seguo il campo aminergico nel quale gli italiani hanno fatto tanto, però mi sembra che anche dal gruppo di Cagliari escano meno lavori. Un campo che io non seguo è quello delle drug addictions, come quella dell’alcool, della cocaina ecc. dove ci sono un po’ più di soldi per la ricerca perché rappresentano un problema sociale oltre che di salute pubblica. Però c’è un disperato bisogno di cambiare l’atteggiamento pubblico nei riguardi della ricerca scientifica e dei suoi finanziamenti. Che poi non occorrono cifre enormi, ma almeno un minimo. Oggi l’Università è in grado di fornire solo poche migliaia di euro ai dipartimenti per aiutare la ricerca. Ottenere fondi europei è un problema perchè la competizione è durissima. Renato Corradetti ha avuto un grant europeo e per qualche anno è stato un uomo felice perché le cifre assegnate sono cospicue. Nell’ultima parte della carriera ho provato a fare una richiesta ma l’Europa non finanzia ricercatori che hanno più di 65 anni. Nel panorama italiano, la SISSA è un posto abbastanza fortunato sia perché fornisce una dote di base abbastanza sostanziosa a tutti i ricercatori (anche se Andrea Nistri negli ultimi si è un po’ lamentato), e perché col nome prestigioso della SISSA è più facile entrare in progetti europei.

Andrebbe esteso a tutti, auguriamocelo.

Non mi faccio illusioni, anche perché l’interesse del Paese del paese e della sua classe dirigente per la ricerca è scarso. Della ricerca si parla continuamente come strumento per lo sviluppo, ma non si capisce che la ricerca deve essere prima di tutto libera, progettata bene ma senza obbiettivi pratici immediati. Questi nasceranno in un secondo momento e sarà il tempo della ricerca applicata. Cito sempre l’esempio del prof. Vittorio Erspamer, farmacologo, altra persona che ho avuto l’onore e il piacere di conoscere bene, che studiando i molluschi scoprì la serotonina che oggi sappiamo avere un ruolo in molte funzioni cerebrali nell’uomo. Non ha influenzato la mia ricerca ma è stato per me un esempio ed un riferimento. Fu membro della commissione nella Commissione del mio concorso a cattedra e poi l’ho incontrato più volte. Era un uomo straordinario per l’acutezza del ragionamento e le capacità sperimentali. Mi ricordo che uno dei pochissimi lavori che ho fatto sulla serotonina in America, che non era neanche sul sistema nervoso centrale ma sul sistema feto placentare, non era piaciuto a Erspamer, per cui andai a trovarlo per sentirmi fare le critiche direttamente [ride].

Grazie mille per aver partecipato.

Spero che quello che ho detto possa servirle. Quello che posso aggiungere è che ho avuto una vita fortunata, divertente e stimolante. Non avrò fatto scoperte sensazionali, però ho contribuito allo sviluppo delle neuroscienze e ho sempre saputo insegnare bene. Ho tirato su diverse brave persone, quindi il mio lavoro di professore l’ho fatto.

Grazie di tutto, professore.

 

 

 

 

 

MEET THE OPINION LEADER: INTERVIEW TO PROF GEOFFREY BURNSTOCK

 

Professor Geoffrey Burnstock completed his PhD at King's College and University College London (UCL). He was head of the Department of Zoology at The University of Melbourne (1964-1975) before moving to London to head the Department of Anatomy and Developmental Biology at UCL until 1997.
His major research interest stems from his seminal discovery of purinergic signalling and definition of purinergic receptor subtypes, their signalling pathways and pathophysiological relevance. As of May 2018 he has over 1550 publications, over 114,170 citations and an h-index of 150.

1) Dear Geoff what was your background like?

I came from a poor, uneducated, boxing family. As a child, I spent the war years evacuated from school and mostly in London during the bombing.

2) What inspired you to do the researcher?

I was initially interested in becoming a doctor, but soon realised that I had the curiosity and inclination to do research.

3) Where did you train and what was it like?

I failed to get into medical school and was finally accepted by King’s College London to take degrees in Zoology and Theology (although I was already an atheist). I started a club called ‘the Sceptics Society’ at the University.

4) Where did you get your inspiration about the “purinergic signalling”?

We did an experiment in Melbourne in the early 1960’s that established autonomic neurotransmission that was neither mediated by the classical acetylcholine nor noradrenaline. We then tried to identify the identity of the non-adrenergic, non-cholinergic neurotransmitter. Neuropeptides, monoamines and amino acids did not satisfy the criteria. Then I was impressed by the work of Szent-Györgyi (1929) and Pamela Holton (1959) to consider ATP, which satisfied the criteria and purinergic signalling was proposed in a Pharmacological Reviews article in 1972.

5) What were the best and worst things about your career?

The worst was the initial widespread rejection of the purinergic hypothesis for the first 20 years. The best has been the current widespread recognition of the therapeutic potential of purinergic signalling.

6) What do you do when you’re not working?

I used to play a lot of tennis and table tennis. I am a keen wood sculptor. I enjoy food and wine. I like to do jigsaw puzzles of paintings by famous artists. I very much enjoy spending time with my daughters and grandchildren.
 

 

 

MEET THE OPINION LEADER: INTERVIEW TO PROF KENNETH JACOBSON

Dr. Kenneth A. Jacobson, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health, Bethesda, MD, USA

1) What was your background like?

I am from a progressive, middle-class family and lived in suburban neighbourhoods in Ohio and California prior to my college education. The public schools that I attended were excellent academically, and my parents encouraged studying and higher education.

2) What inspired you to become a researcher?

Inspired by family members, I had an interest in chemistry from a young age. At that time, science was highly valued in society as a means of improving the human condition. I had a chemistry lab at age 10 in the basement of my home, stocked with reagents and glassware obtained from my grandfather who was an inventor and chemical engineer. On weekends, I used to visit the lab at his small company in Cleveland, to become familiar with basic chemistry. At age 12, I wrote to dyestuff manufacturers asking for samples of organic dyes, including fluorescent dyes, for a school project, and they actually sent me many samples (with no legal formalities) and I learned about their chemical structures and properties. I was known in school for my interest in chemistry, and I had my own exploratory projects at that age.

3) Where did you train and what was it like?

My undergraduate degree is from Reed College, a small liberal arts school in Portland, Oregon. It has the highest rate of STEM (science, technology, engineering and mathematics) undergraduate students going on to earn a PhD of any school in the US. It was very rigorous and required a thesis to be written in the senior year, which I did based on my experimental work in enzymology with Prof. Will Bloch. My PhD studies were with Prof. Murray Goodman at the University of California San Diego, one of the leaders of the peptide field. He introduced me to interdisciplinary science, as he was active in researching the chemical, structural and biological aspects of peptides. It was in Goodman’s lab that I was drawn to designing ligands of G protein-coupled receptors (GPCRs), which is a main focus of my current research. This was long before anything was known about the composition or structure of GPCRs, and adenosine receptors were not yet on the radar. During my PhD studies, I innovated a functionalized congener approach to GPCR ligands (specifically adrenergic receptors), which I later adapted and expanded for purine receptors. By this approach, distal positions of derivatization on a chain-extended pharmacophore can be used to incorporate chemical moieties, such as fluorophores as reporter groups. The distal conjugated moieties can also serve to modulate or enhance the pharmacological profile of the compound by interacting with accessory sites on the ‘hypothetical’ (at that time) receptor. I was making early bitopic ligands for GPCRs, but I didn’t use the term at that time. This was the conceptual framework for the xanthine amine congener (XAC), which I introduced in 1985 (and was the first xanthine that I designed while at NIH) and which has become a key potent adenosine antagonist used in the field. My postdoctoral studies were in the Department of Organic Chemistry at the Weizmann Institute in Israel. My mentor was Prof. Abraham Patchornik, one of the most creative scientists I have known, who was synthesizing peptides using innovative polymeric reagents even before the Merrifield method. He also made the first photosensitive protecting groups – now famous for ‘caging’ bioactive molecules – and I worked on both of those projects. In general, I learned how important it is to have good mentors when embarking on a scientific career, and I have tried to apply that concern to my own trainees (including over 70 postdoctoral fellows). One excellent teacher or supervisor can make an enormous difference in the course of one’s career.

4) What were the best and worst things about your career?

The United States National Institutes of Health (NIH), where I have spent most of my career, is an awesome location for research, with ~7000 basic and clinical scientists on site at the Intramural campus in suburban Maryland, outside Washington, DC. Having arrived at NIH at the end of 1983, I set out to apply chemical solutions to biomedical problems. The environment at NIH is rich in both material and human resources, with experts on every aspect of medicine in close vicinity, and it has been fertile ground for my goals. I passed up several other job opportunities along the way, which would have been more financially rewarding, but I was excited about the research potential at NIH. I value the academic freedom and the ability to start projects on a hunch or by intuition, without having to justify the rationale in advance. Many of these ideas have been fruitful and might not have been funded by standard sources. Consequently, I am grateful for the research support provided by the Government, and by several small companies that have supplemented my lab resources in the context of official collaborative agreements with NIH (known as CRADAs). I am privileged to have had many talented trainees in the lab (from 15 different countries), who have been dedicated to the goals of my Section (Molecular Recognition Section, or ‘MRS’) and many of whom have become internationally recognized researchers in their subsequent careers. I also value the association with international colleagues and friends in the purinergic field. My first meeting with Prof. Geoff Burnstock at the IUPHAR meeting in Sydney, Australia in 1987 convinced me to work on P2 receptors, as well as adenosine receptors, although it took 4-5 years to implement that decision. I was not dissuaded by the misguided scepticism around his work at that time. I have been fortunate in having collaborations with highly inspired and inspiring purinergic researchers, the first of whom was Dr. John Daly at NIH on adenosine receptors. The advancement of several of our molecules into clinical trials has been very gratifying. My dream as a medicinal chemist is to have made a compound that becomes FDA-approved and improves human health, and we have more than a few irons in the fire. We are hopeful that A3 agonists will eventually be approved for use in treating autoimmune inflammatory diseases and liver conditions, including cancer. Future clinical trials of A3 agonists in pain and other conditions are also envisioned. We made it our goal to introduce the first A3 selective agonists and antagonists as soon as the receptor was cloned and before there was any disease target associated, and that objective was achieved in the mid-1990s. Another dream from early days that became a reality is high resolution structures of purine receptors. We are fortunate to have participated in the determination of A2A, P2Y1 and P2Y12 structures, which have become important tools for the rational, structure-based discovery of new ligands. Although I am mostly pleased with my career situation, one negative aspect is related to the fact that NIH is part of a large government bureaucracy, which is not always compatible with the professional needs of basic researchers. I serve on the NIH Assembly of Scientists (AOS) to try to address the shortcomings of life as a scientist at NIH, whether for administrative or other reasons. One important initiative of AOS that I wholly support is to increase diversity in the research and leadership ranks of NIH. In my own group, there is nearly a gender balance cumulatively, and I welcome the opportunity to help underrepresented minorities advance in the field.

5) Do you think that purines can represent an attractive field for the development of new drugs in the near future?

Yes, definitively! Purinergic signalling is now established as a central regulatory system in the body, with relevance to many diseases and conditions, where purinergics can modulate and correct the signalling imbalances created by the pathology. Among small molecules with therapeutic potential, I would include direct receptor modulators, either orthosteric or allosteric, and indirect signalling modulators such as inhibitors of enzymes, e.g. ecto-nucleotidases, or transporters. Use of therapeutic antibodies or nanobodies also has great potential in the field. Thus, there are many possible approaches for purinergic intervention in disease states, and the permutations are enormous. A recent example that we have worked on in collaboration with Daniela Salvemini (St. Louis University) is the use of A3 adenosine agonists for treatment of chronic neuropathic pain of various etiologies. We have honed the most recent generation of A3 agonists to be >10,000-fold selective compared to the other adenosine receptor subtypes, which facilitates the unambiguous pharmacological characterization of their in vivo effects. Since the 1970s, some intended clinical applications of adenosine or P2 receptor ligand (agonists or antagonists) have passed into oblivion, while other new therapeutic concepts (such as A2A/A2B antagonists for cancer immunotherapy) have supplanted them. There seem to be constantly renewed cycles of interest in purinergics. The ubiquitous nature of purinergic signalling in human physiology provides many options for modulation, but at the same time it is challenging to achieve selectivity to avoid side effects. The good news for purinergic drug discovery is that knockout of nearly any protein in these pathways is nonlethal, although it can have beneficial effects in pathological conditions. Thus, pharmacological modulation can be generally tolerated in the body. On that theme, I wish to mention our recent study published in PLoS Biology in collaboration with Marc Reitman (NIDDK), in which all four adenosine receptors were knocked out in a mouse line. The lack of a severe phenotype indicated that extracellular adenosine may be a signal of stress, damage, and/or danger (and as such its modulation can be used in a treatment mode), but adenosine receptors are not essential for homeostasis. Thus, the physiological role of adenosine is better described as correcting allostasis.

6) In your opinion, at the moment what is the most attractive molecular target?

That is a complicated question, because there are many ideas being developed at various stages. I think purinergic immunomodulation, either augmentation for cancer or suppression for inflammatory diseases, is promising. In the cancer field, adenosine receptor blockade (by antagonism or preventing adenosine formation via CD73) is underway in many pharma companies. In the P2 field, mostly antagonists have translational interest, but in some cases, agonists, as well. There’s a lot of room for basic discovery in the P2X and P2Y areas compared to the adenosine receptors, because of the lag in design of selective ligands. Now, antagonists of P2X3 (chronic cough, hypertension), P2X4 (pain) and P2X7 (peripheral and neuro-inflammation) receptors have been heavily explored by pharma. P2Y1 antagonists, an area in which we have had major contributions, have recently become a target for neurodegeneration, which is an intriguing possibility.

 

 

 

MEET THE OPINION LEADER: INTERVIEW TO PROF CHRISTA E. MÜLLER

 

Prof Christa Müller studied pharmacy at the University of Tübingen, Germany, and received her Ph.D. in Pharmaceutical/Medicinal Chemistry from the same university. She completed her habilitation thesis at the University of Tübingen in 1994, and became Associate Professor of Pharmaceutical Chemistry at Würzburg University in the same year. Since 1998 she is full professor of Pharmaceutical Chemistry at Bonn University. She is a co-founder of the Pharma-Center Bonn.

1) Dear Christa what was your background like?

In school, I was interested in many different subjects, including chemistry, mathematics, physics, biology, but also languages, literature, philosophy, history, music and art. I studied pharmacy for two reasons, (1) to have a solid profession which would enable me to be independent earning my own money, and (2) because pharmacy is broad combining several of my areas of interest including my favourite subjects.

2) What inspired you to become a researcher?

It was probably curiosity. Also role models were important, for example one of my favourite (female) school teachers who had earned a PhD.

3) Where did you train and what was it like?

I grew up in a small village in the South of Germany, near Rottweil, where I was born. Rottweil is the oldest city in the state of Baden-Württemberg; it is based on a Roman settlement. After completing my high school degree, I studied at the University of Tübingen, and after graduation, I did the obligatory internship in a public pharmacy in Stuttgart. Then I went back to Tübingen for a PhD in Pharmaceutical/Medicinal Chemistry. Next, I did a postdoctoral stay at the National Institutes of Health in Bethesda, Maryland, USA, in the Laboratory of Bioorganic Chemistry, National Institute of Diabetes, Digestive and Kidney Diseases (NIDDK) with John W. Daly. During that time, I started to work on adenosine
Prof Christa Müller studied pharmacy at the University of Tübingen, Germany, and received her Ph.D. in Pharmaceutical/Medicinal Chemistry from the same university. She completed her habilitation thesis at the University of Tübingen in 1994, and became Associate Professor of Pharmaceutical Chemistry at Würzburg University in the same year. Since 1998 she is full professor of Pharmaceutical Chemistry at Bonn University. She is a co-founder of the Pharma-Center Bonn.
receptors, and I have been progressively fascinated by purine receptors and purinergic signalling. My postdoctoral period in the USA was eye-opening, incredibly inspiring, and highly motivating. I got to know an altogether new world of science, in which everything appeared to be possible. Almost every day I had a chance to attend a talk by a famous researcher. The newest techniques and latest instruments were accessible on campus. I enjoyed exploring a new world of science, and also a great country with phantastic art, music and nature.

4) What were the best and worst things about your career?

The best has been to be relatively independent, to be able to decide on which projects you want to work. It is great to teach young, motivated students, and it is phantastic to get to know so many great colleagues, to visit beautiful places and work in an international environment.
When I started to establish my own group at the University of Tübingen, my first grant proposal submitted to the Deutsche Forschungsgemeinschaft (DFG) was rejected. I should have revised and resubmitted it as soon as possible, but I was so shocked and paralyzed that for a number of years I did not apply to the DFG anymore. All scientists have to live with failures (rejected grant applications or manuscripts…), and we should never take it personally, but try to learn, make it better next time. Maybe the reviewer just did not understand it, so make it clearer, try to get help, collect information, ask colleagues for advice.
The greatest moment in my career was probably the day of my habilitation. It was a tough procedure. The final examination was a lecture in front of the entire Faculty of Chemistry and Pharmacy, all the big professors were present in the lecture hall, trying to test me. I was expected to do drawings on the blackboard in addition to presenting slides (I think I broke the chalk several times due to too much pressure), and afterwards, there were rigorous questions. After the procedure, I invited friends and colleagues into a restaurant for dinner to celebrate the event. When I came home late at night, I found a letter in the postbox with a call for an Associate Professorship of Pharmaceutical Chemistry at the University of Würzburg, Bavaria. This was so incredible, the happiness having survived the habilitation, an event which can hardly be topped, but then, on the very same day, I was offered a professorship!

5) In your opinion, at the moment what is the most attractive molecular target?

There are so many attractive targets out there. I am most interested in novel targets, about which little to almost nothing is known. This is most exciting for me, and it allows amazing discoveries. If possible, I recommend not to go for the easy targets, but for the more demanding projects – it is much more fun.

 

 

MEET THE OPINION LEADER: INTERVIEW TO PROF BRUCE N. CRONSTEIN

 

Prof Cronstein is full professor of Medicine, Department of Medicine at NYU Grossman School of Medicine (NYC, USA) His lab first demonstrated the presence and function of adenosine receptors on human polymorphonuclear leukocytes, including their role in regulating inflammation.

1) What was your background like?

I am originally from Cincinnati, Ohio – a solid Midwesterner. After finishing college (Lake Forest College, outside of Chicago) and medical school in Cincinnati I came to New York City for clinical training in Internal Medicine and Rheumatology. I never had any expectation of becoming a researcher and I thought I would be a practitioner taking care of patients with such diseases as Rheumatoid Arthritis and Systemic Lupus Erythematosus.

2) What inspired you to become a researcher?

During my fellowship in Rheumatology I was required to spend a year in the laboratory. I chose to spend my first year in the lab in Dr. Rochelle Hirschhorn’s laboratory. At the time Dr. Hirschhorn was working on Adenosine Deaminase Deficiency and its therapy and so we designed a project to look at adenosine deaminase and purine nucleotide phosphorylase enzyme activity in CD4 and CD8 T cells from patients with Systemic Erythematosus. While this project did not show much promise and we abandoned it after a year I found that I enjoyed the challenge of laboratory work and hypothesis testing. Interestingly, a genetic polymorphism in the purine nucleotide phosphorylase has been strongly associated with risk for development of Systemic Lupus Erythematosus – obviously we were ahead of our time. Prof Cronstein is full professor of Medicine, Department of Medicine at NYU Grossman School of Medicine (NYC, USA) His lab first demonstrated the presence and function of adenosine receptors on human polymorphonuclear leukocytes, including their role in regulating inflammation.

3) Where did you train and what was it like?

I did all of my laboratory training at NYU School of Medicine under the tutelage of Drs. Rochelle Hirschhorn and Gerald Weissmann. Both imparted to me their enthusiasm for discovery and made the science come alive.

4) What were the best and worst things about your career?

The best thing about my career has been the ability to test new ideas and make new discoveries. SCIENCE IS FUN! On the other hand, the constant scramble for external support, while keeping you on your toes and making sure that your science is up to date, has become a bit wearing. The competitive, winner-take-all stakes in science in the US take a toll after a while.

5) Do you think that purines can represent an attractive field for the development of new drugs in the near future?

As somebody who just started a biotech, Regenosine, to develop and target adenosine receptors I am clearly optimistic about adenosine-related drug development. In addition, the development of adenosine receptor-targeted drugs for the treatment of cancer has been very encouraging. The major difficulty with adenosine receptor targeting is the ontarget toxicity. Adenosine receptors are ubiquitous and are likely to be stimulated in cells and organs in a deleterious fashion. The approach we have taken for treatment with agonists has been to try to target locally (in our case injecting adenosine in liposomes by direct injection into osteoarthritic joints). Adenosine, which has an extremely short half-life in blood and biologic fluids, is unlikely to travel outside of the synovium let alone into the circulation. Similar approaches can be taken for other medical problems.

6) In your opinion, at the moment what is the most attractive molecular target?

Most of our investigations have targeted A2A receptors and this is also true for development of immune modulators for cancer.

 

 

MEET THE OPINION LEADER: INTERVIEW TO PROF JEAN SEVIGN

 

Jean Sevigny is Professor of Microbiology-Infectiology & Immunology, Faculty of Medicine, Université Laval. His research focuses on the functions of extracellular nucleotides and the regulation of signaling through the activation of specific nucleotide receptors, termed P2-receptors. The activation of these receptors is regulated by ectonucleotidases that break down nucleotides at the cell surface. His work on these enzymes has unveiled the physiological roles of endogenous nucleotides that are released by cells in large amount in diverse conditions, especially during inflammation. His PhD studies led to the discovery of the most important family of ectonucleotidases that break down these molecules: the E-NTPDase family. Since then, they have developed tools (antibodies, inhibitors, expression vectors, KO mice, etc.) enabling to study these enzymes and understand their functions. He has a major interest in the control of inflammation by extracellular nucleotides.

1) Jean, what inspired you to become a researcher?

hum… Let me remember… the only thing clear to me at around 14 to 18 years old was that I wanted to get the highest level of studies in a subject that was possible to get at school. So somehow this was kind of certain that I would do a PhD in something. Research appeared the best way for me to achieve the highest University level and biology seemed to be the science that was the most developing when I was at school. Informatic was, but I found it too easy to work on this. I found engineering attracting as well. I decided to go in biology and I never regret it. But at about 16 years old I thought I would be an Engineer which changes a couples of years after.

2) Where did you train and what was it like?

Sherbrooke was a very dynamic University. Harvard as a post doc was kind of something I have to admit.

3) What were the best and worst things about your career?

Getting a grant was at the beginning of my career as an independent researcher (and when it was easier to get those) was marvelous!! A few years ago, when it was difficult for all labs in Canada and in the US, it still is in fact, and when I got only several one year grants, it was rather an immense relief when I got finally got again a couple of 5 year major grants. The feeling was quite different when I got money to hire several new people than when I got a grant to be able to keep the most important people in my lab. Failing this would have been like not providing to my family members. The team work is indeed family also. I do not know if it is a joy or a big deception. This is for sure mixed feeling for me. During my PhD studies I have found the first member of the E-NTPDase family that I then cloned in Simon Robson’s lab as a trainee. While still in Sherbrooke as a PhD student we patented the use of that enzyme (before cloning it) to treat thrombosis and inflammation. The patent was neglected and not taken good care by the people at the time and we lost big opportunities. At the Purines 2016 meeting in Vancouver, Ridong Chen presented his modified and soluble NTPDase3 with improved ADPase activity as a treatment for thrombosis. I felt some how like yes what I did during my PhD is really working ! and somehow I felt that someone else did my work and got the benefit of it. Indeed AstraZeneca is currently recruiting volunteers for a Phase I clinical trial (safety, PK/PD) of AZD3366 (formerly known as APT102; trial NCT04588727), a recombinant form of human NTPDase3 administered intravenously for the treatment of thrombotic diseases. They transformed the original NTPDase3 enzyme into a better ADPase in order to prevent the activation of platelet P2Y1 and P2Y12 which are activated by ADP. This mutation was in the same time making their enzyme more like our NTPDase1. So should I be happy that my PhD led to this ? is it a big deception that someone else did that instead of me? I let you decide. ;-) But getting the found to do that was not possible for me anyway at the time…

4) Do you think that purines can represent an attractive field for the development of new drugs in the near future?

easy answer ! YES !! See also some of my patents in my CV.

5) In your opinion, at the moment what is the most attractive molecular target?

Hum I would say what I am working on at the moment! ;-)

 

 

 

MEET THE OPINION LEADER: INTERVIEW TO PROF FRANCISCO CIRUELA

 

Francisco Ciruela graduated in Biology at the Universitat de Barcelona. He performed his Ph.D. at the Department of Biochemistry and Molecular Biology, Faculty of Chemistry, Universitat de Barcelona under the supervision of Prof. E.I. Canela and Dra. J. Mallol (1995). Throughout this period, he focused on the study of the mechanisms by which adenosine regulates cell functioning through adenosine receptors. During this period, he visited the laboratory of Dr. B. Fredholm at the Karolinska Instituted to learn about the adenosine receptor-associated signalling pathways. Currently, his research work focuses on pathological conditions of GPCR oligomerization in the central nervous system. He received the “ICREA Acadèmia” award from the Catalan Institution for Research and Advanced Studies, a foundation supported by the Catalan Government.

1) What was your background like?

Thank you very much for giving me the opportunity to tell you about my scientific career. I studied Biological Sciences at the University of Barcelona (UB), where I graduated in 1990. As biology did not fulfill me scientific interest, I pursued my PhD at the Chemistry School, at the same university, aiming to apply chemistry in the study and manipulation of biological systems. I finished my PhD in 1995. During this time, I learned quite a lot of chemistry, and I started to get in touch with the adenosine field, focusing on adenosine receptors. Precisely, by this period we had started to study the oligomerization of G protein-coupled receptors, using adenosine A1 receptor as a prototype of GPCR. By this time, there was very little information regarding the structure/activity of those receptors. When I finished my PhD in 1995, I moved to United Kingdom, precisely to the University of Oxford. There, I performed a postdoc until the 2000 while I was awarded with an EMBO long-term fellowship. The first four years, I was working at the MRC Anatomical Neuropharmacology Unit, a UK Medical Research Unit attached to the Department of Pharmacology. Then, I gained a Wellcome Trust Research fellowship at the Department of Pharmacology until the beginning of 2000. Thus, that was my progression towards pharmacology.

2) What inspired you to become a researcher?

When I finished my postdoc, I had this dilemma: to continue my scientific career in United Kingdom with a tenure track position or returning to Spain with much less opportunities and even considering leaving science. So, I decided to come back to Spain and settled in Barcelona again. I returned to the UB and started as an Assistant Professor in Biochemistry, which wasn't my expected scientific/academic interest. I hold this position until 2003, when I just gained a Ramón y Cajal research tenure track position, a really good position in Spain. It's a five-year tenure track position for developing your career as a scientist. I started my own research line at the UB, not without difficulties, of course, because I had to find funding to advance my research. When I finished my Ramón y Cajal research contract in 2007, the UB offered to me the possibility of a permanent position as an Associate Professor within the university. At this point, I decided to move from the Department of Biochemistry of the School of Biology to the Department of Pathology and Experimental Therapeutics (DPET) at the School of Medicine. There I became Associate professor of Pharmacology and I settle my own research group: “Neuropharmacology & Pain” (http://www.ub.edu/neuropharmpain/). This was a crucial and wise decision which had a huge impact into my future academic and research trajectory. Currently, I am a full professor of pharmacology at the UB.

3) Where did you train and what was it like?

Many people know mi research because the adenosine receptors and its oligomerization. Although, I work with other GPCRs for neurotransmitters (e.g., glutamate, dopamine, cannabinoids, opioids, etc.) and biological systems (e.g., cardiac, immune, etc.) apart of adenosine receptors in the brain, but the concepts and hypothesis still are similar. Nevertheless, one of the main topics of my research consist of deciphering the pharmacological impact of GPCR oligomerization, with special emphasis in pathology (e.g., neurodegenerative, and neuropsychiatric diseases). Accordingly, my main working hypothesis suggest that GPCRs form higher ordered structures, called oligomers or macromolecular membrane assemblies which define the receptor’s function. Nowadays, I am trying to demonstrate this hypothesis in vivo (i.e., behaving animals). I came up with this hypothesis by (nearly) accident. In brief, when I was doing my PhD back in 1992, I made an antibody against the adenosine A1 receptor, as this receptor was the topic of my incipient thesis. While I was validating the antibody by performing immunoblots using brain tissue, I observed a protein band of the corresponding expected size (i.e., 37 kDa), but also another band of 75 kDa was constantly detected. Initially, we believed that this was an artifact either by the immunoblot detection or because the antibody I generated was not specific. Nevertheless, after dozens of attempts showing this immunoblot pattern, we arrived at the conclusion that the 75 kDa was a homodimer of A1R. These results were published in 1995 (Ciruela et al., 1995), precisely when some few publications, including the elegant work by Maggio and collaborators (Maggio et al., 1993), started to suggest that GPCRs might oligomerize. A second turning point for our working hypothesis was when we studied the oligomerization between adenosine A1 and A2 receptors, back to the 2006 (Ciruela et al., 2006a). In that case we demonstrated the existence of heteromers containing two receptors recognizing the same transmitter (i.e., adenosine), with opposite signaling (i.e., Gi- vs. Gs- coupling) and within the same endogenous neural context (i.e., striatal glutamatergic nerve terminals). Importantly, when we assessed the functional consequences of this oligomerization, we demonstrated that the A1R/A2AR heteromer formation provides a switch mechanism by which low and high concentrations of adenosine inhibit and stimulate, respectively, glutamate release in the striatum. Thus, we described a molecular device that allowed adenosine to exert a fine-tuning modulation of striatal glutamatergic neurotransmission (Ciruela et al., 2006b). This is an unprecedented concept which may shed light to understand the manner how different signalling systems interact between them providing a harmonized physiological response, thus broadening the basis of the so-called multimodal pharmacology, and refining the pharmacotherapy of some diseases.

4) What were the best and worst things about your career?

The worst thing is the continuous search of fundings for research, specially being in a southern country of Europe. Science funding within Europe is quite unbalanced between countries, because some have large budgets devoted to research (i.e., ≈3% of gross domestic product, or GDP), and others, like ours, have small ones (i.e., ≈1% of GDP). Thus, southern countries within Europe dedicate a limited amount of money for research. Indeed, the European Union (EU) has resources and a shared budget for granting projects. However, it's a bit difficult to get access to as these are quite competitive. Overall, the differences in research investment within EU countries should be harmonized to avoid the continuous brain drain of southern European countries. Nevertheless, we are quite imaginative and collaborative, thus even with the short money we have we are countries with high scientific productivity and holding high ranking positions in the Scimago Journal & Country Rank (https://www.scimagojr.com/countryrank.php), ahead of many other European countries. Of course, we would like to have a larger budged for research (i.e., ≈2-3% of GDP) as to escape from this unsustainable Janus-faced situation (budged vs. productivity) which will be impossible to maintain in long-term basis. The best thing is the personal satisfaction of contributing to the progress of biomedicine (even with a small grain of sand, of course) while helping others to do the same. Also, thanks to developing our research activities in a health Campus composed by two hospitals we have the chance to be close to the patients, and eventually to its associations. Indeed, this forces us to change the way we interpret and communicate our research. I feel we are in debt with the society.

5) In your opinion, at the moment what is the most attractive molecular target?

The demonstration of the existence of GPCR oligomers, first in heterologous expression systems and then in native tissue and in vivo (i.e., behaving animal) has transformed the field of Pharmacology, which historically was based in “one ligand – one receptor”, a very simplistic model of dug action. The existence of GPCRs oligomers in vivo opens the door to understand many pharmacological phenomena which were misunderstood until now. Indeed, this is especially important when we talk about pathology and when the existence of a potential relationship with GPCR oligomerization may exists. In that sense, GPCR oligomers might constitute a superior way to manage some pathologies by elaborating a more refined pharmacology approach (multimodal) built in a GPCR oligomer-based theragnostic approach (i.e., diagnosing and treating the disease using GPCR oligomers as targets).

 

 

MEET THE OPINION LEADER: INTERVIEW TO BRIAN D. GULBRANSEN

 

Brian Gulbransen, professor of physiology and a member of MSU’s Neuroscience Program, focuses his research on understanding how neuroinflammation leads to long-term changes in neural circuitry. His investigations aim to reveal the fundamental rules that govern cell to cell communication in the enteric nervous system (ENS) and elucidate the molecular machinery that contributes to changes in the ENS, a finding that will lead to the development of new therapies for gastrointestinal motility disorders.
Brian received his B.S. in zoology and physiology from University of Wyoming and his Ph.D. in neuroscience from the University of Colorado Anshutz Medical Campus. Prior to joining MSU in 2012, he completed his postdoctoral training at the University of Calgary, where he received several awards to support his research including the Crohn’s and Colitis Foundation of Canada Individual Fellowship, the Achievers in Medical Science Postdoctoral Scholarship, and the Canadian Institutes of Health Research/Canadian Association of Gastroenterology Individual Fellowship.

1) What was your background like?

I’m from a very rural part of the western United States and grew up doing outdoors things like camping, fishing, backpacking, and basically anything else I could find to do outside. I spent a lot of time with animals and exploring the surrounding mountains. My schooling started in a one room schoolhouse that had 8 kids total in grades K-6. This closed when I was in the second grade, and I had to commute to the closest town for the rest of my primary school. After high school, I entered the University of Wyoming and was planning on studying wildlife management and zoology because of my interests in animals and the outdoors.

2) What inspired you to become a researcher?

I was in the Honors Program at the University of Wyoming which required students to complete some undergraduate research experience. They encouraged us to seek this out early on in our undergraduate training so we could develop an independent research project. I was walking around our department and saw a poster outside of a lab describing the enteric nervous system. I thought this sounded really cool so I walked in and asked if I could have a job! Luckily, Dr. Paul Wade was very welcoming and took me under his wing. Under Paul’s mentorship, I developed an undergraduate research project focused on age-related neurodegeneration in the enteric nervous system. This undergraduate research experience got me hooked and I knew this is what I wanted to do.

3) Where did you train and what was it like?

I’ve trained at several places, and all had their positives and negatives. My undergraduate training was at the University of Wyoming and to me, this is home. It is surrounded by mountains and is a really cool spot. For my doctoral training, I went a bit south to the University of Colorado Anschutz Medical Campus in Denver. This was an excellent institution and it was nice to still be close to the mountains but Denver was a bit too big and busy for me. From there, I went north to Canada and did my postdoctoral training with Dr. Keith Sharkey at the University of Calgary in Alberta. This was an amazing place surrounded by unreal scenery. This is complemented by a great city and an outstanding research environment. I absolutely loved it there and would have stayed forever if I didn’t need to find a permanent job.

4) What were the best and worst things about your career?

I like figuring out how things work so having a career where you continually try to understand the underpinnings of how the body works (or doesn’t work) is great. There is always something to study and more questions to ask. This also means that doing what we do never gets old. We have freedom to investigate basically whatever we find interesting and can get funding to do. It’s very cool.
However, it is a demanding career, and it can be stressful - especially when you are responsible for supporting the people you depend on, yourself, and your research. As we progress in our careers, we are gradually removed from doing experiments and focus more on finding support for the lab and managing people. This is both a positive and a negative because it allows you to see the broader picture of what you are doing and to address more questions, but it also means not doing the interesting experiments yourself and spending more time in the office.

5) Do you think that purines can represent an attractive field for the development of new drugs in the near future?

Absolutely. Purines play such a huge role in regulating cell responses in health and disease. We still know relatively little about how varying levels and types affect different cells and this could be a key to developing new drugs. Even the same purinergic receptor type can have opposing effects in different cell types so understanding the whole picture of how purines contribute to disease is very complex. There is huge promise in this area though and drugs targeting purines are important in inflammatory diseases, cancer, and cardiovascular disease to name a few.

6) In your opinion, at the moment what is the most attractive molecular target?

This is a very tough question! In terms of purinergic signaling, I’d have to go with pannexin-1; although I’m a bit biased since I work on this channel. Why I say this is because pannexin-1 plays an integral role in disease processes and mediates communication between multiple cell types including neurons, glia, immune cells, endothelial cells, adipocytes, etc. Modulating the function of this channel seems to have excellent potential to improve diseases such as inflammatory bowel disease, hypertension, obesity, and stroke. We’re still actively investigating the role of neuronal pannexin-1 so stay tuned!

 

 

RICORDO DEL PROF. FRANCESCO CACIAGLI

 

 

Riporto a seguire le bellissime parole della Prof Renata Ciccarelli in ricordo del Prof Francesco Caciagli

Le sue doti sono emerse fin dall'inizio della sua carriera, quando decise di lavorare presso l'Università di Chieti, facendo una scelta difficile. Infatti, decise di lasciare la sua città, che amava, e l'Università di Pisa, che aveva una lunga tradizione accademica e strutture indubbiamente avanzate, per venire in Abruzzo, pur sapendo che l'Università di Chieti era nata da poco e che la Facoltà di Medicina era situata in locali poco idonei con fondi a disposizione praticamente zero. Ma Francesco, da vero pioniere, non si scoraggiò. Con tutte le sue capacità si mise all'opera e con umiltà, ma con determinazione, superando avversità a livello locale e nazionale, riuscì a creare un gruppetto di ricercatori e aprire il filone di ricerca sulle purine, che via via abbiamo portato avanti e ampliato. Così, Francesco è riuscito a farsi strada e a far progredire sé stesso e noi nella carriera accademica. Per noi tutti è stato un Maestro, poiché ci ha insegnato i principi fondamentali da perseguire nella ricerca sperimentale, stimolando al contempo in noi l'interesse e la passione per le tematiche scelte. Credo che il suo impegno scientifico e la sua umanità siano emersi anche nelle riunioni del Purine Club, alle quali partecipava assiduamente insieme con noi, ritenendo che fosse importante contribuire alla sua crescita. Ed in quelle occasioni, si è fatto apprezzare non solo per la capacità di ascoltare gli altri e dare consigli ed aiuti appropriati, ma anche per le sue battute argute, talvolta anche irriverenti, con le quali rallegrava le riunioni sia scientifiche che conviviali. Anche dopo essere andato in pensione ha continuato a dare il suo apporto, sia scientifico che umano, a noi del gruppo, ed anche a colleghi dell'Ateneo che lo richiedevano, e con la determinazione di sempre ha affrontato la sua malattia, lottando strenuamente, fino a che ha potuto.

 

 

 

 

By Dr. Ilaria Dettori
(Department of NEUROFARBA, University of Florence)

 

Article title: Therapeutic effects of polydeoxyribonucleotide in an in vitro neuronal model of ischemia/reperfusion injury
Authors: Jo S, Baek A, Cho Y, Kim SH, Baek D, Hwang J, Cho SR, Kim HJ
Journal: Sci Rep
Year: 2023
Volume: 13
DOI: 10.1038/s41598-023-32744-9 

Main results: In this paper, authors elucidate the therapeutic effects and mechanisms of polydeoxyribonicleotide (PDRN), a DNA-derived drug that increases ADORA2A expression, in an in vitro ischemia/reperfusion injury model. The Neuro-2a cells were put in oxigen-glucose deprivation (OGD) condition per 3h, and then incubated with PDRN for 24h. Results demonstrated that PDRN protected against OGD-induced cell death and decrease inflammatory responses via downregulation of JAK/STAT pathway by regulating ADORA2A expression in the in vitro I/R injury model. Based on these findings, PDRN treatment may be useful in developing more reliable therapeutic strategies for patients with I/R injuries. 

Opinion: This experimental study provides new insights into the therapeutic effects of PDRN on I/R injury, especially for the use of a DNA-derived drug with a peculiar mechanism of action. Even if the study lacks of functional and translational relevance, so to reinforce the conclusions of this study, it is imperative to clarify the detailed mechanisms underlying the protective actions of PDRN treatment with further experiments using an activator or inhibitor of JAK or STAT.

 

By Dr. Vanessa D’Antongiovanni
(Dipartimento di Medicina Clinica e Sperimentale, Università di Pisa)

 

Article title: P2X7 receptor antagonist improves gastrointestinal disorders in spontaneously hypertensive rats
Authors: de Oliveira KBV, Severo JS, da Silva A, Dos Santos B, Mendes PHM, Sabino JPJ, Filho A, Correia-de-Sa P, Dos Santos AAD, da Silva M
Journal: Braz J Med Biol Res
Year: 2023
Volume: 56
DOI: 10.1590/1414-431X2023e12569 

Main results: Hypertension is a pathological condition often associated with gastrointestinal disturbances, such as enteric inflammation and delayed gastric emptying (GE). In this context, purinergic system plays a pivotal role in the control of blood pressure as well as in the maintenance of gastrointestinal homeostasis. The aims of the present study were: 1) to evaluate the effect of the P2X7 receptor antagonist, Brilliant Blue G (BBG), in modulating the GE and the oxidative stress  processes at the level of the gastric fundus, duodenum and colon in spontaneously hypertensive rats (SHR) and 2) to investigate a possible relationship between the above mentioned effects with the renin-angiotensin system. Rats were divided into five groups: control, SHR, SHR+BBG, SHR+BBG+ATP and SHR+BBG+ANG II (angiotensin II). SHR rats showed a significant reduction in GE, as compared with control group. Treatment with BBG in SHR rats normalized the GE values comparable to those observed in control group. Co-administration of ATP or ANG II with BBG abrogate the positive effects of the P2X7 antagonist on GE in SHR rats. In addition, SHR rats showed signs of oxidative stress in the stomach, as documented by an increase in MPO levels, as compared with control rats. This parameter is normalized in SHR rats following BBG administration.

Opinion: The present study indicates that the blockade of P2X7 receptors with BBG, besides counteracting the oxidative stress associated with hypertension, is able to improve the delay of GE. Such beneficial effect of BBG is abrogated following treatment with ANG II or ATP, suggesting that the cross-talk between the renin-angiotensin system and the purinergic system underlies the GE delay associated with hypertension.

 

By Dr. Elisabetta Caiazzo
(Dipartimento di Farmacia, Scuola di Medicina e Chirurgia, Università Degli Studi di Napoli “Federico II”)

 

Article title: IL-12 and IL-27 Promote CD39 Expression on CD8+ T Cells and Differentially Regulate the CD39+CD8+ T Cell Phenotype
Authors: Gerhardt L, Hong MMY, Yousefi Y, Figueredo R, Maleki Vareki S
Journal: J Immunol
Year: 2023
Volume: 210
DOI: 10.4049/jimmunol.2200897 

Main results: Tumor-specific CD8+ tumor-infiltrating lymphocytes (TILs) represent a heterogeneous cell population comprising tumor-specific T cells as well as bystander T cells that play a critical role in tumor control. CD39 enzyme is expressed by tumor-specific CD8+ tumor-infiltrating lymphocytes (TILs), and is not expressed by bystander CD8+ TILs. However, it is unclear whether CD39 expression on tumor-specific T-cells can be regulated by cytokines such as IL-12 and IL-27 that have established roles in promoting effector T-cell differentiation. In this study, the authors used, for in vitro and in vivo experiments, a syngeneic neuroblastoma (neuro-2a) mouse model to examine the ability of IL-12 and IL-27 cytokines to induce CD39 expression on CD8+ T cells. Firstly, CD39 expression was evaluated via flow cytometry after acute activation of splenic CD8+ T cells isolated from naive mice. The authors found elevated frequencies of CD39highCD8+ T cells after stimulation with IL-12 and, to a lesser extent, IL-27 so demonstrating that IL-12 and IL-27 can upregulate CD39 expression on CD8+ T cells, irrespective of previous antigen exposure. Then, splenic CD8+ T cells isolated from immunogenic idMMR neuro-2a–primed mice (where mismatch repair (MMR) deficiency was induced in neuro-2a cells) showed a significantly greater frequency of CD39highCD8+ T cells when activated with IL-12 or IL-27 but this was not observed for CD8+ T cells isolated from the poorly immunogenic pMMR neuro-2a–primed mice in which mismatch repair (MMR) deficiency was not successful. Interestengly, the authors demonstrated that IL-12 and IL-27 could individually induce the expression of CD39 on CD8+ T cells that have encountered immunogenic Ags, whereas those from a poorly immunogenic model required stimulation with both cytokines. The authors found that IL-12 and IL-27 could upregulate CD39 expression on CD8+ T cells independent of anti-CD3/CD28 activation signaling. In addition, they confirmed that upregulation of CD39 on T cells was correlated with coexpression of inhibitory receptors on CD8+ T cells such as inhibitory receptors programmed cell death-1 (PD-1), lymphocyteactivation gene 3 (LAG-3), and T cell Ig and mucin-domain containing-3 (TIM-3) that has been associated with terminal exhaustion of CD8+ T cells but IL-27 attenuated IL-12–induced expression of these inhibitory receptors on CD39+CD8+ T cells. They also demonstrated that CD39+CD8+ T cells have increased effector and cytotoxic functions. Interestingly, IL-27–induced CD39+CD8+ T cells showed the greatest frequency of CD107a expression, but this effect was lost when IL-12 was added in combination and IL-12–induced CD39+CD8+ T cells showed the highest frequency of IFN-γ+ cells, whereas those stimulated with IL-27 had the lowest frequency. Very importantly, neutralizing IL-12 activity in vivo resulted in reduced frequency of CD39+ and exhausted CD8+ TILs.

Opinion: Little is known about how and why tumor-specific CD8+ T cells uniquely express CD39. This study is very interesting because is the first study to identify differential effects of IL-12 and IL-27 on CD39+CD8+ T cell development and function, providing a rationale for the combination of both cytokines to improve future cancer T cell–based immunotherapies. On the other hand, a limitation of this study was the inability to assess the effects of IL-27 neutralization in vivo and confirm the importance of IL-27 on the induction of CD39+CD8+ T cells in vivo.

 

By Dr. Federica Cherchi
(Dipartimento di Neuroscienze, Psicologia, Area del Farmaco e Salute del Bambino (NEUROFARBA), Università degli Studi di Firenze)

 

Article title: Gliotransmission and adenosine signaling promote axon regeneration
Authors: Wang F, Ruppell KT, Zhou S, Qu Y, Gong J, Shang Y, Wu J, Liu X, Diao W, Li Y, Xiang Y
Journal: Dev Cell
Year: 2023
Volume: 58
DOI: 10.1016/j.devcel.2023.03.007 

Main results: In this paper, authors used viral transfection to investigate the glial regulation of neuronal regenerative by adenosine receptors (ARs) in Drosophila larval, which genome encodes no ATP receptors and a single AR coupled to Gs (that increases intracellular Ca2+), and in retinal ganglion cells (RGCs) of adult mice by using viral transfection. Results show that axotomy in Drosphila larval induced Ca2+ spikes in ensheathing glial and lead to the release of ATP/adenosine. Adenosine acts on AR expressed in C4da sensory neurons and induced burst firing, Ca2+ spikes, and Ras activity to promote axon regrowth. Since Drosophila AR shared the highest sequence similarity with A2A AR and A2B AR (54% and 50% respectively), the authors found that the stimulation of the gliotransmitter release also in adult mice induces RGC axon regeneration only if A2B AR was expressed. Moreover, A2B AR activation, but not by adenosine alone, increased RGC survival and axon regeneration through inward rectifier potassium channel Kir2.1, neuronal activity, and Ras.

Opinion: Axon regeneration failure is a major hurdle for functional recovery after a central nervous system lesion and their degeneration is associated with neurodegenerative diseases. With this paper, the authors demonstrated the relevance of neuron-glia interactions by the involvement of ARs. Both in Drosophila larval and in adult mice, the activation of Gs-coupled receptors (A2B AR in mammalian) leads to axon regeneration by enhancing intracellular Ca2+, firing and Ras. These findings suggest that A2B AR signalling, either alone or in combination with other pro-regenerative pathways, could represent a valuable target for mammalian central nervous system repair.

 

 

By Dr. Giulia Magni
(Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano)

 

Article title: Silencing P2X7R alleviates diabetic neuropathic pain involving TRPV1 via PKCε/P38MAPK/NF-κB signaling pathway in rats
Authors: Chen L, Wang H, Xing J, Shi X, Huang H, Huang J, Xu C
Journal: Int J Mol Sci
Year: 2022
Volume: 23
DOI: 10.3390/ijms232214141 

Main results: In this paper, authors investigated the relationship between transient receptor potential vanilloid 1 (TRPV1) and P2X7 receptor channels in neuropathic pain in a rat model of type 2 diabetes induced by high glucose and high-fat diet administration for 4 weeks and a intraperitoneal injection of low-dose streptozocin (35 mg/kg) to destroy islet B cells. Results show that intrathecal administration of P2X7 shRNA and p38 inhibitor relieved hyperalgesia in diabetic rats, with a significant increase in both mechanical and thermal withdrawal threshold, and reverted sciatic nerve conduction velocity. Moreover, P2X7 shRNA reduced the increased RNA and protein receptor expression on satellite glial cells (SGCs) from L4-L6 dorsal root ganglia (DRGs). P2X7 shRNA and P38 inhibitor also significantly reduced neuronal TRPV1 expression, and downregulated the PKCε/P38 MAPK/NF-κB signaling pathway as well as the expression of several pro-inflammatory mediators in DRGs.

Opinion: Peripheral neuropathic pain represents one of the worst consequences of diabetes, characterized by a difficult pharmacological management. With this paper, authors demonstrated the cross-talk among P2X7, P38 and TRPV1. They also showed that blocking P2X7 and p38 attenuated diabethic neuropathy via mechanisms that include the reduction of TRPV1 upregulation and of proinflammatory mediators release, as well as PKCε/P38 MAPK/NF-κB pathway. Therefore, results suggest that P2X7 receptors expressed by SGCs are involved in the described pathogenic process, and targeting the identified pathway could represent a pharmacological approach to treat diabetic neuropathy.

 

By Dr. Mariachiara Zuccarini
(Dipartimento di Scienze Mediche, Orali e Biotecnologiche, Divisione di Farmacologia e Tossicologia, Università degli studi di Chieti-Pescara “G. d’Annunzio”)

 

Article title: Discovery of a Series of Potent, Selective, and Orally Bioavailable Nucleoside
Inhibitors of CD73 That Demonstrates In Vivo Antitumor Activity
Authors: Li J, Chen L, Billedeau RJ, Stanton TF, Chiang JTP, Lee CC, Li W, Steggerda S, Emberley E, Gross M, Bhupathi D, Che X, Chen J, Dang R, Huang T, Ma Y, MacKinnon A, Makkouk A, Marguier G, Neou S, Sotirovska N, Spurlock S, Zhang J, Zhang W, van Zandt M, Yuan L, Savoy J, Parlati F, Sjogren EB
Journal: J Med Chem
Year: 2022
Volume: 66
DOI: 10.1021/acs.jmedchem.2c01287 

Main results: Based on the established immunosuppressive role of Adenosine in the tumor microenvironment, the authors optimized the synthesis of highly potent C-3′-β-alkyne nucleoside analogues as CD73 (ecto-5’-nucleotidase) inhibitors, to be used in the treatment of several types of cancer. The proposed compounds displayed potency gain thanks to the hydrophobic interactions within the binding pocket produced by the thiophene group, whereas a new aryl substitution contributes to increase their lipophilicity. Among others, compound 49 revealed a favorable CD73 inhibitory activity (IC50 = 0.10−0.21 nM) and selectivity for CD73 over the ecto-enzymes ENTPD2 and ENTPD3 (IC50 > 10uM). The inhibitory activity of the hit compound was evaluated against recombinant human CD73 and CD73-expressing SK-MEL-28 cells using a malachite green assay. Inhibition of CD73 in plasma was measured using liquid chromatography-mass spectrometry (LC/MS) to assess the conversion of 15N5-AMP into 15N5-ADO. Noteworthy, the authors took into consideration CD73 activity in the presence of full plasma in order to reflect the plasma protein binding profile. In the in vivo mouse studies, this compound displayed a moderate plasma protein binding profile (42-71% bound), thus allowing a sufficient unbound drug concentration and favorable pharmacodynamic characteristics, despite the limited oral bioavailability ((F = 2.8% at 50 mg/kg dose). A good doseresponse relationship was observed from oral administration with doses ranging from 10 to 50 mg/kg in mice, with a maximal 92% inhibition of plasma CD73 from the highest dose at 2 h post dosing. Moreover, upon the administration of a single oral dose at 50 mg/kg to female mice, the plasma exposure remained above the mouse plasma CD73 IC50 (1.0 nM) and ensured a long half-life with sustained target coverage for 24 h. In order to evaluate in vivo efficacy of this compound, a mouse xenograft model was used. A dose-proportional relationship was assessed at 10, 25, and 50 mg/kg doses via oral BID administration in the EG7 mouse lymphoma tumor model. The antitumor effect was elicited through the activation of anti-tumor immune response, that is activation of tumor-infiltrating CD8+ cells. Of note, this analogue was beneficial either as single-agent or in combination with chemo-therapeutics (doxorubicin and oxaliplatin) and immune-checkpoint inhibitors (anti-PD-L1).

Opinion: In recent years, many research groups took an effort to synthesize nucleoside analogues as CD73 inhibitors able to decrease the immunosuppressive Adenosine levels in the TME. So far, these compounds have been posing many challenges due to the poor bioavailability, as a consequence of the high polarity and anionic charged nature of their structures. In this work, the authors optimized the PK/PD characteristics of these analogues in terms of lipophilicity, systemic unbound drug concentration, half-life, and clearance. These new compounds displayed a good potency profile in the presence of full human plasma, and were able to suppress the production of Adenosine in serum samples from various cancer patients (head and neck squamous cell carcinoma, ovarian, triplenegative breast, and esophageal cancers), thus making them potential candidates for the clinical setting.

 

By Dr. Ilaria Dettori
(Department of NEUROFARBA, University of Florence)

Article title: Continued P2X7 Activation Leads to Mitochondrial Fission and Compromising Microglial Phagocytosis after Subarachnoid Haemorrhage
Authors: Tao T, Chen X, Zhou Y, Zheng Q, Gao S, Wang J, Ding P, Li X, Peng Z, Lu Y, Gao Y, Zhuang Z, Hang CH, Li W
Journal: J Neurochem
Year: 2022
Volume: 163
DOI: 10.1111/jnc.15712 

Main results: Subarachnoid haemorrhage (SAH) accounts for only 9.7% of all strokes, but it has a high rate of disability and fatality. P2X7 activation is a ubiquitous and important phenomenon that occurs after lesions. Damaged cells release high concentrations of ATP as chemokines to recruit microglia to the injury site and initiate a subsequent immune response. Authors focused on the role of P2X7 on microglial phagocytosis, clarifying the mechanisms that are at the base of this phenomenon. Infact, they found that the expression of microglial P2X7 increases after SAH and that the continued activation of P2X7 results in a gradual decrease in phagocytosis, while specifically blocking P2X7 activation reverses the impairment in phagocytosis. In particular, this effect is due to excessive mitochondrial fission, which is mediated by Ca2+/calcineurin/DRP1, so preventing excessive mitochondrial fission could alleviate phagocytic dysfunction. Moreover, they demonstrated that P2X7 inhibition is able to reverse mitochondrial fission after microglial activation and is able to significantly improve microglial phagocytosis, and so restoring brain homeostasis.

Opinion: Authors suggest that increase of P2X7 expression in the early stage of injury, facilitates microglial chemotaxis and proliferation to the lesion site but at the same time compromises microglial phagocytosis in the acute stage. This study deepens the understanding of microglial function after SAH and provides evidence for further regulation.

 

By Dr. Stefano Raffaele
(Università degli Studi di Milano)

Article title: P2Y6 receptor-mediated signaling amplifies TLR-induced pro-inflammatory responses in microglia
Authors: Timmerman R, Zuiderwijk-Sick EA, Bajramovic JJ
Journal: Front Immunol
Year: 2022
Volume: 13
DOI: 10.3389/fimmu.2022.967951 

Main results: Here, the authors used primary microglia cultures from non-human primates (Rhesus macaque) to investigate the effects of P2Y6 receptor inhibition with the selective antagonist MRS2578 on TLRinduced inflammatory response. Results show that P2Y6 blockade significantly reduced the release of pro-inflammatory cytokines from microglia stimulated with different TLR ligands. These data were also confirmed by transcriptomic analysis, revealing a significant modulation of pro inflammatory and immune-related molecular pathways in microglia treated with P2Y6 antagonist + LPS (a TLR4 agonist) as compared to LPS alone, including downregulation of genes related to cytokine production and upregulation of heat shock proteins. Finally, the effects of P2Y6 on the pro-inflammatory response of microglia was shown to involve the transcription factors NFAT, IRFs and NF-κB, as potential targets modulated by P2RY6-mediated signaling. 

Opinion: Growing evidence support the involvement of P2RY6-mediated signaling in neurodegenerative conditions. While the role of microglial P2RY6 in the regulation of phagocytosis has been widely acknowledged, less is known about the impact of this receptor on other functions of microglia, including cytokine production and release. The results from this paper suggest that inhibition of P2Y6 may be a promising strategy to limit the release of harmful pro-inflammatory cytokines by microglia, setting the basis for future in vivo studies evaluating the proper dosage and timing of this approach in experimental models of neurodegeneration.

 

 

By Dr. Ilaria Dettori
(Department of NEUROFARBA, University of Florence)

 

Article title: Continued P2X7 Activation Leads to Mitochondrial Fission and Compromising Microglial Phagocytosis after Subarachnoid Haemorrhage
Authors: Tao T, Chen X, Zhou Y, Zheng Q, Gao S, Wang J, Ding P, Li X, Peng Z, Lu Y, Gao Y, Zhuang Z, Hang CH, Li W
Journal: J Neurochem
Year: 2022
Volume: Online ahead of print
DOI: 10.1111/jnc.15712

Main results: Subarachnoid haemorrhage (SAH) accounts for only 9.7% of all strokes, but it has a high rate of disability and fatality. P2X7 activation is a ubiquitous and important phenomenon that occurs after lesions. Damaged cells release high concentrations of ATP as chemokines to recruit microglia to the injury site and initiate a subsequent immune response. Authors focused on the role of P2X7 on microglial phagocytosis, claryfing the mechanisms that are at the base of this phenomenon. Infact, they found that the expression of microglial P2X7 increases after SAH and that the continued activation of P2X7 results in a gradual decrease in phagocytosis, while specifically blocking P2X7 activation reverses the impairment in phagocytosis. In particular, this effect is due to excessive mitochondrial fission, which is mediated by Ca2+/calcineurin/DRP1, so preventing excessive mitochondrial fission could alleviate phagocytic dysfunction. Moreover, they demonstrated that P2X7 inhibition is able to reverse mitochondrial fission after microglial activation and is able to significantly improve microglial phagocytosis, and so restoring brain homeostasis.

Opinion: Authors suggest that increase of P2X7 expression in the early stage of injury, facilitates microglial chemotaxis and proliferation to the lesion site but at the same time compromises microglial phagocytosis in the acute stage. This study deepens the understanding of microglial function after SAH and provides evidence for further regulation.

 

By Dr. Vanessa D’Antongiovanni
(Dipartimento di Medicina Clinica e Sperimentale, Università di Pisa)

 

Article title: Role of Pannexin-1-P2X7R signaling on cell death and pro-inflammatory mediator expression induced by Clostridioides difficile toxins in enteric glia
Authors: Loureiro AV, Moura-Neto LI, Martins CS, Silva PIM, Lopes MBS, Leitão RFC, Coelho-Aguiar JM, Moura-Neto V, Warren CA, Costa DVS, Brito GAC
Journal: Front Immunol
Year: 2022
Volume: 13
DOI: 10.3389/fimmu.2022.956340

Main results: Clostridioides difficile (C. difficile), a gram-positive anaerobic bacillus, produces toxins A (TcdA) and B (TcdB), both associated with intestinal damage and diarrhea. TcdA and TcdB activate Pannexin-1 (Panx1) channels, releasing ATP, which in turn activates P2X7 receptor (P2X7R) leading to activation of caspase-3/7 pathway and production of pro-inflammatory citokines. In a colitis murine model, activation of P2X7R in enteric glial cells (EGCs) resulted in inflammatory response and cell death. However, it is still unknown the role of Panx1 and P2X7R in EGCs death and inflammatory response induced by C. difficile toxins. Male C57BL/6 mice (8 weeks of age) were infected with C. difficile VPI10463 and the control group received only vehicle per gavage. After three days post-infection, cecum and colon samples were collected to evaluate the expression of Panx1 by immunohistochemistry. To better investigate the role of Panx1 and P2X7R in EGCs death and inflammatory response a set of in vitro experiments were performed in EGCs treated with TcdA or TcdB, in the absence or presence of the Panx1 inhibitor (10Panx trifluoroacetate) or P2X7R antagonist (A438079). C. difficile infection increased the levels of Panx1 in cecum and colon of mice compared to the control group. In in vitro experiments, Panx1 inhibitor decreased caspase-3/7 activity and phosphatidylserine-annexin-V binding, but not interleukin (IL)-6 gene expression in EGCs treated with TcdA and TcdB. Interestingly, P2X7R antagonist reduced both caspase-3/7 activity and phosphatidylserine-annexin-V binding as well as IL-6 gene expression in TcdA and TcdB-challenged EGCs.

Opinion: In the present study Loureiro et al. demonstrated, for the first time, the involvement of Panx1/P2X7R signaling in cell death of EGCs induced by TcdA and TcdB toxins. In particular, the authors observed that C. difficile infection induced a significant increase of Panx1 expression with consequent activation of pro-apoptotic signaling (caspase-3/7 pathway) via P2X7R. In addition, P2X7R activation promotes IL-6 expression with consequent exacerbation of inflammatory response. Therefore, the inhibition of Panx1/P2X7R signaling could represent a promising therapeutic strategy for the treatment of C. difficile infection.

 

By Dr. Stefano Raffaele
(Università degli Studi di Milano)

 

Article title: P2Y6 receptor-mediated signaling amplifies TLR-induced proinflammatory responses in microglia
Authors: Timmerman R, Zuiderwijk-Sick EA, Bajramovic JJ
Journal: Front Immunol
Year: 2022
Volume: 20
DOI: 10.3389/fimmu.2022.967951

Main results: Here, the authors used primary microglia cultures from non-human primates (Rhesus macaque) to investigate the effects of P2Y6 receptor inhibition with the selective antagonist MRS2578 on TLRinduced inflammatory response. Results show that P2Y6 blockade significantly reduced the release of pro-inflammatory cytokines from microglia stimulated with different TLR ligands. These data were also confirmed by transcriptomic analysis, revealing a significant modulation of pro-inflammatory and immune-related molecular pathways in microglia treated with P2Y6 antagonist + LPS (a TLR4 agonist) as compared to LPS alone, including downregulation of genes related to cytokine production and upregulation of heat shock proteins. Finally, the effects of P2Y6 on the pro-inflammatory response of microglia were shown to involve the transcription factors NFAT, IRFs and NF-κB, as potential targets modulated by P2RY6-mediated signaling.

Opinion: Growing evidence support the involvement of P2RY6-mediated signaling in neurodegenerative conditions. While the role of microglial P2RY6 in the regulation of phagocytosis has been widely acknowledged, less is known about the impact of this receptor on other functions of microglia, including cytokine production and release. The results from this paper suggest that inhibition of P2Y6 may be a promising strategy to limit the release of harmful pro-inflammatory cytokines by microglia, setting the basis for future in vivo studies evaluating the proper dosage and timing of this approach in experimental models of neurodegeneration.

 

By Dr. Elisabetta Caiazzo
(Dipartimento di Farmacia, Scuola di Medicina e Chirurgia, Università Degli Studi di Napoli “Federico II”)

 

Article title: CD4+FoxP3+CD73+ regulatory T cell promotes cardiac healing post-myocardial infarction
Authors: Zhuang R, Meng Q, Ma X, Shi S, Gong S, Liu J, Li M, Gu W, Li D, Zhang X, Wang Z, Ge X, Tang J, Lin F, Liang X, Zheng L, Liu Z, Zhou X
Journal: Theranostics
Year: 2022
Volume: 12
DOI: 10.7150/thno.68437

Main results: Growing evidence suggests ecto-5'-nucleotidase/CD73 on immune cells as a potential therapeutic target in cardiovascular diseases. It is well known that the expression of CD73 together with CD39 on Tregs is necessary for their immunosuppressive function. In this study, the authors investigated the contribution of CD73 on Tregs to the resolution of cardiac inflammation and remodeling after myocardial infarction (MI). The authors found that CD4+FoxP3+ regulatory T cell infiltrated into the injured heart and highly expressed CD73 post-murine-MI set up by left anterior descending coronary artery ligation. Then they showed that CD73KO Tregs was associated with more severe cardiac fibrosis, no efficacy on the left ventricular remodeling after 28 days post-MI, a higher percentage of CD4+ gated in CD3+ cells, higher count of CD3+CD4+ T cells, higher protein levels of TNF-α and IFN-γ and less efficiency in reducing T-bet+ T cell infiltration in the heart compared to WT-Tregs. The impairment of the protective function of Tregs in cardiac healing post-MI was attributed to their reduced nuclear levels of FoxP3 and p65. Interestingly, administration of low doses of IL-2/anti-IL-2 complex caused FoxP3+CD73+Tregs expansion in murine MI models contributing to cardiac healing post-MI. In addition, the study revealed reduced frequency of Tregs (CD3+CD4+ CD127-CD25+FoxP3+ T cells in humans) as well as reduced expression of CD73 on Tregs in PBMCs of patients with acute myocardial infarction. Very importantly, the authors demonstrated a negative correlation between the percentage of peripheral blood CD73+CD4+ T cells with the serum levels of NT pro-BNP, troponin, myoglobin, CKI, and ejection fraction value in serum of patients with AMI.

Opinion: Little is known about the contribution and mechanism underlying the action of CD73+Tregs in cardiac repair post-MI. This study is very interesting because highlights the important contribution of CD73 on Tregs to the resolution of cardiac inflammation and remodeling after myocardial infarction through preclinical and clinical data. Very importantly, the authors first demonstrate a reduction of CD73 expression on CD4+ T cells and Tregs in PBMCs of patients with acute myocardial infarction compared to non-MI patients, suggesting that, in patients affected by acute myocardial infarction, CD73 on CD4+ T cells and Tregs may be involved in cardiac healing. Further research is needed to investigate whether CD73+Tregs may be a prognostic parameter in patients.

 

 

By Dr. Giulia Magni
(Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano)

 

Article title: The mGlu5 receptor protomer-mediated dopamine D2 receptor trans-inhibition is dependent on the adenosine A2A receptor protomer: implications for Parkinson’s disease
Authors: Romero-Fernandez W, Taura JJ, Crans RAJ, Lopez-Cano M, Fores-Pons R, Narváez M, Carlsson J, Ciruela F, Fuxe K, Borroto-Escuela DO.
Journal: Mol Neurobiol
Year: 2022
Volume: Online ahead of print
DOI: 10.1007/s12035-022-02946-9

Main results: In the present study, authors demonstrate that adenosine A2A receptor (A2AR) protomer plays a major role in the inhibitory modulation of the density and in the allosteric receptor-receptor interaction within the dopamine D2 receptor (D2R)- metabotropic glutamate receptor type 5 (mGluR5) heteromeric component of the A2AR-D2R-mGluR5 complex in vitro and in vivo. Results show that A2AR expression in HEK293T cells co-expressing D2R and mGluR5 resulted in a significant increase in the formation of the D2R-mGluR5 heteromeric component. Moreover, a significant increase of the high-affinity component of D2R was found upon co-treatment with mGluR5 and A2AR agonists in cells expressing A2AR, D2R and mGluR5. Stimulation of cells co-expressing A2AR, D2R and mGluR5 with an mGluR5 agonist or D2R antagonist fully counteracted the D2R agonist-induced inhibition of cAMP levels, which was not observed in cells only expressing mGluR5 and D2R. Finally, the mGluR5-negative allosteric modulator Raseglurant significantly reduced haloperidol-induced catalepsy in mice, and in A2AR knockout mice haloperidol proved no effect, supporting a functional role for mGluR5 and A2AR in enhancing D2R blockade resulting in catalepsy.

Opinion: The present paper demonstrates that, within the A2AR-D2R-mGluR5 complex, the A2AR protomer enhances the formation of the D2R-mGluR5 component. In vivo, A2AR antagonists targeting the A2AR-D2R-mGluR5 complex in dorsal striatum counteract haloperidol-induced catalepsy, a condition characterized by loss of muscle control and rigid posture as observed in Parkinson’s disease, whose symptoms are mainly caused by dopamine deficiency. Therefore, these findings open the perspective for the development of drugs against neurodegenerative disorders by targeting receptor-receptor interactions.

 

By Dr. Mariachiara Zuccarini 
(Dipartimento di Scienze Mediche, Orali e Biotecnologiche, Divisione di Farmacologia e Tossicologia, Università degli studi di Chieti-Pescara “G. d’Annunzio”)

 

Article title: Purine nucleotide depletion prompts cell migration by stimulating the serine synthesis pathway
Authors: Soflaee MH, Kesavan R, Sahu U, Tasdogan A, Villa E, Djabari Z, Cai F, Tran DH, Vu HS, Ali ES, Rion H, O'Hara BP, Kelekar S, Hallett JH, Martin M, Mathews TP, Gao P, Asara JM, Manning BD, Ben-Sahra I, Hoxhaj G
Journal: Nat Commun
Year: 2022
Volume: 13
DOI: 10.1016/j.chembiol.2021.02.017

Main results: Purine nucleotide synthesis is essential for key biological processes, including cell division. In the present work, prostate, melanoma and lung cancer cells depleted of cellular purine nucleotides, upon treatment with purine synthesis inhibitor MTX (2 μM, 15h), showed an increase in de novo serine synthesis. This effect was reverted by exogenous addition of inosine (50 μM, 1 h) or adenine (50 μM, 1 h). MTX or LTX concomitantly boosted cancer cell migration in a dose-dependent manner as a result of increased intracellular levels of serine as well as a serine synthesis intermediate, 3-phosphoserine. Purine nucleotide depletion was achieved by targeting the adenylate synthesis enzyme phosphoribosylglycinamide formyltransferase (GART) with chemical inhibitors or via CRISPR/Cas9-mediated knockout of GART (ΔGART). Interestingly, the depletion of guanylates by the inosine monophosphate dehydrogenase (IMPDH) inhibitor Mizoribine (15 μM) did not evoke an increase in serine synthesis, thus suggesting that depletion of adenylates was the only responsible for increased cell migration. The selective inhibition of phosphoglycerate dehydrogenase (PHGDH) blocked the purine depletion-dependent serine synthesis. Following treatment with MTX or GART deletion, the increased serine synthesis also correlated with a reduced glycolytic flux (decreased extracellular acidification rate and glucose-derived lactate), with no changes in oxygen consumption rate (OCR). Both the inhibition of serine entry into one-carbon derivatives and formate synthesis reduced MTX-induced cell migration. Moreover, the authors revealed that purine depletion triggered the epithelial-to-mesenchymal transition (EMT) process as evidenced by N-cadherin and Vimentin up-regulation and E-cadherin down-regulation. This event would enhance cell ability to shift towards a pro-migratory phenotype. Finally, the authors examined the metastatic potential of purine depletion by treating mice bearing subcutaneous tumors (derived from murine melanoma) with MTX for 3 weeks. The latter promoted melanoma cell dissemination to lymph nodes and lungs, and again this effect was accompanied by increased serine levels and inhibition of pyruvate kinase M, the final enzyme of glycolysis.

Opinion: Metabolic reprogramming is one of the best characterized hallmarks of cancer and involves biochemical alterations which favor cancer cells to survive and proliferate. This study revealed that purine nucleotide depletion with purine synthesis inhibitors triggered a shunt of glycolytic flux towards a de novo serine synthesis pathway. Extracellular serine supports cell proliferation, being a precursor of essential molecules (glycine and cysteine, sphingolipids, phospholipids, folate, purines). Thus, in the logic of cell homeostasis, the increased serine synthesis would serve to restore, at some point, the nucleotide pool. With regard to the translation impact, purine depletion was also shown to boost tumor cell migration to secondary organs in vivo, this effect being dependent of increased circulating levels of serine. Deciphering the crosstalk between different metabolic fluxes in cancer and in a physiological setting is still a big challenge for the scientific community, therefore investigating the enzymes of purine and serine metabolism could yield a promising set of druggable targets in cancer.

 

By Dr. Ilaria Dettori
(Department of NEUROFARBA, University of Florence)

 

Article title: Adenosine A1R/A3R (Adenosine A1 and A3 Receptor) Agonist AST-004 Reduces Brain Infarction in a Nonhuman Primate Model of Stroke
Authors: Liston TE, Hama A, Boltze J, Poe RB, Natsume T, Hayashi I, Takamatsu H, Korinek WS, Lechleiter JD
Journal: Stroke
Year: 2022
Volume: 53
DOI: 10.1161/STROKEAHA.121.036396

Main results: In this paper, authors investigated the effect of A1R/A3R activation via AST-004, a novel adenosine A1R/A3R receptor agonist, in a Nonhuman Primate model of 4-hour transient cerebral ischemia. AST-004 treatment reduced total infarct volume 24 hours and 5 days after Middle Cerebral Artery occlusion. In addition to reduced total infarct volume, AST-004 treatment reduced the rate of expansion of the infarct volume over time. These data suggest a cerebroprotective effect of AST-004, supported by the finding that penumbra volume decline was reduced under AST-004 treatment. In summary, these results suggest activation of A1R/A3R as a potential cerebroprotective strategy that could be used to prevent brain tissue necrosis and ultimately enhance functional outcome following an acute ischemic stroke.

Opinion: Interestingly, authors demostrated that the efficacy of AST-004 in the reduction of ischemic infarct damage paralleled its concentrations in plasma and in CSF and align with estimated brain A1R/A3R occupancy. These findings suggest further preclinical and clinical investigation of simultaneous activation of A1R and A3R as a novel cerebroprotective strategy, confirming the importance of the newly synthesized mixed compounds as new therapeutic targets thanks to pharmacokinetics advantages.

 

By Dr. Stefano Raffaele
(Università degli Studi di Milano)

 

Article title: The microglial P2Y6 receptor mediates neuronal loss and memory deficits in neurodegeneration
Authors: Puigdellivol M, Milde S, Vilalta A, Cockram TOJ, Allendorf DH, Lee JY, Dundee JM, Pampuscenko K, Borutaite V, Nuthall HN, Brelstaff JH, Spillantini MG, Brown GC.
Journal: Cell Reports
Year: 2021
Volume: 37
DOI: 10.1016/j.celrep.2021.110148

Main results: In this paper, authors investigate the involvement of microglial P2Y6 receptor, known to regulate the phagocytic capacity of these immune cells, in neurodegeneration and cognitive deficits associated with Alzheimer’s disease. Interestingly, knockout of P2Y6 receptor was found to prevent microglial phagocytosis of neurons and subsequent memory loss induced by amyloid-β and tau in two different in vivo models. Moreover, in vitro experiments using microglia-neuron co-cultures demonstrated that microglial P2Y6 specifically mediates the phagocytosis of stressed-but-viable neurons (that release the endogenous receptor agonist UDP in response to damage). Conversely, this mechanism did not affect the engulfment of dead cells and debris.

Opinion: Many researchers in the neuroscience field are currently focusing their efforts on the identification of pharmacological targets able to block the detrimental effects of microglia while preserving their beneficial roles. The results described above highlight microglial P2Y6 receptor as a promising target, as its pharmacological inhibition may help limiting excessive phagocytosis of still viable cells. In parallel, this approach would not affect the clearance capacity of microglia, that is fundamental to initiate repair processes in the central nervous system.

 

By Dr. Vanessa D’Antongiovanni 
(Dipartimento di Medicina Clinica e Sperimentale, Università di Pisa)

 

Article title: Molecular and Pharmacological Evidence for the Expression of Multiple Functional P2 Purinergic Receptors in Human Adipocytes
Authors: Rossato M, Favaretto F, Granzotto M, Crescenzi M, Boscaro A, Di Vincenzo A, Capone F, Dalla Nora E, Zabeo E, Vettor R.
Journal: Molecules
Year: 2022
Volume: 27
DOI: 10.3390/molecules27061913

Main results: Extracellular adenosine 5’-triphosphate (ATP) exerts important functions as an extracellular signaling molecule via the activation of specific P2 purinergic receptors (P2X and P2Y). In particular, the activation of P2Rs in adipocytes triggers several effects, including the inhibition of leptin secretion and glucose transport, the induction of aromatase activity and the stimulation/inhibition of lipogenesis. Currently, only a limited number of studies have well characterized the different expression and functions of P2Rs in human white adipose tissue. Based on this background, the aim of the present study was to investigate the expression of the different P2Rs and their possible functional activation in human adipocytes in primary culture. Human adipocytes express different P2X and P2Y receptor subtypes (P2X1-2-4-5-7 and P2Y1-2-4-6-11-12-13-14). Treatment with extracellular ATP elicited a rise in [Ca2+]i via the activation of P2X and P2Y receptor subtypes. Of note, human adipocytes spontaneously released ATP in the extracellular medium and secreted IL-6 both at rest and after stimulation with ATP. This stimulatory effect of ATP on IL-6 secretion was inhibited by pre-incubation with apyrase, an ATP metabolizing enzyme. These results demonstrate that human adipocytes express different P2Rs functionally activated by extracellular nucleotides. Furthermore, human adipocytes spontaneously release ATP, which can act in an autocrine/paracrine fashion on adipocytes, possibly participating in the regulation of inflammatory cytokine release.

Opinion: In the present study Rossato et al. demonstrated that human adipocytes express different P2X and P2Y receptor subtypes. These receptors are functionally active, being responsive mainly to ATP but also to other purine and pyrimidine analogs. Furthermore, ATP seems to be constitutively released in the extracellular space where it stimulates adipocyte production of IL-6, a well-known pro-inflammatory cytokine inducing a low-grade inflammatory state. The results of the present study open new pathophysiological perspectives on the modulation of adipocyte activity by targeting the adipocyte P2Rs to counteract the inflammatory activity of adipocytes mainly in the presence of adipose tissue expansion, such as in obesity.

 

 

By Dr. Roberta Turiello
(Dipartimento di Farmacia, Università degli Studi di Salerno)

 

Article title: CD73-mediated adenosine production by CD8 T cell-derived extracellular vesicles constitutes an intrinsic mechanism of immune suppression
Authors: Schneider E, Winzer R, Rissiek A, Ricklefs I, Meyer-Schwesinger C, Ricklefs FL, Bauche A, Behrends J, Reimer R, Brenna S, Wasielewski H, Lauten M, Rissiek B, Puig B, Cortesi F, Magnus T, Fliegert R, Müller CE, Gagliani N, Tolosa E.
Journal: Nat Commun
Year: 2021
DOI: 10.1038/s41467-021-26134-w

Main results: The paper here discussed clarifies the role of CD73+ extracellular vesicles in the context of inflammation. Firstly, the authors analysed the expression of CD73 and CD39 in human peripheral blood mononuclear cells, showing that CD73 is expressed only by a low frequency of Tregs, while it is more frequently expressed by CD8 T cells. On the other hand, CD39 is more frequently expressed by Tregs. Importantly, considering the low percentage of Tregs expressing CD73, the co-expression of CD73 and CD39 on these cells is very unlikely to occur. The authors demonstrated that human T cells release enzymatically active CD73, upon activation. The hydrolysis of AMP in supernatants of activated CD8 resulted mostly mediated by CD73-expressing extracellular vesicles, while the contribution of soluble CD73 or other soluble enzymes is negligible. After evaluating the expression of ectonucleotidases, the authors also analysed the ATPase activity in T cell subpopulations, by performing sensitive HPLC-based assay, after treating cells with 1,N6-etheno-ATP. Tregs emerged as the population with the highest ATPase activity, compared with CD8 and CD4con T cells (conventional non-Treg CD4 T cells). This latter population showed the least ATPase activity. Then, the authors performed a suppression assay, using CD73-CD4con T cells as responder cells and different ratios of Tregs. The treatment with Tregs induced only a partial suppression, while the addition of CD73+ extracellular vesicles, isolated from CD73+CD8 T cells, completely suppressed the proliferation and the production of IFN- of effector T cells, in presence of ATP. This effect was not observed when treating cells with extracellular vesicles derived from CD73- CD8 T cells. In order to address the relevance of non-cell bound CD73 in the context of inflammation, the authors isolated extracellular vesicles from the synovial fluids of patients with juvenile idiopathic arthritis. CD73+ extracellular vesicles resulted able to suppress the functions and the proliferation of T cells, in a CD73 dependent manner. These vesicles co-express CD73 and CD8, suggesting that CD8 T cells are the main source of CD73-containing vesicles in the synovial fluid.

Opinion: Adenosine pathway has been described as a potent mechanism of immunosuppression and it exerts a crucial role both in inflammatory diseases and in cancer. During the last years, the circulating enzymes involved in extracellular adenosine production have gained particular interest. This paper provides new interesting details on the role and the source of CD73+ extracellular vesicles in the context of inflammation. The authors showed that, upon activation, human CD8 T cells release CD73+ extracellular vesicles which work in concert with Tregs to produce adenosine. Specifically, the authors propose that CD39 expressed by Tregs is responsible for the hydrolysis of ATP into AMP, but the AMPase activity is provided by CD73+ vesicles, while Tregs alone do not generate sufficient adenosine to supress T cells proliferation and effector functions.

By Dr. Giulia Magni
(Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano)

 

Article title: The ATP-hydrolyzing ectoenzyme E-NTPD8 attenuates colitis through modulation of P2X4 receptor-dependent metabolism in myeloid cells
Authors: Tani H, Li B, Kusu T, Okumura R, Nishimura J, Okuzaki D, Motooka D, Arakawa S, Mori A, Yoshihara T, Ogino T, Tsai SH, Furuta Y, Muneta M, Nakamura S, Fukusaki E, Yamamoto K, Yagita H, Kayama H, Takeda K.
Journal: Proc Natl Acad Sci U S A
Year: 2021
Volume: 118
DOI: 10.1073/pnas.2100594118

Main results: Gut bacteria, composing the microbiota, during their growth produce extracellular ATP in the lumen, which drives host immune responses. To avoid excessive immune reactions in the intestinal mucosa, which leads to pathogenesis of intestinal inflammation, luminal ATP concentration is finely regulated through a yet-to-be discovered molecular mechanism. Authors show that ectonucleoside triphosphate diphosphohydrolase (E-NTPD)8 enzyme is highly expressed in large-intestinal epithelial cells and hydrolyzes microbiota-derived luminal ATP. Compared with wild-type mice, Entpd8-/- mice display higher levels of luminal ATP in the colon and develop more severe dextran sodium sulfate (DSS)-induced colitis, which can be ameliorated by either the depletion of neutrophils and monocytes or by P2rx4 deficiency in hematopoietic cells. Moreover, an increased level of luminal ATP in the colon of Entpd8-/- mice promotes glycolysis in neutrophils through P2X4 receptor-dependent Ca2+ influx, which is linked to prolonged survival and elevated ROS production in these cells. Thus, E-NTPD8 in epithelial cells exerts an immunomodulatory function on intestinal inflammation by controlling metabolic alteration toward glycolysis in myeloid cells via P2X4 receptors.

Opinion: By employing molecular biology and metabolomic techniques, with this paper authors provide the first demonstration that ATP-hydrolyzing enzyme E-NTPD8 acts as an immunomodulator in the colon. Moreover, they show that the clearance of microbiota-derived ATP by E-NTPD8 is essential for inhibiting the prolonged survival of neutrophils and monocytes by discouraging the P2X4 receptor-mediated promotion of glycolysis. Overall, results reveal the mechanism that prevents innate intestinal pathology through the modulation of myeloid cell metabolism and could lead to the identification of therapeutic targets for inflammatory bowel disease (IBD) treatment.

 

By Dr. Mariachiara Zuccarini 
(Dipartimento di Scienze Mediche, Orali e Biotecnologiche, Divisione di Farmacologia e Tossicologia, Università degli studi di Chieti-Pescara “G. d’Annunzio”)

 

Article title: Positive allosteric mechanisms of adenosine A1 receptor-mediated analgesia
Authors: Draper-Joyce CJ, Bhola F, Wang J, Bhattarai A, Nguyen ATN, Cowie-Kent I, O'Sullivan K, Chia LY, Venugopal H, Valant C, Thal DM, Wootten D, Panel N, Carlsson J, Christie MJ, White PJ, Scammells P, May LT, Sexton PM, Danev R, Miao Y, Glukhova A, Imlach WL, Christopoulos A
Journal: Nature
Year: 2021
Volume: 597
DOI: 10.1038/s41586-021-03897-2

Main results: Adenosine is known to evoke pain relief through A1R modulation on peripheral nociceptive neurons. In the present paper, the authors revealed that a positive allosteric modulator (PAM) of the A1R, the [2-amino-4-(3,5-bis(trifluoromethyl)phenyl)thiophen-3-yl)(4-chlorophenyl)methanone] (MIPS521), elicited an analgesic effect by increasing endogenous adenosine binding to A1R in a rodent model of neuropathic pain. The latter was obtained through a partial nerve ligation able to induce sciatic nerve injury in 7-12-week-old male and female Sprague-Dawley rats. The Gaussian accelerated molecular dynamics (GaMD) method demonstrated the bi-directional communication, defined as “positive cooperativity”, between the allosteric modulator (MIPS521) and the endogenous ligand (adenosine) on the A1R-Gi2 heterotrimer. Compared to another PAM, that is VCP171, MIPS521 showed lower allosteric affinity but higher cooperativity with adenosine and more potency in reducing the amplitude and the frequency of electrically evoked excitatory post-synaptic currents (eEPSCs) and spontaneous excitatory post-synaptic currents (sEPSCs) in spinal cord from nerve-injured rats compared to sham surgery controls. By performing molecular dynamics simulations, the authors defined the precise binding site of MIPS521 in the A1R heterotrimer (i.e. the allosteric pocket of transmembrane helices 1, 6 and 7 of the GPCR) and revealed the mechanism by which the allosteric modulator stabilized adenosine-A1R-Gi2 protein complex, thus affecting the ability of the receptor to transition between active and inactive states. The analgesic effect of the intrathecal administration of MIPS521 was reverted by treatment with the A1R antagonist, DPCPX. In addition, MIPS521 reduced mechanical hyperalgesia and spontaneous pain in a conditioned place preference model in vivo. Unlike opioid drugs (e.g. morphine) or traditional A1R orthosteric agonists, both PAMs did not cause acute motor impairment or bradycardia.

Opinion: G protein-coupled receptors (GPCRs) are one of the best characterized receptors to be investigated for drug development. Among others, there is general consensus that A1R/A3R activation or A2A inhibition exert antinociceptive effect. For example, a novel compound, namely AzoAdenosine-3, demonstrated to reduce pain through A1R interaction in a light-dependent manner. However, the ubiquitous expression of A1R receptors makes it difficult to develop highly selective compounds. Indeed, traditional A1R orthosteric agonists may provoke unwanted off-target effects due to the negative chronotropic and inotropic effects in the heart. These off-target effects were not shown in MIPS521-mediated analgesic effect, thus suggesting the therapeutic potential of this highly selective ligand able to target exclusively injured neurons. The recent finding about the structure of the A1R in the active Gi protein complexed state with both Gi2 and adenosine may have important repercussions for structure-based drug design. The discovery of a class of compounds, being effective and safe at the same time, may offer an alternative to opioid analgesic agents for treating neuropathic pain in several chronic diseases such as multiple sclerosis.

 

By Dr. Elisabetta Coppi
(Dipartimento di Neuroscienze, Psicologia, Ricerca del Farmaco e Salute del Bambino (NeuroFarBa), Divisione di Farmacologia e Tossicologia, Università di Firenze)

 

Article title: Adult Stress Promotes Purinergic Signaling to Induce Visceral Pain in Rats with Neonatal Maternal Deprivation
Authors: Hu S, Sun Q, Du WJ, Song J, Li X, Zhang PA, Xu JT, Xu GY
Journal: Neurosci Bull
Year: 2020
Volume: 36
DOI: 10.1007/s12264-020-00575-7

Main results: Il dolore cronico viscerale colpisce fino al 15% della popolazione mondiale e rappresenta un'esigenza medica ancora irrisolta. Anche se i meccanismi di questa patologia rimangono in gran parte poco chiari, evidenze emergenti rilevano una correlazione fra dolore viscerale a condizioni di vita stressanti nell’uomo, in particolar modo durante la prima infanzia. La deprivazione parentale è un modello animale che mima tale condizione di stress. Il presente studio rivela, per la prima volta, un importante ruolo dei recettori purinergici P2X3 nel dolore viscerale cronico indotto da deprivazione parentale nel ratto. Gli Authors: infatti dimostrano che i recettori P2X3 sono sovra-espressi nei neuroni dei gangli delle radici dorsali (DRG) di ratti che hYear: subito deprivazione parentale e che l’ATP induce risposte maggiori in tali neuroni, facilitando l’insorgenza del potenziale d’azione. Tale iper-eccitabilità favorirebbe l’insorgere, in età adulta di ipersensibilità viscerale.

Opinion: Il presente studio dimostra un nuovo meccanismo mediante il quale un neurotrasmettitore da tempo conosciuto come mediatore correlato allo stress, ovvero la noradrenalina, interagisce con il sistema purinergico per dare luogo ad una modulazione, finora inedita, dell’eccitabilità neuronale a livello dei gangli delle radici dorsali, che innervano fra gli altri organi anche il colon (in particolare dai gangli T13 agli L2). In questo caso l’aumentata risposta all’ATP, in seguito alla sovraespressione dei recettori P2X3 dovuta all’evento stressante in età perinatale, sembra essere la causa di quei cambiamenti molecolari ed elettrofisiologici che portano all'ipereccitabilità dei neuroni dei DRG, in particolare di quelli che innervano il colon. Infatti, il blocco di tale recettore purinergico è in grado di prevenire l’instaurarsi dell’ipersensibilità viscerale che si osserva in questi animali nell’età adulta.

 

 

 

By Dr. Ilaria Dettori
(Department of NEUROFARBA, University of Florence)

 

Article title: Erythrocyte adenosine A2B receptor prevents cognitive and auditory dysfunction by promoting hypoxic and metabolic reprogramming
Authors: Qiang Q, Manalo JM, Sun H, Zhang Y, Song A, Wen AQ, Wen YE, Chen C, Liu H, Cui Y, Nemkov T, Reisz JA, Edwards III G, Perreira FA, Kellems RE, Soto C, D’Alessandro A, Xia Y
Journal: PLOS BIOLOGY
Year: 2021
Volume: 19
DOI: 10.1371/journal.pbio.3001239

Main results: Hypoxia drives aging and promotes age-related cognition and hearing functional decline. Recently, studies revealed that signaling through the erythrocyte adenosine A2B receptor (ADORA2B) promotes O2 release to counteract hypoxia at high altitude. Using a erythrocyte-specific deletion of ADORA2B model in mice (eADORA2b-/-), the authors demonstrate that eADORA2b-/- mice show an acceleration of the onset of age-related impairments in spatial learning, memory, and hearing ability, display the early aging-like cellular and molecular features including the proliferation and activation of microglia and macrophages, elevation of pro-inflammatory cytokines, and attenuation of hypoxia-induced glycolytic gene expression to counteract hypoxia in the hippocampus, cortex, or cochlea. Hypoxia sufficiently accelerated early onset of cognitive and cochlear functional decline and inflammatory response in eADORA2b-/- mice. Mechanistically, erythrocyte ADORA2B-mediated activation of AMP activated protein kinase (AMPK) and bisphosphoglycerate mutase (BPGM) promotes hypoxic and metabolic reprogramming to enhance production of 2,3- bisphosphoglycerate (2,3-BPG), an erythrocyte-specific metabolite triggering O2 delivery.

Opinion: These studies indicate a critical role of erythrocyte ADOAR2B–BPGM as an “anti-aging” and “anti- hypoxia” rejuvenating factor by inducing 2,3-BPG production and triggering O2 delivery to attenuate progression to age-related functional decline and immediately suggest novel therapies targeting this signaling axis.

By Dr. Vanessa D’Antongiovanni
(Dipartimento di Medicina Clinica e Sperimentale, Università di Pisa)

 

Article title: P2Y2R Deficiency Ameliorates Hepatic Steatosis by Reducing Lipogenesis and Enhancing Fatty Acid β-Oxidation through AMPK and PGC-1α Induction in High-Fat Diet-Fed Mice
Authors: DusabimanaT, Park EJ, Je J, Jeong K, Yun SP, Kim HJ, Kim H, Park SW
Journal: Int J Mol Sci
Year: 2021
Volume: 22
DOI: 10.3390/ijms22115528

Main results: Non-alcoholic fatty liver disease (NAFLD) is a chronic metabolic liver disease associated with obesity and insulin resistance. Activation of the purinergic receptor P2Y2R has been reported to promote adipogenesis, inflammation and dyslipidemia in adipose tissues in obese mice. However, the role of P2Y2R and its mechanisms in NAFLD remain unknown. Based on this background, the aim of the present study was to investigate the role of P2Y2R in the onset and development of obesity-induced NAFLD. Mice c57bl/6 wild type and P2Y2R knockout (KO) mice were fed with a high-fat diet (HFD) for 12 weeks. Mice P2Y2R KO fed with HFD displayed: 1) an improvement of insulin resistance, 2) a decrease in body weight, plasma insulin and fatty acid synthesis mediators and 3) a reduction in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. Such effects are ascribed to an increase in AMPK activity, pivotally involved in the lipogenesis and fatty acid β- oxidation.

Opinion: In the present study Dusabimana et al. demonstrated that P2Y2R deficiency improved insulin resistance, triglyceride accumulation, and hepatocellular injury by reducing hepatic lipogenesis and promoting fatty acid β-oxidation, thereby ameliorating NAFLD phenotype. Mechanistically, by increasing the AMPK activity, P2Y2R deficiency can regulate de novo lipogenesis and mitochondrial biogenesis. Therefore, P2Y2R represent a promising therapeutic strategy for NAFLD and obesity- induced metabolic disorders.

 

By Dr. Stefano Raffaele
(Università degli Studi di Milano)

 

Article title: Capillary-associated microglia regulate vascular structure and function through PANX1-P2RY12 coupling in mice
Authors: Bisht K, Okojie KA, Sharma K, Lentferink DH, Sun YY, Chen HR, Uweru JO, Amancherla S, Calcuttawala Z, Campos-Salazar AB, Corliss B, Jabbour L, Benderoth J, Friestad B, Mills III WA, Isakson BE, Tremblay ME, Kuan CY, Eyo UB
Journal: Nat Commun
Year: 2021
Volume: 12
DOI: 10.1038/s41467-021-25590-8

Main results: In this paper, authors exploited in vivo two-photon microscopy to unveil a novel homeostatic role of microglia, the resident immune cells of the brain, in the regulation of neurovascular unit structure and permeability. Briefly, results revealed the existence of a subpopulation of capillary-associated microglia (CAMs) that are enriched around brain vasculature (around one third of the total microglia in the healthy brain). These cells were shown to interact with blood vessels using their soma, establishing a more consistent relationship compared to the transient physical contacts typical of microglial processes. CAMs exhibited similar features to parenchymal microglia, suggesting that they are unlikely to be ontogenically or functionally distinct cells. Interestingly, CAM-vessel interactions were found to be regulated by microglial purinergic P2RY12 signaling, activated by purines released from capillaries through pannexin 1 (PANX1) channels. Pharmacological depletion of microglia led to increased capillary diameter, elevated cerebral blood flow, and impaired vasodilative responses, which were recapitulated in P2RY12 and PANX1 knockout mice. Hence, these data demonstrate the importance of microglia in regulating capillary structure and functions, highlighting microglia as a key component of the neurovascular unit.

Opinion: This report provides the first evidence supporting microglial homeostatic contributions to capillary structure and function outside pathology. Increasing our understanding of microglial regulation of cerebrovascular function is particularly relevant for treating microvascular diseases, in which these cellular events might be pathologically altered. Notably, purinergic signaling mediated by PANX1- P2RY12 coupling was found to be essential for microglial interactions with blood vessels. As also suggested by authors, this mechanism could regulate the attraction of microglia to capillaries. However, purines are rapidly degraded in the extracellular space. Purinergic signaling is therefore unlikely to be involved in long-term attachment processes, which could presumably be regulated by other yet-to-be-identified adhesion factors.

 

By Dr. Elisabetta Caiazzo
(Dipartimento di Farmacia, Scuola di Medicina e Chirurgia, Università Degli Studi di Napoli “Federico II”)
 

Article title: Efficacy and Effect of Inhaled Adenosine Treatment in Hospitalized COVID-19 Patients
Authors: Caracciolo M, Correale P, Mangano C, Foti G, Falcone C, Macheda S, Cuzzola M, Conte M, Falzea AC, Iuliano E, Morabito A, Caraglia M, Polimeni N, Ferrarelli A, Labate D, Tescione M, Di Renzo L, Chiricolo G, Romano L, De Lorenzo A
Journal: Front Immunol
Year: 2021
Volume: 12
DOI: 10.3389/fimmu.2021.613070

Main results: The authors of the study investigated the off-label treatment with aerosolized adenosine in hospitalized COVID-19 patients. This is a single-center case-control study that has involved 12 patients in the control group (41.67% females and 58.33% males) and 12 patients in the treatment group (33.33% females and 66.67% males). that were positive for Sars-CoV2 and arrived at the Emergency Department of the “Bianchi Melacrino Morelli” Grande Ospedale Metropolitano (GOM) of Reggio Calabria, Italy, between March 19 and April 13, 2020. The primary endpoints were reduction of length of stay and improvement of prognosis. The secondary endpoints were the reduction of inflammation severity, and timing of lung lesions by chest Computer Tomography. The mean age of the subjects was 56.86 ± 15.65 years and no significant differences were found between the two groups in terms of age, BMI, standard therapy duration, and respiratory parameters. Patients in the treatment group received aerosolized adenosine (Krenosin®), at the dosage of 9 mg every 12 h in the first 24 h and subsequently, every 24 h for 5 days. Treatment with adenosine was limited to 5 days since chronic administration of inhaled adenosine has been demonstrated to cause fibrosis and pulmonary hypertension. All patients received standard therapeutic protocol (low molecular weight heparin, azithromycin, hydroxychloroquine, lopinavir/ritonavir). The authors showed a reduction in days of hospitalization on average 6 days and an improvement in lymphopenia, in PaO2/FiO2, Horowitz index, together with a reduction in inflammation parameters, such as the C-reactive protein level. In addition, they found a reduction in D-dimer concentration as well as a reduction in neutrophils and lymphocytes ratio platelets and lymphocytes ratio. Regarding chest Computer Tomography, the authors found reductions of ground-glass opacities with consolidation and rounded morphology in treated group with a reduction under 50% of pleural effusion and pneumopathy. Use of inhaled adenosine showed a good tolerance with no cardiovascular and respiratory side effects. The authors hypothesized that inhaled adenosine may regulate inflammation through its receptors, A2A and A2B expressed in macrophages, neutrophils, monocytes, lymphocytes, and platelets.
They concluded that the treatment with adenosine, before clinical features of the onset of cytokine storm, seems to be safe and modulates the immune system, allowing an effective response against the progress of the viral infection, reducing days of hospitalization.

Opinion: COVID-19 is still a pandemic with the highest risk of death, especially in unvaccinated people. This study is interesting since provides the first evidence of adenosine use in COVID-19 hospitalized patients. However, it has several limitations. Firstly, this is a single-center case-control study and not a randomized controlled clinical trial. Second, a very small sample size of patients is involved in the study. Third, the authors did not investigate underlying mechanisms to explain the protective effect observed with adenosine administration. In conclusion, even if this study highlights adenosine as a potential therapeutic approach for patients affected by COVID-19 infection, additional trials are needed to have a valid scientific conclusion.

 

 

 

By Dr. Giulia Magni
(Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano)

Article title: Adenosine A3 agonists reverse neuropathic pain via T cell-mediated production of IL-10
Authors: Durante M, Squillace S, Lauro F, Giancotti LA, Coppi E, Cherchi F, Di Cesare Mannelli L, Ghelardini C, Kolar G, Wahlman C, Opejin A, Xiao C, Reitman ML, Tosh DK, Hawiger D, Jacobson KA, Salvemini D
Journal: J Clin Invest
Year: 2021
Volume: 131
DOI: 10.1172/JCI139299

Main results: A3 adenosine receptor (A3AR), a promising therapeutic target to treat neuropathic pain, is expressed by immune cell, and it is known that its activation during pathological conditions modulates cytokine release. The present paper demonstrates that Rag-KO mice, lacking T and B cells, are insensitive to the anti-allodynic effect of A3AR agonists observed in WT mice after chronic constriction injury (CCI) of the sciatic nerve, and the same result was observed in IL-10-KO and IL-10 receptor-KO mice. Adoptive transfer of CD4+ T cells from WT mice infiltrated the dorsal root ganglia (DRG) and restored the anti-allodynic effect of A3AR agonist in Rag-KO mice, while CD4+ T cells from Adora3-KO or IL- 10-KO mice did not. Moreover, CD4+ T cells transfer from WT mice, but not IL-10-KO mice, into IL- 10-KO mice or Adora3-KO mice fully restored the anti-allodynic effect of A3AR activation. Finally, A3AR agonism reduced the excitability of DRG neurons co-cultured with CD4+ T cells in an IL-10- dependent manner, and reduced the phosphorylation of GluN2B-containing NMDA receptors at Tyr1472, a modification associated with the regulation of neuronal hypersensitivity.

Opinion: By using behavioral, genetic, pharmacological and electrophysiological approaches, this study in my opinion represents a very elegant demonstration of the contribution of T cells to the pharmacological actions of A3AR agonists in an established model of neuropathic pain. Results open the perspective for the exploitation of immune cells as a potential cellular substrate for the pharmacological action of A3AR agonists in the treatment of chronic pain conditions.

 

By Dr. Mariachiara Zuccarini
(Dipartimento di Scienze Mediche, Orali e Biotecnologiche, Divisione di Farmacologia e Tossicologia, Università degli studi di Chieti-Pescara “G. d’Annunzio”)

 

Article title: Thermal proteome profiling identifies the membrane-bound purinergic receptor P2X4 as a target of the autophagy inhibitor indophagolin
Authors: Carnero Corrales MA, Zinken S, Konstantinidis G, Rafehi M, Abdelrahman A, Wu YW, Janning P, Muller CE, Laraia L, Waldmann H
Journal: Cell Chem Biol
Year: 2021
Volume: 28
DOI: 10.1016/j.chembiol.2021.02.017 

Main results: In this study, the authors identified the indoline-derivative indophagolin as an inhibitor of autophagy by acting through the P2X4 receptor. Autophagy plays crucial roles in cancer biology and, so far, no evidence has been reported on the role of P2X4 receptor in this cell mechanism. Target identification was achieved by using thermal proteome profiling (TPP), a method that uses cellular thermal shift assay (CETSA) in combination with multiplexed quantitative mass-spectrometry-based proteomics to identify targets of drugs, protein-metabolite and protein-protein interactions by studying protein melting profiles. This method is based on the principle that, upon heat exposure, proteins denature and become insoluble, whereas the soluble proteins are able to change their melting profiles in the presence of protein ligands. The authors successfully applied TPP to identify, for the first time, a membrane-bound receptor, specifically P2X4, as the molecular target of the small molecule, indophagolin, detected through a target-agnostic phenotypic assay. This compound was able to inhibit autophagy (IC50= 140 ± 40 nM) in human breast cancer MCF7 cells stably transfected with eGFP(enhanced green fluorescent protein)-LC3(microtubule-associated protein light chain 3), the latter being a marker for autophagosome formation. The molecular mechanism resulted to be rapamycin (mTOR)-independent and associated to autophagosomes biogenesis rather than maturation. In order to validate this result, the authors performed an isothermal dose- response fingerprinting (ITDRF) experiment with indophagolin in intact cells and demonstrated a dose-dependent thermal stabilization of P2X4 by this compound. Other purinergic receptors were shown to be targeted by indophagolin, namely P2Y4, P2Y6 and P2Y11, although with a lower potency. Interestingly, the selective P2X and P2Y receptor antagonists were not able to inhibit autophagy individually, thereby suggesting that indophagolin could antagonize multiple receptors involved in autophagy so much so that the authors called for a polypharmacological effect.

Opinion: P1 and P2 receptors are known to play key roles in cancer cell survival and death. Autophagy may represent either an obstacle or a protective factor in cancer cells, as it can both promote or suppress tumor development and proliferation. In several studies, the inhibition of autophagy has been shown to increase the efficacy of cancer therapies. Therefore, discovering novel compounds able to target autophagic pathways has a clinical significance in the optimization of antitumor strategies. This study reveals the potential of new target identification techniques relying on mass spectrometry-based proteomics as powerful tools in drug discovery. Although this study positively correlates P2X4 receptor and autophagy modulation, further studies are warranted to unravel the molecular mechanisms underlying this modulation and to further elucidate the role of the P2 selective antagonists in this context.

 

By Dr. Roberta Turiello
(Dipartimento di Farmacia, Università degli Studi di Salerno)

 

Article title: Loss of CD73 shifts transforming growth factor-β1 (TGF-β1) from tumor suppressor to promoter in endometrial cancer
Authors: Kurnit KC, Draisey A, Kazen RC, Chung C, Phan LH, Harvey JB, Feng J, Xie S, Broaddus RR, Bowser JL
Journal: Cancer Lett
Year: 2021
Volume: 505
DOI: 10.1016/j.canlet.2021.01.030

Main results: This work is aimed to clarify the relationship between CD73 and TGF-β1 in endometrial carcinoma (EC). Firstly, the authors injected proestrus stage C57BL/6 mice with TGF-β1 and observed that TGF- β1 promoted epithelial integrity by increasing CD73 expression in normal endometrium. Then the authors verified that TGF-β1 upregulates CD73 also in HEC-1-A cells (non-invasive cancer that is limited to the endometrium). In this case, the increase of CD73 by TGF-β1 also associated with increased cortical F-actin, while this effect was reduced by CD73 siRNA and APCP, a chemical inhibitor of CD73. This result indicates that in early-stage EC cells, TGF- β1 promotes epithelial integrity by inducing CD73 expression. In line with this, the treatment of HEC-1-A with APCP and TGF-β1 led to an increased macromolecular permeability. The authors also verified that CD73 mediated epithelial integrity in an adenosine dependent manner. The use of a pan-adenosine receptor agonist (NECA) rescued the barrier function in HEC-1-A cells treated with APCP and TGF- β1. Furthermore, the inhibition of CD73 increased TGF-β1-mediated migration and invasion in HEC-1-A cells, while NECA reversed this effect. The authors also assessed CD73 mRNA levels in stage I endometrioid EC (EEC) and it resulted reduced in deeply invasive tumors compared to non-invasive and minimally invasive EC. CD73 resulted mainly expressed by the cancer cells of non-invasive tumors, and its expression was significantly lower in deeply invasive tumors. Interestingly, the authors revealed a negative feedback loop between CD73 and TGF-β1 signaling. In HEC-1-A cells, TGF-β1-mediated TGF-β1 canonical signaling resulted increased upon treatment with CD73 siRNA and CD73 inhibitor. CD73 loss also increased cyclin D1, a tumor promoter. Thus, the authors assessed CCND1 mRNA levels in samples of stage I EEC and stratified the tumors as CD73 and CCND1 high and low based on median mRNA levels. Patients with well differentiated (grade 1) tumors have better outcomes as compared to those with poorly differentiated (grade 3) tumors, which are associated with more aggressive clinical features. Interestingly, grade 3 tumors resulted to be CD73Low/CCND1High, while no grade 1 tumors were CD73Low/CCND1High.

Opinion: The critical role of CD73 in cancer progression has widely been confirmed in several human tumors, in which high CD73 expression and activity are associated with poor prognosis and shorter survival of patients. However, a downregulation of CD73 has been observed in some tumors, including endometrial carcinoma, and more studies are required to completely understand the mechanisms that determine the diversity of CD73 expression among human tumors. In this context, this paper investigates the relationship between CD73 and TGF-β1 in endometrial carcinoma, one of the few tumors that downregulate CD73. The authors reveal an interesting new role for CD73 in this type of cancer. They discovered that while TGF-β1 promotes CD73 expression, in turn increasing epithelial integrity, CD73-generated adenosine feeds back to limit TGF-β signaling. The authors also demonstrated that CD73 loss is a mechanism by which endometrial tumors amplify TGF-β signaling and cyclin D1 expression, resulting in a protumor effects.

 

By Dr. Anna Pegoraro
(Dipartimento di Scienze mediche, Università di Ferrara)

 

Article title: Bone marrow niche ATP levels determine leukemia-initiating cell activity via P2X7 in leukemic models
Authors: He X, Wan J, Yang X, Zhang X, Huang D, Li X, Zou Y, Chen C, Yu Z, Xie L, Zhang Y, Liu L, Li S, Zhao Y, Shao H, Yu Y, Zheng J
Journal: J Clin Invest
Year: 2021
Volume: 131
DOI: 10.1172/JCI140242

Main results: Acute myeloid leukemia (AML) is a malignant disease of blood and bone marrow with a high rate of relapse. In this study, He and colleagues correlated ATP and P2X7 receptor with leukemogenic activities of leukemia-initiating cells (LICs), a particular cell population responsible for AML relapse. Thanks to a fluorescent ATP sensor, they demonstrated that ATP preferentially accumulates in the endosteal niche of the bone marrow where LICs reside than in the vascular niche. Interestingly, they found that in LICs the expression of P2X7 is 5-10 fold higher than in other cells present in the bone marrow. Thanks to RNA sequencing, they investigated the potential target of P2X7 activity in LICs and found that LICs proliferation is promoted by CREB and PHGDH activity. In vivo experiments, in both P2X7 null mice and mice injected with AML cells not expressing the P2X7 receptor, they observed that the leukemogenesis was slower than in control mice. In addition, treatment with P2X7 antagonist A740003 reduced leukemia growth in mice, encouraging a new therapeutic strategy with the P2X7 receptor as a pharmacological target.

Opinion: It is well established that ATP accumulates in the tumor microenvironment and through P2X7 receptor can modulate different processes during oncogenesis. In this study, the authors show for the first time that ATP distributes differently within the bone marrow, accumulating in the endosteal niche where cells with active proliferation activity reside. This observation is very important to understand the oncogenic role of ATP in AML considering that LICs localize in the endosteal niche of the bone marrow where can self-renew and re-initiate leukemia after therapy. Moreover, the authors demonstrated that when P2X7 was silencing, the LICs proliferation in the endosteal niche was reduced suggesting that P2X7 is indispensable to promote the self-renew capacity of LICs. In conclusion, this study demonstrated a role for ATP present in the endosteal niche to promote LICs proliferation and migration through the P2X7 receptor and support the use of P2X7 antagonists in the therapy of AML to inhibit the ability of LICs to self-renew and re-initiate leukemia.

 

By Dr. Andrea Spinaci
(Scuola di Scienze del Farmaco e dei Prodotti della Salute, Università di Camerino)

 

Article title: Crystal Structure and Subsequent Ligand Design of a Nonriboside Partial Agonist Bound to the Adenosine A2A Receptor
Authors: Amelia T, van Veldhoven JPD, Falsini M, Liu R, Heitman LH, van Westen GJP, Segala E, Verdon G, Cheng RKY, Cooke RM, van der Es D, IJzerman AP
Journal: J Med Chem
Year: 2021
Volume: 8
DOI: 10.1021/acs.jmedchem.0c01856

Main results: In this interesting study a new series of non-nucleoside adenosine receptor (AR) ligands were designed and synthetized on the basis of molecular docking studies. In particular, it was determined the crystal structure of an engineered human adenosine A2A receptor in complex with the partial agonist LUF5833 (2-(1H-imidazolmethylthio)-6-amino-4-phenylpyridine-3,5-dicarbonitrile) and then it was compared with the structures of the same protein co-crystallized with the full agonist NECA or the antagonist/inverse agonist ZM241385. On the basis of the results obtained from this comparison, a new series of compounds were synthetized in which the position 3 and 5 of the pyridine core of LUF5833 were differently modified by the removal of one or the two cyano groups or by the introduction of endocyclic nitrogens, providing, pyrimidine o triazine cores with variable combinations, maintaining the thiol side chain unmodified. The complex synthesis required for the achievement of the final products consists in 3 different synthetic pathways, based on the type of core to be obtained and on based on the position of the cyano groups on it, and on 26 different types of reaction with yield ranging from 3 to 90%. All the final compounds were tested in binding studies at all the AR subtypes and they showed a substantially unmodified affinity profile with respect to the reference compound LUF5833, except for the derivatives totally devoid of the cyano groups and the endocyclic nitrogens that determined a completely loss of affinity for all the receptors. The new non-nucleoside derivatives were also evaluated for their ability activate the A2AAR in comparison with CGS21680 (full A2AAR agonist) and concentration-response curves were designed. Interestingly the effect evoked on the A2AAR activation is different for each compounds both in term of potency and intrinsic activity, covering the whole range of activities, from partial agonist, to antagonist and inverse agonist. To explain these results further molecular docking studies were performed and what emerged is that probably subtle ligand movements in the binding pocket carried some ligands away from “agonistic” amino acids to remove their activation potential, while other ligands kept a partial agonistic profile. These ligand movements may also be dependent on the presence of explicit water molecules. Therefore, it is conceivable that all the new derivatives arrange themselves in an intermediate position and a various amount of water molecules cause the derivatives to interact or not with the binding pocket, activating or not the receptor.

Opinion: One of the most interesting results, in my opinion, is represented by the switch in activity, from a partial agonist to a potent inverse agonist, evoked by the derivatives in which the cyano group and endocyclic nitrogen where switched from position 3 (called “left handed”) to position 5 (“right handed”) demonstrating how sensitive are the binding pocket of A2AAR also to what we can define constitutional isomers. This study certainly increases the knowledge about ligand-receptor interactions, also revealing how much the presence of water molecules can be decisive in the interactions themselves, and therefore, in the activity.

 

 

By Dr. Elisabetta Coppi
(Dipartimento di Neuroscienze, Psicologia, Ricerca del Farmaco e Salute del Bambino (NeuroFarBa), Divisione di Farmacologia e Tossicologia, Università di Firenze)

Article title: Microglia monitor and protect neuronal function through specialized somatic purinergic junctions
Authors: Cserep C, Posfai B, Lenart N, Fekete R, Laszlo ZI, Lele Z, Orsolits B, Molnar G, Heindl S, Schwarcz AD, Ujvari K, Kornyei Z, Toth K, Szabadits E, Sperlagh B, Baranyi M, Csiba L, Hortobagyi T, Magloczky Z, Martinecz B, Szabo G, Erdelyi F, Szipocs R, Tamkun MM, Gesierich B, Duering M, Katona I, Liesz A, Tamas G, Denes A
Journal: Science
Year: 2020
Volume: 367
DOI: 10.1126/science.aax6752

Main results: Gli Authors: dimostrano, nel presente lavoro, un nuovo tipo di interazione fra neuroni e microglia che avviene grazie a particolari giunzioni somatiche caratterizzate da una citoarchitettura specializzata “ad hoc” per la segnalazione purinergica. In particolare, gli Authors: dimostrano che l’attività elettrica mitocondriale dei neuroni stimola, da parte della microglia, la formazione di giunzioni somatiche con il neurone stesso, e che tale fenomeno è prevenuto dal blocco dei recettori P2Y12 purinergici, attivati da ADP a da ATP. Tale meccanismo è particolarmente importante durante eventi di danno cerebrale poiché l’elevato rilascio di ATP in seguito al danno tissutale, determina l’attivazione dei recettori P2Y12 sulla microglia i quali, attraverso le suddette giunzioni, inibiscono l’upload di calcio a livello neuronale, stabilizzandone l’attività elettrica e svolgendo quindi un ruolo neuroprotettivo.

Opinion: Il presente lavoro è molto interessante sia per l’alta qualità tecnica degli esperimenti, sia per la concettualizzazione del legame di feedback fra evento dannoso (rilascio di ATP come molecola di danno) ed attivazione di meccanismi riparativi innescati dalla microglia. Il tema è ormai ampiamente dibattuto, microglia: protettrice o carnefice del tessuto nervoso? Dal presente lavoro emerge che la risposta a tale quesito va vista in un’ottica più ampia: esiste un “balance” secondo cui molecole che fungono principalmente da “sensori di danno” come ATP, e che generalmente contribuiscono al insulto neurologico, sono in grado tuttavia di attivare risposte di tipo protettivo.

 

By Dr. Ilaria Dettori
(Department of NEUROFARBA, University of Florence)

Article title: In vivo multimodal imaging of adenosine A1 receptors in neuroinflammation after experimental stroke
Authors: Joya A, Ardaya M, Montilla A, Garbizu M, Plaza-Garcia S, Gomez-Vallejo V, Padro D, Gutierrez JJ, Rios X, Ramos-Cabrer P, Cossio U, Pulagam KR, Higuchi M, Domercq M, Cavaliere F, Matute C, Llop J, Martin A
Journal: Theranostics
Year: 2021
Volume: 57
DOI: 10.7150/thno.51046

Main results: In this study, the authors evaluate the expression of adenosine A1 receptors (A1ARs) after transient (90 min) middle cerebral artery occlusion by positrone emission tomography (PET) with [18F]CPFPX, a fluorinated analogue of DPCPX (A1AR antagonist), and immunohistochemistry (IHC). In addition, the role of A1ARs on neuroinflammation reactions is assessed. In the ischemic territory, [18F]CPFPX signal and IHC show the overexpression of A1ARs in microglia and infiltrated leukocytes with a peak at day 3 after ischemia followed by a progressive decline afterwards. The activation of A1ARs with ENBA, A1AR agonist, promotes the reduction of the brain lesion, as measured with T2W-MRI, and the improvement of neurological outcome. Moreover, ENBA declines both the activation and proliferation of microglia and macrophages with [18F]DPA-714 and [18F]FLT radioligands, respectively. Altogether, these results provide novel knowledge regarding the control of A1AR on ischemic damage and inflammation.

Opinion: This is the first study where the A1AR is considered a imaging biomarker of neuroinflammation progress after stroke. From the litterature, it was known the action of A1AR agonists on the first phase of cerebral ischemia, where they reduce the excitotoxic damage. Authors of this paper clarify the role of A1ARs in neuroinflammation after ischemia, suggesting a possible therapeutic target, but without considering the important side effects of the A1 agonists treatment, such as bradycardia and sedation.

 

By Dr. Vanessa D’Antongiovanni
(Dipartimento di Medicina Clinica e Sperimentale, Università di Pisa)

Article title: CD73 promotes colitis-associated tumorigenesis in mice
Authors: Liu XH, Wu XR, Lan N, Zheng XB, Zhou C, Hu T, Chen YF, Cai ZR, Chen ZX, Lan P, Wu XJ
Journal: Oncology Letters
Year: 2020
Volume: 20
DOI: 10.3892/ol.2020.11670

Main results: Patients with inflammatory bowel diseases (IBDs), comprising mainly ulcerative colitis and Crohn’s disease, are 3–6 times more likely to develop colorectal cancer than the general population. In this context, several studies have reported a significant increase in the expression level of ectonucleotidase CD73 in several types of cancer, thus suggesting a pivotal role of this ectonucleotidase in the control of tumor progression. Based on this background, the aim of the present study was to investigate the role of CD73 in IBD-associated tumorigenesis (CAT) in a mouse model of azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced carcinogenesis. Animals were treated with 5'-(α,β-methylene)-diphosphate (APCP), a CD73 inhibitor adenosine, and 1-(6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-β-D-ribofuranuronamide (NECA), a non-selective adenosine receptor agonist. The authors have reported that inhibition of CD73 by APCP suppressed the severity of CAT with attenuated weight loss and smaller tumor size, as compared with AOM/DSS mouse group. By contrast, activation of adenosine receptors by NECA exacerbated CAT. In addition, the authors observed an increased expression of pro-inflammatory cytokines, such as TNF and interleukin-6 (IL-6), in colonic tissue of NECA group. According to RNA sequencing results, potential oncogenes, including arachidonate 15-lipoxygenase (ALOX15), Bcl-2-like protein 15 (Bcl2l15) and N-acetylaspartate synthetase (Nat8l), were downregulated in the APCP group and upregulated in the NECA group compared with AOM/DSS mouse group.

Opinion: In the present study Liu et al. demonstrated that CD73 and its downstream pathway play an important role in CAT. In particular, the inhibition of CD73 attenuated IBD-associated tumorigenesis through the inhibition of pro-inflammatory cytokines, including TNF and IL-6, and the expression of colorectal tumorigenesis-associated genes, such as ALOX15, Bcl2l15 and Nat8l.

 

By Dr. Stefano Raffaele
(Università degli Studi di Milano)

Article title: Spleen glia are a transcriptionally unique glial subtype interposed between immune cells and sympathetic axons
Authors: Lucas TA, Zhu L, Buckwalter MS
Journal: Glia
Year: 2021
Volume: 1-17
DOI: 10.1002/glia.23993

Main results: Glial cells are well known to exert important regulatory activities in the central nervous system (CNS), but a growing body of evidence is showing that they can play important roles also in the gut, as well as in other visceral organs innervated by the peripheral nervous system. In the present paper, the authors focused their attention on a relatively overlooked population of non-myelinating glial cells localized into the spleen, a major immune organ innervated by the sympathetic nervous system. Spleen glia were found to ensheath sympathetic axons localized in lymphocyte-rich areas of the white pulp, thus being at the interface between the immune and the nervous systems, in a privileged position to mediate neuroimmune crosstalk. Transcriptomic profiling of spleen glia confirmed this hypothesis, showing that these cells express several receptors for molecules released by both neuronal and immune cells, including purinergic receptors. In particular, P2Y2 and P2X1 receptors were found to be specifically enriched in spleen glia compared to the other cells of this organ. Furthermore, a comparative analysis showed that spleen glia differentially express genes associated with immune responses as compared to other glial subpopulations in the body, therefore representing a peculiar glial subtype anatomically and transcriptionally poised to participate in neuroimmune communication.

Opinion: The present work represents an essential first step toward understanding the role of peripheral glial cells in neuroimmune communication. Indeed, the anatomical position and transcriptomic profile of spleen glia suggest that these cells are not merely supporting axons, but instead they are most likely active players mediating their bidirectional communication with the immune system. This could be particularly relevant both in the case of a brain injury, when damage signals coming from the neuronal compartment may have a negative impact on the peripheral immune response, and during pathological conditions characterized by systemic inflammation, which might also induce harmful effects on the CNS. Interestingly, purinergic signalling emerges as a pivotal regulator of neuroimmune crosstalk mediated by spleen glia, encouraging further research in this field.

 

By Dr. Elisabetta Caiazzo
(Dipartimento di Farmacia, Scuola di Medicina e Chirurgia, Università Degli Studi di Napoli “Federico II”)

Article title: Defining the CD39/CD73 Axis in SARS-CoV-2 Infection: The CD73- Phenotype Identifies Polyfunctional Cytotoxic Lymphocytes
Authors: Ahmadi P, Hartjen P, Kohsar M, Kummer S, Schmiedel S, Bockmann JH, Fathi A, Huber S, Haag F, Schulze Zur Wiesch J
Journal: Cells
Year: 2020
Volume: 9
DOI: 10.3390/cells9081750

Main results: The authors of the study investigated the immune responses to SARS-CoV-2 and the particular contribution of the two ectonucleotidases CD39 and CD73 to the immune regulation during COVID-19. They isolated peripheral blood mononuclear cells of COVID-19 patients (fourteen patients) and healthy donors (12 uninfected individuals) and stimulated them with phorbol myristate acetate (PMA)/ionomycin for 5 h to analyze the frequency of cytokine-producing cells by flow cytometry. The authors found a significant increase of the proportion of cells co-producing GrB and perforin among CD8+ T cells, NK cells and NKT cells in COVID-19 patients. As already demonstrated in other studies, they found that  COVID-19  patients displayed  low  or reduced  numbers of lymphocytes, including both CD4+ and CD8+ T cells and B cells. In COVID-19 patients, there was a significant decrease in the frequency of CD73- expressing CD8+ T cells, NK cells, and NKT cells. In contrast, the frequency of cells expressing CD39 was elevated among CD8+ T cells, NK cells, and NKT cells although without reaching statistical significance. They did not observe differences in the expression of CD73 and CD39 on CD4+ T cells. Interestingly, CD73- cells were generally more capable of secreting GrB, perforin, TNF-α, or IFN-γ regardless of the disease status, but this effect was stronger in lymphocytes obtained from COVID-19 patients compared to those obtained from healthy donors. COVID-19 patients showed an elevated frequency of CD73- cells and within this cell population, effector functions such as cytokine and toxin secretion were restricted to the CD39- subpopulation. Additionally, the frequencies of CD73+ cells in both CD8+ T cells and NKT cells from COVID-19 patients were negatively correlated with the levels of serum ferritin, underscoring a potential role of ferritin as a prognostic marker for inflammation in COVID-19.

Opinion: COVID-19 is a global pandemic that has resulted in over 2.8 million deaths. This study is interesting since provides the first characterization of CD39 and CD73 on different lymphocyte subsets during SARS-CoV-2 infection; however it has several limitations. Firstly, the small cohort of SARS-CoV-2 infected patients. Second, the authors examined the cytokine/toxin secretion ability of different lymphocyte subsets upon an unspecific stimulation with PMA/ionomycin so without discriminating from antigen exposure or inflammatory bystander activation. Third, a specific mechanism for the observed loss of CD73 on cytotoxic lymphocytes in COVID-19 remains to be elucidated. Fourth, there are no comparisons between severe and mild cases that would provide insights towards a prognostic value of the absence of CD73 in immune activation and disease. Therefore, future studies should investigate differences of cellular CD39 and CD73 expression in patients at different disease stages and their potential as prognostic markers or targets for immunomodulatory therapies.

 

 

By Dr. Roberta Turiello
(Dipartimento di Farmacia, Università degli Studi di Salerno)

 

Article title: Microbiome-derived inosine modulates response to checkpoint inhibitor immunotherapy
Authors: Mager LF, Burkhard R, Pett N, Cooke NCA, Brown K, Ramay H, Paik S, Stagg J, Groves RA, Gallo M, Lewis IA, Geuking MB, McCoy KD
Journal: Science
Year: 2020
Volume: 369
DOI: 10.1126/science.abc3421 

Main results: Recent studies demonstrated that the gut microbiota can influence antitumor immunity and immune checkpoint blockade (ICB) efficacy, therefore the authors of this report intended to explore the mechanisms by which the microbiome enhances ICB therapy. Firstly, the authors isolated bacteria from homogenized murine colorectal tumors and seven bacterial species were found only in ICB-treated tumors. To verify whether these bacterial species influenced the efficacy of ICB therapy, germ-free (GF) mice were monocolonized with the isolated bacteria, then MC38 colorectal cancer cells were implanted and mice were treated with anti-CTLA-4 antibody. Monocolonization with Bifidobacterium pseudolongum, Lactobacillus johnsonii and Olsenella species significantly improved the efficacy of treatment with anti-CTLA-4. Interestingly, B. pseudolongum alone, in absence of anti-CTLA-4 treatment, did not affect the tumor growth or the antitumor immune response but was able to promote the differentiation of mucosal T helper 1 (TH1) cells and CD8+T-bet+ T cells. In presence of anti-CTLA-4 treatment, monocolonization with B. pseudolongum leads to increased splenic TH1 activation and effector function. Gastrointestinal inflammation is a well-known adverse effect of anti CTLA-4 treatment, resulting in impaired integrity of the gut barrier. Thus, the authors hypothesized that the systemic effect of B. pseudolongum may be due to an increased translocation of metabolites into circulation. This hypothesis was confirmed by treating GF mice with serum from anti-CTLA-4-treated B. pseudolongum colonized mice, which led to enhanced antitumor immune response and reduced tumor growth. Interestingly, metabolomic analyses showed that inosine was the metabolite more abundant in sera of B. pseudolongum monocolonized mice compared to controls. In vitro experiments showed that concentrations of inosine measured in sera of B. pseudolongum monocolonized mice were able to induce TH1 activation in a context-dependent manner, via A2A-receptor signaling. This result was also validated in vivo. Furthermore, the authors demonstrated that inosine failed to induce antitumor immune response in tumor-bearing mice adoptively transferred with A2AR deficient T cells. The authors concluded that the ICB-promoting effects of B. pseudolongum is mediated by inosine and is dependent on A2AR signaling in T cells.

Opinion: The results reported in this article are of great interest because they come in addition to compelling evidence indicating that the modification of microbiota may serve as promising adjuvant therapy to ICB in cancer. An interesting point to discuss on is the role of inosine, which emerged as pro-inflammatory mediator in a context-dependent manner. Indeed, the anti-inflammatory and pro tumorigenic effects of A2A receptor stimulation is so well supported by literature that promising agents targeting this receptor have been developed and are under clinical trials. Conversely, the authors of the proposed article show that both oral and systemic administration of inosine resulted in reduced tumor growth and increased immune response, when combined with anti-CTLA-4 agent and CpG. In absence of CpG co-stimulation, inosine led to opposite results, promoting tumor growth and impairing antitumor immune response. These findings open new interesting scenarios which merit to be explored.

 

By Dr. Giulia Magni
(Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano)

 

Article title: Assessment of neuroinflammation in the aging hippocampus using large-molecule microdialysis: Sex differences and role of purinergic receptors.
Authors: Perkins AE, Piazza MK, Vore AS, Deak MM, Varlinskaya EI, Deak T
Journal: Brain Behav Immun
Year: 2020
Volume: 91
DOI: 10.1016/j.bbi.2020.11.013 

Main results: It is known that aging is associated with an enhanced neuroinflammatory response. By employing different approaches in young and aged Fischer 344 rats, the aims of the present study were to investigate age differences in basal expression of a wide range of cytokines and chemokines, to examine evoked cytokine and chemokine release during microdialysis, to unveil sex differences in evoked release of inflammatory factors, and to observe the effect of pharmacological manipulation of the P2X7 receptor on functional release of inflammatory factors. Analysis of inflammatory markers from hippocampus of adult (3 months), middle-aged (12 months), and aged (18 months) male rats revealed no significant differences in total tissue content. Large-molecule in vivo microdialysis analysis of extracellular concentrations of inflammatory factors in the hippocampus from adult and aged male and female rats revealed robust sex differences in extracellular concentrations of CCL3, CCL20, and IL-1α. Reverse dialysis of the P2X7-selective agonist BzATP determined an increase in IL-1α and IL-1β release and rapidly suppressed the release of CXCL1, CCL2, CCL3, CCL20, and IL-6. Overall, the present study provides important new insight into aging and sex differences in neuroinflammation, and their regulation by the P2X7 receptor.

Opinion: Findings included in the present paper add an important piece in the puzzle of “inflammaging”, by extending results to gender differences and to the involvement of the purinergic system. Despite in my opinion further studies are requested to elucidate the molecular mechanisms underlying the observed effects, results show for the first time that pharmacological manipulation of the P2X7 receptor alters the functional release of a broad range of cytokines and chemokines, which might play a role in the regulation of sex differences in neuroinflammation.

 

By Dr. Mariachiara Zuccarini 
(Dipartimento di Scienze Mediche, Orali e Biotecnologiche, Divisione di Farmacologia e Tossicologia, Università degli studi di Chieti-Pescara “G. d’Annunzio”)

 

Article title: Purine metabolism regulates DNA repair and therapy resistance in glioblastoma
Authors: Zhou W, Yao Y, Scott AJ, Wilder-Romans K, Dresser JJ, Werner CK, Sun H, Pratt D, Sajjakulnukit P, Zhao SG, Davis M, Nelson BS, Halbrook CJ, Zhang L, Gatto F, Umemura Y, Walker AK, Kachman M, Sarkaria JN, Xiong J, Morgan MA, Rehemtualla A, Castro MG, Lowenstein P, Chandrasekaran S, Lawrence TS, Lyssiotis CA, Wahl DR
Journal: Nat Commun
Year: 2020
Volume: 11
DOI: 10.1038/s41467-020-17512-x 

Main results: The authors performed an integrative analysis on radiotherapy(RT)-resistant vs. RT-sensitive glioblastoma (GBM) cell lines that revealed different patterns of purine metabolites. Specifically, RT elicited the down-regulation of guanylates, adenylates and glutathione-associated metabolites in RT-sensitive cells. Moreover, when RT-sensitive cells (U118 MG, DBTRG-05MG, and GB-1) were exposed to nucleosides (cytidine, guanosine, uridine and thymidine at concentrations 80–240 μM), the clonogenic assay showed a decreased ability of RT to induce DNA double- stranded breaks (DSBs), referred to phosphorylation of histone H2A variant H2AX, thus favoring DNA repair. Noteworthy, cell treatment with Mycophenolic acid (MPA), an inhibitor of inosine monophosphate dehydrogenase (IMPDH), reduced GTP and increased inosine monophosphate (IMP) and ATP levels. It also radiosensitized RT-resistant cell lines (U87 MG and A172), and this effect was reverted by cell co-treatment with exogenous nucleosides. RT-sensitization relied exclusively on de novo GTP synthesis as HPRT1 silencing (an enzyme of the purine salvage pathway converting hypoxanthine to IMP and guanine to GMP) did not affect RT-resistance. As a step to determine whether GTP depletion was effective also in GBM models in vivo, the authors analyzed the pattern of purine metabolites in flank-xenograft models challenged to three different conditions: i) vehicle control ii) RT alone iii) RT+ mycophenolate mofetil (MMF), the MFA prodrug inhibiting GTP synthesis. RT-induced up-regulation of guanylates was prevented by co-treatment with MMF. Tumor growth was slowed by RT or MMF alone and almost inhibited by RT+MMF. Accordingly, the expression of the cell proliferation marker, Ki-67, resulted to be reduced by combined RT+MMF. Of note, they repeated this set of experiments in an orthotopic PDX model of GBM and again, RT and MMF in combination but not alone, were able to extend mouse survival. Finally, the authors investigated the correlation between inosine monophosphate dehydrogenase 1 (IMPDH1) expression, a rate limiting enzyme in de novo GTP synthesis and target of MPA/MMF, and GBM patient survival. Interestingly, from the analysis of 22 patients in the Pan-Cancer Atlas with IDH mutant GBM, IMPDH1 and IMPDH2 were not prognostic. On the other hand, in IDH wild type GBMs, higher levels of IMPDH correlated with lower survival. Collectively, the authors demonstrated that GTP depletion was able to sensitize GBM cell lines (U87 MG and A172) and primary patient-derived GBM neurospheres to RT by hampering DNA repair, the latter favored by nucleoside supplementation. Interestingly, pyrimidine metabolism had no significant effect on GBM DNA repair and RT-resistance.

Opinion: Glioblastoma is one of the most aggressive and lethal primary brain tumors. Current available treatments (i.e. temozolomide and bevacizumab) are still limited due to tumor genomic heterogeneity. Therefore, unraveling new therapeutic targets able to overcome, among others, RT resistance, represents a great challenge. This study is of great value since the authors employed an integrative analysis (in vitro, in vivo and patient-level data) that enabled them to dissect the intricate network of metabolic pathways activated in RT-sensitive vs. RT-resistant GBM. This is particularly important as metabolic reprogramming is one of the hallmarks of cancer and deciphering key metabolic checkpoints might be a useful therapeutic tool. In this work, increased levels of guanylates assumed a negative connotation in the acquisition of a RT-resistant phenotype due to DNA repair and tumor recurrence. GTP reduction, via inhibition of several purine-converting enzymes (i.e. IMPDH) by MMF, improved in vitro and in vivo response to RT, as well as patient survival. Noteworthy, as MMF is already approved by FDA for treating other diseases, the clinical translation of these data is quite easy. In conclusion, the combination of RT+MMF may represent a novel approach in GBM management.

 

By Prof. Diego Dal Ben 
(Scuola di Scienze del Farmaco e dei Prodotti della Salute, Università di Camerino)

 

Article title: X-Ray crystallography and free energy calculations reveal the binding mechanism of A2A adenosine receptor antagonists 
Authors: Jespers W, Verdon G, Azuaje J, Majellaro M, Keranen H, Garcìa-Mera X, Congreve M, Deflorian F, de Graaf C, Zhukov A, Dorè AS, Mason JS, Acqvist J, Cooke RM, Sotelo E, Gutièrrez-de-Teràn H
Journal: Angew Chem Int Ed
Year: 2020
Volume: 59
DOI: 10.1002/anie.202003788 

Main results: In this work, the authors describe a protocol aimed at designing novel receptor ligands. The protocol consists in the application of molecular dynamics analyses followed by free energy perturbation calculations, obtaining an estimation of the free energy variations related to the ligand-target complex formation in an aqueous environment. The A2A adenosine receptor is chosen as case study, with the development of a novel structural class of receptor ligands and the analysis of their mechanism of action at the A2A target. Three main results were obtained: 1) the development of the computational protocol for the design of novel ligands; 2) the development and optimization of a novel structural class of A2A ligands; 3) novel X-Ray structures of the A2A receptor in complex with antagonists. 

Opinion: The application of the free energy perturbation calculations to the “ligand-target interaction” problem is not a totally novel topic, since its theory and application have been largely tried to date in this field. This work combines the application of these calculations to both the ligand and the receptor structures, considering mutations of the receptor cavity and modifications of the ligand structures, with X-Ray crystallography and binding affinity studies as supports to confirm the hypotheses. The availability of a solid protocol to be applied to the design and optimization of receptor ligands is of key importance, given the powerful computational infrastructures available nowadays and present in an increasing number of molecular modeling laboratories. Considering the purinergic field, this study and analogue works provide further insights in the ligand-receptor interaction mechanisms and novel A2A 3D structures to be employed for the design of novel ligands, besides the novel structural class of A2A receptor ligands.

 

 

By Dr. Ilaria Dettori 
(Department of NEUROFARBA, University of Florence) 

 

Article title: Mesenchymal Stem Cells Attenuated Blood-Brain Barrier Disruption via Downregulation of Aquaporin-4 Expression in EAE Mice 
Authors: Liu Y, Ma Y, Du B, Wang Y,  Yang G-Y, Bi X 
Journal: Mol Neurobiol
Year: 2020 
Volume: 57 
DOI: 10.1007/s12035-020-01998-z 

Main results: Blood-brain barrier (BBB) disruption is one of the hallmarks of multiple sclerosis (MS), aquaporin-4 (AQP4) is a water channel protein, involved in cell edema and distruption of BBB. Mesenchymal stem cells (MSCs) could be an attractive candidate for the treatment of MS, because improved the chronic progressive course of EAE and inhibited AQP4 upregulation in ischemic stroke. So, authors explored whether MSCs could downregulate AQP4 expression and maintain BBB integrity in EAE mice, investigating the underlaying mechanism of MSCs treatment. Results indicated that MSCs therapy improved neurobehavioral outcomes in EAE mice, reduced inflammatory cell infiltration, BBB permeability, and demyelination in spinal cord. In addition, MSCs downregulated AQP4 and A2B adenosine receptor (A2BAR) expression in spinal cord of EAE mice. Authors found that MSCs-conditioned medium (MCM) reduced the expression of inflammatory cytokines, AQP4 and A2BAR in lipopolysaccharide-activated astrocytes. BAY 60-6583 (a selective A2BAR agonist) reversed the MCM-induced AQP4 downregulation and increased p38 MAPK phosphorylation. Furthermore, the upregulation effects of A2BAR agonist were antagonized by p38 MAPK inhibitor, SB203580. 

Opinion: Authors concluded that MSCs alleviated BBB disruption by downregulating AQP4 in MS, possibly through inhibiting the A2BAR/p38 MAPK signaling pathway. It would have been interesting if authors had tested an A2BAR antagonist, thus clarifying A2BAR role in BBB permeability in MS. Since that another study is present in literature, where it is demonstrated that A2BAR block or delete exerts a protective effect in an EAE model, inhibiting Th17 cell differentiation by blocking IL-6 production in EAE model. Thus, A2BAR could represent a therapeutic target for the treatment of MS. 

 

By Dr. Vanessa D’Antongiovanni 
(Dipartimento di Medicina Clinica e Sperimentale, Università di Pisa) 

 

Article title: Adenosine A2A receptor antagonists affects NMDA glutamate receptor function. Potential to address neurodegeneration in Alzheimer’s disease 
Authors: Franco R, Rivas-Santisteban R, Casanovas M, Lillo A, Saura CA, Navarro G 
Journal: Cells 
Year: 2020 
Volume: 9 
DOI: 10.3390/cells9051075 

Main results: N-methyl d-aspartate receptors (NMDARs) are heterotetrameric ion channels that open in response to glutamate. They play a pivotal role in the development of the central nervous system and in the generation of rhythms for breathing and locomotion, as well as in the processes underlying learning, memory, and neuroplasticity. Therefore, an alteration in the expression levels and functions of NMDARs has been implicated in numerous neurological disorders, including in Alzheimer’s disease (AD). In this context, adenosine A2A receptor (A2AR) is emerged as potential targets to prevent neurodegeneration. In the present study, the authors investigated whether the activation of the A2ARs may regulate NMDAR functions in both neurons and microglia cells. The authors reported that NMDARs and A2ARs are able to physically interact forming complexes in both neurons and glial cells. In particular, the authors observed more NMDAR-A2AR complexes in activated microglia than neurons. However, the activation of A2AR receptors resulted in higher NMDAR functionality in neurons, probably by indirect mechanisms. In neurons, the activation of A2ARs triggers an increase in NMDAR function with consequent neuronal death, suggesting that A2AR antagonists may provide neuroprotection via the modulation of NMDAR functionality. 

Opinion: In this study, Franco et al. reported a neuroprotective effect of A2AR antagonists through the modulation of NMDAR function. Indeed, the interaction between NMDARs and A2AR results in a novel functional entity that displayed a cross-antagonism, useful to prevent the exacerbation of NMDAR function by using A2AR antagonists. Taken together, this study demonstrates how the impairment of NMDAR function by A2AR antagonists represents an attractive possibility to provide neuroprotection in AD. 

 

By Dr. Elisabetta Caiazzo 
(Dipartimento di Farmacia, Scuola di Medicina e Chirurgia, Università Degli Studi di Napoli “Federico II”) 

 

Article title: Squalene-based multidrug nanoparticles for improved mitigation of uncontrolled inflammation in rodents 
Authors: Dormont F, Brusini R, Cailleau C, Reynaud F, Peramo A, Gendron A, Mougin J, Gaudin F, Varna M, Couvreur P
Journal: Sci Adv 
Year: 2020 
Volume: 6 
DOI: 10.1126/sciadv.aaz5466 

Main results: The authors of the study developed a novel prodrug-based nanoparticle formulation, made by the conjugation of squalene, to adenosine, and then encapsulating α-tocopherol, enabling the targeted delivery of adenosine and tocopherol (SQAd/ VitE) to the sites of acute inflammation. The authors showed beneficial effect of the formulation both in vitro and in vivo. Specifically, murine cardiac cells treated with SQAd/ VitE showed reduced intracellular ROS production with improved cell survival in a dose-dependent manner compared to controls following incubation with H2O2. Additionally, RAW 264.7 macrophages treated with SQAd/ VitE showed reduced TNF-α levels and NOx production following stimulation with LPS compared to control cells. Mice treatment with SQAd/VitE nanoparticles decreased TNF-α and increased IL-10 plasma levels compared to control groups in models of endotoxemia as well as it reduced MCP-1 and IL-6 pulmonary levels in comparison to nontreated or free drug–treated control. Furthermore, SQAd/VitE nanoparticles treatment reduced the level of lipid peroxidation in the lungs and it did not decrease systolic blood pressure so protecting mice from a side effect caused by adenosine. In addition, in a model of lethal LPS challenge, mice treatment with SQAd/VitE nanoparticles significantly improved the survival rate, compared to control animals. 

Opinion: It is well known that adenosine and some adenosine receptor agonists can promote the resolution of inflammation, but their systemic administration is associated with rapid clearance and side effects. There is evidence of a molecular connection between oxidative stress and inflammatory response. This is the first study with the use of prodrug-based nanocarriers to target the delivery of adenosine with anti-antioxidant for anti-inflammatory therapy. Also, the formulation used can overcome the limitations of both conventional adenosine and antioxidant therapy. Furthermore, it may represent the basis to evaluate adenosine receptor agonist or antagonist delivery together with antioxidants. 

 

by Prof. Stefano Raffaele 
(Università degli Studi di Milano) 

 

Article title: Neuroprotective and neuro-rehabilitative effects of acute purinergic receptor P2X4 (P2X4R) blockade after ischemic stroke 
Authors: Srivastava P, Cronin CG, Scranton VL, Jacobson KA, Liang BT, Verma R
Journal: Exp Neurol
Year: 2020 
Volume: 329 
DOI: 10.1016/j.expneurol.2020.113308 

Main results: Effective regenerative therapies able to enhance brain repair and to improve functional recovery after ischemic stroke still represent an unmet medical need. Here, authors reported the beneficial effects of pharmacological inhibition of the P2X4 receptor, which is known to regulate the inflammatory activation of microglia and infiltrating macrophages, in the middle cerebral artery occlusion (MCAo) murine model of stroke.  Briefly, treatment with the P2X4 antagonist 3-BDBD during the first 3 days post-MCAo resulted in reduced infarct volume and neurological deficits. These effects were attributed to decreased leukocyte infiltration and lower pro-inflammatory activation of microglia/macrophages, accompanied by reduced cell surface levels of P2X4. Moreover, blockade of P2X4 receptor during the acute phase post-stroke provided long-term neuroprotective effects mediated by increased BDNF levels, leading to enhanced functional recovery observed at late disease stages. 

Opinion: The results included in the present paper clearly highlight the beneficial effects of acute P2X4 pharmacological inhibition on brain repair and long-term functional recovery after stroke. Since P2X4 has been demonstrated to finely regulate the phenotype of microglia/macrophages, including the acquisition of pro-resolving and remyelinating properties, P2X4 blockade might also negatively impact the response mediated by these immune cells. Thus, it will be crucial to define the best therapeutic window for such treatment, enabling to exploit the neuroprotective effects of P2X4 inhibitors without affecting the regenerative processes mediated by this receptor.

 

 

by Dr. Giulia Magni
(Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano)

 

Article title: TMEM163 regulates ATP-gated P2X receptor and behavior
Authors: Salm EJ, Dunn PJ, Shan L, Yamasaki M, Malewicz NM, Miyazaki T, Park J, Sumioka A, Hamer RRL, He WW, Morimoto-Tomita M, LaMotte RH, Tomita S
Journal: Cell Reports
Year: 2020
Volume: 31
DOI: 10.1016/j.celrep.2020.107704

Main results: It is well known that ATP and purinergic P2X receptors (P2XRs) are involved in pain transmission, and P2XRs are considered a “druggable” target for novel painkillers. Therefore, a comprehensive knowledge of P2XR functions including the presence of specific modulators is fundamental. In this complex study, authors utilized a genome-wide open reading frame-based screening to identify TMEM163, a six transmembrane domain protein, as a specific modulator of P2XRs. In vitro studies allowed authors to demonstrate that TMEM163 co-expression enhances ATP-evoked current in heterologous cells expressing exogenous P2X3R and P2X4R subtypes. Moreover, TMEM163 modulates the channel properties of P2X3R and P2X4R, and as a consequence potentiates the efficacy of ATP at these receptor sites. Finally, authors performed in vivo studies by generating a TMEM163 KO mouse line, and demonstrated that TMEM163 modulates ATP-evoked behavior and P2XRs activity in the cerebellar granule cells and DRG neurons, including those that convey pain-related information from the peripheral tissue to the spinal cord. Overall, this paper shows that TMEM163 plays an essential role in regulating channel activity and pharmacology of P2X3 and P2X4 receptor subtypes, as well as in modulating mouse behavior evoked by ATP, an agonist implicated in pain.

Opinion: It is widely accepted that purinergic signaling and ATP-gated ionotropic P2XRs are implicated in pain-related behavior. Several modulators of ligand-gated ion channels have recently been identified, thus suggesting the existence of functional modulators for P2XRs as well. The results included in this paper identify TMEM163 as a critical regulator of P2XRs and ATP-evoked pain behavior. In the future, it will be important to investigate whether TMEM163 modulates other properties of P2XRs in vivo and whether TMEM163 could represent an additional interesting target for pain modulation.

 

By Dr. Mariachiara Zuccarini 
(Dipartimento di Scienze Mediche, Orali e Biotecnologiche, Divisione di Farmacologia e Tossicologia, Università degli studi di Chieti-Pescara “G. d’Annunzio”)

 

Article title: CD73 expression on effector T cells sustained by TGF-β facilitates tumor resistance to anti-4-1BB/CD137 therapy
Authors: Chen S, Fan J, Zhang M, Qin L, Dominguez D, Long A, Wang G, Ma R, Li H, Zhang Y, Fang D, Sosman J, Zhang B
Journal: Nat Commun
Year: 2019
Volume: 10
DOI: 10.1038/s41467-018-08123-8

Main results: CD73-Adenosine axis plays a pivotal role in the tumor microenvironment, being responsible of impaired T-cell immunosurveillance and tumor progression in different tumor types. CD73 is a cell surface ecto-enzyme that catalyzes the dephosphorylation of extracellular AMP into adenosine, and is expressed primarily by the cancer cells and the immune cells such as CD4+Foxp3+ regulatory T cells (Tregs) and myeloid-derived suppressor cells recruited by the tumor. 4-1BB (CD137/TNFRSF9), a member of the tumor necrosis factor receptor (TNFR) superfamily expressed by a variety of immune cells (i.e. CD4+ and CD8+ T cells) mediates growth and survival signals for T cells but unfortunately its application as monotherapy has failed in a number of clinical trials. In the present study the authors investigated the antitumor efficacy of a combination therapy, namely anti-CD73 and anti-4-1BB, in a clinically relevant murine adoptive immunotherapy model. The results revealed that anti-CD73 and anti-4-1BB synergize to inhibit tumor growth and increase survival in a B16-SIY melanoma model. These effects rely on the ability of the combination therapy to enhance the infiltration of CD8+ and CD4+ T cells while attenuating recruitment of functional intratumoral Foxp3+ Treg. Anti-4-1BB agonist therapy managed to reduce tumor progression in CD73−/− mice but not in CD73-expressing hosts, driving the expansion of CD73-CD8+ T cell subset. Consistently, anti-4-1BB downregulated the expression levels of Foxp3 in CD73−/− CD4+ T cells, but not in the WT CD4+ T cells. Interestingly, blockade of A2AR activity was able to reduce anti-4-1BB-mediated expansion of Tregs, thus corroborating the positive correlation between CD73 and Foxp3 expression. Noteworthy, TGF-β-rich tumors were found to be resistant to anti-4-1BB therapy, likely due to TGF-β-mediated ability to induce CD73 expression on intratumoral effector CD8+ T cells. Overall, these findings suggest that 4-1BB-induced CD73 downregulation in favor of effector T cell response is regulated via the TGF-β-STAT3 pathway, and involves the release of several cytokines such as IL-2 and IFN-γ. The authors unveiled a new mechanism of action in the TNFR family for agonist therapy enabling the inhibition of melanoma proliferation.

Opinion: The outcome of tumorigenesis is determined, among others, by the balance between anti-tumor immunity and a number of mechanisms of immune escape developed by the tumor. Over the last years, cancer immunotherapy has raised the interest of the scientific community and many clinical trials are ongoing in the attempt to uncover new strategies aimed at boosting anti-cancer immunity. Along with successful therapies based on the regulation of immunological checkpoints (i.e. CTLA-4, PD-1/PD-L1), that of immune-stimulatory receptors has shown lack of therapeutic efficacy in recent clinical trials, mainly when used as monotherapy. In a recent study, 4-1BB–expressing chimeric antigen receptor (CAR T) cells survived longer and increased in number to a greater extent ex vivo compared to CD28-expressing CAR T cells. The authors here revealed the benefit associated with a combination therapy, anti-CD73 and anti-4-1BB, in a B16-SIY melanoma model, as demonstrated by tumor regression and increased survival. The findings also confirmed the active role of Adenosine and its metabolizing enzyme (CD73) in the orchestration of immunosuppressive pathways occurring in tumors. This work is of great clinical relevance assuming the potential application of this therapy in several solid tumors and hematologic malignancies, and garners a new insight into the interaction between CD73 with costimulatory molecules as tumor-specific immunomodulatory targets.

 

by Dr. Roberta Turiello
(Dipartimento di Farmacia, Università degli Studi di Salerno)

Article title: Tumor-derived exosomes promote angiogenesis via adenosine A2B receptor signaling
Authors: Ludwig N, Yerneni SS, Azambuja JH, Gillespie DG, Menshikova EV, Jackson EK, Whiteside TL
Journal: Angiogenesis
Year: 2020
Volume: 23
DOI: 10.1007/s10456-020-09728-8 

Main results: The authors isolated exosomes from head and neck squamous cell carcinoma line (UMSCC47) and observed that these vesicles carry CD73 and CD39 on the surface of their membrane, while CD39 is also present inside the vesicles. Interestingly, analysis with UPLC-MS/MS revealed the presence of adenosine and inosine inside the exosomes. Thus, exosomes are both producer and shuttle of immunosuppressive adenosine. In vitro experiments showed that the incubation of tumor-derived exosomes with macrophages leads to M2 macrophages differentiation, while the use of A2BR antagonist, but not A1R or A2AR antagonist, abolishes this effect. These results were validated using exosomes isolated from cancer patients, while exosomes from healthy donors did not impair the differentiation of macrophages. Of note, under A2BR stimulation, M2 macrophages produce pro-angiogenic factors which in turn stimulate the growth of endothelial cells. In addition, endothelial cells can be directly stimulated by tumor-derived exosomes, via the activation of A2BR. In line with these results, the injection of tumor-derived exosomes resulted in enhanced vascularization in A1R-/-, A2AR-/- but not in A2BR-/- rats. All these data clearly demonstrate that tumor-derived exosomes are able to promote angiogenesis via A2BR stimulation, both on macrophages that on endothelial cells.

Opinion: Exosomes are small extracellular vesicles able to circulate in body fluids, playing a relevant role in the intercellular communication. Great interest is currently growing on these vesicles, due to their potential to promote tumor growth and metastases dissemination. Exosomes isolated from cancer patients have already been observed to carry active CD39 and CD73 on their membrane. The authors of this publication observed that tumor-derived exosomes not only are able to produce adenosine, via CD39 and CD73, but they also carry adenosine and inosine in their inner compartment. Thus, exosomes can shuttle adenosine and inosine far from the site where they are released, protecting these nucleotides from metabolism or uptake mechanisms. This represents a new important finding in purinergic field. Furthermore, the authors demonstrate for the first time that tumor-derived exosomes promote angiogenesis by their direct interaction with endothelial cells and by stimulating the production of pro-angiogenic factors by macrophages. These findings underline the importance of the adenosine pathway as potential therapeutic target in cancer patients.

 

By Prof. Diego Dal Ben 
(Scuola di Scienze del Farmaco e dei Prodotti della Salute, Università di Camerino)

Article title: Docking Screens for Dual Inhibitors of Disparate Drug Targets for Parkinson’s Disease
Authors: Jaiteh M, Zeifman A, Saarinen M, Svenningsson P, Bréa J, Loza MI, Carlsson J
Journal: J Med Chem
Year: 2018
Volume: 61
DOI: 10.1021/acs.jmedchem.8b00204

Main results: In this work, the authors describe a virtual screening study aimed to identify potential inhibitors of the A2A adenosine receptor and the monoaminoxidase (MAO) B enzyme, as novel anti-Parkinson agents. Databases of millions of compounds are docked into the binding sites of the two proteins. Results suggest 24 molecules with promising interaction profile with the A2A and MAO-B. In vitro evaluation of these molecules at the two targets shows that some of them are nanomolar inhibitors at the two proteins. Further optimization of two of these molecules leads to the design and synthesis of further derivatives presenting nanomolar affinity at the two targets.

Opinion: The “multitarget ligand” approach represents a powerful strategy to develop compounds of therapeutic interest. Beside the great potentialities, this strategy presents some difficulties, like the design of compounds optimized to interact with (at least) two significantly different proteins, or the increased number of receptor/enzyme subtypes to be considered to evaluate the compound selectivity. As authors report in the article, the availability of experimental 3D structures of the targets and a significant number of known ligands is of great help to perform reliable studies, to better interpret the affinity data, and to more efficiently identify novel ligands and predict their activity. This study is one among several articles reported in the last years describing synthesis and evaluation of novel dual A2A-MAO-B ligands as anti-Parkinson agents. The interest for this topic is high since the combination of the activity at the two targets could provide novel therapeutic tools of good potential for this neurodegenerative disease.

 

 

By Dr. Ilaria Dettori
(Dipartimento di Neuroscienze, Psicologia, Ricerca del Farmaco e Salute del Bambino (NeuroFarBa), Divisione di Farmacologia e Tossicologia, Università di Firenze)

Article title: Inactivation of endothelial adenosine A2A receptors protects mice from cerebral ischaemia‐induced brain injury
Authors: Zhou Y, Zeng X, Li G, Yang Q, Xu J, Zhang M, Mao X, Cao Y, Wang L, Xu Y, Wang Y, Zhang Y, Xu Z, Wu C, Chen JF, Hoda MN, Liu Z, Hong M, Huo Y
Journal: Br J Pharmacol
Year: 2019
Volume: 176
DOI: 10.1111/bph.14673

Main results: Inactivation of the gene for adenosine A2A receptors (Adora2a) protects against brain injury in experimental stroke. However, the cell‐specific pathogenic effects of A2A receptors in thromboembolic stroke and the underlying mechanisms remain undefined. Aims of this study were to explore that the inhibition of endothelial A2A receptors after thromboembolic stroke improves post‐stroke outcomes via down‐regulation of inflammation, in in vivo model of thromboembolic stroke and with in vitro experiments. Authors demonstrated that eMCAo induced Adora2a expression in brain, mice globally deficent for Adora2a exhibited a riduced neurological deficit and reuced infarct Volume: . Mice specifically deficient in endothelial Adora2a showed a reduced leukocyte infiltration, BBB leakage, and oedema after stroke, data confirmed also in vitro. They obtained the same results with the application of A2A receptor antagonist, KW6002. Finally, they reported that the inactivation of Adora2a inhibited endothelial inflammation via suppression of the NLRP3 inflammasome. In conclusion, they demonstrated that A2A receptors in brain endothelial cells mediate inflammation after thromboembolic stroke, through down‐regulation of the endothelial NLRP3 inflammasome, suggesting new approaches to vascular protection after ischaemic stroke.

Opinion: This study explored for the first time the role of endothelial A2A receptor after stroke. Studies are present in litterature about the role of central A2A receptors after cerebral ischemia, where the early administration of an A2A receptor antagonist exerted a protective effects, reducing excitotoxicity. So, taken together, these papers suggest the use of A2A receptor antagonists early after cerebral ischemia as protective agents in order to contrast both excitotoxicity and inflammation after stroke.

 

By Dr. Stefano Raffaele
(Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano)

Article title: Microglia monitor and protect neuronal function via specialized somatic purinergic junctions
Authors: Cserép C, Pósfai B, Lénárt N, Fekete R, László ZI, Lele Z, Orsolits B, Molnár G, Heindl S, Schwarcz AD, Ujvári K, Környei Z, Tóth K, Szabadits E, Sperlágh B, Baranyi M, Csiba L, Hortobágyi T, Maglóczky Z, Martinecz B, Szabó G, Erdélyi F, Szipőcs R, Tamkun MM, Gesierich B, Duering M, Katona I, Liesz A, Tamás G, Dénes Á
Journal: Science
Year: 2020
Volume: 367
DOI: 10.1126/science.aax6752

Main results: Microglia represent the immunocompetent cells of the central nervous system, exerting a restless surveillance of the microenvironment in order to preserve brain homeostasis and to rapidly react to detrimental stimuli. Unveiling the mechanisms at the basis of the interaction between microglia and the other brain cells is crucial to develop novel therapeutic approaches capable to shape microglial response and to support its beneficial functions. In the present paper, the authors exploited cutting-edge techniques to demonstrate the existence of specialized interaction sites between microglial processes and neuronal cell bodies, called somatic microglia-neuron junctions. The purinergic P2Y12 receptor emerged as a key player in the formation and maintenance of these interactions, being selectively enriched in the microglial compartment of the somatic junctions to sense neuronal mitochondria activity. The alteration of this process, occurring after acute injury like cerebral ischemia, resulted in the disruption of somatic junctions, triggering protective microglial activation in order to restore the physiological neuronal function or to promote the phagocytosis of irreversibly damaged cells. Accordingly, pharmacological inhibition of P2Y12 prior to ischemia induction interfered with microglial surveillance, leading to increased size of the ischemic damage and worse neurological outcome.

Opinion: The existence, in both rodent and human brain, of ‘platforms’ for cell-to-cell communication, optimized for purinergic signaling, opens novel intriguing opportunities to understand the mechanisms underlying brain damage and the consequent repair mechanisms. In this context, the microglial purinergic P2Y12 receptor emerged as a sensor of the metabolic state of neighboring cells and as a master regulator of microglial response to brain injury.

 

By Dr. Elisabetta Coppi
(Dipartimento di Neuroscienze, Psicologia, Ricerca del Farmaco e Salute del Bambino (NeuroFarBa), Divisione di Farmacologia e Tossicologia, Università di Firenze)

Article title: The P2Y14 receptor in the trigeminal ganglion contributes to the maintenance of inflammatory pain
Authors: Lin J, Zhang YY, Liu F, Fang XY, Liu MK, Huang CL, Wang H, Liao DQ, Zhou C, Shen JF
Journal: Neurochem Int
Year: 2019
Volume:  131
DOI: 10.1016/j.neuint.2019.104567

Main results: The authors describe, for the first time, the involvement of P2Y14 receptor in pain, in particular in a rat model of inflammatory orofacial pain. They injected an irritant, i.e. complete Freund's adjuvant (CFA), into the right whisker pad area of the rat face and they measured mechanical hyperalgesia induced ipsilateral to injury by the von Frey test. They also measured biochemical changes in the trigeminal ganglion (TG). They found significant upregulation of P2Y14 receptor, glial fibrillary acidic protein (GFAP) and pro-inflamatory cytokines such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), C–C chemokine CCL2 in the TG, as well as enhanced activation of p-ERK1/2 and p-p38 proteins. Importantly, they also demonstrate that mechanical allodynia was attenuated by the selective P2Y14 receptor antagonist PPTN thus unveiling a critical role of this receptor subtype in orofacial inflammatory pain.

Opinion: The present study demonstrates, for the first time, a new mechanism that contributes to the onset and maintenance of inflammatory pain. The P2Y14 receptor is still under investigation and there is still a paucity of data in the literature describing their functional role/s in cells and tissues. The present paper is of potential interest for a number of scientists and may represent the starting point for a brand new area of investigation, i.e. the possible use of P2Y14 receptor antagonists as efficacious non-opioid pain killers

 

By Dr. Elisabetta Caiazzo
(Dipartimento di Farmacia, Scuola di Medicina e Chirurgia, Università Degli Studi di Napoli “Federico II”)

Article title: Adenosine receptor agonism protects against NETosis and thrombosis in antiphospholipid syndrome
Authors: Ali RA, Gandhi AA, Meng H, Yalavarthi S, Vreede AP, Estes SK, Palmer OR, Bockenstedt PL, Pinsky DJ, Greve JM, Diaz JA, Kanthi Y, Knight JS
Journal: Nat Commun
Year: 2019
Volume: 10
DOI: 10.1038/s41467-019-09801-x

Main results:In this study Ali et al., have focused on the role of A2A receptor in antiphospholipid syndrome and neutrophil activity. The authors found that the specific A2A receptor agonist, CGS21680, inhibited neutrophil extracellular trap (NET) release from neutrophils treated with antiphospholipid IgG antibodies (pooled from four patients with primary antiphospholipid syndrome) by triggering cyclic AMP formation. They also found that CGS21680 reduced reactive oxygen species formation in neutrophils activated by phorbol 12-myristate 13-acetate. Besides, they observed that in a murine model of thrombosis induced by flow-restriction of inferior vena cava, the administration of CGS21680 reduced thrombus incidence, weight, length and plasma NET levels. Interestingly, CGS21680 also reduced thrombus size and NET release following the administration of antiphospholipid IgG antibodies. So, they concluded that the A2A receptor activation appears to be beneficial against NETosis and thrombosis in antiphospholipid syndrome.

Opinion: Antiphospholipid syndrome is an autoimmune disease characterized by venous or arterial thrombosis and/or pregnancy complications with the persistent positivity of antiphospholipid antibodies. Recently, the release of NET by neutrophils has been recognized as a prothrombotic mechanism in antiphospholipid syndrome. It has been suggested to target inflammation other than to limit thrombosis for proper management of antiphospholipid syndrome. Many of the anti-inflammatory effects of adenosine are attributed to the stimulation of the A2A receptor subtype. This is the first study that analyzes A2A agonism in antiphospholipid syndrome and neutrophil activity as well as the first evidence of the protective effect of A2A receptor stimulation in venous thrombosis. In addition, these findings highlight the beneficial effect of A2A receptor in limiting thrombosis and inflammation.

 

By Dr. Vanessa D’Antongiovanni
(Dipartimento di Medicina Clinica e Sperimentale, Università di Pisa)

Article title: Adenosine-5'-triphosphate suppresses proliferation and migration capacity of human endometrial stem cells
Authors:  Semenova S, Shatrova A, Vassilieva I, Shamatova M, Pugovkina N, Negulyaev Y.
Journal: J Cell Mol Med
Year: 2020
Volume: 24
DOI: 10.1111/jcmm.15115 

Main results: Extracellular ATP, through the activation of P2X and P2Y purinergic receptors, regulates proliferation and migratory capacity of several types of cells, including stem cells. The capacity of stem cells to migrate to the specific organs is essential for normal tissue homeostasis and repair as well as for the creation of stem cell-based regenerative medicines. In the present study, the authors investigated the impact of extracellular ATP on migration and proliferation capacity of endometrial stem cells (eMSCs), pivotally involved in the endometrial recovery during each menstrual cycle. The authors reported that ATP suppresses the proliferation and migration ability of eMSCs, via purinergic receptors. In particular, the authors observed a marked presence of P2X7 receptor subtype in eMSCs. Despite this, treatment with specific P2X7 ligands did not affect the cell migration of eMSCs, suggesting that inhibition of cell migration occurs without the involvement of P2X7 receptors.

Opinion: In this study, Semenova et al. reported that ATP suppresses proliferative and migratory capacity of eMSCs, thus affecting the regenerative potential of these cells. The lost of function or the reduction in the number of eMSCs can lead to the formation of thin endometrium, that is unable to maintain gestation. Taken together, this study highlights how monitoring of processes associated with ATP-dependent regulation of cell migration and proliferation will allow to significantly improve the regenerative potential of eMSCs for use in tissue engineering and regenerative medicine.

 

 

by Dr. Roberta Turiello
(Dipartimento di Farmacia, Università degli Studi di Salerno)

 

Article title: CD73 Immune Checkpoint Defines Regulatory NK-cells within the Tumor Microenvironment
Authors: Neo SY, Yang Y, Record J, Ma R, Chen X, Chen Z, Tobin N, Blake E, Seitz C, Thomas R, Wagner AK, Andersson J, de Boniface J, Bergh J, Murray S, Alici E, Childs R, Johansson M, Westerberg L, Haglund F, Hartman J, Lundqvist A
Journal: J Clin Invest
Year: 2020
Volume: 130
DOI: 10.1172/JCI128895

Main results: In the study, the authors investigate how peripheral blood and tumor-infiltrating NK cells differ in patients with breast cancer and sarcoma. They found out that tumor-infiltrating NK cells express higher level of surface CD73. They also observed that the frequency of CD73+ NK cells in tumor microenvironment correlates with larger tumor size in patients with breast cancer. Flow cytometry analysis showed that tumor-infiltrating CD73+ NK upregulate also other immune checkpoints including LAG-3, TIM-3, PD-1, and PD-L1. The expression of these proteins resulted significantly lower in CD73- NK cell. Then the authors investigated the mechanism involved in CD73 overexpression in NK cells. To do this, peripheral blood NK cells from healthy individuals were co-cultured with fresh sarcoma and breast tumor resections or with tumor cell lines. In both conditions, NK cells upregulate the expression of CD73. This regulation depends on physical cell contact as NK cells did not acquire the expression of CD73 when co-cultured with tumor cells separated by transwell or treated with tumor-conditioned culture media. Furthermore, the CD73 expression resulted driven by STAT3. The authors observed that CD73 expressed by NK cells is active and can hydrolyze AMP into adenosine, however CD73+ NK cells can suppress CD4+ T cells in absence of AMP, thus suggesting an immunosuppressive mechanism independent from adenosine production. In contrast to CD73- NK cells, CD73+ NK cells produce higher levels of IL-10 and TGF-β, upon co-culture with tumor cells. The production of these cytokines leads to CD4+ T cells suppression and resulted significantly reduced in the presence of GPB730, a STAT3 inhibitor.

Opinion: The adenosine-CD73 axis has become an attractive target for developing new agents able to restore anti-tumor immune response. However, CD73 regulation is still not well understood. This study brilliantly clarifies some mechanisms involved in CD73 regulation in tumor-infiltrating NK cells in breast cancer and sarcoma. The authors showed that CD73 is upregulated in NK cells after physical contact with tumor cells and that the overexpression is driven by STAT3. Furthermore, tumor-infiltrating NK cells produce high levels of IL-10 and TGF-β.
The study highlights some new interesting mechanisms involved in CD73 expression and supports the use of IL-10 and TGF-β inhibitors to complement current immunotherapy. In addition, the frequency of CD73+ NK cells resulted related to tumor size in breast cancer. Very importantly, these results could be the starting point for further investigations on NK cells in patients with other solid tumors.

 

by Dr. Giulia Magni
(Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano)

 

Article title: Microglial P2Y12 receptor regulates seizure-induced neurogenesis and immature neuronal projections
Authors: Mo M, Eyo UB, Xie M, Peng J, Bosco DB, Umpierre AD, Zhu X, Tian DS, Xu P, Wu LJ.
Journal: J Neurosci
Year: 2019
Volume: 39
DOI: 10.1523/JNEUROSCI.0487-19.2019

Main results: Seizures are a common pathological condition in humans, characterized by alterations of the normal balance of brain excitation and inhibition, leading to aberrant neurogenesis and increased immature neuronal projections. It is well known that microglial cells contribute to neurogenesis and regulate seizure phenotypes, but their role in aberrant neurogenic seizure contexts has not been adequately investigated. In this paper, authors combined the intracerebroventricular kainic acid model of seizures with pharmacogenetic approaches to ablate microglia in male mice. Results show that microglia promote seizure-induced neurogenesis and subsequent seizure-induced immature neuronal projections in the adult dentate gyrus. More importantly, authors identified microglial P2Y12 receptors as key regulators in promoting neurogenesis and immature neuronal projections after seizures. Authors hypothesized that, following an insult leading to aberrant neurogenesis and immature neuronal projections during epileptogenesis, neuronal cell death in the hippocampus triggers the release of ADP, which, acting on microglial P2Y12 receptors, promotes both seizure-induced neurogenesis and sprouting.

Opinion: Several processes driving epileptogenesis in the brain have been identified so far, including aberrant neurogenesis and increased immature neuronal projections. Understanding the mechanisms promoting such changes is therefore critical for the development of new therapies to successfully block epileptogenesis. In this paper, author demonstrate, for the first time, that microglia in general, and specifically microglial P2Y12 receptors, promote both aberrant neurogenesis and increased immature neuronal projections. Therefore, targeting this pathway could be a novel therapeutic strategy to be translated into the clinic.

 

by Prof. Diego Dal Ben 
(Scuola di Scienze del Farmaco e dei Prodotti della Salute, Università di Camerino)

 

Article title: Full-Length P2X7 Structures Reveal How Palmitoylation Prevents Channel Desensitization 
Authors: McCarthy E, Yoshioka C, Mansoor SE
Journal: Cell
Year: 2019
Volume: 179
DOI: 10.1016/j.cell.2019.09.017  

Main results: In this work, the authors describe two cryoelectron microscopy structures of the full-length rat P2X7 receptor in the apo and ATP-bound states. A key result is the first description of the intracellular domains of the P2X7 receptor, which were absent in the previously reported P2X7 structures. Furthermore, the reported structures allow a comparison of the inactive (closed) state and the activated (ATP-bound) state, depicting the receptor rearrangement due to interaction with agonist. In addition, two structural features of the cytoplasmic segments are observed: on the one hand, a cysteine-rich domain (named C-cys) with a key role in receptor desensitization upon agonist stimulation; on the other hand, a long segment (named cytoplasmic ballast) with a key role in pore formation and containing a zinc ion complex and a guanine nucleotide binding region.

Opinion: The P2X7 is a key player in a number of physiological and pathological processes, and it represents a potential target for several diseases. The depiction of the inactive and active arrangements of this protein is of critical importance since it allows to interpret the protein-agonist interaction at a molecular level, to interpret also the effect of mutagenesis studies reported to date, and to identify novel sites as targets of non-competitive ligands potentially able to modulate the protein function. The description of the intracellular domains is of importance since truncated forms of this protein present specific features in terms of receptor activation, ability to give the pore, and cellular roles. Finally, the identification of intracellular zinc and nucleotides binding domains opens novel fields for the study of the roles of this critical membrane protein in modulating the function of intracellular enzymes.

 

by Dr. Elisabetta Caiazzo
(Dipartimento di Farmacia, Scuola di Medicina e Chirurgia, Università Degli Studi di Napoli “Federico II”)

 

Article title: ENTPD-1 disrupts inflammasome IL-1β–driven venous thrombosis
Authors: Yadav V, Chi L, Zhao R, Tourdot BE, Yalavarthi S, Jacobs BN, Banka A, Liao H, Koonse S, Anyanwu AC, Visovatti SH, Holinstat MA, Kahlenberg JM, Knight JS, Pinsky DJ, Kanthi Y
Journal: J Clin Invest
Year: 2019
Volume: 129
DOI: 10.1172/JCI124804

Main results: In this study the authors investigate the effect of CD39 haploinsufficiency (Cd39+/–) in venous thrombosis in a restricted-flow model of murine inferior vena cava stenosis. Yadav and co-workers found that the loss of a single Cd39 allele increases thrombus weights and thrombus frequency 2 days after inferior vena cava stenosis with sometimes extending into the iliac veins. In addition, Cd39+/– mice show enhanced fibrin accumulation and tissue factor in thrombus compared to wild type (WT) mice. Furthermore, they observed a significantly neutrophil recruitment to the thrombus venous in Cd39+/–. Moreover, they detected enhanced phosphorylation of the transactivator serine534 site of the NF-κB p65 subunit in Cd39+/– thrombi, as well as increased expression of the NOD-like receptor pyrin domain–containing 3 protein (NLRP3), increased expression of the apoptosis-associated speck-like protein containing a CARD (ASC), and increased expression of active caspase-1 in Cd39+/– mice. In addition, they found enhanced IL-1β content in venous thrombi from Cd39+/– following venous thrombosis compared with WT thrombi and elevated plasma levels of IL-1β in Cd39+/– mice compared with WT mice. Therefore, they show a critical role for Cd39 in inhibiting NLRP3 inflammasome activation and IL-1β signaling in venous thrombogenesis.

Opinion: Deep vein thrombosis represents a major preventable cause of morbidity and mortality worldwide. This is the first preclinical study that demonstrates a protective role for CD39 enzyme in venous thrombosis by regulating NLRP3 inflammasome activation. The study highlights the important role of CD39 at interface between inflammation and vascular thrombosis. Even if additional studies are required, it represents a good starting point to investigate in more detail CD39 role in deep vein thrombosis and its related diseases.

by Dr. Elisabetta Coppi
(University of Florence)

 

Article title: P2Y13 receptors regulate microglial morphology, surveillance, and resting levels of interleukin 1β release
Authors: Kyrargyri V, Madry C, Rifat A, Arancibia-Carcamo L, Jones SP, Chan VTT, Xu Y, Robaye B, Attwell D
Journal: GLIA
Year: 2019
Volume: Ahead of print
DOI: 10.1002/glia.23719

Main results: P2Y12 receptors are known stimulate motility of microglial cells to the site of damage. However, little is known about the role of P2Y13 receptor in these cells, even if transcriptome data suggest that this is the second most expressed neurotransmitter receptor in microglia. The authors show, by the use of P2Y13 KO mice, that P2Y13 receptors potentiate the chemotactic response of P2Y12 and also potentiate P2Y12-activated THIK-1 K+ currents evoked by exogenous ADP application in these cells in culture. Furthermore, KO mice, under basal non-inflammatory conditions, have fivefold higher levels of interleukin 1β (IL-1β) and their microglia is less ramified with a shorter process length. Finally, Microglial surveillance still occurs in conditions of P2Y12 receptor block in P2Y13 KO mice. The authors conclude that P2Y13 receptors regulate microglial morphology and resting IL-1β levels and cooperate with P2Y12 in the chemotactic function of these cells.

Opinion: It is surprising that so little is known about the role of P2Y13 in microglia, even if it is the second most expressed neurotransmitter receptor in these cells. This work is of paramount importance in the field of purines because it provides the first demonstration for the role of this highly expressed receptor in these cells. The authors, by taking advantage of P2Y13 KO mice, elegantly demonstrate that this receptor participates to the chemotactic response induced by P2Y12 receptor activation, driving cells towards a source of ADP. The fact that microglia morphology is also dramatically changed under basal conditions of P2Y13 KO mice is another important information confirming a key role of this purinergic receptor in the neuroinflamatory response.

by Dr. Vanessa D’Antongiovanni
(Dipartimento di Medicina Clinica e Sperimentale, Università di Pisa)

 

Article title: Resveratrol Modulates and Reverses the Age-Related Effect on Adenosine-Mediated Signalling in SAMP8 Mice
Authors: Sánchez-Melgar A, Albasanz JL, Palomera-Ávalos V, Pallàs M, Martín M.
Journal: Mol Neurobiol
Year: 2019
Volume: 56
DOI: 10.1007/s12035-018-1281-8

Main results: Adenosine A1 and A2A receptors play a critical role in the control of neuronal dysfunction and neurodegeneration. Several studies reported that these receptor subtypes are altered in neurodegenerative disease, including Alzheimer’s disease. In the present study, the authors investigated the effect of Resveratrol (RVS), a natural polyphenol present in berries, peanuts, grape skin and red wine, on adenosinergic system in SAMP8 mice, widely considered as an Alzheimer’s model. The authors reported a strong down-regulation of A1 receptors as very early event during aging in SAMP8 mice, whereas A2A receptors were found to be significantly increased in older mice. The treatment with RSV reverse the age-related loss of adenosine A1 receptors and its signaling pathway. By contrast, adenosine A2A receptors were not modulated by RSV treatment, but A2A-mediated signaling was completely desensitized as compared with untreated mice. In addition, the authors observed a significant reduction of 5'-nucleotidase and adenosine deaminase, enzymes involved on adenosine metabolism, after RVS treatment although the levels of adenosine remained unchanged.

Opinion: In this study, Sánchez-Melgar et al. explain the molecular mechanism by which RSV affects adenosine signaling pathways in SAMP8 mice. The desensitization of A2A-mediated signaling along with the agonistic effect observed in A1 functionality suggest neuroprotective properties of this molecule on cognitive decline via adenosine signaling. Taken together, this study highlight that targeting adenosine-mediated signaling could be a therapeutic strategy for neurodegenerative diseases, and the role that RVS plays via adenosinergic system should be considered.

by Dr. Ilaria Dettori
(Department of NEUROFARBA, University of Florence)

 

Article title: Ecto-5′-nucleotidase (CD73) attenuates inflammation after spinal cord injury by promoting macrophages/microglia M2 polarization in mice
Authors: Xu S, Zhu W, Shao M, Zhang F, Guo J, Xu H, Jiang J, Ma X, Xia X, Zhi X, Zhou P, Lu F
Journal: J Neuroinflammation
Year: 2018
Volume: 15
DOI: 10.1186/s12974-018-1183-8

Main results: The aim of this study was to determine the role of ecto-5′-nucleotidase (CD73) in the modulation of macrophages/microglia trasformation between the M1 (anti-inflammatory) and the M2 (pro-inflammatory) phenotypes. For the in vivo experiments, the authors used CD73-knock out (KO) mice in a spinal cord injury (SCI) model. They demonstrated an upregulation of CD73 expression after SCI and a poorer locomotor recovery, neuronal disorganization and higher levels of M1 markers in CD73-KO mice comparison to wild type mice. In order to futher discern the CD73 effect on microglia activation, they conducted in vitro experiments with BV2 cells, that either downregulated or upregulated CD73 expression. They reported that CD73 overexpression augmented microglial M2 polarization through p38 phosphorilation following A2B receptors activation. So, they concluded that CD73 had an anti-inflammatory role in SCI attributable to its facilitation of macrophages/microglia M2 polarization.

Opinion: This is the first study about the neuroprotective effects of CD73 through A2B receptors activation. It also allows to better understand the role of modulatory factors involved on macrophages/microglia polarization that characterizes the progression of Central Nervous System disorders, proposing new target molecules for the development of novel therapeutic methods for neuroinflammatory diseases.

by Dr. Stefano Raffaele
(Università degli Studi di Milano)

 

Article title: P2X7 Receptors Drive Poly(I:C) Induced Autism-like Behavior in Mice
Authors: Horváth G, Otrokocsi L, Beko K, Baranyi M, Kittel Á, Fritz-Ruenes PA, Sperlágh B
Journal: J Neurosci
Year: 2019
Volume: 39
DOI: 10.1523/JNEUROSCI.1895-18.2019

Main results: Increasing evidence suggest that perinatal risk factors, including maternal immune activation (MIA), are strongly related to the development of Autism Spectrum Disorder (ASD) in the affected children. Here, authors exploited a mouse model of MIA induced by injection of the non-pathogenic agent poly(I:C) during pregnancy to investigate the role of the P2X7 receptor in the generation of ASD in the offspring. Briefly, authors demonstrated that poly(I:C) injection in P2X7 wild-type mouse dams induced an autism-like phenotype in their offspring, while behavioral and histological alterations were not observed after genetic knockout or pharmacological inhibition of maternal P2X7. Moreover, authors showed that postnatal pharmacological inhibition of P2X7 effectively alleviated MIA-induced behavioral and morphological deficits in the offspring. Finally, authors demonstrated that ATP administration to P2X7 wild-type dams, but not to P2X7 knockout animals, was able to generate an autism-like phenotype in the offspring, suggesting that perinatal activation of P2X7 by ATP is sufficient to induce ASD.

Opinion: ASD is a neurodevelopmental psychiatric disorder gaining attention because of its rapidly increasing prevalence world-wide. With the present paper, authors highlighted the P2X7 receptor as a pivotal player in MIA-induced generation of ASD in the offspring. They suggested that P2X7 activation may act as a trigger for the maternal immune system, with consequent increase of pro-inflammatory mediators in the fetal brain leading to disrupted neurodevelopment. On this basis, authors provided strong evidence indicating that pharmacological inhibition of maternal or offspring P2X7 receptors could be an effective strategy for the prevention and treatment of ASD.

 

 

by Prof. Francesco Di Virgilio 
(Dipartimento di Morfologia, Chirurgia e Medicina Sperimentale, Università di Ferrara)

 
Article title: Potentiation of P2RX7 as a host-directed strategy for control of mycobacterial infection 
Authors: Matty MA, Knudsen DR, Walton EM, Beerman RW, Cronan MR, Pyle CJ, Hernandez RE, 
Tobin DM 
Journal: eLife 
Year: 2019 
Volume: 8 
DOI: 10.7554/eLife.39123 

Main results: In this study Tobin and co-workers show that potentiation of P2X7R or (P2RX7) functions by a positive allosteric modulator (i.e. the drug clemastine) achieves a remarkable anti-mycobacterial effect in a model of zebrafish tuberculosis infection. Tuberculosis is a leading cause of death worldwide, and as yet a poorly curable disease. Mycobacterium tuberculosis survives within macrophage phagocytic vacuoles, requires long lasting treatments to be eradicated, and often develops antibiotic resistance. The pharma industry is struggling to develop novel and more efficient anti-mycobacterial drugs, but the alternative approach based on potentiation of host defenses is also possible. 
The P2X7R is eminently expressed by mononuclear phagocytes, the main immune cells involved in anti-mycobacterial defense. When overactivated, the P2X7R promotes macrophage apoptosis as well as death of mycobacteria resident within phagocytic vacuoles; thus, it was proposed several years ago that P2X7R stimulation might help the host to get rid of mycobacterial infections by inducing death of the intracellular pathogen. However, P2X7R exploitation to trigger macrophage cell death is very impractical since its only physiological agonist so far known is ATP (and only at high concentrations), and the pharmacological agonist commonly used (benzoyl ATP) is poorly selective. Nevertheless, over the years a few positive allosteric compounds have been described, e.g. the antibiotic polymyxin B, the antibacterial peptide LL 37, and the antihistaminic drug clemastine. These compounds are much more handy that receptor agonists since they are inactive under normal physiological conditions, but become stimulants when the extracellular ATP concentration is increased, as is the case at inflammatory and tumor sites sites.

Opinion: Tobin and co-workers demonstrate that clemastine is a powerful anti-mycobacterial agent in a zebrafish model of tuberculosis thanks to its positive allosteric effect on the P2X7R, thus leading to substantial reduction of the mycobacterial load. This is a very interesting example of how host directed therapies might help fight poorly curable infectious diseases such as tuberculosis. 

 

By Dr. Giulia Magni 
(Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano) 

Article title: Overexpression of P2X4 receptor in Schwann cells promotes motor and sensory functional recovery and remyelination via BDNF secretion after nerve injury 
Authors: Su WF, Wu F, Jin ZH, Gu Y, Chen YT, Fei Y, Chen H, Wang YX, Xing LY, Zhao YY, Yuan Y, Tang X, Chen G 
Journal: Glia 
Year: 2019 
Volume: 67 
DOI: 10.1002/glia.23527  

Main results: The role of P2X4 receptor expressed in the central nervous system in regulating several physiological and pathological functions, including neuropathic pain, is well documented. On the other hand, little is known about its distribution and functions in the peripheral nervous system. In this paper, authors show that P2X4 receptor protein is mainly localized in the lysosomes of Schwann cells in mouse sciatic nerve. In cultured Schwann cells, TNF-α increases P2X4 receptor protein expression and promotes its trafficking to Schwann cells surface, and TNF-α-induced BDNF secretion is P2X4-dependent. In an in vivo model of sciatic nerve crush injury, authors show that P2X4 protein expression in Schwann cells is strongly upregulated, intra-sciatic injection of a P2X4 receptor blocker retards functional recovery of injured nerves, and overexpression of the receptor in Schwann cells by genetic manipulation promotes motor and sensory functional recovery and accelerates nerve remyelination, by increasing BDNF release. 

Opinion: The pro-nociceptive role of P2X4 receptor in the central nervous system has been widely demonstrated, in different models of neuropathic pain. With this study authors show, for the first time, that promoting P2X4 receptor expression in Schwann cells after peripheral nerve injury could represent an innovative approach to foster remyelination and nerve repair and, in turn, recovery from neuropathic pain. 

 

By Dr. Mariachiara Zuccarini 
(Dipartimento di Scienze Mediche, Orali e Biotecnologiche, Divisione di Farmacologia e Tossicologia, Università degli studi di Chieti-Pescara “G. d’Annunzio”) 

Article title: Guanosine prevents depressive-like behaviors in rats following bilateral dorsolateral striatum lesion induced by 6-hydroxydopamine 
Authors: Marques NF, Binder LB, Roversi K, Sampaio TB, Constantino LC, Prediger RD, Tasca CI
Journal: Behav Brain Res 
Year: 2019 
Volume: 15 
DOI: 10.1016/j.bbr.2019.112014 

Main results: The present study demonstrates the antidepressant-like effects of Guanosine (GUO) in an animal model of Parkinson’s disease (PD). At early stages, PD is characterized by neuropathological changes, namely anhedonic- and depressive-like behaviors, with no manifestations of motor symptoms. The effects of GUO were evaluated by a battery of behavioral tasks and biochemical analyses in rats submitted to a stereotaxic bilateral infusion of 6-hydroxydopamine (6-OHDA) in the dorsolateral striatum. In summary, 24 h after the surgery, rats were divided in 4 groups: i) SHAM; ii) SHAM+GUO (7.5 mg/kg, i.p, daily for 21 days); iii) 6-OHDA (10 μg/hemisphere); iv) 6-OHDA+GUO. Treatment with GUO in the 6-OHDA+GUO group vs 6-OHDA group significantly increased sucrose consumption and decreased immobility time in the forced swimming test (FST), thus suggesting a protective role of GUO in reducing 6-OHDA-induced anhedonic-like behavior in these tests performed at the 5th or 21st day of GUO treatment, respectively. The locomotor activity in the open field test (OFT), evaluated for motor deficits, as well as social interaction behavior, did not reveal any significant effect of GUO. Moreover, cortical, striatal and hippocampal slices were evaluated for Reactive Oxygen Species (ROS) production and mitochondrial membrane potential. The results demonstrated that 6-OHDA did not induce ROS generation in the investigated brain regions, while eliciting a mitochondrial potential disruption in the hippocampus that was prevented by GUO treatment. However, the authors reported an antioxidant effect of GUO in a previous in vitro study where GUO was shown to decrease 6-OHDA-induced ROS production. Collectively, these results prompted the proposal that treatment with GUO caused an overall protective effect towards 6-OHDA-induced non-motor symptoms associated with early phase of PD.

Opinion: It is now widely accepted that guanine-based purines (GBPs) play a central role in the neuromodulation under physiological and pathological conditions. In particular, extracellular GUO revealed neurotrophic and neuroprotective effects in a large number of in vitro and in vivo models of ischemia and neurodegenerative disorders such as AD and PD. Indeed, GUO has been shown to decrease neuronal cell death and increase dopaminergic neurons at the substantia nigra pars compacta, thus reducing dyskinesia in several models of PD. In this work the authors showed that GUO prevented also non-motor deficits, likely activating PI3K/Akt/mTOR signaling pathway. Noteworthy, acute and chronic treatment with GUO may be considered as promising antiparkinson therapeutic strategies, characterized by a low toxicity profile, for both motor and depressive-like symptoms relief. Given the high incidence of depression in PD patients, it is of great importance to find novel agents able to manage these comorbidities hardly impacting on patients’ quality of life.

 

By Prof. Diego Dal Ben 
(Scuola di Scienze del Farmaco e dei Prodotti della Salute, Università di Camerino) 

Article title: Structural basis for subtype-specific inhibition of the P2X7 receptor 
Authors: Karasawa A, Kawate T
Journal: eLife 
Year: 2016 
Volume: 5 
DOI: 10.7554/eLife.22153.001 

Main results: In this work, the authors report the first crystal structures of the mammalian P2X7 receptor obtained and solved to date. The various crystal structures contain the protein in complex with non-competitive inhibitors, some of them known as reference ligands for this protein and often used as pharmacological tools. Even if these ligands are structurally unrelated to each other, they bind to the same pocket that is located at the interface between two monomers and not in strict proximity to the ATP binding site. A set of ligand-protein interactions is conserved among the various ligands, suggesting a common mechanism of action even at molecular level. One of the crystal structures contains also the protein in complex with a non-competitive inhibitor and the endogenous agonist ATP; this structure shows how the non-competitive ligand stabilizes the protein in an arrangement that does not lead to the gate opening even if in the presence of the agonist. 

Opinion: This work has various kinds of impacts. First, the three-dimensional structures of one of the hottest medchem targets are reported and provide an overview of the general architecture of the protein; in this sense, the arrangement of the P2X7 receptor is in accordance with the reported structures of the P2X4 and P2X3 receptors, confirming again the general structure of the P2X receptors. Second, these structures show the mechanism of action of a set of key P2X7 ligands, providing insights on the mechanism of non-competitive inhibition of this protein (first structural data ever reported on a P2X receptor about this kind of mechanism of action). Third, the molecular detail of the ligand-protein interaction and the comparison of the intermolecular contacts for the various ligands with the protein are of key relevance to provide a solid platform for structure-based design and development of novel classes of non-competitive P2X7 inhibitors.

 

By Dr. Roberta Turiello 
(Dipartimento di Farmacia, Università degli Studi di Salerno) 

Article title: Specific Decrease in B-Cell-Derived Extracellular Vesicles Enhances Post-Chemotherapeutic CD8+ T Cell Responses 
Authors: Zhang F, Li R, Yang Y, Shi C, Shen Y, Lu C, Chen Y, Zhou W, Lin A, Yu L, Zhang W, Xue Z, Wang J, Cai Z 
Journal: Immunity 
Year: 2019 
Volume: 50 
DOI: 10.1016/j.immuni.2019.01.010  

Main results: The authors studied the immunosuppressive activity of CD19+ extracellular vesicles (EVs), produced by B cells. These EVs express both CD39 and CD73 and are able to hydrolyze ATP and AMP, producing adenosine. Because of chemotherapy can promote ATP release, the authors investigated whether CD19+ EVs could alter the antitumor effects of cytotoxic agents, by hydrolyzing ATP. In vivo experiments showed that treatment with CD19+ EVs can reduce the chemotherapy efficacy in different models of murine tumors, while pretreatment of CD19+ EVs with a CD39 inhibitor (POM-1), a CD73 inhibitor (APCP) or an anti-CD73 mAb prevented this effect. Furthermore, treatments of tumor-bearing mice (nude mice, CD4+-depleted mice and CD8+-depleted mice) demonstrated that CD19+ EVs can impair the chemotherapeutic efficacy by inhibiting CD8+ T cells activation and proliferation. Then the authors extended their investigations to cancer patients. Specifically, CD19+ EVs were isolated from serum of gastric or colon cancer patients. The CD19+ EVs levels in serum resulted to be negatively correlated with the progression-free survival period (PFSPs). Stratification analysis indicated that patients with lower serum CD19+ EVs had longer PFSPs. Surgical removal of tumor resulted in decreased levels of CD19+ EVs. The authors showed that specific silencing of Rab27a in B Cells by inactivated Epstein-Barr virus carrying Rab27a siRNA leads to inhibition of CD19+ EVs production, improving thus the chemotherapy efficacy. 

Opinion: This study led to very interesting results, highlighting the key role of EVs in immunosuppression. The authors showed that CD19+ EVs contain both CD73 and CD39 and are able to reduce chemotherapy efficacy by producing adenosine. Additionally, the study shows that chemotherapy effects can be improved by silencing Rab27a expression, whose upregulation enhances the production of CD19+ EVs. Altogether the results of this work revealed the possibility to consider CD19+ EVs as valuable factor for diagnosis and monitoring of antitumor chemotherapeutic effect in cancer patients. Furthermore, reducing CD19+ EVs production could represent a new attractive strategy for anticancer therapy.

 

 

 

By Dr. Elisabetta Coppi
(Dipartimento di Neuroscienze, Psicologia, Ricerca del Farmaco e Salute del Bambino (NeuroFarBa), Divisione di Farmacologia e Tossicologia, Università di Firenze)

 

Article title: CaV2.2 Gates Calcium-Independent but Voltage-Dependent Secretion in Mammalian Sensory Neurons
Authors: Chai Z, Wang C, Huang R, Wang Y, Zhang X, Wu Q, Wang Y, Wu X, Zheng L, Zhang C, Guo W, Xiong W, Ding J, Zhu F, Zhou Z
Journal: Neuron
Year: 2017
Volume: 96
DOI: 10.1016/j.neuron.2017.10.028

Main results: The authors describe, for the first time, a voltage-dependent, Ca2+-independent, mechanism of vesicular release that occurs purely at the somatic level in neurons of the dorsal root ganglia (dorsal root ganglia: DRG). In particular, the study shows that action potential induction by current injection in DRG neuronal cells in vitro directly activate the SNARE complex thus leading to the fusion of vesicles with the plasma membrane. So, differently to what observed at synapses, this extra-synaptic mechanism or release is not dependent upon intracellular Ca2+ rise. Of note, the authors also demonstrate that the content of vesicles released by this particular mechanism is ATP, as demonstrated by the luciferin-luciferase technique.

Opinion: The present study demonstrates, for the first time, a new mechanism of ATP release at extra-synaptic level, i.e. at the soma of DRG neurons, which does not depend on intracellular Ca2+ rise. This mechanism, only described for ATP up do now, may be relevant in conditions of "pathological" electrical activation of these neurons, such as extra-firing or “burst” activity described during neuropathic pain states. In these pathologies, DRG neurons could release an extra-amount of ATP thus contributing to the establishment of the chronic pain state.

 

By Dr. Ilaria Dettori
(Dipartimento di Neuroscienze, Psicologia, Ricerca del Farmaco e Salute del Bambino (NeuroFarBa), Divisione di Farmacologia e Tossicologia, Università di Firenze)

 

Article title: Adora2b Signaling on Bone Marrow Derived Cells Dampens Myocardial Ischemia-Reperfusion Injury
Authors: Koeppen M, Harter PN, Bonney S, Bonney M, Reithel S, Zachskorn C, Mittelbronn M, Eckle T
Journal: Anesthesiology
Year: 2012
Volume: 116
DOI: 10.1097/ALN.0b013e318255793c

Main results: Wild Type (WT) or A2B receptor KO (Adora2b-/-) mice were subjected to 60 min of myocardial ischemia followed by 120 min of reperfusion. Adora2b-/- mice showed an increased myocardial damage compared with WT mice and the WT mice treated with Adora2b agonist, BAY 60-6583, at onset of reperfusion, showed a decreased myocardial injury, demonstrating a putative cardioprotective role of Adora2b in myocardial injury after ischemia-reperfusion (I-R). To study on which cell type Adora2b needs to be activated to be cardioprotective, the authors generated bone marrow chimeric mice. The chimeric mice underwent I-R injury, the Adora2b-/- mice transplanted with WT bone marrow, showed a myocardial injury significantly decreased, similar to WT mice, demonstrating that Adora2b needed to be present on bone marrow derived cells to elicit cardioprotective effect. Then, they reported that polymorphonuclear leukocytes (PMNs) were the dominant bone marrow derived population in myocardial ischemia and that the Adora2b had no influence on the number of PMNs infiltrating the myocardial tissue after I-R injury, but their activation inhibited TNF-α release. Finally, to confirm that Adora2b directly inhibited TNF-α release from PMNs, the authors showed that the pretreatment with BAY 60-6583 of human activated PMNs, abrogated TNF-α release.

Opinion: This study shows the contribution of Adora2b on inflammatory cells to cardioprotection in myocardial ischemia-reperfusion model. The study provides some clarification on the peripheral anti-inflammatory role of Adora2b, isolating a direct Adora2b action on inhibition of TNF-α release by PMNs. Even if the authors don’t explain why the Adora2b has no influence on the number of PMNs invading myocardium after I-R injury, the article has a very good experimental approach and represents one of the few article in scientific literature that explore enigmatic roles of Adora2b.

 

By Dr. Stefano Raffaele 
(Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano)

 

Article title: P2X4 receptor controls microglia activation and favors remyelination in autoimmune encephalitis
Authors: Zabala A, Vazquez-Villoldo N, Rissiek B, Gejo J, Martin A, Palomino A, Perez-Samartín A, Pulagam KR, Lukowiak M, Capetillo-Zarate E, Llop J, Magnus T, Koch-Nolte F, Rassendren F, Matute C, Domercq M
Journal: EMBO Mol Med
Year: 2018
Volume: 10
DOI: 10.15252/emmm.201708743

Main results: Fostering endogenous myelin repair represents a promising approach for the treatment of demyelinating diseases such as Multiple Sclerosis (MS). In this respect, it has been shown that CNS-resident microglia and blood-borne macrophages are involved not only in detrimental response to signals of injury, but also in tissue recovery, by acquiring multiple activated phenotypes.
Interestingly, the presence of reactive microglia/macrophages expressing the P2X4 receptor has been demonstrated both in MS demyelinating lesions and in the experimental autoimmune encephalomyelitis (EAE) animal model of MS. In the present paper, authors showed that P2X4 blockade in EAE mice, by either pharmacological treatment with the antagonist TNP-ATP and genetic knockout of the receptor, exacerbated EAE neurological symptoms. Conversely, enhancing P2X4 function by treatment with the allosteric modulator ivermectin ameliorated disease outcome. In particular, ivermectin beneficial effects have been associated with microglial switch toward pro-regenerative functions, such as phagocytosis of myelin debris and remyelination, unveiling a new role for P2X4 in the regulation of microglial polarization.

Opinion: Nowadays, the most widely used therapies for MS consist in immunomodulators that are effective in reducing relapses, but they do not affect neuronal or myelin damage. For this reason, understanding the cellular and molecular mechanisms underlying chronic disease progression is relevant to develop alternative therapeutic interventions aimed at promoting tissue repair. In this respect, the present paper highlights the P2X4 receptor as a novel potential pharmacological target capable to foster myelin regeneration by pushing microglia to acquire beneficial properties.

 

By Dr. Vanessa D’Antongiovanni
(Dipartimento di Medicina Clinica e Sperimentale, Università di Pisa)

 

Article title: Blockade of Pannexin-1 channels and Purinergic P2X7 receptors shows protective effects against cytokines-induced colitis of human colonic mucosa
Authors: Diezmos EF, Markus I, Perera DS, Gan S, Zhang L, Sandow SL, Bertrand PP, Liu L.
Journal: Front Pharmacol
Year: 2018
Volume: 9
DOI: 10.3389/fphar.2018.00865 

Main results: The authors investigated the role of pannexin-1 (Panx1) channels, implicated in the release of small molecules such as ATP, and of purinergic P2X7 receptors (P2X7Rs) in an ex vivo colitis model developed from human colonic mucosal explants. Proinflammatory cytokines, TNFα and IL-1β, were used to induce colitis in mucosal strips, and the effects of Panx1 and P2X7Rs on cytokines-induced tissue damage were determined in the presence of the Panx1 channel blocker (10Panx1) and P2X7R antagonist (A438079). The study showed that the blockade of Panx1 channels and P2X7R reduced the inflammatory cytokines-induced crypt damage, loss of tight junctions and increase in cell permeability of Caco-2 cells, thus suppressing colonic inflammation. Thus, Panx1 and P2X7R are involved in mucosal damage, a common clinical feature of inflammatory bowel disease.

Opinion: The present study supports the theory that Panx1 and P2X7R have roles in mediating inflammation and cell death, and the likely mechanism of action is via the activation of the Panx1 channels and P2X7R from the multitude of cell types in the colonic mucosal layer. Taken together, these findings demonstrate how Panx1 and P2X7R may be a potential therapeutic target for the treatment of inflammatory bowel disease.

 

By Dr. Elisabetta Caiazzo
(Dipartimento di Farmacia, Scuola di Medicina e Chirurgia, Università Degli Studi di Napoli “Federico II”)

 

Article title: Imbalance in the Expression of Genes Associated with Purinergic Signalling in the Lung and Systemic Arteries of COPD Patients
Authors: Careta O, Cuevas E, Muñoz-Esquerre M, López-Sánchez M, Pascual-González Y, Dorca J, Aliagas E, Santos S
Journal: Sci Rep
Year: 2019
Volume: 9
DOI: 10.1038/s41598-019-39233-y

Main results: The study focuses on the expression of 17 genes involved in purinergic signalling and inflammation on pulmonary tissue and intercostal arteries of never smokers (NS), non-obstructed smokers (NOS) and stable COPD patients. All the results are presented by the authors as fold change relative to NS. The authors found that the ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1/CD39) and ectonucleoside triphosphate diphosphohydrolase 2 (ENTPD2) expression in the lung were downregulated in both NOS and COPD patients, but upregulated or unaffected in the intercostal arteries of COPD patients compared to NOS. No changes were found in the expression of ectonucleoside triphosphate diphosphohydrolase 3 (ENTPD3) in the lung while a downregulation was observed in the systemic arteries of both COPD patients and NOS. Ectonucleotide Pyrophosphatase/Phosphodiesterase 1 (ENPP1) was upregulated in the intercostal arteries and in the lung of both NOS and COPD patients. The ecto-5'-nucleotidase (CD73) expression was downregulated in both tissues in COPD patients compared to NOS, while adenosine deaminase (ADA) and Dipeptidyl Peptidase 4 (DPP4/CD26) expression was downregulated in the lung and upregulated in the intercostal arteries. Among P1 and P2 receptors, P2RX2, P2RX7, P2RY2 and ADORA1 gene expression in the lung was downregulated in both COPD and NOS patients. ADORA2A was upregulated only in the lung of COPD patients while ADORA3 was upregulated in both groups. In the intercostal arteries, P2RX2 expression was upregulated in COPD patients and down-regulated in NOS, while P2RY2 was upregulated in both groups. ADORA1 expression was upregulated in the intercostal arteries of both COPD patients and NOS with a statistical significance in COPD group. The expression of IL-13 was increased in the lung of both NOS and COPD patients, but it was not expressed in the intercostal arteries. CCL18 gene expression was downregulated in both tissues only in COPD patients.
In conclusion, the authors observed that in the early stage of COPD and in NOS patients the changes in the expression of the genes associated with purinergic signalling in the lung led to ATP accumulation and decreased adenosine levels that may promote inflammation. In addition, the authors suggest that the upregulation of CD73 observed in NOS patients may be a compensatory mechanism.

Opinion: This is the first study that describes changes in purinergic signalling both in the lungs and intercostal arteries of COPD patients in the early stage of the pathology.
Although further research is needed, the results emerging of this study highlight the great importance to establish the role of purinergic signalling machinery in the early stage of the pathology and in the vascular changes that exist in COPD. Additional investigations may help to explore the underlying mechanisms of the frequent association between COPD and cardiovascular disease.

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