Purinergic signaling changes in mesial temporal lobe epilepsy (MTLE)

Purinergic signaling

changes in mesial

temporal lobe epilepsy

(MTLE)

Ângela Daniela Abreu Oliveira

Mestrado em Bioquímica

Departamento de Imuno-Fisiologia e Farmacologia (ICBAS-UP) Center for Drug Discovery and Innovative Medicines - MedInUP 2016

Orientador

Professora Doutora Maria Graça Lobo, Professora Auxiliar, ICBAS/UP

Coorientadores

Professor Doutor Paulo Correia-de-Sá, Professor Catedrático, ICBAS/UP

Doutora Aurora Barros-Barbosa, Investigadora, ICBAS-UP (agora no I3S)

Todas as correções determinadas pelo júri, e só essas, foram efetuadas. O Presidente do Júri,

Acknowledgements

I would like to acknowledge Professor Paulo Correia de Sá for giving me the opportunity to perform this work in the Laboratory of Pharmacology and Neurobiology of ICBAS and also for his patience, deep knowledge and precious scientific advices through this year.

I especially thank Professor Graça Lobo for her guidance, sympathy and care during this work.

I would like to acknowledge Aurora Barros-Barbosa for her guidance, affection, patience and the huge availability during this work. Thanks for helping me grow as a scientist and as a person, without her this work would not have been possible.

I would also like to thank to Doctor João Miguel Cordeiro for his knowledge and support during this work.

I would also like to thank all my colleagues and friends at the Laboratory of Pharmacology and Neurobiology of ICBAS for the support, the scientific sharing and the great time that we had together. I am very grateful to Doctor Fátima Ferreirinha for the precious help in this work, regarding confocal microscopy and western blot experiments. I acknowledge Mrs. Belmira’s wise words, good wave and contagious mood and for always being there when I needed. I would also like to thank Mrs. Helena and Mrs. Milaydis for all the support, help, sympathy and availability during this work.

I would like to thank Mafalda, Adriana, Inês, Filipa, Isabel Calejo and Ana Filipa the valuable support, friendship, help and the great laughter that they gave me. I especially thank Liliana who accompanied me during all these years at university and always helped me, even in the most difficult times. Thanks for the support, patience, care, confidence and friendship.

I would also like to thank Diana, Carla, Patricia, Daniela, Cristina and Augusto for the support, friendship, care and for being present, even several miles apart.

I wish also to acknowledge my mother in law for her sympathy, support and for helping me with this dissertation.

I would like to thank my mother's family for the support, care and attention they have given me over the years, and especially for supporting my parents in these last five years.

I would like to thank my parents for the love, unconditional support, being always present in my life without asking anything in return, and for making possible the completion of this academic path.

Lastly but not least, a special thanks to my boyfriend João for the love, patience, confidentiality, friendship, for always being present during all these years, and the tireless support in the last year that allowed me to finish this important task of my life. I know it was not always easy to deal with me, but your presence in my life was crucial in this final stage. Thank you for being such a wonderful person my love.

Brief-note

Part of this work was accepted for publication in a peer-review scientific journal (Epub ahead of print) and was publicly presented at two scientific meetings as a poster and an oral communication:

Purinergic Signaling (2016) – published online ahead of print in 20 September 2016. doi: 10.1007/s11302-016-9535-2.

“Adenosine A2A receptor and ecto-5'-nucleotidase/CD73 are upregulated in hippocampal astrocytes of human patients with mesial temporal lobe epilepsy (MTLE)”

Barros-Barbosa A.R., Ferreirinha F., Oliveira Â., Mendes M., Lobo M.G., Santos A., Rangel R., Pelletier J., Sévigny J., Cordeiro J.M., Correia-de-Sá P.

ISN’16 – 7th _{ISN Special Neurochemistry Conference, Coimbra-Portugal, 1-4 June}

2016, poster presentation:

“Upregulation of the adenosine A2A receptor and CD73 in hippocampal astocytes of patients with mesial temporal lobe epilepsy (MTLE)”

Barros-Barbosa A. R., Ferreirinha F., Oliveira

Â., Mendes M., Lobo, M.G., Santos A.,

IJUP’16 – Investigação Jovem na Universidade do Porto, 9º Encontro de Jovens Investigadores da Universidade do Porto, Porto-Portugal, 17-19 February 2016, oral communication:

“Overexpression of P2X7 and A2A receptors in the hippocampus of patients with mesial temporal lobe epilepsy (MTLE)”

Oliveira Â., Barros-Barbosa A.R., Ferreirinha F., Lobo M.G., Santos A., Rangel R., Cordeiro J.M., Correia-de-Sá P.

Resumo

Apesar da epilepsia ser uma das doenças mais antigas do mundo esta ainda não possui uma cura definitiva, dado que a sua base celular e molecular não é ainda completamente conhecida. De facto, cerca de 30% dos pacientes epiléticos são refratários à terapia e a maioria destes sofre de epilepsia do lobo mesial temporal (MTLE). Apenas uma parte dos doentes refratários à terapêutica medicamentosa satisfazem os critérios para a remoção cirúrgica do tecido danificado como tratamento de último recurso, ficando os restantes doentes sujeitos a uma terapia anticonvulsiva pouco eficaz. Isto realça a necessidade de pesquisar novos alvos farmacológicos capazes de controlar as convulsões e/ou o processo epileptogénico.

O glutamato e o ácido -aminobutírico (GABA) são neurotransmissores-chave no sistema nervoso central (CNS). O controlo minucioso da ação destes dois neurotransmissores é crucial para a manutenção da normalidade da transmissão sináptica no cérebro, já que o descontrolo entre a neurotransmissão glutamatérgica e GABAérgica tem sido considerado uma das causas da epilepsia. Nos últimos anos, a sinalização purinérgica tem surgido como um potencial alvo terapêutico para modular os níveis sinápticos destes neurotransmissores tendo em consideração que os níveis extracelulares do trifosfato de adenosina (ATP) e do seu metabolito, a adenosina, aumentam drasticamente no cérebro durante a atividade neuronal de elevada frequência e/ou lesões cerebrais. Por exemplo, a coabitação espacial e temporal destas duas purinas (ATP e adenosina) com os dois neurotransmissores, glutamato e GABA, tem sido frequentemente reportada durante crises convulsivas.

Existem evidências sugerindo que durante as convulsões, a formação exagerada de adenosina no meio extracelular pode ter um papel pró-convulsivo através da ativação do recetor excitatório A2A, em consequência do desequilíbrio dos

níveis extracelulares de glutamato e de GABA. Além disso, alguns trabalhos têm mostrado que o recetor A2A é preferencialmente ativado pela adenosina proveniente do

catabolismo extracelular do ATP libertado, em que a enzima ecto-5’-nucleotidase/CD73 desempenha um papel limitante. Tendo em conta resultados anteriores do nosso grupo mostrando que o recetor A2A se encontra mais expresso nos

astrócitos do hipocampo de doentes com MTLE, avaliámos a expressão da enzima ecto-5’-nucleotidase/CD73 e a sua relação com os recetores A2A em amostras de

hipocampo de indivíduos controlo e de doentes com MTLE usando técnicas de Western blot e de imunofluorescência acoplada à microscopia confocal, respetivamente. Os resultados obtidos mostram que a ecto-5'-nucleotidase/CD73 está mais expressa (37 vezes) nos astrócitos de hipocampo de doentes com MTLE e que a

mesma colocaliza, pelo menos parcialmente, com o recetor A2A da adenosina,

sugerindo que a ecto-5'-nucleotidase/CD73 está idealmente posicionada para gerar adenosina a partir do ATP, promovendo assim a ativação imediata do recetor A2A.

Resultados anteriores do grupo de investigação do Laboratório de Farmacologia e Neurobiologia do ICBAS mostraram que a ativação do recetor pré-sináptico P2X7 pelo ATP favorece a libertação de GABA e glutamato a partir de terminais nervosos isolados (sinaptossomas) de córtex cerebral de ratazana na ausência de Ca2+_{. Apesar destas evidências, os mecanismos moleculares subjacentes}

a este efeito do ATP permanecem por esclarecer. Este trabalho foi delineado para estudar, em paralelo e sob as mesmas condições experimentais, os mecanismos envolvidos na libertação de [3_{H]GABA e [}14_{C]glutamato por sinaptossomas corticais de}

ratazana causada pela ativação do recetor inotrópico P2X7 na ausência e na presença (condições fisiológicas) de Ca2+ _{no meio extracelular. A acumulação de GABA e}

glutamato foi avaliada por espectrometria de cintilação líquida. Os resultados mostram que o agonista prototípico do recetor P2X7, BzATP (100-300 μM), favoreceu a libertação de [3_{H]GABA e [}14_{C]glutamato de um modo dependente da concentração,}

sendo o efeito na libertação de [14_{C]glutamato 2 vezes superior ao efeito sobre a}

libertação de [3_{H]GABA. A libertação de [}14_{C]glutamato e [}3_{H]GABA induzida pelo}

BzATP foi completamente prevenida pelo antagonista seletivo do recetor P2X7, A-438079 (3-10 μM), confirmando o envolvimento do recetor P2X7 na libertação destes dois neurotransmissores. Na ausência de Ca2+_{, a libertação de [}3_{H]GABA induzida}

pela ativação do recetor P2X7 foi significativamente atenuada pela inibição do transportador de GABA do tipo 1 (GAT1) com o SKF 89976A (40 μM), indicando que a libertação é mediada pelo reversão do GAT1. Este resultado é suportado por outro mostrando que o efeito facilitatório do BzATP sobre a libertação de GABA é inibido pela substituição do Na+ _{no meio extracelular por NMDG}+_{. Estes resultados indicam}

que a reversão do transportador GAT1 promove a saída de GABA devido ao influxo de Na+ _{através do canal do recetor P2X7. Paradoxalmente, o mesmo efeito não foi}

observado na presença de Ca2+ _{em concentrações fisiológicas (2.2 mM CaCl} 2) no

meio extracelular. Neste caso, o efeito facilitatório do BzATP foi inibido quase na totalidade pela depleção intracelular do Ca2+ _{livre com um quelante rápido de cálcio,}

BAPTA-AM (50 μM), sugerindo que a libertação de [3_{H]GABA induzida pela ativação}

do recetor P2X7 a partir de sinaptossomas corticais de ratazana ocorre através de um mecanismo dependente de Ca2+_.

Relativamente à libertação de glutamato, o efeito facilitatório do BzATP não foi alterado pela inibição dos transportadores de aminoácidos excitatórios (EAATs) com DL-TBOA (100 μM), tanto na presença como na ausência de Ca2+ _{no meio}

extracelular, nem pela depleção intracelular de Ca2+ livre com BAPTA-AM (50 μM). O

efeito facilitatório do BzATP sobre a libertação de [14_{C]glutamato foi atenuado pela}

carbenoxolona (10 μM), um inibidor não seletivo de hemicanais contendo panexina-1, tanto na presença como na ausência de Ca2+ _{no meio extracelular. Estes resultados}

sugerem que a libertação de [14_{C]glutamato induzida pela ativação do recetor P2X7}

parece ocorrer, pelo menos em parte, através de hemicanais contendo panexina-1. O efeito facilitatório do BzATP sobre a libertação de [14_{C]glutamato também foi atenuado}

após a substituição extracelular do Na+ _{por NMDG}+_{, mas esta situação foi verificada}

apenas na ausência extracelular de Ca2+_{. Tendo em conta resultados de outros}

autores mostrando que o glutamato pode ser libertado diretamente através do recetor P2X7, não podemos excluir esta última hipótese para a qual são necessários mais estudos para clarificar essa questão.

Os resultados também mostram que a ativação dos canais de sódio sensíveis à voltagem pela veratridina (VT) favoreceu a libertação de [3_{H]GABA e [}14_C]glutamato,

sendo este efeito completamente prevenido pelo inibidor dos canais de sódio sensíveis à voltagem, tetrodotoxina (TTX). O efeito da VT na libertação de [3_{H]GABA foi 2 vezes}

superior ao efeito sobre a libertação de [14_{C]glutamato, contrariamente ao efeito do}

recetor P2X7. Na ausência de Ca2+_{, a libertação de [}3_{H]GABA e [}14_C]glutamato

induzida pela VT foi significativamente atenuada pelo SKF 89976A (40 μM) e DL-TBOA (100 μM), respetivamente, indicando que a dissipação do gradiente de Na+

devido à ativação dos canais de sódio sensíveis à voltagem favorece a libertação destes neurotransmissores pela reversão dos transportadores GAT1 e EAATs. Contudo, em concentrações fisiológicas de Ca2+ _{a libertação de [}3_{H]GABA e}

[14_{C]glutamato induzida pela VT não foi significativamente atenuada pelo SKF 89976A}

(40 μM) e DL-TBOA (100 μM), sugerindo que nestas condições a VT promove a libertação de [3_{H]GABA e [}14_{C]glutamato por um mecanismo dependente de Ca}2+_.

Os resultados apresentados sugerem que a ativação do recetor P2X7 causa a libertação diferencial de GABA e de glutamato dependendo da concentração extracelular de Ca2+_{. A libertação de glutamato induzida pelo BzATP pelos}

sinaptossomas corticais da ratazana parece ocorrer através de hemicanais contendo panexina-1, não sendo de excluir a possibilidade deste aminoácido ser libertado através do próprio recetor P2X7, enquanto a VT promove a libertação deste neurotransmissor por um mecanismo dependente de Ca2+ _{se este ião estiver}

disponível no meio extracelular, podendo a mesma libertação processar-se pela reversão do transportadores EAATs, na ausência de Ca2+ _{extracelularmente. Por outro}

lado, a libertação de GABA induzida pelo BzATP e pela VT parece ser muito mais sensível a alterações na concentração extracelular de Ca2+ _{no microambiente}

sináptico. Concluímos que em condições fisiológicas, a ativação do recetor P2X7 e dos canais de sódio sensíveis à voltagem promove a libertação de GABA por um mecanismo dependente de Ca2+_{, enquanto na ausência de Ca}2+_{, a libertação de GABA}

ocorre através da reversão do transportador de GABA, GAT1.

Os resultados apresentados neste trabalho, em conjunto com dados experimentais anteriores, sugerem que a ativação dos recetores P2X7 e A2A pode

influenciar a excitabilidade neuronal através da facilitação da neurotransmissão glutamatérgica local enquanto promove, embora em menor extensão, a persistência da neurotransmissão GABAérgica necessária para assegurar o controlo difuso da ativação neuronal. Além da sua relevância em processos de aquisição de memória e aprendizagem, esta modulação purinérgica dos níveis extracelulares de GABA e glutamato pode adquirir um significado diferente em situações patológicas, tais como a MTLE, onde a expressão dos recetores P2X7 e A2A está significativamente

aumentada. Neste contexto, a ativação dos recetores P2X7 e A2A pode resultar numa

sinalização pro-convulsiva capaz de potenciar a excitotoxidade neuronal através do aumento excessivo dos níveis de glutamato na sinapse comparativamente com os níveis de GABA.

Em conclusão, este trabalho reforça a ideia de que o aumento massivo dos níveis extracelulares de ATP e do seu metabolito, adenosina, durante a atividade neuronal de elevada frequência e/ou em situações patológicas, tais como convulsões prolongadas ou repetidas, pode exercer um efeito deletério através da ativação de recetores ionotrópicos P2X7 e metabotrópicos A2A, respetivamente. Deste modo,

propomos que o bloqueio dos recetores P2X7 e A2A, bem como da atividade da

ecto-5'-nucleotidase/CD73, no cérebro epilético possa ser considerado uma nova estratégia para o tratamento dos doentes com MTLE resistentes à terapia medicamentosa habitual devido à sua capacidade para restaurar os níveis de glutamato e GABA nas sinapses corticais.

Abstract

Epilepsy is one of the world oldest recognized disorders, but there is still no cure for this disease, since its cellular and molecular basis is still largely unknown. In fact, nearly 30% of epileptic patients are drug refractory and most of them exhibit mesial temporal lobe epilepsy (MTLE). Only a subset of these patients meet the criteria for surgical ablation of damaged tissue as the last resource treatment, leaving the remaining patients with an unmet medical need. This calls for investigating new pharmacological targets to control seizures and/or epileptogenesis.

Glutamate and -aminobutyric acid (GABA) are key neurotransmitters in the central nervous system (CNS). Fine-tuning between these two neurotransmitters is crucial for the maintenance of normal synaptic transmission in the brain, since imbalance between the glutamatergic and the GABAergic neurotransmission is a hallmark of epilepsy. Purinergic signaling emerge as potential therapeutic target to modulate synaptic levels of these neurotransmitters, since extracellular levels of adenosine triphosphate (ATP) and its metabolite, adenosine, dramatically increase in the brain during high-frequency neuronal firing and/or upon noxious brain conditions, such as epileptic seizures, allowing the spatial and temporal co-habitation of these two purines (ATP and adenosine) with glutamate and GABA.

Several lines of evidence suggest that high extracellular adenosine levels occurring during epileptic seizures may exert a pro-convulsant role via the activation of the excitatory A2A receptor and consequent imbalance of extracellular glutamate and

GABA levels. Additionally, some evidences suggest that the A2A receptor is

preferentially activated by adenosine resulting from the extracellular catabolism of released ATP, where ecto-5’-nucleotidase/CD73 enzyme has a key role. Taking into account previous results from our group showing that the A2A receptor is upregulated in

human hippocampal astrocytes of MTLE patients, we evaluated the expression of ecto-5’-nucleotidase/CD73 enzyme and its co-localization with the A2A receptor in human

hippocampal specimens from control individuals and MTLE patients by Western blot analysis and immunofluorescence confocal microscopy, respectively. The present results show that ecto-5'-nucleotidase/CD73 is overexpressed (37-fold) in astrocytes of the hippocampus of human MTLE patients and that this enzyme co-localizes, at least partially, with the A2A receptor, suggesting that ecto-5'-nucleotidase/CD73 is ideally

positioned to promote A2A receptor activation after ATP metabolism into adenosine.

Previous data from our group also showed that presynaptic activation of the ionotropic P2X7 receptor in Ca2+_{-free conditions favors the release of GABA and}

however the underlying mechanisms remain unclear. Therefore, the present work aimed at studying, in parallel and under the same experimental conditions, the mechanisms underlying [3_{H]GABA and [}14_{C]glutamate release from rat cortical}

synaptosomes triggered by P2X7 receptor activation in the absence and in the presence (physiologic conditions) of extracellular Ca2+_{. GABA and glutamate}

accumulation was evaluated by liquid scintillation spectrometry. Data show that the prototypical P2X7 receptor agonist, 2’(3’)-O-(4-Benzoylbenzoyl)ATP (BzATP; 100-300 μM), triggered [3_{H]GABA and [}14_{C]glutamate release in a concentration-dependent}

manner, being the effect on [14_{C]glutamate release 2-fold higher than the effect on}

[3_{H]GABA outflow. BzATP-induced [}14_{C]glutamate and [}3_{H]GABA release was fully}

prevented by the selective P2X7 receptor antagonist, A-438079 (3-10 μM), confirming the involvement of the P2X7 receptor on the release of these two neurotransmitters. In Ca2+_{-free media, P2X7 receptor-triggered [}3_{H]GABA release was significantly}

attenuated by GABA transporter 1 (GAT1) inhibition with SKF 89976A (40 μM), indicating that this release is mediated by GAT1 transport reversal. This result is supported by another finding showing that the facilitatory effect of BzATP on [3_H]GABA

release is inhibited when extracellular Na+ _{is replaced by NMDG}+_{, thus suggesting that}

GAT1 transport reversal is secondary to Na+ _{influx through the P2X7 receptor.}

However, the same did not occur under normal Ca2+ _{conditions (2.2 mM CaCl}

2). In this

case, the facilitatory effect of BzATP was inhibited almost completely by the intracellular Ca2+ _{chelating compound, BAPTA-AM (50 μM), thus indicating that in the}

presence of extracellular Ca2+ _{the P2X7-induced [}3_{H]GABA release from rat cortical}

synaptosomes occurs through a Ca2+_{-dependent mechanism.}

Regarding glutamate release, the facilitatory effect of BzATP was not altered by the inhibition of excitatory amino acid transporters (EAATs) with DL-TBOA (100 μM), both in the presence and in the absence of extracellular Ca2+_{, nor by intracellular Ca}2+

chelation with BAPTA-AM (50 μM). Inhibition of pannexin-1 hemichannels with carbenoxolone (10 μM) decreased the facilitatory effect of BzATP on [14_C]glutamate

release independently of extracellular Ca2+ _{concentrations, thus suggesting that}

P2X7-induced [14_{C]glutamate release may occur, at least in part, through pannexin-1}

hemichannels. The facilitatory effect of BzATP on [14_{C]glutamate release was also}

inhibited by replacing extracellular Na+ _{by NMDG}+_{, but this was only verified in Ca}2+

-free conditions. At this time, one cannot rule out the possibility that glutamate is being released directly via the P2X7 receptor pore as proposed by other authors.

Data also show that a voltage-sensitive Na+ _{channels activator, veratridine (VT),}

triggered [3_{H]GABA and [}14_{C]glutamate release, being this effect fully prevented by a}

release was 2-fold higher than VT-induced [14_{C]glutamate release, contrary to the}

effect of the P2X7 receptor. In a Ca2+_{-free media, VT-triggered [}3_{H]GABA and}

[14_{C]glutamate release was partially attenuated by SKF 89976A (40 μM) and DL-TBOA}

(100 μM), respectively, indicating that the dissipation of the Na+ _{gradient due to}

activation of voltage-sensitive Na+ _{channels favors the release of these two}

neurotransmitters, at least in part, through the reversal of GAT1 and EAATs transporters. However, under normal Ca2+ _{conditions neither the release of [}3_H]GABA

nor of [14_{C]glutamate induced by VT were affected in the presence of SKF 89976A (40}

μM) and DL-TBOA (100 μM), suggesting that, in these conditions, VT triggers [3_{H]GABA and [}14_{C]glutamate release via Ca}2+_{-dependent mechanisms.}

The results presented here show that activation of the P2X7 receptor triggers a differential release of GABA and glutamate depending on extracellular Ca2+

concentrations. BzATP-induced glutamate release from rat cortical synaptosomes seems to occur through pannexin-1-containing hemichannels and possibly via the P2X7 receptor pore independently of the presence of extracellular Ca2+_{, while VT}

triggers the release of this neurotransmitter through a Ca2+_{-dependent mechanism}

when this ion is readily available in the extracellular milieu, but it favors glutamate outflow through reversal of EAATs transporters in the absence of extracellular Ca2+_{. On}

the other hand, BzATP- and VT-induced GABA release are much more sensitive to changes in the Ca2+ _{concentration in the synaptic microenvironment. In physiological}

conditions, activation of the P2X7 receptor and voltage-sensitive Na+ _{channels promote}

the release of GABA via Ca2+_{-dependent mechanism, while in Ca}2+_{-free conditions}

GABA outflow occurs through reversal of GAT1 transporter.

The present results, together with previous findings, suggest that activation of P2X7 and A2A receptors may influence neuronal excitation through facilitation of local

glutamatergic neurotransmission while promoting, albeit to a lesser extent, the endurance of GABAergic neurotransmission ensuring tonic and more diffuse neuro-inhibition following neuronal activation. Besides its relevance in processes such as memory and learning, the purinergic modulation of extracellular levels of GABA and glutamate may acquire a different meaning under pathological conditions, such as MTLE, where the expression of P2X7 and A2A receptors are significantly enhanced.

Therefore, activation of these purinoceptors may result into a pro-convulsive signaling that leads to neuronal excitotoxicity through excessive rise of glutamate levels in the synapse comparing to GABA levels.

In summary, data presented here strengthen the idea that massive increase in extracellular ATP and its metabolite, adenosine, during high-frequency neuronal firing or under pathological conditions, such as prolonged or repeated seizures, may exert a

deleterious role via ionotropic P2X7 and metabotropic A2A receptors activation,

respectively. Therefore, P2X7 and A2A purinoceptors blockade, as well as

ecto-5'-nucleotidase/CD73 blockade, in the epileptic brain may be a novel and valuable strategy for the treatment of drug-refractory MTLE patients by restoring glutamate and GABA levels in the synapse.

Contents

Acknowledgements ...I

Brief-note ...III

Resumo... V

Abstract ... IX

List of Figures and Tables ... XV

List of Symbols and Abbreviations... XVII

Chapter I – Introduction ... 1

1.1. Epilepsy ...3

1.1.1. Mesial temporal lobe epilepsy ... 5

1.2. Purinergic signaling...5

1.2.1 Purinergic receptors ... 8

P1 recept ors ... 9

P2X receptors ... 11

P2Y receptors ... 13

1.2.2. Purines and epilepsy ... 14

1.3. Neurotransmission in CNS: glutamate and GABA ...16

1.3.1. Glutamat ergic neurotransmission ... 16

Glutamat e transporters ... 20

1.3.2. GABAergic neurotransmission ... 22

GABA transporters ... 26

1.3.3. The role of purines on glutamatergic and GABAergic neurotransmission ... 28

Chapter II – Aims... 31

Chapter III – Materials and Methods ... 35

3.1. Origin and manipulation of brain tissue ...37

3.1.1. Rat brain tissue ... 37

3.1.2. Human brain tissue... 37

3.2. Isolation of synaptosomes ...38

3.2.1. Protein quantification ... 39

3.3. Evaluation of the release of [3_{H]GABA and [}14_{C]glutamate by synaptosomes} from the rat cerebral cortex ...40

3.4. Western blot analysis (WB)...41

3.4.1. Handling and preparation of samples ... 41

3.4.3. Immunological and quantification of protein ... 43

3.5. Immunofluorescence staining and confocal microscopy...44

3.6. Data presentation and statistical analysis ...46

Chapter

IV

– Ecto-5’-nucleotidase/CD73 is upregulated in the

hippocampus of MTLE human patients ... 49

4.1. State of the art...51

4.2. Results...52

4.2.1. Ecto-5’-nucleotidase/CD73 is overexpressed in total lysates of the hippocampus of MTLE human patients ... 52

4.2.2. Ecto-5’-nucleotidase/CD73 is localized in the proximity of astrocytic adenosine A2A receptors ... 53

4.3. Discussion and conclusions...55

Chapter V

– Role of P2X7 receptors activation on GABA and glutamate

release... 57

5.1. State of the art...59

5.2. Results...60

5.2.1. P2X7 activation promotes [3_{H]GABA and [}14_{C]glutamate release from rat cerebral} cortex synaptosomes ... 60

5.2.2. Blockage of GAT1 prevents the facilitatory effect of BzATP on [3_{H]GABA release} in absenc e of extracellular calcium, but not in normal calcium conditions ... 63

5.2.3. The role of extracellular Na+ _{on the P2X7 receptor-triggered GABA and glutamate} release... 64

5.2.4. Activation of the P2X7 receptor can trigger the release of GABA through a Ca2+ -dependent mechanism when this ion is available in the extracellular fluid ... 66

5.2.5. P2X7-induced glutamate release is partially dependent on the activity of pannexin-1-c ontaining hemichannels ... 67

5.2.6. Activation of voltage-gated Na+ _{channels triggers [}3_{H]GABA and [}14_C]glutamate release through the reversal of high-affinity amino acid transporters in Ca2+_{-free media} ... 69

5.3. Discussion and conclusions...72

Chapter VI – Final Discussion and Conclusions ... 79

List of Figures and Tables

Figure 1 – Schematic representation of ATP metabolism inside and outside the cell ... 7 Figure 2 – Schematic representation of the subunits of the three different purinergic receptors and their transduction mechanisms: metabotropic P1 receptors, ionotropic P2X receptors and metabotropic P2Y receptors. P2X receptors are comprised by three subunits, while P1 and P2Y receptors are composed by a single subunit... 9 Figure 3 – Schematic representation of glutamatergic synapse... 17 Figure 4 – Schematic representation of two different families of glutamatergic receptors: ionotropic (AMPA, kainate and NMDA receptors) and metabotropic (mGlu receptor) receptors ... 18 Figure 5 – Schematic representation of ionic fluxes coupled to glutamate (panel of left) and GABA (panel of right) uptake ... 20 Figure 6 – Schematic representation of a GABAergic synapse ... 22 Figure 7 – Schematic representation of two different families of GABAergic receptors: ionotropic GABAA and metabotropic GABAB receptors ... 24

Figure 8 – Schematic representation of “GABA shift” in developmental neurons and its relation with alterations of intracellular Cl- _{concentrations ... 26}

Figure 9 – A: Electron microscopy image of a synaptosomal fraction from a mouse brain; 92000 × magnification; scale bar = 200 m). B: Electron microscopy image of a synaptosomal fraction from a Octopus vulgar brain where it is possible to identify synaptic vesicles (sv), mitochondria, larger vesicles (Iv), tubule, post-synaptic processes and outer membrane. Adapted from Jones (1967) ... 39 Figure 10 – BioTek Synergy TM _{HT microplate reader ... 40}

Figure 11 – A: Semi-automated 12-sample superfusion system (SF-12 Suprafusion 1000, Brandel, Gaithersburg, MD, USA). B: TriCarb2900TR Perkin Elmer spectrometer ... 41 Figure 12 – Schematic protocol of release of [3_{H]GABA and [}14_{C]glutamate from}

synaptosomes... 41 Figure 13 – A: Eletropforesis system (Bio-Rad, California, USA). B: Schematic representation of Wet Transfer process ... 42 Figure 14 – A: Schematic representation of chemiluminescence detection by ClarityTM

Western ECL Substrate (Bio-Rad, California, USA): the secondary antibody that binds to the primary antibody (specific for the protein of interest) is conjugated to HRP, which acts on the ECL substrate and emits light (adapted from Bio-Rad, 2016); B: ChemiDoc MP imaging system (Bio-Rad California, U.S.A.) ... 44 Figure 15 – Laser scanning confocal microscope Olympus FV1000 (Tokyo, Japan) .. 46

Figure 16 – Ecto-5’-nucleotidase/CD73 is overexpressed in total lysates of the

hippocampus of MTLE human patients... 52

Figure 17 – Ecto-5’-nucleotidase/CD73 partially co-localizes with the A2A receptor and with the astrocytic cell marker (GFAP) in the human hippocampus... 55

Figure 18 – BzATP, a prototypical P2X7 receptor agonist, promotes GABA and glutamate release from rat cerebral cortex synaptosomes... 62

Figure 19 – BzATP-induced release of both neurotransmitters – GABA and glutamate – depends on the activation of P2X7 receptor ... 62

Figure 20 – GAT1 revert its function upon P2X7 receptor activation and releases GABA in the absence of extracellular calcium ... 64

Figure 21 – BzATP-triggered release of GABA and glutamate from synaptosomes of the rat cerebral cortex, in a Ca2+_{-free media, depends on the presence of Na}+ _{in the} extracellular media... 65

Figure 22 – BzATP-induced GABA release occurs through a Ca2+_-dependent mechanism when this ion is available in the extracellular fluid ... 67

Figure 23 – BzATP-triggered glutamate release from synaptosomes of the rat cerebral cortex is mediated, at least in part, through pannexin-1-containing hemichannels ... 68

Figure 24 – Voltage-gated Na+ _{channels activation triggers GABA and glutamate} release from rat cerebral cortex synaptosomes... 70

Figure 25 – Activation of voltage-gated Na+ channels with VT triggers [3_{H]GABA and} [14_{C]glutamate release through the reversal of high-affinity amino acid transporters in} Ca2+_{-free media, but not when Ca}2+ _{is available in the extracellular fluid ... 71}

Figure 26 – Schematic diagram representing the role of P2X7 receptor and VT on GABA and glutamate release under physiological conditions and under high-frequency nerve stimulus (low extracellular Ca2+ _{levels) in rat cortical nerve terminals ... 76}

Table 1 – Endogenous ligands and signaling mechanism of purinergic receptors ... 10

Table 2 – Nomenclature and location of the glutamate transporter subtypes ... 21

Table 3 – Nomenclature and location of different GABA transporter subtypes ... 27

Table 4 – Comparison of clinical variables of control and MTLE patients ... 38

Table 5 – Primary and secondary antibodies used in the western blot analysis ... 43

Table 6 – Primary and secondary antibodies used in immunofluorescence staining and confocal microscopy ... 45

List of Symbols and Abbreviations

A-438079, 3-[[5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl]methyl]pyridine hydrochloride ABC, ATP-binding cassette proteins

AC, Adenylyl cyclase

ADA, Adenosine deaminase ADK, Adenosine kinase ADP, Adenosine diphosphate AEDs, Antiepileptic drugs

AMP, Adenosine monophosphate

AMPA, -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ANOVA, Analysis of variance

asct1, Alanine-serine-cysteine transporters AOAA, Aminooxyacetic acid

ATP, Adenosine triphosphate

BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid tetrakis (acetoxymethyl ester)

BCA, Bicinchoninic acid

BDNF, Brain derived neurotrophic factor BGT1, Betaine-GABA transporter BSA, Bovine serum albumin

BzATP, 2’(3’)-O-(4-Benzoylbenzoyl)ATP CA, Ammon’s horn or Cornu Ammonis Ca2+_{, Calcium}

CaM, Calmodulin

CaMKII, Calmodulin kinase II cAMP, Cyclic AMP

CD73, Ecto-5'-nucleotidase

CFTR, Cystic fibrosis transmembrane conductance regulator protein CHP-HGSA, Centro Hospitalar do Porto – Hospital Geral de Santo António Cl-_{, Chloride}

CNS, Central nervous system CPMs, Cintilations per minute DAG, Diacylglycerol

DAPI, 4',6-diamidino-2-phenylindole DG, Dentate gyrus

dk, Donkey

DL-TBOA, DL-threo-β-benzyloxyaspartic acid DPMs, Disintegrations per minute

EAATs, Excitatory amino acid transporters

EC50, Effective concentration that produces 50% of maximal effect EGTA, Ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid E-NPPs, Ectonucleotide pyrophosphatase and/or phosphodiesterases E-NTPDases, Ectonucleoside triphosphate diphosphohydrolases EPSC, Excitatory postsynaptic current

GABA, -aminobutyric acid GABAT, GABA transaminase GAD, Glutamate decarboxylase

GAPDH, Glyceraldehyde 3-phosphate dehydrogenase GATs, GABA transporters

GFAP, Glial fibrillary acidic protein gp, Guinea pig

GPCR, G-protein coupled receptors GTP, Guanosine triphosphate HCO3-_{, Bicarbonate}

He, Helium

HEPES, 2-[4-(2-Hydroxyethyl)piperazin-1-yl]ethanesulfonic acid HRP, Horseradish peroxidase

IgG, Immunoglobulin G

INMLCF-DN, Instituto Nacional de Medicina Legal e Ciências Forenses – Delegação do Norte

IP3, Inositol 1,4,5-trisphosphate

IPSPs, Inhibitory postsynaptic currents K+_{, Potassium}

KCC2, K+_-Cl- _{cotransporter}

Kir, Inwardly rectifying potassium channels KO, Knock-out

LTD, Long-term depression LTP, Long term potentiation Mg2+_{, Magnesium}

mGluR, Metabotropic glutamate receptor min, Minutes

MTLE, Mesial temporal lobe epilepsy Na+_{, Sodium}

NAADP+_{, Nicotinic acid adenine dinucleotide phosphate} NAD+_{, Nicotinamide adenine dinucleotide}

Ne, Neon

NKCC1, Na+_-K+_-Cl- _{cotransporter}

NMDA, N-methyl-D-aspartate NMDG, N-methyl-D-glucamine PBS, Phosphate-buffered saline PKA, Protein kinase A

PKC, Protein kinase C PLC-, Phospholipase C- PNS, Peripheral nervous system PVDF, Polyvinylidene difluoride rb, Rabbit

RIPA, Radio-Immunoprecipitation Assay Buffer RPMs, Revolutions per minute

SE, Status epilepticus slc1, Solute carrier family SDS, Sodium dodecyl sulphate

SCH 58261,

2-(2-Furanyl)-7-(2-phenylethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine

SKF 89976A, 1-(4,4-diphenyl-3-butenyl)-3-piperidinecarboxylic acid hydrochloride) TCA,Tricarboxylic acid

TM, Transmembrane

TLE, Temporal lobe epilepsy TTX, Tetrodotoxin

UDP, Uridine diphosphate UTP, Uridine triphosphate

VGLUTs, Vesicular glutamate transporters VGATs, Vesicular GABA transporters VNUT, Vesicular nucleotide transporters VT, Veratridine

Zn2+_{, Zinc}

Δψ, Electrochemical potential ρ, Pearson’s coefficient

CHAPTER I

INTRODUCTION

1.1. Epilepsy

Epilepsy is one of the most common neurological diseases, affecting approximately 50 million people worldwide. This neurological disease is characterized by unpredictable recurrence of seizures caused by episodes of abnormal and hypersynchronous neuronal network activity in the forebrain (Delorenzo et al., 2005; Engelborghs et al., 2000; Madsen et al., 2010; Pitkänen and Lukasiuk, 2011; Rassendren and Audinat, 2016). The type of seizures varies with brain region where the changes are triggered, according to the etiology (primary cause) and the speed at which electrical discharges propagate. Therefore, the magnitude of seizures may vary from light and nearly undetectable symptoms to brief lapses of consciousness or severe muscle spasms and convulsions, culminating in status epilepticus (SE) – a state of continuous seizures that is considered the most extreme form of seizure (Trinka et al., 2015).

Considering the classification established by Berg and Scheffer (2011) on the basis of etiology, epilepsy can be classified into three groups: genetic, structural/metabolic and unknown (the nature of the underlying cause is unidentified). Furthermore, it is known that several types of brain injuries are causes of acquired epilepsy, including traumatic brain injury, stroke, prolonged febrile seizure, congenital brain malformations, cerebrovascular disorders, cerebral hypoxia, infectious diseases or cerebral tumor (Hunt et al., 2013; Timofeev et al., 2013). In cases of acquired epilepsy, it is possible to be proceeded by a period free of symptoms or complications after the occurrence of insult. This period, called latency period, can be associated with epileptogenesis, a dynamic process that involves progressively structural and biochemical changes, and establishes critical interconnections that lead to spontaneous seizure onset (Engel, 2001; Engel and Pedley, 2007; O'Dell et al., 2012). These changes may continue to accumulate with each new insult over the course of the disease, and include neurodegeneration, neurogenesis, astrogliosis, axonal damage or aberrant sprouting of mossy fiber, dendritic plasticity, reorganization of the molecular architecture and the extracellular matrix of individual brain cells, recruitment of inflammatory cells into brain tissue and blood-brain barrier damage, (Bae et al., 2010; de Lanerolle et al., 2003; O'Dell et al., 2012; Sharma et al., 2007; Yang et al., 2010).

The cellular and molecular basis of this disease is still largely unknown. However, it is believed that the cause is multifactorial and involves: an imbalance between glutamatergic and GABAergic neurotransmission; changes in the function and/or composition of ionotropic receptors (leading to alterations on ionic gradients of

Na+_{, Ca}2+_{, Cl}- _{or K}+_{); changes in Ca}2+ _{activity as a second messenger or changes in}

endogenous neuroprotective and anticonvulsive activities (Delorenzo et al., 2005; Engelborghs et al., 2000; Lerche et al., 2013; Miles et al., 2012; Pavlov and Walker, 2013; Waszkielewicz et al., 2013). Ignorance of the mechanisms involved in the development of this neuropathology and the existence of different forms of epilepsy or epileptic syndromes made necessary the development of various rodent animal models of epilepsy. These models can be induced by neurochemical agents (e.g. kainic acid and pilocarpine), hyperthermal or hypoxic insults, traumatic injuries, electrical stimulation protocols (e.g. after discharges, electroshock-induced seizures and kindling), optogenetics and rodent strains with idiopathic or audiogenic -induced seizures (Curia et al., 2008; Kandratavicius et al., 2014; O'Dell et al., 2012; Pitkänen and Lukasiuk, 2011).

Anti-epileptic drugs (AEDs) are the frontline treatment for epilepsy (Wiebe and Jette, 2012), and there are over twenty AEDs in clinical use. The current available AEDs have different mechanisms of action which include: (1) inhibition of excitatory neurotransmission (Beck and Yaari, 2012; Engelborghs et al., 2000; O'Dell et al., 2012) essentially by decreasing membrane excitability through the interaction with ionic channel conductance – Ca2+ _{(e.g. ethosuximide), K}+ _{(e.g. retigabine) and Na}+ _(e.g.

lacosamide and carbamazepine) – or with neurotransmitter receptors and (2) enhancement of inhibitory neurotransmission (e.g. barbiturates and benzodiazepines). Additionally, newer generation drugs, such as levetiracetam (can affect exocytosis upon binding selectively to synaptic vesicle protein SV2A), seem to influence the prevention or modification of epileptogenesis (Löscher et al., 2009; Russo et al., 2010; Yan et al., 2005).

Although there are plenty of drugs for the treatment of epilepsy, as well as several lines of research, only 50% of the patients are adequately treated for their symptoms with AEDs, and 30% of the remaining patients are refractory to medication, even when treated with three or more different drugs (Beck and Yaari, 2012; Madsen et al., 2009; White, 1999). For refractory patients, the only therapeutic approach is the surgical ablation of epileptogenic zone, i.e. the area responsible for seizures (Rosenow and Lüders, 2001). However, epileptogenic focus and its boundaries must be accurately mapped with support of neuroimaging techniques, electrophysiological recordings, functional testing and analysis of seizure semiology. Unfortunately, only patients who undergo these requirements and which epileptogenic zone can be safely removed are able to profit from this type of treatment, leaving the remaining patients with an unmet medical need. This calls for investigating new pharmacological targets able to control seizures and/or epileptogenesis.

1.1.1. Mesial temporal lobe epilepsy (MTLE)

MTLE is the most common form of drug-resistant epilepsy and is characterized by spontaneous and progressive seizures. Normally, this pathology is associated with previous injuries, such as trauma, SE and febrile seizures (Kharatishvili and Pitkanen, 2010; Yang et al., 2010). Histology studies showed changes in the hippocampus (O'Dell et al., 2012), as well as in several neocortical regions (Alhusaini et al., 2012; Bartolomei et al., 2005; Biagini et al., 2013; Di Maio, 2014; Doherty et al., 2003; Kandratavicius et al., 2014; Scanlon et al., 2011). Hippocampal changes are mostly associated with hippocampal sclerosis characterized by neuronal loss of the Ammon’s horn or Cornu Ammonis (CA; hence the subdivisions CA1 through CA4), concomitant astrogliosis (Cendes et al., 2014; Thom, 2014), and axonal sprouting of the granule neurons of the dentate gyrus (DG) (Sloviter, 1996; Wieser, 2004). These findings are consistent with MTLE characteristic seizures that involve temporal cortex and certain limbic structures, such as hippocampus, entorhinal cortex and amygdala (Maillard et al., 2004; Spencer and Spencer, 1994). However, the biochemical pathways leading to neuron degeneration, gliosis, and mossy fiber sprouting remain unclear. Interestingly, according to the International League Against Epilepsy, approximately 20% of MTLE patients do not have hippocampal sclerosis (Cendes et al., 2014).

As mentioned before, MTLE is associated with previous injuries that remain invisible over 5–15 years (latency period). During this period, the patient suffers irreversible structural and biochemical changes through an epileptogenic mechanism. After this latency period, the patient begins to suffer from recurrent spontaneous seizures which are usually controllable with medication at the beginning (silent period). However, as the disease progresses, patients frequently develop intractable symptoms that cannot be handled with any AEDs (Boison, 2008; Di Maio, 2014; O'Dell et al., 2012; Sharma et al., 2007; Wieser, 2004). Therefore, amygdalo-hypocampectomy surgery appears as the only available treatment of last resource if the epileptogenic zone is conveniently located. In approximately 85% of cases, patients have their epileptic seizures reduced, and these can be controlled by current AEDs (de Lanerolle et al., 2003). Nervertheless, it remains the need to find new pharmacological targets for therapeutic approach to drug-refractory MTLE patients.

1.2. Purinergic signaling

ATP has been long recognized as an intracellular energy source in the brain and multiple other tissues, with a potent long-term (trophic) role in growth and cell

proliferation, and acts as a chemoattractant for immune cells in the extracellular milieu (reviewed by Abbracchio et al., 2009; reviewed by Sáez-Orellana et al., 2015). In 1976, Burnstock pioneered the recognition that purines (namely ATP and adenosine) also exert powerful modulatory influences in the mammalian CNS as neurotransmitters / neuromodulators (Abbracchio et al., 2009; Burnstock, 2016; reviewed by Frenguelli et al., 2007; Sáez-Orellana et al., 2015). Subsequently, nowadays it is recognized that ATP also acts as a neurotransmitter and a neuromodulator in peripheral nervous system (PNS), and it participates in long-term synaptic plasticity events and regulates the survival of neurons and the following repair process under pathological conditions, such as ischemia and inflammation (reviewed by Boison et al., 2010; reviewed by Burnstock, 2007; reviewed by Dale and Frenguelli, 2009; Frenguelli et al., 2007; reviewed by Heinrich et al., 2012).Therefore, it seems that the purinergic signaling has an important role in neurodegeneration, neuroprotection and neuroregeneration (reviewed by Burnstock, 2016; Miras-Portugal et al., 2016; Rodrigues et al., 2015; Sperlágh and Illes, 2014).

ATP is stored in synaptic and astrocyte secretory vesicles and in chromaffin granules through the Cl-_{-dependent vesicular nucleotide transporter (VNUT), which}

also recognizes guanosine triphosphate (GTP) and adenosine diphosphate (ADP). This transporter uses the electrochemical potential (Δψ) and a pH gradient provided by the V-type H+_{-ATPase (V/H}+_{-ATPase) (Abbracchio et al., 2009; Lazarowski, 2012;}

Rodrigues et al., 2015). ATP can be released or co-released with other classical neurotransmitters (e.g. glutamate, GABA, serotonin) from different cell types, namely neurons (dendrites, axons and nerve terminals), astrocytes and microglia through several pathways: (1) constitutive and regulated Ca2+_{-dependent vesicular release}

(exocytosis); (2) ATP transporters (cystic fibrosis transmembrane conductance regulator protein (CFTR), ATP-binding cassette proteins (ABC), P-glycoprotein; (3) hemichannels containing pannexins and connexins; (4) lysosome exocytosis; (5) ATP-sensitive P2X7 receptor, and (6) volume ATP-sensitive anion channels (Abbracchio et al., 2009; Boison et al., 2010; Burnstock et al., 2011b; Heinrich et al., 2012; Lazarowski, 2012; Sáez-Orellana et al., 2015; Scemes et al., 2007). Under basal conditions, the intracellular concentration of ATP is much higher (5-8 milimolar) than the extracellular concentration of the nucleotide (nano to micromolar). The extracellular levels of ATP dramatically increase (reaching millimolar levels) during high-frequency neuronal firing or upon noxious brain conditions, such as trauma, hypoxia/ischemia and epilepsy-associated seizures. This is indicative of regulated mechanisms of ATP release rather than simple ATP leakage (Cunha et al., 1996; Dale and Frenguelli, 2009; Frenguelli et al., 2007; Heinrich et al., 2012; Rodrigues et al., 2015; Sáez-Orellana et al., 2015).

ADK

ADK

Once released, ATP exerts its role through activation of several subtypes of purinoceptors (see next section). In the extracellular milieu, ATP has a short lifetime ( 200 ms) being quickly hydrolysed by a cascade of ectonucleotidases (Figure 1). These enzymes are very important not just because they control the lifetime and the production of ATP metabolites, but because they also act as signaling modulators of the activity of many purinoceptor subtypes by regulating the production and/or metabolism of their endogenous activators. There are four major ectonucleotidase groups – ectonucleoside triphosphate diphosphohydrolases (E-NTPDases), ectonucleotide pyrophosphatase and/or phosphodiesterases (E-NPPs), alkaline phosphatases, and ecto-5'-nucleotidase (CD73) – and they are all expressed in the brain (Abbracchio et al., 2009; Chikahisa and Sei, 2011; Heinrich et al., 2012; Sáez-Orellana et al., 2015). These enzymes were grouped taking into account their specificity for substrates and products formation. E-NTPDases and E-NPPs hydrolyze ATP and ADP to adenosine monophosphate (AMP), which is further hydrolyzed to adenosine by CD73. Alkaline phosphatases hydrolyse equally well nucleoside tri, di and monophosphates, while E-NPPs are the only enzymes that can metabolize dinucleoside polyphosphates, nicotinamide adenine dinucleotide (NAD+_{) and uridine}

diphosphate (UDP) sugars (Abbracchio et al., 2009).

Extracellular adenosine is produced by the enzymatic breakdown of released ATP, but it can also be directly released to the extracellular media via equilibrative

Figure 1 – Schematic representation of ATP metabolism inside and outside the cell. Adapted from Abbracchio et al.

nucleoside transporters once the intracellular levels of the nucleoside increase for instance as a result from the catabolism of ATP in stressed cells (Abbracchio et al., 2009; Boison et al., 2010; Dale and Frenguelli, 2009; Heinrich et al., 2012; Siegel et al., 2006; Sims and Dale, 2014; Wall and Dale, 2013). Once in the extracellular space, adenosine can be deaminated to inosine by adenosine deaminase (ADA, mainly cytosolic but also occuring at the cell surface). Phosphorylation back to AMP is most common in the cytosol via intracellular adenosine kinase (ADK). The extracellular removal of adenosine by cellular uptake via equilibrative nucleoside transporters followed by phosphorylation of the nucleoside by ADK existing in neurons and neighboring non-neuronal cells is the most efficient mechanism to control adenosine concentrations in synapses of the CNS (Chikahisa and Sei, 2011; Correia-de-Sá and Ribeiro, 1996; Latini and Pedata, 2001; Siegel et al., 2006). Therefore, ADK, ADA and equilibrative nucleoside transporters are key players in the regulation of intracellular and extracellular adenosine levels.

1.2.1. Purinergic receptors

The concept of purinergic neurotransmission was firstly proposed by Burnstock in 1972. However, only in 1976 was established the concept of purinergic receptors (Burnstock, 1976). Two purinergic receptor families were classified (Burnstock, 1978), P1 and P2 receptors, depending on the affinity for adenosine and ATP/ADP, respectively (Figure 2). Later, Abbracchio and Burnstock (1994) proposed that P2 purinoceptors should belong to two major families based on studies of transduction mechanisms, pharmacology and cloning of nucleotide receptors: a P2X family of ligand-gated ion channel receptors, and a P2Y family of G-protein coupled receptors (GPCR). Curiously, purinoceptors might be the most abundant receptors in mammalian tissues, since they are expressed in all types of cells (Burnstock and Knight, 2004).

P1 receptors

There are four metabotropic adenosine receptors subtypes, which are coupled to G-proteins (Table 1): A1, A2A, A2B, and A3. These receptors have seven putative

transmembrane (TM) domains of hydrophobic amino acids, being the COOH terminal on the cytoplasmatic side, while the NH2 terminal of the protein is in the extracellular

side of the membrane (Figure 2). The residues of the distal (carboxyl) region of the second extracellular loop are essential for ligand binding and specificity, and the intracellular segment of the receptor interacts with the appropriate G-protein, with subsequent activation of the intracellular signal transduction mechanism (Burnstock, 2007). A1 and A3 receptors are coupled to Gi/0 protein and mediate inhibition of adenylyl

cyclase (AC), activation of several types of K+ _{channels, inactivation of Ca}2+ _channels

and activation of phospholipase C- (PLC-). Additionally, A3 receptor may also be

coupled to Gq/11 protein. On the other hand, A2A and A2B receptors are coupled to Gs

protein, mediate the formation of cyclic AMP (cAMP) through stimulation of AC, and can also be associated with intracellular Ca2+ _{mobilization (Abbracchio et al., 2009;}

reviewed by Boison, 2008; Burnstock et al., 2011a; reviewed by Tomé et al., 2010).

Figure 2 – Schematic representation of the subunits of the three different purinergic receptors and their transduction

mechanisms: metabotropic P1 receptors, ionotropic P2X receptors and metabotropic P2Y receptors. P2X receptors are comprised by three subunits, w hile P1 and P2Y receptors are composed by a single subunit. Adapted from Abbracchio et al. (2009), Baroja-Mazo et al. (2013) and Burnstock (2007).

ADP UDP

Adenosine

ATP UTP

Table 1 – Endogenous ligands and signaling mechanism of purinergic receptors. Adapted from Burnstock (2007) and

Rang et al. (2016).

In the brain, extracellular adenosine mainly controls excitatory neurotransmission through a coordinated action of inhibitory A1 receptors and

facilitatory A2A receptors, despite A2B and A3 receptors have also been identified in

some brain regions (Latini and Pedata, 2001). Actually, A1 receptors are the most

abundant adenosine receptors in the brain and display a widespread distribution (located mainly in the synapses), whereas A2A receptors are most abundant in the

basal ganglia, but are also present at lower density in other brain regions, namely the hippocampus (reviewed by Augusto et al., 2013; Burnstock et al., 2011a; Dixon et al., 1996; Schiffmann et al., 1991; Tomé et al., 2010). Furthermore, recent studies evidenced that A2A receptors, in addition of being expressed in synaptic terminals, are

Family Subtypes Signal mechanism Ligand

P1 receptors

A1 Gi/G0: ↓ cAMP Adenosine

A2A Gs: ↑ cAMP Adenosine

A2B Gs: ↑ cAMP Adenosine

A3 Gi/G0, Gq/G11: ↓ cAMP, PLC-β activation Adenosine

P2X receptors

P2X1 Ion channel (Ca2+ _{and Na}+_{) ATP}

P2X2 Ion channel (mainly Ca2+_{) ATP}

P2X3 Ion channel ATP

P3X4 Ion channel (mainly Ca2+_{) ATP}

P2X5 Ion channel ATP

P2X6 Ion channel ATP

P2X7 Ion channel and large pore with

prolonged activation ATP

P2Y receptors

P2Y1 Gq/G11, Gi/G0: PLC-β activation, ion

channel (K+ _{and Ca}2+₎ ADP

P2Y2 Gq/G11 and possibly Gi/G0: PLC-β

activation ATP, UTP

P2Y4 Gq/G11 and possibly Gi: PLC-β activation,

PLA2 stimulation ATP, GTP, UTP

P2Y6 Gq/G11: PLC-β activation UDP

P2Y11 Gq/G11 and Gs: PLC-β activation, ↑ cAMP

ATP, ADP, NAD+_,

NAADP+

P2Y12 Giα: ↓ cAMP ATP, ADP

P2Y13 Gi/G0: ↓ cAMP ADP

P2Y14 Gi/G0: ↓ cAMP, PLC stimulation

glucose, UDP-galactose

also located in astrocytes (Boison et al., 2010; Kanno and Nishizaki, 2012; Matos et al., 2013; Matos et al., 2012; Orr et al., 2015). Adenosine plays several roles in the CNS involving an inhibitory tonus of neurotransmission and neuroprotective role. However, the unbalance between A1 and A2A receptors activation may result in neurological

dysfunctions. In fact, the A2A receptor has been implicated in diverse pathological

conditions of the CNS, such as epilepsy (Huicong et al., 2013; Tomé et al., 2010), Huntington's disease (reviewed by Burnstock, 2016), Parkinson’s disease (Uchida et al., 2015), and Alzheimer’s disease (Orr et al., 2015).

P2X receptors

P2X receptors are ligand-gated cation channels (ionotropic receptors) that open a pore permeable to Na+_{, K}+ _{and Ca}2+ _{upon ATP binding (Figure 2 and Table 1),}

leading to depolarization of neurons and smooth muscle. These receptors are assembled by three individual subunits encoded by seven distintic genes (P2X1 to P2X7). Each subunit of P2X receptors has two transmembrane domains, a large glycosylated extracellular ligand-binding loop with several conserved cysteines and an intracellular COOH and NH2 terminals which contain consensus binding motifs for

protein kinases (Abbracchio et al., 2009; Burnstock and Knight, 2004; North, 2002; Roberts et al., 2006; Sáez-Orellana et al., 2015). Furthermore, all subunits have consensus sequences for N-linked glycosylation (Asn-X-Ser/Thr) that are essential for trafficking to the plasma membrane (North, 2002; Robinson and Murrell-Lagnado, 2013; Sáez-Orellana et al., 2015). P2X1-6 subunits comprise 384 amino acids and have an EC50 for ATP of 1-10 µM, whereas P2X7 subunits have 595 amino acids

because of the long intracellular C-terminus with 239 amino acids and are activated by 10-1000 µM ATP concentrations. P2X receptors are 30-50% pairwise identical at the peptide level (Abbracchio et al., 2009; North, 2002; Skaper et al., 2010; Sperlágh and Illes, 2014). Currently it is known that P2X receptors can be homo- or heterotrimerics complexes, and that their phenotypes are determined by assembly of individual subunits. Curiously, the P2X6 receptor only exists in the form of heteromeric channels. Presently, seven functional heterotrimeric receptors have been identified: P2X1/2, P2X1/4, P2X1/5, P2X2/3, P2X2/6, P2X4/6 and P2X4/7; apparently, the most physiologically relevant are P2X1/2, P2X1/4, P2X1/5 and P2X2/3 receptors (Abbracchio et al., 2009; Baroja-Mazo et al., 2013; Burnstock and Knight, 2004; Roberts et al., 2006; Sáez-Orellana et al., 2015). All the P2X receptors subunits are expressed in neurons, but the expression is heterogeneous in distinct brain regions

and cell types, allowing a great variability of responses to ATP (Abbracchio et al., 2009; Sáez-Orellana et al., 2015).

“Synaptic plasticity is a term used to describe long-term changes in synaptic connectivity and efficacy, either following physiological alterations in neuronal activity (such as learning and memory), or resulting from pathological disturbances (epilepsy, chronic pain or drug dependence). Long-term potentiation (LTP) and its counterpart termed long-term depression (LTD) have a crucial role in synaptic plasticity” (Rang et al., 2016). Remarkably, evidence has shown that over-activation of P2X receptors may trigger either LTP or LTD (Burnstock et al., 2011a). Furthermore, in most CNS neurons presynaptic P2X receptors can modulate the release of several neurotransmitters such as glutamate, dopamine and GABA, whereas postsynaptic P2X receptors interact with many ionotropic receptors including the nicotinic acetylcholine receptors, GABAA and

N-methyl-D-aspartate (NMDA) receptors. These interactions may be mediated by Ca2+_,

Ca2+_{-activated kinases phosphorylating the receptors and/or their anchoring proteins,}

or through direct interactions between receptors (Abbracchio et al., 2009; Burnstock et al., 2011a; Sáez-Orellana et al., 2015).

The P2X7 receptor has particular characteristics compared to other P2X receptors; the activity of this receptor is enhanced by low extracellular concentrations of divalent cations, such as Ca2+ _{and Mg}2+ _{(Jiang, 2009; North, 2002; Virginio et al.,}

1997; Yan et al., 2011). Although this receptor displays an unusually large ionic conductance, repeated or prolonged activation opens a non-selective pore that allows the permeation of large molecular weight organic cations up to 600-800 Da (North, 2002; Sperlágh and Illes, 2014). However, this mechanism is not clear yet, since: (1) evidences show that the mechanism responsible for this permeation is the progressive channel dilation of the P2X7 receptor pore, and the carboxyl terminus domais and the TM2 region of this receptor are essential for the pore formation (Cervetto et al., 2013; Marcoli et al., 2008; Sun et al., 2013); (2) studies also demostrate that the open channel conformation of the P2X7 receptor can allow the passage of negatively charged fluorescent dyes (Browne et al., 2013), and (3) other studies have shown that the P2X7 receptor may recruit additional pore-forming proteins, usually pannexin-1 (Baroja-Mazo et al., 2013; Locovei et al., 2007; Pelegrin and Surprenant, 2006; Sperlágh and Illes, 2014). Besides the P2X7 receptor being associated with LTP phenomena (Chu et al., 2010), evidence has shown that it is also involved in many pathological conditions of neurological disorders, such as neurotrauma (Kimbler et al., 2012), epilepsy (Engel et al., 2012; Henshall et al., 2013; Jimenez-Pacheco et al., 2013; Sperlágh and Illes, 2014), neuropathic pain (Itoh et al., 2011; North and Jarvis, 2013), multiple sclerosis (Gu et al., 2015; Matute et al., 2007), amyotrophic lateral

sclerosis (Yiangou et al., 2006), Alzheimer’s disease (McLarnon et al., 2006; Miras-Portugal et al., 2015; Sáez-Orellana et al., 2015), Parkinson’s disease (Carmo et al., 2014; Marcellino et al., 2010), and Huntington’s disease (Diaz-Hernandez et al., 2009).

P2Y receptors

There are currently known eight metabotropic P2Y receptor subtypes: P2Y1,

P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13 and P2Y14. All receptors have a similar subunit

topology of seven transmembrane domains, an extracellular NH2 terminal, and an

intracellular COOH terminal with consensus binding motifs for protein kinases (Figure 2). Additionally, the extracellular loops have four cysteine residues essential for proper trafficking of the receptor to the cell surface, and molecular studies shows that some positively charged residues in TM3, TM6 and TM7 are crucial for receptor activation by nucleotides. However, these receptors show a low level of homology (19-55% identical at peptide level), resulting in significant differences in their pharmacological and operational profiles (Abbracchio et al., 2009; Baroja-Mazo et al., 2013; Burnstock, 2007; Burnstock and Knight, 2004). The most remarkable difference between these receptors is their ability to use different endogenous nucleotides as agonists (Table 1): P2Y1, P2Y11, P2Y12 and P2Y13 receptors are only activated by adenine nucleotides;

P2Y2 and P2Y4 receptors preferentially react to triphosphate nucleotides; the P2Y6

receptor binds preferentially to UDP, and the P2Y14 receptor is only activated by

UDP-sugars (UDP-glucose and UDP-galactose) (Burnstock, 2007).

P2Y receptors can also be grouped in two distinct subgroups based on phylogenetic similarity, presence of amino acids critical for ligand binding and selectivity of G-protein coupling: (1) the P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 subgroup

is most often coupled to Gq protein, which activates PLC-, and (2) the P2Y12, P2Y13,

and P2Y14 subgroup use Gi protein to inhibit adenylyl cyclase and modulate ion

channels (Abbracchio et al., 2006; Abbracchio et al., 2009; Burnstock, 2007). The P2Y11 receptor can also be coupled to Gs protein, leading to activation of AC and

regulation of ion fluxes. Evidence suggests that receptors activation by several agonists results in the coupling to different G-proteins (biased receptors), indicating an agonist-specific signaling pathway (Abbracchio et al., 2006; Abbracchio et al., 2009; White et al., 2003).

P2Y receptors are widely distributed on both neurons and glia, being responsible for initiation of intracellular Ca2+ _{signals and regulation of ion channels in}

neurons (Abbracchio et al., 2006; Abbracchio et al., 2009; Burnstock et al., 2011a; Burnstock and Knight, 2004; Burnstock et al., 2011b). Interestingly, P2Y receptors may