POTENTIAL OF ADENOSINE-SYSTEM-BASED THERAPIES IN THE TREATMENT OF NEUROIMMUNE DISORDERS

Dissertação Mestrado Integrado em Medicina POTENTIAL OF ADENOSINE-SYSTEM-BASED THERAPIES IN THE TREATMENT OF NEUROIMMUNE DISORDERS Ivo Marcos Pinto Peixoto Orientadora: Prof. Doutora Laura Joana Fevereiro Oliveira Porto 2012 POTENTIAL OF ADENOSINE-SYSTEM-BASED THERAPIES IN THE TREATMENT OF NEUROIMMUNE DISORDERS 1 Instituto de Ciências Biomédicas Abel Salazar – U. Porto Dissertação Mestrado Integrado em Medicina POTENTIAL OF ADENOSINE-SYSTEM-BASED THERAPIES IN THE TREATMENT OF NEUROIMMUNE DISORDERS Ivo Peixoto1; Laura Oliveira2 1. Instituto de Ciências Biomédicas Abel Salazar – Universidade do Porto Endereço: Avenida Araújo e Silva, 34 4560-451 Penafiel, Portugal E-mail: [email protected] 2. Professora auxiliar, Instituto de Ciências Biomédicas Abel Salazar – Universidade do Porto, Departamento de Imunofisiologia e Farmacologia, Laboratório de Farmacologia e Neurobiologia. Porto 2012 POTENTIAL OF ADENOSINE-SYSTEM-BASED THERAPIES IN THE TREATMENT OF NEUROIMMUNE DISORDERS 2 Acknowledgements To Prof. Laura Oliveira for the remarkable support and enthusiasm. A special thanks for introducing me to the fascinating world of neuropharmacology. To my family for the notable presence and everlasting encouragement throughout the years. POTENTIAL OF ADENOSINE-SYSTEM-BASED THERAPIES IN THE TREATMENT OF NEUROIMMUNE DISORDERS 3 Contents Abstract............................................................................................................. 4 Resumo ............................................................................................................. 5 Introduction ...................................................................................................... 7 Objectives ......................................................................................................... 8 Adenosine in the Brain .................................................................................... 9 Adenosine in Neuroglia / Neuroinflammation.............................................. 11 Adenosine in different brain disorders......................................................... 15 a) Alzheimer’s Disease............................................................................... 15 b) Parkinson’s Disease............................................................................... 17 c) Huntington’s Disease.............................................................................. 20 d) Multiple Sclerosis.................................................................................... 22 Discussion ...................................................................................................... 24 Conclusions.................................................................................................... 28 References...................................................................................................... 29 Annex A........................................................................................................... 42 POTENTIAL OF ADENOSINE-SYSTEM-BASED THERAPIES IN THE TREATMENT OF NEUROIMMUNE DISORDERS 4 Abstract One of the grand challenges in the 21st century is to unravel the molecular elements associated to the dynamic crosstalk between immune and neuronal cells in neurologic diseases, and then use that knowledge to design effective neuroimmunemediated therapeutic strategies. In light of the critical ties between the nervous and immune systems, adenosine pathways may be a key element in the control of neuroimmune homeostasies since adenosine receptors (ARs) play an important role in the modulation of neuronal activity and inflammatory/immune responses. Indeed, the omnipresence of ARs in neurons, glia and immune cells makes adenosine a key regulator of many facets of brain function and, therefore, a valuable target under brain pathological conditions. Disorders under the umbrella of neuroimmunology are not just the prototypic immune-mediated central (CNS) and peripheral nervous system diseases, such as Multiple Sclerosis and Myasthenia Gravis. It is worth noting that virtually all of the major neurologic conditions (including Alzheimer’s disease, cerebrovascular disease, epilepsy, Parkinson’s, and CNS infection) are now recognized to have immune/inflammatory components. In the presence of inflammation and immune dysfunction, the adenosine system acts as a sensor, which through dynamic modification in the expression of ecto-enzymes and ARs, adapts neuronal tissue metabolism status and contributes to mechanisms deputed to neuroprotection. In keeping with these concepts it is becoming increasingly appreciated that drugs targeted on adenosine pathways can exert beneficial effects in neuroimmune disorders. This review aims to discuss the role of adenosine in the pathophysiology of neurodegenerative disorders in which the immune system plays a key role, such as Multiple Sclerosis, Alzheimer’s, Parkinson’s and Huntington’s disease, as well as to highlight the mechanisms through which pharmacological modulation of the adenosine pathway may have potential application in their therapeutic management. Keywords: Adenosine; Adenosine Receptors; Neurodegenerative disorders; Neuroimmune disorders; Adenosine-based therapies. POTENTIAL OF ADENOSINE-SYSTEM-BASED THERAPIES IN THE TREATMENT OF NEUROIMMUNE DISORDERS 5 Resumo Um dos grandes desafios no século XXI é a descoberta dos elementos moleculares envolvidos na interação dinâmica entre as células imunológicas e neuronais em doenças neurológicas e aplicar esse conhecimento no desenvolvimento de estratégias terapêuticas eficazes com mediação neuro-imunológica. Tendo em conta as conexões entre os sistemas nervoso e imunológico, o sistema adenosinérgico parece ser um elemento chave no controlo da homeostasia neuro-imunológica uma vez que os recetores de adenosina têm um papel importante na modulação tanto da atividade neuronal como das respostas inflamatórias/imunológicas. De fato, a omnipresença destes recetores nos neurónios, glia e em células do sistema imunológico faz da adenosina um regulador importante de vários aspetos da função cerebral e, portanto, um alvo terapêutico potencial nas patologias cerebrais. A área da neuroimunologia não abrange apenas as doenças de mediação imunológica prototípicas que afetam o sistema nervoso central e periférico, tais como a Esclerose Múltipla e a Miastenia Gravis. Na verdade, virtualmente todas as doenças neurológicas (tais como a doença cerebrovascular, infeção cerebral, doenças de Alzheimer e Parkinson e epilepsia) têm hoje uma componente inflamatória/imunológica reconhecida. Na presença de inflamação e disfunção imunológica, o sistema adenosinérgico atua como um sensor que através de modificações dinâmicas na expressão de ectoenzimas e dos seus recetores consegue adaptar o metabolismo dos neurónios e contribuir com efeitos neuroprotetores. Posto isto, tem vindo a crescer o interesse sobre os efeitos benéficos do controlo das vias da adenosina em doenças neuro-imunológicas. Este artigo de revisão tem objetivo de discutir o papel da adenosina na fisiopatologia de doenças neurodegenerativas em que o sistema imunológico tem um papel preponderante, tais como a Esclerose Múltipla e as doenças de Alzheimer, Parkinson e Huntington, assim como esclarecer os mecanismos pelos quais a modulação farmacológica das vias da adenosina poderá ter um lugar importante no seu tratamento. Palavras-Chave: Adenosina; Receptores de Adenosina; Doenças Neurodegenerativas; Doenças Neuroimunes; Terapias baseadas na adenosina. POTENTIAL OF ADENOSINE-SYSTEM-BASED THERAPIES IN THE TREATMENT OF NEUROIMMUNE DISORDERS 6 Abbreviation List: AD – Alzheimer’s Disease NO – Nitric Oxide ADA – Adenosine Deaminase PD – Parkinson’s Disease AK – Adenosine Kinase SNc – Substantia Nigra pars AMP – Adenosine 5’-monophosphate compacta APP – Amyloid Precursor Protein SNr – Substantia Nigra pars AR – Adenosine Receptor reticulata ATP – Adenosine 5’-triphosphate TGF-β – Transforming Growth BDNF – Brain-derived neurotrophic factor Factor-beta CB – Cannabinoid System TNF-α – Tumor necrosis factor alpha CCL2 – Chemokine (C-C motif) ligand 2 Trk receptors – Tropomyosin-related CD39 – Ecto-apyrase kinase receptors CD73 – Ecto-‘5-nucleotidase UPS – Ubiquitin Proteossome CNS – Central Nervous System COX - Ciclooxygenase DR – Dopamine Receptor EAE –Experimental Autoimmune Encephalomyelitis ENT – Equilibrative nucleoside transporter FOXP3 – Forkhead Box P3 protein GABA –γ-Aminobutyric acid GLT – Glutamate transporter Gpi – Globus Pallidus, internal segment HD – Huntington’s Disease IFN-γ– Interferon gamma IL – Interleukin iNOS – Inducible nitric oxide synthase L-DOPA – 3,4-Dihydroxy-L-phenylalanine MAO-B – Monoamine Oxidase B mGlu – Metabotropic glutamate receptor MHC – Major histocompatibility complex MS – Multiple Sclerosis MSN – Medium spiny neuron NFT – Neurofibrillary Tangles NGF – Nerve growth factor NMDA – N-Methyl-D-aspartate POTENTIAL OF ADENOSINE-SYSTEM-BASED THERAPIES IN THE TREATMENT OF NEUROIMMUNE DISORDERS 7 Introduction Adenosine has emerged as an extracellular signalling molecule able to coordinate homeostasis in all cells with effects on tissue protection and repair (Linden 2005). However, in the nervous system, adenosine also exerts a rather specific neuromodulatory role, controlling synaptic transmission and synaptic plasticity (Gomes 2011) as well as coordinating neural networks (Sperlagh and Vizi 2011). The double action of this system led to the consideration of adenosine as a therapeutic target in the management of neurologic disorders. Part of the role of adenosine seems to be related to the regulation of neuroinflammatory processes and modulation of immune responses, especially in pathological settings, offering neuroprotection potential and a means of control of neurodegenerative and neuroimmune conditions (Stone et al. 2009). The combined effects of adenosine on neuronal viability and immune responses propelled extensive research in this field and a large body of evidence encouraging adenosine-system-based therapies has emerged. POTENTIAL OF ADENOSINE-SYSTEM-BASED THERAPIES IN THE TREATMENT OF NEUROIMMUNE DISORDERS 8 Objectives This review article aims to summarize the current evidence on the adenosinemediated mechanisms in the pathophysiology of neuroimmune disorders, focusing on neurodegenerative and autoimmune diseases and to highlight therapeutical rationale and future potential of the adenosine system. A short overview of the adenosine role in the nervous system function and in the brain immune system signaling will ensue, followed by clinical implications in particular neurologic disorders. POTENTIAL OF ADENOSINE-SYSTEM-BASED THERAPIES IN THE TREATMENT OF NEUROIMMUNE DISORDERS 15 Adenosine in different brain disorders a) Alzheimer’s Disease Alzheimer’s disease (AD) is the most common form of dementia in the elderly, and is characterized by progressive memory loss and cognitive deterioration (Blennow et al. 2006). Defining neuropathological features are: a selective synaptic failure and neuronal loss in several brain regions, especially the cerebral cortex and the hippocampus; the deposition of extracellular amyloid plaques containing the Aβpeptide and the formation of intraneuronal neurofibrillary tangles (NFT) (Querfurth and La Ferla 2010). It is believed, particularly from genetic studies, that an imbalance between production, clearance and aggregation of Aβpeptides is crucial to AD onset (Tanzi and Bertram 2005). In addition, the severity of the cognitive impairment correlates with the levels of oligomers rather than with the total Aβburden (Lue LF et al. 1999), proving to be synaptotoxic (Klyubin et al. 2008). Moreover, aggregation of Aβoligomers into fibrils gives rise to diffuse amyloid plaques which are associated with neuronal death, neuritic dystrophy, dendritic spine loss and abnormal axons (Knowles et al. 1999), leading to synaptic loss and disruption (Stern et al. 2004). This is an early change and structurally correlates with the cognitive impairment (Arendt 2005 for review). Furthermore, there is an apparent contribution of neuroinflammatory processes in AD pathophysiology (Eikelenboom et al. 2006; Heneka and O’Banion 2007) and activated microglia and astrocytes were found to be associated with amyloid plaques (Wisniewski and Wiegel 1991;Stalder et al. 1999). There is also conflicting evidence concerning whether Aβ activates a glial inflammatory response or if glial dysfunction precedes amyloidogenesis (Polazzi and Monti 2010). Adenosine, acting mainly through A1and A2A ARs, can modulate cognition and memory (Sebastião and Ribeiro 2009a). There is increasing evidence suggesting redistribution and changes in the density of adenosine receptors in AD (Gomes et al. 2011). Autoradiography and binding studies in humans with AD have consistently shown a loss of A1ARs in the hippocampus, especially in the dentate gyrus (Jansen et al. 1990;Kalaria et al. 1990;Ulas et al. 1993;Deckert et al. 1998). There are also studies demonstrating decreases in A1ARs in the striatum (Ikeda et al. 1993), the temporal cortex and thalamus of AD patients (Fukumitsu et al. 2008). In contrast, the levels and functional activity of both A1and A2A ARs seem to be increased in the frontal POTENTIAL OF ADENOSINE-SYSTEM-BASED THERAPIES IN THE TREATMENT OF NEUROIMMUNE DISORDERS 16 cortex of these patients (Albasanz et al. 2008), which is in agreement with observations from a transgenic mouse model of AD (Arendash et al. 2006). Notwithstanding, an immunolocalization study revealed increased immunoreactivity of A1ARs in neurons with NFT and in dystrophic neurites of senile plaques in the hippocampus and frontal cortex (Angulo et al. 2003). Moreover, the same study exposed the positive influences of the A1AR on amyloid precursor protein (APP) processing and in tau phosphorylation, slowing down the progression of the disease (Angulo et al. 2003). There is more limited data about the distribution of A2A ARs in AD, but interestingly it has been found an increased expression of A2A ARs in microglial cells in the hippocampus and cerebral cortex (Angulo et al. 2003). As previously stated, chronic stressful conditions elicit upregulation of A2A ARs (Rebola et al. 2005), encouraging the modulation of these receptors as a neuroprotective strategy. In vitro and in vivo studies using A2A AR antagonists almost completely prevented Aβ-induced synaptic loss and neurotoxicity (Dall’Igna et al. 2003;Dall’Igna et al. 2007;Canas et al. 2009), raising the possibility of reversing neuronal deficits. The precise mechanism of this neuroprotection is still to be unveiled, but the brain anti-inflammatory properties (Geiger et al. 2006), the protection against free radicals (Leite et al. 2011) and the control of glutamate excitotoxicity (Cunha 2005) derived from A2A AR blockade may all have an influence. Nevertheless, it is somewhat surprising the cognitive enhancement derived from A2A AR blockade, since A2A AR stimulation in vitro is known to facilitate acetylcholine secretion and enhances glutamatergic transmission in the hippocampus (e.g. Jin and Fredholm 1997). Indeed, currently the most used drugs in AD induce cholinergic enhancement. A considerable amount of information has arisen, regarding the benefits of adenosine receptor manipulation in AD, and this derives greatly from the impact of caffeine in both animal AD experimental models and epidemiological studies. Caffeine is a natural methylxanthine and a non-selective A1and A2A AR antagonist (Fredholm et al. 1999), and neuroprotective benefits from long-term intake have been reported. In AD transgenic mice, long-term administration of caffeine improved cognitive performance, and reduced the levels of soluble Aβ fragments and of Aβ peptides (Arendash et al. 2006). More recently, a similar study has demonstrated sustained reductions in plasma Aβ and decreases in both soluble and deposited Aβ in hippocampus and cortex (Cao et al. 2009). The same group has also shown that caffeine has the ability to reverse the pre-installed memory deficits and the pre-existing considerable Aβ burden in these models (Arendash et al. 2009). These effects are POTENTIAL OF ADENOSINE-SYSTEM-BASED THERAPIES IN THE TREATMENT OF NEUROIMMUNE DISORDERS 17 mimicked by selective A2A AR antagonists, strengthening the idea that A2A ARs are responsible for these beneficial effects. There is also a limited number of epidemiological studies conducted to determine the association between long-term (years to decades) caffeine consumption and the incidence of AD. Recent systematic reviews and meta-analyses from cohort and case-control studies have found a trend towards the protection against cognitive decline in AD, with highest benefit among the eldest age group (Barranco Quintana et al. 2007;Santos et al. 2010). However, the heterogeneous methodologies and results preclude definite statements in this topic. Besides these insights on the role of adenosine in AD pathophysiology there is also strong evidence concerning the possible role of caffeine and ARs in other conditions of memory impairment. Of note, caffeine has also influence on brain acetylcholine, leading to concentration increases in the prefrontal cortex of rats (Acquas et al. 2002), an effect likely mediated by the A1AR (Maemoto et al. 2004). Moreover, caffeine prevents in both rodents and humans the acute memory loss induced by scopolamine (Riedel et al. 1995;Botton et al. 2010). This highlights the specific correction of the cholinergic pathway involved in caffeine memory-enhancing effects. However, selective A2A AR blockade did not reproduce these results suggesting that these receptors do not affect general processes of memory impairment but instead play a crucial role restricted to neurodegenerative conditions involving an insidious synaptic deterioration leading to memory dysfunction (Cunha et al. 2008). There is clearly a need of more basic research on the role of the adenosinergic system in AD pathophysiology and associated cognitive impairment in order to understand its prophylactic and/or therapeutic potential. b) Parkinson’s Disease Parkinson’s Disease (PD) is a common neurodegenerative disorder characterized by a progressive loss of the dopaminergic neurons from the substantia nigra pars compacta (SNc) in the striatum, which leads to the typical motor symptoms of akinesia, bradykinesia, rigidity, resting tremor and postural abnormalities (Morelli et al. 2009). The etiology of the disease is still unknown but factors such as protein misfolding and aggregation and oxidative stress associated to mitochondrial dysfunction and excitotoxicity are important features in PD pathogenesis (Dauer and Przedborski 2003). POTENTIAL OF ADENOSINE-SYSTEM-BASED THERAPIES IN THE TREATMENT OF NEUROIMMUNE DISORDERS 18 Currently, it is accepted that movement is regulated through integration of corticothalamic information from the striatum and output from the internal globus pallidus (Gpi) and substantia nigra pars reticulata (SNr) under tonic dopaminergic conditions (Albin et al. 1989). The GABAergic MSNs make up the major striatal output pathways – the monosynaptic D1R-expressing direct pathway and the striatopallidal D2R-expressing indirect pathway. Thus, dopamine acting through the different receptors in the direct and indirect pathways leads to movement facilitation and inhibition, respectively (Kulisevsky and Poyurovsky 2012). The important roles of dopamine in this control are lost in PD. Therefore, the introduction of 3,4-dihydroxy-L- phenylalanine (L-DOPA) and of dopamine agonists was a successful strategy to restore the deficits in the motor loop. However this only provides a symptomatic relief, with no influence on disease progression and has troubling long-term consequences such as motor fluctuations and L-DOPA-induced dyskinesias (Stacy and Galbreath 2008;Jankovic 2005). ARs, especially the A2A subtype, have a prominent role in several aspects of PD pathophysiology. A2A ARs show the ability to control D2R function through both the formation of heteromers and intracellular signaling pathways (Schiffmann et al. 2007) and therefore can alter each other’s pharmacological properties such as affinity and desensitization in an antagonistic manner (Xie et al. 2007). The strong expression of A2A ARs in the striatum, namely the post-synaptic co-localization with D2R in GABAergic striatopallidal MSNs (Hillion et al. 2002) pinpoints a rationale for the use of A2A AR antagonists as an alternative or adjunctive to the dopamine-based therapies. In addition, A2A ARs are also expressed in GP and their blockade reduces extracellular GABA concentration, potentiating motor activity induced by L-DOPA (Rosin et al. 2003; Simola et al. 2006). There is a large number of epidemiological and experimental studies in rodent and primate non-human models of PD, which confirm the motor activity enhancement effects mediated by acute A2A AR blockade (Morelli et al. 2010; Wei et al. 2011) and can ameliorate symptoms even as monotherapy (Pinna et al. 2007). Moreover, synergistic interactions between L-DOPA and A2A AR antagonists have also been described suggesting that they may be co-administered to potentiate the motor stimulant effects (Rose et al. 2006; Hodgson et al. 2010). Similar studies on the effects of chronic A2A AR antagonism in animal models have shown analogous findings. With the co-administration of A2A AR antagonists after the onset of motor fluctuations or L– DOPA-induced dyskinesia, it was allowed a reduction in the doses of dopaminomimetic compounds with potentiation of motor improvements without worsening dyskinesia POTENTIAL OF ADENOSINE-SYSTEM-BASED THERAPIES IN THE TREATMENT OF NEUROIMMUNE DISORDERS 19 (Armentero et al. 2011 for review). More recently, evidence from knockout studies pointed towards a possible contribution of A2A ARs in different brain regions and cellular elements other than the post-synaptic striatopallidal neurons in the attenuation or reversal of L-DOPA induced motor complications, a subject that requires further investigation (Wei et al. 2011). Taken together, this data from non-clinical studies propelled clinical trials testing the use of selective A2A AR antagonists in PD patients. Indeed, five clinical trials are currently underway (phases I to III) to analyze the therapeutic potential of adenosine A2A AR (Armentero et al. 2011 for review). The most compelling evidence from these studies comes from the highly selective A2A AR antagonist istradefylline. Bara-Jimenez and coworkers (2003) reported that at low doses of L-DOPA, istradefylline potentiated the antiparkinsonian response with less dyskinesia compared with that induced by optimal dose L-DOPA alone. In patients with dyskinesia, istradefylline has recently shown consistent and sustained increases in OFF time (Factor et al. 2010) but most studies in the field reported increased (although not troublesome) dyskinesias. However, changes in the design of the clinical trials such as dose selection, eventual concomitant caffeine intake and disease stage of the target population may provide different outcomes in the future. In regard to the neurodegenerative process, A2A AR blockade also reduces dopaminergic cell loss, although the mechanism for this neuroprotective role has not been uncovered yet (Chen et al. 2007). The previously stated ability of the A2A AR to control glutamate release may be implicated, as well as the aforementioned modulation of glia-mediated neuroinflammation in PD. Moreover, antagonism of A2A AR also influences the blood-brain barrier permeability and the traffic of peripheral immune cells (Varani et al. 2001). Neuroprotection may also be related with the inhibition of monoamine oxidase B (MAO-B) reported with A2A AR antagonists (Petzer et al. 2009), decreasing oxidative stress and hazardous byproducts (Sagi et al. 2007). Additionally, there is increasing evidence that neurodegeneration begins with a synaptic dysfunction, which later evolves into an overt damage of neurons (Dauer and Przedborski 2003). Accordingly, the recent work of Gomes and colleagues has shed some light on the role of the A2A AR in the control of corticostriatal circuits through glial cell line-derived neurotrophic factor (GDNF) modulation of glutamatergic and dopaminergic nerve terminals (Gomes et al. 2006; Gomes et al. 2009). Hence, A2A AR antagonism has a plethora of beneficial effects, not only relieving motor deficits in established PD but also showing a broader disease-modifying potential, arresting neurodegeneration and slowing progression. Nonetheless, concise POTENTIAL OF ADENOSINE-SYSTEM-BASED THERAPIES IN THE TREATMENT OF NEUROIMMUNE DISORDERS 20 evidence on the specific mechanisms involved and their relevance is still scarce. In spite of the obstacles there is an exciting prospect for A2A AR antagonists as alternative or adjunctive therapies in PD, as further basic and clinical investigation goes on. c) Huntington’s Disease Huntington’s Disease (HD) is an autossomal dominant neurodegenerative disease characterized by progressively worsening chorea, psychiatric disturbances and cognitive impairment. The pathological hallmark of HD is the degeneration of striatal GABAergic enkephalinergic MSN’s (Mitchell et al. 1999). The molecular origin of this degeneration has been ascribed to a genetic mutation that leads to the production of an abnormal form of the protein Huntingtin (The Huntington’s Disease Collaborative Research Group 1993). The mechanisms of pathogenicity remain unknown but it has been suggested both a loss of function of the normal Huntingtin, as well as a gain of function related to the toxic properties of the mutated protein (Cattaneo et al. 2005). Indeed, several lines of evidence support the idea that the mutated Huntingtin induces corticostriatal glutamatergic dysfunction. This includes increased glutamate release and decreased astrocytic glutamate clearance, together with increases in activation of NMDA receptors and triggering of mitochondrial dysfunction (Popolli et al. 2007). Moreover, recent evidence demonstrated increased immune activation of macrophages and microglia in HD, pointing towards a role for immune dysfunction in this brain pathology (Björkqvist et al. 2008). The high density of A2A ARs in the striatum, either pre-synaptically in the corticostriatal afferents modulating glutamate release as well as in the post-synaptical MSNs, supports pathophysiological relevance of this receptor in HD (Rosin et al. 1999; Hettinger et al. 2001). The observation of decreased expression of A2A ARs in the striatum of early HD patients (Glass et al. 2000) and of transgenic mice (Blum et al. 2003) further reinforced their contribution to pathogenesis. Several HD animal model studies (both pharmacological and genetic knockout) in different phases of the disease revealed changes in A2A AR expression, density and/or signaling. Although expression of the A2A ARs in the striatum is markedly decreased during HD progression (Chiang et al. 2005), striatal cells expressing the mutant Huntingtin have shown aberrant increases in A2A AR signaling (Varani et al. 2010;Chou et al. 2005;Tarditi et al. 2006). Despite such controversies, A2A ARs remain a potential target in HD. POTENTIAL OF ADENOSINE-SYSTEM-BASED THERAPIES IN THE TREATMENT OF NEUROIMMUNE DISORDERS 21 As previously stated, adenosine acting on A2A ARs at pre-synaptic sites, promotes glutamate release (Popoli et al. 2002) and is thus detrimental to neurons, while at post-synaptic, extra-synaptic sites and glial cells there is a speculative neuroprotective potential. Studies from HD animal models, regarding activation and blockade of A2A ARs have yielded conflicting findings in relation to neurodegeneration since both A2A AR agonists and antagonists were capable of producing neuroprotection (Popoli et al. 2007 for review). A recent study in A2A AR knockout mice showed worsened motor performance and survival, supporting the idea that early and chronic blockade of A2A ARs may not be beneficial (Mievis et al. 2011). This might reflect the preversus post-synaptic effects of A2A ARs, and is in line with the unexpected ability of A2A AR antagonists to potentiate NMDA-mediated toxicity (Popoli et al. 2007). The same controversy exists in regard to mitochondrial and metabolic dysfunction with findings of either protection or increase of striatal MSN lesion (Alfinito et al. 2003;Fink et al. 2004). Moreover, it has been shown that A2A AR agonists can enhance ubiquitin proteossome system (UPS) activity (Chiang et al. 2009), which may be of value since Huntingtin aggregate formation and UPS dysfunction are major features of HD (Wang et al. 2008). In addition, normal Huntingtin has beneficial antiapoptotic actions and contributes to the production and delivery of BDNF, namely to striatal targets, and this supply is decreased in HD (Zuccato and Cattaneo 2007). BDNF is particularly important for the survival and plasticity phenomena of corticostriatal synapses (Cattaneo et al. 2005) and A2A AR agonists are able to transactivate the TrkB BDNF receptor and regulate BDNF levels (Sebastião and Ribeiro 2009b). Indeed, A2A ARs have shown a crucial role in this regulation (Tebano et al. 2010), which provides another neuroprotective mechanism afforded by adenosine. However, BDNF can also contribute to cell death, depending on the receptor activated (Gomes et al. 2011), which further complicates this controversy. Finally, A2A AR antagonism is generally accepted as a neuroprotective strategy, through the control of the neuroinflammatory component of neurodegenerative diseases, including HD. Furthermore, A2A ARs expressed in bone marrow-derived inflammatory cells have proved to be important contributors to striatal damage in transgenic models of HD (Huang et al. 2006). It appears that the disease stage and time-frame, the drug administration protocol, and the clinical manifestations might play critical roles in evaluating the future therapeutic potential of A2A AR ligands (Popoli et al. 2008). HD is thus a special case POTENTIAL OF ADENOSINE-SYSTEM-BASED THERAPIES IN THE TREATMENT OF NEUROIMMUNE DISORDERS 22 where the complexity of the disease combines with the complexity of A2A AR pharmacology, possibly yielding biphasic neurotoxic-neuroprotective effects and making it difficult to establish if this is a target of interest. d) Multiple Sclerosis Multiple Sclerosis (MS) is an inflammatory demyelinating disease of the CNS, classically defined as an auto-reactive T-cell-driven damage directed towards oligodendrocytes and the myelin sheath surrounding central axons, with features of mononuclear cell infiltration and inflammatory damage (Prat and Antel 2005). However, more recent work has indicated prominent and widespread neuronal damage early in the disease (Vogt et al. 2009). This led to the hypothesis of MS as an inflammatory demyelinating and neurodegenerative disease but it remains elusive if there is a sequence of events or distinct pathological processes culminating in disease (Herz et al. 2010). Research focus has thus shifted towards the involvement of cellular and molecular mechanisms in this neuroimmune crosstalk. In addition, astrocytes and microglia have shown a dual role in MS, both promoting damage and inflammation as well as restoring the damaged tissues (Williams et al. 2007;Muzio et al. 2007). Besides the roles in neuro-glial and glia-glial communication, not much is known regarding purinergic signaling in MS. Adenosine levels are decreased in the blood of MS patients, together with an increase of TNF-α (Mayne et al. 1999). In addition, blood immune cells and glial cels from MS patients show decreased expression of the A1AR, suggesting a dysfunction of this receptor in the pathogenesis of the disease (Johnston et al. 2001). Consistently, studies using experimental autoimmune encephalomyelitis (EAE), the most widely used animal model of MS, found that A1AR stimulation protects against neuroinflammation, demyelination and oligodendrocyte cytotoxicity (Tsutsui et al. 2004). This is further supported by the recent finding that suppression of EAE- induced neuroinflammation and neurobehavioral deficits by glucocorticoid treatment is accompanied by a concurrent increase in A1AR expression in monocytoid cells (Tsutsui et al. 2008). Moreover, adenosine directly promotes oligodendrocyte progenitor cell differentiation and myelination (Stevens et al. 2002) and stimulates their migration via A1ARs (Othman et al. 2003). More recently, chronic intake of caffeine has shown the surprising ability to upregulate A1ARs, transforming growth factor-beta (TGF-β) and to suppress interferongamma (IFN-γ) in EAE guinea pigs (Chen et al. 2010). This data supports the view of A1AR-mediated shift from Th1 to Th2 cell function in the attenuation of pathology POTENTIAL OF ADENOSINE-SYSTEM-BASED THERAPIES IN THE TREATMENT OF NEUROIMMUNE DISORDERS 23 (Chen et al. 2010) and provides a neurobiological basis for epidemiological investigation into the possible relationship between caffeine consumption and development of multiple sclerosis in humans. Furthermore, pharmacological blockade of A2A AR protected mice from EAE induction, highlighting the critical role of adenosine in the modulation of EAE, although the exact mechanisms remain to be clarified (Mills et al. 2008). In the murine EAE model of MS, adenosine can also exacerbate immunopathological mechanisms. Infiltrating lymphocytes into the CNS requires the expression of CD73 either on T cells or in the CNS for disease induction (Mills et al. 2008). This means that the anti-inflammatory effects of adenosine can be offset by the effect on the access of pathogenic T-cells to the CNS. Moreover, it was demonstrated that CD39 is expressed in a subset of forkhead box P3 (FOXP3)-expressing regulatory T cells, the numbers of which are strikingly reduced in patients with relapsing-remitting MS (Borsellino et al. 2007). These cells are suggested to play a role in the suppression of autoimmune damage, and their reduction in MS might impair the control of IL-17-mediated inflammation (Fletcher et al. 2009). In addition, lymphocytes from MS patients have shown an increased CD39 activity, which may provide a protective mechanism by fostering AR-mediated anti-inflammatory actions (Spanevello et al. 2010). Besides CD73 and CD39, also ADA is involved in lymphocyte functioning in MS. ADA is known to control growth, proliferation, differentiation and migration of lymphocytes (Pérez-Aguilar et al. 2010). Since lymphocytes from MS patients exhibit reduced ADA activity (Vivekanandhan et al. 2005), this may further contribute to their infiltration in the CNS. There is mounting evidence on the link between adenosine and the etiology of MS. The multifaceted role of this molecule and the myriad of integrative mechanisms in the control of immune responses open new avenues for treatments to be pursued in the future, as additional understanding is provided. POTENTIAL OF ADENOSINE-SYSTEM-BASED THERAPIES IN THE TREATMENT OF NEUROIMMUNE DISORDERS 24 Discussion The future prospects for adenosine-system-based therapies in chronic neurodegenerative diseases seem promising. However, in spite of the vast contributions of ARs to neuroprotection mechanisms and to the regulation of inflammation in the last few years, there are still several open questions which deserve special attention. Adenosine elicits a multitude of effects in different cells, and factors such as expression, density, localization, transducing systems, and functions of ARs (particularly the A2A) are subject to modifications upon brain activity or pathology. The same promiscuity occurs with the institution of pharmacological modulation of ARs or adenosine metabolism pathways and with caffeine intake, especially in a long-term basis. Moreover, the sources and pathways of generation of extracellular adenosine are still not fully uncovered in order to understand the dynamics of these receptors. In regard to the experimental models of neurodegeneration, it is difficult to draw conclusions on the precise role of ARs in chronic conditions, since evidence derives in great part from acute injury models and some do not take into account the age of the animal. This is particularly relevant since conditions like AD and PD are strikingly prevalent in the elderly. Furthermore it is uncertain the translational potential of such studies into the human brain. It seems well established that A1AR activation affords neuroprotection while blockade of A2A ARs attenuates the burden of most brain disorders. As previously proposed, this would mean that a combination of AK inhibitors and antagonists of A2A ARs would achieve the most neuroprotective potential (Cunha 2005). However this impact can only be predicted if more light is shed on when and how extracellular adenosine levels change in the brain (including synapse, neuro-glial domain, glia-glial domain or gliovascular domain), the blood-brain barrier and in infiltrating myeloid cells. In addition, these fluctuations may vary with disease stage and between diseases, further complicating the scenario. The recognition of the dual role of neuroinflammation on neurodegeneration and neuroprotection and the importance of the adenosinergic system in the control of the balance between tissue injury and repair also turned attention towards ARs as modulators of glial and immune functions. The apparent primordial physiological protective function of adenosine following acute insults may be overshadowed by its reduced ability to protect against more chronic injury. Adenosine signals are endogenously protective, but imperfect and inconsistent. 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Ulas J, Brunner LC, Nguyen L, Cotman CW (1993) Reduced density of adenosine A1 receptors and preserved coupling of adenosine A1 receptors to G proteins in Alzheimer hippocampus: a quantitative autoradiographic study. Neuroscience 52: 843-854. POTENTIAL OF ADENOSINE-SYSTEM-BASED THERAPIES IN THE TREATMENT OF NEUROIMMUNE DISORDERS 47 estratégia terapêutica a adotar, contrabalançando os efeitos anti-inflamatórios com a infiltração de células T patogénicas. Discussão As perspetivas do futuro de terapias baseadas na adenosina em doenças neurodegenerativas crónicas é animadora. No entanto, apesar das descobertas nos últimos anos acerca da vasta contribuição dos recetores de adenosina para mecanismos neuroprotetores e na regulação da inflamação, existem ainda várias questões em aberto que merecem especial atenção. A adenosina desencadeia uma multiplicidade de efeitos em diferentes células, e fatores tais como a expressão, densidade, localização, sistema de transdução de sinal, e funções dos recetores da adenosina estão sujeitos a modificações consoante a atividade cerebral ou na presença de patologia. A mesma promiscuidade ocorre com a instituição de modulação farmacológica dos recetores ou das vias de metabolização da adenosina assim como após o consumo de cafeína, particularmente a longo-termo. Além disso, as fontes e as vias de geração de adenosina extracelular ainda não estão completamente caracterizadas de forma a compreender a dinâmica destes recetores. Relativamente aos modelos experimentais de neurodegeneração, é difícil tecer conclusões sobre o papel específico dos recetores da adenosina em doenças crónicas, visto que grande parte das evidências é derivada de modelos de lesão aguda e por vezes não levam em conta a idade do animal. Isto tem particular relevância uma vez que doenças como AD e PD são altamente prevalentes no idoso. Parece bem estabelecido que a ativação dos recetores A1confere neuroprotecção enquanto que o bloqueio dos recetores A2A atenua a patologia na maioria das doenças cerebrais. Isto significaria que uma combinação de inibidores da adenosina cinase e antagonistas A2A resultaria no maior potencial neuroprotector. Contudo, este impacto só poderá ser previsto assim que seja clarificado como e quando é que se alteram os níveis cerebrais (incluindo sinapse e domínios neuro-glial, glia-glial e gliovascular) de adenosina extracelular. Além disso, tais flutuações podem variar consoante a doença e o seu estadio, o que complica este cenário. O reconhecimento do papel duplo da neuroinflamação na neurodegeneração e neuroproteção, aliado à função de controlo do balanço entre dano e reparação tecidular do sistema adenosinérgico, levou à investigação das potencialidades da adenosina na modulação das funções gliais e imunes. O papel primordial aparente da adenosina de proteção fisiológica em danos agudos contrasta com a capacidade reduzida de proteção em condições crónicas. Os sinais mediados pela adenosina são POTENTIAL OF ADENOSINE-SYSTEM-BASED THERAPIES IN THE TREATMENT OF NEUROIMMUNE DISORDERS 48 endogenamente protetores , mas imperfeitos e inconsistentes. Por exemplo, o bloqueio dos recetores A2A cerebrais para suprimir inflamação pode exacerbar efeitos proinflamatórios periféricos. Aliás, as interações com astrócitos e microglia têm provavelmente uma grande importância nesta modulação, visto que cada uma destas células gliais apresenta recetores de adenosina e têm efeitos opostos de promoção das respostas imunes e de inibição da inflamação do sistema nervoso central. No que concerne às doenças neurodegenerativas em específico, existem também alguns aspetos que merecem discussão. Na AD ainda é difícil distinguir os impactos do bloqueio A2A no défice cognitivo daqueles que estão relacionados com a fisiopatologia da doença em si. Um alvo putativo a ser testado está relacionado com a função colinérgica. Foi observado que os recetores α-7 nicotínicos da acetilcolina estão aumentados no início da doença e que podem ser controlados pela ativação dos recetores A2A. Visto que há um défice de projeções colinérgicas em AD, o bloqueio de recetores A2A pode assim ter ainda mais um mecanismo de regulação de fenómenos de plasticidade neuronial. Os últimos anos de investigação revolucionaram a visão clássica da PD. Na verdade, a neurodegeneração estende-se a outros locais e outros terminais e os circuitos de controlo motor dos gânglios da base são muito mais intrincados, dificultando a compreensão do papel da adenosina na sua fisiopatologia. Além disso, a caracterização e desenvolvimento de ligandos que tenham como alvo heterómeros que contenham o recetor A2A devem ser considerados no futuro. Estes têm propriedades farmacológicas únicas e são mais seletivos em localização do que cada um dos recetores constituintes, permitindo uma melhor perceção dos mecanismos envolvidos na doença. As discrepâncias dos efeitos dos recetores A2A na HD têm sido o maior entrave no desenvolvimento de novas terapias. Agonistas A2A parecem apresentar maior benefício em estadios tardios de degeneração enquanto que os efeitos protetores do bloqueio A2A dependem do grau de inibição pós-sináptica. Também aqui a formação de heterómeros que contenham o recetor A2A constitui um alvo mais específico e oportuno. A investigação do papel específico do sistema adenosinérgico na fisiopatologia da MS está ainda no seu início. A MS pode ser vista como um modelo de autoimunidade protetora, em que os mecanismos naturais de reparação cerebral estão impedidos. Os recetores A1parecem ser importantes na restauração desses mecanismos e a indução de aumentos de adenosina extracelular parece uma estratégia plausível. POTENTIAL OF ADENOSINE-SYSTEM-BASED THERAPIES IN THE TREATMENT OF NEUROIMMUNE DISORDERS 49 As múltiplas questões em aberto poderão obter resposta com a exploração das potencialidades dos recetores A2B e A3, assim como através do uso de novos agonistas e antagonistas, moduladores alostéricos e de heterómeros com diferentes afinidades e propriedades, criando nomeadamente um balanço adequado entre efeitos pré- e pós-sinápticos. A ação das ectonucleotidases também tem um papel importante na sinalização purinérgica de recetores de ATP versus adenosina, após se ter verificada a formação de complexos oligoméricos entre estes. Também foi observado que a astrogliose reativa presente em muitas patologias cerebrais está associada a um aumento patológico da expressão de adenosina cinase. O uso lógico de inibidores desta enzima gerou, porém, efeitos laterais sistémicos significativos que incentivaram uma nova era de abordagens de aumento focal de adenosina. Aqui estão incluídas estratégias inteligentes como a distribuíção por polímeros, a distribuíção por células estaminais e a terapia génica. Conclusões Existe, portanto, um espectro inteiro de mecanismos fisio(pato)lógicos que estão sujeitos a regulação adenosinérgica. Terapias baseadas nesta abordagem irão depender do timing de tratamento, no que diz respeito à janela terapeutica, ao estadio e progressão dos danos e à duração e monitorização dos efeitos benéficos e adversos. Esta coincidência tempo-espaço será somente alcançada através de uma melhor compreensão sobre o modo como os sinais da adenosina são percebidos, discriminados, sustentados e terminados.