The Intranigral Infusion of Human-Alpha Synuclein Oligomers Induces a Cognitive Impairment in Rats Associated with Changes in Neuronal Firing and Neuroinflammation in the Anterior Cingulate Cortex.

Parkinson's disease (PD) is a complex pathology causing a plethora of non-motor symptoms besides classical motor impairments, including cognitive disturbances. Recent studies in the PD human brain have reported microgliosis in limbic and neocortical structures, suggesting a role for neuroinflammation in the development of cognitive decline. Yet, the mechanism underlying the cognitive pathology is under investigated, mainly for the lack of a valid preclinical neuropathological model reproducing the disease's motor and non-motor aspects. Here, we show that the bilateral intracerebral infusion of pre-formed human alpha synuclein oligomers (H-αSynOs) within the substantia nigra pars compacta (SNpc) offers a valid model for studying the cognitive symptoms of PD, which adds to the classical motor aspects previously described in the same model. Indeed, H-αSynOs-infused rats displayed memory deficits in the two-trial recognition task in a Y maze and the novel object recognition (NOR) test performed three months after the oligomer infusion. In the anterior cingulate cortex (ACC) of H-αSynOs-infused rats the in vivo electrophysiological activity was altered and the expression of the neuron-specific immediate early gene (IEG) Npas4 (Neuronal PAS domain protein 4) and the AMPA receptor subunit GluR1 were decreased. The histological analysis of the brain of cognitively impaired rats showed a neuroinflammatory response in cognition-related regions such as the ACC and discrete subareas of the hippocampus, in the absence of any evident neuronal loss, supporting a role of neuroinflammation in cognitive decline. We found an increased GFAP reactivity and the acquisition of a proinflammatory phenotype by microglia, as indicated by the increased levels of microglial Tumor Necrosis Factor alpha (TNF-α) as compared to vehicle-infused rats. Moreover, diffused deposits of phospho-alpha synuclein (p-αSyn) and Lewy neurite-like aggregates were found in the SNpc and striatum, suggesting the spreading of toxic protein within anatomically interconnected areas. Altogether, we present a neuropathological rat model of PD that is relevant for the study of cognitive dysfunction featuring the disease. The intranigral infusion of toxic oligomeric species of alpha-synuclein (α-Syn) induced spreading and neuroinflammation in distant cognition-relevant regions, which may drive the altered neuronal activity underlying cognitive deficits.

Repurposing Pomalidomide as a Neuroprotective Drug: Efficacy in an Alpha-Synuclein-Based Model of Parkinson's Disease.

Marketed drugs for Parkinson's disease (PD) treat disease motor symptoms but are ineffective in stopping or slowing disease progression. In the quest of novel pharmacological approaches that may target disease progression, drug-repurposing provides a strategy to accelerate the preclinical and clinical testing of drugs already approved for other medical indications. Here, we targeted the inflammatory component of PD pathology, by testing for the first time the disease-modifying properties of the immunomodulatory imide drug (IMiD) pomalidomide in a translational rat model of PD neuropathology based on the intranigral bilateral infusion of toxic preformed oligomers of human α-synuclein (H-αSynOs). The neuroprotective effect of pomalidomide (20 mg/kg; i.p. three times/week 48 h apart) was tested in the first stage of disease progression by means of a chronic two-month administration, starting 1 month after H-αSynOs infusion, when an already ongoing neuroinflammation is observed. The intracerebral infusion of H-αSynOs induced an impairment in motor and coordination performance that was fully rescued by pomalidomide, as assessed via a battery of motor tests three months after infusion. Moreover, H-αSynOs-infused rats displayed a 40-45% cell loss within the bilateral substantia nigra, as measured by stereological counting of TH + and Nissl-stained neurons, that was largely abolished by pomalidomide. The inflammatory response to H-αSynOs infusion and the pomalidomide treatment was evaluated both in CNS affected areas and peripherally in the serum. A reactive microgliosis, measured as the volume occupied by the microglial marker Iba-1, was present in the substantia nigra three months after H-αSynOs infusion as well as after H-αSynOs plus pomalidomide treatment. However, microglia differed for their phenotype among experimental groups. After H-αSynOs infusion, microglia displayed a proinflammatory profile, producing a large amount of the proinflammatory cytokine TNF-α. In contrast, pomalidomide inhibited the TNF-α overproduction and elevated the anti-inflammatory cytokine IL-10. Moreover, the H-αSynOs infusion induced a systemic inflammation with overproduction of serum proinflammatory cytokines and chemokines, that was largely mitigated by pomalidomide. Results provide evidence of the disease modifying potential of pomalidomide in a neuropathological rodent model of PD and support the repurposing of this drug for clinical testing in PD patients.

Nicotine, cocaine, amphetamine, morphine, and ethanol increase norepinephrine output in the bed nucleus of stria terminalis of freely moving rats.

The bed nucleus of stria terminalis (BNST) is a complex limbic area involved in neuroendocrine and behavioural responses and, in particular, in the modulation of the stress response. BNST is innervated by dopamine and norepinephrine, which are known to be involved in drug addiction. It is also known that several drugs of abuse increase dopamine transmission in the BNST, but there has been less research regarding the effect on norepinephrine transmission. Here, we have used the microdialysis technique to investigate the effect of several drugs of abuse on norepinephrine transmission in the BNST of freely moving rats. We observed that nicotine (0.2-0.4 mg/kg), cocaine (2.5-5 mg/kg), amphetamine (0.25-0.5 mg/kg), and ethanol (0.5-1.0 g/kg), dose-dependently increased norepinephrine output while the effect of morphine at 3.0 was lower than that of 1.0 mg/kg. These results suggest that many drugs of abuse, though possessing diverse mechanisms of action, share the property of increasing norepinephrine transmission in the BNST. Furthermore, we suggest that the recurring activation of NE transmission in the BNST, due to drug administration, contributes to the alteration of the function that BNST assumes in how the behavioural response to stress manifests, favouring the establishment of the stress-induced drug seeking.

Can pioglitazone be potentially useful therapeutically in treating patients with COVID-19?

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a pandemic disease (COVID-19) that has spread globally causing more than 30,000 deaths. Despite the immense and ongoing global effort, no efficacious drugs to fight this plague have been identified and patients admitted to the intensive care units (ICU), for respiratory distress, are managed mostly by means of supportive care based on oxygen maintenance. Several authors have reported that the prevalence of hypertension, diabetes, cardiovascular and cerebrovascular diseases comorbidities were indeed frequent among patients with COVID-19, which suggests that these conditions are likely to aggravate and complicate the prognosis. What the aforementioned diseases have in common is a latent chronic inflammatory state that may be associated with the alteration of laboratory parameters that are typical of the metabolic syndrome and insulin resistance. In severe COVID-19 patients laboratory markers of inflammation such as C-reactive protein, IL-6, D-dimer, serum ferritin and lactate dehydrogenase are elevated in many patients; assessed since the 4th-6th day of illness onset, such increases seem to be predictive of an adverse prognosis. Our hypothesis is that drugs belonging to the family of thiazolidinediones (TZD) such as pioglitazone or rosiglitazone, approved for treating the condition of insulin resistance and the accompanying inflammation, could ameliorate the prognosis of those COVID-19 patients with diabetes, hypertension and cardiovascular disorders comorbidities. TZD are PPARγ agonists that act on nuclear receptors, thereby triggering certain transcription factors. TZD were widely used for type-2 diabetes in the first decade of this century and although concerns have been raised for possible side effects associated with long-term treatment, their use has been recently revaluated for their anti-inflammatory properties in numerous medical conditions.

Neuroprotection by the Immunomodulatory Drug Pomalidomide in the Drosophila LRRK2WD40 Genetic Model of Parkinson's Disease.

The search for new disease-modifying drugs for Parkinson's disease (PD) is a slow and highly expensive process, and the repurposing of drugs already approved for different medical indications is becoming a compelling alternative option for researchers. Genetic variables represent a predisposing factor to the disease and mutations in leucine-rich repeat kinase 2 (LRRK2) locus have been correlated to late-onset autosomal-dominant PD. The common fruit fly Drosophila melanogaster carrying the mutation LRRK2 loss-of-function in the WD40 domain (LRRK2WD40), is a simple in vivo model of PD and is a valid tool to first evaluate novel therapeutic approaches to the disease. Recent studies have suggested a neuroprotective activity of immunomodulatory agents in PD models. Here the immunomodulatory drug Pomalidomide (POM), a Thalidomide derivative, was examined in the Drosophila LRRK2WD40 genetic model of PD. Mutant and wild type flies received increasing POM doses (1, 0.5, 0.25 mM) through their diet from day 1 post eclosion, until postnatal day (PN) 7 or 14, when POM's actions were evaluated by quantifying changes in climbing behavior as a measure of motor performance, the number of brain dopaminergic neurons and T-bars, mitochondria integrity. LRRK2WD40 flies displayed a spontaneous age-related impairment of climbing activity, and POM significantly and dose-dependently improved climbing performance both at PN 7 and PN 14. LRRK2WD40 fly motor disability was underpinned by a progressive loss of dopaminergic neurons in posterior clusters of the protocerebrum, which are involved in the control of locomotion, by a low number of T-bars density in the presynaptic bouton active zones. POM treatment fully rescued the cell loss in all posterior clusters at PN 7 and PN 14 and significantly increased the T-bars density. Moreover, several damaged mitochondria with dilated cristae were observed in LRRK2WD40 flies treated with vehicle but not following POM. This study demonstrates the neuroprotective activity of the immunomodulatory agent POM in a genetic model of PD. POM is an FDA-approved clinically available and well-tolerated drug used for the treatment of multiple myeloma. If further validated in mammalian models of PD, POM could rapidly be clinically tested in humans.

Beneficial effects of curtailing immune susceptibility in an Alzheimer's disease model.

BACKGROUND: Currently, there are no effective therapeutic options for Alzheimer's disease, the most common, multifactorial form of dementia, characterized by anomalous amyloid accumulation in the brain. Growing evidence points to neuroinflammation as a major promoter of AD. We have previously shown that the proinflammatory cytokine TNFSF10 fuels AD neuroinflammation, and that its immunoneutralization results in improved cognition in the 3xTg-AD mouse. METHODS: Here, we hypothesize that inflammatory hallmarks of AD might parallel with central and peripheral immune response dysfunction. To verify such hypothesis, we used a triple transgenic mouse model of AD. 3xTg-AD mice were treated for 12 months with an anti-TNFSF10 antibody, and thereafter immune/inflammatory markers including COX2, iNOS, IL-1ß and TNF-α, CD3, GITR, and FoxP3 (markers of regulatory T cells) were measured in the spleen as well as in the hippocampus. RESULTS: Spleens displayed accumulation of amyloid-ß1-42 (Aß1-42), as well as high expression of Treg cell markers FoxP3 and GITR, in parallel with the increased levels of inflammatory markers COX2, iNOS, IL-1ß and TNF-α, and blunted IL-10 expression. Moreover, CD3 expression was increased in the hippocampus, consistently with FoxP3 and GITR. After chronic treatment of 3xTg-AD mice with an anti-TNFSF10 antibody, splenic FoxP3, GITR, and the above-mentioned inflammatory markers expression was restored to basal levels, while expression of IL-10 was increased. A similar picture was observed in the hippocampus. Such improvement of peripheral and CNS inflammatory/immune response was associated with decreased microglial activity in terms of TNFα production, as well as decreased expression of both amyloid and phosphorylated tau protein in the hippocampus of treated 3xTg-AD mice. Interestingly, we also reported an increased expression of both CD3 and FoxP3, in sections from human AD brain. CONCLUSIONS: We suggest that neuroinflammation in the brain of 3xTg-AD mice triggered by TNFSF10 might result in a more general overshooting of the immune response. Treatment with an anti-TNFSF10 antibody blunted inflammatory processes both in the spleen and hippocampus. These data confirm the detrimental role of TNFSF10 in neurodegeneration, and corroborate the hypothesis of the anti-TNFSF10 strategy as a potential treatment to improve outcomes in AD.

Immunomodulatory drugs alleviate l-dopa-induced dyskinesia in a rat model of Parkinson's disease.

BACKGROUND: Thalidomide and closely related analogues are used clinically for their immunomodulatory and antiangiogenic properties mediated by the inhibition of the proinflammatory cytokine tumor necrosis factor α. Neuroinflammation and angiogenesis contribute to classical neuronal mechanisms underpinning the pathophysiology of l-dopa-induced dyskinesia, a motor complication associated with l-dopa therapy in Parkinson's disease. The efficacy of thalidomide and the more potent derivative 3,6'-dithiothalidomide on dyskinesia was tested in the 6-hydroxydopamine Parkinson's disease model. METHODS: Three weeks after 6-hydroxydopamine infusion, rats received 10 days of treatment with l-dopa plus benserazide (6 mg/kg each) and thalidomide (70 mg/kg) or 3,6'-dithiothalidomide (56 mg/kg), and dyskinesia and contralateral turning were recorded daily. Rats were euthanized 1 hour after the last l-dopa injection, and levels of tumor necrosis factor-α, interleukin-10, OX-42, vimentin, and vascular endothelial growth factor immunoreactivity were measured in their striatum and substantia nigra reticulata to evaluate neuroinflammation and angiogenesis. Striatal levels of GLUR1 were measured as a l-dopa-induced postsynaptic change that is under tumor necrosis factor-α control. RESULTS: Thalidomide and 3,6'-dithiothalidomide significantly attenuated the severity of l-dopa-induced dyskinesia while not affecting contralateral turning. Moreover, both compounds inhibited the l-dopa-induced microgliosis and excessive tumor necrosis factor-α in the striatum and substantia nigra reticulata, while restoring physiological levels of the anti-inflammatory cytokine interleukin-10. l-Dopa-induced angiogenesis was inhibited in both basal ganglia nuclei, and l-dopa-induced GLUR1 overexpression in the dorsolateral striatum was restored to normal levels. CONCLUSIONS: These data suggest that decreasing tumor necrosis factor-α levels may be useful to reduce the appearance of dyskinesia, and thalidomide, and more potent derivatives may provide an effective therapeutic approach to dyskinesia. © 2019 International Parkinson and Movement Disorder Society.

Neuroinflammation in L-DOPA-induced dyskinesia: beyond the immune function.

Neuroinflammation is a main component of Parkinson's disease (PD) neuropathology, where unremitting reactive microglia and microglia-secreted soluble molecules such as cytokines, contribute to the neurodegenerative process as part of an aberrant immune reaction. Besides, pro-inflammatory cytokines, predominantly TNF-α, play an important neuromodulatory role in the healthy and diseased brain, being involved in neurotransmitter metabolism, synaptic scaling and brain plasticity. Recent preclinical studies have evidenced an exacerbated neuroinflammatory reaction in the striatum of parkinsonian rats that developed dyskinetic responses following L-DOPA administration. These findings prompted investigation of non-neuronal mechanisms of L-DOPA-induced dyskinesia (LID) involving glial cells and glial-secreted soluble molecules. Hence, besides the classical mechanisms of LID that include abnormal corticostriatal neurotransmission and maladaptive changes in striatal medium spiny neurons (MSNs), here we review studies supporting a role of striatal neuroinflammation in the development of LID, with a focus on microglia and the pro-inflammatory cytokine TNF-α. Moreover, we discuss several mechanisms that have been involved in the development of LID, which are directly or indirectly under the control of TNF-α, and might be abnormally affected by its chronic overproduction and release by microglia in PD. It is proposed that TNF-α may contribute to the altered neuronal responses occurring in LID by targeting receptor trafficking and function in MSNs, but also dopamine synthesis in preserved dopaminergic terminals and serotonin metabolism in serotonergic neurons. Therapeutic approaches specifically targeting glial-secreted cytokines may represent a novel target for preventing or treating LID.

l-DOPA-induced dyskinesia and neuroinflammation: do microglia and astrocytes play a role?

In Parkinson's disease (PD), l-DOPA therapy leads to the emergence of motor complications including l-DOPA-induced dyskinesia (LID). LID relies on a sequence of pre- and postsynaptic neuronal events, leading to abnormal corticostriatal neurotransmission and maladaptive changes in striatal projection neurons. In recent years, additional non-neuronal mechanisms have been proposed to contribute to LID. Among these mechanisms, considerable attention has been focused on l-DOPA-induced inflammatory responses. Microglia and astrocytes are the main actors in neuroinflammatory responses, and their double role at the interface between immune and neurophysiological responses is starting to be elucidated. Both microglia and astrocytes express a multitude of neurotransmitter receptors and via the release of several soluble molecules modulate synaptic function in neuronal networks. Here we review preclinical and clinical evidence of glial overactivation by l-DOPA, supporting a role of microglia and astrocytes in the development of LID. We propose that in PD, chronically and abnormally activated microglia and astrocytes lead to an aberrant neuron-glia communication, which affect synaptic activity and neuroplasticity contributing to the development of LID.

Differential induction of dyskinesia and neuroinflammation by pulsatile versus continuous l-DOPA delivery in the 6-OHDA model of Parkinson's disease.

Neuroinflammation is associated with l-DOPA treatment in Parkinson's disease (PD), suggesting a role in l-DOPA-induced dyskinesia (LID), however it is unclear whether increased inflammation is specifically related to the dyskinetic outcome of l-DOPA treatment. Diversely from oral l-DOPA, continuous intrajejunal l-DOPA infusion is associated with very low dyskinetic outcome in PD patients. We reproduced these regimens of administration in 6-OHDA-lesioned hemiparkinsonian rats, where dyskinetic responses and striatal neuroinflammation induced by chronic pulsatile (DOPAp) or continuous (DOPAc) l-DOPA were compared. Moreover, we investigated the contribution of a peripheral inflammatory challenge with lipopolysaccharide (LPS), to DOPAp-induced dyskinetic and neuroinflammatory responses. Rats 6-OHDA-infused in the medial forebrain bundle received two weeks treatment with DOPAp, DOPAc via subcutaneous osmotic minipumps, or DOPAp followed by DOPAc. l-DOPA plasma levels were measured in all experimental groups. An independent group of rats received one peripheral dose of LPS 24h before DOPAp treatment. Abnormal involuntary movements (AIMs) were evaluated as a rat model of LID. Immunoreactivity (IR) for OX-42, microglial and neuronal TNF-α, iNOS and GFAP was quantified in denervated and contralateral striatum. In addition, serum TNF-α was measured. The 6-OHDA denervation induced a mild microgliosis in the striatum two weeks after neurotoxin infusion, and increased TNF-α IR in microglia. Rats receiving the DOPAp treatment developed AIMs and displayed increased striatal OX-42, microglial TNF-α, iNOS and GFAP. Moreover, TNF-α IR was also increased in a subpopulation of striatal neurons. Conversely, DOPAc did not induce AIMs or inflammatory responses in either drug-naïve animals or rats that were previously dyskinetic when exposed to DOPAp. Serum TNF-α was not altered by any l-DOPA treatment. LPS pre-treatment increased the degree of DOPAp-induced AIMs and striatal IR for OX-42, TNF-α, iNOS and GFAP. Altogether the present findings indicate that in the 6-OHDA model, chronic l-DOPA induces striatal inflammatory responses, which however depend upon the administration regimen and the dyskinetic outcome of drug treatment. The potentiation of dyskinetic responses by LPS suggests a reciprocal causal link between neuroinflammation and LID.

Dynamic changes in pro- and anti-inflammatory cytokines in microglia after PPAR-γ agonist neuroprotective treatment in the MPTPp mouse model of progressive Parkinson's disease.

Neuroinflammatory changes play a pivotal role in the progression of Parkinson's disease (PD) pathogenesis. Recent findings have suggested that activated microglia may polarize similarly to peripheral macrophages in the central nervous system (CNS), assuming a pro-inflammatory M1 phenotype or the alternative anti-inflammatory M2 phenotype via cytokine production. A skewed M1 activation over M2 has been related to disease progression in Alzheimer disease, and modulation of microglia polarization may be a therapeutic target for neuroprotection. By using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-probenecid (MPTPp) mouse model of progressive PD, we investigated dynamic changes in the production of pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α and interleukin (IL)-1ß, and anti-inflammatory cytokines, such as transforming growth factor (TGF)-ß and IL-10, within Iba-1-positive cells in the substantia nigra compacta (SNc). In addition, to further characterize changes in the M2 phenotype, we measured CD206 in microglia. Moreover, in order to target microglia polarization, we evaluated the effect of the peroxisome-proliferator-activated receptor (PPAR)-γ agonist rosiglitazone, which has been shown to exert neuroprotective effects on nigral dopaminergic neurons in PD models, and acts as a modulator of cytokine production and phenotype in peripheral macrophages. Chronic treatment with MPTPp induced a progressive degeneration of SNc neurons. The neurotoxin treatment was associated with a gradual increase in both TNF-α and IL-1ß colocalization with Iba-1-positive cells, suggesting an increase in pro-inflammatory microglia. In contrast, TGF-ß colocalization was reduced by the neurotoxin treatment, while IL-10 was mostly unchanged. Administration of rosiglitazone during the full duration of MPTPp treatment reverted both TNF-α and IL-1ß colocalization with Iba-1 to control levels. Moreover, rosiglitazone induced an increase in TGF-ß and IL-10 colocalization compared with the MPTPp treatment. CD206 was gradually reduced by the chronic MPTPp treatment, while rosiglitazone restored control levels, suggesting that M2 anti-inflammatory microglia were stimulated and inflammatory microglia were inhibited by the neuroprotective treatment. The results show that the dopaminergic degeneration was associated with a gradual microglia polarization to the inflammatory over the anti-inflammatory phenotype in a chronic mouse model of PD. Neuroprotective treatment with rosiglitazone modulated microglia polarization, boosting the M2 over the pro-inflammatory phenotype. PPAR-γ agonists may offer a novel approach to neuroprotection, acting as disease-modifying drugs through an immunomodulatory action in the CNS.

Pathophysiological roles for purines: adenosine, caffeine and urate.

The motor symptoms of Parkinson's disease (PD) are primarily due to the degeneration of the dopaminergic neurons in the nigrostriatal pathway. However, several other brain areas and neurotransmitters other than dopamine such as noradrenaline, 5-hydroxytryptamine and acetylcholine are affected in the disease. Moreover, adenosine because of the extensive interaction of its receptors with the dopaminergic system has been implicated in the pathophysiology of the disease. Based on the involvement of these non-dopaminergic neurotransmitters in PD and the sometimes severe adverse effects that limit the mainstay use of dopamine-based anti-parkinsonian treatments, recent assessments have called for a broadening of therapeutic options beyond the traditional dopaminergic drug arsenal. In this review we describe the interactions between dopamine and adenosine receptors that underpin the pre-clinical and clinical rationale for pursuing adenosine A(2A) receptor antagonists as symptomatic and potentially neuroprotective treatment of PD. The review will pay particular attention to recent results regarding specific A(2A) receptor-receptor interactions and recent findings identifying urate, the end product of purine metabolism, as a novel prognostic biomarker and candidate neuroprotectant in PD.

Inactivation of neuronal forebrain A receptors protects dopaminergic neurons in a mouse model of Parkinson's disease.

Adenosine A(2A) receptors antagonists produce neuroprotective effects in animal models of Parkinson's disease (PD). As neuroinflammation is involved in PD pathogenesis, both neuronal and glial A(2A) receptors might participate to neuroprotection. We employed complementary pharmacologic and genetic approaches to A(2A) receptor inactivation, in a multiple MPTP mouse model of PD, to investigate the cellular basis of neuroprotection by A(2A) antagonism. MPTP.HCl (20 mg/kg daily for 4 days) was administered in mice treated with the A(2A) antagonist SCH58261, or in conditional knockout mice lacking A(2A) receptors on forebrain neurons (fbnA(2A)KO mice). MPTP-induced partial loss of dopamine neurons in substantia nigra pars compacta (SNc) and striatum (Str), associated with increased astroglial and microglial immunoreactivity in these areas. Astroglia was similarly activated 1, 3, and 7 days after MPTP administration, whereas maximal microglial reactivity was detected on day 1, returning to baseline 7 days after MPTP administration. SCH58261 attenuated dopamine cell loss and gliosis in SNc and Str. Selective depletion of A(2A) receptors in fbnA(2A)KO mice completely prevented MPTP-induced dopamine neuron degeneration and gliosis in SNc, and partially counteracted gliosis in Str. Results provide evidence of a primary role played by neuronal A(2A) receptors in neuroprotective effects of A(2A) antagonists in a multiple MPTP injections model of PD. With the symptomatic antiparkinsonian potential of several A(2A) receptor antagonists being pursued in clinical trials, this study adds to the rationale for broader clinical benefit and use of these drugs early in the treatment of PD.

Adenosine A2A receptors and Parkinson's disease.

The drug treatment of Parkinson's disease (PD) is accompanied by a loss of drug efficacy, the onset of motor complications, lack of effect on non-motor symptoms, and a failure to modify disease progression. As a consequence, novel approaches to therapy are sought, and adenosine A(2A) receptors (A(2A)ARs) provide a viable target. A(2A)ARs are highly localized to the basal ganglia and specifically to the indirect output pathway, which is highly important in the control of voluntary movement. A(2A)AR antagonists can modulate gamma-aminobutyric acid (GABA) and glutamate release in basal ganglia and other key neurotransmitters that modulate motor activity. In both rodent and primate models of PD, A(2A)AR antagonists produce alterations in motor behavior, either alone or in combination with dopaminergic drugs, which suggest that they will be effective in the symptomatic treatment of PD. In clinical trials, the A(2A)AR antagonist istradefylline reduces "off" time in patients with PD receiving optimal dopaminergic therapy. However, these effects have proven difficult to demonstrate on a consistent basis, and further clinical trials are required to establish the clinical utility of this drug class. Based on preclinical studies, A(2A)AR antagonists may also be neuroprotective and have utility in the treatment of neuropsychiatric disorders. We are only now starting to explore the range of potential uses of A(2A)AR antagonists in central nervous system disorders, and their full utility is still to be uncovered.

Potentiation of amphetamine-mediated responses in caffeine-sensitized rats involves modifications in A2A receptors and zif-268 mRNAs in striatal neurons.

Subchronic intermittent administration of caffeine induces sensitization of motor behaviour and promotes cross-sensitization to amphetamine motor activity. In order to evaluate the possible mechanisms at the basis of these effects, modifications in A(2A) receptor and zif-268 mRNAs were evaluated in rats subchronically treated with caffeine (15 mg/kg i.p.) and challenged with caffeine (15 mg/kg i.p.) or amphetamine (0.5, 1 mg/kg s.c.) 3 days after discontinuation of treatment. Results showed that the sensitized motor response to caffeine was associated with a decrease of adenosine A(2A) receptor and zif-268 mRNA levels in the striatum and nucleus accumbens, whereas cross-sensitization to amphetamine was linked to a more pronounced increase of zif-268 mRNA levels in the striatum, but not in the nucleus accumbens. Single-cell analysis showed that zif-268 mRNA modifications occurred in Enk(+) striatopallidal neurons after acute or subchronic treatment with caffeine and in Enk(-) striatonigral neurons after acute amphetamine administration. Potentiation of amphetamine effects was not associated with modifications of amphetamine-induced dopamine release in nucleus accumbens in caffeine-pretreated rats compared with vehicle-pretreated rats. Results demonstrate that sensitization to caffeine and cross-sensitization to amphetamine are associated with post-synaptic neuroadaptive changes in selective neuronal populations of the striatum.

Adenosine A2A and dopamine receptor interactions in basal ganglia of dopamine denervated rats.

In the unilateral 6-hydroxydopamine-lesioned rat model of Parkinson's disease, blockade of A2A receptors facilitates L-dopa-induced turning behavior by antagonism of A2A transmission, which is increased after dopamine depletion. After long-term intermittent administration of doses that produced the same effect on turning behavior, SCH 58261 (5 mg/kg) + L-dopa (3 mg/kg) induced a stable turning behavior, whereas L-dopa (6 mg/kg) alone induced a sensitized turning behavior. Behavioral studies were correlated to changes in dynorphin and enkephalin mRNAs in the striatum and in glutamic acid decarboxylase 67 (GAD67) mRNA in the striatum, globus pallidus, and substantia nigra. The expression of dynorphin and, to a lesser extent, enkephalin mRNAs was increased in the lesioned striatum of rats that received long-term L-dopa treatment but not in rats that received long-term SCH 58261 + L-dopa treatment. Similarly, GAD67 mRNA was increased in the striatum and globus pallidus by long-term L-dopa administration but not by long-term SCH 58261 + L-dopa administration. GAD67 mRNA was strongly reduced in the lesioned substantia nigra after long-term L-dopa treatment, whereas the reduction of GAD67 mRNA was less marked after SCH 58261 + L-dopa treatment. By increasing L-dopa turning behavior, A2A receptor antagonism allows the utilization of doses of L-dopa that do not produce sensitization of turning behavior, an effect correlated with the dyskinetic potential of dopamine agonist drugs. Moreover, the combination of SCH 58261 + L-dopa produces little or no change in the striatal, pallidal, and nigral expression of markers correlated with dopamine agonist dyskinetic potential.

Blockade of A2A receptors plus l-DOPA after nigrostriatal lesion results in GAD67 mRNA changes different from l-DOPA alone in the rat globus pallidus and substantia nigra reticulata.

Studies in animal models of Parkinson's disease (PD) suggest the potential utility of adenosine A(2A) antagonists in the treatment of this disease. In the present study, unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rats received chronic intermittent treatment with the adenosine A(2A) antagonist SCH58261 (5 mg/kg) plus l-DOPA (3 mg/kg) or l-DOPA (6 mg/kg) alone, at doses producing the same intensity of contralateral turning on first administration. Three days after discontinuation of treatments, GABA synthesizing enzyme glutamic acid decarboxylase (GAD67) mRNA was evaluated at cellular level in the globus pallidus (GP) and substantia nigra pars reticulata (SNr) by in situ hybridization. 6-OHDA lesion significantly increased GAD67 mRNA levels in both the GP and SNr ipsilateral to the lesion. Chronic l-DOPA (6 mg/kg), in contrast to SCH58261 plus l-DOPA (3 mg/kg), produced a sensitized contralateral turning indicative of dyskinetic potential and further increased GAD67 mRNA in the GP. In the SNr, a significant decrease in GAD67 mRNA was observed after either treatments. However, while l-DOPA (6 mg/kg) decreased SNr GAD67 mRNA below the intact side, SCH58261 plus l-DOPA (3 mg/kg) brought GAD67 mRNA to the same level of the intact SNr. l-DOPA (3 mg/kg) or SCH58261 (5 mg/kg) alone failed to modify GAD67 mRNA. Results suggest that an increase in GAD67 mRNA in GP and a decrease in SNr might underlie dyskinetic movements induced by chronic l-DOPA. In contrast, the lack of GAD67 mRNA changes in the GP and a less marked inhibition of SNr might correlate with the absence of dyskinetic potential observed after SCH58261 plus l-DOPA.

Differential regulation of GAD67, enkephalin and dynorphin mRNAs by chronic-intermittent L-dopa and A2A receptor blockade plus L-dopa in dopamine-denervated rats.

Adenosine A2A receptor antagonists have been proposed as an effective therapy in the treatment of Parkinson's disease. In the present study, we compared the modifications on striatal glutamate decarboxylase (GAD67), enkephalin, and dynorphin mRNA levels produced by a chronic-intermittent administration of L-3,4-dihydroxyphenyl-alanine (L-dopa) (6 mg/kg) with those produced by the adenosine A2A receptor antagonist SCH 58261 (5 mg/kg) plus L-dopa (3 mg/kg) in unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rats. As previously reported, L-dopa (6 mg/kg) and SCH 58261 (5 mg/kg) plus L-dopa (3 mg/kg) produced the same degree of turning behavior after the first administration. However, while L-dopa (6 mg/kg) induced a sensitized turning behavior response during the course of the treatment, which indicated a dyskinetic potential, SCH 58261 (5 mg/kg) plus L-dopa (3 mg/kg) produced a stable turning behavior response, which was predictive of absence of dyskinetic side effects. Unilateral 6-OHDA lesion produced an elevation in striatal GAD67 and enkephalin mRNA levels and to a decrease in dynorphin mRNA levels. Chronic-intermittent L-dopa (6 mg/kg) treatment increased the striatal levels of GAD67, dynorphin, and enkephalin mRNA in the lesioned side as compared to the vehicle treatment. Chronic-intermittent SCH 58261 (5 mg/kg) plus L-dopa (3 mg/kg) as well as L-dopa (3 mg/kg) or SCH 58261 (5 mg/kg) alone did not produce any significant modification in GAD67, dynorphin, or enkephalin mRNA levels in the lesioned striatum as compared to the striatum of vehicle-treated rats. The results show that combined SCH 58261 plus L-dopa did not produce long-term changes in markers of striatal efferent neurons activity and suggest that the lack of modifications in GAD67 and dynorphin mRNA after SCH 58261 plus L-dopa might correlate with the lack of turning behavior sensitization which predicts drug dyskinetic potential.