Nigrostriatal degeneration determines dynamics of glial inflammatory and phagocytic activity.

Glial cells are key players in the initiation of innate immunity in neurodegeneration. Upon damage, they switch their basal activation state and acquire new functions in a context and time-dependent manner. Since modulation of neuroinflammation is becoming an interesting approach for the treatment of neurodegenerative diseases, it is crucial to understand the specific contribution of these cells to the inflammatory reaction and to select experimental models that recapitulate what occurs in the human disease. Previously, we have characterized a region-specific activation pattern of CD11b+ cells and astrocytes in the α-synuclein overexpression mouse model of Parkinson´s disease (PD). In this study we hypothesized that the time and the intensity of dopaminergic neuronal death would promote different glial activation states. Dopaminergic degeneration was induced with two administration regimens of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), subacute (sMPTP) and chronic (cMPTP). Our results show that in the sMPTP mouse model, the pro-inflammatory phenotype of striatal CD11b+ cells was counteracted by an anti-inflammatory astrocytic profile. In the midbrain the roles were inverted, CD11b+ cells exhibited an anti-inflammatory profile and astrocytes were pro-inflammatory. The overall response generated resulted in decreased CD4 T cell infiltration in both regions. Chronic MPTP exposure resulted in a mild and prolonged neuronal degeneration that generated a pro-inflammatory response and increased CD4 T cell infiltration in both regions. At the onset of the neurodegenerative process, microglia and astrocytes cooperated in the removal of dopaminergic terminals. With time, only microglia maintained the phagocytic activity. In the ventral midbrain, astrocytes were the main phagocytic mediators at early stages of degeneration while microglia were the major phagocytic cells in the chronic state. In this scenario, we questioned which activation pattern recapitulates better the features of glial activation in PD. Glial activation in the cMPTP mouse model reflects many pathways of their corresponding counterparts in the human brain with advanced PD. Altogether, our results point toward a context-dependent cooperativity of microglia/myeloid cells and astrocytes in response to neuronal damage and the relevance of selecting the right experimental models for the study of neuroinflammation.

Microglia and astrocyte activation is region-dependent in the α-synuclein mouse model of Parkinson's disease.

Inflammation is a common feature in neurodegenerative diseases that contributes to neuronal loss. Previously, we demonstrated that the basal inflammatory tone differed between brain regions and, consequently, the reaction generated to a pro-inflammatory stimulus was different. In this study, we assessed the innate immune reaction in the midbrain and in the striatum using an experimental model of Parkinson's disease. An adeno-associated virus serotype 9 expressing the α-synuclein and mCherry genes or the mCherry gene was administered into the substantia nigra. Myeloid cells (CD11b+ ) and astrocytes (ACSA2+ ) were purified from the midbrain and striatum for bulk RNA sequencing. In the parkinsonian midbrain, CD11b+ cells presented a unique anti-inflammatory transcriptomic profile that differed from degenerative microglia signatures described in experimental models for other neurodegenerative conditions. By contrast, striatal CD11b+ cells showed a pro-inflammatory state and were similar to disease-associated microglia. In the midbrain, a prominent increase of infiltrated monocytes/macrophages was observed and, together with microglia, participated actively in the phagocytosis of dopaminergic neuronal bodies. Although striatal microglia presented a phagocytic transcriptomic profile, morphology and cell density was preserved and no active phagocytosis was detected. Interestingly, astrocytes presented a pro-inflammatory fingerprint in the midbrain and a low number of differentially displayed transcripts in the striatum. During α-synuclein-dependent degeneration, microglia and astrocytes experience context-dependent activation states with a different contribution to the inflammatory reaction. Our results point towards the relevance of selecting appropriate cell targets to design neuroprotective strategies aimed to modulate the innate immune system during the active phase of dopaminergic degeneration.

Expression of Endothelial NOX5 Alters the Integrity of the Blood-Brain Barrier and Causes Loss of Memory in Aging Mice.

Blood-Brain barrier (BBB) disruption is a hallmark of central nervous system (CNS) dysfunction, and oxidative stress is one of the molecular mechanisms that may underlie this process. NADPH oxidases (NOX) are involved in oxidative stress-mediated vascular dysfunction and participate in the pathophysiology of its target organs. The NADPH oxidase 5 (NOX5) isoform is absent in rodents, and although little is known about the role it may play in disrupting the BBB, it has recently been implicated in experimental stroke. Our aim was to investigate the role of NADPH oxidase 5 (NOX5) in promoting vascular alterations and to identify its impact on the cognitive status of aged mice. No differences were detected in the arterial blood pressure or body weight between knock-in mice expressing endothelial NOX5 and the control mice. The Morris water maze test showed memory impairments in the aged knock-in mice expressing NOX5 compared with their control littermates. For assessing the BBB integrity, we studied the protein expression of two tight junction (TJ) proteins: Zonula occludens-1 (ZO-1) and occludin. Compared to the control animals, Aged NOX5 mice exhibited reduced levels of both proteins, demonstrating an alteration of the BBB integrity. Our data indicate that vascular NOX5 may favor behavioral changes with aging through oxidative stress-mediated BBB breakdown.

Understanding the Potential Role of Sirtuin 2 on Aging: Consequences of SIRT2.3 Overexpression in Senescence.

Sirtuin 2 (SIRT2) has been associated to aging and age-related pathologies. Specifically, an age-dependent accumulation of isoform 3 of SIRT2 in the CNS has been demonstrated; however, no study has addressed the behavioral or molecular consequences that this could have on aging. In the present study, we have designed an adeno-associated virus vector (AAV-CAG-Sirt2.3-eGFP) for the overexpression of SIRT2.3 in the hippocampus of 2 month-old SAMR1 and SAMP8 mice. Our results show that the specific overexpression of this isoform does not induce significant behavioral or molecular effects at short or long term in the control strain. Only a tendency towards a worsening in the performance in acquisition phase of the Morris Water Maze was found in SAMP8 mice, together with a significant increase in the pro-inflammatory cytokine Il-1ß. These results suggest that the age-related increase of SIRT2.3 found in the brain is not responsible for induction or prevention of senescence. Nevertheless, in combination with other risk factors, it could contribute to the progression of age-related processes. Understanding the specific role of SIRT2 on aging and the underlying molecular mechanisms is essential to design new and more successful therapies for the treatment of age-related diseases.

Gut microbial dysbiosis after traumatic brain injury modulates the immune response and impairs neurogenesis.

The influence of the gut microbiota on traumatic brain injury (TBI) is presently unknown. This knowledge gap is of paramount clinical significance as TBI patients are highly susceptible to alterations in the gut microbiota by antibiotic exposure. Antibiotic-induced gut microbial dysbiosis established prior to TBI significantly worsened neuronal loss and reduced microglia activation in the injured hippocampus with concomitant changes in fear memory response. Importantly, antibiotic exposure for 1 week after TBI reduced cortical infiltration of Ly6Chigh monocytes, increased microglial pro-inflammatory markers, and decreased T lymphocyte infiltration, which persisted through 1 month post-injury. Moreover, microbial dysbiosis was associated with reduced neurogenesis in the dentate gyrus 1 week after TBI. By 3 months after injury (11 weeks after discontinuation of the antibiotics), we observed increased microglial proliferation, increased hippocampal neuronal loss, and modulation of fear memory response. These data demonstrate that antibiotic-induced gut microbial dysbiosis after TBI impacts neuroinflammation, neurogenesis, and fear memory and implicate gut microbial modulation as a potential therapeutic intervention for TBI.

CB2 Receptors and Neuron-Glia Interactions Modulate Neurotoxicity Generated by MAGL Inhibition.

Monoacylglycerol lipase inhibition (MAGL) has emerged as an interesting therapeutic target for neurodegenerative disease treatment due to its ability to modulate the endocannabinoid system and to prevent the production of proinflammatory mediators. To obtain a beneficial response, it is necessary to understand how this inhibition affects the neuron-glia crosstalk and neuron viability. In this study, the effect of MAGL inhibition by KML29 was evaluated in two types of rat cortical primary cultures; mixed cultures, including neuron and glial cells, and neuron-enriched cultures. The risk of neuronal death was estimated by longitudinal survival analysis. The spontaneous neuronal risk of death in culture was higher in the absence of glial cells, a process that was enhanced by KML29 addition. In contrast, neuronal survival was not compromised by MAGL inhibition in the presence of glial cells. Blockade of cannabinoid type 2 (CB2) receptors expressed mainly by microglial cells did not affect the spontaneous neuronal death risk but decreased neuronal survival when KML29 was added. Modulation of cannabinoid type 1 (CB1) receptors did not affect neuronal survival. Our results show that neuron-glia interactions are essential for neuronal survival. CB2 receptors play a key role in these protective interactions when neurons are exposed to toxic conditions.

Midbrain microglia mediate a specific immunosuppressive response under inflammatory conditions.

BACKGROUND: Inflammation is a critical process for the progression of neuronal death in neurodegenerative disorders. Microglia play a central role in neuroinflammation and may affect neuron vulnerability. Next generation sequencing has shown the molecular heterogeneity of microglial cells; however, the variability in their response to pathological inputs remains unknown. METHODS: To determine the effect of an inflammatory stimulus on microglial cells, lipopolysaccharide (LPS) was administered peripherally to mice and the inflammatory status of the cortex, hippocampus, midbrain, and striatum was assessed. Microglial activation and interaction with the immune system were analyzed in single cell suspensions obtained from the different brain regions by fluorescence-activated cell sorting, next generation RNA sequencing, real-time PCR, and immunohistochemical techniques. Antigen-presenting properties of microglia were evaluated by the ability of isolated cells to induce a clonal expansion of CD4+ T cells purified from OT-II transgenic mice. RESULTS: Under steady-state conditions, the midbrain presented a high immune-alert state characterized by the presence of two unique microglial subpopulations, one expressing the major histocompatibility complex class II (MHC-II) and acting as antigen-presenting cells and another expressing the toll-like receptor 4 (TLR4), and by the presence of a higher proportion of infiltrating CD4+ T cells. This state was not detected in the cortex, hippocampus, or striatum. Systemic LPS administration induced a general increase in classic pro-inflammatory cytokines, in co-inhibitory programmed death ligand 1 (PD-L1), and in cytotoxic T lymphocyte antigen 4 (CTLA-4) receptors, as well as a decrease in infiltrating effector T cells in all brain regions. Interestingly, a specific immune-suppressive response was observed in the midbrain which was characterized by the downregulation of MHC-II microglial expression, the upregulation of the anti-inflammatory cytokines IL10 and TGFß, and the increase in infiltrating regulatory T cells. CONCLUSIONS: These data show that the midbrain presents a high immune-alert state under steady-state conditions that elicits a specific immune-suppressive response when exposed to an inflammatory stimulus. This specific inflammatory tone and response may have an impact in neuronal viability.

Dopamine Receptor D3 Expression Is Altered in CD4+ T-Cells From Parkinson's Disease Patients and Its Pharmacologic Inhibition Attenuates the Motor Impairment in a Mouse Model.

Neuroinflammation constitutes a fundamental process involved in Parkinson's disease (PD). Microglial cells play a central role in the outcome of neuroinflammation and consequent neurodegeneration of dopaminergic neurons in the substantia nigra. Current evidence indicates that CD4+ T-cells infiltrate the brain in PD, where they play a critical role determining the functional phenotype of microglia, thus regulating the progression of the disease. We previously demonstrated that mice bearing dopamine receptor D3 (DRD3)-deficient CD4+ T-cells are completely refractory to neuroinflammation and consequent neurodegeneration induced by the administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In this study we aimed to determine whether DRD3-signalling is altered in peripheral blood CD4+ T-cells obtained from PD patients in comparison to healthy controls (HC). Furthermore, we evaluated the therapeutic potential of targeting DRD3 confined to CD4+ T-cells by inducing the pharmacologic antagonism or the transcriptional inhibition of DRD3-signalling in a mouse model of PD induced by the chronic administration of MPTP and probenecid (MPTPp). In vitro analyses performed in human cells showed that the frequency of peripheral blood Th1 and Th17 cells, two phenotypes favoured by DRD3-signalling, were significantly increased in PD patients. Moreover, naïve CD4+ T-cells obtained from PD patients displayed a significant higher Th1-biased differentiation in comparison with those naïve CD4+ T-cells obtained from HC. Nevertheless, DRD3 expression was selectively reduced in CD4+ T-cells obtained from PD patients. The results obtained from in vivo experiments performed in mice show that the transference of CD4+ T-cells treated ex vivo with the DRD3-selective antagonist PG01037 into MPTPp-mice resulted in a significant reduction of motor impairment, although without significant effect in neurodegeneration. Conversely, the transference of CD4+ T-cells transduced ex vivo with retroviral particles codifying for an shRNA for DRD3 into MPTPp-mice had no effects neither in motor impairment nor in neurodegeneration. Notably, the systemic antagonism of DRD3 significantly reduced both motor impairment and neurodegeneration in MPTPp mice. Our findings show a selective alteration of DRD3-signalling in CD4+ T-cells from PD patients and indicate that the selective DRD3-antagonism in this subset of lymphocytes exerts a therapeutic effect in parkinsonian animals dampening motor impairment.

Cannabinoid pharmacology/therapeutics in chronic degenerative disorders affecting the central nervous system.

The endocannabinoid system (ECS) exerts a modulatory effect of important functions such as neurotransmission, glial activation, oxidative stress, or protein homeostasis. Dysregulation of these cellular processes is a common neuropathological hallmark in aging and in neurodegenerative diseases of the central nervous system (CNS). The broad spectrum of actions of cannabinoids allows targeting different aspects of these multifactorial diseases. In this review, we examine the therapeutic potential of the ECS for the treatment of chronic neurodegenerative diseases of the CNS focusing on Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. First, we describe the localization of the molecular components of the ECS and how they are altered under neurodegenerative conditions, either contributing to or protecting cells from degeneration. Second, we address recent advances in the modulation of the ECS using experimental models through different strategies including the direct targeting of cannabinoid receptors with agonists or antagonists, increasing the endocannabinoid tone by the inhibition of endocannabinoid hydrolysis, and activation of cannabinoid receptor-independent effects. Preclinical evidence indicates that cannabinoid pharmacology is complex but supports the therapeutic potential of targeting the ECS. Third, we review the clinical evidence and discuss the future perspectives on how to bridge human and animal studies to develop cannabinoid-based therapies for each neurodegenerative disorder. Finally, we summarize the most relevant opportunities of cannabinoid pharmacology related to each disease and the multiple unexplored pathways in cannabinoid pharmacology that could be useful for the treatment of neurodegenerative diseases.

Stereological Estimates of Glutamatergic, GABAergic, and Cholinergic Neurons in the Pedunculopontine and Laterodorsal Tegmental Nuclei in the Rat.

The pedunculopontine tegmental nucleus (PPN) and laterodorsal tegmental nucleus (LDT) are functionally associated brainstem structures implicated in behavioral state control and sensorimotor integration. The PPN is also involved in gait and posture, while the LDT plays a role in reward. Both nuclei comprise characteristic cholinergic neurons intermingled with glutamatergic and GABAergic cells whose absolute numbers in the rat have been only partly established. Here we sought to determine the complete phenotypical profile of each nucleus to investigate potential differences between them. Counts were obtained using stereological methods after the simultaneous visualization of cholinergic and either glutamatergic or GABAergic cells. The two isoforms of glutamic acid decarboxylase (GAD), GAD65 and GAD67, were separately analyzed. Dual in situ hybridization revealed coexpression of GAD65 and GAD67 mRNAs in ∼90% of GAD-positive cells in both nuclei; thus, the estimated mean numbers of (1) cholinergic, (2) glutamatergic, and (3) GABAergic cells in PPN and LDT, respectively, were (1) 3,360 and 3,650; (2) 5,910 and 5,190; and (3) 4,439 and 7,599. These data reveal significant differences between PPN and LDT in their relative phenotypical composition, which may underlie some of the functional differences observed between them. The estimation of glutamatergic cells was significantly higher in the caudal PPN, supporting the reported functional rostrocaudal segregation in this nucleus. Finally, a small subset of cholinergic neurons (8% in PPN and 5% in LDT) also expressed the glutamatergic marker Vglut2, providing anatomical evidence for a potential corelease of transmitters at specific target areas.

The expression of cannabinoid type 1 receptor and 2-arachidonoyl glycerol synthesizing/degrading enzymes is altered in basal ganglia during the active phase of levodopa-induced dyskinesia.

Management of levodopa-induced dyskinesias (LID) is one of the main challenges in the treatment of Parkinson's disease patients. Mechanisms involved in the appearance of these involuntary movements are not well known but modifications in the activity of different neurotransmitter pathways seem to play an important role. The objective of this study was to determine differences in the expression levels of the endocannabinoid system (ECS) elements that would support a role in LID. The basal ganglia nuclei, putamen, external segment of the globus pallidus (GPe), internal segment of the globus pallidus (GPi), subthalamic nucleus (STN) and substantia nigra (SN) were dissected out from cryostat sections obtained from two groups of parkinsonian monkeys treated with levodopa to induce dyskinesias. One group of dyskinetic animals was sacrificed under the effect of levodopa, during the active phase of LID, and the other group 24 h after the last levodopa dose (OFF levodopa). Biochemical analysis by real-time PCR for ECS elements was performed. CB1 receptor expression was upregulated in the putamen, GPe and STN during the active phase of dyskinesia and downregulated in the same nuclei and in the SN when dyskinetic animals were OFF levodopa. Changes in the 2-arachidonoyl glycerol (2-AG) synthesizing/degrading enzymes affecting the pallidal-subthalamic projections in dyskinetic animals OFF levodopa would suggest that 2-AG may play a role in LID. Anandamide (AEA) synthesizing/degrading enzymes were altered specifically in the GPe of untreated parkinsonian monkeys, suggesting that increased AEA levels may be a compensatory mechanism. These results indicate that the expression of the ECS elements is influenced by alterations in dopaminergic neurotransmission. On one hand, changes in CB1 receptor expression and in the 2-AG synthesizing/degrading enzymes suggest that they could be a therapeutic target for the active phase of LID. On the other hand, AEA metabolism could provide a non-dopaminergic target for symptomatic relief. However, further research is needed to unravel the mechanism of action of the ECS and how they could be modulated for a therapeutic purpose.

Impact of Neurodegenerative Diseases on Drug Binding to Brain Tissues: From Animal Models to Human Samples.

Drug efficacy in the central nervous system (CNS) requires an additional step after crossing the blood-brain barrier. Therapeutic agents must reach their targets in the brain to modulate them; thus, the free drug concentration hypothesis is a key parameter for in vivo pharmacology. Here, we report the impact of neurodegeneration (Alzheimer's disease (AD) and Parkinson's disease (PD) compared with healthy controls) on the binding of 10 known drugs to postmortem brain tissues from animal models and humans. Unbound drug fractions, for some drugs, are significantly different between healthy and injured brain tissues (AD or PD). In addition, drugs binding to brain tissues from AD and PD animal models do not always recapitulate their binding to the corresponding human injured brain tissues. These results reveal potentially relevant implications for CNS drug discovery.

GPR55: A therapeutic target for Parkinson's disease?

The GPR55 receptor is expressed abundantly in the brain, especially in the striatum, suggesting it might fulfill a role in motor function. Indeed, motor behavior is impaired in mice lacking GPR55, which also display dampened inflammatory responses. Abnormal-cannabidiol (Abn-CBD), a synthetic cannabidiol (CBD) isomer, is a GPR55 agonist that may serve as a therapeutic agent in the treatment of inflammatory diseases. In this study, we explored whether modulating GPR55 could also represent a therapeutic approach for the treatment of Parkinson's disease (PD). The distribution of GPR55 mRNA was first analyzed by in situ hybridization, localizing GPR55 transcripts to neurons in brain nuclei related to movement control, striatum, globus pallidus, subthalamic nucleus, substantia nigra and cortex. Striatal expression of GPR55 was downregulated in parkinsonian conditions. When Abn-CBD and CBD (5 mg/kg) were chronically administered to mice treated over 5 weeks with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and probenecid (MPTPp), Abn-CBD but not CBD prevented MPTPp induced motor impairment. Although Abn-CBD protected dopaminergic cell bodies, it failed to prevent degeneration of the terminals or preserve dopamine levels in the striatum. Both compounds induced morphological changes in microglia that were compatible with an anti-inflammatory phenotype that did not correlate with a neuroprotective activity. The symptomatic relief of Abn-CBD was further studied in the haloperidol-induced catalepsy mouse model. Abn-CBD had an anti-cataleptic effect that was reversed by CBD and PSB1216, a newly synthesized GPR55 antagonist, and indeed, two other GPR55 agonists also displayed anti-cataleptic effects (CID1792197 and CID2440433). These results demonstrate for the first time that activation of GPR55 might be beneficial in combating PD.

Pharmacologic antagonism of dopamine receptor D3 attenuates neurodegeneration and motor impairment in a mouse model of Parkinson's disease.

Neuroinflammation involves the activation of glial cells, which is associated to the progression of neurodegeneration in Parkinson's disease. Recently, we and other researchers demonstrated that dopamine receptor D3 (D3R)-deficient mice are completely refractory to neuroinflammation and consequent neurodegeneration associated to the acute intoxication with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In this study we examined the therapeutic potential and underlying mechanism of a D3R-selective antagonist, PG01037, in mice intoxicated with a chronic regime of administration of MPTP and probenecid (MPTPp). Biodistribution analysis indicated that intraperitoneally administered PG01037 crosses the blood-brain barrier and reaches the highest concentration in the brain 40 min after the injection. Furthermore, the drug was preferentially distributed to the brain in comparison to the plasma. Treatment of MPTPp-intoxicated mice with PG01037 (30 mg/kg, administrated twice a week for five weeks) attenuated the loss of dopaminergic neurons in the substantia nigra pars compacta, as evaluated by stereological analysis, and the loss of striatal dopaminergic terminals, as determined by densitometric analyses of tyrosine hydroxylase and dopamine transporter immunoreactivities. Accordingly, the treatment resulted in significant improvement of motor performance of injured animals. Interestingly, the therapeutic dose of PG01037 exacerbated astrogliosis and resulted in increased ramification density of microglial cells in the striatum of MPTPp-intoxicated mice. Further analyses suggested that D3R expressed in astrocytes favours a beneficial astrogliosis with anti-inflammatory consequences on microglia. Our findings indicate that D3R-antagonism exerts a therapeutic effect in parkinsonian animals by reducing the loss of dopaminergic neurons in the nigrostriatal pathway, alleviating motor impairments and modifying the pro-inflammatory phenotype of glial cells.

Seeing through the smoke: Human and animal studies of cannabis use and endocannabinoid signalling in corticolimbic networks.

Public opinion surrounding the recreational use and therapeutic potential of cannabis is shifting. This review describes new work examining the behavioural and neural effects of cannabis and the endocannabinoid system, highlighting key regions within corticolimbic brain circuits. First, we consider the role of human genetic factors and cannabis strain chemotypic differences in contributing to interindividual variation in the response to cannabinoids, such as THC, and review studies demonstrating that THC-induced impairments in decision-making processes are mediated by actions at prefrontal CB1 receptors. We further describe evidence that signalling through prefrontal or ventral hippocampal CB1 receptors modulates mesolimbic dopamine activity, aberrations of which may contribute to emotional processing deficits in schizophrenia. Lastly, we review studies suggesting that endocannabinoid tone in the amygdala is a critical regulator of anxiety, and report new data showing that FAAH activity is integral to this response. Together, these findings underscore the importance of cannabinoid signalling in the regulation of cognitive and affective behaviours, and encourage further research given their social, political, and therapeutic implications.

Fatty acid amide hydrolase inhibition for the symptomatic relief of Parkinson's disease.

Elements of the endocannabinoid system are strongly expressed in the basal ganglia where they suffer profound rearrangements after dopamine depletion. Modulation of the levels of the endocannabinoid 2-arachidonoyl-glycerol by inhibiting monoacylglycerol lipase alters glial phenotypes and provides neuroprotection in a mouse model of Parkinson's disease. In this study, we assessed whether inhibiting fatty acid amide hydrolase could also provide beneficial effects on the time course of this disease. The fatty acid amide hydrolase inhibitor, URB597, was administered chronically to mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and probenecid (MPTPp) over 5weeks. URB597 (1mg/kg) prevented MPTPp induced motor impairment but it did not preserve the dopamine levels in the nigrostriatal pathway or regulate glial cell activation. The symptomatic relief of URB597 was confirmed in haloperidol-induced catalepsy assays, where its anti-cataleptic effects were both blocked by antagonists of the two cannabinoid receptors (CB1 and CB2), and abolished in animals deficient in these receptors. Other fatty acid amide hydrolase inhibitors, JNJ1661010 and TCF2, also had anti-cataleptic properties. Together, these results demonstrate an effect of fatty acid amide hydrolase inhibition on the motor symptoms of Parkinson's disease in two distinct experimental models that is mediated by cannabinoid receptors.

Neuroprotective Effect of JZL184 in MPP(+)-Treated SH-SY5Y Cells Through CB2 Receptors.

Growing evidence suggests that the endocannabinoid system plays a role in neuroprotection in Parkinson's disease. Recently, we have shown the neuroprotective effect of monoacylglycerol lipase (MAGL) inhibition with JZL184 in the chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model. However, further investigation is needed to determine the neuroprotective mechanisms of the endocannabinoid system on the nigrostriatal pathway. The aim of this work was to investigate whether the neuroprotective effect of JZL184 in mice could be extended to an in vitro cellular model to further understand the mechanism of action of the drug. The SH-SY5Y cell line was selected based on its dopaminergic-like phenotype and its susceptibility to 1-methyl-4-phenylpyridinium iodide (MPP(+)) toxicity. Furthermore, SH-SY5Y cells express both cannabinoid receptors, CB1 and CB2. The present study describes the neuroprotective effect of MAGL inhibition with JZL184 in SH-SY5Y cells treated with MPP(+). The effect of JZL184 in cell survival was blocked by AM630, a CB2 receptor antagonist, and it was mimicked with JWH133, a CB2 receptor agonist. Rimonabant, a CB1 receptor antagonist, did not affect JZL184-induced cell survival. These results demonstrate that the neuroprotective effect of MAGL inhibition with JZL184 described in animal models of Parkinson's disease could be extended to in vitro models such as SH-SY5Y cells treated with MPP(+). This represents a useful tool to study mechanisms of neuroprotection mediated by MAGL inhibition, and we provide evidence for the possible involvement of CB2 receptors in the improvement of cell survival.

Structures for G-Protein-Coupled Receptor Tetramers in Complex with G Proteins.

G-Protein-coupled receptors (GPCRs) were classically described as monomers. We now appreciate that they also function as homo- and hetero-oligomers, for which structural information is lacking. Here, we use available 3D structures and biochemical considerations to present and evaluate experimentally testable structural models for GPCR oligomers and associated G proteins.

Monoacylglycerol lipase inhibitor JZL184 is neuroprotective and alters glial cell phenotype in the chronic MPTP mouse model.

Changes in cannabinoid receptor expression and concentration of endocannabinoids have been described in Parkinson's disease; however, it remains unclear whether they contribute to, or result from, the disease process. To evaluate whether targeting the endocannabinoid system could provide potential benefits in the treatment of the disease, the effect of a monoacylglycerol lipase inhibitor that prevents degradation of 2-arachidonyl-glycerol was tested in mice treated chronically with probenecid and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTPp). Chronic administration of the compound, JZL184 (8 mg/kg), prevented MPTPp-induced motor impairment and preserved the nigrostriatal pathway. Furthermore, none of the hypokinetic effects associated with cannabinoid receptor agonism were observed. In the striatum and substantia nigra pars compacta, MPTPp animals treated with JZL184 exhibited astroglial and microglial phenotypic changes that were accompanied by increases in TGFß messenger RNA expression and in glial cell-derived neurotrophic factor messenger RNA and protein levels. JZL184 induced an increase in ß-catenin translocation to the nucleus, implicating the Wnt/catenin pathway. Together, these results demonstrate a potent neuroprotective effect of JZL184 on the nigrostriatal pathway of parkinsonian animals, likely involving restorative astroglia and microglia activation and the release of neuroprotective and antiinflammatory molecules.

GPR40 activation leads to CREB and ERK phosphorylation in primary cultures of neurons from the mouse CNS and in human neuroblastoma cells.

GPR40, the free fatty acid receptor 1, is expressed strongly in the primate pancreas and brain. While the role of pancreatic GPR40 in glucose homeostasis has been extensively studied, the absence of this G-protein-coupled receptor from the brain of rodents has hampered studies into its role in the central nervous system. However, we found intense GPR40 mRNA expression by in situ hybridization in mouse hippocampal and motor cortex neurons. Furthermore, in a neuroblastoma cell GPR40 was activated by docosahexaenoic acid and selective agonists, yet not by palmitic acid. Significantly, the activation of GPR40 provoked the phosphorylation of the cAMP response element-binding protein, CREB. The receptor was also functional in primary cultures of murine neurons, in which its activation by a selective agonist produced the phosphorylation of CREB and of extracellular signal-regulated kinases, ERK1/2. These results suggest that mice represent a suitable model for elucidating the role of GPR40 in brain function.