Cell-Type Specific Regulation of Cholesterogenesis by CYP46A1 Re-Expression in zQ175 HD Mouse Striatum.

Cholesterol metabolism dysregulation is associated with several neurological disorders. In Huntington's disease (HD), several enzymes involved in cholesterol metabolism are downregulated, among which the neuronal cholesterol 24-hydroxylase, CYP46A1, is of particular interest. The restoration of CYP46A1 expression in striatal neurons of HD mouse models is beneficial for motor behavior, cholesterol metabolism, transcriptomic activity, and alleviates neuropathological hallmarks induced by mHTT. Among the genes regulated after CYP46A1 restoration, those involved in cholesterol synthesis and efflux may explain the positive effect of CYP46A1 on cholesterol precursor metabolites. Since cholesterol homeostasis results from a fine-tuning between neurons and astrocytes, we quantified the distribution of key genes regulating cholesterol metabolism and efflux in astrocytes and neurons using in situ hybridization coupled with S100ß and NeuN immunostaining, respectively. Neuronal expression of CYP46A1 in the striatum of HD zQ175 mice increased key cholesterol synthesis driver genes (Hmgcr, Dhcr24), specifically in neurons. This effect was associated with an increase of the srebp2 transcription factor gene that regulates most of the genes encoding for cholesterol enzymes. However, the cholesterol efflux gene, ApoE, was specifically upregulated in astrocytes by CYP46A1, probably though a paracrine effect. In summary, the neuronal expression of CYP46A1 has a dual and specific effect on neurons and astrocytes, regulating cholesterol metabolism. The neuronal restoration of CYP46A1 in HD paves the way for future strategies to compensate for mHTT toxicity.

A positive allosteric modulator of mGlu4 receptors restores striatal plasticity in an animal model of l-Dopa-induced dyskinesia.

By decreasing glutamate transmission, mGlu4 receptor positive allosteric modulators (mGlu4-PAM), in combination with levodopa (l-DOPA) may restore the synergy between glutamatergic and dopaminergic transmissions, thus maximizing the improvement of motor function in Parkinson's disease (PD). This study aimed to clarify the effects of foliglurax, a selective mGlu4-PAM, on the loss of bidirectional synaptic plasticity associated with l-DOPA-induced dyskinesia (LID). Behavioral assessments compared dyskinesia intensity in 6-hydroxydopamine (6-OHDA)-lesioned rats treated with l-DOPA or l-DOPA plus foliglurax. In slices from the same rats, patch-clamp techniques were used to examine electrophysiological differences in glutamatergic synapses, evaluating the EPSCs mediated by NMDA and AMPA receptors in striatal spiny projection neurons. High-frequency stimulation of corticostriatal fibers was used as long-term potentiation (LTP)-inducing protocol. Conversely, 15 min of low-frequency stimulation was applied to depotentiate LTP. The density of dendritic spines was measured in striatal slices in the same experimental conditions. Our results show that, in corticostriatal slices, foliglurax decreased spontaneous glutamatergic transmission in both sham-operated and 6-OHDA lesioned rats. When co-administered with l-DOPA in 6-OHDA-lesioned rats, foliglurax fully restored dendritic spine density in a dose-dependent manner. Moreover, this co-treatment rescued striatal bidirectional plasticity and attenuated the intensity of l-DOPA-induced dyskinesia. This is the first demonstration in an animal model of PD and dyskinesia that a mGlu4 PAM can restore striatal synaptic plasticity.

Altered Cholesterol Homeostasis in Huntington's Disease.

Huntington's disease (HD) is an autosomal dominant genetic disorder caused by an expansion of the CAG repeat in the first exon of Huntingtin's gene. The associated neurodegeneration mainly affects the striatum and the cortex at early stages and progressively spreads to other brain structures. Targeting HD at its earlier stages is under intense investigation. Numerous drugs were tested, with a rate of success of only 3.5% approved molecules used as symptomatic treatment. The restoration of cholesterol metabolism, which is central to the brain homeostasis and strongly altered in HD, could be an interesting disease-modifying strategy. Cholesterol is an essential membrane component in the central nervous system (CNS); alterations of its homeostasis have deleterious consequences on neuronal functions. The levels of several sterols, upstream of cholesterol, are markedly decreased within the striatum of HD mouse model. Transcription of cholesterol biosynthetic genes is reduced in HD cell and mouse models as well as post-mortem striatal and cortical tissues from HD patients. Since the dynamic of brain cholesterol metabolism is complex, it is essential to establish the best method to target it in HD. Cholesterol, which does not cross the blood-brain-barrier, is locally synthesized and renewed within the brain. All cell types in the CNS synthesize cholesterol during development but as they progress through adulthood, neurons down-regulate their cholesterol synthesis and turn to astrocytes for their full supply. Cellular levels of cholesterol reflect the dynamic balance between synthesis, uptake and export, all integrated into the context of the cross talk between neurons and glial cells. In this review, we describe the latest advances regarding the role of cholesterol deregulation in neuronal functions and how this could be a determinant factor in neuronal degeneration and HD progression. The pathways and major mechanisms by which cholesterol and sterols are regulated in the CNS will be described. From this overview, we discuss the main clinical strategies for manipulating cholesterol metabolism in the CNS, and how to reinstate a proper balance in HD.

Cell-type- and region-specific modulation of cocaine seeking by micro-RNA-1 in striatal projection neurons.

The persistent and experience-dependent nature of drug addiction may result in part from epigenetic alterations, including non-coding micro-RNAs (miRNAs), which are both critical for neuronal function and modulated by cocaine in the striatum. Two major striatal cell populations, the striato-nigral and striato-pallidal projection neurons, express, respectively, the D1 (D1-SPNs) and D2 (D2-SPNs) dopamine receptor, and display distinct but complementary functions in drug-evoked responses. However, a cell-type-specific role for miRNAs action has yet to be clarified. Here, we evaluated the expression of a subset of miRNAs proposed to modulate cocaine effects in the nucleus accumbens (NAc) and dorsal striatum (DS) upon sustained cocaine exposure in mice and showed that these selected miRNAs were preferentially upregulated in the NAc. We focused on miR-1 considering the important role of some of its predicted mRNA targets, Fosb and Npas4, in the effects of cocaine. We validated these targets in vitro and in vivo. We explored the potential of miR-1 to regulate cocaine-induced behavior by overexpressing it in specific striatal cell populations. In DS D1-SPNs miR-1 overexpression downregulated Fosb and Npas4 and reduced cocaine-induced CPP reinstatement, but increased cue-induced cocaine seeking. In DS D2-SPNs miR-1 overexpression reduced the motivation to self-administer cocaine. Our results indicate a role of miR1 and its target genes, Fosb and Npas4, in these behaviors and highlight a precise cell-type- and region-specific modulatory role of miR-1, illustrating the importance of cell-specific investigations.

Disrupting D1-NMDA or D2-NMDA receptor heteromerization prevents cocaine's rewarding effects but preserves natural reward processing.

Addictive drugs increase dopamine in the nucleus accumbens (NAc), where it persistently shapes excitatory glutamate transmission and hijacks natural reward processing. Here, we provide evidence, from mice to humans, that an underlying mechanism relies on drug-evoked heteromerization of glutamate N-methyl-d-aspartate receptors (NMDAR) with dopamine receptor 1 (D1R) or 2 (D2R). Using temporally controlled inhibition of D1R-NMDAR heteromerization, we unraveled their selective implication in early phases of cocaine-mediated synaptic, morphological, and behavioral responses. In contrast, preventing D2R-NMDAR heteromerization blocked the persistence of these adaptations. Interfering with these heteromers spared natural reward processing. Notably, we established that D2R-NMDAR complexes exist in human samples and showed that, despite a decreased D2R protein expression in the NAc, individuals with psychostimulant use disorder display a higher proportion of D2R forming heteromers with NMDAR. These findings contribute to a better understanding of molecular mechanisms underlying addiction and uncover D2R-NMDAR heteromers as targets with potential therapeutic value.

Npas4 regulates medium spiny neuron physiology and gates cocaine-induced hyperlocomotion.

We show here that the transcription factor Npas4 is an important regulator of medium spiny neuron spine density and electrophysiological parameters and that it determines the magnitude of cocaine-induced hyperlocomotion in mice. Npas4 is induced by synaptic stimuli that cause calcium influx, but not dopaminergic or PKA-stimulating input, in mouse medium spiny neurons and human iPSC-derived forebrain organoids. This induction is independent of ubiquitous kinase pathways such as PKA and MAPK cascades, and instead depends on calcineurin and nuclear calcium signalling. Npas4 controls a large regulon containing transcripts for synaptic molecules, such as NMDA receptors and VDCC subunits, and determines in vivo MSN spine density, firing rate, I/O gain function and paired-pulse facilitation. These functions at the molecular and cellular levels control the locomotor response to drugs of abuse, as Npas4 knockdown in the nucleus accumbens decreases hyperlocomotion in response to cocaine in male mice while leaving basal locomotor behaviour unchanged.

The cholesterol 24-hydroxylase activates autophagy and decreases mutant huntingtin build-up in a neuroblastoma culture model of Huntington's disease.

OBJECTIVE: Compromised brain cholesterol turnover and altered regulation of brain cholesterol metabolism have been allied with some neurodegenerative diseases, including Huntington's disease (HD). Following our previous studies in HD, in this study we aim to investigate in vitro in a neuroblastoma cellular model of HD, the effect of CYP46A1 overexpression, an essential enzyme in cholesterol metabolism, on huntingtin aggregation and levels. RESULTS: We found that CYP46A1 reduces the quantity and size of mutant huntingtin aggregates in cells, as well as the levels of mutant huntingtin protein. Additionally, our results suggest that the observed beneficial effects of CYP46A1 in HD cells are linked to the activation of autophagy. Taken together, our results further demonstrate that CYP46A1 is a pertinent target to counteract HD progression.

From Signaling Molecules to Circuits and Behaviors: Cell-Type-Specific Adaptations to Psychostimulant Exposure in the Striatum.

Addiction is characterized by a compulsive pattern of drug seeking and consumption and a high risk of relapse after withdrawal that are thought to result from persistent adaptations within brain reward circuits. Drugs of abuse increase dopamine (DA) concentration in these brain areas, including the striatum, which shapes an abnormal memory trace of drug consumption that virtually highjacks reward processing. Long-term neuronal adaptations of gamma-aminobutyric acidergic striatal projection neurons (SPNs) evoked by drugs of abuse are critical for the development of addiction. These neurons form two mostly segregated populations, depending on the DA receptor they express and their output projections, constituting the so-called direct (D1 receptor) and indirect (D2 receptor) SPN pathways. Both SPN subtypes receive converging glutamate inputs from limbic and cortical regions, encoding contextual and emotional information, together with DA, which mediates reward prediction and incentive values. DA differentially modulates the efficacy of glutamate synapses onto direct and indirect SPN pathways by recruiting distinct striatal signaling pathways, epigenetic and genetic responses likely involved in the transition from casual drug use to addiction. Herein we focus on recent studies that have assessed psychostimulant-induced alterations in a cell-type-specific manner, from remodeling of input projections to the characterization of specific molecular events in each SPN subtype and their impact on long-lasting behavioral adaptations. We discuss recent evidence revealing the complex and concerted action of both SPN populations on drug-induced behavioral responses, as these studies can contribute to the design of future strategies to alleviate specific behavioral components of addiction.

Early epigenomic and transcriptional changes reveal Elk-1 transcription factor as a therapeutic target in Huntington's disease.

Huntington's disease (HD) is a chronic neurodegenerative disorder characterized by a late clinical onset despite ubiquitous expression of the mutant Huntingtin gene (HTT) from birth. Transcriptional dysregulation is a pivotal feature of HD. Yet, the genes that are altered in the prodromal period and their regulators, which present opportunities for therapeutic intervention, remain to be elucidated. Using transcriptional and chromatin profiling, we found aberrant transcription and changes in histone H3K27acetylation in the striatum of R6/1 mice during the presymptomatic disease stages. Integrating these data, we identified the Elk-1 transcription factor as a candidate regulator of prodromal changes in HD. Exogenous expression of Elk-1 exerted beneficial effects in a primary striatal cell culture model of HD, and adeno-associated virus-mediated Elk-1 overexpression alleviated transcriptional dysregulation in R6/1 mice. Collectively, our work demonstrates that aberrant gene expression precedes overt disease onset in HD, identifies the Elk-1 transcription factor as a key regulator linked to early epigenetic and transcriptional changes in HD, and presents evidence for Elk-1 as a target for alleviating molecular pathology in HD.

CYP46A1 gene therapy deciphers the role of brain cholesterol metabolism in Huntington's disease.

Dysfunctions in brain cholesterol homeostasis have been extensively related to brain disorders. The main pathway for brain cholesterol elimination is its hydroxylation into 24S-hydroxycholesterol by the cholesterol 24-hydrolase, CYP46A1. Increasing evidence suggests that CYP46A1 has a role in the pathogenesis and progression of neurodegenerative disorders, and that increasing its levels in the brain is neuroprotective. However, the mechanisms underlying this neuroprotection remain to be fully understood. Huntington's disease is a fatal autosomal dominant neurodegenerative disease caused by an abnormal CAG expansion in huntingtin's gene. Among the multiple cellular and molecular dysfunctions caused by this mutation, altered brain cholesterol homeostasis has been described in patients and animal models as a critical event in Huntington's disease. Here, we demonstrate that a gene therapy approach based on the delivery of CYP46A1, the rate-limiting enzyme for cholesterol degradation in the brain, has a long-lasting neuroprotective effect in Huntington's disease and counteracts multiple detrimental effects of the mutated huntingtin. In zQ175 Huntington's disease knock-in mice, CYP46A1 prevented neuronal dysfunctions and restored cholesterol homeostasis. These events were associated to a specific striatal transcriptomic signature that compensates for multiple mHTT-induced dysfunctions. We thus explored the mechanisms for these compensations and showed an improvement of synaptic activity and connectivity along with the stimulation of the proteasome and autophagy machineries, which participate to the clearance of mutant huntingtin (mHTT) aggregates. Furthermore, BDNF vesicle axonal transport and TrkB endosome trafficking were restored in a cellular model of Huntington's disease. These results highlight the large-scale beneficial effect of restoring cholesterol homeostasis in neurodegenerative diseases and give new opportunities for developing innovative disease-modifying strategies in Huntington's disease.

Cocaine conditioned place preference: unexpected suppression of preference due to testing combined with strong conditioning.

Conditioned place preference (CPP) is widely used for evaluating the rewarding effects of drugs. Like other memories, CPP is proposed to undergo reconsolidation during which it is unstable and sensitive to pharmacological inhibition. Previous studies have shown that cocaine CPP can be apparently erased by extracellular signal-regulated kinase (ERK) pathway inhibition during cocaine reconditioning (re-exposure to the drug-paired environment in the presence of the drug). Here, we show that blockade of D1 receptors during reconditioning prevented ERK activation and induced a loss of CPP. However, we also unexpectedly observed a CPP disappearance in mice that underwent testing and reconditioning with cocaine alone, specifically in strong conditioning conditions. The loss was due to the intermediate test. CPP was not recovered with reconditioning or priming in the short term, but it spontaneously reappeared after a month. When we challenged the D1 antagonist-mediated erasure, we observed that both a high dose of cocaine and a first CPP test were required for this effect. Our results also suggest a balance between D1-dependent ERK pathway activation and an A2a-dependent mechanism in D2 striatal neurons in controlling CPP expression. Our data reveal that, paradoxically, a simple CPP test can induce a complete (but transient) loss of place preference following strong but not weak cocaine conditioning. This study emphasizes the complex nature of CPP memory and the importance of multiple parameters that must be taken into consideration when investigating reconsolidation.

CYP46A1 protects against NMDA-mediated excitotoxicity in Huntington's disease: Analysis of lipid raft content.

Huntington's Disease (HD) is an autosomal dominant neurodegenerative disease caused by abnormal polyglutamine expansion in huntingtin (mHtt) protein leading to degeneration of striatal neurons. Excitotoxicity, consecutive to overstimulation of N-methyl d-aspartate receptors (NMDARs) has a pivotal role in many neurological disorders including HD. Mutant Htt causes enhanced NMDA sensitivity, alteration of NMDAR expression and localization in neurons. Excitotoxic events initiate neuronal death in numerous ways, including activation of apoptotic cascades. Among the NMDAR subunits involved in glutamatergic-mediated excitotoxicity, GluN2B has been extensively reported. In addition to excitotoxicity, alteration of cholesterol metabolism has been observed in HD, with a decrease of cholesterol precursor synthesis along with an increase of cholesterol accumulation, which is deleterious for neurons. Expression of Cholesterol Hydroxylase enzyme, CYP46A1, which converts cholesterol into 24 S-hydroxycholesterol is down-regulated in HD. We found that CYP46A1 overexpression is beneficial in HD neurons and mouse model, but the mechanisms involved still remain unclear. In this study we addressed the effect of CYP46A1 on NMDAR-mediated excitotoxicity in HD primary neurons and its role in modulating cholesterol and localization of GLUN2B in lipid rafts. We showed that CYP46A1 is protective against NMDAR-mediated excitotoxicity in two different HD neuronal cell models. Cholesterol as well as GluN2B level in lipid raft, are significantly increased by mHtt. Despite a clear effect of CYP46A1 in reducing cholesterol content in lipid raft extracts from wild type neurons, CYP46A1 overexpression in HD neurons could not normalize the increased cholesterol levels in lipid rafts. This study highlights the beneficial role of CYP46A1 against NMDAR-mediated excitotoxicity and gives further insights into the cellular mechanisms underlying CYP46A1-mediated neuroprotection.

Antidepressive effects of targeting ELK-1 signal transduction.

Depression, a devastating psychiatric disorder, is a leading cause of disability worldwide. Current antidepressants address specific symptoms of the disease, but there is vast room for improvement 1 . In this respect, new compounds that act beyond classical antidepressants to target signal transduction pathways governing synaptic plasticity and cellular resilience are highly warranted2-4. The extracellular signal-regulated kinase (ERK) pathway is implicated in mood regulation5-7, but its pleiotropic functions and lack of target specificity prohibit optimal drug development. Here, we identified the transcription factor ELK-1, an ERK downstream partner 8 , as a specific signaling module in the pathophysiology and treatment of depression that can be targeted independently of ERK. ELK1 mRNA was upregulated in postmortem hippocampal tissues from depressed suicides; in blood samples from depressed individuals, failure to reduce ELK1 expression was associated with resistance to treatment. In mice, hippocampal ELK-1 overexpression per se produced depressive behaviors; conversely, the selective inhibition of ELK-1 activation prevented depression-like molecular, plasticity and behavioral states induced by stress. Our work stresses the importance of target selectivity for a successful approach for signal-transduction-based antidepressants, singles out ELK-1 as a depression-relevant transducer downstream of ERK and brings proof-of-concept evidence for the druggability of ELK-1.

Serotonergic Signaling Controls Input-Specific Synaptic Plasticity at Striatal Circuits.

Monoaminergic modulation of cortical and thalamic inputs to the dorsal striatum (DS) is crucial for reward-based learning and action control. While dopamine has been extensively investigated in this context, the synaptic effects of serotonin (5-HT) have been largely unexplored. Here, we investigated how serotonergic signaling affects associative plasticity at glutamatergic synapses on the striatal projection neurons of the direct pathway (dSPNs). Combining chemogenetic and optogenetic approaches reveals that impeding serotonergic signaling preferentially gates spike-timing-dependent long-term depression (t-LTD) at thalamostriatal synapses. This t-LTD requires dampened activity of the 5-HT4 receptor subtype, which we demonstrate controls dendritic Ca2+ signals by regulating BK channel activity, and which preferentially localizes at the dendritic shaft. The synaptic effects of 5-HT signaling at thalamostriatal inputs provide insights into how changes in serotonergic levels associated with behavioral states or pathology affect striatal-dependent processes.

Cocaine increases dopaminergic connectivity in the nucleus accumbens.

The development of addictive behavior is associated with functional and structural plasticity in the mesocorticolimbic pathway. Increased connectivity upon cocaine administration has been inferred from increases in dendritic spine density, but without observations of presynaptic elements. Recently, we established a method that enables analyses of both dendritic spines and glutamatergic boutons and presented evidence that cocaine induces changes in striatal connectivity. As the pharmacological and behavioral effects of cocaine directly implicate dopaminergic neurons and their afferents, a remaining question is whether dopaminergic striatal innervations also undergo structural plasticity. To address this issue, we generated transgenic mice in which the fluorophore tdTomato is expressed under the promoter of the dopamine transporter gene. In these mice, specific labeling of dopaminergic boutons was observed in the striatum. Of note, the accordance of our results for control mice with previous electron microscopy studies confirms that our method can be used to decipher the spatial organization of boutons in relation to dendritic elements. Following repeated cocaine administration that led to behavioral locomotor sensitization, an increased density of dopaminergic boutons was observed 1 day later in the nucleus accumbens shell specifically, and not in other striatal regions. Combined labeling of dopaminergic boutons and striatal dendrites showed that cocaine significantly increased the percentage of dendritic spines associated with a dopaminergic bouton. Our results show that chronic cocaine administration induces structural plasticity of dopaminergic boutons that could participate in dopamine-dependent neuronal adaptations in the striatum.

Rapid Synaptogenesis in the Nucleus Accumbens Is Induced by a Single Cocaine Administration and Stabilized by Mitogen-Activated Protein Kinase Interacting Kinase-1 Activity.

BACKGROUND: Repeated cocaine exposure produces new spine formation in striatal projection neurons (SPNs) of the nucleus accumbens. However, an acute exposure to cocaine can trigger long-lasting synaptic plasticity in SPNs leading to behavioral alterations. This raises the intriguing question as to whether a single administration of cocaine could enduringly modify striatal connectivity. METHODS: A three-dimensional morphometric analysis of presynaptic glutamatergic boutons and dendritic spines was performed on SPNs 1 hour and 1 week after a single cocaine administration. Time-lapse two-photon microscopy in adult slices was used to determine the precise molecular-events sequence responsible for the rapid spine formation. RESULTS: A single injection triggered a rapid synaptogenesis and persistent increase in glutamatergic connectivity in SPNs from the shell part of the nucleus accumbens, specifically. Synapse formation occurred through clustered growth of active spines contacting pre-existing axonal boutons. Spine growth required extracellular signal-regulated kinase activation, while spine stabilization involved transcription-independent protein synthesis driven by mitogen-activated protein kinase interacting kinase-1, downstream from extracellular signal-regulated kinase. The maintenance of new spines driven by mitogen-activated protein kinase interacting kinase-1 was essential for long-term connectivity changes induced by cocaine in vivo. CONCLUSIONS: Our study originally demonstrates that a single administration of cocaine is able to induce stable synaptic rewiring in the nucleus accumbens, which will likely influence responses to subsequent drug exposure. It also unravels a new functional role for cocaine-induced extracellular signal-regulated kinase pathway independently of nuclear targets. Finally, it reveals that mitogen-activated protein kinase interacting kinase-1 has a pivotal role in cocaine-induced connectivity.

Activity-Regulated Cytoskeleton-Associated Protein Accumulates in the Nucleus in Response to Cocaine and Acts as a Brake on Chromatin Remodeling and Long-Term Behavioral Alterations.

BACKGROUND: Addiction relies on persistent alterations of neuronal properties, which depends on gene regulation. Activity-regulated cytoskeleton-associated protein (Arc) is an immediate early gene that modulates neuronal plasticity underlying learning and memory. Its role in cocaine-induced neuronal and behavioral adaptations remains elusive. METHODS: Acute cocaine-treated mice were used for quantitative reverse-transcriptase polymerase chain reaction, immunocytochemistry, and confocal imaging from striatum. Live imaging and transfection assays for Arc overexpression were performed from primary cultures. Molecular and behavioral adaptations to cocaine were studied from Arc-deficient mice and their wild-type littermates. RESULTS: Arc messenger RNA and proteins are rapidly induced in the striatum after acute cocaine administration, via an extracellular-signal regulated kinase-dependent de novo protein synthesis. Although detected in dendrites, Arc accumulates in the nucleus in active zones of transcription, where it colocalizes with phospho-Ser10-histone H3, an important component of nucleosomal response. In vitro, Arc overexpression downregulates phospho-Ser10-histone H3 without modifying extracellular-signal regulated kinase phosphorylation in the nucleus. In vivo, Arc-deficient mice display decreased heterochromatin domains, a high RNA-polymerase II activity and enhanced c-Fos expression. These mice presented an exacerbated psychomotor sensitization and conditioned place preference induced by low doses of cocaine. CONCLUSIONS: Cocaine induces the rapid induction of Arc and its nuclear accumulation in striatal neurons. Locally, it alters the nucleosomal response, and acts as a brake on chromatin remodeling and gene regulation. These original observations posit Arc as a major homeostatic modulator of molecular and behavioral responses to cocaine. Thus, modulating Arc levels may provide promising therapeutic approaches in drug addiction.

Coupling of D2R Short but not D2R Long receptor isoform to the Rho/ROCK signaling pathway renders striatal neurons vulnerable to mutant huntingtin.

Huntington's disease, an inherited neurodegenerative disorder, results from abnormal polyglutamine extension in the N-terminal region of the huntingtin protein. This mutation causes preferential degeneration of striatal projection neurons. We previously demonstrated, in vitro, that dopaminergic D2 receptor stimulation acted in synergy with expanded huntingtin to increase aggregates formation and striatal death through activation of the Rho/ROCK signaling pathway. In vivo, in a lentiviral-mediated model of expanded huntingtin expression in the rat striatum, we found that the D2 antagonist haloperidol protects striatal neurons against expanded huntingtin-mediated toxicity. Two variant transcripts are generated by alternative splicing of the of D2 receptor gene, the D2R-Long and the D2R-Short, which are thought to play different functional roles. We show herein that overexpression of D2R-Short, but not D2R-Long in cell lines is associated with activation of the RhoA/ROCK signaling pathway. In striatal neurons in culture, the selective D2 agonist Quinpirole triggers phosphorylation of cofilin, a downstream effector of ROCK, which is abrogated by siRNAs that knockdown both D2R-Long and D2R-Short, but not by siRNAs targeting D2R-Long alone. Aggregate formation and neuronal death induced by expanded huntingtin, were potentiated by Quinpirole. This D2 agonist-mediated effect was selectively inhibited by the siRNA targeting both D2R-Long and D2R-Short but not D2R-Long alone. Our data provide evidence for a specific coupling of D2R-Short to the RhoA/ROCK/cofilin pathway, and its involvement in striatal vulnerability to expanded huntingtin. A new route for targeting Rho-ROCK signaling in Huntington's disease is unraveled with our findings.

CYP46A1, the rate-limiting enzyme for cholesterol degradation, is neuroprotective in Huntington's disease.

Huntington's disease is an autosomal dominant neurodegenerative disease caused by abnormal polyglutamine expansion in huntingtin (Exp-HTT) leading to degeneration of striatal neurons. Altered brain cholesterol homeostasis has been implicated in Huntington's disease, with increased accumulation of cholesterol in striatal neurons yet reduced levels of cholesterol metabolic precursors. To elucidate these two seemingly opposing dysregulations, we investigated the expression of cholesterol 24-hydroxylase (CYP46A1), the neuronal-specific and rate-limiting enzyme for cholesterol conversion to 24S-hydroxycholesterol (24S-OHC). CYP46A1 protein levels were decreased in the putamen, but not cerebral cortex samples, of post-mortem Huntington's disease patients when compared to controls. Cyp46A1 mRNA and CYP46A1 protein levels were also decreased in the striatum of the R6/2 Huntington's disease mouse model and in SThdhQ111 cell lines. In vivo, in a wild-type context, knocking down CYP46A1 expression in the striatum, via an adeno-associated virus-mediated delivery of selective shCYP46A1, reproduced the Huntington's disease phenotype, with spontaneous striatal neuron degeneration and motor deficits, as assessed by rotarod. In vitro, CYP46A1 restoration protected SThdhQ111 and Exp-HTT-expressing striatal neurons in culture from cell death. In the R6/2 Huntington's disease mouse model, adeno-associated virus-mediated delivery of CYP46A1 into the striatum decreased neuronal atrophy, decreased the number, intensity level and size of Exp-HTT aggregates and improved motor deficits, as assessed by rotarod and clasping behavioural tests. Adeno-associated virus-CYP46A1 infection in R6/2 mice also restored levels of cholesterol and lanosterol and increased levels of desmosterol. In vitro, lanosterol and desmosterol were found to protect striatal neurons expressing Exp-HTT from death. We conclude that restoring CYP46A1 activity in the striatum promises a new therapeutic approach in Huntington's disease.