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.This work was supported by Brainvectis and E.rare: E-Rare Joint Transnational Call for Proposals 2017 “Transnational Research Projects for Innovative Therapeutic Approaches for Rare Diseases”. CN laboratory is supported by the French Muscular Dystrophy Association (AFM-Téléthon), the Ataxia UK, and the Fundação para a Ciência e Tecnologia (project ALG-01-0145-FEDER-29480 “SeGrPolyQ”). AM is supported by a Ph.D. fellowship from FCT (SFRH/BD/133192/2017)info:eu-repo/semantics/publishedVersio

A deconvolution method to improve automated 3D-analysis of dendritic spines: application to a mouse model of Huntington's disease

International audienceDendritic spines are postsynaptic structures the morphology of which correlates with the strength of synaptic efficacy. Measurements of spine density and spine morphology are achievable using recent imaging and bioinformatics tools. The three-dimensional automated analysis requires optimization of image acquisition and treatment. Here, we studied the critical steps for optimal confocal microscopy imaging of dendritic spines. We characterize the deconvolution process and show that it improves spine morphology analysis. With this method, images of dendritic spines from medium spiny neurons are automatically detected by the software Neuronstudio, which retrieves spine density as well as spine diameter and volume. This approach is illustrated with three-dimensional analysis of dendritic spines in a mouse model of Huntington's disease: the transgenic R6/2 mice. In symptomatic mutant mice, we confirm the decrease in spine density, and the method brings further information and show a decrease in spine volume and dendrite diameter. Moreover, we show a significant decrease in spine density at presymptomatic age which so far has gone unnoticed

Altered Cholesterol Homeostasis in Huntington’s Disease

International audienceHuntington'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-brainbarrier, 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

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

International audienceHuntington'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

Mutations of SPG4 are responsible for a loss of function of spastin, an abundant neuronal protein localized in the nucleus

Mutations of spastin are responsible for the most common autosomal dominant form of hereditary spastic paraplegia (AD-HSP), a disease characterized by axonal degeneration of corticospinal tracts and posterior columns. Generation of polyclonal antibodies specific to spastin has revealed two isoforms of 75 and 80 kDa in both human and mouse tissues with a tissue-specific variability of the isoform ratio. Spastin is an abundant protein in neural tissues and immunolabeling experiments have shown that spastin is expressed in neurons but not in glial cells. These data indicate that axonal degeneration linked to spastin mutations is caused by a primary defect of neurons. Protein and transcript analyses of patients carrying either nonsense or frameshift spastin mutations revealed neither truncated protein nor mutated transcripts, providing evidence that these mutations are responsible for a loss of spastin function. Identifying agents able to induce the expression of the non-mutated spastin allele should represent an attractive therapeutic strategy in this disease

Mitogen and Stress-activated Kinase-1 deficiency and transcriptional dysregulation in Huntington's Disease

Collaborative Congress of the European-Society-of-Gene-and-Cell-Therapy/French-Society-of-Cell-and-Gen e-Therapy, Versailles, FRANCE, OCT 25-29, 2012International audienceno abstrac

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

International audienc

Beneficial effects of striatal restoration of CYP46A1 expression using AAVrh10 serotype in Huntington's disease mice model (R6/2)

Collaborative Congress of the European-Society-of-Gene-and-Cell-Therapy/French-Society-of-Cell-and-Gen e-Therapy, Versailles, FRANCE, OCT 25-29, 2012International audienceno abstrac

Rapid synaptogenesis in the Nucleus Accumbens is induced by a single cocaine administration and stabilized by MAP Kinase interacting kinase 1 activity

International audienceBackground: Repeated cocaine exposure produces new spine formation in Striatal Projection Neurons (SPNs) of the Nucleus Accumbens (NAc). However, an acute exposure to cocaine can trigger long-lasting synaptic plasticity in SPN leading to behavioral alterations. This raises the intriguing question as to whether acute cocaine could modify enduringly striatal connectivity.Methods: A 3D morphometric analysis of presynaptic glutamatergic boutons and dendritic spines was performed on SPN one hour and one 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 SPN from the shell part of the NAc, specifically. Synapse formation occurred through clustered growth of active spines contacting pre-existing axonal boutons. Spine growth required ERK activation, while spine stabilization involved transcription-independent protein synthesis driven by MAP kinase interacting kinase-1 (MNK-1), downstream from ERK. The maintenance of new spines driven by MNK-1 was essential for long-term connectivity changes induced by cocaine in vivo.Conclusions: Our study originally demonstrates that an acute administration of cocaine is able to induce stable synaptic rewiring in the NAc, which will likely influence responses to subsequent drug exposure. It also unravels a new functional role for cocaine-induced ERK pathway independently of nuclear targets. Finally, it reveals that MNK-1 has a pivotal role in cocaine-induced connectivity