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.

Impairment in cholesterol metabolism is associated with many neurodegenerative disorders including Alzheimer's disease (AD). However, the lipid alterations underlying neurodegeneration and the connection between altered cholesterol levels and AD remains not fully understood. We recently showed that cholesterol accumulation in hippocampal neurons, induced by silencing Cyp46a1 gene expression, leads to neurodegeneration with a progressive neuronal loss associated with AD-like phenotype in wild-type mice. We used a targeted and non-targeted lipidomics approach by liquid chromatography coupled to high-resolution mass spectrometry to further characterize lipid modifications associated to neurodegeneration and cholesterol accumulation induced by CYP46A1 inhibition. Hippocampus lipidome of normal mice was profiled 4 weeks after cholesterol accumulation due to Cyp46a1 gene expression down-regulation at the onset of neurodegeneration. We showed that major membrane lipids, sphingolipids and specific enzymes involved in phosphatidylcholine and sphingolipid metabolism, were rapidly increased in the hippocampus of AAV-shCYP46A1 injected mice. This lipid accumulation was associated with alterations in the lysosomal cargoe, accumulation of phagolysosomes and impairment of endosome-lysosome trafficking. Altogether, we demonstrated that inhibition of cholesterol 24-hydroxylase, key enzyme of cholesterol metabolism leads to a complex dysregulation of lipid homeostasis. Our results contribute to dissect the potential role of lipids in severe neurodegenerative diseases like AD.

Amyloid precursor protein (APP), a key molecule of Alzheimer disease, is metabolized in 2 antagonist pathways generating the soluble APP alpha (sAPPα) having neuroprotective properties and the beta amyloid (Aβ) peptide at the origin of neurotoxic oligomers, particularly Aβ1-42. Whether extracellular Aβ1-42 oligomers modulate the formation and secretion of sAPPα is not known. We report here that the addition of Aβ1-42 oligomers to primary cortical neurons induced a transient increase in α-secretase activity and secreted sAPPα 6-9 hours later. Preventing the generation of sAPPα by using small interfering RNAs (siRNAs) for the α-secretases ADAM10 and ADAM17 or for APP led to increased Aβ1-42 oligomer-induced cell death after 24 hours. Neuronal injuries due to oxidative stress or growth factor deprivation also generated sAPPα 7 hours later. Finally, acute injection of Aβ1-42 oligomers into wild-type mouse hippocampi induced transient secretion of sAPPα 48-72 hours later. Altogether, these data suggest that neurons respond to stress by generating sAPPα for their survival. These data must be taken into account when interpreting sAPPα levels as a biomarker in neurological disorders.

X-linked adrenoleukodystrophy (ALD), an inherited demyelinating disorder of the central nervous system, can be corrected by allogeneic bone marrow transplantation, likely due to the turnover of brain macrophages that are bone marrow derived. ALD is characterized by an accumulation of very long chain fatty acids (VLCFA) due to the deficiency of an ATP binding cassette transporter that imports these fatty acids in peroxisomes. Murine retroviral transduction results in metabolic correction of ALD CD34(+) cells in vitro but reinfusion of these cells into ALD patients would not provide clinical benefit owing to the absence of selective advantage conferred by transgene expression. High-efficiency transduction of ALD CD34(+) peripheral blood mobilized cells was achieved using an HIV-based vector driving ALD gene expression under the elongation factor 1 alpha promoter and a protocol without prestimulation of CD34(+) cells with cytokines prior to transduction to preserve their stem cell properties. Efficient expression of the ALD gene was demonstrated in monocytes/macrophages derived from cultures of transduced ALD CD34(+) cells and in long-term culture initiating cells. VLCFA metabolism was corrected in transduced CD34(+), CFU-derived, and LTC-derived cells, indicating that the vector-encoded ALD protein was fully functional. Transplantation of transduced ALD CD34(+) cells into NOD/SCID mice resulted in long-term expression of ALD protein in monocytes/macrophages derived from engrafted stem cells.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder characterized by accumulation of sulfatides in both glial cells and neurons. MLD results from an inherited deficiency of arylsulfatase A (ARSA) and myelin degeneration in the central and peripheral nervous systems. Currently, no effective treatment is available for the most frequent late infantile (LI) form of MLD after symptom onset. The LI form results in rapid neurological degradation and early death. ARSA enzyme must be rapidly and efficiently delivered to brain and spinal cord oligodendrocytes of patients with LI MLD in order to potentially stop the progression of the disease. We previously showed that brain gene therapy with adeno-associated virus serotype rh10 (AAVrh10) driving the expression of human ARSA cDNA alleviated most long-term disease manifestations in MLD mice but was not sufficient in MLD patient to improve disease progression. Herein, we evaluated the short-term effects of intravenous AAVPHP.eB delivery driving the expression of human ARSA cDNA under the control of the cytomegalovirus/b-actin hybrid (CAG) promoter in 6-month-old MLD mice that already show marked sulfatide accumulation and brain pathology. Within 3 months, a single intravenous injection of AAVPHP.eB-hARSA-HA resulted in correction of brain and spinal cord sulfatide storage, and improvement of astrogliosis and microgliosis in brain and spinal cord of treated animals. These results strongly support to consider the use of AAVPHP.eB-hARSA vector for intravenous gene therapy in symptomatic rapidly progressing forms of MLD.

Exosomes represent a strategy for optimizing the adeno-associated virus (AAV) toward the development of novel therapeutic options for neurodegenerative disorders. However, in vivo spreading of exosomes and AAVs after intracerebral administration is poorly understood. This study provides an assessment and comparison of the spreading into the brain of exosome-enveloped AAVs (exo-AAVs) or unassociated AAVs (std-AAVs) through in vivo optical imaging techniques like probe-based confocal laser endomicroscopy (pCLE) and ex vivo fluorescence microscopy. The std-AAV serotypes (AAV6 and AAV9) encoding the GFP were enveloped in exosomes and injected into the ipsilateral hippocampus. At 3 months post-injection, pCLE detected enhanced GFP expression of both exo-AAV serotypes in contralateral hemispheres compared to std-AAVs. Although sparse GFP-positive astrocytes were observed using exo-AAVs, our results show that the enhancement of the transgene expression resulting from exo-AAVs was largely restricted to neurons and oligodendrocytes. Our results suggest (1) the possibility of combining gene therapy with an endoscopic approach to enable tracking of exo-AAV spread, and (2) exo-AAVs allow for widespread, long-term gene expression in the CNS, supporting the use of exo-AAVs as an efficient gene delivery tool.

Recombinant adeno-associated viral (AAV) vectors have advanced to the vanguard of gene therapy. Numerous naturally occurring serotypes have been used to target cells in various tissues. There is a strong need for fast and dynamic methods which efficiently unravel viral tropism in whole organs. Ultramicroscopy (UM) is a novel fluorescence microscopy technique that images optically cleared undissected specimens, achieving good resolutions at high penetration depths while being non-destructive. UM was applied to obtain high-resolution 3D analysis of AAV transduction in adult mouse brains, especially in the hippocampus, a region of interest for Alzheimer's disease therapy. We separately or simultaneously compared transduction efficacies for commonly used serotypes (AAV9 and AAVrh10) using fluorescent reporter expression. We provide a detailed comparative and quantitative analysis of the transduction profiles. UM allowed a rapid analysis of marker fluorescence expression in neurons with intact projections deep inside the brain, in defined anatomical structures. Major hippocampal neuronal transduction was observed with both vectors, with slightly better efficacy for AAV9 in UM. Glial response and synaptic marker expression did not change post transduction.We propose UM as a novel valuable complementary tool to efficiently and simultaneously unravel tropism of different viruses in a single non-dissected adult rodent brain.

We used lentiviral vectors (LVs) to generate a new SCA7 animal model overexpressing a truncated mutant ataxin-7 (MUT ATXN7) fragment in the mouse cerebellum, in order to characterize the specific neuropathological and behavioral consequences of the genetic defect in this brain structure.

Elevations in neuronal cholesterol have been associated with several degenerative diseases. An enhanced excitability and synchronous firing in surviving neurons are among the sequels of neuronal death in these diseases and also in some epileptic syndromes. Here, we attempted to increase neuronal cholesterol levels, using a short hairpin RNA to suppress expression of the enzyme cytochrome P450 family 46, subfamily A, polypeptide 1 gene (CYP46A1). This protein hydroxylates cholesterol and so facilitates transmembrane extrusion. A short hairpin RNA CYP46A1construction coupled to the adeno-associated virus type 5 was injected focally and unilaterally into mouse hippocampus. It was selectively expressed first in neurons of the cornu ammonis (hippocampus) (CA)3a region. Cytoplasmic and membrane cholesterol increased, and the neuronal soma volume increased and then decreased before pyramidal cells died. As CA3a pyramidal cells died, interictal electroencephalographic (EEG) events occurred during exploration and non-rapid eye movement sleep. With time, neuronal death spread to involve pyramidal cells and interneurons of the CA1 region. CA1 neuronal death was correlated with a delayed local expression of phosphorylated tau. Astrocytes were activated throughout the hippocampus and microglial activation was specific to regions of neuronal death. CA1 neuronal death was correlated with distinct aberrant EEG activity. During exploratory behaviour and rapid eye movement sleep, EEG oscillations at 7-10 Hz (theta) could accelerate to 14-21 Hz (beta) waves. They were accompanied by low-amplitude, high-frequency oscillations of peak power at ~300 Hz and a range of 250-350 Hz. Although episodes of EEG acceleration were not correlated with changes in exploratory behaviour, they were followed in some animals by structured seizure-like discharges. These data strengthen links between increased cholesterol, neuronal sclerosis and epileptic behaviour.

Preventing neurodegeneration-associated disability progression in patients with multiple sclerosis (MS) remains an unmet therapeutic need. As remyelination prevents axonal degeneration, promoting this process in patients might enhance neuroprotection. In demyelinating mouse lesions, local overexpression of semaphorin 3F (Sema3F), an oligodendrocyte progenitor cell (OPC) attractant, increases remyelination. However, molecular targeting to MS lesions is a challenge. A clinically relevant paradigm for delivering Sema3F to demyelinating lesions could be to use blood-derived macrophages as vehicles. Thus, we chose transplantation of genetically modified hematopoietic stem cells (HSCs) as means of obtaining chimeric mice with circulating Sema3F-overexpressing monocytes. We demonstrated that Sema3F-transduced HSCs stimulate OPC migration in a neuropilin 2 (Nrp2, Sema3F receptor)-dependent fashion, which was conserved in middle-aged OPCs. While demyelinating lesions induced in mice with Sema3F-expressing blood cells showed no changes in inflammation and OPC survival, OPC recruitment was enhanced which accelerated the onset of remyelination. Our results provide a proof of concept that blood cells, particularly monocytes/macrophages, can be used to deliver pro-remyelinating agents "at the right time and place," suggesting novel means for remyelination-promoting strategies in MS.

Wireless powered optogenetic cell-based implant provides a strategy to deliver subcutaneously therapeutic proteins. Immortalize Human Mesenchymal Stem Cells (hMSC-TERT) expressing the bacteriophytochrome diguanylate cyclase (DGCL) were validated for optogenetic controlled interferon-β delivery (Optoferon cells) in a bioelectronic cell-based implant. Optoferon cells transcriptomic profiling was used to elaborate an in-silico model of the recombinant interferon-β production. Wireless optoelectronic device integration was developed using additive manufacturing and injection molding. Implant cell-based optoelectronic interface manufacturing was established to integrate industrial flexible compact low-resistance screen-printed Near Field Communication (NFC) coil antenna. Optogenetic cell-based implant biocompatibility, and device performances were evaluated in the Experimental Autoimmune Encephalomyelitis (EAE) mouse model of multiple sclerosis.

Recent evidences suggest the involvement of DYRK1A (dual specificity tyrosine phosphorylation-regulated kinase 1 A) in Alzheimer's disease (AD). Here we showed that DYRK1A undergoes a proteolytic processing in AD patients hippocampus without consequences on its kinase activity. Resulting truncated forms accumulate in astrocytes and exhibit increased affinity towards STAT3ɑ, a regulator of inflammatory process. These findings were confirmed in APP/PS1 mice, an amyloid model of AD, suggesting that this DYRK1A cleavage is a consequence of the amyloid pathology. We identified in vitro the Leucettine L41 as a compound able to prevent DYRK1A proteolysis in both human and mouse protein extracts. We then showed that intraperitoneal injections of L41 in aged APP/PS1 mice inhibit STAT3ɑ phosphorylation and reduce pro-inflammatory cytokines levels (IL1- β, TNF-ɑ and IL-12) associated to an increased microglial recruitment around amyloid plaques and decreased amyloid-β plaque burden. Importantly, L41 treatment improved synaptic plasticity and rescued memory functions in APP/PS1 mice. Collectively, our results suggest that DYRK1A may contribute to AD pathology through its proteolytic process, reducing its kinase specificity. Further evaluation of inhibitors of DYRK1A truncation promises a new therapeutic approach for AD.

Alzheimer's disease (AD) is the most frequent form of dementia in the elderly and no effective treatment is currently available. The mechanisms triggering AD onset and progression are still imperfectly dissected. We aimed at deciphering the modifications occurring in vivo during the very early stages of AD, before the development of amyloid deposits, neurofibrillary tangles, neuronal death and inflammation. Most current AD models based on Amyloid Precursor Protein (APP) overproduction beginning from in utero, to rapidly reproduce the histological and behavioral features of the disease within a few months, are not appropriate to study the early steps of AD development. As a means to mimic in vivo amyloid APP processing closer to the human situation in AD, we used an adeno-associated virus (AAV)-based transfer of human mutant APP and Presenilin 1 (PS1) genes to the hippocampi of two-month-old C57Bl/6 J mice to express human APP, without significant overexpression and to specifically induce its amyloid processing.

It is suspected that excess of brain cholesterol plays a role in Alzheimer's disease (AD). Membrane-associated cholesterol was shown to be increased in the brain of individuals with sporadic AD and to correlate with the severity of the disease. We hypothesized that an increase of membrane cholesterol could trigger sporadic AD early phenotypes.

The identification of the most efficient method for whole central nervous system targeting that is translatable to humans and the safest route of adeno-associated virus (AAV) administration is a major concern for future applications in clinics. Additionally, as many AAV serotypes were identified for gene introduction into the brain and the spinal cord, another key to human gene-therapy success is to determine the most efficient serotype. In this study, we compared lumbar intrathecal administration through catheter implantation and intracerebroventricular administration in the cynomolgus macaque. We also evaluated and compared two AAV serotypes that are currently used in clinical trials: AAV9 and AAVrh10. We demonstrated that AAV9 lumbar intrathecal delivery using a catheter achieved consistent transgene expression in the motor neurons of the spinal cord and in the neurons/glial cells of several brain regions, whereas AAV9 intracerebroventricular delivery led to a consistent transgene expression in the brain. In contrast, AAVrh10 lumbar intrathecal delivery led to rare motor neuron targeting. Finally, we found that AAV9 efficiently targets respiratory and skeletal muscles after injection into the cerebrospinal fluid (CSF), which represents an outstanding new property that can be useful for the treatment of diseases affecting both the central nervous system and muscle.

Cholesterol, an essential component of the brain, and its local metabolism are involved in many neurodegenerative diseases. The blood-brain barrier is impermeable to cholesterol; hence, cholesterol homeostasis in the central nervous system represents a balance between in situ biosynthesis and elimination. Cytochrome P450 46A1 (CYP46A1), a central nervous system-specific enzyme, converts cholesterol to 24-hydroxycholesterol, which can freely cross the blood-brain barrier and be degraded in the liver. By the dual action of initiating cholesterol efflux and activating the cholesterol synthesis pathway, CYP46A1 is the key enzyme that ensures brain cholesterol turnover. In humans and mouse models, CYP46A1 activity is altered in Alzheimer's and Huntington's diseases, spinocerebellar ataxias, glioblastoma, and autism spectrum disorders. In mouse models, modulations of CYP46A1 activity mitigate the manifestations of Alzheimer's, Huntington's, Nieman-Pick type C, and Machao-Joseph (spinocerebellar ataxia type 3) diseases as well as amyotrophic lateral sclerosis, epilepsy, glioblastoma, and prion infection. Animal studies revealed that the CYP46A1 activity effects are not limited to cholesterol maintenance but also involve critical cellular pathways, like gene transcription, endocytosis, misfolded protein clearance, vesicular transport, and synaptic transmission. How CYP46A1 can exert central control of such essential brain functions is a pressing question under investigation. The potential therapeutic role of CYP46A1, demonstrated in numerous models of brain disorders, is currently being evaluated in early clinical trials. This review summarizes the past 70 years of research that has led to the identification of CYP46A1 and brain cholesterol homeostasis as powerful therapeutic targets for severe pathologies of the CNS.

Improving a drug delivery system is critical to treat central nervous system disorders. Here we studied an innovative approach based on implantation of a wireless-powered cell-based device in mice. This device, coupling biologic material and electronics, is the first of its kind. The advantage of this technology is its ability to control the secretion of a therapeutic molecule and to switch the classical permanent delivery to activation on demand. In diseases with relapsing-remitting phases such as multiple sclerosis, such activation could be selectively achieved in relapsing phases. However, the safety (tolerance to biomaterials and surgical procedure) of such a clinical device needs to be verified. Therefore, the development of tools to assess the biocompatibility of the system in animal models is an essential step. We present the development of this new therapeutic approach, the challenges we encountered during the different steps of its development (such as cell loading in the chamber, surgery protocol for subcutaneous implantation of the device) and the tools we used to evaluate cell viability and biocompatibility of the device.