Restoring brain cholesterol turnover improves autophagy and has therapeutic potential in mouse models of spinocerebellar ataxia.

Spinocerebellar ataxias (SCAs) are devastating neurodegenerative disorders for which no curative or preventive therapies are available. Deregulation of brain cholesterol metabolism and impaired brain cholesterol turnover have been associated with several neurodegenerative diseases. SCA3 or Machado-Joseph disease (MJD) is the most prevalent ataxia worldwide. We show that cholesterol 24-hydroxylase (CYP46A1), the key enzyme allowing efflux of brain cholesterol and activating brain cholesterol turnover, is decreased in cerebellar extracts from SCA3 patients and SCA3 mice. We investigated whether reinstating CYP46A1 expression would improve the disease phenotype of SCA3 mouse models. We show that administration of adeno-associated viral vectors encoding CYP46A1 to a lentiviral-based SCA3 mouse model reduces mutant ataxin-3 accumulation, which is a hallmark of SCA3, and preserves neuronal markers. In a transgenic SCA3 model with a severe motor phenotype we confirm that cerebellar delivery of AAVrh10-CYP46A1 is strongly neuroprotective in adult mice with established pathology. CYP46A1 significantly decreases ataxin-3 protein aggregation, alleviates motor impairments and improves SCA3-associated neuropathology. In particular, improvement in Purkinje cell number and reduction of cerebellar atrophy are observed in AAVrh10-CYP46A1-treated mice. Conversely, we show that knocking-down CYP46A1 in normal mouse brain impairs cholesterol metabolism, induces motor deficits and produces strong neurodegeneration with impairment of the endosomal-lysosomal pathway, a phenotype closely resembling that of SCA3. Remarkably, we demonstrate for the first time both in vitro, in a SCA3 cellular model, and in vivo, in mouse brain, that CYP46A1 activates autophagy, which is impaired in SCA3, leading to decreased mutant ataxin-3 deposition. More broadly, we show that the beneficial effect of CYP46A1 is also observed with mutant ataxin-2 aggregates. Altogether, our results confirm a pivotal role for CYP46A1 and brain cholesterol metabolism in neuronal function, pointing to a key contribution of the neuronal cholesterol pathway in mechanisms mediating clearance of aggregate-prone proteins. This study identifies CYP46A1 as a relevant therapeutic target not only for SCA3 but also for other SCAs.

ßAPP Processing Drives Gradual Tau Pathology in an Age-Dependent Amyloid Rat Model of Alzheimer's Disease.

The treatment of Alzheimer's disease (AD) remains challenging and requires a better in depth understanding of AD progression. Particularly, the link between amyloid protein precursor (APP) processing and Tau pathology development remains poorly understood. Growing evidences suggest that APP processing and amyloid-ß (Aß) release are upstream of Tau pathology but the lack of animal models mimicking the slow progression of human AD raised questions around this mechanism. Here, we described that an AD-like ßAPP processing in adults wild-type rats, yielding to human APP, ßCTF and Aß levels similar to those observed in AD patients, is sufficient to trigger gradual Tauopathy. The Tau hyperphosphorylation begins several months before the formation of both amyloid plaques and tangle-like aggregates in aged rats and without associated inflammation. Based on a longitudinal characterization over 30 months, we showed that extrasynaptic and emotional impairments appear before long-term potentiation deficits and memory decline and so before Aß and Tau aggregations. These compelling data allowed us to (1) experimentally confirm the causal relationship between ßAPP processing and Tau pathology in vivo and without Tau transgene overexpression, (2) support the amyloidogenic cascade and (3) propose a 4-step hypothesis of prodromal AD progression.

Interleukin-2 improves amyloid pathology, synaptic failure and memory in Alzheimer's disease mice.

Interleukin-2 (IL-2)-deficient mice have cytoarchitectural hippocampal modifications and impaired learning and memory ability reminiscent of Alzheimer's disease. IL-2 stimulates regulatory T cells whose role is to control inflammation. As neuroinflammation contributes to neurodegeneration, we investigated IL-2 in Alzheimer's disease. Therefore, we investigated IL-2 levels in hippocampal biopsies of patients with Alzheimer's disease relative to age-matched control individuals. We then treated APP/PS1ΔE9 mice having established Alzheimer's disease with IL-2 for 5 months using single administration of an AAV-IL-2 vector. We first found decreased IL-2 levels in hippocampal biopsies of patients with Alzheimer's disease. In mice, IL-2-induced systemic and brain regulatory T cells expansion and activation. In the hippocampus, IL-2 induced astrocytic activation and recruitment of astrocytes around amyloid plaques, decreased amyloid-ß42/40 ratio and amyloid plaque load, improved synaptic plasticity and significantly rescued spine density. Of note, this tissue remodelling was associated with recovery of memory deficits, as assessed in the Morris water maze task. Altogether, our data strongly suggest that IL-2 can alleviate Alzheimer's disease hallmarks in APP/PS1ΔE9 mice with established pathology. Therefore, this should prompt the investigation of low-dose IL-2 in Alzheimer's disease and other neuroinflammatory/neurodegenerative disorders.

Deregulation of autophagy in postmortem brains of Machado-Joseph disease patients.

Autophagy, the major pathway for protein turnover, is critical to maintain cellular homeostasis and has been implicated in neurodegenerative diseases. The aim of this research was to analyze the expression of autophagy markers in postmortem brains from Machado-Joseph disease (MJD) patients. The expression of autophagy markers in the cerebellum and the oculomotor nucleus from MJD patients and age-matched controls with no signs of neuropathology was inspected postmortem by immunohistochemistry (IHC) and Western blot. Furthermore, autophagy was examined by means of transmission electron microscopy (TEM). Western blot and IHC revealed nuclear accumulation of misfolded ataxin-3 (ATXN3) and the presence of ubiquitin- and p62-positive aggregates in MJD patients as compared to controls. Moreover, the autophagic proteins, autophagy-related gene (Atg) protein (ATG)-7, ATG-12, ATG16L2 and autophagosomal microtubule-associated protein light chain 3 (LC3) were significantly increased in MJD brains relative to controls, while beclin-1 levels were reduced in MJD patients. Increase in the levels of lysosomal-associated membrane protein 2 (LAMP-2) and of the endosomal markers (Rab7 and Rab1A) were observed in MJD patients relatively to controls. In addition, these findings were further confirmed by TEM in brain tissue where large vesicles accumulating electron-dense materials were highly enriched in MJD patients. Postmortem brains with MJD exhibit increased markers of autophagy relative to age-matched control brains, therefore suggesting strong dysregulation of autophagy that may have an important role in the course of MJD pathogenesis.

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.

CYP46A1 inhibition, brain cholesterol accumulation and neurodegeneration pave the way for Alzheimer's disease.

Abnormalities in neuronal cholesterol homeostasis have been suspected or observed in several neurodegenerative disorders including Alzheimer's disease, Parkinson's disease and Huntington's disease. However, it has not been demonstrated whether an increased abundance of cholesterol in neurons in vivo contributes to neurodegeneration. To address this issue, we used RNA interference methodology to inhibit the expression of cholesterol 24-hydroxylase, encoded by the Cyp46a1 gene, in the hippocampus of normal mice. Cholesterol 24-hydroxylase controls cholesterol efflux from the brain and thereby plays a major role in regulating brain cholesterol homeostasis. We used an adeno-associated virus vector encoding short hairpin RNA directed against the mouse Cyp46a1 mRNA to decrease the expression of the Cyp46a1 gene in hippocampal neurons of normal mice. This increased the cholesterol concentration in neurons, followed by cognitive deficits and hippocampal atrophy due to apoptotic neuronal death. Prior to neuronal death, the recruitment of the amyloid protein precursor to lipid rafts was enhanced leading to the production of ß-C-terminal fragment and amyloid-ß peptides. Abnormal phosphorylation of tau and endoplasmic reticulum stress were also observed. In the APP23 mouse model of Alzheimer's disease, the abundance of amyloid-ß peptides increased following inhibition of Cyp46a1 expression, and neuronal death was more widespread than in normal mice. Altogether, these results suggest that increased amounts of neuronal cholesterol within the brain may contribute to inducing and/or aggravating Alzheimer's disease.

The autophagy/lysosome pathway is impaired in SCA7 patients and SCA7 knock-in mice.

There is still no treatment for polyglutamine disorders, but clearance of mutant proteins might represent a potential therapeutic strategy. Autophagy, the major pathway for organelle and protein turnover, has been implicated in these diseases. To determine whether the autophagy/lysosome system contributes to the pathogenesis of spinocerebellar ataxia type 7 (SCA7), caused by expansion of a polyglutamine tract in the ataxin-7 protein, we looked for biochemical, histological and transcriptomic abnormalities in components of the autophagy/lysosome pathway in a knock-in mouse model of the disease, postmortem brain and peripheral blood mononuclear cells (PBMC) from patients. In the mouse model, mutant ataxin-7 accumulated in inclusions immunoreactive for the autophagy-associated proteins mTOR, beclin-1, p62 and ubiquitin. Atypical accumulations of the autophagosome/lysosome markers LC3, LAMP-1, LAMP2 and cathepsin-D were also found in the cerebellum of the SCA7 knock-in mice. In patients, abnormal accumulations of autophagy markers were detected in the cerebellum and cerebral cortex of patients, but not in the striatum that is spared in SCA7, suggesting that autophagy might be impaired by the selective accumulation of mutant ataxin-7. In vitro studies demonstrated that the autophagic flux was impaired in cells overexpressing full-length mutant ataxin-7. Interestingly, the expression of the early autophagy-associated gene ATG12 was increased in PBMC from SCA7 patients in correlation with disease severity. These results provide evidence that the autophagy/lysosome pathway is impaired in neurons undergoing degeneration in SCA7. Autophagy/lysosome-associated molecules might, therefore, be useful markers for monitoring the effects of potential therapeutic approaches using modulators of autophagy in SCA7 and other autophagy/lysosome-associated neurodegenerative disorders.

Interferon ß induces clearance of mutant ataxin 7 and improves locomotion in SCA7 knock-in mice.

We showed previously, in a cell model of spinocerebellar ataxia 7, that interferon beta induces the expression of PML protein and the formation of PML protein nuclear bodies that degrade mutant ataxin 7, suggesting that the cytokine, used to treat multiple sclerosis, might have therapeutic value in spinocerebellar ataxia 7. We now show that interferon beta also induces PML-dependent clearance of ataxin 7 in a preclinical model, SCA7(266Q/5Q) knock-in mice, and improves motor function. Interestingly, the presence of mutant ataxin 7 in the mice induces itself the expression of endogenous interferon beta and its receptor. Immunohistological studies in brains from two patients with spinocerebellar ataxia 7 confirmed that these modifications are also caused by the disease in humans. Interferon beta, administered intraperitoneally three times a week in the knock-in mice, was internalized with its receptor in Purkinje and other cells and translocated to the nucleus. The treatment induced PML protein expression and the formation of PML protein nuclear bodies and decreased mutant ataxin 7 in neuronal intranuclear inclusions, the hallmark of the disease. No reactive gliosis or other signs of toxicity were observed in the brain or internal organs. The performance of the SCA7(266Q/5Q) knock-in mice was significantly improved on two behavioural tests sensitive to cerebellar function: the Locotronic® Test of locomotor function and the Beam Walking Test of balance, motor coordination and fine movements, which are affected in patients with spinocerebellar ataxia 7. In addition to motor dysfunction, SCA7(266Q/5Q) mice present abnormalities in the retina as in patients: ataxin 7-positive neuronal intranuclear inclusions that were reduced by interferon beta treatment. Finally, since neuronal death does not occur in the cerebellum of SCA7(266Q/5Q) mice, we showed in primary cell cultures expressing mutant ataxin 7 that interferon beta treatment improves Purkinje cell survival.

Modulation of Brain Cholesterol Metabolism through CYP46A1 Overexpression for Rett Syndrome.

Rett syndrome (RTT) is a rare neurodevelopmental disorder caused by mutation in the X-linked gene methyl-CpG-binding protein 2 (Mecp2), a ubiquitously expressed transcriptional regulator. RTT results in mental retardation and developmental regression that affects approximately 1 in 10,000 females. Currently, there is no curative treatment for RTT. Thus, it is crucial to develop new therapeutic approaches for children suffering from RTT. Several studies suggested that RTT is linked with defects in cholesterol homeostasis, but for the first time, therapeutic evaluation is carried out by modulating this pathway. Moreover, AAV-based CYP46A1 overexpression, the enzyme involved in cholesterol pathway, has been demonstrated to be efficient in several neurodegenerative diseases. Based on these data, we strongly believe that CYP46A1 could be a relevant therapeutic target for RTT. Herein, we evaluated the effects of intravenous AAVPHP.eB-hCYP46A1-HA delivery in male and female Mecp2-deficient mice. The applied AAVPHP.eB-hCYP46A1 transduced essential neurons of the central nervous system (CNS). CYP46A1 overexpression alleviates behavioral alterations in both male and female Mecp2 knockout mice and extends the lifespan in Mecp2-deficient males. Several parameters related to cholesterol pathway are improved and correction of mitochondrial activity is demonstrated in treated mice, which highlighted the clear therapeutic benefit of CYP46A1 through the neuroprotection effect. IV delivery of AAVPHP.eB-CYP46A1 is perfectly well tolerated with no inflammation observed in the CNS of the treated mice. Altogether, our results strongly suggest that CYP46A1 is a relevant target and overexpression could alleviate the phenotype of Rett patients.

Silencing ataxin-3 mitigates degeneration in a rat model of Machado-Joseph disease: no role for wild-type ataxin-3?

Machado-Joseph disease or spinocerebellar ataxia type 3 (MJD/SCA3) is a fatal, autosomal dominant disorder caused by a cytosine-adenine-guanine expansion in the coding region of the MJD1 gene. RNA interference has potential as a therapeutic approach but raises the issue of the role of wild-type ataxin-3 (WT ATX3) in MJD and of whether the expression of the wild-type protein must be maintained. To address this issue, we both overexpressed and silenced WT ATX3 in a rat model of MJD. We showed that (i) overexpression of WT ATX3 did not protect against MJD pathology, (ii) knockdown of WT ATX3 did not aggravate MJD pathology and that (iii) non-allele-specific silencing of ataxin-3 strongly reduced neuropathology in a rat model of MJD. Our findings indicate that therapeutic strategies involving non-allele-specific silencing to treat MJD patients may be safe and effective.

Overexpression of the autophagic beclin-1 protein clears mutant ataxin-3 and alleviates Machado-Joseph disease.

Machado-Joseph disease, also known as spinocerebellar ataxia type 3, is the most common of the dominantly inherited ataxias worldwide and is characterized by mutant ataxin-3 misfolding, intracellular accumulation of aggregates and neuronal degeneration. Here we investigated the implication of autophagy, the major pathway for organelle and protein turnover, in the accumulation of mutant ataxin-3 aggregates and neurodegeneration found in Machado-Joseph disease and we assessed whether specific stimulation of this pathway could mitigate the disease. Using tissue from patients with Machado-Joseph disease, transgenic mice and a lentiviral-based rat model, we found an abnormal expression of endogenous autophagic markers, accumulation of autophagosomes and decreased levels of beclin-1, a crucial protein in the early nucleation step of autophagy. Lentiviral vector-mediated overexpression of beclin-1 led to stimulation of autophagic flux, mutant ataxin-3 clearance and overall neuroprotective effects in neuronal cultures and in a lentiviral-based rat model of Machado-Joseph disease. These data demonstrate that autophagy is a key degradation pathway, with beclin-1 playing a significant role in alleviating Machado-Joseph disease pathogenesis.

The Challenge of Gene Therapy for Neurological Diseases: Strategies and Tools to Achieve Efficient Delivery to the Central Nervous System.

For more than 10 years, gene therapy for neurological diseases has experienced intensive research growth and more recently therapeutic interventions for multiple indications. Beneficial results in several phase 1/2 clinical studies, together with improved vector technology have advanced gene therapy for the central nervous system (CNS) in a new era of development. Although most initial strategies have focused on orphan genetic diseases, such as lysosomal storage diseases, more complex and widespread conditions like Alzheimer's disease, Parkinson's disease, epilepsy, or chronic pain are increasingly targeted for gene therapy. Increasing numbers of applications and patients to be treated will require improvement and simplification of gene therapy protocols to make them accessible to the largest number of affected people. Although vectors and manufacturing are a major field of academic research and industrial development, there is a growing need to improve, standardize, and simplify delivery methods. Delivery is the major issue for CNS therapies in general, and particularly for gene therapy. The blood-brain barrier restricts the passage of vectors; strategies to bypass this obstacle are a central focus of research. In this study, we present the different ways that can be used to deliver gene therapy products to the CNS. We focus on results obtained in large animals that have allowed the transfer of protocols to human patients and have resulted in the generation of clinical data. We discuss the different routes of administration, their advantages, and their limitations. We describe techniques, equipment, and protocols and how they should be selected for safe delivery and improved efficiency for the next generation of gene therapy trials for CNS diseases.

Striatal and nigral pathology in a lentiviral rat model of Machado-Joseph disease.

Machado-Joseph disease (MJD) is a fatal, dominant neurodegenerative disorder. MJD results from polyglutamine repeat expansion in the MJD-1 gene, conferring a toxic gain of function to the ataxin-3 protein. In this study, we aimed at overexpressing ataxin-3 in the rat brain using lentiviral vectors (LV), to generate an in vivo MJD genetic model and, to study the disorder in defined brain regions: substantia nigra, an area affected in MJD, cortex and striatum, regions not previously reported to be affected in MJD. LV encoding mutant or wild-type human ataxin-3 was injected in the brain of adult rats and the animals were tested for behavioral deficits and neuropathological abnormalities. Striatal pathology was confirmed in transgenic mice and human tissue. In substantia nigra, unilateral overexpression of mutant ataxin-3 led to: apomorphine-induced turning behavior; formation of ubiquitinated ataxin-3 aggregates; alpha-synuclein immunoreactivity; and loss of dopaminergic markers (TH and VMAT2). No neuropathological changes were observed upon wild-type ataxin-3 overexpression. Mutant ataxin-3 expression in striatum and cortex, resulted in accumulation of misfolded ataxin-3, and within striatum, loss of neuronal markers. Striatal pathology was confirmed by observation in MJD transgenic mice of ataxin-3 aggregates and substantial reduction of DARPP-32 immunoreactivity and, in human striata, by ataxin-3 inclusions, immunoreactive for ubiquitin and alpha-synuclein. This study demonstrates the use of LV encoding mutant ataxin-3 to produce a model of MJD and brings evidence of striatal pathology, suggesting that this region may contribute to dystonia and chorea observed in some MJD patients and may represent a target for therapies.

Sustained effects of nonallele-specific Huntingtin silencing.

OBJECTIVE: Huntington's disease (HD) is a fatal autosomal dominant neurodegenerative disorder caused by a polyglutamine expansion in the huntingtin (htt) protein. No cure is available to date to alleviate neurodegeneration. Recent studies have demonstrated that RNA interference represents a promising approach for the treatment of autosomal dominant disorders. But whether an allele-specific silencing of mutant htt or a nonallele-specific silencing should be considered has not been addressed. METHODS: We developed small hairpin RNA targeting mutant or wild-type htt transcripts, or both. RESULTS: We confirmed the therapeutic potential of sihtt administered with lentiviral vectors in rodent models of HD and showed that initiation of small interfering RNA treatment after the onset of HD symptoms is still efficacious and reduces the HD-like pathology. We then addressed the question of the impact of nonallele-specific silencing and demonstrated that silencing of endogenous htt to 25 to 35% in vivo is altering several pathways associated with known htt functions but is not inducing overt toxicity or increasing striatal vulnerability up to 9 months after treatment. INTERPRETATION: These data indicate that the coincident silencing of the wild-type and mutant htt may be considered as a therapeutic tool for HD.

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.

Real-Time Monitoring of Exosome Enveloped-AAV Spreading by Endomicroscopy Approach: A New Tool for Gene Delivery in the Brain.

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.

SUMOylation by SUMO2 is implicated in the degradation of misfolded ataxin-7 via RNF4 in SCA7 models.

Perturbation of protein homeostasis and aggregation of misfolded proteins is a major cause of many human diseases. A hallmark of the neurodegenerative disease spinocerebellar ataxia type 7 (SCA7) is the intranuclear accumulation of mutant, misfolded ataxin-7 (polyQ-ATXN7). Here, we show that endogenous ATXN7 is modified by SUMO proteins, thus also suggesting a physiological role for this modification under conditions of proteotoxic stress caused by the accumulation of polyQ-ATXN7. Co-immunoprecipitation experiments, immunofluorescence microscopy and proximity ligation assays confirmed the colocalization and interaction of polyQ-ATXN7 with SUMO2 in cells. Moreover, upon inhibition of the proteasome, both endogenous SUMO2/3 and the RNF4 ubiquitin ligase surround large polyQ-ATXN7 intranuclear inclusions. Overexpression of RNF4 and/or SUMO2 significantly decreased levels of polyQ-ATXN7 and, upon proteasomal inhibition, led to a marked increase in the polyubiquitination of polyQ-ATXN7. This provides a mechanism for the clearance of polyQ-ATXN7 from affected cells that involves the recruitment of RNF4 by SUMO2/3-modified polyQ-ATXN7, thus leading to its ubiquitination and proteasomal degradation. In a SCA7 knock-in mouse model, we similarly observed colocalization of SUMO2/3 with polyQ-ATXN7 inclusions in the cerebellum and retina. Furthermore, we detected accumulation of SUMO2/3 high-molecular-mass species in the cerebellum of SCA7 knock-in mice, compared with their wild-type littermates, and changes in SUMO-related transcripts. Immunohistochemical analysis showed the accumulation of SUMO proteins and RNF4 in the cerebellum of SCA7 patients. Taken together, our results show that the SUMO pathway contributes to the clearance of aggregated ATXN7 and suggest that its deregulation might be associated with SCA7 disease progression.

Inhibition of DYRK1A proteolysis modifies its kinase specificity and rescues Alzheimer phenotype in APP/PS1 mice.

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- ß, TNF-ɑ and IL-12) associated to an increased microglial recruitment around amyloid plaques and decreased amyloid-ß 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.

Clinical Gene Therapy for Neurodegenerative Diseases: Past, Present, and Future.

Clinical gene therapy has made important advances over the last decade. Among neurological diseases, severe genetic neurodegenerative conditions have been the focus of initial clinical applications. Gene therapy has also addressed complex neurodegenerative diseases, particularly Parkinson's disease, with encouraging results in human patients, demonstrating that specific targeting of central nervous system (CNS) cells is a relevant strategy for severe pathologies and that efficient access to the CNS with viral vectors is an achievable goal. The purpose of this review is to summarize the gene therapy clinical applications that have been conducted for neurodegenerative diseases. Limitations and hurdles to obtain and demonstrate benefit in patients, and the new developments that should allow new clinical applications with high beneficial potential are discussed.