ASOs are an effective treatment for disease-associated oligodendrocyte signatures in premanifest and symptomatic SCA3 mice.

Spinocerebellar ataxia type 3 (SCA3) is the most common dominantly inherited ataxia. Currently, no preventive or disease-modifying treatments exist for this progressive neurodegenerative disorder, although efforts using gene silencing approaches are under clinical trial investigation. The disease is caused by a CAG repeat expansion in the mutant gene, ATXN3, producing an enlarged polyglutamine tract in the mutant protein. Similar to other paradigmatic neurodegenerative diseases, studies evaluating the pathogenic mechanism focus primarily on neuronal implications. Consequently, therapeutic interventions often overlook non-neuronal contributions to disease. Our lab recently reported that oligodendrocytes display some of the earliest and most progressive dysfunction in SCA3 mice. Evidence of disease-associated oligodendrocyte signatures has also been reported in other neurodegenerative diseases, including Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, and Huntington's disease. Here, we assess the effects of anti-ATXN3 antisense oligonucleotide (ASO) treatment on oligodendrocyte dysfunction in premanifest and symptomatic SCA3 mice. We report a severe, but modifiable, deficit in oligodendrocyte maturation caused by the toxic gain-of-function of mutant ATXN3 early in SCA3 disease that is transcriptionally, biochemically, and functionally rescued with anti-ATXN3 ASO. Our results highlight the promising use of an ASO therapy across neurodegenerative diseases that requires glial targeting in addition to affected neuronal populations.

Extracellular vesicle-based delivery of silencing sequences for the treatment of Machado-Joseph disease/spinocerebellar ataxia type 3.

Machado-Joseph disease (MJD)/spinocerebellar ataxia type 3 (SCA3) is the most common autosomal dominantly inherited ataxia worldwide. It is caused by an over-repetition of the trinucleotide CAG within the ATXN3 gene, which confers toxic properties to ataxin-3 (ATXN3) species. RNA interference technology has shown promising therapeutic outcomes but still lacks a non-invasive delivery method to the brain. Extracellular vesicles (EVs) emerged as promising delivery vehicles due to their capacity to deliver small nucleic acids, such as microRNAs (miRNAs). miRNAs were found to be enriched into EVs due to specific signal motifs designated as ExoMotifs. In this study, we aimed at investigating whether ExoMotifs would promote the packaging of artificial miRNAs into EVs to be used as non-invasive therapeutic delivery vehicles to treat MJD/SCA3. We found that miRNA-based silencing sequences, associated with ExoMotif GGAG and ribonucleoprotein A2B1 (hnRNPA2B1), retained the capacity to silence mutant ATXN3 (mutATXN3) and were 3-fold enriched into EVs. Bioengineered EVs containing the neuronal targeting peptide RVG on the surface significantly decreased mutATXN3 mRNA in primary cerebellar neurons from MJD YAC 84.2 and in a novel dual-luciferase MJD mouse model upon daily intranasal administration. Altogether, these findings indicate that bioengineered EVs carrying miRNA-based silencing sequences are a promising delivery vehicle for brain therapy.

Effectiveness of High-Frequency Repetitive Transcranial Magnetic Stimulation in Patients With Spinocerebellar Ataxia Type 3.

OBJECTIVE: To investigate the effectiveness of high-frequency repetitive transcranial magnetic stimulation (HF-rTMS) on improvement of clinical symptoms in patients with spinocerebellar ataxia type 3 (SCA3). METHODS: Sixteen SCA3 participants diagnosed by genetic testing were enrolled in this sham-controlled and double-blind trial. They received either a 2-week 10-Hz rTMS intervention or sham stimulation targeting the vermis and cerebellum. The Scale for Assessment and Rating of Ataxia and the International Cooperative Ataxia Rating Scale were completed at baseline and poststimulation. RESULTS: Compared with baseline, the HF-rTMS group demonstrated a significant improvement in the total Scale for Assessment and Rating of Ataxia ( P < 0.0001) and the International Cooperative Ataxia Rating Scale scores ( P = 0.002). After 2-week treatment, the real group exhibited decreasing pattern in 3 subgroups, especially for limb kinetic function ( P < 0.0001). CONCLUSIONS: Short-term HF-rTMS treatment is a potentially promising and feasible tool for rehabilitation in patients with SCA3. Studies with long-term follow-up need to be carried out in the future and further need to assess gait, limb kinetic function, speech and oculomotor disorders.

Production of Spinocerebellar Ataxia Type 3 Model Mice by Intravenous Injection of AAV-PHP.B Vectors.

We aimed to produce a mouse model of spinocerebellar ataxia type 3 (SCA3) using the mouse blood-brain barrier (BBB)-penetrating adeno-associated virus (AAV)-PHP.B. Four-to-five-week-old C57BL/6 mice received injections of high-dose (2.0 × 1011 vg/mouse) or low-dose (5.0 × 1010 vg/mouse) AAV-PHP.B encoding a SCA3 causative gene containing abnormally long 89 CAG repeats [ATXN3(Q89)] under the control of the ubiquitous chicken ß-actin hybrid (CBh) promoter. Control mice received high doses of AAV-PHP.B encoding ATXN3 with non-pathogenic 15 CAG repeats [ATXN3(Q15)] or phosphate-buffered saline (PBS) alone. More than half of the mice injected with high doses of AAV-PHP.B encoding ATXN3(Q89) died within 4 weeks after the injection. No mice in other groups died during the 12-week observation period. Mice injected with low doses of AAV-PHP.B encoding ATXN3(Q89) exhibited progressive motor uncoordination starting 4 weeks and a shorter stride in footprint analysis performed at 12 weeks post-AAV injection. Immunohistochemistry showed thinning of the molecular layer and the formation of nuclear inclusions in Purkinje cells from mice injected with low doses of AAV-PHP.B encoding ATXN3(Q89). Moreover, ATXN3(Q89) expression significantly reduced the number of large projection neurons in the cerebellar nuclei to one third of that observed in mice expressing ATXN3(Q15). This AAV-based approach is superior to conventional methods in that the required number of model mice can be created simply by injecting AAV, and the expression levels of the responsible gene can be adjusted by changing the amount of AAV injected. Moreover, this method may be applied to produce SCA3 models in non-human primates.

Gene editing as a therapeutic strategy for spinocerebellar ataxia type-3.

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease, is a neurodegenerative disease caused by expanded polyglutamine repeats in exon 10 of the ataxin-3 gene, ATXN3. The accumulation of mutant ATXN3 protein leads to severe clinical manifestations and premature death. Clinically, SCA3 pathology is characterized by progressive ataxia leading to motor incoordination that may affect balance, gait and speech, and neuropathologically by a progressive degeneration of the spinal cord and cerebellum, as well as the cerebral cortex and basal ganglia. Although SCA3 is a rare disease, it is the most common autosomal dominant spinocerebellar ataxia worldwide. Its geographical distribution varies worldwide, with peak prevalence in certain regions of Brazil, Portugal and China. In 1994, the identification of the polyglutamine expansion in the ATXN3 gene made it possible not only to diagnose this pathology but also to dissect the mechanisms leading to cellular degeneration. As a monogenic disease for which only symptomatic treatment is available, the ATXN3 gene represents an attractive therapeutic target for gene editing strategies.

Therapeutic effects of engineered exosome-based miR-25 and miR-181a treatment in spinocerebellar ataxia type 3 mice by silencing ATXN3.

BACKGROUND: Spinocerebellar ataxia type 3 (SCA3) is the most common autosomal dominant hereditary ataxia worldwide, which is however in a lack of effective treatment. In view of that engineered exosomes are a promising non-invasive gene therapy transporter that can overcome the traditional problem of poor drug delivery, the aim of this study was to evaluate, for the first time, the value of exosome-based microRNA therapy in SCA3 and the therapeutic effects of intravenously administrated ATXN3 targeting microRNAs in transgenic SCA3 mouse models. METHODS: The rabies virus glycoprotein (RVG) peptide-modified exosomes loaded with miR-25 or miR-181a were peripherally injected to enable targeted delivery of miRNAs to the brain of SCA3 mice. The behaviors, ATXN3 level, purkinje cell and other neuronal loss, and neuroinflammation were evaluated 4 weeks after initial treatment. RESULTS: The targeted and efficient delivery of miR-25 and miR-181a by modified exosomes substantially inhibited the mutant ATXN3 expression, reduced neuron apoptosis and induced motor improvements in SCA3 mouse models without increasing the neuroinflammatory response. CONCLUSIONS: Our study confirmed the therapeutic potential of engineered exosome-based miR-25 and miR-181a treatment in substantially reducing ATXN3 aggregation and cytotoxicity by relying on its targeted and efficient drug delivery performance in SCA3 mice. This treatment method shows a promising prospect for future clinical applications in SCA3.

ULK overexpression mitigates motor deficits and neuropathology in mouse models of Machado-Joseph disease.

Machado-Joseph disease (MJD) is a fatal neurodegenerative disorder clinically characterized by prominent ataxia. It is caused by an expansion of a CAG trinucleotide in ATXN3, translating into an expanded polyglutamine (polyQ) tract in the ATXN3 protein, that becomes prone to misfolding and aggregation. The pathogenesis of the disease has been associated with the dysfunction of several cellular mechanisms, including autophagy and transcription regulation. In this study, we investigated the transcriptional modifications of the autophagy pathway in models of MJD and assessed whether modulating the levels of the affected autophagy-associated transcripts (AATs) would alleviate MJD-associated pathology. Our results show that autophagy is impaired at the transcriptional level in MJD, affecting multiple AATs, including Unc-51 like autophagy activating kinase 1 and 2 (ULK1 and ULK2), two homologs involved in autophagy induction. Reinstating ULK1/2 levels by adeno-associated virus (AAV)-mediated gene transfer significantly improved motor performance while preventing neuropathology in two in vivo models of MJD. Moreover, in vitro studies showed that the observed positive effects may be mainly attributed to ULK1 activity. This study provides strong evidence of the beneficial effect of overexpression of ULK homologs, suggesting these as promising instruments for the treatment of MJD and other neurodegenerative disorders.

Autophagy in Spinocerebellar Ataxia Type 3: From Pathogenesis to Therapeutics.

Machado-Joseph disease (MJD) or spinocerebellar ataxia 3 (SCA3) is a rare, inherited, monogenic, neurodegenerative disease, and the most common SCA worldwide. MJD/SCA3 causative mutation is an abnormal expansion of the triplet CAG at exon 10 within the ATXN3 gene. The gene encodes for ataxin-3, which is a deubiquitinating protein that is also involved in transcriptional regulation. In normal conditions, the ataxin-3 protein polyglutamine stretch has between 13 and 49 glutamines. However, in MJD/SCA3 patients, the size of the stretch increases from 55 to 87, contributing to abnormal protein conformation, insolubility, and aggregation. The formation of aggregates, which is a hallmark of MJD/SCA3, compromises different cell pathways, leading to an impairment of cell clearance mechanisms, such as autophagy. MJD/SCA3 patients display several signals and symptoms in which the most prominent is ataxia. Neuropathologically, the regions most affected are the cerebellum and the pons. Currently, there are no disease-modifying therapies, and patients rely only on supportive and symptomatic treatments. Due to these facts, there is a huge research effort to develop therapeutic strategies for this incurable disease. This review aims to bring together current state-of-the-art strategies regarding the autophagy pathway in MJD/SCA3, focusing on evidence for its impairment in the disease context and, importantly, its targeting for the development of pharmacological and gene-based therapies.

Identification of the calpain-generated toxic fragment of ataxin-3 protein provides new avenues for therapy of Machado-Joseph disease| Spinocerebellar ataxia type 3.

AIMS: Machado-Joseph disease (MJD) is the most frequent dominantly inherited cerebellar ataxia worldwide. Expansion of a CAG trinucleotide in the MJD1 gene translates into a polyglutamine tract within ataxin-3, which upon proteolysis may lead to MJD. The aim of this work was to understand the in vivo contribution of calpain proteases to the pathogenesis of MJD. Therefore, we investigated (a) the calpain cleavage sites in ataxin-3 protein, (b) the most toxic ataxin-3 fragment generated by calpain cleavage and (c) whether targeting calpain cleavage sites of mutant ataxin-3 could be a therapeutic strategy for MJD. METHODS: We generated truncated and calpain-resistant constructs at the predicted cleavage sites of ataxin-3 using inverse PCR mutagenesis. Lentiviral vectors encoding these constructs were transduced in the adult mouse brain prior to western blot and immunohistochemical analysis 5 and 8 weeks later. RESULTS: We identified the putative calpain cleavage sites for both wild-type and mutant ataxin-3 proteins. The mutation of these sites eliminated the formation of the toxic fragments, namely, the 26-kDa fragment, the major contributor for striatal degeneration. Nonetheless, reducing the formation of both the 26- and 34-kDa fragments was required to preclude the intranuclear localisation of ataxin-3. A neuroprotective effect was observed upon mutagenesis of calpain cleavage sites within mutant ataxin-3 protein. CONCLUSIONS: These findings suggest that the calpain system should be considered a target for MJD therapy. The identified calpain cleavage sites will contribute to the design of targeted drugs and genome editing systems for those specific locations.

Mesenchymal stem cells for lysosomal storage and polyglutamine disorders: Possible shared mechanisms.

BACKGROUND: Mesenchymal stem cells' (MSC) therapeutic potential has been investigated for the treatment of several neurodegenerative diseases. The fact these cells can mediate a beneficial effect in different neurodegenerative contexts strengthens their competence to target diverse mechanisms. On the other hand, distinct disorders may share similar mechanisms despite having singular neuropathological characteristics. METHODS: We have previously shown that MSC can be beneficial for two disorders, one belonging to the groups of Lysosomal Storage Disorders (LSDs) - the Krabbe Disease or Globoid Cell Leukodystrophy, and the other to the family of Polyglutamine diseases (PolyQs) - the Machado-Joseph Disease or Spinocerebellar ataxia type 3. We gave also input into disease characterization since neuropathology and MSC's effects are intrinsically associated. This review aims at describing MSC's multimode of action in these disorders while emphasizing to possible mechanistic alterations they must share due to the accumulation of cellular toxic products. RESULTS: Lysosomal storage disorders and PolyQs have different aetiology and associated symptoms, but both result from the accumulation of undegradable products inside neuronal cells due to inefficient clearance by the endosomal/lysosomal pathway. Moreover, numerous cellular mechanisms that become compromised latter are also shared by these two disease groups. CONCLUSIONS: Here, we emphasize MSC's effect in improving proteostasis and autophagy cycling turnover, neuronal survival, synaptic activity and axonal transport. LSDs and PolyQs, though rare in their predominance, collectively affect many people and require our utmost dedication and efforts to get successful therapies due to their tremendous impact on patient s' lives and society.

Machado-Joseph disease/spinocerebellar ataxia type 3: lessons from disease pathogenesis and clues into therapy.

Machado-Joseph disease (MJD), also known as spinocerebellar ataxia type 3 (SCA3), is an incurable disorder, widely regarded as the most common form of spinocerebellar ataxia in the world. MJD/SCA3 arises from mutation of the ATXN3 gene, but this simple monogenic cause contrasts with the complexity of the pathogenic mechanisms that are currently admitted to underlie neuronal dysfunction and death. The aberrantly expanded protein product - ataxin-3 - is known to aggregate and generate toxic species that disrupt several cell systems, including autophagy, proteostasis, transcription, mitochondrial function and signalling. Over the years, research into putative therapeutic approaches has often been devoted to the development of strategies that counteract disease at different stages of cellular pathogenesis. Silencing the pathogenic protein, blocking aggregation, inhibiting toxic proteolytic processing and counteracting dysfunctions of the cellular systems affected have yielded promising ameliorating results in studies with cellular and animal models. The current review analyses the available studies dedicated to the investigation of MJD/SCA3 pathogenesis and the exploration of possible therapeutic strategies, focusing primarily on gene therapy and pharmacological approaches rooted on the molecular and cellular mechanisms of disease.

Cerebellar transcranial direct current stimulation in spinocerebellar ataxia type 3 (SCA3-tDCS): rationale and protocol of a randomized, double-blind, sham-controlled study.

BACKGROUND: Spinocerebellar ataxia type 3 (SCA3) is the most common subtype among the autosomal dominant cerebellar ataxias, a group of neurodegenerative disorders for which currently no disease-specific therapy is available. Evidence-based options for symptomatic treatment of ataxia are also limited. Recent investigations in a heterogeneous group of hereditary and acquired ataxias showed promising, prolonged effects of a two-week course with daily sessions of cerebellar anodal transcranial direct current stimulation (tDCS) on ataxia severity, gait speed, and upper limb dexterity. The aim of the SCA3-tDCS study is to further examine whether tDCS improves ataxia severity and various (cerebellar) non-motor symptoms in a homogeneous cohort of SCA3 patients and to explore the time course of these effects. METHODS/DESIGN: An investigator-initiated, double-blind, randomized, sham-controlled, single-center trial will be conducted. Twenty mildly to moderately affected SCA3 patients (Scale for the Assessment and Rating of Ataxia score between 3 and 20) will be included and randomly assigned in a 1:1 ratio to either cerebellar anodal tDCS or sham cerebellar tDCS. Patients, investigators, and outcome assessors are unaware of treatment allocation. Cerebellar tDCS (20 min, 2 mA, ramp-up and down periods of 30 s each) will be delivered over ten sessions, distributed in two groups of five consecutive days with a two-day break in between. Outcomes are assessed after a single session of tDCS, after the tenth stimulation (T1), and after three, six, and twelve months. The primary outcome measure is the absolute change of the SARA score between baseline and T1. In addition, effects on a variety of other motor and neuropsychological functions in which the cerebellum is known to be involved will be evaluated using quantitative motor tests, static posturography, neurophysiological measurements, cognitive assessment, and questionnaires. DISCUSSION: The results of this study will inform us whether repeated sessions of cerebellar anodal tDCS benefit SCA3 patients and whether this form of non-invasive stimulation might be a novel therapeutic approach to consider in a neurorehabilitation setting. Combined with two earlier controlled trials, a positive effect of the SCA3-tDCS study will encourage implementation of this intervention and stimulate further research in other SCAs and heredodegenerative ataxias. TRIAL REGISTRATION: NL7321 , registered October 8, 2018.

Repeated Mesenchymal Stromal Cell Treatment Sustainably Alleviates Machado-Joseph Disease.

Machado-Joseph disease (MJD) or spinocerebellar ataxia type 3, the most common dominant spinocerebellar ataxia (SCA) worldwide, is caused by over-repetition of a CAG repeat in the ATXN3/MJD1 gene, which translates into a polyglutamine tract within the ataxin-3 protein. There is no treatment for this fatal disorder. Despite evidence of the safety and efficacy of mesenchymal stromal cells (MSCs) in delaying SCA disease progression in exploratory clinical trials, unanticipated regression of patients to the status prior to treatment makes the investigation of causes and solutions urgent and imperative. In the present study, we compared the efficacy of a single intracranial injection with repeated systemic MSC administration in alleviating the MJD phenotype of two strongly severe genetic rodent models. We found that a single MSC transplantation only produces transient effects, whereas periodic administration promotes sustained motor behavior and neuropathology alleviation, suggesting that MSC therapies should be re-designed to get sustained beneficial results in clinical practice. Furthermore, MSC promoted neuroprotection, increased the levels of GABA and glutamate, and decreased the levels of Myo-inositol, which correlated with motor improvements, indicating that these metabolites may serve as valid neurospectroscopic biomarkers of disease and treatment. This study makes important contributions to the design of new clinical approaches for MJD and other SCAs/polyglutamine disorders.

Planning Future Clinical Trials for Machado-Joseph Disease.

Spinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD) is an autosomal dominant multiple neurological systems degenerative disorder caused by a CAG repeat expansion at ATXN3 gene. Only a few treatments were evaluated in randomized clinical trials (RCT) in SCA3/MJD patients, with a lack of evidence for both disease-modifying and symptomatic therapies. The present chapter discuss in detail major methodological issues for planning future RCT for SCA3/MJD. There are several potential therapies for SCA3/MJD with encouraging preclinical results. Route of treatment, dosage titration and potential therapy biomarkers might differ among candidate drugs; however, the core study design and protocol will be mostly the same. RCT against placebo group is the best study design to test a disease-modifying therapy; the same cannot be stated for some symptomatic treatments. Main outcomes for future RCT are clinical scales: the Scale for the Assessment and Rating of ataxia (SARA) is currently the instrument of choice to prove efficacy of disease-modifying or symptomatic treatments against ataxia, the most important disease feature. Ataxia quantitative scales or its composite scores can be used as primary outcomes to provide preliminary evidence of efficacy in phase 2 RCT, due to a greater sensitivity to change. Details regarding eligibility criteria, randomization, sample size estimation, duration and type of analysis for both disease modifying and symptomatic treatment trials, were also discussed. Finally, a section anticipates the methodological issues for testing novel drugs when an effective treatment is already available. We conclude emphasizing four points, the first being the need of RCT for a number of different aims in the care of SCA3/MJD. Due to large sample sizes needed to warrant power, RCT for disease-modifying therapies should be multicenter enterprises. There is an urge need for surrogate markers validated for several drug classes. Finally, engagement of at risk or presymptomatic individuals in future trials will enable major advances on treatment research for SCA3/MJD.

Neuropeptide Y mitigates neuropathology and motor deficits in mouse models of Machado-Joseph disease.

Machado-Joseph disease (MJD) is a fatal, dominantly inherited neurodegenerative disorder associated with an expanded polyglutamine tract within the ataxin-3 protein, and characterized by progressive impairment of motor coordination, associated with neurodegeneration of specific brain regions, including cerebellum and striatum. The currently available therapies do not allow modification of disease progression. Neuropeptide Y (NPY) has been shown to exert potent neuroprotective effects by multiple pathways associated with the MJD mechanisms of disease. Thus, we evaluated NPY levels in MJD and investigated whether raising NPY by gene transfer would alleviate neuropathological and behavioural deficits in cerebellar and striatal mouse models of the disease. For that, a cerebellar transgenic and a striatal lentiviral-based models of MJD were used. NPY overexpression in the affected brain regions in these two mouse models was obtained by stereotaxic injection of adeno-associated viral vectors encoding NPY. Up to 8 weeks after viral injection, balance and motor coordination behaviour and neuropathology were analysed. We observed that NPY levels were decreased in two MJD patients' cerebella and in striata and cerebella of disease mouse models. Furthermore, overexpression of NPY alleviated the motor coordination impairments and attenuated the related neuropathological parameters, preserving cerebellar volume and granular layer thickness, reducing striatal lesion and decreasing mutant ataxin-3 aggregation. Additionally, NPY mediated increase of brain-derived neurotrophic factor levels and decreased neuroinflammation markers. Our data suggest that NPY is a potential therapeutic strategy for MJD.

Re-establishing ataxin-2 downregulates translation of mutant ataxin-3 and alleviates Machado-Joseph disease.

Machado-Joseph disease is a progressive neurodegenerative disorder associated with the polyQ-expanded ataxin-3 (encoded by ATXN3), for which no therapy is available. With the aim of clarifying the mechanism of neurodegeneration, we hypothesized that the abnormally long polyQ tract would interact aberrantly with ataxin-2 (encoded by ATXN2), another polyQ protein whose function has recently been linked to translational regulation. Using patient's samples and cellular and animal's models we found that in Machado-Joseph disease: (i) ataxin-2 levels are reduced; and (ii) its subcellular localization is changed towards the nucleus. Restoring ataxin-2 levels by lentiviral-mediated overexpression: (i) reduced mutant ataxin-3 levels; and (ii) rescued behaviour defects and neuropathology in a transgenic mouse model of Machado-Joseph disease. Conversely (i) mutating the ataxin-2 motif that enables binding to its natural interactor and translation activator poly(A)-binding protein; or (ii) overexpressing poly(A)-binding protein, had opposite effects, increasing mutant ataxin-3 translation and aggregation. This work suggests that in Machado-Joseph disease, mutant ataxin-3 drives an abnormal reduction of ataxin-2 levels, which overactivates poly(A)-binding protein, increases translation of mutant ataxin-3 and other proteins and aggravates Machado-Joseph disease. Re-establishment of ataxin-2 levels reduces mutant ataxin-3 and alleviates Machado-Joseph disease pathogenesis opening a new avenue for therapeutic intervention in this and potentially other polyQ disorders.

Transplantation of cerebellar neural stem cells improves motor coordination and neuropathology in Machado-Joseph disease mice.

Machado-Joseph disease is a neurodegenerative disease without effective treatment. Patients with Machado-Joseph disease exhibit significant motor impairments such as gait ataxia, associated with multiple neuropathological changes including mutant ATXN3 inclusions, marked neuronal loss and atrophy of the cerebellum. Thus, an effective treatment of symptomatic patients with Machado-Joseph disease may require cell replacement, which we investigated in this study. For this purpose, we injected cerebellar neural stem cells into the cerebellum of adult Machado-Joseph disease transgenic mice and assessed the effect on the neuropathology, neuroinflammation mediators and neurotrophic factor levels and motor coordination. We found that upon transplantation into the cerebellum of adult Machado-Joseph disease mice, cerebellar neural stem cells differentiate into neurons, astrocytes and oligodendrocytes. Importantly, cerebellar neural stem cell transplantation mediated a significant and robust alleviation of the motor behaviour impairments, which correlated with preservation from Machado-Joseph disease-associated neuropathology, namely reduction of Purkinje cell loss, reduction of cellular layer shrinkage and mutant ATXN3 aggregates. Additionally, a significant reduction of neuroinflammation and an increase of neurotrophic factors levels was observed, indicating that transplantation of cerebellar neural stem cells also triggers important neuroprotective effects. Thus, cerebellar neural stem cells have the potential to be used as a cell replacement and neuroprotective approach for Machado-Joseph disease therapy.

Hsp104 suppresses polyglutamine-induced degeneration post onset in a drosophila MJD/SCA3 model.

There are no effective therapeutics that antagonize or reverse the protein-misfolding events underpinning polyglutamine (PolyQ) disorders, including Spinocerebellar Ataxia Type-3 (SCA3). Here, we augment the proteostasis network of Drosophila SCA3 models with Hsp104, a powerful protein disaggregase from yeast, which is bafflingly absent from metazoa. Hsp104 suppressed eye degeneration caused by a C-terminal ataxin-3 (MJD) fragment containing the pathogenic expanded PolyQ tract, but unexpectedly enhanced aggregation and toxicity of full-length pathogenic MJD. Hsp104 suppressed toxicity of MJD variants lacking a portion of the N-terminal deubiquitylase domain and full-length MJD variants unable to engage polyubiquitin, indicating that MJD-ubiquitin interactions hinder protective Hsp104 modalities. Importantly, in staging experiments, Hsp104 suppressed toxicity of a C-terminal MJD fragment when expressed after the onset of PolyQ-induced degeneration, whereas Hsp70 was ineffective. Thus, we establish the first disaggregase or chaperone treatment administered after the onset of pathogenic protein-induced degeneration that mitigates disease progression.

An insight into advances in the pathogenesis and therapeutic strategies of spinocerebellar ataxia type 3.

Abstract Spinocerebellar ataxia type 3 (SCA3) is the most common type of spinocerebellar ataxia, which are inherited neurodegenerative diseases. CAG repeat expansions that translate into an abnormal length of glutamine residues are considered to be the disease-causing mutation. The pathological mechanisms of SCA3 are not fully elucidated but may include aggregate or inclusion formation, imbalance of cellular protein homeostasis, axonal transportation dysfunction, translation dysregulation, mitochondrial damage and oxidative stress, abnormal neural signaling pathways, etc. Currently, symptom relief is the only available therapeutic route; however, promising therapeutic targets have been discovered, such as decreasing the mutant protein through RNA interference (RNAi) and antisense oligonucleotides (AONs) and replacement therapy using stem cell transplantation. Other potential targets can inhibit the previously mentioned pathological mechanisms. However, additional efforts are necessary before these strategies can be used clinically.

Toward RNAi therapy for the polyglutamine disease Machado-Joseph disease.

Machado-Joseph disease (MJD) is a dominantly inherited ataxia caused by a polyglutamine-coding expansion in the ATXN3 gene. Suppressing expression of the toxic gene product represents a promising approach to therapy for MJD and other polyglutamine diseases. We performed an extended therapeutic trial of RNA interference (RNAi) targeting ATXN3 in a mouse model expressing the full human disease gene and recapitulating key disease features. Adeno-associated virus (AAV) encoding a microRNA (miRNA)-like molecule, miRATXN3, was delivered bilaterally into the cerebellum of 6- to 8-week-old MJD mice, which were then followed up to end-stage disease to assess the safety and efficacy of anti-ATXN3 RNAi. Despite effective, lifelong suppression of ATXN3 in the cerebellum and the apparent safety of miRATXN3, motor impairment was not ameliorated in treated MJD mice and survival was not prolonged. These results with an otherwise effective RNAi agent suggest that targeting a large extent of the cerebellum alone may not be sufficient for effective human therapy. Artificial miRNAs or other nucleotide-based suppression strategies targeting ATXN3 more widely in the brain should be considered in future preclinical tests.