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.

Machado-Joseph disease (MJD) or spinocerebellar ataxia type 3 (SCA3) is an autosomal dominantly-inherited neurodegenerative disorder caused by the over-repetition of a CAG codon in the MJD1 gene. This expansion translates into a polyglutamine tract that confers a toxic gain-of-function to the mutant protein – ataxin-3, leading to neurodegeneration in specific brain regions, with particular severity in the cerebellum. No treatment able to modify the disease progression is available. However, gene silencing by RNA interference has shown promising results. Therefore, in this study we investigated whether lentiviral-mediated allele-specific silencing of the mutant ataxin-3 gene, after disease onset, would rescue the motor behavior deficits and neuropathological features in a severely impaired transgenic mouse model of MJD. For this purpose, we injected lentiviral vectors encoding allele-specific silencing-sequences (shAtx3) into the cerebellum of diseased transgenic mice expressing the targeted C-variant of mutant ataxin-3 present in 70% of MJD patients. This variation permits to discriminate between the wild-type and mutant forms, maintaining the normal function of the wild-type allele and silencing only the mutant form. Quantitative analysis of rotarod performance, footprint and activity patterns revealed significant and robust alleviation of gait, balance (average 3-fold increase of rotarod test time), locomotor and exploratory activity impairments in shAtx3-injected mice, as compared to control ones injected with shGFP. An important improvement of neuropathology was also observed, regarding the number of intranuclear inclusions, calbindin and DARPP-32 immunoreactivity, fluorojade B and Golgi staining and molecular and granular layers thickness. These data demonstrate for the first time the efficacy of gene silencing in blocking the MJD-associated motor-behavior and neuropathological abnormalities after the onset of the disease, supporting the use of this strategy for therapy of MJD.

Machado-Joseph disease or spinocerebellar ataxia type 3, the most common dominantly-inherited spinocerebellar ataxia, results from translation of the polyglutamine-expanded and aggregation prone ataxin 3 protein. Clinical manifestations include cerebellar ataxia and pyramidal signs and there is no therapy to delay disease progression. Beclin 1, an autophagy-related protein and essential gene for cell survival, is decreased in several neurodegenerative disorders. This study aimed at evaluating if lentiviral-mediated beclin 1 overexpression would rescue motor and neuropathological impairments when administered to pre- and post-symptomatic lentiviral-based and transgenic mouse models of Machado-Joseph disease. Beclin 1-mediated significant improvements in motor coordination, balance and gait with beclin 1-treated mice equilibrating longer periods in the Rotarod and presenting longer and narrower footprints. Furthermore, in agreement with the improvements observed in motor function beclin 1 overexpression prevented neuronal dysfunction and neurodegeneration, decreasing formation of polyglutamine-expanded aggregates, preserving Purkinje cell arborization and immunoreactivity for neuronal markers. These data show that overexpression of beclin 1 in the mouse cerebellum is able to rescue and hinder the progression of motor deficits when administered to pre- and post-symptomatic stages of the disease.

Machado Joseph Disease (MJD) is the most frequent autosomal dominantly inherited cerebellar ataxia caused by the over-repetition of a CAG trinucleotide in the ATXN3 gene. This expansion translates into a polyglutamine tract within the ataxin-3 protein that confers a toxic gain-of-function to the mutant protein ataxin-3, contributing to protein misfolding and intracellular accumulation of aggregates and neuronal degeneration. Autophagy impairment has been shown to be one of the mechanisms that contribute for the MJD phenotype. Here we investigated whether this phenotype was present in patient-derived fibroblasts, a common somatic cell type used in the derivation of induced pluripotent stem cells and subsequent differentiation into neurons, for in vitro disease modeling. We generated and studied adult dermal fibroblasts from 5 MJD patients and 4 healthy individuals and we found that early passage MJD fibroblasts exhibited autophagy impairment with an underlying mechanism of decreased autophagosome production. The overexpression of beclin-1 on MJD fibroblasts reverted partially autophagy impairment by increasing the autophagic flux but failed to increase the levels of autophagosome production. Overall, our results provide a well-characterized MJD fibroblast resource for neurodegenerative disease research and contribute for the understanding of mutant ataxin-3 biology and its molecular consequences.

Machado-Joseph disease (MJD), also known as spinocerebellar ataxia type 3 (SCA3), is a fatal, dominant neurodegenerative disorder caused by the polyglutamine-expanded protein ataxin-3. Clinical manifestations include cerebellar ataxia and pyramidal signs culminating in severe neuronal degeneration. Currently, there is no therapy able to modify disease progression. In the present study, we aimed at investigating one of the most severely affected brain regions in the disorder--the cerebellum--and the behavioral defects associated with the neuropathology in this region. For this purpose, we injected lentiviral vectors encoding full-length human mutant ataxin-3 in the mouse cerebellum of 3-week-old C57/BL6 mice. We show that circumscribed expression of human mutant ataxin-3 in the cerebellum mediates within a short time frame--6 weeks, the development of a behavioral phenotype including reduced motor coordination, wide-based ataxic gait, and hyperactivity. Furthermore, the expression of mutant ataxin-3 resulted in the accumulation of intranuclear inclusions, neuropathological abnormalities, and neuronal death. These data show that lentiviral-based expression of mutant ataxin-3 in the mouse cerebellum induces localized neuropathology, which is sufficient to generate a behavioral ataxic phenotype. Moreover, this approach provides a physiologically relevant, cost-effective and time-effective animal model to gain further insights into the pathogenesis of MJD and for the evaluation of experimental therapeutics of MJD.

Mitochondrial deregulation has gained increasing support as a pathological mechanism in Huntington’s disease (HD), a genetic-based neurodegenerative disorder caused by CAG expansion in the HTT gene. In this study, we thoroughly investigated mitochondrial-based mechanisms in HD patient-derived iPSC (HD-iPSC) and differentiated neural stem cells (NSC) versus control cells, as well as in cells subjected to CRISPR/Cas9-CAG repeat deletion. We analyzed mitochondrial morphology, function and biogenesis, linked to exosomal release of mitochondrial components, glycolytic flux, ATP generation and cellular redox status. Mitochondria in HD cells exhibited round shape and fragmented morphology. Functionally, HD-iPSC and HD-NSC displayed lower mitochondrial respiration, exosomal release of cytochrome c, decreased ATP/ADP, reduced PGC-1α and complex III subunit expression and activity, and were highly dependent on glycolysis, supported by pyruvate dehydrogenase (PDH) inactivation. HD-iPSC and HD-NSC mitochondria showed ATP synthase reversal and increased calcium retention. Enhanced mitochondrial reactive oxygen species (ROS) were also observed in HD-iPSC and HD-NSC, along with decreased UCP2 mRNA levels. CRISPR/Cas9-CAG repeat deletion in HD-iPSC and derived HD-NSC ameliorated mitochondrial phenotypes. Data attests for intricate metabolic and mitochondrial dysfunction linked to transcriptional deregulation as early events in HD pathogenesis, which are alleviated following CAG deletion.

Machado-Joseph disease or Spinocerebellar ataxia type 3 is a progressive fatal neurodegenerative disorder caused by the polyglutamine-expanded protein ataxin-3. Recent studies demonstrate that RNA interference is a promising approach for the treatment of Machado-Joseph disease. However, whether gene silencing at an early time-point is able to prevent the appearance of motor behavior deficits typical of the disease when initiated before onset of the disease had not been explored. Here, using a lentiviral-mediated allele-specific silencing of mutant ataxin-3 in an early pre-symptomatic cerebellar mouse model of Machado-Joseph disease we show that this strategy hampers the development of the motor and neuropathological phenotypic characteristics of the disease. At the histological level, the RNA-specific silencing of mutant ataxin-3 decreased formation of mutant ataxin-3 aggregates, preserved Purkinje cell morphology and expression of neuronal markers while reducing cell death. Importantly, gene silencing prevented the development of impairments in balance, motor coordination, gait and hyperactivity observed in control mice. These data support the therapeutic potential of RNA interference for Machado-Joseph disease and constitute a proof of principle of the beneficial effects of early allele-specific silencing for therapy of this disease.

Machado-Joseph disease (MJD) is a genetic neurodegenerative disease caused by an expanded polyglutamine tract within the protein ataxin-3 (ATXN3). Despite current efforts, MJD's mechanism of pathogenesis remains unclear and no disease-modifying treatment is available. Therefore, in this study, we investigated (1) the role of the 3' UTR of ATXN3, a putative microRNA (miRNA) target, (2) whether miRNA biogenesis and machinery are dysfunctional in MJD, and (3) which specific miRNAs target ATXN3-3' UTR and whether they can alleviate MJD neuropathology in vivo. Our results demonstrate that endogenous miRNAs, by targeting sequences in the 3' UTR, robustly reduce ATXN3 expression and aggregation in vitro and neurodegeneration and neuroinflammation in vivo. Importantly, we found an abnormal MJD-associated downregulation of genes involved in miRNA biogenesis and silencing activity. Finally, we identified three miRNAs-mir-9, mir-181a, and mir-494-that interact with the ATXN3-3' UTR and whose expression is dysregulated in human MJD neurons and in other MJD cell and animal models. Furthermore, overexpression of these miRNAs in mice resulted in reduction of mutATXN3 levels, aggregate counts, and neuronal dysfunction. Altogether, these findings indicate that endogenous miRNAs and the 3' UTR of ATXN3 play a crucial role in MJD pathogenesis and provide a promising opportunity for MJD treatment.