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Spinocerebellar ataxia type 3 (SCA3)/Machado-Joseph disease (MJD) is a heritable proteinopathy disorder, whose causative gene, ATXN3, undergoes alternative splicing. Ataxin-3 protein isoforms differ in their toxicity, suggesting that certain ATXN3 splice variants may be crucial in driving the selective toxicity in SCA3. Using RNA-seq datasets we identified and determined the abundance of annotated ATXN3 transcripts in blood (n = 60) and cerebellum (n = 12) of SCA3 subjects and controls. The reference transcript (ATXN3-251), translating into an ataxin-3 isoform harbouring three ubiquitin-interacting motifs (UIMs), showed the highest abundance in blood, while the most abundant transcript in the cerebellum (ATXN3-208) was of unclear function. Noteworthy, two of the four transcripts that encode full-length ataxin-3 isoforms but differ in the C-terminus were strongly related with tissue expression specificity: ATXN3-251 (3UIM) was expressed in blood 50-fold more than in the cerebellum, whereas ATXN3-214 (2UIM) was expressed in the cerebellum 20-fold more than in the blood. These findings shed light on ATXN3 alternative splicing, aiding in the comprehension of SCA3 pathogenesis and providing guidance in the design of future ATXN3 mRNA-lowering therapies., Competing Interests: Declaration of competing interest TK is receiving research support from the Bundesministerium für Bildung und Forschung (BMBF), the National Institutes of Health (NIH) and Servier. Within the last 24 months, he has received consulting fees from Biogen, UCB and Vico Therapeutics. BvdW is supported by grants from ZonMw, NOW, Hersenstichting, Gossweiler Foundation, and Radboud university medical center; he has served on the scientific advisory boards or steering committees of uniQure, VICO Therapeutics, and Servier. LS is receiving research support from the European Commission, the Bundesministerium für Bildung und Forschung (BMBF) and the Bundesministerium für Gesundheit (BMG) as well as the Deutsche Forschungsgemeinschaft (DFG), Servier and Vigil Neuroscience. Within the last 24 months, he has received consulting fees from Vico Therapeutics. The remaining authors declare that they have nothing to report., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Transcriptional dysregulation has been described in spinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD), an autosomal dominant ataxia caused by a polyglutamine expansion in the ataxin-3 protein. As ataxin-3 is ubiquitously expressed, transcriptional alterations in blood may reflect early changes that start before clinical onset and might serve as peripheral biomarkers in clinical and research settings. Our goal was to describe enriched pathways and report dysregulated genes, which can track disease onset, severity or progression in carriers of the ATXN3 mutation (pre-ataxic subjects and patients). Global dysregulation patterns were identified by RNA sequencing of blood samples from 40 carriers of ATXN3 mutation and 20 controls and further compared with transcriptomic data from post-mortem cerebellum samples of MJD patients and controls. Ten genes-ABCA1, CEP72, PTGDS, SAFB2, SFSWAP, CCDC88C, SH2B1, LTBP4, MEG3 and TSPOAP1-whose expression in blood was altered in the pre-ataxic stage and simultaneously, correlated with ataxia severity in the overt disease stage, were analysed by quantitative real-time PCR in blood samples from an independent set of 170 SCA3/MJD subjects and 57 controls. Pathway enrichment analysis indicated the Gαi signalling and the oestrogen receptor signalling to be similarly affected in blood and cerebellum. SAFB2, SFSWAP and LTBP4 were consistently dysregulated in pre-ataxic subjects compared to controls, displaying a combined discriminatory ability of 79%. In patients, ataxia severity was associated with higher levels of MEG3 and TSPOAP1. We propose expression levels of SAFB2, SFSWAP and LTBP4 as well as MEG3 and TSPOAP1 as stratification markers of SCA3/MJD progression, deserving further validation in longitudinal studies and in independent cohorts., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominantly inherited neurodegenerative disease, which belongs to the trinucleotide repeat disease group with a CAG repeat expansion in exon 1 of the ATXN2 gene resulting in an ataxin-2 protein with an expanded polyglutamine (polyQ)-stretch. The disease is late manifesting leading to early death. Today, therapeutic interventions to cure the disease or even to decelerate disease progression are not available yet. Furthermore, primary readout parameter for disease progression and therapeutic intervention studies are limited. Thus, there is an urgent need for quantifiable molecular biomarkers such as ataxin-2 becoming even more important due to numerous potential protein-lowering therapeutic intervention strategies. The aim of this study was to establish a sensitive technique to measure the amount of soluble polyQ-expanded ataxin-2 in human biofluids to evaluate ataxin-2 protein levels as prognostic and/or therapeutic biomarker in SCA2. Time-resolved fluorescence energy transfer (TR-FRET) was used to establish a polyQ-expanded ataxin-2-specific immunoassay. Two different ataxin-2 antibodies and two different polyQ-binding antibodies were validated in three different concentrations and tested in cellular and animal tissue as well as in human cell lines, comparing different buffer conditions to evaluate the best assay conditions. We established a TR-FRET-based immunoassay for soluble polyQ-expanded ataxin-2 and validated measurements in human cell lines including iPSC-derived cortical neurons. Additionally, our immunoassay was sensitive enough to monitor small ataxin-2 expression changes by siRNA or starvation treatment. We successfully established the first sensitive ataxin-2 immunoassay to measure specifically soluble polyQ-expanded ataxin-2 in human biomaterials., (© 2023. The Author(s).)

Background: Non-motor symptoms (NMS) are a substantial burden for patients with SCA3. There are limited data on their frequency, and their relation with disease severity and activities of daily living is not clear. In addition, lifestyle may either influence or be affected by the occurrence of NMS., Objective: To characterize NMS in SCA3 and investigate possible associations with disease severity and lifestyle factors., Methods: In a prospective cohort study, we performed a cross-sectional analysis of NMS in 227 SCA3 patients, 42 pre-ataxic mutation carriers, and 112 controls and tested for associations with SARA score, activities of daily living, and the lifestyle factors alcohol consumption, smoking and physical activity., Results: Sleep disturbance, restless legs syndrome, mild cognitive impairment, depression, bladder dysfunction and pallhypesthesia were frequent among SCA3 patients, while mainly absent in pre-ataxic mutation carriers. Except for restless legs syndrome, NMS correlated significantly with disease severity and activities of daily living. Alcohol abstinence was associated with bladder dysfunction. Patients with higher physical activity showed less cognitive impairment and fewer depressive symptoms, but these differences were not significant., Conclusion: This study revealed a clear association between disease severity and NMS, likely driven by the progression of the widespread neurodegenerative process. Associations between lifestyle and NMS can probably be attributed to the influence of NMS on lifestyle., (© 2022. The Author(s).)

Background: Disease severity in spinocerebellar ataxia type 3 (SCA3) is commonly defined by the Scale for the Assessment and Rating of Ataxia (SARA) sum score, but little is known about the contributions and progression patterns of individual items., Objectives: To investigate the temporal dynamics of SARA item scores in SCA3 patients and evaluate if clinical and demographic factors are differentially associated with evolution of axial and appendicular ataxia., Methods: In a prospective, multinational cohort study involving 11 European and 2 US sites, SARA scores were determined longitudinally in 223 SCA3 patients with a follow-up assessment after 1 year., Results: An increase in SARA score from 10 to 20 points was mainly driven by axial and speech items, with a markedly smaller contribution of appendicular items. Finger chase and nose-finger test scores not only showed the lowest variability at baseline, but also the least deterioration at follow-up. Compared with the full set of SARA items, omission of both tests would result in lower sample size requirements for therapeutic trials. Sex was associated with change in SARA sum score and appendicular, but not axial, subscore, with a significantly faster progression in men. Despite considerable interindividual variability, the average annual progression rate of SARA score was approximately three times higher in subjects with a disease duration over 10 years than in those within 10 years from onset., Conclusion: Our findings provide evidence for a difference in temporal dynamics between axial and appendicular ataxia in SCA3 patients, which will help inform the design of clinical trials and development of new (etiology-specific) outcome measures. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society., (© 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.)

Background and Purpose: Clinical trials in spinocerebellar ataxia type 3 (SCA3) will require biomarkers for use as outcome measures., Methods: To evaluate total tau (t-tau), glial fibrillary acidic protein (GFAP), ubiquitin carboxy-terminal hydrolase L1 (UCHL1) and neurofilament light-chain (NfL) as fluid biomarkers in SCA3, ATXN3 mutation carriers (n = 143) and controls (n = 172) were clinically assessed, and the plasma concentrations of the four proteins were analysed on the Simoa HD-1 platform. Eleven ATXN3 mutation carrier cerebrospinal fluid samples were analysed for t-tau and phosphorylated tau (p-tau 181 ). A transgenic SCA3 mouse model (MJDTg) was used to measure cerebellar t-tau levels., Results: Plasma t-tau levels were higher in mutation carriers below the age of 50 compared to controls, and the Inventory of Non-Ataxia Signs was associated with t-tau in ataxic patients (p = 0.004). Pre-ataxic carriers showed higher cerebrospinal fluid t-tau and p-tau 181 concentrations compared to ataxic patients (p = 0.025 and p = 0.014, respectively). Cerebellar t-tau was elevated in MJDTg mice compared to wild-type (p = 0.033) only in the early stages of the disease. GFAP and UCHL1 did not show higher levels in mutation carriers compared to controls. Plasma NfL concentrations were higher in mutation carriers compared to controls, and differences were greater for younger carriers. The Scale for the Assessment and Rating of Ataxia was the strongest predictor of NfL in ataxic patients (p < 0.001)., Conclusion: Our results suggest that tau might be a marker of early disease stages in SCA3. NfL can discriminate mutation carriers from controls and is associated with different clinical variables. Longitudinal studies are required to confirm their potential role as biomarkers in clinical trials., (© 2022 The Authors. European Journal of Neurology published by John Wiley & Sons Ltd on behalf of European Academy of Neurology.)

Machado-Joseph disease (MJD) is characterized by a pathological expansion of the polyglutamine (polyQ) tract within the ataxin-3 protein. Despite its primarily cytoplasmic localization, polyQ-expanded ataxin-3 accumulates in the nucleus and forms intranuclear aggregates in the affected neurons. Due to these histopathological hallmarks, the nucleocytoplasmic transport machinery has garnered attention as an important disease relevant mechanism. Here, we report on MJD cell model-based analysis of the nuclear transport receptor karyopherin subunit beta-1 (KPNB1) and its implications in the molecular pathogenesis of MJD. Although directly interacting with both wild-type and polyQ-expanded ataxin-3, modulating KPNB1 did not alter the intracellular localization of ataxin-3. Instead, overexpression of KPNB1 reduced ataxin-3 protein levels and the aggregate load, thereby improving cell viability. On the other hand, its knockdown and inhibition resulted in the accumulation of soluble and insoluble ataxin-3. Interestingly, the reduction of ataxin-3 was apparently based on protein fragmentation independent of the classical MJD-associated proteolytic pathways. Label-free quantitative proteomics and knockdown experiments identified mitochondrial protease CLPP as a potential mediator of the ataxin-3-degrading effect induced by KPNB1. We confirmed reduction of KPNB1 protein levels in MJD by analyzing two MJD transgenic mouse models and induced pluripotent stem cells (iPSCs) derived from MJD patients. Our results reveal a yet undescribed regulatory function of KPNB1 in controlling the turnover of ataxin-3, thereby highlighting a new potential target of therapeutic value for MJD., (© 2022. The Author(s).)

Dysfunctional mitochondria are linked to several neurodegenerative diseases. Metabolic defects, a symptom which can result from dysfunctional mitochondria, are also present in spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease, the most frequent, dominantly inherited neurodegenerative ataxia worldwide. Mitochondrial dysfunction has been reported for several neurodegenerative disorders and ataxin-3 is known to deubiquitinylate parkin, a key protein required for canonical mitophagy. In this study, we analyzed mitochondrial function and mitophagy in a patient-derived SCA3 cell model. Human fibroblast lines isolated from SCA3 patients were immortalized and characterized. SCA3 patient fibroblasts revealed circular, ring-shaped mitochondria and featured reduced OXPHOS complexes, ATP production and cell viability. We show that wildtype ataxin-3 deubiquitinates VDAC1 (voltage-dependent anion channel 1), a member of the mitochondrial permeability transition pore and a parkin substrate. In SCA3 patients, VDAC1 deubiquitination and parkin recruitment to the depolarized mitochondria is inhibited. Increased p62-linked mitophagy, autophagosome formation and autophagy is observed under disease conditions, which is in line with mitochondrial fission. SCA3 fibroblast lines demonstrated a mitochondrial phenotype and dysregulation of parkin-VDAC1-mediated mitophagy, thereby promoting mitochondrial quality control via alternative pathways.

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph Disease, is a progressive neurodegenerative disorder characterized by loss of neuronal matter due to the expansion of the CAG repeat in the ATXN3/MJD1 gene and subsequent ataxin-3 protein. Although the underlying pathogenic protein expansion has been known for more than 20 years, the complexity of its effects is still under exploration. The ataxin-3 protein in its expanded form is known to aggregate and disrupt cellular processes in neuronal tissue but the role of the protein on populations of immune cells is unknown. Recently, mast cells have emerged as potential key players in neuroinflammation and neurodegeneration. Here, we examined the mast cell-related effects of ataxin-3 expansion in the brain tissues of 304Q ataxin-3 knock-in mice and SCA3 patients. We also established cultures of mast cells from the 304Q knock-in mice and examined the effects of 304Q ataxin-3 knock-in on the immune responses of these cells and on markers involved in mast cell growth, development and function. Specifically, our results point to a role for expanded ataxin-3 in suppression of mast cell marker CD117/c-Kit, pro-inflammatory cytokine TNF-α and NF-κB inhibitor IκBα along with an increased expression of the granulocyte-attracting chemokine CXCL1. These results are the beginning of a more holistic understanding of ataxin-3 and could point to the development of novel therapeutic targets which act on inflammation to mitigate symptoms of SCA3., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sowa, Haas, Hübener-Schmid and Lorentz.)

Background: Lifestyle could influence the course of hereditary ataxias, but representative data are missing., Objective: The objective of this study was to characterize lifestyle in spinocerebellar ataxia type 3 (SCA3) and investigate possible associations with disease parameters., Methods: In a prospective cohort study, data on smoking, alcohol consumption, physical activity, physiotherapy, and body mass index (BMI) were collected from 243 patients with SCA3 and 119 controls and tested for associations with age of onset, disease severity, and progression., Results: Compared with controls, patients with SCA3 were less active and consumed less alcohol. Less physical activity and alcohol abstinence were associated with more severe disease, but not with progression rates or age of onset. Smoking, BMI, or physiotherapy did not correlate with disease parameters., Conclusion: Differences in lifestyle factors of patients with SCA3 and controls as well as associations of lifestyle factors with disease severity are likely driven by the influence of symptoms on behavior. No association between lifestyle and disease progression was detected. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society., (© 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.)

Spinocerebellar ataxia type 3 is the most common autosomal dominant inherited ataxia worldwide, caused by a CAG repeat expansion in the Ataxin-3 gene resulting in a polyglutamine (polyQ)-expansion in the corresponding protein. The disease is characterized by neuropathological, phenotypical, and specific transcriptional changes in affected brain regions. So far, there is no mouse model available representing all the different aspects of the disease, yet highly needed for a better understanding of the disease pathomechanisms. Here, we characterized a novel Ataxin-3 knock-in mouse model, expressing a heterozygous or homozygous expansion of 304 CAACAGs in the murine Ataxin-3 locus using biochemical, behavioral, and transcriptomic approaches. We compared neuropathological, and behavioral features of the new knock-in model with the in SCA3 research mostly used YAC84Q mouse model. Further, we compared transcriptional changes found in cerebellar samples of the SCA3 knock-in mice and post-mortem human SCA3 patients. The novel knock-in mouse is characterized by the expression of a polyQ-expansion in the murine Ataxin-3 protein, leading to aggregate formation, especially in brain regions known to be vulnerable in SCA3 patients, and impairment of Purkinje cells. Along these neuropathological changes, the mice showed a reduction in body weight accompanied by gait and balance instability. Transcriptomic analysis of cerebellar tissue revealed age-dependent differential expression, enriched for genes attributed to myelinating oligodendrocytes. Comparing these changes with those found in cerebellar tissue of SCA3 patients, we discovered an overlap of differentially expressed genes pointing towards similar gene expression perturbances in several genes linked to myelin sheaths and myelinating oligodendrocytes., (© 2021. The Author(s).)

Aberrant O -GlcNAcylation, a protein posttranslational modification defined by the O -linked attachment of the monosaccharide N -acetylglucosamine ( O -GlcNAc), has been implicated in neurodegenerative diseases. However, although many neuronal proteins are substrates for O -GlcNAcylation, this process has not been extensively investigated in polyglutamine disorders. We aimed to evaluate the enzyme O -GlcNAc transferase (OGT), which attaches O -GlcNAc to target proteins, in Machado-Joseph disease (MJD). MJD is a neurodegenerative condition characterized by ataxia and caused by the expansion of a polyglutamine stretch within the deubiquitinase ataxin-3, which then present increased propensity to aggregate. By analyzing MJD cell and animal models, we provide evidence that OGT is dysregulated in MJD, therefore compromising the O -GlcNAc cycle. Moreover, we demonstrate that wild-type ataxin-3 modulates OGT protein levels in a proteasome-dependent manner, and we present OGT as a substrate for ataxin-3. Targeting OGT levels and activity reduced ataxin-3 aggregates, improved protein clearance and cell viability, and alleviated motor impairment reminiscent of ataxia of MJD patients in zebrafish model of the disease. Taken together, our results point to a direct interaction between OGT and ataxin-3 in health and disease and propose the O -GlcNAc cycle as a promising target for the development of therapeutics in the yet incurable MJD., Competing Interests: The authors declare no competing interest.

Spinocerebellar ataxia type 3 (SCA3) is caused by an expanded polyglutamine stretch in ataxin-3. While wild-type ataxin-3 has important functions, e.g., as a deubiquitinase, downregulation of mutant ataxin-3 is likely to slow down the course of this fatal disease. We established a screening platform with human neurons of patients and controls derived from induced pluripotent stem cells to test antisense oligonucleotides (ASOs) for their effects on ataxin-3 expression. We identified an ASO that suppressed mutant and wild-type ataxin-3 levels by >90% after a singular treatment. Next, we screened pairs of ASOs designed to selectively target the mutant or the wild-type allele by taking advantage of a SNP (c.987G > C) in ATXN3 that is present in most SCA3 patients. We found ASOmut4 to reduce levels of mutant ataxin-3 by 80% after 10 days while leaving expression of wild-type ataxin-3 largely unaffected. In a long-term study we proved this effect to last for about 4 weeks after a single treatment without signs of neurotoxicity. This study provides proof of principle that allele-specific lowering of poly(Q)-expanded ataxin-3 by selective ASOs is feasible and long lasting, with sparing of wild-type ataxin-3 expression in a human cell culture model that is genetically identical to SCA3 patients., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

Background: Spinocerebellar ataxia type 3 is a rare neurodegenerative disease caused by a CAG repeat expansion in the ataxin-3 gene. Although no curative therapy is yet available, preclinical gene-silencing approaches to reduce polyglutamine (polyQ) toxicity demonstrate promising results. In view of upcoming clinical trials, quantitative and easily accessible molecular markers are of critical importance as pharmacodynamic and particularly as target engagement markers., Objective: We aimed at developing an ultrasensitive immunoassay to measure specifically polyQ-expanded ataxin-3 in plasma and cerebrospinal fluid (CSF)., Methods: Using the novel single molecule counting ataxin-3 immunoassay, we analyzed cross-sectional and longitudinal patient biomaterials., Results: Statistical analyses revealed a correlation with clinical parameters and a stability of polyQ-expanded ataxin-3 during conversion from the pre-ataxic to the ataxic phases., Conclusions: The novel immunoassay is able to quantify polyQ-expanded ataxin-3 in plasma and CSF, whereas ataxin-3 levels in plasma correlate with disease severity. Longitudinal analyses demonstrated a high stability of polyQ-expanded ataxin-3 over a short period. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society., (© 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.)

The worldwide development of twin cohorts began after World War II. These cohorts now include around 1.5 million twins, and more than 2748 twin studies have been published between 1950 and 2012. Each year, the number of twin publications increases by another 500 to 1000. The underrepresentation of German twin studies cannot be solely explained by the abuse of medical research under National Socialism. Developing and expanding large twin cohorts is a challenge in terms of both ethics and data protection. However, twin cohorts enable long-term and real-time research on many medical issues and contribute to answer the question of predisposition or environment as possible disease triggers - even after the sequencing of the human genome.There are currently two German twin cohorts: the biomedical cohort HealthTwiSt, with around 1500 pairs of twins, and TwinLife, a sociological-psychological cohort with around 4000 pairs of twins. There are also disease-specific cohorts. The TwinHealth Consortium in the Faculty of Medicine at the University of Tübingen was established in 2016 with the aim of enabling open-ended and sustainable twin research in Tübingen to answer various scientific questions.With the help of systematic literature research and medical history, this article gives an overview of the worldwide development of twin studies and databases over the last 100 years. The example of the Tübingen TwinHealth Initiative illuminates the structure of a twin cohort and its legal, ethical, and data protection aspects., (© 2021. The Author(s).)

In view of upcoming clinical trials, quantitative molecular markers accessible in peripheral blood are of critical importance as prognostic or pharmacodynamic markers in genetic neurodegenerative diseases such as Spinocerebellar Ataxia Type 3 (SCA3), in particular for signaling target engagement. In this pilot study, we focused on the quantification of ataxin-3, the protein altered in SCA3, in human peripheral blood mononuclear cells (PBMCs) acquired from preataxic and ataxic SCA3 mutation carriers as well as healthy controls, as a molecular marker directly related to SCA3 pathophysiology. We established two different highly sensitive TR-FRET-based immunoassays to measure the protein levels of either total full-length, non-expanded and expanded, ataxin-3 or specifically polyQ-expanded ataxin-3. In PBMCs, a clear discrimination between SCA3 mutation carrier and controls were seen measuring polyQ-expanded ataxin-3 protein level. Additionally, polyQ-expanded ataxin-3 protein levels correlated with disease progression and clinical severity as assessed by the Scale for the Assessment and Rating of Ataxia. Total full-length ataxin-3 protein levels were directly influenced by the expression levels of the polyQ-expanded ataxin-3 protein, but were not correlated with clinical parameters. Assessment of ataxin-3 levels in fibroblasts or induced pluripotent stem cells allowed to distinguish mutation carriers from controls, thus providing proof-of-principle validation of our PBMC findings across cell lines. Total full-length or polyQ-expanded ataxin-3 protein was not detectable by TR-FRET assays in other biofluids like plasma or cerebrospinal fluid, indicating the need for ultra-sensitive assays for these biofluids. Standardization studies revealed that tube systems, blood sampling, and PBMC preparation may influence ataxin-3 protein levels indicating a high demand for standardized protocols in biomarker studies. In conclusion, the polyQ-expanded ataxin-3 protein is a promising candidate as a molecular target engagement marker in SCA3 in future clinical trials, determinable even in-easily accessible-peripheral blood biomaterials. These results, however, require validation in a larger cohort and further standardization of modifying conditions.

Spinocerebellar ataxia type 3 (SCA3) is a rare neurodegenerative disorder resulting from an aberrant expansion of a polyglutamine stretch in the ataxin-3 protein and subsequent neuronal death. The underlying intracellular signaling pathways are currently unknown. We applied the Reverse-phase Protein MicroArray (RPMA) technology to assess the levels of 50 signaling proteins (in phosphorylated and total forms) using three in vitro and in vivo models expressing expanded ataxin-3: (i) human embryonic kidney (HEK293T) cells stably transfected with human ataxin-3 constructs, (ii) mouse embryonic fibroblasts (MEF) from SCA3 transgenic mice, and (iii) whole brains from SCA3 transgenic mice. All three models demonstrated a high degree of similarity sharing a subset of phosphorylated proteins involved in the PI3K/AKT/GSK3/mTOR pathway. Expanded ataxin-3 strongly interfered (by stimulation or suppression) with normal ataxin-3 signaling consistent with the pathogenic role of the polyglutamine expansion. In comparison with normal ataxin-3, expanded ataxin-3 caused a pro-survival stimulation of the ERK pathway along with reduced pro-apoptotic and transcriptional responses.

Parkinson's disease (PD) is an age-dependent neurodegenerative disorder. Besides characteristic motor symptoms, patients suffer from cognitive impairments linked to pathology in cortical areas. Due to obvious challenges in tracing the underlying molecular perturbations in human brain over time, we took advantage of a well-characterized PD rat model. Using RNA sequencing, we profiled the frontocortical transcriptome of post-mortem patient samples and aligned expression changes with perturbation patterns obtained in the model at 5 and 12 months of age reflecting a presymptomatic and symptomatic time point. Integrating cell type-specific reference data, we identified a shared expression signature between both species that pointed to oligodendrocyte-specific, myelin-associated genes. Drawing on longitudinal information from the model, their nearly identical upregulation in both species could be traced to two distinctive perturbance modes. While one mode exhibited age-independent alterations that affected genes including proteolipid protein 1 ( PLP1 ), the other mode, impacting on genes like myelin-associated glycoprotein ( MAG ), was characterized by interferences of disease gene and adequate expression adaptations along aging. Our results highlight that even for a group of functionally linked genes distinct interference mechanisms may underlie disease progression that cannot be distinguished by examining the terminal point alone but instead require longitudinal interrogation of the system.

With molecular treatments coming into reach for spinocerebellar ataxia type 3 (SCA3), easily accessible, cross-species validated biomarkers for human and preclinical trials are warranted, particularly for the preataxic disease stage. We assessed serum levels of neurofilament light (NfL) and phosphorylated neurofilament heavy (pNfH) in ataxic and preataxic subjects of two independent multicentric SCA3 cohorts and in a SCA3 knock-in mouse model. Ataxic SCA3 subjects showed increased levels of both NfL and pNfH. In preataxic subjects, NfL levels increased with proximity to the individual expected onset of ataxia, with significant NfL elevations already 7.5 years before onset. Cross-sectional NfL levels correlated with both disease severity and longitudinal disease progression. Blood NfL and pNfH increases in human SCA3 were each paralleled by similar changes in SCA3 knock-in mice, here also starting already at the presymptomatic stage, closely following ataxin-3 aggregation and preceding Purkinje cell loss in the brain. Blood neurofilaments, particularly NfL, might thus provide easily accessible, cross-species validated biomarkers in both ataxic and preataxic SCA3, associated with earliest neuropathological changes, and serve as progression, proximity-to-onset and, potentially, treatment-response markers in both human and preclinical SCA3 trials., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

Proteolytic fragmentation of polyglutamine-expanded ataxin-3 is a concomitant and modifier of the molecular pathogenesis of Machado-Joseph disease (MJD), the most common autosomal dominant cerebellar ataxia. Calpains, a group of calcium-dependent cysteine proteases, are important mediators of ataxin-3 cleavage and implicated in multiple neurodegenerative conditions. Pharmacologic and genetic approaches lowering calpain activity showed beneficial effects on molecular and behavioural disease characteristics in MJD model organisms. However, specifically targeting one of the calpain isoforms by genetic means has not yet been evaluated as a potential therapeutic strategy. In our study, we tested whether calpains are overactivated in the MJD context and if reduction or ablation of calpain-1 expression ameliorates the disease-associated phenotype in MJD cells and mice. In all analysed MJD models, we detected an elevated calpain activity at baseline. Lowering or removal of calpain-1 in cells or mice counteracted calpain system overactivation and led to reduced cleavage of ataxin-3 without affecting its aggregation. Moreover, calpain-1 knockout in YAC84Q mice alleviated excessive fragmentation of important synaptic proteins. Despite worsening some motor characteristics, YAC84Q mice showed a rescue of body weight loss and extended survival upon calpain-1 knockout. Together, our findings emphasize the general potential of calpains as a therapeutic target in MJD and other neurodegenerative diseases., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Hereditary spastic paraplegia type 5 is a neurodegenerative disease caused by loss-of-function mutations in the CYP7B1 gene encoding the oxysterol 7-α-hydroxylase involved in bile acid synthesis in the liver. Lack of CYP7B1 leads to an accumulation of its oxysterol substrates, in particular 25-hydroxycholesterol and 27-hydroxycholesterol that are able to cross the blood-brain barrier and have neurotoxic properties. A potential therapeutic strategy for SPG5 is the replacement of CYP7B1 by administration of mRNA. Here, we studied the intravenous application of formulated mouse and human CYP7B1 mRNA in mice lacking the endogenous Cyp7b1 gene. A single-dose injection of either mouse or human CYP7B1 mRNA led to a pronounced degradation of oxysterols in liver and serum within 2 days of treatment. Pharmacokinetics indicate a single injection of human CYP7B1 mRNA to be effective in reducing oxysterols for at least 5 days. Repetitive applications of mRNA were safe for at least 17 days and resulted in a significant reduction of neurotoxic oxysterols not only in liver and serum but also to some extent in the brain. Our study highlights the potential to use mRNA as a novel therapy to treat patients with SPG5 disease., (© 2019 The Author(s).)

Spinocerebellar ataxia type 3 (SCA3), or Machado-Joseph disease (MJD), is a progressive neurodegenerative disorder caused by a CAG expansion in the ATXN3 gene. The expanded CAG repeat is translated into a prolonged polyglutamine repeat in the ataxin-3 protein and accumulates within inclusions, acquiring toxic properties, which results in degeneration of the cerebellum and brain stem. In the current study, a non-allele-specific ATXN3 silencing approach was investigated using artificial microRNAs engineered to target various regions of the ATXN3 gene (miATXN3). The mi ATXN3 candidates were screened in vitro based on their silencing efficacy on a luciferase (Luc) reporter co-expressing ATXN3 . The three best miATXN3 candidates were further tested for target engagement and potential off-target activity in induced pluripotent stem cells (iPSCs) differentiated into frontal brain-like neurons and in a SCA3 knockin mouse model. Besides a strong reduction of ATXN3 mRNA and protein, small RNA sequencing revealed efficient guide strand processing without passenger strands being produced. We used different methods to predict alteration of off-target genes upon AAV5-mi ATXN3 treatment and found no evidence for unwanted effects. Furthermore, we demonstrated in a large animal model, the minipig, that intrathecal delivery of AAV5 can transduce the main areas affected in SCA3 patients. These results proved a strong basis to move forward to investigate distribution, efficacy, and safety of AAV5-mi ATXN3 in large animals., (© 2019 The Author(s).)

Alterations in mitochondrial morphology and function have been linked to neurodegenerative diseases, including Parkinson disease, Alzheimer disease and Huntington disease. Metabolic defects, resulting from dysfunctional mitochondria, have been reported in patients and respective animal models of all those diseases. Spinocerebellar Ataxia Type 3 (SCA3), another neurodegenerative disorder, also presents with metabolic defects and loss of body weight in early disease stages although the possible role of mitochondrial dysfunction in SCA3 pathology is still to be determined. Interestingly, the SCA3 disease protein ataxin-3, which is predominantly localized in cytoplasm and nucleus, has also been associated with mitochondria in both its mutant and wildtype form. This observation provides an interesting link to a potential mitochondrial involvement of mutant ataxin-3 in SCA3 pathogenesis. Furthermore, proteolytic cleavage of ataxin-3 has been shown to produce toxic fragments and even overexpression of artificially truncated forms of ataxin-3 resulted in mitochondria deficits. Therefore, we analyzed the repercussions of expressing a naturally occurring N-terminal cleavage fragment of ataxin-3 and the influence of an endogenous expression of the S256 cleavage fragment in vitro and in vivo . In our study, expression of a fragment derived from calpain cleavage induced mitochondrial fragmentation and cristae alterations leading to a significantly decreased capacity of mitochondrial respiration and contributing to an increased susceptibility to apoptosis. Furthermore, analyzing mitophagy revealed activation of autophagy in the early pathogenesis with reduced lysosomal activity. In conclusion, our findings indicate that cleavage of ataxin-3 by calpains results in fragments which interfere with mitochondrial function and mitochondrial degradation processes.

Ataxin-3, the disease protein in Machado-Joseph disease, is known to be proteolytically modified by various enzymes including two major families of proteases, caspases and calpains. This processing results in the generation of toxic fragments of the polyglutamine-expanded protein. Although various approaches were undertaken to identify cleavage sites within ataxin-3 and to evaluate the impact of fragments on the molecular pathogenesis of Machado-Joseph disease, calpain-mediated cleavage of the disease protein and the localization of cleavage sites remained unclear. Here, we report on the first precise localization of calpain cleavage sites in ataxin-3 and on the characterization of the resulting breakdown products. After confirming the occurrence of calpain-derived fragmentation of ataxin-3 in patient-derived cell lines and post-mortem brain tissue, we combined in silico prediction tools, western blot analysis, mass spectrometry, and peptide overlay assays to identify calpain cleavage sites. We found that ataxin-3 is primarily cleaved at two sites, namely at amino acid positions D208 and S256 and mutating amino acids at both cleavage sites to tryptophan nearly abolished ataxin-3 fragmentation. Furthermore, analysis of calpain cleavage-derived fragments showed distinct aggregation propensities and toxicities of C-terminal polyglutamine-containing breakdown products. Our data elucidate the important role of ataxin-3 proteolysis in the pathogenesis of Machado-Joseph disease and further emphasize the relevance of targeting this disease pathway as a treatment strategy in neurodegenerative disorders., (© The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Spinocerebellar ataxia type 3 (SCA3) is an autosomal dominantly inherited neurodegenerative disorder for which no curative therapy is available. The cause of this disease is the expansion of a CAG repeat in the so-called ATXN3 gene leading to an expanded polyglutamine stretch in the ataxin-3 protein. Although the function of ataxin-3 has been defined as a deubiquitinating enzyme, the pathogenic pathway underlying SCA3 remains to be deciphered. Besides others, also the glutamatergic system seems to be altered in SCA3. The antiglutamatergic substance riluzole has thus been suggested as a potential therapeutic agent for SCA3. To assess whether riluzole is effective in the treatment of SCA3 in vivo, we used a phenotypically well-characterized conditional mouse model previously generated by us. Treatment with 10 mg/kg riluzole in the drinking water was started when mice showed impairment in rotarod performance. Post-symptomatic treatment with riluzole carried out for a period of 10 months led to reduction of the soluble ataxin-3 level and an increase in ataxin-3 positive accumulations, but did not improve motor deficits measured by rotarod. There was also no positive effect on home cage behavior or body weight. We even observed a pronounced reduction of calbindin expression in Purkinje cells in riluzole-treated mice. Thus, long-term treatment with riluzole was not able to alleviate disease symptoms observed in transgenic SCA3 mice and should be considered with caution in the treatment of human patients. Assessing riluzole as a potential treatment for spinocerebellar ataxia type 3 (SCA3) had no beneficial, but rather a worsening effect on our transgenic SCA3 mouse model. We hypothesize that: Riluzole treatment enhanced glutamate release in ATXN3-expressing cells leading to an increased Ca(2+) influx resulting in Purkinje cell damage shown by loss of calbindin expression., (© 2016 International Society for Neurochemistry.)