Spinocerebellar Ataxia Type 1 Characteristics in Patient-Derived Fibroblast and iPSC-Derived Neuronal Cultures.

BACKGROUND: Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disease caused by a polyglutamine expansion in the ataxin-1 protein resulting in neuropathology including mutant ataxin-1 protein aggregation, aberrant neurodevelopment, and mitochondrial dysfunction. OBJECTIVES: Identify SCA1-relevant phenotypes in patient-specific fibroblasts and SCA1 induced pluripotent stem cells (iPSCs) neuronal cultures. METHODS: SCA1 iPSCs were generated and differentiated into neuronal cultures. Protein aggregation and neuronal morphology were evaluated using fluorescent microscopy. Mitochondrial respiration was measured using the Seahorse Analyzer. The multi-electrode array (MEA) was used to identify network activity. Finally, gene expression changes were studied using RNA-seq to identify disease-specific mechanisms. RESULTS: Bioenergetics deficits in patient-derived fibroblasts and SCA1 neuronal cultures showed altered oxygen consumption rate, suggesting involvement of mitochondrial dysfunction in SCA1. In SCA1 hiPSC-derived neuronal cells, nuclear and cytoplasmic aggregates were identified similar in localization as aggregates in SCA1 postmortem brain tissue. SCA1 hiPSC-derived neuronal cells showed reduced dendrite length and number of branching points while MEA recordings identified delayed development in network activity in SCA1 hiPSC-derived neuronal cells. Transcriptome analysis identified 1050 differentially expressed genes in SCA1 hiPSC-derived neuronal cells associated with synapse organization and neuron projection guidance, where a subgroup of 151 genes was highly associated with SCA1 phenotypes and linked to SCA1 relevant signaling pathways. CONCLUSIONS: Patient-derived cells recapitulate key pathological features of SCA1 pathogenesis providing a valuable tool for the identification of novel disease-specific processes. This model can be used for high throughput screenings to identify compounds, which may prevent or rescue neurodegeneration in this devastating disease. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Identification of the ataxin-1 interaction network and its impact on spinocerebellar ataxia type 1.

BACKGROUND: Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disease caused by a polyglutamine expansion in the ataxin-1 protein. The pathogenic mechanism resulting in SCA1 is still unclear. Protein-protein interactions affect the function and stability of ataxin-1. METHODS: Wild-type and mutant ataxin-1 were expressed in HEK-293T cells. The levels of expression were assessed using real-time polymerase chain reaction (PCR) and Western blots. Co-immunoprecipitation was done in HEK-293T cells expressing exogenous wild-type and mutant ataxin-1 using anti-Flag antibody following by tandem affinity purification in order to study protein-protein interactions. The candidate interacting proteins were validated by immunoprecipitation. Chromatin immunoprecipitation and high-throughput sequencing and RNA immunoprecipitation and high-throughput sequencing were performed using HEK-293T cells expressing wild-type or mutant ataxin-1. RESULTS: In this study using HEK-293T cells, we found that wild-type ataxin-1 interacted with MCM2, GNAS, and TMEM206, while mutant ataxin-1 lost its interaction with MCM2, GNAS, and TMEM206. Two ataxin-1 binding targets containing the core GGAG or AAAT were identified in HEK-293T cells using ChIP-seq. Gene Ontology analysis of the top ataxin-1 binding genes identified SLC6A15, NTF3, KCNC3, and DNAJC6 as functional genes in neurons in vitro. Ataxin-1 also was identified as an RNA-binding protein in HEK-293T cells using RIP-seq, but the polyglutamine expansion in the ataxin-1 had no direct effects on the RNA-binding activity of ataxin-1. CONCLUSIONS: An expanded polyglutamine tract in ataxin-1 might interfere with protein-protein or protein-DNA interactions but had little effect on protein-RNA interactions. This study suggested that the dysfunction of protein-protein or protein-DNA interactions is involved in the pathogenesis of SCA1.

RNA Toxicity and Perturbation of rRNA Processing in Spinocerebellar Ataxia Type 2.

BACKGROUND: Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative disease caused by expansion of a CAG repeat in Ataxin-2 (ATXN2) gene. The mutant ATXN2 protein with a polyglutamine tract is known to be toxic and contributes to the SCA2 pathogenesis. OBJECTIVE: Here, we tested the hypothesis that the mutant ATXN2 transcript with an expanded CAG repeat (expATXN2) is also toxic and contributes to SCA2 pathogenesis. METHODS: The toxic effect of expATXN2 transcripts on SK-N-MC neuroblastoma cells and primary mouse cortical neurons was evaluated by caspase 3/7 activity and nuclear condensation assay, respectively. RNA immunoprecipitation assay was performed to identify RNA binding proteins (RBPs) that bind to expATXN2 RNA. Quantitative PCR was used to examine if ribosomal RNA (rRNA) processing is disrupted in SCA2 and Huntington's disease (HD) human brain tissue. RESULTS: expATXN2 RNA induces neuronal cell death, and aberrantly interacts with RBPs involved in RNA metabolism. One of the RBPs, transducin ß-like protein 3 (TBL3), involved in rRNA processing, binds to both expATXN2 and expanded huntingtin (expHTT) RNA in vitro. rRNA processing is disrupted in both SCA2 and HD human brain tissue. CONCLUSION: These findings provide the first evidence of a contributory role of expATXN2 transcripts in SCA2 pathogenesis, and further support the role of expHTT transcripts in HD pathogenesis. The disruption of rRNA processing, mediated by aberrant interaction of RBPs with expATXN2 and expHTT transcripts, suggest a point of convergence in the pathogeneses of repeat expansion diseases with potential therapeutic implications. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Destabilizing the AXH Tetramer by Mutations: Mechanisms and Potential Antiaggregation Strategies.

The AXH domain of protein Ataxin 1 is thought to play a key role in the misfolding and aggregation pathway responsible for Spinocerebellar ataxia 1. For this reason, a molecular level understanding of AXH oligomerization pathway is crucial to elucidate the aggregation mechanism, which is thought to trigger the disease. This study employs classical and enhanced molecular dynamics to identify the structural and energetic basis of AXH tetramer stability. Results of this work elucidate molecular mechanisms behind the destabilizing effect of protein mutations, which consequently affect the AXH tetramer assembly. Moreover, results of the study draw attention for the first time, to our knowledge, to the R638 protein residue, which is shown to play a key role in AXH tetramer stability. Therefore, R638 might be also implicated in the AXH oligomerization pathway and stands out as a target for future experimental studies focused on self-association mechanisms and fibril formation of full-length ATX1.

In vivo neurometabolic profiling in patients with spinocerebellar ataxia types 1, 2, 3, and 7.

Spinocerebellar ataxias (SCAs) belong to polyglutamine repeat disorders and are characterized by a predominant atrophy of the cerebellum and the pons. Proton magnetic resonance spectroscopy ((1) H MRS) using an optimized semiadiabatic localization by adiabatic selective refocusing (semi-LASER) protocol was performed at 3 T to determine metabolite concentrations in the cerebellar vermis and pons of a cohort of patients with SCA1 (n=16), SCA2 (n=12), SCA3 (n=21), and SCA7 (n=12) and healthy controls (n=33). Compared with controls, patients displayed lower total N-acetylaspartate and, to a lesser extent, lower glutamate, reflecting neuronal loss/dysfunction, whereas the glial marker, myoinositol (myo-Ins), was elevated. Patients also showed higher total creatine as reported in Huntington's disease, another polyglutamine repeat disorder. A strong correlation was found between the Scale for the Assessment and Rating of Ataxia and the neurometabolites in both affected regions of patients. Principal component analyses confirmed that neuronal metabolites (total N-acetylaspartate and glutamate) were inversely correlated in the vermis and the pons to glial (myo-Ins) and energetic (total creatine) metabolites, as well as to disease severity (motor scales). Neurochemical plots with selected metabolites also allowed the separation of SCA2 and SCA3 from controls. The neurometabolic profiles detected in patients underlie cell-specific changes in neuronal and astrocytic compartments that cannot be assessed by other neuroimaging modalities. The inverse correlation between metabolites from these two compartments suggests a metabolic attempt to compensate for neuronal damage in SCAs. Because these biomarkers reflect dynamic aspects of cellular metabolism, they are good candidates for proof-of-concept therapeutic trials. © 2015 International Parkinson and Movement Disorder Society.

Intercellular Propagation and Aggregate Seeding of Mutant Ataxin-1.

Intercellular propagation of aggregated protein inclusions along actin-based tunneling nanotubes (TNTs) has been reported as a means of pathogenic spread in Alzheimer's, Parkinson's, and Huntington's diseases. Propagation of oligomeric-structured polyglutamine-expanded ataxin-1 (Atxn1[154Q]) has been reported in the cerebellum of a Spinocerebellar ataxia type 1 (SCA1) knock-in mouse to correlate with disease propagation. In this study, we investigated whether a physiologically relevant polyglutamine-expanded ATXN1 protein (ATXN1[82Q]) could propagate intercellularly. Using a cerebellar-derived live cell model, we observed ATXN1 aggregates form in the nucleus, subsequently form in the cytoplasm, and finally, propagate to neighboring cells along actin-based intercellular connections. Additionally, we observed the facilitation of aggregate-resistant proteins into aggregates given the presence of aggregation-prone proteins within cells. Taken together, our results support a pathogenic role of intercellular propagation of polyglutamine-expanded ATXN1 inclusions.

Analysis of (CAG)n expansion in ATXN1, ATXN2 and ATXN3 in Chinese patients with multiple system atrophy.

Multiple system atrophy (MSA) is a complex and multifactorial neurodegenerative disease, and its pathogenesis remains uncertain. Patients with MSA or spinocerebellar ataxia (SCA) show overlapping clinical phenotypes. Previous studies have reported that intermediate or long CAG expansions in SCA genes have been associated with other neurodegenerative disease. In this study, we screened for the number of CAG repeats in ATXN1, 2 and 3 in 200 patients with MSA and 314 healthy controls to evaluate possible associations between (CAG)n in these three polyQ-related genes and MSA. Our findings indicated that longer repeat lengths in ATXN2 were associated with increased risk for MSA in Chinese individuals. No relationship was observed between CAG repeat length in the three examined genes and age at onset (AO) of MSA.

Large-scale assessment of polyglutamine repeat expansions in Parkinson disease.

OBJECTIVES: We aim to clarify the pathogenic role of intermediate size repeat expansions of SCA2, SCA3, SCA6, and SCA17 as risk factors for idiopathic Parkinson disease (PD). METHODS: We invited researchers from the Genetic Epidemiology of Parkinson's Disease Consortium to participate in the study. There were 12,346 cases and 8,164 controls genotyped, for a total of 4 repeats within the SCA2, SCA3, SCA6, and SCA17 genes. Fixed- and random-effects models were used to estimate the summary risk estimates for the genes. We investigated between-study heterogeneity and heterogeneity between different ethnic populations. RESULTS: We did not observe any definite pathogenic repeat expansions for SCA2, SCA3, SCA6, and SCA17 genes in patients with idiopathic PD from Caucasian and Asian populations. Furthermore, overall analysis did not reveal any significant association between intermediate repeats and PD. The effect estimates (odds ratio) ranged from 0.93 to 1.01 in the overall cohort for the SCA2, SCA3, SCA6, and SCA17 loci. CONCLUSIONS: Our study did not support a major role for definite pathogenic repeat expansions in SCA2, SCA3, SCA6, and SCA17 genes for idiopathic PD. Thus, results of this large study do not support diagnostic screening of SCA2, SCA3, SCA6, and SCA17 gene repeats in the common idiopathic form of PD. Likewise, this largest multicentered study performed to date excludes the role of intermediate repeats of these genes as a risk factor for PD.