Antagonistic roles of canonical and Alternative-RPA in disease-associated tandem CAG repeat instability.

Expansions of repeat DNA tracts cause >70 diseases, and ongoing expansions in brains exacerbate disease. During expansion mutations, single-stranded DNAs (ssDNAs) form slipped-DNAs. We find the ssDNA-binding complexes canonical replication protein A (RPA1, RPA2, and RPA3) and Alternative-RPA (RPA1, RPA3, and primate-specific RPA4) are upregulated in Huntington disease and spinocerebellar ataxia type 1 (SCA1) patient brains. Protein interactomes of RPA and Alt-RPA reveal unique and shared partners, including modifiers of CAG instability and disease presentation. RPA enhances in vitro melting, FAN1 excision, and repair of slipped-CAGs and protects against CAG expansions in human cells. RPA overexpression in SCA1 mouse brains ablates expansions, coincident with decreased ATXN1 aggregation, reduced brain DNA damage, improved neuron morphology, and rescued motor phenotypes. In contrast, Alt-RPA inhibits melting, FAN1 excision, and repair of slipped-CAGs and promotes CAG expansions. These findings suggest a functional interplay between the two RPAs where Alt-RPA may antagonistically offset RPA's suppression of disease-associated repeat expansions, which may extend to other DNA processes.

Loss of Ataxin-1 Potentiates Alzheimer's Pathogenesis by Elevating Cerebral BACE1 Transcription.

Expansion of CAG trinucleotide repeats in ATXN1 causes spinocerebellar ataxia type 1 (SCA1), a neurodegenerative disease that impairs coordination and cognition. While ATXN1 is associated with increased Alzheimer's disease (AD) risk, CAG repeat number in AD patients is not changed. Here, we investigated the consequences of ataxin-1 loss of function and discovered that knockout of Atxn1 reduced CIC-ETV4/5-mediated inhibition of Bace1 transcription, leading to increased BACE1 levels and enhanced amyloidogenic cleavage of APP, selectively in AD-vulnerable brain regions. Elevated BACE1 expression exacerbated Aß deposition and gliosis in AD mouse models and impaired hippocampal neurogenesis and olfactory axonal targeting. In SCA1 mice, polyglutamine-expanded mutant ataxin-1 led to the increase of BACE1 post-transcriptionally, both in cerebrum and cerebellum, and caused axonal-targeting deficit and neurodegeneration in the hippocampal CA2 region. These findings suggest that loss of ataxin-1 elevates BACE1 expression and Aß pathology, rendering it a potential contributor to AD risk and pathogenesis.

Dysregulation of alternative splicing in spinocerebellar ataxia type 1.

Spinocerebellar ataxia type 1 is caused by an expansion of the polyglutamine tract in ATAXIN-1. Ataxin-1 is broadly expressed throughout the brain and is involved in regulating gene expression. However, it is not yet known if mutant ataxin-1 can impact the regulation of alternative splicing events. We performed RNA sequencing in mouse models of spinocerebellar ataxia type 1 and identified that mutant ataxin-1 expression abnormally leads to diverse splicing events in the mouse cerebellum of spinocerebellar ataxia type 1. We found that the diverse splicing events occurred in a predominantly cell autonomous manner. A majority of the transcripts with misregulated alternative splicing events were previously unknown, thus allowing us to identify overall new biological pathways that are distinctive to those affected by differential gene expression in spinocerebellar ataxia type 1. We also provide evidence that the splicing factor Rbfox1 mediates the effect of mutant ataxin-1 on misregulated alternative splicing and that genetic manipulation of Rbfox1 expression modifies neurodegenerative phenotypes in a Drosophila model of spinocerebellar ataxia type 1 in vivo. Together, this study provides novel molecular mechanistic insight into the pathogenesis of spinocerebellar ataxia type 1 and identifies potential therapeutic strategies for spinocerebellar ataxia type 1.

Dual targeting of brain region-specific kinases potentiates neurological rescue in Spinocerebellar ataxia type 1.

A critical question in neurodegeneration is why the accumulation of disease-driving proteins causes selective neuronal loss despite their brain-wide expression. In Spinocerebellar ataxia type 1 (SCA1), accumulation of polyglutamine-expanded Ataxin-1 (ATXN1) causes selective degeneration of cerebellar and brainstem neurons. Previous studies revealed that inhibiting Msk1 reduces phosphorylation of ATXN1 at S776 as well as its levels leading to improved cerebellar function. However, there are no regulators that modulate ATXN1 in the brainstem-the brain region whose pathology is most closely linked to premature death. To identify new regulators of ATXN1, we performed genetic screens and identified a transcription factor-kinase axis (ZBTB7B-RSK3) that regulates ATXN1 levels. Unlike MSK1, RSK3 is highly expressed in the human and mouse brainstems where it regulates Atxn1 by phosphorylating S776. Reducing Rsk3 rescues brainstem-associated pathologies and deficits, and lowering Rsk3 and Msk1 together improves cerebellar and brainstem function in an SCA1 mouse model. Our results demonstrate that selective vulnerability of brain regions in SCA1 is governed by region-specific regulators of ATXN1, and targeting multiple regulators could rescue multiple degenerating brain areas.

Differential effects of Wnt-ß-catenin signaling in Purkinje cells and Bergmann glia in spinocerebellar ataxia type 1.

Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited neurodegenerative disease characterized by progressive ataxia and degeneration of specific neuronal populations, including Purkinje cells (PCs) in the cerebellum. Previous studies have demonstrated a critical role for various evolutionarily conserved signaling pathways in cerebellar patterning, such as the Wnt-ß-catenin pathway; however, the roles of these pathways in adult cerebellar function and cerebellar neurodegeneration are largely unknown. In this study, we found that Wnt-ß-catenin signaling activity was progressively enhanced in multiple cell types in the adult SCA1 mouse cerebellum, and that activation of this signaling occurs in an ataxin-1 polyglutamine (polyQ) expansion-dependent manner. Genetic manipulation of the Wnt-ß-catenin signaling pathway in specific cerebellar cell populations revealed that activation of Wnt-ß-catenin signaling in PCs alone was not sufficient to induce SCA1-like phenotypes, while its activation in astrocytes, including Bergmann glia (BG), resulted in gliosis and disrupted BG localization, which was replicated in SCA1 mouse models. Our studies identify a mechanism in which polyQ-expanded ataxin-1 positively regulates Wnt-ß-catenin signaling and demonstrate that different cell types have distinct responses to the enhanced Wnt-ß-catenin signaling in the SCA1 cerebellum, underscoring an important role of BG in SCA1 pathogenesis.

Perceptual and Acoustic Analysis of Speech in Spinocerebellar ataxia Type 1.

This study characterizes the speech phenotype of spinocerebellar ataxia type 1 (SCA1) using both perceptual and objective acoustic analysis of speech in a cohort of SCA1 patients. Twenty-seven symptomatic SCA1 patients in various disease stages (SARA score range: 3-32 points) and 18 sex and age matched healthy controls underwent a clinical assessment addressing ataxia severity, non-ataxia signs, cognitive functioning, and speech. Speech samples were perceptually rated by trained speech therapists, and acoustic metrics representing speech timing, vocal control, and voice quality were extracted. Perceptual analysis revealed reduced intelligibility and naturalness in speech samples of SCA1 patients. Acoustically, SCA1 patients presented with slower speech rate and diadochokinetic rate as well as longer syllable duration compared to healthy controls. No distinct abnormalities in voice quality in the acoustic analysis were detected at group level. Both the affected perceptual and acoustic variables correlated with ataxia severity. Longitudinal assessment of speech is needed to place changes in speech in the context of disease progression and potential response to treatment.

Quantitative Evaluation of Stance as a Sensitive Biomarker of Postural Ataxia Development in Preclinical SCA1 Mutation Carriers.

The aim of this study was to determine the time between the first detection of postural control impairments and the evident manifestation of ataxia in preclinical SCA1 individuals. Twenty five preclinical SCA1 mutation carriers: 13 with estimated disease onset ≤ 6 years (SCA1 +) aged 27.8 ± 8.1 years; 12 with expected disease onset > 6 years (SCA1-) aged 26.6 ± 3.1 years and 26 age and sex matched healthy controls (HCs) underwent static posturography during 5 years of observation. The movements of the centre of feet pressure (COP) during quiet standing with eyes open (EO) and closed (EC) were quantified by calculating the mean radius (R), developed surface area (A) and mean COP movement velocity (V). Ataxia was evaluated by use of the Scale for Assessment and Rating of Ataxia (SARA).SCA1 + exhibited significantly worse quality of stance with EC vs. SCA1- (p < 0.05 for V) and HCs (p < 0.001) even 5 to 6 years before estimated disease onset. There were no statistically significant differences between SCA1- and HCs. A slow increase in Cohen's d effect size was observed for VEO up to the clinical manifestation of ataxia. VEO and AEC recorded in preclinical SCA1 individuals correlated slightly but statistically significantly with SARA (r = 0.47).The study confirms that static posturography detects COP sway changes in SCA1 preclinical gene carriers even 5 to 6 years before estimated disease onset. The quantitative evaluation of stance in preclinical SCA is a sensitive biomarker for the monitoring of the disease progression and may be useful in clinical trials.

PGT-M for spinocerebellar ataxia type 1: development of a STR panel and a report of two clinical cases.

PURPOSE: To present the developed preimplantation genetic testing (PGT) for spinocerebellar ataxia type 1 (SCA1) and the outcomes of IVF with PGT. METHODS: PGT was performed for two unrelated couples from the Republic of Sakha (Yakutia) with the risk of SCA1 in one spouse. We have developed a system for PGT of a monogenic disease (PGT-M) for SCA1, which includes the analysis of a panel of 11 polymorphic STR markers linked to the ATXN1 gene and a pathogenic variant of the ATXN1 gene using nested PCR and fragment analysis. IVF/ICSI programs were performed according to standard protocols. Multiple displacement amplification (MDA) was used for whole genome amplification (WGA) and array comparative genomic hybridization (aCGH) for aneuploidy testing (PGT-A). RESULTS: Eight STRs were informative for the first couple and ten for the second. Similarity of the haplotypes carrying pathogenic variants of the ATXN1 gene was noted. In the first case, during IVF/ICSI-PGT, three embryos reached the blastocyst stage and were biopsied. One embryo was diagnosed as normal by maternal STR haplotype and the ATXN1 allele. PGT-A revealed euploidy. The embryo transfer resulted in a singleton pregnancy, and a healthy boy was born. Postnatal diagnosis confirmed normal ATXN1. In the second case, two blastocysts were biopsied. Both were diagnosed as normal by PGT-M, but PGT-A revealed aneuploidy. CONCLUSION: Birth of a healthy child after PGT for SCA1 was the first case of successful preimplantation prevention of SCA1 for the Yakut couple and the first case of successful PGT for SCA1 in Russia.

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.

CAT Interruption as a Protective Factor in Chinese Patients with Spinocerebellar Ataxia Type 1.

Spinocerebellar ataxia type 1 (SCA1) is the third most common type of spinocerebellar ataxias in China. CAT interruptions in the pathogenic alleles of SCA1 patients had only been reported by limited documents and there was a lack of data based on the Chinese population. In this study, we detected CAT interrupted pathogenic alleles in SCA1 patients from 4 out of 79 (5.1%) Chinese families. Their total CAG repeats were larger (median 58 vs. 47, p < 0.001) but ages at onset were later (median 46 vs. 38, p = 0.020). The longest uninterrupted CAG repeats could explain 65.4% of the AAO variance, making an increase of 28.0% compared to the total CAG repeats. The interruption pattern was greatly different between Chinese cohort and Caucasian cohort, indicating the effect of race.

The extra-cerebellar effects of spinocerebellar ataxia type 1 (SCA1): looking beyond the cerebellum.

Spinocerebellar ataxia type 1 (SCA1) is one of nine polyglutamine (polyQ) diseases and is characterized as an adult late-onset, progressive, dominantly inherited genetic disease. SCA1 is caused by an increase in the number of CAG repeats in the ATXN1 gene leading to an expanded polyQ tract in the ATAXIN-1 protein. ATAXIN-1 is broadly expressed throughout the brain. However, until recently, SCA1 research has primarily centered on the cerebellum, given the characteristic cerebellar Purkinje cell loss observed in patients, as well as the progressive motor deficits, including gait and limb incoordination, that SCA1 patients present with. There are, however, also other symptoms such as respiratory problems, cognitive defects and memory impairment, anxiety, and depression observed in SCA1 patients and mouse models, which indicate that there are extra-cerebellar effects of SCA1 that cannot be explained solely through changes in the cerebellar region of the brain alone. The existing gap between human and mouse model studies of extra-cerebellar regions in SCA1 makes it difficult to answer many important questions in the field. This review will cover both the cerebellar and extra-cerebellar effects of SCA1 and highlight the need for further investigations into the impact of mutant ATXN1 expression in these regions. This review will also discuss implications of extra-cerebellar effects not only for SCA1 but other neurodegenerative diseases showing diverse pathology as well.

Electrophysiological and neuropsychological assessment of cognition in spinocerebellar ataxia type 1 patients: a pilot study.

BACKGROUND: Event-related potentials (ERPs) reflect cognitive processing: negative early components (N100, N200) are involved in the sensory and perceptual processing of a stimulus, whereas late positive component P300 requires conscious attention. Both neuropsychological and affective disorders are present in patients with spinocerebellar ataxia type 1 (SCA1), but the underlying mechanisms need further clarification. MATERIALS AND METHODS: In this pilot study, we assessed cognitive processing by recording auditory ERPs in 16 consecutive SCA1 patients and 16 healthy controls (HC) matched for age and sex. Motor and nonmotor symptoms were evaluated using the Scale for the Assessment and Rating of Ataxia (SARA) and an extensive neuropsychological battery. ERPs were recorded using an oddball paradigm, and peak latency and amplitude of N100, N200, and P300 were measured in the averaged responses to target tones. RESULTS: We found in SCA1 significantly increased latencies of N200 and P300 (p=0.033, p=0.007) and decreased amplitudes of N100 and P300 (p=0.024, p=0.038) compared with HC. Furthermore, P300 latency had the highest AUC in the discrimination of SCA1 in ROC analysis. The expansion of trinucleotide repeats correlated with P300 latency (r=-0.607, p=0.048), whereas both P300 and N100 amplitudes correlated with the severity of motor symptoms (r=-0.692, p=0.003; r=-0.621; p=0.010). Significant correlations between P300 latency and the scores of Emotion Attribution Task (r=-0.633, p=0.027), as well as between N200 latency and the scores of Frontal Assessment Battery and Stroop test (r=-0.520, p=0.047; r=0.538, p=0.039), were observed. CONCLUSIONS: This research provides for the first time an extensive characterization of ERPs as useful electrophysiological markers to identify early cognitive dysfunction in SCA1.

Experimental Treatment with Edaravone in a Mouse Model of Spinocerebellar Ataxia 1.

Edaravone is a mitochondrially targeted drug with a suggested capability to modify the course of diverse neurological diseases. Nevertheless, edaravone has not been tested yet in the context of spinocerebellar ataxia 1 (SCA1), an incurable neurodegenerative disease characterized mainly by cerebellar disorder, with a strong contribution of inflammation and mitochondrial dysfunction. This study aimed to address this gap, exploring the potential of edaravone to slow down SCA1 progression in a mouse knock-in SCA1 model. SCA1154Q/2Q and healthy SCA12Q/2Q mice were administered either edaravone or saline daily for more than 13 weeks. The functional impairments were assessed via a wide spectrum of behavioral assays reflecting motor and cognitive deficits and behavioral abnormalities. Moreover, we used high-resolution respirometry to explore mitochondrial function, and immunohistochemical and biochemical tools to assess the magnitude of neurodegeneration, inflammation, and neuroplasticity. Data were analyzed using (hierarchical) Bayesian regression models, combined with the methods of multivariate statistics. Our analysis pointed out various previously documented neurological and behavioral deficits of SCA1 mice. However, we did not detect any plausible therapeutic effect of edaravone on either behavioral dysfunctions or other disease hallmarks in SCA1 mice. Thus, our results did not provide support for the therapeutic potential of edaravone in SCA1.

Chronic optogenetic stimulation of Bergman glia leads to dysfunction of EAAT1 and Purkinje cell death, mimicking the events caused by expression of pathogenic ataxin-1.

Bergmann glia (BG) are highly specialized radial astrocytes of the cerebellar cortex, which play a key role in the uptake of synaptic glutamate via the excitatory amino acid transporter EAAT1. Multiple lines of evidence suggest that in cerebellar neurodegenerative diseases reactive BG has a negative impact on neuronal function and survival through compromised EAAT activity. A family of such diseases are those caused by expansion of CAG repeats in genes of the ataxin family, resulting in spinocerebellar ataxias (SCA). We investigated the contribution of BG to the pathogenesis of cerebellar neurodegeneration in a model of SCA1, which was induced by expression of a polyglutamine mutant of ataxin-1 (ATXN1[Q85]) in BG specifically. We compared the outcomes with a novel model where we triggered excitotoxicity by a chronic optogenetic activation of BG with channelrhodopsin-2 (ChR2). In both cases we detected evidence of reduced glutamate uptake manifested by prolongation of excitatory postsynaptic currents in Purkinje cells which is consistent with documented reduction of expression and/or function of EAAT1. In both models we detected astroglyosis and Purkinje cells atrophy. Finally, the same pattern was detected in a knock-in mouse which expresses a polyglutamine mutant ataxin-1 ATXN1[Q154] in a non-cell-selective manner. Our results suggest that ATXN1[Q85] and ChR2-induced insult targeted to BG closely mimics SCA1 pathology, where excessive glutamate signaling appears to be a common feature likely being an important contributor to cerebellar neurodegeneration.

Progress of macular atrophy during 30 months' follow-up in a patient with spinocerebellar ataxia type1 (SCA1).

PURPOSE: To report the 30-months' course of macular dystrophy in a patient with genetically confirmed spinocerebellar ataxia type1 (SCA1). METHODS: Detailed ophthalmological examinations including best-corrected visual acuity (BCVA), perimetry, multimodal fundus imaging, and electrophysiological recordings were performed on a 52-year-old woman with SCA1. The number of CAG sequence repeats of the candidate gene was verified. RESULTS: The baseline decimal BCVA was 0.2 OD and 0.3 OS. Goldman perimetry showed relative central scotomas and slight enlargements of Mariotte blind spot bilaterally. Ophthalmoscopy revealed no abnormalities in the macula and optic disk. Fundus autofluorescence (FAF) showed a circular hyperautofluorescence and round-shaped hypoautofluorescence in the macula. Optical coherence tomography (OCT) showed a loss of the interdigitation zone and ellipsoid zone (EZ) in the macula. Full-field scotopic and photopic Full-field electroretinograms (ERGs) were normal, and multifocal ERGs were decreased in the central area. After 30 months, the BCVA had not changed, but the FAF showed a spark-like hypoautofluorescence in the macula. The abnormal area of the EZ had expanded toward the periphery, and the rate of EZ loss was 199.7%/year OD and 206.8%/year OS. Genetic examinations revealed an increase in the number of heterozygous CAG repeats in the ATXN1 gene, and the CAG repeat number of the mutant allele ranged from 43 to 48. CONCLUSIONS: The full-field scotopic and photopic ERGs were normal. The mfERGs were significantly smaller in the central region. OCT demonstrated bilateral photoreceptor atrophy in the macula, and the rate of EZ loss was more rapid than in other macular dystrophies. Spark-like hypoautofluorescence appeared during the course of the disease process which might be a specific feature of SCA1-related retinopathy.

Genetic Modeling of the Neurodegenerative Disease Spinocerebellar Ataxia Type 1 in Zebrafish.

Dominant spinocerebellar ataxias (SCAs) are progredient neurodegenerative diseases commonly affecting the survival of Purkinje cells (PCs) in the human cerebellum. Spinocerebellar ataxia type 1 (SCA1) is caused by the mutated ataxin1 (Atx1) gene product, in which a polyglutamine stretch encoded by CAG repeats is extended in affected SCA1 patients. As a monogenetic disease with the Atx1-polyQ protein exerting a gain of function, SCA1 can be genetically modelled in animals by cell type-specific overexpression. We have established a transgenic PC-specific SCA1 model in zebrafish coexpressing the fluorescent reporter protein mScarlet together with either human wild type Atx1[30Q] as control or SCA1 patient-derived Atx1[82Q]. SCA1 zebrafish display an age-dependent PC degeneration starting at larval stages around six weeks postfertilization, which continuously progresses during further juvenile and young adult stages. Interestingly, PC degeneration is observed more severely in rostral than in caudal regions of the PC population. Although such a neuropathology resulted in no gross locomotor control deficits, SCA1-fish with advanced PC loss display a reduced exploratory behaviour. In vivo imaging in this SCA1 model may help to better understand such patterned PC death known from PC neurodegeneration diseases, to elucidate disease mechanisms and to provide access to neuroprotective compound characterization in vivo.

5'UTR-mediated regulation of Ataxin-1 expression.

Expression of mutant Ataxin-1 with an abnormally expanded polyglutamine domain is necessary for the onset and progression of spinocerebellar ataxia type 1 (SCA1). Understanding how Ataxin-1 expression is regulated in the human brain could inspire novel molecular therapies for this fatal, dominantly inherited neurodegenerative disease. Previous studies have shown that the ATXN1 3'UTR plays a key role in regulating the Ataxin-1 cellular pool via diverse post-transcriptional mechanisms. Here we show that elements within the ATXN1 5'UTR also participate in the regulation of Ataxin-1 expression. PCR and PacBio sequencing analysis of cDNA obtained from control and SCA1 human brain samples revealed the presence of three major, alternatively spliced ATXN1 5'UTR variants. In cell-based assays, fusion of these variants upstream of an EGFP reporter construct revealed significant and differential impacts on total EGFP protein output, uncovering a type of genetic rheostat-like function of the ATXN1 5'UTR. We identified ribosomal scanning of upstream AUG codons and increased transcript instability as potential mechanisms of regulation. Importantly, transcript-based analyses revealed significant differences in the expression pattern of ATXN1 5'UTR variants between control and SCA1 cerebellum. Together, the data presented here shed light into a previously unknown role for the ATXN1 5'UTR in the regulation of Ataxin-1 and provide new opportunities for the development of SCA1 therapeutics.

Purkinje cell COX deficiency and mtDNA depletion in an animal model of spinocerebellar ataxia type 1.

Spinocerebellar ataxias (SCAs) are a genetically heterogeneous group of cerebellar degenerative disorders, characterized by progressive gait unsteadiness, hand incoordination, and dysarthria. Ataxia type 1 (SCA1) is caused by the expansion of a CAG trinucleotide repeat in the SCA1 gene resulting in the atypical extension of a polyglutamine (polyQ) tract within the ataxin-1 protein. Our main objective was to investigate the mitochondrial oxidative metabolism in the cerebellum of transgenic SCA1 mice. SCA1 transgenic mice develop clinical features in the early life stages (around 5 weeks of age) presenting pathological cerebellar signs with concomitant progressive Purkinje neuron atrophy and relatively little cell loss; this evidence suggests that the SCA1 phenotype is not the result of cell death per se, but a possible effect of cellular dysfunction that occurs before neuronal demise. We studied the mitochondrial oxidative metabolism in cerebellar cells from both homozygous and heterozygous transgenic SCA1 mice, aged 2 and 6 months. Histochemical examination showed a cytochrome-c-oxidase (COX) deficiency in the Purkinje cells (PCs) of both heterozygous and homozygous mice, the oxidative defect being more prominent in older mice, in which the percentage of COX-deficient PC was up to 30%. Using a laser-microdissector, we evaluated the mitochondrial DNA (mtDNA) content on selectively isolated COX-competent and COX-deficient PC by quantitative Polymerase Chain Reaction and we found mtDNA depletion in those with oxidative dysfunction. In conclusion, the selective oxidative metabolism defect observed in neuronal PC expressing mutant ataxin occurs as early as 8 weeks of age thus representing an early step in the PC degeneration process in SCA1 disease.

Diminishing return for mechanistic therapeutics with neurodegenerative disease duration?: There may be a point in the course of a neurodegenerative condition where therapeutics targeting disease-causing mechanisms are futile.

The conventional approach to developing disease-modifying treatments for neurodegenerative conditions has been to identify drivers of pathology and inhibit such pathways. Here we discuss the possibility that the efficacy of such approaches may be increasingly attenuated as disease progresses. This is based on experiments using mouse models of spinocerebellar ataxia type 1 and Huntington's disease (HD), where expression of the dominantly acting mutations could be switched off, as well as studies in human HD, which suggest that the primary genetic driver of age-of-onset of disease is a much weaker determinant of disease progression in affected individuals. The idea that one may approach a point in the disease course where such rational therapeutic strategies based on targets which determine onset of disease have minimal efficacy, suggests that one needs to consider other approaches to therapies and clinical trial design, including initiation of therapies in presymptomatic individuals.