Progressive Cerebrocerebellar Uncoupling in Sporadic and Genetic Forms of Amyotrophic Lateral Sclerosis.

BACKGROUND AND OBJECTIVES: Amyotrophic lateral sclerosis (ALS) is predominantly associated with motor cortex, corticospinal tract (CST), brainstem, and spinal cord degeneration, and cerebellar involvement is much less well characterized. However, some of the cardinal clinical features of ALS, such as dysarthria, dysphagia, gait impairment, falls, and impaired dexterity, are believed to be exacerbated by coexisting cerebellar pathology. Cerebellar pathology may also contribute to cognitive, behavioral, and pseudobulbar manifestations. Our objective was to systematically assess both intracerebellar pathology and cerebrocerebellar connectivity alterations in a genetically stratified cohort of ALS. METHODS: A prospective, multimodal neuroimaging study was conducted to evaluate the longitudinal evolution of intracerebellar pathology and cerebrocerebellar connectivity, using structural and functional measures. RESULTS: A total of 113 healthy controls and 212 genetically stratified individuals with ALS were included: (1) C9orf72 hexanucleotide carriers ("C9POS"), (2) sporadic patients who tested negative for ALS-associated genetic variants, and (3) intermediate-length CAG trinucleotide carriers in ATXN2 ("ATXN2"). Flocculonodular lobule (padj = 0.014, 95% CI -5.06e-5 to -3.98e-6) and crura (padj = 0.031, 95% CI -1.63e-3 to -5.55e-5) volume reductions were detected at baseline in sporadic patients. Cerebellofrontal and cerebelloparietal structural connectivity impairment was observed in both C9POS and sporadic patients at baseline, and both projections deteriorated further over time in sporadic patients (padj = 0.003, t(249) = 3.04 and padj = 0.05, t(249) = 1.93). Functional cerebelloparietal uncoupling was evident in sporadic patients at baseline (padj = 0.004, 95% CI -0.19 to -0.03). ATXN2 patients exhibited decreased cerebello-occipital functional connectivity at baseline (padj = 0.004, 95% CI -0.63 to -0.06), progressive cerebellotemporal functional disconnection (padj = 0.025, t(199) = -2.26), and progressive flocculonodular lobule degeneration (padj = 0.017, t(249) = -2.24). C9POS patients showed progressive ventral dentate atrophy (padj = 0.007, t(249) = -2.75). The CSTs (padj < 0.001, 95% CI 4.89e-5 to 1.14e-4) and transcallosal interhemispheric fibers (padj < 0.001, 95% CI 5.21e-5 to 1.31e-4) were affected at baseline in C9POS and exhibited rapid degeneration over the 4 time points. The rate of decline in CST and corpus callosum integrity was faster than the rate of cerebrocerebellar disconnection (padj = 0.001, t(190) = 6.93). DISCUSSION: ALS is associated with accruing intracerebellar disease burden as well as progressive corticocerebellar uncoupling. Contrary to previous suggestions, we have not detected evidence of compensatory structural or functional changes in response to supratentorial degeneration. The contribution of cerebellar disease burden to dysarthria, dysphagia, gait impairment, pseudobulbar affect, and cognitive deficits should be carefully considered in clinical assessments, monitoring, and multidisciplinary interventions.

The polyglutamine protein ATXN2: from its molecular functions to its involvement in disease.

A CAG repeat sequence in the ATXN2 gene encodes a polyglutamine (polyQ) tract within the ataxin-2 (ATXN2) protein, showcasing a complex landscape of functions that have been progressively unveiled over recent decades. Despite significant progresses in the field, a comprehensive overview of the mechanisms governed by ATXN2 remains elusive. This multifaceted protein emerges as a key player in RNA metabolism, stress granules dynamics, endocytosis, calcium signaling, and the regulation of the circadian rhythm. The CAG overexpansion within the ATXN2 gene produces a protein with an extended poly(Q) tract, inducing consequential alterations in conformational dynamics which confer a toxic gain and/or partial loss of function. Although overexpanded ATXN2 is predominantly linked to spinocerebellar ataxia type 2 (SCA2), intermediate expansions are also implicated in amyotrophic lateral sclerosis (ALS) and parkinsonism. While the molecular intricacies await full elucidation, SCA2 presents ATXN2-associated pathological features, encompassing autophagy impairment, RNA-mediated toxicity, heightened oxidative stress, and disruption of calcium homeostasis. Presently, SCA2 remains incurable, with patients reliant on symptomatic and supportive treatments. In the pursuit of therapeutic solutions, various studies have explored avenues ranging from pharmacological drugs to advanced therapies, including cell or gene-based approaches. These endeavours aim to address the root causes or counteract distinct pathological features of SCA2. This review is intended to provide an updated compendium of ATXN2 functions, delineate the associated pathological mechanisms, and present current perspectives on the development of innovative therapeutic strategies.

Structured and disordered regions of Ataxin-2 contribute differently to the specificity and efficiency of mRNP granule formation.

Ataxin-2 (ATXN2) is a gene implicated in spinocerebellar ataxia type II (SCA2), amyotrophic lateral sclerosis (ALS) and Parkinsonism. The encoded protein is a therapeutic target for ALS and related conditions. ATXN2 (or Atx2 in insects) can function in translational activation, translational repression, mRNA stability and in the assembly of mRNP-granules, a process mediated by intrinsically disordered regions (IDRs). Previous work has shown that the LSm (Like-Sm) domain of Atx2, which can help stimulate mRNA translation, antagonizes mRNP-granule assembly. Here we advance these findings through a series of experiments on Drosophila and human Ataxin-2 proteins. Results of Targets of RNA Binding Proteins Identified by Editing (TRIBE), co-localization and immunoprecipitation experiments indicate that a polyA-binding protein (PABP) interacting, PAM2 motif of Ataxin-2 may be a major determinant of the mRNA and protein content of Ataxin-2 mRNP granules. Experiments with transgenic Drosophila indicate that while the Atx2-LSm domain may protect against neurodegeneration, structured PAM2- and unstructured IDR- interactions both support Atx2-induced cytotoxicity. Taken together, the data lead to a proposal for how Ataxin-2 interactions are remodelled during translational control and how structured and non-structured interactions contribute differently to the specificity and efficiency of RNP granule condensation as well as to neurodegeneration.

Clinical features, disease progression, and nuclear imaging in ATXN2-related parkinsonism in a longitudinal cohort.

BACKGROUND: Spinocerebellar ataxia 2 (SCA2) with a low range of CAG repeat expansion of ATXN2 gene can present with predominant or isolated parkinsonism that closely resembles Parkinson's disease (PD). This study is aimed at comparing clinical features, disease progression, and nuclear imaging between ATXN2-related parkinsonism (ATXN2-P) and PD. METHODS: Three hundred and seventy-seven clinically diagnosed PD with family history were screened by multiplex ligation-dependent probe amplification, whole-exome sequencing or target sequencing, and dynamic mutation testing of 10 SCA subtypes. The baseline and longitudinal clinical features as well as the dual-tracer positron emission tomography (PET) imaging were compared between ATXN2-P and genetically undefined familial PD (GU-fPD). RESULTS: Fifteen ATXN2-P patients from 7 families and 50 randomly selected GU-fPD patients were evaluated. Significantly less resting tremor and more symmetric signs were observed in ATXN2-P than GU-fPD. No significant difference was found in motor progression and duration from onset to occurrence of fluctuation, dyskinesia, and recurrent falls between the two groups. Cognitive impairment and rapid-eye-movement sleep behavior disorder were more common in ATXN2-P. During follow-up, olfaction was relatively spared, and no obvious progression of cognition dysfunction evaluated by Mini-Mental State Examination scores was found in ATXN2-P. PET results of ATXN2-P demonstrated a symmetric, diffuse, and homogenous dopamine transporter loss of bilateral striatum and a glucose metabolism pattern inconsistent with that in PD. CONCLUSIONS: Symmetric motor signs and unique nuclear imaging might be the clues to distinguish ATXN2-P from GU-fPD.

Digital Gait Measures Capture 1-Year Progression in Early-Stage Spinocerebellar Ataxia Type 2.

BACKGROUND: With disease-modifying drugs in reach for cerebellar ataxias, fine-grained digital health measures are highly warranted to complement clinical and patient-reported outcome measures in upcoming treatment trials and treatment monitoring. These measures need to demonstrate sensitivity to capture change, in particular in the early stages of the disease. OBJECTIVE: Our aim is to unravel gait measures sensitive to longitudinal change in the-particularly trial-relevant-early stage of spinocerebellar ataxia type 2 (SCA2). METHODS: We performed a multicenter longitudinal study with combined cross-sectional and 1-year interval longitudinal analysis in early-stage SCA2 participants (n = 23, including nine pre-ataxic expansion carriers; median, ATXN2 CAG repeat expansion 38 ± 2; median, Scale for the Assessment and Rating of Ataxia [SARA] score 4.8 ± 4.3). Gait was assessed using three wearable motion sensors during a 2-minute walk, with analyses focused on gait measures of spatio-temporal variability that have shown sensitivity to ataxia severity (eg, lateral step deviation). RESULTS: We found significant changes for gait measures between baseline and 1-year follow-up with large effect sizes (lateral step deviation P = 0.0001, effect size rprb = 0.78), whereas the SARA score showed no change (P = 0.67). Sample size estimation indicates a required cohort size of n = 43 to detect a 50% reduction in natural progression. Test-retest reliability and minimal detectable change analysis confirm the accuracy of detecting 50% of the identified 1-year change. CONCLUSIONS: Gait measures assessed by wearable sensors can capture natural progression in early-stage SCA2 within just 1 year-in contrast to a clinical ataxia outcome. Lateral step deviation represents a promising outcome measure for upcoming multicenter interventional trials, particularly in the early stages of cerebellar ataxia. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Ataxin-2 sequesters Raptor into aggregates and impairs cellular mTORC1 signaling.

Ataxin-2 (Atx2) is a polyglutamine (polyQ) protein, in which abnormal expansion of the polyQ tract can trigger protein aggregation and consequently cause spinocerebellar ataxia type 2 (SCA2), but the mechanism underlying how Atx2 aggregation leads to proteinopathy remains elusive. Here, we investigate the molecular mechanism and cellular consequences of Atx2 aggregation by molecular cell biology approaches. We have revealed that either normal or polyQ-expanded Atx2 can sequester Raptor, a component of mammalian target of rapamycin complex 1 (mTORC1), into aggregates based on their specific interaction. Further research indicates that the polyQ tract and the N-terminal region (residues 1-784) of Atx2 are responsible for the specific sequestration. Moreover, this sequestration leads to suppression of the mTORC1 activity as represented by down-regulation of phosphorylated P70S6K, which can be reversed by overexpression of Raptor. As mTORC1 is a key regulator of autophagy, Atx2 aggregation and sequestration also induces autophagy by upregulating LC3-II and reducing phosphorylated ULK1 levels. This study proposes that Atx2 sequesters Raptor into aggregates, thereby impairing cellular mTORC1 signaling and inducing autophagy, and will be beneficial for a better understanding of the pathogenesis of SCA2 and other polyQ diseases.

TDP-43 impairs sleep in Drosophila through Ataxin-2-dependent metabolic disturbance.

Neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal dementia are associated with substantial sleep disruption, which may accelerate cognitive decline and brain degeneration. Here, we define a role for trans-activation response element (TAR) DNA binding protein 43 (TDP-43), a protein associated with human neurodegenerative disease, in regulating sleep using Drosophila. Expression of TDP-43 severely disrupts sleep, and the sleep deficit is rescued by Atx2 knockdown. Brain RNA sequencing revealed that Atx2 RNA interference regulates transcripts enriched for small-molecule metabolic signaling in TDP-43 brains. Focusing on these Atx2-regulated genes, we identified suppressors of the TDP-43 sleep phenotype enriched for metabolism pathways. Knockdown of Atx2 or treatment with rapamycin attenuated the sleep phenotype and mitigated the disruption of small-molecule glycogen metabolism caused by TDP-43. Our findings provide a connection between toxicity of TDP-43 and sleep disturbances and highlight key aspects of metabolism that interplay with TDP-43 toxicity upon Atx2 rescue.

ATXN2 is a target of N-terminal proteolysis.

Spinocerebellar ataxia 2 (SCA2) is a neurodegenerative disorder caused by the expansion of the poly-glutamine (polyQ) tract of Ataxin-2 (ATXN2). Other polyQ-containing proteins such as ATXN7 and huntingtin are associated with the development of neurodegenerative diseases when their N-terminal polyQ domains are expanded. Furthermore, they undergo proteolytic processing events that produce N-terminal fragments that include the polyQ stretch, which are implicated in pathogenesis. Interestingly, N-terminal ATXN2 fragments were reported in a brain extract from a SCA2 patient, but it is currently unknown whether an expanded polyQ domain contributes to ATXN2 proteolytic susceptibility. Here, we used transient expression in HEK293 cells to determine whether ATXN2 is a target for specific N-terminal proteolysis. We found that ATXN2 proteins with either normal or expanded polyQ stretches undergo proteolytic cleavage releasing an N-terminal polyQ-containing fragment. We identified a short amino acid sequence downstream of the polyQ domain that is necessary for N-terminal cleavage of full-length ATXN2 and sufficient to induce proteolysis of a heterologous protein. However, this sequence is not required for cleavage of a short ATXN2 isoform produced from an alternative start codon located just upstream of the CAG repeats encoding the polyQ domain. Our study extends our understanding of ATXN2 posttranslational regulation by revealing that this protein can be the target of specific proteolytic cleavage events releasing polyQ-containing products that are modulated by the N-terminal domain of ATXN2. N-terminal ATXN2 proteolysis of expanded polyQ domains might contribute to SCA2 pathology, as observed in other neurodegenerative disorders caused by polyQ domain expansion.

Mitigating a TDP-43 proteinopathy by targeting ataxin-2 using RNA-targeting CRISPR effector proteins.

The TDP-43 proteinopathies, which include amyotrophic lateral sclerosis and frontotemporal dementia, are a devastating group of neurodegenerative disorders that are characterized by the mislocalization and aggregation of TDP-43. Here we demonstrate that RNA-targeting CRISPR effector proteins, a programmable class of gene silencing agents that includes the Cas13 family of enzymes and Cas7-11, can be used to mitigate TDP-43 pathology when programmed to target ataxin-2, a modifier of TDP-43-associated toxicity. In addition to inhibiting the aggregation and transit of TDP-43 to stress granules, we find that the in vivo delivery of an ataxin-2-targeting Cas13 system to a mouse model of TDP-43 proteinopathy improved functional deficits, extended survival, and reduced the severity of neuropathological hallmarks. Further, we benchmark RNA-targeting CRISPR platforms against ataxin-2 and find that high-fidelity forms of Cas13 possess improved transcriptome-wide specificity compared to Cas7-11 and a first-generation effector. Our results demonstrate the potential of CRISPR technology for TDP-43 proteinopathies.

A spatially defined human Notch receptor interaction network reveals Notch intracellular storage and Ataxin-2-mediated fast recycling.

The Notch signaling pathway controls cell growth, differentiation, and fate decisions. Dysregulation of Notch signaling has been linked to various human diseases. Notch receptor resides in multiple cellular compartments, and its translocation plays a central role in pathway activation. However, the spatial regulation of Notch receptor functions remains largely elusive. Using TurboID-based proximity labeling followed by affinity purification and mass spectrometry, we establish a spatially defined human Notch receptor interaction network. Notch receptors interact with different proteins in distinct subcellular compartments to perform specific cellular functions. This spatially defined interaction network also reveals that a large fraction of NOTCH is stored at the endoplasmic reticulum (ER)-Golgi intermediate compartment and recruits Ataxin-2-dependent recycling machinery for rapid recycling, Notch signaling activation, and leukemogenesis. Our work provides insights into dynamic Notch receptor complexes with exquisite spatial resolution, which will help in elucidating the detailed regulation of Notch receptors and highlight potential therapeutic targets for Notch-related pathogenesis.

Poly(A)-binding protein is an ataxin-2 chaperone that regulates biomolecular condensates.

Biomolecular condensation underlies the biogenesis of an expanding array of membraneless assemblies, including stress granules (SGs), which form under a variety of cellular stresses. Advances have been made in understanding the molecular grammar of a few scaffold proteins that make up these phases, but how the partitioning of hundreds of SG proteins is regulated remains largely unresolved. While investigating the rules that govern the condensation of ataxin-2, an SG protein implicated in neurodegenerative disease, we unexpectedly identified a short 14 aa sequence that acts as a condensation switch and is conserved across the eukaryote lineage. We identify poly(A)-binding proteins as unconventional RNA-dependent chaperones that control this regulatory switch. Our results uncover a hierarchy of cis and trans interactions that fine-tune ataxin-2 condensation and reveal an unexpected molecular function for ancient poly(A)-binding proteins as regulators of biomolecular condensate proteins. These findings may inspire approaches to therapeutically target aberrant phases in disease.

Stressing the role of a short linear motif in ataxin-2 condensation.

Ataxin-2, an RNA-binding protein that is conserved across eukaryotes, is involved in stress granule assembly and age-associated neurodegenerative diseases. In this issue of Molecular Cell, Boeynaems et al.1 identify a short linear motif in ataxin-2 as a condensation switch, providing molecular insights into its essential role in cellular stress response.

Pbp1 associates with Puf3 and promotes translation of its target mRNAs involved in mitochondrial biogenesis.

Pbp1 (poly(A)-binding protein-binding protein 1) is a cytoplasmic stress granule marker that is capable of forming condensates that function in the negative regulation of TORC1 signaling under respiratory conditions. Polyglutamine expansions in its mammalian ortholog ataxin-2 lead to spinocerebellar dysfunction due to toxic protein aggregation. Here, we show that loss of Pbp1 in S. cerevisiae leads to decreased amounts of mRNAs and mitochondrial proteins which are targets of Puf3, a member of the PUF (Pumilio and FBF) family of RNA-binding proteins. We found that Pbp1 supports the translation of Puf3-target mRNAs in respiratory conditions, such as those involved in the assembly of cytochrome c oxidase and subunits of mitochondrial ribosomes. We further show that Pbp1 and Puf3 interact through their respective low complexity domains, which is required for Puf3-target mRNA translation. Our findings reveal a key role for Pbp1-containing assemblies in enabling the translation of mRNAs critical for mitochondrial biogenesis and respiration. They may further explain prior associations of Pbp1/ataxin-2 with RNA, stress granule biology, mitochondrial function, and neuronal health.

Association of variants in the ATXN2 (rs7137828), FOXC1 (rs2745572) and TXNRD2 (rs35934224) genes as risk factors for primary open-angle glaucoma development in a Brazilian cohort.

BACKGROUND: Primary open-angle glaucoma (POAG), the world's main cause of irreversible blindness, is an asymptomatic and neurodegenerative disease of multifactorial etiology with ethnic and geographic disparities. Multiethnic genome-wide association studies (GWAS) identified single nucleotide variants (SNVs) in ATXN2, FOXC1, and TXNRD2 loci as risk factors for POAG pathophysiology and/or endophenotypes. The aim of this case-control study was to investigate the association of the variants rs7137828 (ATXN2), rs2745572 (FOXC1), and rs35934224 (TXNRD2), as risk factors for POAG development, additionally to rs7137828 association with glaucoma clinical parameters in a Brazilian cohort from the Southeast and South regions. METHODS: This investigation comprised 506 cases and 501 controls. Variants rs2745572 and rs35934224 were genotyped through TaqMan® assays and validated by Sanger sequencing. Variant rs7137828 was genotyped exclusively by Sanger sequencing. RESULTS: The primary research outcome revealed that the variant rs7137828 (ATXN2) was associated with an increased risk for the development of POAG in the presence of the TT genotype compared to the CC genotype (p = 0.006; Odds Ratio [OR] = 1.717; Confidence Interval [CI] 95% = 1.169-2.535). There was no significant association of rs2745572 and rs35934224 genotypes with POAG. The CT genotype of the rs7137828 was associated with the vertical cup-to-disk ratio (VCDR) (p = .023) but not with the age at diagnosis or the mean deviation. CONCLUSION: Our data indicate the rs7137828 associated with increased risk for the development of POAG and VCDR in a Brazilian cohort. If validated in additional populations, these findings may enable the development of relevant strategies for early diagnosis of glaucoma in the future.

TR-FRET-Based Immunoassay to Measure Ataxin-2 as a Target Engagement Marker in Spinocerebellar Ataxia Type 2.

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.

ATXN2-Mediated PI3K/AKT Activation Confers Gastric Cancer Chemoresistance and Attenuates CD8+ T Cell Cytotoxicity.

As one of the primary therapeutic choices, chemotherapy is widely adopted for progressive gastric cancer (GC), but the development of chemoresistance has limited chemotherapy efficacy and partly contributes to poor prognosis. Immunotherapy is increasingly being applied in the clinical treatment of GC and is also benefitting patients. To ascertain whether ATXN2 affects chemotherapy efficacy in GC cells and its role in GC immune escape, we performed high-throughput sequencing to clarify genes differentially expressed between 5-FU-resistant and 5-FU-sensitive GC cells and then conducted qRT-PCR to assess ATXN2 expression in GC tissues. Furthermore, the influence of ATXN2 on resistance was studied in vitro and in vivo, ATXN2 and other protein expression levels were detected using Western blotting and immunohistochemistry (IHC), and the direct association of SP1 and ATXN2 was confirmed through luciferase reporter gene analysis. We found elevated ATXN2 in GC tumors and a negative correlation between ATXN2 levels and the prognosis of GC. Furthermore, by activating the PI3K/AKT pathway, ATXN2 was found to promote chemoresistance in GC, facilitating BCL2L1 expression. In GC cells, ATXN2 further stimulated PD-L1 expression and provided better immunotherapy efficacy. Finally, we demonstrated that SP1 transcriptionally regulated the expression of ATXN2 and prompted GC chemoresistance and immune escape. In conclusion, our study reveals the important roles of the SP1/ATXN2/PI3K-AKT/BCL2L1 signalling pathway in GC chemoresistance and of the SP1/ATXN2/PI3K-AKT/PD-L1 signalling pathway in GC immunotherapy. Our findings provide new theories and experimental references for overcoming chemotherapy resistance in GC and enhancing the efficacy of immunotherapy for GC.

Mutant Ataxin-2 Expression in Aged Animals Aggravates Neuropathological Features Associated with Spinocerebellar Ataxia Type 2.

Spinocerebellar ataxia type 2 (SCA2) is a rare autosomal, dominantly inherited disease, in which the affected individuals have a disease onset around their third life decade. The molecular mechanisms underlying SCA2 are not yet completely understood, for which we hypothesize that aging plays a role in SCA2 molecular pathogenesis. In this study, we performed a striatal injection of mutant ataxin-2 mediated by lentiviral vectors, in young and aged animals. Twelve weeks post-injection, we analyzed the striatum for SCA2 neuropathological features and specific aging hallmarks. Our results show that aged animals had a higher number of mutant ataxin-2 aggregates and more neuronal marker loss, compared to young animals. Apoptosis markers, cleaved caspase-3, and cresyl violet staining also indicated increased neuronal death in the aged animal group. Additionally, mRNA levels of microtubule-associated protein 1 light-chain 3B (LC3) and sequestosome-1 (SQSTM1/p62) were altered in the aged animal group, suggesting autophagic pathway dysfunction. This work provides evidence that aged animals injected with expanded ataxin-2 had aggravated SCA2 disease phenotype, suggesting that aging plays an important role in SCA2 disease onset and disease progression.

Genome-wide CRISPR screen reveals v-ATPase as a drug target to lower levels of ALS protein ataxin-2.

Mutations in the ataxin-2 gene (ATXN2) cause the neurodegenerative disorders amyotrophic lateral sclerosis (ALS) and spinocerebellar ataxia type 2 (SCA2). A therapeutic strategy using antisense oligonucleotides targeting ATXN2 has entered clinical trial in humans. Additional ways to decrease ataxin-2 levels could lead to cheaper or less invasive therapies and elucidate how ataxin-2 is normally regulated. Here, we perform a genome-wide fluorescence-activated cell sorting (FACS)-based CRISPR-Cas9 screen in human cells and identify genes encoding components of the lysosomal vacuolar ATPase (v-ATPase) as modifiers of endogenous ataxin-2 protein levels. Multiple FDA-approved small molecule v-ATPase inhibitors lower ataxin-2 protein levels in mouse and human neurons, and oral administration of at least one of these drugs-etidronate-is sufficient to decrease ataxin-2 in the brains of mice. Together, we propose v-ATPase as a drug target for ALS and SCA2 and demonstrate the value of FACS-based screens in identifying genetic-and potentially druggable-modifiers of human disease proteins.

Targeting RTN4/NoGo-Receptor reduces levels of ALS protein ataxin-2.

Gene-based therapeutic strategies to lower ataxin-2 levels are emerging for the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and spinocerebellar ataxia type 2 (SCA2). Additional strategies to lower levels of ataxin-2 could be beneficial. Here, we perform a genome-wide arrayed small interfering RNA (siRNA) screen in human cells and identify RTN4R, the gene encoding the RTN4/NoGo-Receptor, as a potent modifier of ataxin-2 levels. RTN4R knockdown, or treatment with a peptide inhibitor, is sufficient to lower ataxin-2 protein levels in mouse and human neurons in vitro, and Rtn4r knockout mice have reduced ataxin-2 levels in vivo. We provide evidence that ataxin-2 shares a role with the RTN4/NoGo-Receptor in limiting axonal regeneration. Reduction of either protein increases axonal regrowth following axotomy. These data define the RTN4/NoGo-Receptor as a novel therapeutic target for ALS and SCA2 and implicate the targeting of ataxin-2 as a potential treatment following nerve injury.

Ataxin-2, Twenty-four, and Dicer-2 are components of a noncanonical cytoplasmic polyadenylation complex.

Cytoplasmic polyadenylation is a mechanism to promote mRNA translation in a wide variety of biological contexts. A canonical complex centered around the conserved RNA-binding protein family CPEB has been shown to be responsible for this process. We have previously reported evidence for an alternative noncanonical, CPEB-independent complex in <i>Drosophila</i>, of which the RNA-interference factor Dicer-2 is a component. Here, we investigate Dicer-2 mRNA targets and protein cofactors in cytoplasmic polyadenylation. Using RIP-Seq analysis, we identify hundreds of potential Dicer-2 target transcripts, ∼60% of which were previously found as targets of the cytoplasmic poly(A) polymerase Wispy, suggesting widespread roles of Dicer-2 in cytoplasmic polyadenylation. Large-scale immunoprecipitation revealed Ataxin-2 and Twenty-four among the high-confidence interactors of Dicer-2. Complex analyses indicated that both factors form an RNA-independent complex with Dicer-2 and mediate interactions of Dicer-2 with Wispy. Functional poly(A)-test analyses showed that Twenty-four and Ataxin-2 are required for cytoplasmic polyadenylation of a subset of Dicer-2 targets. Our results reveal components of a novel cytoplasmic polyadenylation complex that operates during <i>Drosophila</i> early embryogenesis.