Slc9a6 mutation causes Purkinje cell loss and ataxia in the shaker rat.

The shaker rat carries a naturally occurring mutation leading to progressive ataxia characterized by Purkinje cell (PC) loss. We previously reported on fine-mapping the shaker locus to the long arm of the rat X chromosome. In this work, we sought to identify the mutated gene underlying the shaker phenotype and confirm its identity by functional complementation. We fine-mapped the candidate region and analyzed cerebellar transcriptomes, identifying a XM_217630.9 (Slc9a6):c.[191_195delinsA] variant in the Slc9a6 gene that segregated with disease. We generated an adeno-associated virus (AAV) targeting Slc9a6 expression to PCs using the mouse L7-6 (L7) promoter. We administered the AAV prior to the onset of PC degeneration through intracerebroventricular injection and found that it reduced the shaker motor, molecular and cellular phenotypes. Therefore, Slc9a6 is mutated in shaker and AAV-based gene therapy may be a viable therapeutic strategy for Christianson syndrome, also caused by Slc9a6 mutation.

Staufen Impairs Autophagy in Neurodegeneration.

OBJECTIVE: The mechanistic target of rapamycin (mTOR) kinase is one of the master coordinators of cellular stress responses, regulating metabolism, autophagy, and apoptosis. We recently reported that staufen1 (STAU1), a stress granule (SG) protein, was overabundant in fibroblast cell lines from patients with spinocerebellar ataxia type 2 (SCA2), amyotrophic lateral sclerosis, frontotemporal degeneration, Huntington's, Alzheimer's, and Parkinson's diseases as well as animal models, and patient tissues. STAU1 overabundance is associated with mTOR hyperactivation and links SG formation with autophagy. Our objective was to determine the mechanism of mTOR regulation by STAU1. METHODS: We determined STAU1 abundance with disease- and chemical-induced cellular stressors in patient cells and animal models. We also used RNA-binding assays to contextualize STAU1 interaction with MTOR mRNA. RESULTS: STAU1 and mTOR were overabundant in bacterial artificial chromosome (BAC)-C9ORF72, ATXN2Q127 , and Thy1-TDP-43 transgenic mouse models. Reducing STAU1 levels in these mice normalized mTOR levels and activity and autophagy-related marker proteins. We also saw increased STAU1 levels in HEK293 cells transfected to express C9ORF72-relevant dipeptide repeats (DPRs). Conversely, DPR accumulations were not observed in cells treated by STAU1 RNA interference (RNAi). Overexpression of STAU1 in HEK293 cells increased mTOR levels through direct MTOR mRNA interaction, activating downstream targets and impairing autophagic flux. Targeting mTOR by rapamycin or RNAi normalized STAU1 abundance in an SCA2 cellular model. INTERPRETATION: STAU1 interaction with mTOR drives its hyperactivation and inhibits autophagic flux in multiple models of neurodegeneration. Staufen, therefore, constitutes a novel target to modulate mTOR activity and autophagy, and for the treatment of neurodegenerative diseases. ANN NEUROL 2023;93:398-416.

A quantitative high-throughput screen identifies compounds that lower expression of the SCA2-and ALS-associated gene ATXN2.

CAG repeat expansions in the ATXN2 (ataxin-2) gene can cause the autosomal dominant disorder spinocerebellar ataxia type 2 (SCA2) as well as increase the risk of ALS. Abnormal molecular, motor, and neurophysiological phenotypes in SCA2 mouse models are normalized by lowering ATXN2 transcription, and reduction of nonmutant Atxn2 expression has been shown to increase the life span of mice overexpressing the TDP-43 (transactive response DNA-binding protein 43 kDa) ALS protein, demonstrating the potential benefits of targeting ATXN2 transcription in humans. Here, we describe a quantitative high-throughput screen to identify compounds that lower ATXN2 transcription. We screened 428,759 compounds in a multiplexed assay using an ATXN2-luciferase reporter in human embryonic kidney 293 (HEK-293) cells and identified a diverse set of compounds capable of lowering ATXN2 transcription. We observed dose-dependent reductions of endogenous ATXN2 in HEK-293 cells treated with procillaridin A, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), and heat shock protein 990 (HSP990), known inhibitors of HSP90 and Na+/K+-ATPases. Furthermore, HEK-293 cells expressing polyglutamine-expanded ATXN2-Q58 treated with 17-DMAG had minimally detectable ATXN2, as well as normalized markers of autophagy and endoplasmic reticulum stress, including STAU1 (Staufen 1), molecular target of rapamycin, p62, LC3-II (microtubule-associated protein 1A/1B-light chain 3II), CHOP (C/EBP homologous protein), and phospho-eIF2α (eukaryotic initiation factor 2α). Finally, bacterial artificial chromosome ATXN2-Q22 mice treated with 17-DMAG or HSP990 exhibited highly reduced ATXN2 protein abundance in the cerebellum. Taken together, our study demonstrates inhibition of HSP90 or Na+/K+-ATPases as potentially effective therapeutic strategies for treating SCA2 and ALS.

Core transcriptional networks in Williams syndrome: IGF1-PI3K-AKT-mTOR, MAPK and actin signaling at the synapse echo autism.

Gene networks for disorders of social behavior provide the mechanisms critical for identifying therapeutic targets and biomarkers. Large behavioral phenotypic effects of small human deletions make the positive sociality of Williams syndrome (WS) ideal for determining transcriptional networks for social dysfunction currently based on DNA variations for disorders such as autistic spectrum disorder (ASD) and schizophrenia (SCHZ). Consensus on WS networks has been elusive due to the need for larger cohort size, sensitive genome-wide detection and analytic tools. We report a core set of WS network perturbations in a cohort of 58 individuals (34 with typical, 6 atypical deletions and 18 controls). Genome-wide exon-level expression arrays robustly detected changes in differentially expressed gene (DEG) transcripts from WS deleted genes that ranked in the top 11 of 12 122 transcripts, validated by quantitative reverse transcription PCR, RNASeq and western blots. WS DEG's were strictly dosed in the full but not the atypical deletions that revealed a breakpoint position effect on non-deleted CLIP2, a caveat for current phenotypic mapping based on copy number variants. Network analyses tested the top WS DEG's role in the dendritic spine, employing GeneMANIA to harmonize WS DEGs with comparable query gene-sets. The results indicate perturbed actin cytoskeletal signaling analogous to the excitatory dendritic spines. Independent protein-protein interaction analyses of top WS DEGs generated a 100-node graph annotated topologically revealing three interacting pathways, MAPK, IGF1-PI3K-AKT-mTOR/insulin and actin signaling at the synapse. The results indicate striking similarity of WS transcriptional networks to genome-wide association study-based ASD and SCHZ risk suggesting common network dysfunction for these disorders of divergent sociality.

Antisense oligonucleotide therapy for spinocerebellar ataxia type 2.

There are no disease-modifying treatments for adult human neurodegenerative diseases. Here we test RNA-targeted therapies in two mouse models of spinocerebellar ataxia type 2 (SCA2), an autosomal dominant polyglutamine disease. Both models recreate the progressive adult-onset dysfunction and degeneration of a neuronal network that are seen in patients, including decreased firing frequency of cerebellar Purkinje cells and a decline in motor function. We developed a potential therapy directed at the ATXN2 gene by screening 152 antisense oligonucleotides (ASOs). The most promising oligonucleotide, ASO7, downregulated ATXN2 mRNA and protein, which resulted in delayed onset of the SCA2 phenotype. After delivery by intracerebroventricular injection to ATXN2-Q127 mice, ASO7 localized to Purkinje cells, reduced cerebellar ATXN2 expression below 75% for more than 10 weeks without microglial activation, and reduced the levels of cerebellar ATXN2. Treatment of symptomatic mice with ASO7 improved motor function compared to saline-treated mice. ASO7 had a similar effect in the BAC-Q72 SCA2 mouse model, and in both mouse models it normalized protein levels of several SCA2-related proteins expressed in Purkinje cells, including Rgs8, Pcp2, Pcp4, Homer3, Cep76 and Fam107b. Notably, the firing frequency of Purkinje cells returned to normal even when treatment was initiated more than 12 weeks after the onset of the motor phenotype in BAC-Q72 mice. These findings support ASOs as a promising approach for treating some human neurodegenerative diseases.

The AKT modulator A-443654 reduces α-synuclein expression and normalizes ER stress and autophagy.

Accumulation of α-synuclein is a main underlying pathological feature of Parkinson's disease and α-synucleinopathies, for which lowering expression of the α-synuclein gene (SNCA) is a potential therapeutic avenue. Using a cell-based luciferase reporter of SNCA expression we performed a quantitative high-throughput screen of 155,885 compounds and identified A-443654, an inhibitor of the multiple functional kinase AKT, as a potent inhibitor of SNCA. HEK-293 cells with CAG repeat expanded ATXN2 (ATXN2-Q58 cells) have increased levels of α-synuclein. We found that A-443654 normalized levels of both SNCA mRNA and α-synuclein monomers and oligomers in ATXN2-Q58 cells. A-443654 also normalized levels of α-synuclein in fibroblasts and iPSC-derived dopaminergic neurons from a patient carrying a triplication of the SNCA gene. Analysis of autophagy and endoplasmic reticulum stress markers showed that A-443654 successfully prevented α-synuclein toxicity and restored cell function in ATXN2-Q58 cells, normalizing the levels of mTOR, LC3-II, p62, STAU1, BiP, and CHOP. A-443654 also decreased the expression of DCLK1, an inhibitor of α-synuclein lysosomal degradation. Our study identifies A-443654 and AKT inhibition as a potential strategy for reducing SNCA expression and treating Parkinson's disease pathology.

ALS-associated genes in SCA2 mouse spinal cord transcriptomes.

The spinocerebellar ataxia type 2 (SCA2) gene ATXN2 has a prominent role in the pathogenesis and treatment of amyotrophic lateral sclerosis (ALS). In addition to cerebellar ataxia, motor neuron disease is often seen in SCA2, and ATXN2 CAG repeat expansions in the long normal range increase ALS risk. Also, lowering ATXN2 expression in TDP-43 ALS mice prolongs their survival. Here we investigated the ATXN2 relationship with motor neuron dysfunction in vivo by comparing spinal cord (SC) transcriptomes reported from TDP-43 and SOD1 ALS mice and ALS patients with those from SCA2 mice. SC transcriptomes were determined using an SCA2 bacterial artificial chromosome mouse model expressing polyglutamine expanded ATXN2. SCA2 cerebellar transcriptomes were also determined, and we also investigated the modification of gene expression following treatment of SCA2 mice with an antisense oligonucleotide (ASO) lowering ATXN2 expression. Differentially expressed genes (DEGs) defined three interconnected pathways (innate immunity, fatty acid biosynthesis and cholesterol biosynthesis) in separate modules identified by weighted gene co-expression network analysis. Other key pathways included the complement system and lysosome/phagosome pathways. Of all DEGs in SC, 12.6% were also dysregulated in the cerebellum. Treatment of mice with an ATXN2 ASO also modified innate immunity, the complement system and lysosome/phagosome pathways. This study provides new insights into the underlying molecular basis of SCA2 SC phenotypes and demonstrates annotated pathways shared with TDP-43 and SOD1 ALS mice and ALS patients. It also emphasizes the importance of ATXN2 in motor neuron degeneration and confirms ATXN2 as a therapeutic target.

Staufen1 in Human Neurodegeneration.

OBJECTIVE: Mutations in the ATXN2 gene (CAG expansions ≥32 repeats) can be a rare cause of Parkinson's disease and amyotrophic lateral sclerosis (ALS). We recently reported that the stress granule (SG) protein Staufen1 (STAU1) was overabundant in neurodegenerative disorder spinocerebellar ataxia type 2 (SCA2) patient cells, animal models, and ALS-TDP-43 fibroblasts, and provided a link between SG formation and autophagy. We aimed to test if STAU1 overabundance has a role in the pathogenesis of other neurodegenerative diseases. METHODS: With multiple neurodegenerative patient-derived cell models, animal models, and human postmortem ALS tissue, we evaluate STAU1 function using biochemical and immunohistological analyses. RESULTS: We demonstrate STAU1 overabundance and increased total and phosphorylated mammalian target of rapamycin (mTOR) in fibroblast cells from patients with ALS with mutations in TDP-43, patients with dementia with PSEN1 mutations, a patient with parkinsonism with MAPT mutation, Huntington's disease (HD) mutations, and SCA2 mutations. Increased STAU1 levels and mTOR activity were seen in human ALS spinal cord tissues as well as in animal models. Changes in STAU1 and mTOR protein levels were post-transcriptional. Exogenous expression of STAU1 in wildtype cells was sufficient to activate mTOR and downstream targets and form SGs. Targeting STAU1 by RNAi normalized mTOR, suggesting a potential role for therapy in diseases associated with STAU1 overabundance. INTERPRETATION: STAU1 overabundance in neurodegeneration is a common phenomenon associated with hyperactive mTOR. Targeting STAU1 with ASOs or miRNA viral vectors may represent a novel, efficacious therapy for neurodegenerative diseases characterized by overabundant STAU1. ANN NEUROL 2021;89:1114-1128.

MTSS1/Src family kinase dysregulation underlies multiple inherited ataxias.

The genetically heterogeneous spinocerebellar ataxias (SCAs) are caused by Purkinje neuron dysfunction and degeneration, but their underlying pathological mechanisms remain elusive. The Src family of nonreceptor tyrosine kinases (SFK) are essential for nervous system homeostasis and are increasingly implicated in degenerative disease. Here we reveal that the SFK suppressor Missing-in-metastasis (MTSS1) is an ataxia locus that links multiple SCAs. MTSS1 loss results in increased SFK activity, reduced Purkinje neuron arborization, and low basal firing rates, followed by cell death. Surprisingly, mouse models for SCA1, SCA2, and SCA5 show elevated SFK activity, with SCA1 and SCA2 displaying dramatically reduced MTSS1 protein levels through reduced gene expression and protein translation, respectively. Treatment of each SCA model with a clinically approved Src inhibitor corrects Purkinje neuron basal firing and delays ataxia progression in MTSS1 mutants. Our results identify a common SCA therapeutic target and demonstrate a key role for MTSS1/SFK in Purkinje neuron survival and ataxia progression.

Gene co-expression network analysis for identifying modules and functionally enriched pathways in SCA2.

Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant neurodegenerative disease caused by CAG repeat expansion in the ATXN2 gene. The repeat resides in an encoded region of the gene resulting in polyglutamine (polyQ) expansion which has been assumed to result in gain of function, predominantly, for the ATXN2 protein. We evaluated temporal cerebellar expression profiles by RNA sequencing of ATXN2Q127 mice versus wild-type (WT) littermates. ATXN2Q127 mice are characterized by a progressive motor phenotype onset, and have progressive cerebellar molecular and neurophysiological (Purkinje cell firing frequency) phenotypes. Our analysis revealed previously uncharacterized early and progressive abnormal patterning of cerebellar gene expression. Weighted Gene Coexpression Network Analysis revealed four gene modules that were significantly correlated with disease status, composed primarily of genes associated with GTPase signaling, calcium signaling and cell death. Of these genes, few overlapped with differentially expressed cerebellar genes that we identified in Atxn2-/- knockout mice versus WT littermates, suggesting that loss-of-function is not a significant component of disease pathology. We conclude that SCA2 is a disease characterized by gain of function for ATXN2.

Ataxin-2 regulates RGS8 translation in a new BAC-SCA2 transgenic mouse model.

Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant disorder with progressive degeneration of cerebellar Purkinje cells (PCs) and other neurons caused by expansion of a glutamine (Q) tract in the ATXN2 protein. We generated BAC transgenic lines in which the full-length human ATXN2 gene was transcribed using its endogenous regulatory machinery. Mice with the ATXN2 BAC transgene with an expanded CAG repeat (BAC-Q72) developed a progressive cellular and motor phenotype, whereas BAC mice expressing wild-type human ATXN2 (BAC-Q22) were indistinguishable from control mice. Expression analysis of laser-capture microdissected (LCM) fractions and regional expression confirmed that the BAC transgene was expressed in PCs and in other neuronal groups such as granule cells (GCs) and neurons in deep cerebellar nuclei as well as in spinal cord. Transcriptome analysis by deep RNA-sequencing revealed that BAC-Q72 mice had progressive changes in steady-state levels of specific mRNAs including Rgs8, one of the earliest down-regulated transcripts in the Pcp2-ATXN2[Q127] mouse line. Consistent with LCM analysis, transcriptome changes analyzed by deep RNA-sequencing were not restricted to PCs, but were also seen in transcripts enriched in GCs such as Neurod1. BAC-Q72, but not BAC-Q22 mice had reduced Rgs8 mRNA levels and even more severely reduced steady-state protein levels. Using RNA immunoprecipitation we showed that ATXN2 interacted selectively with RGS8 mRNA. This interaction was impaired when ATXN2 harbored an expanded polyglutamine. Mutant ATXN2 also reduced RGS8 expression in an in vitro coupled translation assay when compared with equal expression of wild-type ATXN2-Q22. Reduced abundance of Rgs8 in Pcp2-ATXN2[Q127] and BAC-Q72 mice supports our observations of a hyper-excitable mGluR1-ITPR1 signaling axis in SCA2, as RGS proteins are linked to attenuating mGluR1 signaling.

ATXN2-AS, a gene antisense to ATXN2, is associated with spinocerebellar ataxia type 2 and amyotrophic lateral sclerosis.

OBJECTIVE: Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative disease caused by a CAG repeat expansion in the gene ataxin-2 (ATXN2). ATXN2 intermediate-length CAG expansions were identified as a risk factor for amyotrophic lateral sclerosis (ALS). The ATXN2 CAG repeat is translated into polyglutamine, and SCA2 pathogenesis has been thought to derive from ATXN2 protein containing an expanded polyglutamine tract. However, recent evidence of bidirectional transcription at multiple CAG/CTG disease loci has led us to test whether additional mechanisms of pathogenesis may contribute to SCA2. METHODS: In this work, using human postmortem tissue, various cell models, and animal models, we provide the first evidence that an antisense transcript at the SCA2 locus contributes to SCA2 pathogenesis. RESULTS: We demonstrate the expression of a transcript, containing the repeat as a CUG tract, derived from a gene (ATXN2-AS) directly antisense to ATXN2. ATXN2-AS transcripts with normal and expanded CUG repeats are expressed in human postmortem SCA2 brains, human SCA2 fibroblasts, induced SCA2 pluripotent stem cells, SCA2 neural stem cells, and lymphoblastoid lines containing an expanded ATXN2 allele associated with ALS. ATXN2-AS transcripts with a CUG repeat expansion are toxic in an SCA2 cell model and form RNA foci in SCA2 cerebellar Purkinje cells. Finally, we detected missplicing of amyloid beta precursor protein and N-methyl-D-aspartate receptor 1 in SCA2 brains, consistent with findings in other diseases characterized by RNA-mediated pathogenesis. INTERPRETATION: These results suggest that ATXN2-AS has a role in SCA2 and possibly ALS pathogenesis, and may therefore provide a novel therapeutic target for these diseases. Ann Neurol 2016;80:600-615.

Cerebellar Micro-RNA Profile in a Mouse Model of Spinocerebellar Ataxia Type 2.

Background and Objectives: Micro-RNAs (miRNAs) are critical for regulating the expression of genes in multiple neurodegenerative diseases, but miRNAs have not been investigated in spinocerebellar ataxia type 2 (SCA2). SCA2, a dominantly inherited progressive neurodegenerative polyglutamine (polyQ) disease, is caused by a CAG repeat expansion in the ataxin-2 (ATXN2) gene. In this study, we determined miRNA transcriptomes in SCA2-BAC-ATXN2[Q72] transgenic mice. Methods: We assessed the expression of miRNAs in SCA2 transgenic mouse cerebella using the HiSeq Illumina sequencer. We used the miRNA target filter tool in Qiagen Ingenuity Pathway Analysis (IPA) to identify target genes of differentially expressed miRNAs (DEmiRs) within in the SCA2 mouse transcriptomes and then performed pathway analyses. Results: Our analysis revealed significant changes in the expression levels of multiple miRNAs in mice with SCA2. We identified 81 DEmiRs in mice with SCA2, with 52 miRNAs upregulated and 29 miRNAs downregulated after onset of rotarod deficit. Subsequent IPA processing enabled us to establish connections between these DEmiRs and specific biological regulatory functions. Furthermore, by using the IPA miRNA target filter, we identified target genes of DEmiRs in the SCA2-BAC-ATXN2[Q72] transcriptome data set and demonstrated their significant impact on several biological functional and disease pathways. Discussion: Our study establishes the role of both DEmiRs and their targets in SCA2 pathogenesis. By expressing mutant ATXN2 under the control of its endogenous regulatory elements in the SCA2-BAC-ATXN2[Q72] mouse model, we identified a set of DEmiRs that are shared across multiple neurodegenerative diseases including other SCAs, Alzheimer disease (AD), Parkinson disease (PD), and amyotrophic lateral sclerosis (ALS). There was a significant overlap of both DEmiRs and their targets of BAC-ATXN2[Q72] transcriptomes in dysregulated pathways that characterize SCA2. This observation also extended to dysregulated pathways in ALS, AD, and PD. DEmiRs identified in this study may represent therapeutic targets for neurodegeneration or lead to biomarkers for characterizing various neurodegenerative diseases.

A GGC-repeat expansion in ZFHX3 encoding polyglycine causes spinocerebellar ataxia type 4 and impairs autophagy.

Despite linkage to chromosome 16q in 1996, the mutation causing spinocerebellar ataxia type 4 (SCA4), a late-onset sensory and cerebellar ataxia, remained unknown. Here, using long-read single-strand whole-genome sequencing (LR-GS), we identified a heterozygous GGC-repeat expansion in a large Utah pedigree encoding polyglycine (polyG) in zinc finger homeobox protein 3 (ZFHX3), also known as AT-binding transcription factor 1 (ATBF1). We queried 6,495 genome sequencing datasets and identified the repeat expansion in seven additional pedigrees. Ultrarare DNA variants near the repeat expansion indicate a common distant founder event in Sweden. Intranuclear ZFHX3-p62-ubiquitin aggregates were abundant in SCA4 basis pontis neurons. In fibroblasts and induced pluripotent stem cells, the GGC expansion led to increased ZFHX3 protein levels and abnormal autophagy, which were normalized with small interfering RNA-mediated ZFHX3 knockdown in both cell types. Improving autophagy points to a therapeutic avenue for this novel polyG disease. The coding GGC-repeat expansion in an extremely G+C-rich region was not detectable by short-read whole-exome sequencing, which demonstrates the power of LR-GS for variant discovery.

RNA steady-state defects in myotonic dystrophy are linked to nuclear exclusion of SHARP.

We describe a new mechanism by which CTG tract expansion affects myotonic dystrophy (DM1). Changes to the levels of a panel of RNAs involved in muscle development and function that are downregulated in DM1 are due to aberrant localization of the transcription factor SHARP (SMART/HDAC1-associated repressor protein). Mislocalization of SHARP in DM1 is consistent with increased CRM1-mediated export of SHARP to the cytoplasm. A direct link between CTG repeat expression and SHARP mislocalization is demonstrated as expression of expanded CTG repeats in normal cells recapitulates cytoplasmic SHARP localization. These results demonstrate a role for the inactivation of SHARP transcription in DM1 biology.

Effects of STAU1/staufen1 on autophagy in neurodegenerative diseases.

The double-stranded RNA-binding protein, STAU1 (staufen double-stranded RNA binding protein 1) is a multifunctional protein that localizes to stress granules (SGs). We had previously found that STAU1 is overabundant in fibroblast cell lines from patients with spinocerebellar ataxia type 2 (SCA2) or amyotrophic lateral sclerosis (ALS)-frontotemporal dementia (FTD) as well as in animal models of these diseases. STAU1 overabundance is post-transcriptional and associated with MTOR hyperactivation and links SG formation with macroautophagy/autophagy. Reducing STAU1 levels in mice with ALS mutations normalizes MTOR activity and autophagy-related marker proteins. We also see increased STAU1 levels in HEK293 cells expressing C9orf72-relevant dipeptide repeats (DPRs), and DPRs are not observed in cells where STAU1 is targeted by RNAi. Overexpression of STAU1 in HEK293 cells increases MTOR translation by directly interacting with the MTOR mRNA 5'UTR, activating downstream targets and impairing autophagic flux. STAU1 may constitute a novel target to modulate MTOR activity and autophagy and for the treatment of neurodegenerative diseases.

Expanded CUG repeats Dysregulate RNA splicing by altering the stoichiometry of the muscleblind 1 complex.

To understand the role of the splice regulator muscleblind 1 (MBNL1) in the development of RNA splice defects in myotonic dystrophy I (DM1), we purified RNA-independent MBNL1 complexes from normal human myoblasts and examined the behavior of these complexes in DM1 myoblasts. Antibodies recognizing MBNL1 variants (MBNL1(CUG)), which can sequester in the toxic CUG RNA foci that develop in DM1 nuclei, were used to purify MBNL1(CUG) complexes from normal myoblasts. In normal myoblasts, MBNL1(CUG) bind 10 proteins involved in remodeling ribonucleoprotein complexes including hnRNP H, H2, H3, F, A2/B1, K, L, DDX5, DDX17, and DHX9. Of these proteins, only MBNL1(CUG) colocalizes extensively with DM1 CUG foci (>80% of foci) with its partners being present in <10% of foci. Importantly, the stoichiometry of MBNL1(CUG) complexes is altered in DM1 myoblasts, demonstrating an increase in the steady state levels of nine of its partner proteins. These changes are recapitulated by the expression of expanded CUG repeat RNA in Cos7 cells. Altered stoichiometry of MBNL1(CUG) complexes results from aberrant protein synthesis or stability and is unlinked to PKCα function. Modeling these changes in normal myoblasts demonstrates that increased levels of hnRNP H, H2, H3, F, and DDX5 independently dysregulate splicing in overlapping RNA subsets. Thus expression of expanded CUG repeats alters the stoichiometry of MBNL1(CUG) complexes to allow both the reinforcement and expansion of RNA processing defects.

The RNA-binding protein and stress granule component ATAXIN-2 is expressed in mouse and human tissues associated with glaucoma pathogenesis.

Polyglutamine repeat expansions in the Ataxin-2 (ATXN2) gene were first implicated in Spinocerebellar Ataxia Type 2, a disease associated with degeneration of motor neurons and Purkinje cells. Recent studies linked single nucleotide polymorphisms in the gene to elevated intraocular pressure in primary open angle glaucoma (POAG); yet, the localization of ATXN2 across glaucoma-relevant tissues of the vertebrate eye has not been thoroughly examined. This study characterizes ATXN2 expression in the mouse and human retina, and anterior eye, using an antibody validated in ATXN2-/- retinas. ATXN2-ir was localized to cytosolic sub compartments in retinal ganglion cell (RGC) somata and proximal dendrites in addition to GABAergic, glycinergic, and cholinergic amacrine cells in the inner plexiform layer (IPL) and displaced amacrine cells. Human, but not mouse retinas showed modest immunolabeling of bipolar cells. ATXN2 immunofluorescence was prominent in the trabecular meshwork and pigmented and nonpigmented cells of the ciliary body, with analyses of primary human trabecular meshwork cells confirming the finding. The expression of ATXN2 in key POAG-relevant ocular tissues supports the potential role in autophagy and stress granule formation in response to ocular hypertension.

Splicing Control of Pontocerebellar Development.

In this issue of Neuron, Chai et al. (2021) analyze several families with neurodegeneration and marked pontocerebellar hypoplasia and microcephaly and identify recessive (bi-allelic) mutations in peptidyl-prolyl isomerase-like 1 (PPIL1) and pre-RNA-processing-17 (PPR17). PPIL1 patient mutation knockin mice develop neuronal apoptosis. Loss of either protein affects splicing predominantly involving GC-rich and short introns.

Correction: Generation of SNCA Cell Models Using Zinc Finger Nuclease (ZFN) Technology for Efficient High-Throughput Drug Screening.

[This corrects the article DOI: 10.1371/journal.pone.0136930.].