CGG repeat expansion in LRP12 in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the degeneration of motor neurons. Although repeat expansion in C9orf72 is its most common cause, the pathogenesis of ALS isn't fully clear. In this study, we show that repeat expansion in LRP12, a causative variant of oculopharyngodistal myopathy type 1 (OPDM1), is a cause of ALS. We identify CGG repeat expansion in LRP12 in five families and two simplex individuals. These ALS individuals (LRP12-ALS) have 61-100 repeats, which contrasts with most OPDM individuals with repeat expansion in LRP12 (LRP12-OPDM), who have 100-200 repeats. Phosphorylated TDP-43 is present in the cytoplasm of iPS cell-derived motor neurons (iPSMNs) in LRP12-ALS, a finding that reproduces the pathological hallmark of ALS. RNA foci are more prominent in muscle and iPSMNs in LRP12-ALS than in LRP12-OPDM. Muscleblind-like 1 aggregates are observed only in OPDM muscle. In conclusion, CGG repeat expansions in LRP12 cause ALS and OPDM, depending on the length of the repeat. Our findings provide insight into the repeat length-dependent switching of phenotypes.

Repeat RNA expansion disorders of the nervous system: post-transcriptional mechanisms and therapeutic strategies.

Dozens of incurable neurological disorders result from expansion of short repeat sequences in both coding and non-coding regions of the transcriptome. Short repeat expansions underlie microsatellite repeat expansion (MRE) disorders including myotonic dystrophy (DM1, CUG50-3,500 in DMPK; DM2, CCTG75-11,000 in ZNF9), fragile X tremor ataxia syndrome (FXTAS, CGG50-200 in FMR1), spinal bulbar muscular atrophy (SBMA, CAG40-55 in AR), Huntington's disease (HD, CAG36-121 in HTT), C9ORF72- amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD and C9-ALS/FTD, GGGGCC in C9ORF72), and many others, like ataxias. Recent research has highlighted several mechanisms that may contribute to pathology in this heterogeneous class of neurological MRE disorders - bidirectional transcription, intranuclear RNA foci, and repeat associated non-AUG (RAN) translation - which are the subject of this review. Additionally, many MRE disorders share similar underlying molecular pathologies that have been recently targeted in experimental and preclinical contexts. We discuss the therapeutic potential of versatile therapeutic strategies that may selectively target disrupted RNA-based processes and may be readily adaptable for the treatment of multiple MRE disorders. Collectively, the strategies under consideration for treatment of multiple MRE disorders include reducing levels of toxic RNA, preventing RNA foci formation, and eliminating the downstream cellular toxicity associated with peptide repeats produced by RAN translation. While treatments are still lacking for the majority of MRE disorders, several promising therapeutic strategies have emerged and will be evaluated within this review.

SFPQ regulates the accumulation of RNA foci and dipeptide repeat proteins from the expanded repeat mutation in C9orf72.

The expanded GGGGCC repeat mutation in the C9orf72 gene is the most common genetic cause of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The expansion is transcribed to sense and antisense RNA, which form RNA foci and bind cellular proteins. This mechanism of action is considered cytotoxic. Translation of the expanded RNA transcripts also leads to the accumulation of toxic dipeptide repeat proteins (DPRs). The RNA-binding protein splicing factor proline and glutamine rich (SFPQ), which is being increasingly associated with ALS and FTD pathology, binds to sense RNA foci. Here, we show that SFPQ plays an important role in the C9orf72 mutation. Overexpression of SFPQ resulted in higher numbers of both sense and antisense RNA foci and DPRs in transfected human embryonic kidney (HEK) cells. Conversely, reduced SPFQ levels resulted in lower numbers of RNA foci and DPRs in both transfected HEK cells and C9orf72 mutation-positive patient-derived fibroblasts and lymphoblasts. Therefore, we have revealed a role of SFPQ in regulating the C9orf72 mutation that has implications for understanding and developing novel therapeutic targets for ALS and FTD.This article has an associated First Person interview with the first author of the paper.

G-Quadruplexes in Repeat Expansion Disorders.

The repeat expansions are the main genetic cause of various neurodegeneration diseases. More than ten kinds of repeat sequences with different lengths, locations, and structures have been confirmed in the past two decades. G-rich repeat sequences, such as CGG and GGGGCC, are reported to form functional G-quadruplexes, participating in many important bioprocesses. In this review, we conducted an overview concerning the contribution of G-quadruplex in repeat expansion disorders and summarized related mechanisms in current pathological studies, including the increasing genetic instabilities in replication and transcription, the toxic RNA foci formed in neurons, and the loss/gain function of proteins and peptides. Furthermore, novel strategies targeting G-quadruplex repeats were developed based on the understanding of disease mechanism. Small molecules and proteins binding to G-quadruplex in repeat expansions were investigated to protect neurons from dysfunction and delay the progression of neurodegeneration. In addition, the effects of environment on the stability of G-quadruplex were discussed, which might be critical factors in the pathological study of repeat expansion disorders.

C9orf72 hexanucleotide repeat expansion leads to altered neuronal and dendritic spine morphology and synaptic dysfunction.

Frontotemporal lobar degeneration (FTLD) comprises a heterogenous group of progressive neurodegenerative syndromes. To date, no validated biomarkers or effective disease-modifying therapies exist for the different clinical or genetic subtypes of FTLD. The most common genetic cause underlying FTLD and amyotrophic lateral sclerosis (ALS) is a hexanucleotide repeat expansion in the C9orf72 gene (C9-HRE). FTLD is accompanied by changes in several neurotransmitter systems, including the glutamatergic, GABAergic, dopaminergic, and serotonergic systems and many clinical symptoms can be explained by disturbances in these systems. Here, we aimed to elucidate the effects of the C9-HRE on synaptic function, molecular composition of synapses, and dendritic spine morphology. We overexpressed the pathological C9-HRE in cultured E18 mouse primary hippocampal neurons and characterized the pathological, morphological, and functional changes by biochemical methods, confocal microscopy, and live cell calcium imaging. The C9-HRE-expressing neurons were confirmed to display the pathological RNA foci and DPR proteins. C9-HRE expression led to significant changes in dendritic spine morphologies, as indicated by decreased number of mushroom-type spines and increased number of stubby and thin spines, as well as diminished neuronal branching. These morphological changes were accompanied by concomitantly enhanced susceptibility of the neurons to glutamate-induced excitotoxicity as well as augmented and prolonged responses to excitatory stimuli by glutamate and depolarizing potassium chloride as compared to control neurons. Mechanistically, the hyperexcitation phenotype in the C9-HRE-expressing neurons was found to be underlain by increased activity of extrasynaptic GluN2B-containing N-methyl-d-aspartate (NMDA) receptors. Our results are in accordance with the idea suggesting that C9-HRE is associated with enhanced excitotoxicity and synaptic dysfunction. Thus, therapeutic interventions targeted to alleviate synaptic disturbances might offer efficient avenues for the treatment of patients with C9-HRE-associated FTLD.

Nuclear RNA foci from C9ORF72 expansion mutation form paraspeckle-like bodies.

The GGGGCC (G4C2) repeat expansion mutation in the C9ORF72 gene is the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Transcription of the repeat and formation of nuclear RNA foci, which sequester specific RNA-binding proteins, is one of the possible pathological mechanisms. Here, we show that (G4C2) n repeat RNA predominantly associates with essential paraspeckle proteins SFPQ, NONO, RBM14, FUS and hnRNPH and colocalizes with known paraspeckle-associated RNA hLinc-p21. As formation of paraspeckles in motor neurons has been associated with early phases of ALS, we investigated the extent of similarity between paraspeckles and (G4C2) n RNA foci. Overexpression of (G4C2)72 RNA results in their increased number and colocalization with SFPQ-stained nuclear bodies. These paraspeckle-like (G4C2)72 RNA foci form independently of the known paraspeckle scaffold, the long non-coding RNA NEAT1 Moreover, the knockdown of SFPQ protein in C9ORF72 expansion mutation-positive fibroblasts significantly reduces the number of (G4C2) n RNA foci. In conclusion, (G4C2) n RNA foci have characteristics of paraspeckles, which suggests that both RNA foci and paraspeckles play roles in FTD and ALS, and implies approaches for regulation of their formation.

Cardiac Pathology in Myotonic Dystrophy Type 1.

Myotonic dystrophy type 1 (DM1), the most common muscular dystrophy affecting adults and children, is a multi-systemic disorder affecting skeletal, cardiac, and smooth muscles as well as neurologic, endocrine and other systems. This review is on the cardiac pathology associated with DM1. The heart is one of the primary organs affected in DM1. Cardiac conduction defects are seen in up to 75% of adult DM1 cases and sudden death due to cardiac arrhythmias is one of the most common causes of death in DM1. Unfortunately, the pathogenesis of cardiac manifestations in DM1 is ill defined. In this review, we provide an overview of the history of cardiac studies in DM1, clinical manifestations, and pathology of the heart in DM1. This is followed by a discussion of emerging data about the utility of cardiac magnetic resonance imaging (CMR) as a biomarker for cardiac disease in DM1, and ends with a discussion on models of cardiac RNA toxicity in DM1 and recent clinical guidelines for cardiologic management of individuals with DM1.

Reprogramming fibroblasts and peripheral blood cells from a C9ORF72 patient: A proof-of-principle study.

As for the majority of neurodegenerative diseases, pathological mechanisms of amyotrophic lateral sclerosis (ALS) have been challenging to study due to the difficult access to alive patients' cells. Induced pluripotent stem cells (iPSCs) offer a useful in vitro system for modelling human diseases. iPSCs can be theoretically obtained by reprogramming any somatic tissue although fibroblasts (FB) remain the most used cells. However, reprogramming peripheral blood cells (PB) may offer significant advantages. In order to investigate whether the choice of starting cells may affect reprogramming and motor neuron (MNs) differentiation potential, we used both FB and PB from a same C9ORF72-mutated ALS patient to obtain iPSCs and compared several hallmarks of the pathology. We found that both iPSCs and MNs derived from the two tissues showed identical properties and features and can therefore be used interchangeably, giving the opportunity to easily obtain iPSCs from a more manageable source of cells, such as PB.

RNA-mediated toxicity in C9orf72 ALS and FTD.

A GGGGCC hexanucleotide repeat expansion in the first intron of C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Compelling evidence suggests that gain of toxicity from the bidirectionally transcribed repeat expanded RNAs plays a central role in disease pathogenesis. Two potential mechanisms have been proposed including RNA-mediated toxicity and/or the production of toxic dipeptide repeat proteins. In this review, we focus on the role of RNA mediated toxicity in ALS/FTD caused by the C9orf72 mutation and discuss arguments for and against this mechanism. In addition, we summarize how G4C2 repeat RNAs can elicit toxicity and potential therapeutic strategies to mitigate RNA-mediated toxicity.

C9orf72-FTD/ALS pathogenesis: evidence from human neuropathological studies.

What are the most important and treatable pathogenic mechanisms in C9orf72-FTD/ALS? Model-based efforts to address this question are forging ahead at a blistering pace, often with conflicting results. But what does the human neuropathological literature reveal? Here, we provide a critical review of the human studies to date, seeking to highlight key gaps or uncertainties in our knowledge. First, we engage the C9orf72-specific mechanisms, including C9orf72 haploinsufficiency, repeat RNA foci, and dipeptide repeat protein inclusions. We then turn to some of the most prominent C9orf72-associated features, such as TDP-43 loss-of-function, TDP-43 aggregation, and nuclear transport defects. Finally, we review potential disease-modifying epigenetic and genetic factors and the natural history of the disease across the lifespan. Throughout, we emphasize the importance of anatomical precision when studying how candidate mechanisms relate to neuronal, regional, and behavioral findings. We further highlight methodological approaches that may help address lingering knowledge gaps and uncertainties, as well as other logical next steps for the field. We conclude that anatomically oriented human neuropathological studies have a critical role to play in guiding this fast-moving field toward effective new therapies.

[The Role of Mutant RNA in the Pathogenesis of Huntington's Disease and Other Polyglutamine Diseases].

Polyglutamine diseases are rare, inherited neurodegenerative pathologies that arise as a result of expansion of trinucleotide CAG repeats in the coding segment of certain genes. This expansion leads to the appearance of mRNA with abnormally long repetitive CAG triplets (mCAG-RNA) and proteins with polyglutamine (PolyQ) tracts in the cells, which is why these pathologies are commonly termed polyglutamine diseases, or PolyQ diseases. To date, nine PolyQ diseases have been described: Huntington's disease, dentatorubral pallidoluysian atrophy (DRPLA), spinal and bulbar muscular atrophy (SBMA), and six different types of spinocerebellar ataxia (SCA 1,2,3,6,7, and 17). PolyQ diseases lead to serious, constantly progressing dysfunctions of the nervous and/or muscular systems, and there currently exists no efficacious therapy for any of them. Recent studies have convincingly shown that mCAG-RNA can actively participate in the pathological process during the development of PolyQ diseases. Mutant RNA is involved in a wide range of molecular mechanisms, ultimately leading to disruption of the functions of transcription, splicing, translation, cytosol structure, RNA transport from the nucleus to the cytoplasm, and, finally, to neurodegeneration. This review discusses the involvement of mutant mCAG-RNA in neurodegenerative processes in PolyQ diseases.

Repeat Expansion Disease Models.

Repeat expansion disorders are a group of inherited neuromuscular diseases, which are caused by expansion mutations of repeat sequences in the disease-causing genes. Repeat expansion disorders include a class of diseases caused by repeat expansions in the coding region of the genes, producing mutant proteins with amino acid repeats, mostly the polyglutamine (polyQ) diseases, and another class of diseases caused by repeat expansions in the noncoding regions, producing aberrant RNA with expanded repeats, which are called noncoding repeat expansion diseases. A variety of Drosophila disease models have been established for both types of diseases, and they have made significant contributions toward elucidating the molecular mechanisms of and developing therapies for these neuromuscular diseases.

Reduction of toxic RNAs in myotonic dystrophies type 1 and type 2 by the RNA helicase p68/DDX5.

Myotonic dystrophies type 1 (DM1) and type 2 (DM2) are neuromuscular diseases, caused by accumulation of CUG and CCUG RNAs in toxic aggregates. Here we report that the increased stability of the mutant RNAs in both types of DM is caused by deficiency of RNA helicase p68. We have identified p68 by studying CCUG-binding proteins associated with degradation of the mutant CCUG repeats. Protein levels of p68 are reduced in DM1 and DM2 biopsied skeletal muscle. Delivery of p68 in DM1/2 cells causes degradation of the mutant RNAs, whereas delivery of p68 in skeletal muscle of DM1 mouse model reduces skeletal muscle myopathy and atrophy. Our study shows that correction of p68 may reduce toxicity of the mutant RNAs in DM1 and in DM2.

In-depth clinico-pathological examination of RNA foci in a large cohort of C9ORF72 expansion carriers.

A growing body of evidence suggests that a loss of chromosome 9 open reading frame 72 (C9ORF72) expression, formation of dipeptide-repeat proteins, and generation of RNA foci contribute to disease pathogenesis in amyotrophic lateral sclerosis and frontotemporal dementia. Although the levels of C9ORF72 transcripts and dipeptide-repeat proteins have already been examined thoroughly, much remains unknown about the role of RNA foci in C9ORF72-linked diseases. As such, we performed a comprehensive RNA foci study in an extensive pathological cohort of C9ORF72 expansion carriers (n = 63). We evaluated two brain regions using a newly developed computer-automated pipeline allowing recognition of cell nuclei and RNA foci (sense and antisense) supplemented by manual counting. In the frontal cortex, the percentage of cells with sense or antisense RNA foci was 26 or 12%, respectively. In the cerebellum, 23% of granule cells contained sense RNA foci and 1% antisense RNA foci. Interestingly, the highest percentage of cells with RNA foci was observed in cerebellar Purkinje cells (~70%). In general, more cells contained sense RNA foci than antisense RNA foci; however, when antisense RNA foci were present, they were usually more abundant. We also observed that an increase in the percentage of cells with antisense RNA foci was associated with a delayed age at onset in the frontal cortex (r = 0.43, p = 0.003), whereas no other associations with clinico-pathological features were seen. Importantly, our large-scale study is the first to provide conclusive evidence that RNA foci are not the determining factor of the clinico-pathological variability observed in C9ORF72 expansion carriers and it emphasizes that the distribution of RNA foci does not follow the pattern of neurodegeneration, stressing the complex interplay between different aspects of C9ORF72-related diseases.

RNA toxicity and missplicing in the common eye disease fuchs endothelial corneal dystrophy.

Fuchs endothelial corneal dystrophy (FECD) is an inherited degenerative disease that affects the internal endothelial cell monolayer of the cornea and can result in corneal edema and vision loss in severe cases. FECD affects ∼5% of middle-aged Caucasians in the United States and accounts for >14,000 corneal transplantations annually. Among the several genes and loci associated with FECD, the strongest association is with an intronic (CTG·CAG)n trinucleotide repeat expansion in the TCF4 gene, which is found in the majority of affected patients. Corneal endothelial cells from FECD patients harbor a poly(CUG)n RNA that can be visualized as RNA foci containing this condensed RNA and associated proteins. Similar to myotonic dystrophy type 1, the poly(CUG)n RNA co-localizes with and sequesters the mRNA-splicing factor MBNL1, leading to missplicing of essential MBNL1-regulated mRNAs. Such foci and missplicing are not observed in similar cells from FECD patients who lack the repeat expansion. RNA-Seq splicing data from the corneal endothelia of FECD patients and controls reveal hundreds of differential alternative splicing events. These include events previously characterized in the context of myotonic dystrophy type 1 and epithelial-to-mesenchymal transition, as well as splicing changes in genes related to proposed mechanisms of FECD pathogenesis. We report the first instance of RNA toxicity and missplicing in a common non-neurological/neuromuscular disease associated with a repeat expansion. The FECD patient population with this (CTG·CAG)n trinucleotide repeat expansion exceeds that of the combined number of patients in all other microsatellite expansion disorders.

Insights into the pathogenic mechanisms of Chromosome 9 open reading frame 72 (C9orf72) repeat expansions.

The identification of a hexanucleotide repeat expansion in a non-coding region of C9orf72 as a major cause of both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) drastically changed the field of research on both of these conditions. Yet, despite the vast amount of work aimed at elucidating the molecular mechanisms underlying the role of this repeat in disease, the exact pathomechanisms are still unclear. A reduction in the expression of the C9orf72 gene is observed in patients, but a gain-of-function model is now preferred. The hexanucleotide repeat expansion forms RNA foci in the central nervous system (CNS) of repeat-positive FTD and ALS patients, and these foci are believed to sequester RNA-binding proteins (RBPs) and impair their function in RNA processing. At the same time, the repeat undergoes repeat-associated non-ATG translation to produce dipeptide repeat proteins that also form inclusions in the patient CNS. Studies from cells and flies suggest that these proteins may also be an important factor in the disease. Finally, the hexanucleotide repeat also induces the mislocalization and aggregation of TAR DNA-binding protein 43 (TDP-43) through an as yet unknown mechanism. This review covers the different potential pathogenic factors that have been put forth for C9orf72-repeat-associated FTD and ALS (C9-FTD/ALS), while highlighting some remaining questions. A repeat expansion in C9orf72 is a common cause of both frontal temporal dementia and amyotrophic lateral sclerosis. Although there is a decrease in C9orf72 expression in patients, this repeat is believed to induce disease primarily through an unknown gain-of-function mechanism involving the RNA, repeat-associated non-AUG translation, or both. This review summarizes and discusses current knowledge on C9orf72 repeat-associated pathophysiology. This article is part of the Frontotemporal Dementia special issue.

Frontotemporal lobar dementia and amyotrophic lateral sclerosis associated with c9orf72 expansion.

An intronic GGGGCC repeat expansion in c9orf72 gene has been identified as the most common genetic cause of frontotemporal lobar dementia (FTLD), amyotrophic lateral sclerosis (ALS) and FTLD-ALS. The discovery of c9orf72 gene has led to important scientific progresses and has considerably changed our clinical practice over the last few years. This paper summarizes the common and less typical phenotypes associated with c9orf72 expansion, the complex pathological pattern characterized by p62/dipeptide repeat aggregates, as well as the pathological mechanisms by which the expansion might produce neurodegeneration implicating loss-of-function, RNA toxicity, RNA-binding protein sequestration and accumulation of dipeptide repeats. We also discuss the recommendations and limits for genetic testing and counseling in clinical practice.

Characteristic RNA foci of the abnormal hexanucleotide GGCCUG repeat expansion in spinocerebellar ataxia type 36 (Asidan).

BACKGROUND AND PURPOSE: Spinocerebellar ataxia type 36 (SCA36), also called Asidan, is an autosomal-dominant neurodegenerative disorder identified as a hexanucleotide GGCCTG repeat expansion in the first intron 1 of the NOP56 gene. In the present study, for the first time an autopsy sample from an Asidan patient was examined and cytoplasmic inclusions and (GGCCUG)n repeat RNA foci were detected. METHODS: Hematoxylin and eosin staining, immunohistochemical staining, as well as fluorescence in situ hybridization were used to investigate the cytoplasmic inclusions of ubiquitin and p62 and the (GGCCUG)n repeat RNA foci. RESULTS: The present study showed both ubiquitin- and p62-positive inclusions in the cytoplasm of the inferior olivary nucleus of the Asidan patient, (GGCCUG)n RNA foci in neuronal nuclei of the cerebrum, cerebellum, inferior olive, spinal cord and temporal muscle, and three types of RNA foci, i.e. single small, multiple small and giant. Of interest is that the giant RNA foci, nearly 10 µm in diameter, that were detected in Purkinje cells, spinal motor neurons and most frequently in the inferior olivary nucleus, may be responsible for pivotal clinical symptoms of Asidan. CONCLUSIONS: The present study is the first report to show neuronal cytoplasmic inclusion bodies and giant RNA foci in an Asidan patient. The relationships between the giant RNA foci and neurodegeneration have yet to be studied.

RNA-mediated toxicity in neurodegenerative disease.

Cellular viability depends upon the well-orchestrated functions carried out by numerous protein-coding and non-coding RNAs, as well as RNA-binding proteins. During the last decade, it has become increasingly evident that abnormalities in RNA processing represent a common feature among many neurodegenerative diseases. In "RNAopathies", which include diseases caused by non-coding repeat expansions, RNAs exert toxicity via diverse mechanisms: RNA foci formation, bidirectional transcription, and the production of toxic RNAs and proteins by repeat associated non-ATG translation. The mechanisms of toxicity in "RNA-binding proteinopathies", diseases in which RNA-binding proteins like TDP-43 and FUS play a prominent role, have yet to be fully elucidated. Nonetheless, both loss of function of the RNA binding protein, and a toxic gain of function resulting from its aggregation, are thought to be involved in disease pathogenesis. As part of the special issue on RNA and Splicing Regulation in Neurodegeneration, this review intends to explore the diverse RNA-related mechanisms contributing to neurodegeneration, with a special emphasis on findings emerging from animal models.

Abnormal RNA structures (RNA foci) containing a penta-nucleotide repeat (UGGAA)n in the Purkinje cell nucleus is associated with spinocerebellar ataxia type 31 pathogenesis.

Spinocerebellar ataxia type 31 (SCA31) is an autosomal-dominant cerebellar ataxia showing a Purkinje cell (PC)-predominant neurodegeneration in humans. The mutation is a complex penta-nucleotide repeat containing (TGGAA)n , (TAGAA)n , (TAAAA)n and (TAGAATAAAA)n inserted in an intron shared by two different genes BEAN1 and TK2 located in the long arm of the human chromosome 16. Previous studies have shown that (TGGAA)n is the critical component of SCA31 pathogenesis while the three other repeats, also present in normal Japanese, are not essential. Importantly, it has been shown that BEAN1 and TK2 are transcribed in mutually opposite directions in the human brain. Furthermore, abnormal RNA structures called "RNA foci" are observed by a probe against (UAGAAUAAAA)n in SCA31 patients' PC nuclei, indicating that the BEAN1-direction mutant transcript appears instrumental for the pathogenesis. However, it is not known whether the critical repeat (TGGAA)n contributes to the formation of RNA foci, neither do we understand how the RNA foci formation is relevant to the pathogenesis. To address these issues, we investigated two SCA31 cerebella by fluorescence in situ hybridization using a probe against (UGGAA)n . We also asked whether the mutant BEAN1-transcript containing (UGGAA)n exerts toxicity compared to the other three repeats in cultured cells. Histopathologically, we confirm that the PC is the main target of SCA31 pathogenesis. We find that the RNA foci containing (UGGAA)n are indeed observed in PC nuclei of both SCA31 patients, whereas similar foci were not observed in control individuals. In both transiently and stably expressed cultured cell models, we also find that the mutation transcribed in the BEAN1-direction yields more toxicity than control transcripts and forms RNA foci detected with probes against (UGGAA)n and (UAGAAUAAAA)n . Taking these findings together, we conclude that the RNA foci containing BEAN1-direction transcript (UGGAA)n are associated with PC degeneration in SCA31.