FUS regulates RAN translation through modulating the G-quadruplex structure of GGGGCC repeat RNA in C9orf72-linked ALS/FTD.

Abnormal expansions of GGGGCC repeat sequence in the noncoding region of the C9orf72 gene is the most common cause of familial amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD). The expanded repeat sequence is translated into dipeptide repeat proteins (DPRs) by noncanonical repeat-associated non-AUG (RAN) translation. Since DPRs play central roles in the pathogenesis of C9-ALS/FTD, we here investigate the regulatory mechanisms of RAN translation, focusing on the effects of RNA-binding proteins (RBPs) targeting GGGGCC repeat RNAs. Using C9-ALS/FTD model flies, we demonstrated that the ALS/FTD-linked RBP FUS suppresses RAN translation and neurodegeneration in an RNA-binding activity-dependent manner. Moreover, we found that FUS directly binds to and modulates the G-quadruplex structure of GGGGCC repeat RNA as an RNA chaperone, resulting in the suppression of RAN translation in vitro. These results reveal a previously unrecognized regulatory mechanism of RAN translation by G-quadruplex-targeting RBPs, providing therapeutic insights for C9-ALS/FTD and other repeat expansion diseases.

Insight Into Spinocerebellar Ataxia Type 31 (SCA31) From Drosophila Model.

Spinocerebellar ataxia type 31 (SCA31) is a progressive neurodegenerative disease characterized by degeneration of Purkinje cells in the cerebellum. Its genetic cause is a 2.5- to 3.8-kb-long complex pentanucleotide repeat insertion containing (TGGAA)n, (TAGAA)n, (TAAAA)n, and (TAAAATAGAA)n located in an intron shared by two different genes: brain expressed associated with NEDD4-1 (BEAN1) and thymidine kinase 2 (TK2). Among these repeat sequences, (TGGAA)n repeat was the only sequence segregating with SCA31, which strongly suggests its pathogenicity. In SCA31 patient brains, the mutant BEAN1 transcript containing expanded UGGAA repeats (UGGAAexp) was found to form abnormal RNA structures called RNA foci in cerebellar Purkinje cell nuclei. In addition, the deposition of pentapeptide repeat (PPR) proteins, poly(Trp-Asn-Gly-Met-Glu), translated from UGGAAexp RNA, was detected in the cytoplasm of Purkinje cells. To uncover the pathogenesis of UGGAAexp in SCA31, we generated Drosophila models of SCA31 expressing UGGAAexp RNA. The toxicity of UGGAAexp depended on its length and expression level, which was accompanied by the accumulation of RNA foci and translation of repeat-associated PPR proteins in Drosophila, consistent with the observation in SCA31 patient brains. We also revealed that TDP-43, FUS, and hnRNPA2B1, motor neuron disease-linked RNA-binding proteins bound to UGGAAexp RNA, act as RNA chaperones to regulate the formation of RNA foci and repeat-associated translation. Further research on the role of RNA-binding proteins as RNA chaperones may also provide a novel therapeutic strategy for other microsatellite repeat expansion diseases besides SCA31.

Dupilumab for Atopic Dermatitis, a Possible Risk Factor of Juvenile Ischemic Stroke: A Case Report.

Dupilumab, a dual inhibitor of IL-4 and IL-13 cytokine signaling, is indicated for the treatment of moderate-to-severe atopic dermatitis, which leads to the control of atopic dermatitis. The cytokines IL-4 and IL-13 are related to vascular inflammation, which is mediated by vascular endothelial cells. We report the case of a 20-year-old man with atopic dermatitis treated with dupilumab for half a year, who presented with sudden onset of dizziness, nausea, and slight cerebellar ataxia. Brain magnetic resonance imaging revealed acute infarction in the bicerebellar hemispheres. No risk factors known to be associated with ischemic stroke in young adults were detected. We suspected this ischemic stroke might be related to dupilumab. The administration of dupilumab was discontinued, and he had no recurrence subsequently. IL-4 and IL-13, anti-inflammatory cytokines secreted from T helper 2 cells, suppress proinflammatory cytokines. Therefore, dupilumab, a dual inhibitor of IL-4 and IL-13 cytokine signaling, leads to the promotion of coagulation and thrombosis. We speculate that the activation of proinflammatory cytokines in vascular endothelial cells by the inhibition of IL-4 and IL-13 signaling by dupilumab led to ischemic stroke even at a young age.

Regulatory Role of RNA Chaperone TDP-43 for RNA Misfolding and Repeat-Associated Translation in SCA31.

Microsatellite expansion disorders are pathologically characterized by RNA foci formation and repeat-associated non-AUG (RAN) translation. However, their underlying pathomechanisms and regulation of RAN translation remain unknown. We report that expression of expanded UGGAA (UGGAAexp) repeats, responsible for spinocerebellar ataxia type 31 (SCA31) in Drosophila, causes neurodegeneration accompanied by accumulation of UGGAAexp RNA foci and translation of repeat-associated pentapeptide repeat (PPR) proteins, consistent with observations in SCA31 patient brains. We revealed that motor-neuron disease (MND)-linked RNA-binding proteins (RBPs), TDP-43, FUS, and hnRNPA2B1, bind to and induce structural alteration of UGGAAexp. These RBPs suppress UGGAAexp-mediated toxicity in Drosophila by functioning as RNA chaperones for proper UGGAAexp folding and regulation of PPR translation. Furthermore, nontoxic short UGGAA repeat RNA suppressed mutated RBP aggregation and toxicity in MND Drosophila models. Thus, functional crosstalk of the RNA/RBP network regulates their own quality and balance, suggesting convergence of pathomechanisms in microsatellite expansion disorders and RBP proteinopathies.

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.

Cytoplasmic location of α1A voltage-gated calcium channel C-terminal fragment (Cav2.1-CTF) aggregate is sufficient to cause cell death.

The human α1A voltage-dependent calcium channel (Cav2.1) is a pore-forming essential subunit embedded in the plasma membrane. Its cytoplasmic carboxyl(C)-tail contains a small poly-glutamine (Q) tract, whose length is normally 4∼19 Q, but when expanded up to 20∼33Q, the tract causes an autosomal-dominant neurodegenerative disorder, spinocerebellar ataxia type 6 (SCA6). A recent study has shown that a 75-kDa C-terminal fragment (CTF) containing the polyQ tract remains soluble in normal brains, but becomes insoluble mainly in the cytoplasm with additional localization to the nuclei of human SCA6 Purkinje cells. However, the mechanism by which the CTF aggregation leads to neurodegeneration is completely elusive, particularly whether the CTF exerts more toxicity in the nucleus or in the cytoplasm. We tagged recombinant (r)CTF with either nuclear-localization or nuclear-export signal, created doxycyclin-inducible rat pheochromocytoma (PC12) cell lines, and found that the CTF is more toxic in the cytoplasm than in the nucleus, the observations being more obvious with Q28 (disease range) than with Q13 (normal-length). Surprisingly, the CTF aggregates co-localized both with cAMP response element-binding protein (CREB) and phosphorylated-CREB (p-CREB) in the cytoplasm, and Western blot analysis showed that the quantity of CREB and p-CREB were both decreased in the nucleus when the rCTF formed aggregates in the cytoplasm. In human brains, polyQ aggregates also co-localized with CREB in the cytoplasm of SCA6 Purkinje cells, but not in other conditions. Collectively, the cytoplasmic Cav2.1-CTF aggregates are sufficient to cause cell death, and one of the pathogenic mechanisms may be abnormal CREB trafficking in the cytoplasm and reduced CREB and p-CREB levels in the nuclei.

Reduced brain-derived neurotrophic factor (BDNF) mRNA expression and presence of BDNF-immunoreactive granules in the spinocerebellar ataxia type 6 (SCA6) cerebellum.

Spinocerebellar ataxia type 6 (SCA6) is an autosomal-dominant neurodegenerative disorder caused by a small expansion of tri-nucleotide (CAG) repeat encoding polyglutamine (polyQ) in the gene for α(1A) voltage-dependent calcium channel (Ca(v) 2.1). Thus, this disease is one of the nine neurodegenerative disorders called polyQ diseases. The Purkinje cell predominant neuronal loss is the characteristic neuropathology of SCA6, and a 75-kDa carboxy-terminal fragment (CTF) of Ca(v) 2.1 containing polyQ, which remains soluble in normal brains, becomes insoluble in the cytoplasm of SCA6 Purkinje cells. Because the suppression of the brain-derived neurotrophic factor (BDNF) expression is a potentially momentous phenomenon in many other polyQ diseases, we implemented BDNF expression analysis in SCA6 human cerebellum using quantitative RT-PCR for the BDNF mRNA, and by immunohistochemistry for the BDNF protein. We observed significantly reduced BDNF mRNA levels in SCA6 cerebellum (n = 3) compared to controls (n = 6) (Mann-Whitney U-test, P = 0.0201). On immunohistochemistry, BDNF protein was only weakly stained in control cerebellum. On the other hand, we found numerous BDNF-immunoreactive granules in dendrites of SCA6 Purkinje cells. We did not observe similar BDNF-immunoreactive granules in other polyQ diseases, such as Huntington's disease or SCA2. As we often observed that the 1C2-positive Ca(v) 2.1 aggregates existed more proximally than the BDNF-positive granules in the dendrites, we speculated that the BDNF protein trafficking in dendrites may be disturbed by Ca(v) 2.1 aggregates in SCA6 Purkinje cells. We conclude that the SCA6 pathogenic mechanism associates with the BDNF mRNA expression reduction and abnormal localization of BDNF protein.

The carboxy-terminal fragment of alpha(1A) calcium channel preferentially aggregates in the cytoplasm of human spinocerebellar ataxia type 6 Purkinje cells.

Spinocerebellar ataxia type 6 (SCA6) is an autosomal dominant neurodegenerative disease caused by a small polyglutamine (polyQ) expansion (control: 4-20Q; SCA6: 20-33Q) in the carboxyl(C)-terminal cytoplasmic domain of the alpha(1A) voltage-dependent calcium channel (Ca(v)2.1). Although a 75-85-kDa Ca(v)2.1 C-terminal fragment (CTF) is toxic in cultured cells, its existence in human brains and its role in SCA6 pathogenesis remains unknown. Here, we investigated whether the small polyQ expansion alters the expression pattern and intracellular distribution of Ca(v)2.1 in human SCA6 brains. New antibodies against the Ca(v)2.1 C-terminus were used in immunoblotting and immunohistochemistry. In the cerebella of six control individuals, the CTF was detected in sucrose- and SDS-soluble cytosolic fractions; in the cerebella of two SCA6 patients, it was additionally detected in SDS-insoluble cytosolic and sucrose-soluble nuclear fractions. In contrast, however, the CTF was not detected either in the nuclear fraction or in the SDS-insoluble cytosolic fraction of SCA6 extracerebellar tissues, indicating that the CTF being insoluble in the cytoplasm or mislocalized to the nucleus only in the SCA6 cerebellum. Immunohistochemistry revealed abundant aggregates in cell bodies and dendrites of SCA6 Purkinje cells (seven patients) but not in controls (n = 6). Recombinant CTF with a small polyQ expansion (rCTF-Q28) aggregated in cultured PC12 cells, but neither rCTF-Q13 (normal-length polyQ) nor full-length Ca(v)2.1 with Q28 did. We conclude that SCA6 pathogenesis may be associated with the CTF, normally found in the cytoplasm, being aggregated in the cytoplasm and additionally distributed in the nucleus.

[Molecular genetic approach to spinocerebellar ataxias].

Spinocerebellar ataxia (SCA) is a group of degenerative ataxias with autosomal dominant inheritance. The most common form of mutation that causes SCA is the expansion of trinucleotide (CAG) repeat encoding polyglutamine. These "polyglutamine disorders" are, SCA1, SCA2, Machado-Joseph disease, SCA6, SCA7, SCA17 and DRPLA. Another dynamic mutation, yet a non-coding one, has been identified as the cause of SCA8, SCA10 and SCA12. This mutation includes, trinucleotide (CAG/CTG) expansion causing SCA8 and SCA12, and pentanuclotide (ATTCT) expansion leading SCA10. In addition to these dynamic mutations, static mutations, such as missense mutations and deletions, have been identified to cause SCA5, SCA11, SCA13, SCA14, SCA15 and SCA27. Since 1992, authors have been involved in identifying the mutation (s) of autosomal dominant cerebellar ataxia with rather pure cerebellar syndrome (ADCAIII). About a half of our cohort with ADCAIII were SCA6, caused by a small CAG repeat expansion in the alpha1A-voltage-dependent calcium channel gene. Recent study in patients' brains suggested that a small polyglutamine expansion leads a portion of this channel protein to aggregate in the Purkinje cell. Another type of ADCAIII is the chromosome 16q22.1-linked ADCA. By a comprehensive positional cloning strategy, we have found a genetic change that segregate with the disease. Identifying the mutation of 16q-ADCA is imperative for understanding molecular basis of this disease.

Spinocerebellar ataxia type 31 is associated with "inserted" penta-nucleotide repeats containing (TGGAA)n.

Spinocerebellar ataxia type 31 (SCA31) is an adult-onset autosomal-dominant neurodegenerative disorder showing progressive cerebellar ataxia mainly affecting Purkinje cells. The SCA31 critical region was tracked down to a 900 kb interval in chromosome 16q22.1, where the disease shows a strong founder effect. By performing comprehensive Southern blot analysis and BAC- and fosmid-based sequencing, we isolated two genetic changes segregating with SCA31. One was a single-nucleotide change in an intron of the thymidine kinase 2 gene (TK2). However, this did not appear to affect splicing or expression patterns. The other was an insertion, from 2.5-3.8 kb long, consisting of complex penta-nucleotide repeats including a long (TGGAA)n stretch. In controls, shorter (1.5-2.0 kb) insertions lacking (TGGAA)n were found only rarely. The SCA31 repeat insertion's length inversely correlated with patient age of onset, and an expansion was documented in a single family showing anticipation. The repeat insertion was located in introns of TK2 and BEAN (brain expressed, associated with Nedd4) expressed in the brain and formed RNA foci in the nuclei of patients' Purkinje cells. An electrophoretic mobility-shift assay showed that essential splicing factors, serine/arginine-rich splicing factors SFRS1 and SFRS9, bind to (UGGAA)n in vitro. Because (TGGAA)n is a characteristic sequence of paracentromeric heterochromatin, we speculate that the insertion might have originated from heterochromatin. SCA31 is important because it exemplifies human diseases associated with "inserted" microsatellite repeats that can expand through transmission. Our finding suggests that the ectopic microsatellite repeat, when transcribed, might cause a disease involving the essential splicing factors.

Spinocerebellar ataxia type 6 knockin mice develop a progressive neuronal dysfunction with age-dependent accumulation of mutant CaV2.1 channels.

Spinocerebellar ataxia type 6 (SCA6) is a neurodegenerative disorder caused by CAG repeat expansions within the voltage-gated calcium (Ca(V)) 2.1 channel gene. It remains controversial whether the mutation exerts neurotoxicity by changing the function of Ca(V)2.1 channel or through a gain-of-function mechanism associated with accumulation of the expanded polyglutamine protein. We generated three strains of knockin (KI) mice carrying normal, expanded, or hyperexpanded CAG repeat tracts in the Cacna1a locus. The mice expressing hyperexpanded polyglutamine (Sca6(84Q)) developed progressive motor impairment and aggregation of mutant Ca(V)2.1 channels. Electrophysiological analysis of cerebellar Purkinje cells revealed similar Ca(2+) channel current density among the three KI models. Neither voltage sensitivity of activation nor inactivation was altered in the Sca6(84Q) neurons, suggesting that expanded CAG repeat per se does not affect the intrinsic electrophysiological properties of the channels. The pathogenesis of SCA6 is apparently linked to an age-dependent process accompanied by accumulation of mutant Ca(V)2.1 channels.

Direct and accurate measurement of CAG repeat configuration in the ataxin-1 (ATXN-1) gene by "dual-fluorescence labeled PCR-restriction fragment length analysis".

Spinocerebellar ataxia type 1 (SCA1; OMIM: #164400) is an autosomal dominant cerebellar ataxia caused by an expansion of CAG repeat, which encodes polyglutamine, in the ataxin-1 (ATXN1) gene. Length of polyglutamine in the ATXN1 protein is the critical determinant of pathogenesis of this disease. Molecular diagnosis of SCA1 is usually undertaken by assessing the length of CAG repeat configuration using primers spanning this configuration. However, this conventional method may potentially lead to misdiagnosis in assessing polyglutamine-encoding CAG repeat length, since CAT interruptions may be present within the CAG repeat configuration, not only in normal controls but also in neurologically symptomatic subjects. We developed a new method for assessing actual CAG repeat numbers not interrupted by CAT sequences. Polymerase chain reaction using a primer pair labeled with two different fluorescences followed by restriction enzyme digestion with SfaNI which recognizes the sequence "GCATC(N)(5)", lengths of actual CAG repeats that encode polyglutamine were directly detected. We named this method "dual fluorescence labeled PCR-restriction fragment length analysis". We found that numbers of actual CAG repeat encoding polyglutamine do not overlap between our cohorts of normal chromosomes (n=385) and SCA1 chromosomes (n=5). We conclude that the present method is a useful way for molecular diagnosis of SCA1.

Analyses of copy number and mRNA expression level of the alpha-synuclein gene in multiple system atrophy.

Multiple system atrophy (MSA) is a sporadic neurodegenerative disease manifested clinically by progressive ataxia, parkinsonism, and autonomic dysfunction. Its cause is unknown, and there is no curative therapy. Alpha-synuclein is an important protein forming aggregations called glial cytoplasmic inclusions (GCIs) in oligodendroglia; these aggregations are considered important in MSA pathogenesis. Overexpression of the human alpha-synuclein gene in mice induces the formation of GCI-like aggregations in oligodendrocytes, leading mice to exhibit neurological signs similar to those in MSA patients. However, previous studies have excluded mutations within the coding region of the alpha-synuclein gene in MSA patients. To determine whether alteration in the expression level of the alpha-synuclein gene is associated with MSA pathogenesis, we used TaqMan quantitative PCR assay to analyze the alpha-synuclein gene copy number in patients' genomes. We also used quantitative RT-PCR and in situ hybridization to analyze alpha-synuclein mRNA expression in MSA patients' brain tissues. We found no alteration in the alpha-synuclein gene copy number in the patients' genomes (n = 50). Quantitative analysis for alpha-synuclein mRNA by the TaqMan method showed that alpha-synuclein mRNA levels were comparable between control (n = 3) and MSA (n = 3) cerebella. On in situ hybridization, the number of neurons with alpha-synuclein mRNA expression was no greater in the cerebella of MSA patients (n = 3) than in the controls (n = 3). However, GCIs were seen in these MSA specimens on immunohistochemistry for alpha-synuclein. These results suggest that alpha-synuclein gene expression is not the fundamental cause of MSA.

Redefining the disease locus of 16q22.1-linked autosomal dominant cerebellar ataxia.

The 16q22.1-linked autosomal dominant cerebellar ataxia (16q-ADCA; Online Mendelian Inheritance in Man [OMIN] #117210) is one of the most common ADCAs in Japan. Previously, we had reported that the patients share a common haplotype by founder effect and that a C-to-T substitution (-16C>T) in the puratrophin-1 gene was strongly associated with the disease. However, recently, an exceptional patient without the substitution was reported, indicating that a true pathogenic mutation might be present elsewhere. In this study, we clarified the disease locus more definitely by the haplotype analysis of families showing pure cerebellar ataxia. In addition to microsatellite markers, the single nucleotide polymorphisms (SNPs) that we identified on the disease chromosome were examined to confirm the borders of the disease locus. The analysis of 64 families with the -16C>T substitution in the puratrophin-1 gene revealed one family showing an ancestral recombination event between SNP04 and SNP05 on the disease chromosome. The analysis of 22 families without identifiable genetic mutations revealed another family carrying the common haplotype centromeric to the puratrophin-1 gene, but lacking the -16C>T substitution in this gene. We concluded that the disease locus of 16q-ADCA was definitely confined to a 900-kb genomic region between the SNP04 and the -16C>T substitution in the puratrophin-1 gene in 16q22.1.