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.

Identification of hub molecules of FUS-ALS by Bayesian gene regulatory network analysis of iPSC model: iBRN.

Fused in sarcoma/translated in liposarcoma (FUS) is a causative gene of amyotrophic lateral sclerosis (ALS). Mutated FUS causes accumulation of DNA damage and cytosolic stress granule (SG) formation, thereby motor neuron (MN) death. However, key molecular aetiology remains unclear. Here, we applied a novel platform technology, iBRN, "Non- biased" Bayesian gene regulatory network analysis based on induced pluripotent stem cell (iPSC)-derived cell model, to elucidate the molecular aetiology using transcriptome of iPSC-derived MNs harboring FUSH517D. iBRN revealed "hub molecules", which strongly influenced transcriptome network, such as miR-125b-5p-TIMELESS axis and PRKDC for the molecular aetiology. Next, we confirmed miR-125b-5p-TIMELESS axis in FUSH517D MNs such that miR-125b-5p regulated several DNA repair-related genes including TIMELESS. In addition, we validated both introduction of miR-125b-5p and knocking down of TIMELESS caused DNA damage in the cell culture model. Furthermore, PRKDC was strongly associated with FUS mis-localization into SGs by DNA damage under impaired DNA-PK activity. Collectively, our iBRN strategy provides the first compelling evidence to elucidate molecular aetiology in neurodegenerative diseases.

Aberrant axon branching via Fos-B dysregulation in FUS-ALS motor neurons.

BACKGROUND: The characteristic structure of motor neurons (MNs), particularly of the long axons, becomes damaged in the early stages of amyotrophic lateral sclerosis (ALS). However, the molecular pathophysiology of axonal degeneration remains to be fully elucidated. METHOD: Two sets of isogenic human-induced pluripotent stem cell (hiPSCs)-derived MNs possessing the single amino acid difference (p.H517D) in the fused in sarcoma (FUS) were constructed. By combining MN reporter lentivirus, MN specific phenotype was analyzed. Moreover, RNA profiling of isolated axons were conducted by applying the microfluidic devices that enable axon bundles to be produced for omics analysis. The relationship between the target gene, which was identified as a pathological candidate in ALS with RNA-sequencing, and the MN phenotype was confirmed by intervention with si-RNA or overexpression to hiPSCs-derived MNs and even in vivo. The commonality was further confirmed with other ALS-causative mutant hiPSCs-derived MNs and human pathology. FINDINGS: We identified aberrant increasing of axon branchings in FUS-mutant hiPSCs-derived MN axons compared with isogenic controls as a novel phenotype. We identified increased level of Fos-B mRNA, the binding target of FUS, in FUS-mutant MNs. While Fos-B reduction using si-RNA or an inhibitor ameliorated the observed aberrant axon branching, Fos-B overexpression resulted in aberrant axon branching even in vivo. The commonality of those phenotypes was further confirmed with other ALS causative mutation than FUS. INTERPRETATION: Analyzing the axonal fraction of hiPSC-derived MNs using microfluidic devices revealed that Fos-B is a key regulator of FUS-mutant axon branching. FUND: Japan Agency for Medical Research and development; Japanese Ministry of Education, Culture, Sports, Science and Technology Clinical Research, Innovation and Education Center, Tohoku University Hospital; Japan Intractable Diseases (Nanbyo) Research Foundation; the Kanae Foundation for the Promotion of Medical Science; and "Inochi-no-Iro" ALS research grant.

Aberrant astrocytic expression of chondroitin sulfate proteoglycan receptors in a rat model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. Progressive and systemic loss of motor neurons with gliosis in the central nervous system (CNS) is a neuropathological hallmark of ALS. Chondroitin sulfate proteoglycans (CSPGs) are the major components of the extracellular matrix of the mammalian CNS, and they inhibit axonal regeneration physically by participating to form the glial scar. Recently, protein tyrosine phosphatase sigma (PTPσ) and leukocyte common antigen-related protein were discovered as CSPG receptors that play roles in inhibiting regeneration. Here we examined the expression of CSPG receptors in transgenic female rats overexpressing an ALS-linked mutant cytosolic Cu/Zn superoxide dismutase gene (SOD1). In contrast to controls, multiple immunofluorescence analyses revealed aberrant expression of CSPG receptors dominantly in reactive astrocytes, while PTPσ expression in neurons decreased in the spinal ventral horns of ALS transgenic rats. The aberrant and progressive astrocytic expression of CSPG receptors and reactive astrocytes themselves may be therapeutic targets for reconstructing a regeneration-supportive microenvironment under neurodegenerative conditions such as ALS.

Comprehensive targeted next-generation sequencing in Japanese familial amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by loss of motor neurons. We have recently identified SOD1 and FUS mutations as the most common causes in a consecutive series of 111 familial ALS pedigrees in Japan. To reveal possible genetic causes for the remaining 51 patients with familial ALS (45 pedigrees), we performed targeted next-generation sequencing of 35 known ALS/motor neuron diseases-related genes. Known variants in ANG, OPTN, SETX, and TARDBP were identified in 6 patients. A novel likely pathogenic homozygous variant in ALS2 was identified in 1 patient. In addition, 18 patients harbored 1-3 novel variants of uncertain significance, whereas hexanucleotide repeat expansions in C9ORF72 were not detected using repeat-primed polymerase chain reaction. Collectively, in our Japanese cohort, the frequencies of SOD1, FUS, SETX, TARDBP, ANG, and OPTN variants were 32%, 11%, 2%, 2%, 1%, and 1%, respectively. These findings indicate considerable differences in the genetic variations associated with familial ALS across populations. Further genetic analyses and functional studies of novel variants are warranted.

Establishment of In Vitro FUS-Associated Familial Amyotrophic Lateral Sclerosis Model Using Human Induced Pluripotent Stem Cells.

Amyotrophic lateral sclerosis (ALS) is a late-onset motor neuron disorder. Although its neuropathology is well understood, the cellular and molecular mechanisms are yet to be elucidated due to limitations in the currently available human genetic data. In this study, we generated induced pluripotent stem cells (iPSC) from two familial ALS (FALS) patients with a missense mutation in the fused-in sarcoma (FUS) gene carrying the heterozygous FUS H517D mutation, and isogenic iPSCs with the homozygous FUS H517D mutation by genome editing technology. These cell-derived motor neurons mimicked several neurodegenerative phenotypes including mis-localization of FUS into cytosolic and stress granules under stress conditions, and cellular vulnerability. Moreover, exon array analysis using motor neuron precursor cells (MPCs) combined with CLIP-seq datasets revealed aberrant gene expression and/or splicing pattern in FALS MPCs. These results suggest that iPSC-derived motor neurons are a useful tool for analyzing the pathogenesis of human motor neuron disorders.

Genotype-phenotype relationships in familial amyotrophic lateral sclerosis with FUS/TLS mutations in Japan.

INTRODUCTION: We investigated possible genotype-phenotype correlations in Japanese patients with familial amyotrophic lateral sclerosis (FALS) carrying fused in sarcoma/translated in liposarcoma (FUS/TLS) gene mutations. METHODS: A consecutive series of 111 Japanese FALS pedigrees were screened for copper/zinc superoxide dismutase 1 (SOD1) and FUS/TLS gene mutations. Clinical data, including onset age, onset site, disease duration, and extramotor symptoms, were collected. RESULTS: Nine different FUS/TLS mutations were found in 12 pedigrees. Most of the patients with FUS/TLS-linked FALS demonstrated early onset in the brainstem/upper cervical region, and relatively short disease duration. A few mutations exhibited phenotypes that were distinct from typical cases. Frontotemporal dementia was present in 1 patient. CONCLUSIONS: This study revealed a characteristic phenotype in FUS/TLS-linked FALS patients in Japan. FUS/TLS screening is recommended in patients with FALS with this phenotype. Muscle Nerve 54: 398-404, 2016.

FALS with FUS mutation in Japan, with early onset, rapid progress and basophilic inclusion.

Mutations in the fused in sarcoma (FUS, also known as translated in liposarcoma) gene have been recently discovered to be associated with familial amyotrophic lateral sclerosis (FALS) in African, European and American populations. In a Japanese family with FALS, we found the R521C FUS mutation, which has been reported to be found in various ethnic backgrounds. The family history revealed 23 patients with FALS among 46 family members, suggesting a 100% penetrance rate. They developed muscle weakness at an average age of 35.3 years, followed by dysarthria, dysphagia, spasticity and muscle atrophy. The average age of death was 37.2 years. Neuropathological examination of the index case revealed remarkable atrophy of the brainstem tegmentum characterized by cytoplasmic basophilic inclusion bodies in the neurons of the brainstem. We screened 40 FALS families in Japan and found 4 mutations (S513P, K510E, R514S, H517P) in exon 14 and 15 of FUS. Even in Asian races, FALS with FUS mutations may have the common characteristics of early onset, rapid progress and high penetrance rate, although in patients with the S513P mutation it was late-onset. Degeneration in multiple systems and cytoplasmic basophilic inclusion bodies were found in the autopsied cases.