TDP-43 nuclear loss in FTD/ALS causes widespread alternative polyadenylation changes.

In frontotemporal dementia and amyotrophic lateral sclerosis, the RNA-binding protein TDP-43 is depleted from the nucleus. TDP-43 loss leads to cryptic exon inclusion but a role in other RNA processing events remains unresolved. Here, we show that loss of TDP-43 causes widespread changes in alternative polyadenylation, impacting expression of disease-relevant genes (e.g., ELP1, NEFL, and TMEM106B) and providing evidence that alternative polyadenylation is a new facet of TDP-43 pathology.

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

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

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

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

Cracking the cryptic code in amyotrophic lateral sclerosis and frontotemporal dementia: Towards therapeutic targets and biomarkers.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two devastating human neurodegenerative diseases. A hallmark pathological feature of both diseases is the depletion of the RNA-binding protein TDP-43 from the nucleus in the brain and spinal cord of patients. A major function of TDP-43 is to repress the inclusion of cryptic exons during RNA splicing. When it becomes depleted from the nucleus in disease, this function is lost, and recently, several key cryptic splicing targets of TDP-43 have emerged, including STMN2, UNC13A, and others. UNC13A is a major ALS/FTD risk gene, and the genetic variations that increase the risk for disease seem to do so by making the gene more susceptible to cryptic exon inclusion when TDP-43 function is impaired. Here, we discuss the prospects and challenges of harnessing these cryptic splicing events as novel therapeutic targets and biomarkers. Deciphering this new cryptic code may be a touchstone for ALS and FTD diagnosis and treatment.

TDP-43 represses cryptic exon inclusion in the FTD-ALS gene UNC13A.

A hallmark pathological feature of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is the depletion of RNA-binding protein TDP-43 from the nucleus of neurons in the brain and spinal cord1. A major function of TDP-43 is as a repressor of cryptic exon inclusion during RNA splicing2-4. Single nucleotide polymorphisms in UNC13A are among the strongest hits associated with FTD and ALS in human genome-wide association studies5,6, but how those variants increase risk for disease is unknown. Here we show that TDP-43 represses a cryptic exon-splicing event in UNC13A. Loss of TDP-43 from the nucleus in human brain, neuronal cell lines and motor neurons derived from induced pluripotent stem cells resulted in the inclusion of a cryptic exon in UNC13A mRNA and reduced UNC13A protein expression. The top variants associated with FTD or ALS risk in humans are located in the intron harbouring the cryptic exon, and we show that they increase UNC13A cryptic exon splicing in the face of TDP-43 dysfunction. Together, our data provide a direct functional link between one of the strongest genetic risk factors for FTD and ALS (UNC13A genetic variants), and loss of TDP-43 function.

Reduced PHOX2B stability causes axonal growth impairment in motor neurons with TARDBP mutations.

Amyotrophic lateral sclerosis (ALS) is an adult-onset incurable motor neuron (MN) disease. The reasons for selective MN vulnerability in ALS are unknown. Axonal pathology is among the earliest signs of ALS. We searched for novel modulatory genes in human MN axon shortening affected by TARDBP mutations. In transcriptome analysis of RNA present in the axon compartment of human-derived induced pluripotent stem cell (iPSC)-derived MNs, PHOX2B (paired-like homeobox protein 2B) showed lower expression in TARDBP mutant axons, which was consistent with axon qPCR and in situ hybridization. PHOX2B mRNA stability was reduced in TARDBP mutant MNs. Furthermore, PHOX2B knockdown reduced neurite length in human MNs. Finally, phox2b knockdown in zebrafish induced short spinal axons and impaired escape response. PHOX2B is known to be highly express in other types of neurons maintained after ALS progression. Collectively, TARDBP mutations induced loss of axonal resilience, which is an important ALS-related phenotype mediated by PHOX2B downregulation.

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.

An Amyotrophic Lateral Sclerosis-Associated Mutant of C21ORF2 Is Stabilized by NEK1-Mediated Hyperphosphorylation and the Inability to Bind FBXO3.

C21ORF2 and NEK1 have been identified as amyotrophic lateral sclerosis (ALS)-associated genes. Both genes are also mutated in certain ciliopathies, suggesting that they might contribute to the same signaling pathways. Here we show that FBXO3, the substrate receptor of an SCF ubiquitin ligase complex, binds and ubiquitylates C21ORF2, thereby targeting it for proteasomal degradation. C21ORF2 stabilizes the kinase NEK1, with the result that loss of FBXO3 stabilizes not only C21ORF2 but also NEK1. Conversely, NEK1-mediated phosphorylation stabilizes C21ORF2 by attenuating its interaction with FBXO3. We found that the ALS-associated V58L mutant of C21ORF2 is more susceptible to phosphorylation by NEK1, with the result that it is not ubiquitylated by FBXO3 and therefore accumulates together with NEK1. Expression of C21ORF2(V58L) in motor neurons induced from mouse embryonic stem cells impaired neurite outgrowth. We suggest that inhibition of NEK1 activity is a potential therapeutic approach to ALS associated with C21ORF2 mutation.

Generation of an ALS human iPSC line KEIOi001-A from peripheral blood of a Charcot disease-affected patient carrying TARDBP p.N345K heterozygous SNP mutation.

Amyotrophic Lateral Sclerosis is the most common motor neuron degenerative disease in adults, and TARDBP gene mutations have been reported to be involved in the pathogenesis. We present here how we generated the human induced pluripotent stem cell (hiPSC) line KEIOi001-A/SM4-4-5 from the peripheral blood of a 63-year-old male patient presenting the c.1035C > G heterozygous SNP mutation in the TARDBP gene locus. The established hiPSC line does not express the exogenous reprogramming factors oriP nor EBNA1 and shows no karyotypic abnormalities, while it expresses pluripotent stem cell markers, presents the SNP mutation and is capable of three-germ layers differentiation in vitro.

Omics Approach to Axonal Dysfunction of Motor Neurons in Amyotrophic Lateral Sclerosis (ALS).

Amyotrophic lateral sclerosis (ALS) is an intractable adult-onset neurodegenerative disease that leads to the loss of upper and lower motor neurons (MNs). The long axons of MNs become damaged during the early stages of ALS. Genetic and pathological analyses of ALS patients have revealed dysfunction in the MN axon homeostasis. However, the molecular pathomechanism for the degeneration of axons in ALS has not been fully elucidated. This review provides an overview of the proposed axonal pathomechanisms in ALS, including those involving the neuronal cytoskeleton, cargo transport within axons, axonal energy supply, clearance of junk protein, neuromuscular junctions (NMJs), and aberrant axonal branching. To improve understanding of the global changes in axons, the review summarizes omics analyses of the axonal compartments of neurons in vitro and in vivo, including a motor nerve organoid approach that utilizes microfluidic devices developed by this research group. The review also discusses the relevance of intra-axonal transcription factors frequently identified in these omics analyses. Local axonal translation and the relationship among these pathomechanisms should be pursued further. The development of novel strategies to analyze axon fractions provides a new approach to establishing a detailed understanding of resilience of long MN and MN pathology in ALS.

AMPK Complex Activation Promotes Sarcolemmal Repair in Dysferlinopathy.

Mutations in dysferlin are responsible for a group of progressive, recessively inherited muscular dystrophies known as dysferlinopathies. Using recombinant proteins and affinity purification methods combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS), we found that AMP-activated protein kinase (AMPK)γ1 was bound to a region of dysferlin located between the third and fourth C2 domains. Using ex vivo laser injury experiments, we demonstrated that the AMPK complex was vital for the sarcolemmal damage repair of skeletal muscle fibers. Injury-induced AMPK complex accumulation was dependent on the presence of Ca2+, and the rate of accumulation was regulated by dysferlin. Furthermore, it was found that the phosphorylation of AMPKα was essential for plasma membrane repair, and treatment with an AMPK activator rescued the membrane-repair impairment observed in immortalized human myotubes with reduced expression of dysferlin and dysferlin-null mouse fibers. Finally, it was determined that treatment with the AMPK activator metformin improved the muscle phenotype in zebrafish and mouse models of dysferlin deficiency. These findings indicate that the AMPK complex is essential for plasma membrane repair and is a potential therapeutic target for dysferlinopathy.

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.

Extracellular α-synuclein enters dopaminergic cells by modulating flotillin-1-assisted dopamine transporter endocytosis.

The neuropathological hallmarks of Parkinson's disease (PD) include the appearance of α-synuclein (α-SYN)-positive Lewy bodies (LBs) and the loss of catecholaminergic neurons. Thus, a potential mechanism promoting the uptake of extracellular α-SYN may exist in susceptible neurons. Of the various differentially expressed proteins, we are interested in flotillin (FLOT)-1 because this protein is highly expressed in the brainstem catecholaminergic neurons and is strikingly up-regulated in PD brains. In this study, we found that extracellular monomeric and fibrillar α-SYN can potentiate FLOT1-dopamine transporter (DAT) binding and pre-endocytic clustering of DAT on the cell surface, thereby facilitating DAT endocytosis and down-regulating its transporter activity. Moreover, we demonstrated that α-SYN itself exploited the DAT endocytic process to enter dopaminergic neuron-like cells, and both FLOT1 and DAT were found to be the components of LBs. Altogether, these findings revealed a novel role of extracellular α-SYN on cellular trafficking of DAT and may provide a rationale for the cell type-specific, functional, and pathologic alterations in PD.-Kobayashi, J., Hasegawa, T., Sugeno, N., Yoshida, S., Akiyama, T., Fujimori, K., Hatakeyama, H., Miki, Y., Tomiyama, A., Kawata, Y., Fukuda, M., Kawahata, I., Yamakuni, T., Ezura, M., Kikuchi, A., Baba, T., Takeda, A., Kanzaki, M., Wakabayashi, K., Okano, H., Aoki, M. Extracellular α-synuclein enters dopaminergic cells by modulating flotillin-1-assisted dopamine transporter endocytosis.

Modeling sporadic ALS in iPSC-derived motor neurons identifies a potential therapeutic agent.

Amyotrophic lateral sclerosis (ALS) is a heterogeneous motor neuron disease for which no effective treatment is available, despite decades of research into SOD1-mutant familial ALS (FALS). The majority of ALS patients have no familial history, making the modeling of sporadic ALS (SALS) essential to the development of ALS therapeutics. However, as mutations underlying ALS pathogenesis have not yet been identified, it remains difficult to establish useful models of SALS. Using induced pluripotent stem cell (iPSC) technology to generate stem and differentiated cells retaining the patients' full genetic information, we have established a large number of in vitro cellular models of SALS. These models showed phenotypic differences in their pattern of neuronal degeneration, types of abnormal protein aggregates, cell death mechanisms, and onset and progression of these phenotypes in vitro among cases. We therefore developed a system for case clustering capable of subdividing these heterogeneous SALS models by their in vitro characteristics. We further evaluated multiple-phenotype rescue of these subclassified SALS models using agents selected from non-SOD1 FALS models, and identified ropinirole as a potential therapeutic candidate. Integration of the datasets acquired in this study permitted the visualization of molecular pathologies shared across a wide range of SALS models.

Antagonizing bone morphogenetic protein 4 attenuates disease progression in a rat model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is an adult-onset, fatal neurodegenerative syndrome characterized by the systemic loss of motor neurons with prominent astrocytosis and microgliosis in the spinal cord and brain. Astrocytes play an essential role in maintaining extracellular microenvironments that surround motor neurons, and are activated by various insults. Growing evidence points to a non-cell autonomous neurotoxicity caused by chronic and sustained astrocytic activation in patients with neurodegenerative diseases, including ALS. However, the mechanisms that underlie the harmful effects of astrocytosis in patients with ALS remain unresolved. We focused on bone morphogenetic proteins as a major soluble factor that promotes astrocytogenesis and its activation in the adult spinal cord. In a transgenic rat model with ALS-linked mutant Cu/Zn superoxide dismutase gene, BMP4 was progressively up-regulated in reactive astrocytes of the spinal ventral horns, whereas the BMP-antagonist noggin was decreased in association with neuronal degeneration. Continuous intrathecal noggin supplementation after disease onset significantly ameliorated motor dysfunction symptoms, neurogenic muscle atrophy, and extended survival of symptomatic ALS model rats, despite lack of deterrence against neuronal death itself. The exogenous noggin inhibited astrocytic hypertrophy, astrocytogenesis, and neuroinflammation by inactivating both Smad1/5/8 and p38 mitogen-activated protein kinase pathways. Moreover, intrathecal infusion of a Bmp4-targeted antisense oligonucleotides and provided selective Bmp4 knockdown in vivo, which suppressed astrocyte and microglia activation, reproducing the aforementioned results by noggin treatment. Collectively, we clarified the involvement of BMP4 in the processes of excessive gliosis that exacerbate the disease progression of the ALS model rats. Our study demonstrated that BMP4, with its downstream signaling, might be a novel therapeutic target for disease-modifying therapies in ALS.

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.