The RNA-binding protein FUS/TLS undergoes calcium-mediated nuclear egress during excitotoxic stress and is required for GRIA2 mRNA processing.

Excitotoxic levels of glutamate represent a physiological stress that is strongly linked to amyotrophic lateral sclerosis (ALS) and other neurological disorders. Emerging evidence indicates a role for neurodegenerative disease-linked RNA-binding proteins (RBPs) in the cellular stress response. However, the relationships between excitotoxicity, RBP function, and disease have not been explored. Here, using primary cortical and motor neurons, we found that excitotoxicity induced the translocation of select ALS-linked RBPs from the nucleus to the cytoplasm within neurons. RBPs affected by excitotoxicity included TAR DNA-binding protein 43 (TDP-43) and, most robustly, fused in sarcoma/translocated in liposarcoma (FUS/TLS or FUS). We noted that FUS is translocated through a calcium-dependent mechanism and that its translocation coincides with striking alterations in nucleocytoplasmic transport. Furthermore, glutamate-induced up-regulation of glutamate ionotropic receptor α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type subunit 2 (GRIA2) in neurons depended on FUS expression, consistent with a functional role for FUS in excitotoxic stress. These findings reveal molecular links among prominent factors in neurodegenerative diseases, namely excitotoxicity, disease-associated RBPs, and nucleocytoplasmic transport.

Traffic jam at the nuclear pore: All roads lead to nucleocytoplasmic transport defects in ALS/FTD.

Amyotrophic lateral sclerosis (ALS) is a fatal late-onset neurodegenerative disease that specifically affects the function and survival of spinal and cortical motor neurons. ALS shares many genetic, clinical, and pathological characteristics with frontotemporal dementia (FTD), and these diseases are now recognized as presentations of a disease spectrum known as ALS/FTD. The molecular determinants of neuronal loss in ALS/FTD are still debated, but the recent discovery of nucleocytoplasmic transport defects as a common denominator of most if not all forms of ALS/FTD has dramatically changed our understanding of the pathogenic mechanisms of this disease. Loss of nuclear pores and nucleoporin aggregation, altered nuclear morphology, and impaired nuclear transport are some of the most prominent features that have been identified using a variety of animal, cellular, and human models of disease. Here, we review the experimental evidence linking nucleocytoplasmic transport defects to the pathogenesis of ALS/FTD and propose a unifying view on how these defects may lead to a vicious cycle that eventually causes neuronal death.

A Drosophila model of ALS reveals a partial loss of function of causative human PFN1 mutants.

Mutations in the profilin 1 (PFN1) gene are causative for familial amyotrophic lateral sclerosis (fALS). However, it is still not fully understood how these mutations lead to neurodegeneration. To address this question, we generated a novel Drosophila model expressing human wild-type and ALS-causative PFN1 mutants. We show that at larval neuromuscular junctions (NMJ), motor neuron expression of wild-type human PFN1 increases the number of ghost boutons, active zone density, F-actin content, and the formation of filopodia. In contrast, the expression of ALS-causative human PFN1 mutants causes a less pronounced phenotype, suggesting a loss of function of these mutants in promoting NMJ remodeling. Importantly, expression of human PFN1 in motor neurons results in progressive locomotion defects and shorter lifespan in adult flies, while ALS-causative PFN1 mutants display a less toxic effect. In summary, our study provides evidence that PFN1 is important in regulating NMJ morphology and influences survival and locomotion in Drosophila. Furthermore, our results suggest ALS-causative human PFN1 mutants display a partial loss of function relative to wild-type hPFN1 that may contribute to human disease pathogenesis.

Mutations in the profilin 1 gene cause familial amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a late-onset neurodegenerative disorder resulting from motor neuron death. Approximately 10% of cases are familial (FALS), typically with a dominant inheritance mode. Despite numerous advances in recent years, nearly 50% of FALS cases have unknown genetic aetiology. Here we show that mutations within the profilin 1 (PFN1) gene can cause FALS. PFN1 is crucial for the conversion of monomeric (G)-actin to filamentous (F)-actin. Exome sequencing of two large ALS families showed different mutations within the PFN1 gene. Further sequence analysis identified 4 mutations in 7 out of 274 FALS cases. Cells expressing PFN1 mutants contain ubiquitinated, insoluble aggregates that in many cases contain the ALS-associated protein TDP-43. PFN1 mutants also display decreased bound actin levels and can inhibit axon outgrowth. Furthermore, primary motor neurons expressing mutant PFN1 display smaller growth cones with a reduced F/G-actin ratio. These observations further document that cytoskeletal pathway alterations contribute to ALS pathogenesis.

Deficiency of the Survival of Motor Neuron Protein Impairs mRNA Localization and Local Translation in the Growth Cone of Motor Neurons.

Spinal muscular atrophy (SMA) is a neurodegenerative disease primarily affecting spinal motor neurons. It is caused by reduced levels of the survival of motor neuron (SMN) protein, which plays an essential role in the biogenesis of spliceosomal small nuclear ribonucleoproteins in all tissues. The etiology of the specific defects in the motor circuitry in SMA is still unclear, but SMN has also been implicated in mediating the axonal localization of mRNA-protein complexes, which may contribute to the axonal degeneration observed in SMA. Here, we report that SMN deficiency severely disrupts local protein synthesis within neuronal growth cones. We also identify the cytoskeleton-associated growth-associated protein 43 (GAP43) mRNA as a new target of SMN and show that motor neurons from SMA mouse models have reduced levels ofGAP43mRNA and protein in axons and growth cones. Importantly, overexpression of two mRNA-binding proteins, HuD and IMP1, restoresGAP43mRNA and protein levels in growth cones and rescues axon outgrowth defects in SMA neurons. These findings demonstrate that SMN plays an important role in the localization and local translation of mRNAs with important axonal functions and suggest that disruption of this function may contribute to the axonal defects observed in SMA. SIGNIFICANCE STATEMENT: The motor neuron disease spinal muscular atrophy (SMA) is caused by reduced levels of the survival of motor neuron (SMN) protein, which plays a key role in assembling RNA/protein complexes that are essential for mRNA splicing. It remains unclear whether defects in this well characterized housekeeping function cause the specific degeneration of spinal motor neurons observed in SMA. Here, we describe an additional role of SMN in regulating the axonal localization and local translation of the mRNA encoding growth-associated protein 43 (GAP43). This study supports a model whereby SMN deficiency impedes transport and local translation of mRNAs important for neurite outgrowth and stabilization, thus contributing to axon degeneration, muscle denervation, and motor neuron cell death in SMA.

The ALS disease protein TDP-43 is actively transported in motor neuron axons and regulates axon outgrowth.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease specifically affecting cortical and spinal motor neurons. Cytoplasmic inclusions containing hyperphosphorylated and ubiquitinated TDP-43 are a pathological hallmark of ALS, and mutations in the gene encoding TDP-43 have been directly linked to the development of the disease. TDP-43 is a ubiquitous DNA/RNA-binding protein with a nuclear role in pre-mRNA splicing. However, the selective vulnerability and axonal degeneration of motor neurons in ALS pose the question of whether TDP-43 may have an additional role in the regulation of the cytoplasmic and axonal fate of mRNAs, processes important for neuron function. To investigate this possibility, we have characterized TDP-43 localization and dynamics in primary cultured motor neurons. Using a combination of cell imaging and biochemical techniques, we demonstrate that TDP-43 is localized and actively transported in live motor neuron axons, and that it co-localizes with well-studied axonal mRNA-binding proteins. Expression of the TDP-43 C-terminal fragment led to the formation of hyperphosphorylated and ubiquitinated inclusions in motor neuron cell bodies and neurites, and these inclusions specifically sequestered the mRNA-binding protein HuD. Additionally, we showed that overexpression of full-length or mutant TDP-43 in motor neurons caused a severe impairment in axon outgrowth, which was dependent on the C-terminal protein-interacting domain of TDP-43. Taken together, our results suggest a role of TDP-43 in the regulation of axonal growth, and suggest that impairment in the post-transcriptional regulation of mRNAs in the cytoplasm of motor neurons may be a major factor in the development of ALS.

LINC complex alterations are a hallmark of sporadic and familial ALS/FTD.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that primarily affects motor neurons, leading to progressive muscle weakness and loss of voluntary muscle control. While the exact cause of ALS is not fully understood, emerging research suggests that dysfunction of the nuclear envelope (NE) may contribute to disease pathogenesis and progression. The NE plays a role in ALS through several mechanisms, including nuclear pore defects, nucleocytoplasmic transport impairment, accumulation of mislocalized proteins, and nuclear morphology abnormalities. The LINC complex is the second biggest multi-protein complex in the NE and consists of the SUN1/2 proteins spanning the inner nuclear membrane and Nesprin proteins embedded in the outer membrane. The LINC complex, by interacting with both the nuclear lamina and the cytoskeleton, transmits mechanical forces to the nucleus regulating its morphology and functional homeostasis. In this study we show extensive alterations to the LINC complex in motor and cortical iPSC-derived neurons and spinal cord organoids carrying the ALS causative mutation in the C9ORF72 gene (C9). Importantly, we show that such alterations are present in vivo in a cohort of sporadic ALS and C9-ALS postmortem spinal cord and motor cortex biopsies. We also found that LINC complex disruption strongly correlated with nuclear morphological alterations occurring in ALS neurons, independently of TDP43 mislocalization. Altogether, our data establish morphological and functional alterations to the LINC complex as important events in ALS pathogenic cascade, making this pathway a possible target for both biomarker and therapy development.

LINC complex alterations are a key feature of sporadic and familial ALS/FTD.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that primarily affects motor neurons, leading to progressive muscle weakness and loss of voluntary muscle control. While the exact cause of ALS is not fully understood, emerging research suggests that dysfunction of the nuclear envelope (NE) may contribute to disease pathogenesis and progression. The NE plays a role in ALS through several mechanisms, including nuclear pore defects, nucleocytoplasmic transport impairment, accumulation of mislocalized proteins, and nuclear morphology abnormalities. The LINC complex is the second biggest multi-protein complex in the NE and consists of the SUN1/2 proteins spanning the inner nuclear membrane and Nesprin proteins embedded in the outer membrane. The LINC complex, by interacting with both the nuclear lamina and the cytoskeleton, transmits mechanical forces to the nucleus regulating its morphology and functional homeostasis. In this study we show extensive alterations to the LINC complex in motor and cortical iPSC-derived neurons and spinal cord organoids carrying the ALS causative mutation in the C9ORF72 gene (C9). Importantly, we show that such alterations are present in vivo in a cohort of sporadic ALS and C9-ALS postmortem spinal cord and motor cortex specimens. We also found that LINC complex disruption strongly correlated with nuclear morphological alterations occurring in ALS neurons, independently of TDP43 mislocalization. Altogether, our data establish morphological and functional alterations to the LINC complex as important events in ALS pathogenic cascade, making this pathway a possible target for both biomarker and therapy development.

Altered nuclear envelope homeostasis is a key pathogenic event in C9ORF72-linked ALS/FTD.

ALS and FTD are complex neurodegenerative disorders that primarily affects motor neurons in the brain and spinal cord, and cortical neurons in the frontal lobe. Although the pathogenesis of ALS/FTD is unclear, recent research spotlights nucleocytoplasmic transport impairment, DNA damage, and nuclear abnormalities as drivers of neuronal death. In this study, we show that loss of nuclear envelope (NE) integrity is a key pathology associated with nuclear pore complex (NPC) injury in C9ORF72 mutant neurons. Importantly, we show that mechanical stresses generated by cytoskeletal forces on the NE can lead to NPC injury, loss of nuclear integrity, and accumulation of DNA damage. Importantly, we demonstrate that restoring NE tensional homeostasis, by disconnecting the nucleus from the cytoskeleton, can rescue NPC injury and reduce DNA damage in C9ORF72 mutant cells. Together, our data suggest that modulation of NE homeostasis and repair may represent a novel and promising therapeutic target for ALS/FTD.

The survival of motor neuron (SMN) protein interacts with the mRNA-binding protein HuD and regulates localization of poly(A) mRNA in primary motor neuron axons.

Spinal muscular atrophy (SMA) results from reduced levels of the survival of motor neuron (SMN) protein, which has a well characterized function in spliceosomal small nuclear ribonucleoprotein assembly. Currently, it is not understood how deficiency of a housekeeping protein leads to the selective degeneration of spinal cord motor neurons. Numerous studies have shown that SMN is present in neuronal processes and has many interaction partners, including mRNA-binding proteins, suggesting a potential noncanonical role in axonal mRNA metabolism. In this study, we have established a novel technological approach using bimolecular fluorescence complementation (BiFC) and quantitative image analysis to characterize SMN-protein interactions in primary motor neurons. Consistent with biochemical studies on the SMN complex, BiFC analysis revealed that SMN dimerizes and interacts with Gemin2 in nuclear gems and axonal granules. In addition, using pull down assays, immunofluorescence, cell transfection, and BiFC, we characterized a novel interaction between SMN and the neuronal mRNA-binding protein HuD, which was dependent on the Tudor domain of SMN. A missense mutation in the SMN Tudor domain, which is known to cause SMA, impaired the interaction with HuD, but did not affect SMN axonal localization or self-association. Furthermore, time-lapse microscopy revealed SMN cotransport with HuD in live motor neurons. Importantly, SMN knockdown in primary motor neurons resulted in a specific reduction of both HuD protein and poly(A) mRNA levels in the axonal compartment. These findings reveal a noncanonical role for SMN whereby its interaction with mRNA-binding proteins may facilitate the localization of associated poly(A) mRNAs into axons.

ALS-associated KIF5A mutations abolish autoinhibition resulting in a toxic gain of function.

Understanding the pathogenic mechanisms of disease mutations is critical to advancing treatments. ALS-associated mutations in the gene encoding the microtubule motor KIF5A result in skipping of exon 27 (KIF5AΔExon27) and the encoding of a protein with a novel 39 amino acid residue C-terminal sequence. Here, we report that expression of ALS-linked mutant KIF5A results in dysregulated motor activity, cellular mislocalization, altered axonal transport, and decreased neuronal survival. Single-molecule analysis revealed that the altered C terminus of mutant KIF5A results in a constitutively active state. Furthermore, mutant KIF5A possesses altered protein and RNA interactions and its expression results in altered gene expression/splicing. Taken together, our data support the hypothesis that causative ALS mutations result in a toxic gain of function in the intracellular motor KIF5A that disrupts intracellular trafficking and neuronal homeostasis.

Novel TUBA4A Variant Associated With Familial Frontotemporal Dementia.

OBJECTIVE: Despite the strong genetic component of frontotemporal dementia (FTD), a substantial proportion of patients remain genetically unresolved. We performed an in-depth study of a family with an autosomal dominant form of FTD to investigate the underlying genetic cause. METHODS: Following clinical and pathologic characterization of the family, genetic studies included haplotype sharing analysis and exome sequencing. Subsequently, we performed immunohistochemistry, immunoblotting, and a microtubule repolymerization assay to investigate the potential impact of the candidate variant in tubulin alpha 4a (TUBA4A). RESULTS: The clinical presentation in this family is heterogeneous, including behavioral changes, parkinsonian features, and uncharacterized dementia. Neuropathologic examination of 2 patients revealed TAR DNA binding protein 43 (TDP-43) pathology with abundant dystrophic neurites and neuronal intranuclear inclusions, consistent with frontotemporal lobar degeneration-TDP type A. We identified a likely pathogenic variant in TUBA4A segregating with disease. TUBA4A encodes for α-tubulin, which is a major component of the microtubule network. Variants in TUBA4A have been suggested as a rare genetic cause of amyotrophic lateral sclerosis (ALS) and have sporadically been reported in patients with FTD without supporting genetic segregation. A decreased trend of TUBA4A protein abundance was observed in patients compared with controls, and a microtubule repolymerization assay demonstrated disrupted α-tubulin function. As opposed to variants found in ALS, TUBA4A variants associated with FTD appear more localized to the N-terminus, indicating different pathogenic mechanisms. CONCLUSIONS: Our findings support the role of TUBA4A variants as rare genetic cause of familial FTD.

Association of Variants in the SPTLC1 Gene With Juvenile Amyotrophic Lateral Sclerosis.

Importance: Juvenile amyotrophic lateral sclerosis (ALS) is a rare form of ALS characterized by age of symptom onset less than 25 years and a variable presentation. Objective: To identify the genetic variants associated with juvenile ALS. Design, Setting, and Participants: In this multicenter family-based genetic study, trio whole-exome sequencing was performed to identify the disease-associated gene in a case series of unrelated patients diagnosed with juvenile ALS and severe growth retardation. The patients and their family members were enrolled at academic hospitals and a government research facility between March 1, 2016, and March 13, 2020, and were observed until October 1, 2020. Whole-exome sequencing was also performed in a series of patients with juvenile ALS. A total of 66 patients with juvenile ALS and 6258 adult patients with ALS participated in the study. Patients were selected for the study based on their diagnosis, and all eligible participants were enrolled in the study. None of the participants had a family history of neurological disorders, suggesting de novo variants as the underlying genetic mechanism. Main Outcomes and Measures: De novo variants present only in the index case and not in unaffected family members. Results: Trio whole-exome sequencing was performed in 3 patients diagnosed with juvenile ALS and their parents. An additional 63 patients with juvenile ALS and 6258 adult patients with ALS were subsequently screened for variants in the SPTLC1 gene. De novo variants in SPTLC1 (p.Ala20Ser in 2 patients and p.Ser331Tyr in 1 patient) were identified in 3 unrelated patients diagnosed with juvenile ALS and failure to thrive. A fourth variant (p.Leu39del) was identified in a patient with juvenile ALS where parental DNA was unavailable. Variants in this gene have been previously shown to be associated with autosomal-dominant hereditary sensory autonomic neuropathy, type 1A, by disrupting an essential enzyme complex in the sphingolipid synthesis pathway. Conclusions and Relevance: These data broaden the phenotype associated with SPTLC1 and suggest that patients presenting with juvenile ALS should be screened for variants in this gene.

TDP-43 is recruited to stress granules in conditions of oxidative insult.

Transactive response DNA-binding protein 43 (TDP-43) forms abnormal ubiquitinated and phosphorylated inclusions in brain tissues from patients with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration. TDP-43 is a DNA/RNA-binding protein involved in RNA processing, such as transcription, pre-mRNA splicing, mRNA stabilization and transport to dendrites. We found that in response to oxidative stress and to environmental insults of different types TDP-43 is capable to assemble into stress granules (SGs), ribonucleoprotein complexes where protein synthesis is temporarily arrested. We demonstrated that a specific aminoacidic interval (216-315) in the C-terminal region and the RNA-recognition motif 1 domain are both implicated in TDP-43 participation in SGs as their deletion prevented the recruitment of TDP-43 into SGs. Our data show that TDP-43 is a specific component of SGs and not of processing bodies, although we proved that TDP-43 is not necessary for SG formation, and its gene silencing does not impair cell survival during stress. The analysis of spinal cord tissue from ALS patients showed that SG markers are not entrapped in TDP-43 pathological inclusions. Although SGs were not evident in ALS brains, we speculate that an altered control of mRNA translation in stressful conditions may trigger motor neuron degeneration at early stages of the disease.

TDP-43 and NOVA-1 RNA-binding proteins as competitive splicing regulators of the schizophrenia-associated TNIK gene.

The RNA-binding protein TDP-43, associated to amyotrophic lateral sclerosis and frontotemporal dementia, regulates the alternative splicing of several genes, including the skipping of TNIK exon 15. TNIK, a genetic risk factor for schizophrenia and causative for intellectual disability, encodes for a Ser/Thr kinase regulating negatively F-actin dynamics. Here we show that in the human adult nervous system TNIK exon 15 is mostly included compared to the other tissues and that, during neuronal differentiation of human induced pluripotent stem cells and of human neuroblastoma cells, TNIK exon 15 inclusion increases independently of TDP-43 protein content. By studying the possible molecular interplay of TDP-43 with brain-specific splicing factors, we found that the neuronal NOVA-1 protein competitively inhibits both TDP-43 and hnRNPA2/B1 skipping activity on TNIK by means of a RNA-dependent interaction and that this competitive mechanism is common to other TDP-43 RNA targets. We also show that the TNIK protein isoforms including/excluding exon 15 differently regulate cell spreading in non-neuronal cells and neuritogenesis in primary cortical neurons. Our data suggest a complex regulation between the ubiquitous TDP-43 and the neuron-specific NOVA-1 splicing factors in the brain that may help better understand the pathobiology of both neurodegenerative diseases and schizophrenia.

Modulation of actin polymerization affects nucleocytoplasmic transport in multiple forms of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of unknown etiology. Although defects in nucleocytoplasmic transport (NCT) may be central to the pathogenesis of ALS and other neurodegenerative diseases, the molecular mechanisms modulating the nuclear pore function are still largely unknown. Here we show that genetic and pharmacological modulation of actin polymerization disrupts nuclear pore integrity, nuclear import, and downstream pathways such as mRNA post-transcriptional regulation. Importantly, we demonstrate that modulation of actin homeostasis can rescue nuclear pore instability and dysfunction caused by mutant PFN1 as well as by C9ORF72 repeat expansion, the most common mutation in ALS patients. Collectively, our data link NCT defects to ALS-associated cellular pathology and propose the regulation of actin homeostasis as a novel therapeutic strategy for ALS and other neurodegenerative diseases.

The 3' untranslated region of human Cyclin-Dependent Kinase 5 Regulatory subunit 1 contains regulatory elements affecting transcript stability.

BACKGROUND: CDK5R1 plays a central role in neuronal migration and differentiation during central nervous system development. CDK5R1 has been implicated in neurodegenerative disorders and proposed as a candidate gene for mental retardation. The remarkable size of CDK5R1 3'-untranslated region (3'-UTR) suggests a role in post-transcriptional regulation of CDK5R1 expression. RESULTS: The bioinformatic study shows a high conservation degree in mammals and predicts several AU-Rich Elements (AREs). The insertion of CDK5R1 3'-UTR into luciferase 3'-UTR causes a decreased luciferase activity in four transfected cell lines. We identified 3'-UTR subregions which tend to reduce the reporter gene expression, sometimes in a cell line-dependent manner. In most cases the quantitative analysis of luciferase mRNA suggests that CDK5R1 3'-UTR affects mRNA stability. A region, leading to a very strong mRNA destabilization, showed a significantly low half-life, indicating an accelerated mRNA degradation. The 3' end of the transcript, containing a class I ARE, specifically displays a stabilizing effect in neuroblastoma cell lines. We also observed the interaction of the stabilizing neuronal RNA-binding proteins ELAV with the CDK5R1 transcript in SH-SY5Y cells and identified three 3'-UTR sub-regions showing affinity for ELAV proteins. CONCLUSION: Our findings evince the presence of both destabilizing and stabilizing regulatory elements in CDK5R1 3'-UTR and support the hypothesis that CDK5R1 gene expression is post-transcriptionally controlled in neurons by ELAV-mediated mechanisms. This is the first evidence of the involvement of 3'-UTR in the modulation of CDK5R1 expression. The fine tuning of CDK5R1 expression by 3'-UTR may have a role in central nervous system development and functioning, with potential implications in neurodegenerative and cognitive disorders.

The Survival of Motor Neuron Protein Acts as a Molecular Chaperone for mRNP Assembly.

Spinal muscular atrophy (SMA) is a motor neuron disease caused by reduced levels of the survival of motor neuron (SMN) protein. SMN is part of a multiprotein complex that facilitates the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs). SMN has also been found to associate with mRNA-binding proteins, but the nature of this association was unknown. Here, we have employed a combination of biochemical and advanced imaging methods to demonstrate that SMN promotes the molecular interaction between IMP1 protein and the 3' UTR zipcode region of ß-actin mRNA, leading to assembly of messenger ribonucleoprotein (mRNP) complexes that associate with the cytoskeleton to facilitate trafficking. We have identified defects in mRNP assembly in cells and tissues from SMA disease models and patients that depend on the SMN Tudor domain and explain the observed deficiency in mRNA localization and local translation, providing insight into SMA pathogenesis as a ribonucleoprotein (RNP)-assembly disorder.

ALS-linked FUS exerts a gain of toxic function involving aberrant p38 MAPK activation.

Mutations in Fused in Sarcoma/Translocated in Liposarcoma (FUS) cause familial forms of amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by progressive axonal degeneration mainly affecting motor neurons. Evidence from transgenic mouse models suggests mutant forms of FUS exert an unknown gain-of-toxic function in motor neurons, but mechanisms underlying this effect remain unknown. Towards this end, we studied the effect of wild type FUS (FUS WT) and three ALS-linked variants (G230C, R521G and R495X) on fast axonal transport (FAT), a cellular process critical for appropriate maintenance of axonal connectivity. All ALS-FUS variants impaired anterograde and retrograde FAT in squid axoplasm, whereas FUS WT had no effect. Misfolding of mutant FUS is implicated in this process, as the molecular chaperone Hsp110 mitigated these toxic effects. Interestingly, mutant FUS-induced impairment of FAT in squid axoplasm and of axonal outgrowth in mammalian primary motor neurons involved aberrant activation of the p38 MAPK pathway, as also reported for ALS-linked forms of Cu, Zn superoxide dismutase (SOD1). Accordingly, increased levels of active p38 MAPK were detected in post-mortem human ALS-FUS brain tissues. These data provide evidence for a novel gain-of-toxic function for ALS-linked FUS involving p38 MAPK activation.

Mutations in the vesicular trafficking protein annexin A11 are associated with amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder. We screened 751 familial ALS patient whole-exome sequences and identified six mutations including p.D40G in the ANXA11 gene in 13 individuals. The p.D40G mutation was absent from 70,000 control whole-exome sequences. This mutation segregated with disease in two kindreds and was present in another two unrelated cases (P = 0.0102), and all mutation carriers shared a common founder haplotype. Annexin A11-positive protein aggregates were abundant in spinal cord motor neurons and hippocampal neuronal axons in an ALS patient carrying the p.D40G mutation. Transfected human embryonic kidney cells expressing ANXA11 with the p.D40G mutation and other N-terminal mutations showed altered binding to calcyclin, and the p.R235Q mutant protein formed insoluble aggregates. We conclude that mutations in ANXA11 are associated with ALS and implicate defective intracellular protein trafficking in disease pathogenesis.