HuD (ELAVL4) gain-of-function impairs neuromuscular junctions and induces apoptosis in in vitro and in vivo models of amyotrophic lateral sclerosis.

Early defects at the neuromuscular junction (NMJ) are among the first hallmarks of the progressive neurodegenerative disease amyotrophic lateral sclerosis (ALS). According to the "dying back" hypothesis, disruption of the NMJ not only precedes, but is also a trigger for the subsequent degeneration of the motoneuron in both sporadic and familial ALS, including ALS caused by the severe FUS pathogenic variant P525L. However, the mechanisms linking genetic and environmental factors to NMJ defects remain elusive. By taking advantage of co-cultures of motoneurons and skeletal muscle derived from human induced pluripotent stem cells (iPSCs), we show that the neural RNA binding protein HuD (ELAVL4) may underlie NMJ defects and apoptosis in FUS-ALS. HuD overexpression in motoneurons phenocopies the severe FUSP525L mutation, while HuD knockdown in FUSP525L co-cultures produces phenotypic rescue. We validated these findings in vivo in a Drosophila FUS-ALS model. Neuronal-restricted overexpression of the HuD-related gene, elav, produces per se a motor phenotype, while neuronal-restricted elav knockdown significantly rescues motor dysfunction caused by FUS. Finally, we show that HuD levels increase upon oxidative stress in human motoneurons and in sporadic ALS patients with an oxidative stress signature. On these bases, we propose HuD as an important player downstream of FUS mutation in familial ALS, with potential implications for sporadic ALS related to oxidative stress.

Mutations of GEMIN5 are associated with coenzyme Q10 deficiency: long-term follow-up after treatment.

GEMIN5 exerts key biological functions regulating pre-mRNAs intron removal to generate mature mRNAs. A series of patients were reported harboring mutations in GEMIN5. No treatments are currently available for this disease. We treated two of these patients with oral Coenzyme Q10 (CoQ10), which resulted in neurological improvements, although MRI abnormalities remained. Whole Exome Sequencing demonstrated compound heterozygosity at the GEMIN5 gene in both cases: Case one: p.Lys742* and p.Arg1016Cys; Case two: p.Arg1016Cys and p.Ser411Hisfs*6. Functional studies in fibroblasts revealed a decrease in CoQ10 biosynthesis compared to controls. Supplementation with exogenous CoQ10 restored it to control intracellular CoQ10 levels. Mitochondrial function was compromised, as indicated by the decrease in oxygen consumption, restored by CoQ10 supplementation. Transcriptomic analysis of GEMIN5 patients compared with controls showed general repression of genes involved in CoQ10 biosynthesis. In the rigor mortis defective flies, CoQ10 levels were decreased, and CoQ10 supplementation led to an improvement in the adult climbing assay performance, a reduction in the number of motionless flies, and partial restoration of survival. Overall, we report the association between GEMIN5 dysfunction and CoQ10 deficiency for the first time. This association opens the possibility of oral CoQ10 therapy, which is safe and has no observed side effects after long-term therapy.

MATR3 pathogenic variants differentially impair its cryptic splicing repression function.

Matrin-3 (MATR3) is an RNA-binding protein implicated in neurodegenerative and neurodevelopmental diseases. However, little is known regarding the role of MATR3 in cryptic splicing within the context of functional genes and how disease-associated variants impact this function. We show that loss of MATR3 leads to cryptic exon inclusion in many transcripts. We reveal that ALS-linked S85C pathogenic variant reduces MATR3 solubility but does not impair RNA binding. In parallel, we report a novel neurodevelopmental disease-associated M548T variant, located in the RRM2 domain, which reduces protein solubility and impairs RNA binding and cryptic splicing repression functions of MATR3. Altogether, our research identifies cryptic events within functional genes and demonstrates how disease-associated variants impact MATR3 cryptic splicing repression function.

Drosha-dependent microRNAs modulate FUS-mediated neurodegeneration in vivo.

Mutations in the Fused in Sarcoma (FUS) gene cause the familial and progressive form of amyotrophic lateral sclerosis (ALS). FUS is a nuclear RNA-binding protein involved in RNA processing and the biogenesis of a specific set of microRNAs. Here we report that Drosha and two previously uncharacterized Drosha-dependent miRNAs are strong modulators of FUS expression and prevent the cytoplasmic segregation of insoluble mutant FUS in vivo. We demonstrate that depletion of Drosha mitigates FUS-mediated degeneration, survival and motor defects in Drosophila. Mutant FUS strongly interacts with Drosha and causes its cytoplasmic mis-localization into the insoluble FUS inclusions. Reduction in Drosha levels increases the solubility of mutant FUS. Interestingly, we found two Drosha dependent microRNAs, miR-378i and miR-6832-5p, which differentially regulate the expression, solubility and cytoplasmic aggregation of mutant FUS in iPSC neurons and mammalian cells. More importantly, we report different modes of action of these miRNAs against mutant FUS. Whereas miR-378i may regulate mutant FUS inclusions by preventing G3BP-mediated stress granule formation, miR-6832-5p may affect FUS expression via other proteins or pathways. Overall, our research reveals a possible association between ALS-linked FUS mutations and the Drosha-dependent miRNA regulatory circuit, as well as a useful perspective on potential ALS treatment via microRNAs.

NgR1 binding to reovirus reveals an unusual bivalent interaction and a new viral attachment protein.

Nogo-66 receptor 1 (NgR1) binds a variety of structurally dissimilar ligands in the adult central nervous system to inhibit axon extension. Disruption of ligand binding to NgR1 and subsequent signaling can improve neuron outgrowth, making NgR1 an important therapeutic target for diverse neurological conditions such as spinal crush injuries and Alzheimer's disease. Human NgR1 serves as a receptor for mammalian orthoreovirus (reovirus), but the mechanism of virus-receptor engagement is unknown. To elucidate how NgR1 mediates cell binding and entry of reovirus, we defined the affinity of interaction between virus and receptor, determined the structure of the virus-receptor complex, and identified residues in the receptor required for virus binding and infection. These studies revealed that central NgR1 surfaces form a bridge between two copies of viral capsid protein σ3, establishing that σ3 serves as a receptor ligand for reovirus. This unusual binding interface produces high-avidity interactions between virus and receptor to prime early entry steps. These studies refine models of reovirus cell-attachment and highlight the evolution of viruses to engage multiple receptors using distinct capsid components.

SMN regulates GEMIN5 expression and acts as a modifier of GEMIN5-mediated neurodegeneration.

GEMIN5 is essential for core assembly of small nuclear Ribonucleoproteins (snRNPs), the building blocks of spliceosome formation. Loss-of-function mutations in GEMIN5 lead to a neurodevelopmental syndrome among patients presenting with developmental delay, motor dysfunction, and cerebellar atrophy by perturbing SMN complex protein expression and assembly. Currently, molecular determinants of GEMIN5-mediated disease have yet to be explored. Here, we identified SMN as a genetic suppressor of GEMIN5-mediated neurodegeneration in vivo. We discovered that an increase in SMN expression by either SMN gene therapy replacement or the antisense oligonucleotide (ASO), Nusinersen, significantly upregulated the endogenous levels of GEMIN5 in mammalian cells and mutant GEMIN5-derived iPSC neurons. Further, we identified a strong functional association between the expression patterns of SMN and GEMIN5 in patient Spinal Muscular Atrophy (SMA)-derived motor neurons harboring loss-of-function mutations in the SMN gene. Interestingly, SMN binds to the C-terminus of GEMIN5 and requires the Tudor domain for GEMIN5 binding and expression regulation. Finally, we show that SMN upregulation ameliorates defective snRNP biogenesis and alternative splicing defects caused by loss of GEMIN5 in iPSC neurons and in vivo. Collectively, these studies indicate that SMN acts as a regulator of GEMIN5 expression and neuropathologies.

Axon guidance genes modulate neurotoxicity of ALS-associated UBQLN2.

Mutations in the ubiquitin (Ub) chaperone Ubiquilin 2 (UBQLN2) cause X-linked forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) through unknown mechanisms. Here, we show that aggregation-prone, ALS-associated mutants of UBQLN2 (UBQLN2ALS) trigger heat stress-dependent neurodegeneration in Drosophila. A genetic modifier screen implicated endolysosomal and axon guidance genes, including the netrin receptor, Unc-5, as key modulators of UBQLN2 toxicity. Reduced gene dosage of Unc-5 or its coreceptor Dcc/frazzled diminished neurodegenerative phenotypes, including motor dysfunction, neuromuscular junction defects, and shortened lifespan, in flies expressing UBQLN2ALS alleles. Induced pluripotent stem cells (iPSCs) harboring UBQLN2ALS knockin mutations exhibited lysosomal defects while inducible motor neurons (iMNs) expressing UBQLN2ALS alleles exhibited cytosolic UBQLN2 inclusions, reduced neurite complexity, and growth cone defects that were partially reversed by silencing of UNC5B and DCC. The combined findings suggest that altered growth cone dynamics are a conserved pathomechanism in UBQLN2-associated ALS/FTD.

LSD1/PRMT6-targeting gene therapy to attenuate androgen receptor toxic gain-of-function ameliorates spinobulbar muscular atrophy phenotypes in flies and mice.

Spinobulbar muscular atrophy (SBMA) is caused by CAG expansions in the androgen receptor gene. Androgen binding to polyQ-expanded androgen receptor triggers SBMA through a combination of toxic gain-of-function and loss-of-function mechanisms. Leveraging cell lines, mice, and patient-derived specimens, we show that androgen receptor co-regulators lysine-specific demethylase 1 (LSD1) and protein arginine methyltransferase 6 (PRMT6) are overexpressed in an androgen-dependent manner specifically in the skeletal muscle of SBMA patients and mice. LSD1 and PRMT6 cooperatively and synergistically transactivate androgen receptor, and their effect is enhanced by expanded polyQ. Pharmacological and genetic silencing of LSD1 and PRMT6 attenuates polyQ-expanded androgen receptor transactivation in SBMA cells and suppresses toxicity in SBMA flies, and a preclinical approach based on miRNA-mediated silencing of LSD1 and PRMT6 attenuates disease manifestations in SBMA mice. These observations suggest that targeting overexpressed co-regulators can attenuate androgen receptor toxic gain-of-function without exacerbating loss-of-function, highlighting a potential therapeutic strategy for patients with SBMA.

WNK kinases sense molecular crowding and rescue cell volume via phase separation.

When challenged by hypertonicity, dehydrated cells must recover their volume to survive. This process requires the phosphorylation-dependent regulation of SLC12 cation chloride transporters by WNK kinases, but how these kinases are activated by cell shrinkage remains unknown. Within seconds of cell exposure to hypertonicity, WNK1 concentrates into membraneless condensates, initiating a phosphorylation-dependent signal that drives net ion influx via the SLC12 cotransporters to restore cell volume. WNK1 condensate formation is driven by its intrinsically disordered C terminus, whose evolutionarily conserved signatures are necessary for efficient phase separation and volume recovery. This disorder-encoded phase behavior occurs within physiological constraints and is activated in vivo by molecular crowding rather than changes in cell size. This allows kinase activity despite an inhibitory ionic milieu and permits cell volume recovery through condensate-mediated signal amplification. Thus, WNK kinases are physiological crowding sensors that phase separate to coordinate a cell volume rescue response.

Further delineation of GEMIN4 related neurodevelopmental disorder with microcephaly, cataract, and renal abnormalities syndrome.

Pathogenic variants in GEMIN4 have recently been linked to an inherited autosomal recessive neurodevelopmental disorder characterized with microcephaly, cataracts, and renal abnormalities (NEDMCR syndrome). This report provides a retrospective review of 16 patients from 11 unrelated Saudi consanguineous families with GEMIN4 mutations. The cohort comprises 11 new and unpublished clinical details from five previously described patients. Only two missense, homozygous, pathogenic variants were found in all affected patients, suggesting a founder effect. All patients shared global developmental delay with variable ophthalmological, renal, and skeletal manifestations. In addition, we knocked down endogenous Drosophila GEMIN4 in neurons to further investigate the mechanism of the functional defects in affected patients. Our fly model findings demonstrated developmental defects and motor dysfunction suggesting that loss of GEMIN4 function is detrimental in vivo; likely similar to human patients. To date, this study presents the largest cohort of patients affected with GEMIN4 mutations. Considering that identifying GEMIN4 defects in patients presenting with neurodevelopmental delay and congenital cataract will help in early diagnosis, appropriate management and prevention plans that can be made for affected families.

Skeletal Muscle Pathogenesis in Polyglutamine Diseases.

Polyglutamine diseases are characterized by selective dysfunction and degeneration of specific types of neurons in the central nervous system. In addition, nonneuronal cells can also be affected as a consequence of primary degeneration or due to neuronal dysfunction. Skeletal muscle is a primary site of toxicity of polyglutamine-expanded androgen receptor, but it is also affected in other polyglutamine diseases, more likely due to neuronal dysfunction and death. Nonetheless, pathological processes occurring in skeletal muscle atrophy impact the entire body metabolism, thus actively contributing to the inexorable progression towards the late and final stages of disease. Skeletal muscle atrophy is well recapitulated in animal models of polyglutamine disease. In this review, we discuss the impact and relevance of skeletal muscle in patients affected by polyglutamine diseases and we review evidence obtained in animal models and patient-derived cells modeling skeletal muscle.

Functional and structural deficiencies of Gemin5 variants associated with neurological disorders.

Dysfunction of RNA-binding proteins is often linked to a wide range of human disease, particularly with neurological conditions. Gemin5 is a member of the survival of the motor neurons (SMN) complex, a ribosome-binding protein and a translation reprogramming factor. Recently, pathogenic mutations in Gemin5 have been reported, but the functional consequences of these variants remain elusive. Here, we report functional and structural deficiencies associated with compound heterozygosity variants within the Gemin5 gene found in patients with neurodevelopmental disorders. These clinical variants are located in key domains of Gemin5, the tetratricopeptide repeat (TPR)-like dimerization module and the noncanonical RNA-binding site 1 (RBS1). We show that the TPR-like variants disrupt protein dimerization, whereas the RBS1 variant confers protein instability. All mutants are defective in the interaction with protein networks involved in translation and RNA-driven pathways. Importantly, the TPR-like variants fail to associate with native ribosomes, hampering its involvement in translation control and establishing a functional difference with the wild-type protein. Our study provides insights into the molecular basis of disease associated with malfunction of the Gemin5 protein.

Autosomal Recessive Cerebellar Atrophy and Spastic Ataxia in Patients With Pathogenic Biallelic Variants in GEMIN5.

The hereditary ataxias are a heterogenous group of disorders with an increasing number of causative genes being described. Due to the clinical and genetic heterogeneity seen in these conditions, the majority of such individuals endure a diagnostic odyssey or remain undiagnosed. Defining the molecular etiology can bring insights into the responsible molecular pathways and eventually the identification of therapeutic targets. Here, we describe the identification of biallelic variants in the GEMIN5 gene among seven unrelated families with nine affected individuals presenting with spastic ataxia and cerebellar atrophy. GEMIN5, an RNA-binding protein, has been shown to regulate transcription and translation machinery. GEMIN5 is a component of small nuclear ribonucleoprotein (snRNP) complexes and helps in the assembly of the spliceosome complexes. We found that biallelic GEMIN5 variants cause structural abnormalities in the encoded protein and reduce expression of snRNP complex proteins in patient cells compared with unaffected controls. Finally, knocking out endogenous Gemin5 in mice caused early embryonic lethality, suggesting that Gemin5 expression is crucial for normal development. Our work further expands on the phenotypic spectrum associated with GEMIN5-related disease and implicates the role of GEMIN5 among patients with spastic ataxia, cerebellar atrophy, and motor predominant developmental delay.

DDX17 is involved in DNA damage repair and modifies FUS toxicity in an RGG-domain dependent manner.

Mutations in the RNA binding protein, Fused in Sarcoma (FUS), lead to amyotrophic lateral sclerosis (ALS), the most frequent form of motor neuron disease. Cytoplasmic aggregation and defective DNA repair machinery are etiologically linked to mutant FUS-associated ALS. Although FUS is involved in numerous aspects of RNA processing, little is understood about the pathophysiological mechanisms of mutant FUS. Here, we employed RNA-sequencing technology in Drosophila brains expressing FUS to identify significantly altered genes and pathways involved in FUS-mediated neurodegeneration. We observed the expression levels of DEAD-Box Helicase 17 (DDX17) to be significantly downregulated in response to mutant FUS in Drosophila and human cell lines. Mutant FUS recruits nuclear DDX17 into cytoplasmic stress granules and physically interacts with DDX17 through the RGG1 domain of FUS. Ectopic expression of DDX17 reduces cytoplasmic mislocalization and sequestration of mutant FUS into cytoplasmic stress granules. We identified DDX17 as a novel regulator of the DNA damage response pathway whose upregulation repairs defective DNA damage repair machinery caused by mutant neuronal FUS ALS. In addition, we show DDX17 is a novel modifier of FUS-mediated neurodegeneration in vivo. Our findings indicate DDX17 is downregulated in response to mutant FUS, and restoration of DDX17 levels suppresses FUS-mediated neuropathogenesis and toxicity in vivo.

Traumatic injury compromises nucleocytoplasmic transport and leads to TDP-43 pathology.

Traumatic brain injury (TBI) is a predisposing factor for many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), and chronic traumatic encephalopathy (CTE). Although defects in nucleocytoplasmic transport (NCT) is reported ALS and other neurodegenerative diseases, whether defects in NCT occur in TBI remains unknown. We performed proteomic analysis on Drosophila exposed to repeated TBI and identified resultant alterations in several novel molecular pathways. TBI upregulated nuclear pore complex (NPC) and nucleocytoplasmic transport (NCT) proteins as well as alter nucleoporin stability. Traumatic injury disrupted RanGAP1 and NPC protein distribution in flies and a rat model and led to coaggregation of NPC components and TDP-43. In addition, trauma-mediated NCT defects and lethality are rescued by nuclear export inhibitors. Importantly, genetic upregulation of nucleoporins in vivo and in vitro triggered TDP-43 cytoplasmic mislocalization, aggregation, and altered solubility and reduced motor function and lifespan of animals. We also found NUP62 pathology and elevated NUP62 concentrations in postmortem brain tissues of patients with mild or severe CTE as well as co-localization of NUP62 and TDP-43 in CTE. These findings indicate that TBI leads to NCT defects, which potentially mediate the TDP-43 pathology in CTE.

RNA dependent suppression of C9orf72 ALS/FTD associated neurodegeneration by Matrin-3.

The most common genetic cause of amyotrophic lateral sclerosis (ALS) is a GGGGCC (G4C2) hexanucleotide repeat expansions in first intron of the C9orf72 gene. The accumulation of repetitive RNA sequences can mediate toxicity potentially through the formation of intranuclear RNA foci that sequester key RNA-binding proteins (RBPs), and non-ATG mediated translation into toxic dipeptide protein repeats. However, the contribution of RBP sequestration to the mechanisms underlying RNA-mediated toxicity remain unknown. Here we show that the ALS-associated RNA-binding protein, Matrin-3 (MATR3), colocalizes with G4C2 RNA foci in patient tissues as well as iPSC-derived motor neurons harboring the C9orf72 mutation. Hyperexpansion of C9 repeats perturbed subcellular distribution and levels of endogenous MATR3 in C9-ALS patient-derived motor neurons. Interestingly, we observed that ectopic expression of human MATR3 strongly mitigates G4C2-mediated neurodegeneration in vivo. MATR3-mediated suppression of C9 toxicity was dependent on the RNA-binding domain of MATR3. Importantly, we found that expression of MATR3 reduced the levels of RAN-translation products in mammalian cells in an RNA-dependent manner. Finally, we have shown that knocking down endogenous MATR3 in C9-ALS patient-derived iPSC neurons decreased the presence of G4C2 RNA foci in the nucleus. Overall, these studies suggest that MATR3 genetically modifies the neuropathological and the pathobiology of C9orf72 ALS through modulating the RNA foci and RAN translation.

RNA-recognition motif in Matrin-3 mediates neurodegeneration through interaction with hnRNPM.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is an adult-onset, fatal neurodegenerative disease characterized by progressive loss of upper and lower motor neurons. While pathogenic mutations in the DNA/RNA-binding protein Matrin-3 (MATR3) are linked to ALS and distal myopathy, the molecular mechanisms underlying MATR3-mediated neuromuscular degeneration remain unclear. METHODS: We generated Drosophila lines with transgenic insertion of human MATR3 wildtype, disease-associated variants F115C and S85C, and deletion variants in functional domains, ΔRRM1, ΔRRM2, ΔZNF1 and ΔZNF2. We utilized genetic, behavioral and biochemical tools for comprehensive characterization of our models in vivo and in vitro. Additionally, we employed in silico approaches to find transcriptomic targets of MATR3 and hnRNPM from publicly available eCLIP datasets. RESULTS: We found that targeted expression of MATR3 in Drosophila muscles or motor neurons shorten lifespan and produces progressive motor defects, muscle degeneration and atrophy. Strikingly, deletion of its RNA-recognition motif (RRM2) mitigates MATR3 toxicity. We identified rump, the Drosophila homolog of human RNA-binding protein hnRNPM, as a modifier of mutant MATR3 toxicity in vivo. Interestingly, hnRNPM physically and functionally interacts with MATR3 in an RNA-dependent manner in mammalian cells. Furthermore, common RNA targets of MATR3 and hnRNPM converge in biological processes important for neuronal health and survival. CONCLUSIONS: We propose a model of MATR3-mediated neuromuscular degeneration governed by its RNA-binding domains and modulated by interaction with splicing factor hnRNPM.