RESUMEN
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.
Asunto(s)
Proteínas Serina-Treonina Quinasas , Fosforilación , Tamaño de la CélulaRESUMEN
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.
Asunto(s)
Proteínas de Drosophila , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H , MicroARNs , Ribonucleasa III , Animales , Esclerosis Amiotrófica Lateral/metabolismo , Drosophila/genética , Drosophila/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Mutación , Neuronas/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/metabolismo , Humanos , Ribonucleasa III/metabolismo , Proteínas de Drosophila/metabolismoRESUMEN
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.
Asunto(s)
Atrofia Muscular Espinal , Proteínas de Unión al ARN , Humanos , Neuronas Motoras/metabolismo , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/genética , Ribonucleoproteínas Nucleares Pequeñas/química , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas del Complejo SMN/genética , Dominio TudorRESUMEN
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.
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Anomalías Múltiples , Catarata , Microcefalia , Trastornos del Neurodesarrollo , Anomalías Múltiples/diagnóstico , Anomalías Múltiples/genética , Catarata/patología , Homocigoto , Humanos , Riñón/anomalías , Microcefalia/diagnóstico , Microcefalia/genética , Microcefalia/patología , Antígenos de Histocompatibilidad Menor , Trastornos del Neurodesarrollo/genética , Linaje , Ribonucleoproteínas Nucleares Pequeñas/genética , Síndrome , Anomalías UrogenitalesRESUMEN
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.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , ARN Helicasas DEAD-box/genética , Reparación del ADN/genética , Proteína FUS de Unión a ARN/toxicidad , Animales , Línea Celular , Gránulos Citoplasmáticos/química , Daño del ADN , Drosophila , Femenino , Humanos , Masculino , Enfermedades Neurodegenerativas/genética , Análisis de Secuencia de ARNRESUMEN
Traumatic brain injury (TBI) has been predicted to be a predisposing factor for amyotrophic lateral sclerosis (ALS) and other neurological disorders. Despite the importance of TBI in ALS progression, the underlying cellular and molecular mechanisms are still an enigma. Here, we examined the contribution of TBI as an extrinsic factor and investigated whether TBI influences the susceptibility of developing neurodegenerative symptoms. To evaluate the effects of TBI in vivo, we applied mild to severe trauma to Drosophila and found that TBI leads to the induction of stress granules (SGs) in the brain. The degree of SGs induction directly correlates with the level of trauma. Furthermore, we observed that the level of mortality is directly proportional to the number of traumatic hits. Interestingly, trauma-induced SGs are ubiquitin, p62 and TDP-43 positive, and persistently remain over time suggesting that SGs might be aggregates and exert toxicity in our fly models. Intriguingly, TBI on animals expressing ALS-linked genes increased mortality and locomotion dysfunction suggesting that mild trauma might aggravate neurodegenerative symptoms associated with ALS. Furthermore, we found elevated levels of high molecular weight ubiquitinated proteins and p62 in animals expressing ALS-causing genes with TBI, suggesting that TBI may lead to the defects in protein degradation pathways. Finally, we observed that genetic and pharmacological induction of autophagy enhanced the clearance of SGs and promoted survival of flies in vivo. Together, our study demonstrates that trauma can induce SG formation in vivo and might enhance neurodegenerative phenotypes in the fly models of ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Lesiones Traumáticas del Encéfalo/genética , Encéfalo/metabolismo , Gránulos Citoplasmáticos/metabolismo , Drosophila melanogaster/genética , Demencia Frontotemporal/genética , Procesamiento Proteico-Postraduccional , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Animales Modificados Genéticamente , Autofagia/genética , Encéfalo/patología , Lesiones Traumáticas del Encéfalo/metabolismo , Lesiones Traumáticas del Encéfalo/patología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Gránulos Citoplasmáticos/patología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Demencia Frontotemporal/metabolismo , Demencia Frontotemporal/patología , Humanos , Locomoción/fisiología , Longevidad , Neuronas/metabolismo , Neuronas/patología , Factores Asociados con la Proteína de Unión a TATA/genética , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Factor de Transcripción TFIID/genética , Factor de Transcripción TFIID/metabolismo , Índices de Gravedad del Trauma , Ubiquitina/genética , Ubiquitina/metabolismo , UbiquitinaciónAsunto(s)
Encéfalo/crecimiento & desarrollo , Glicina Hidroximetiltransferasa/genética , Corazón/crecimiento & desarrollo , Malformaciones del Desarrollo Cortical/genética , Mitocondrias/metabolismo , Encéfalo/patología , Carbono/metabolismo , Femenino , Humanos , Imagen por Resonancia Magnética/métodos , Masculino , SíndromeRESUMEN
Loss-of-function variants in NIMA-related kinase 1 (NEK1) constitute a major genetic cause of amyotrophic lateral sclerosis (ALS), accounting for 2 to 3% of all cases. However, how NEK1 mutations cause motor neuron (MN) dysfunction is unknown. Using mass spectrometry analyses for NEK1 interactors and NEK1-dependent expression changes, we find functional enrichment for proteins involved in the microtubule cytoskeleton and nucleocytoplasmic transport. We show that α-tubulin and importin-ß1, two key proteins involved in these processes, are phosphorylated by NEK1 in vitro. NEK1 is essential for motor control and survival in Drosophila models in vivo, while using several induced pluripotent stem cell (iPSC)-MN models, including NEK1 knockdown, kinase inhibition, and a patient mutation, we find evidence for disruptions in microtubule homeostasis and nuclear import. Notably, stabilizing microtubules with two distinct classes of drugs restored NEK1-dependent deficits in both pathways. The capacity of NEK1 to modulate these processes that are critically involved in ALS pathophysiology renders this kinase a formidable therapeutic candidate.
Asunto(s)
Esclerosis Amiotrófica Lateral , Humanos , Esclerosis Amiotrófica Lateral/genética , Transporte Activo de Núcleo Celular , Quinasa 1 Relacionada con NIMA/genética , Proteínas , Neuronas Motoras , Microtúbulos , HomeostasisRESUMEN
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.
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Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Humanos , Esclerosis Amiotrófica Lateral/genética , Demencia Frontotemporal/genética , Orientación del Axón , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Mutación , Factores de Transcripción/genética , Ubiquitinas/metabolismo , Receptores de Netrina/genéticaRESUMEN
Amyotrophic lateral sclerosis and frontotemporal dementia patients with a hexanucleotide repeat expansion in C9ORF72 (C9-HRE) accumulate poly-GR and poly-PR aggregates. The pathogenicity of these arginine-rich dipeptide repeats (R-DPRs) is thought to be driven by their propensity to bind low-complexity domains of multivalent proteins. However, the ability of R-DPRs to bind native RNA and the significance of this interaction remain unclear. Here, we used computational and experimental approaches to characterize the physicochemical properties of R-DPRs and their interaction with RNA. We find that poly-GR predominantly binds ribosomal RNA (rRNA) in cells and exhibits an interaction that is predicted to be energetically stronger than that for associated ribosomal proteins. Critically, modified rRNA "bait" oligonucleotides restore poly-GR-associated ribosomal deficits and ameliorate poly-GR toxicity in patient neurons and Drosophila models. Our work strengthens the hypothesis that ribosomal function is impaired by R-DPRs, highlights a role for direct rRNA binding in mediating ribosomal dysfunction, and presents a strategy for protecting against C9-HRE pathophysiological mechanisms.
Asunto(s)
Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Animales , Humanos , Demencia Frontotemporal/genética , Esclerosis Amiotrófica Lateral/genética , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , ARN Ribosómico/genética , Secuenciación de Inmunoprecipitación de Cromatina , ARN/genética , Drosophila/genética , Drosophila/metabolismo , Expansión de las Repeticiones de ADNRESUMEN
Spinal and bulbar muscular atrophy is caused by polyglutamine (polyQ) expansions in androgen receptor (AR), generating gain-of-function toxicity that may involve phosphorylation. Using cellular and animal models, we investigated what kinases and phosphatases target polyQ-expanded AR, whether polyQ expansions modify AR phosphorylation, and how this contributes to neurodegeneration. Mass spectrometry showed that polyQ expansions preserve native phosphorylation and increase phosphorylation at conserved sites controlling AR stability and transactivation. In small-molecule screening, we identified that CDC25/CDK2 signaling could enhance AR phosphorylation, and the calcium-sensitive phosphatase calcineurin had opposite effects. Pharmacologic and genetic manipulation of these kinases and phosphatases modified polyQ-expanded AR function and toxicity in cells, flies, and mice. Ablation of CDK2 reduced AR phosphorylation in the brainstem and restored expression of Myc and other genes involved in DNA damage, senescence, and apoptosis, indicating that the cell cycle-regulated kinase plays more than a bystander role in SBMA-vulnerable postmitotic cells.
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Calcio , Receptores Androgénicos , Ratones , Animales , Receptores Androgénicos/química , Mutación con Ganancia de Función , Quinasas Ciclina-Dependientes/genética , Monoéster Fosfórico Hidrolasas/genéticaRESUMEN
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.
Asunto(s)
Atrofia Bulboespinal Ligada al X , Dípteros , Trastornos Musculares Atróficos , Ratones , Animales , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Atrofia Bulboespinal Ligada al X/genética , Andrógenos , Mutación con Ganancia de Función , Fenotipo , Histona Demetilasas/genética , Trastornos Musculares Atróficos/genética , Trastornos Musculares Atróficos/metabolismoRESUMEN
A G4C2 hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of ALS and FTLD (C9-ALS/FTLD) with cytoplasmic TDP-43 inclusions observed in regions of neurodegeneration. The accumulation of repetitive RNAs and dipeptide repeat protein (DPR) are two proposed mechanisms of toxicity in C9-ALS/FTLD and linked to impaired nucleocytoplasmic transport. Nucleocytoplasmic transport is regulated by the phenylalanine-glycine nucleoporins (FG nups) that comprise the nuclear pore complex (NPC) permeability barrier. However, the relationship between FG nups and TDP-43 pathology remains elusive. Our studies show that nuclear depletion and cytoplasmic mislocalization of one FG nup, NUP62, is linked to TDP-43 mislocalization in C9-ALS/FTLD iPSC neurons. Poly-glycine arginine (GR) DPR accumulation initiates the formation of cytoplasmic RNA granules that recruit NUP62 and TDP-43. Cytoplasmic NUP62 and TDP-43 interactions promotes their insolubility and NUP62:TDP-43 inclusions are frequently found in C9orf72 ALS/FTLD as well as sporadic ALS/FTLD postmortem CNS tissue. Our findings indicate NUP62 cytoplasmic mislocalization contributes to TDP-43 proteinopathy in ALS/FTLD.
Asunto(s)
Esclerosis Amiotrófica Lateral , Degeneración Lobar Frontotemporal , Esclerosis Amiotrófica Lateral/metabolismo , Proteína C9orf72/genética , Expansión de las Repeticiones de ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Dipéptidos/metabolismo , Degeneración Lobar Frontotemporal/metabolismo , Glicina/genética , HumanosRESUMEN
The huntingtin (HTT) protein transports various organelles, including vesicles containing neurotrophic factors, from embryonic development throughout life. To better understand how HTT mediates axonal transport and why this function is disrupted in Huntington's disease (HD), we study vesicle-associated HTT and find that it is dimethylated at a highly conserved arginine residue (R118) by the protein arginine methyltransferase 6 (PRMT6). Without R118 methylation, HTT associates less with vesicles, anterograde trafficking is diminished, and neuronal death ensues-very similar to what occurs in HD. Inhibiting PRMT6 in HD cells and neurons exacerbates mutant HTT (mHTT) toxicity and impairs axonal trafficking, whereas overexpressing PRMT6 restores axonal transport and neuronal viability, except in the presence of a methylation-defective variant of mHTT. In HD flies, overexpressing PRMT6 rescues axonal defects and eclosion. Arginine methylation thus regulates HTT-mediated vesicular transport along the axon, and increasing HTT methylation could be of therapeutic interest for HD.
Asunto(s)
Transporte Axonal/genética , Epigénesis Genética , Proteína Huntingtina/genética , Enfermedad de Huntington/genética , Proteínas Nucleares/genética , Proteína-Arginina N-Metiltransferasas/genética , Vesículas Transportadoras/metabolismo , Secuencia de Aminoácidos , Animales , Arginina/metabolismo , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Muerte Celular , Modelos Animales de Enfermedad , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Proteína Huntingtina/metabolismo , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/patología , Metilación , Ratones , Ratones Transgénicos , Unión Neuromuscular/genética , Unión Neuromuscular/metabolismo , Unión Neuromuscular/patología , Neuronas/metabolismo , Neuronas/patología , Proteínas Nucleares/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Vesículas Transportadoras/genética , Vesículas Transportadoras/patologíaRESUMEN
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.
Asunto(s)
Transporte Activo de Núcleo Celular , Lesiones Traumáticas del Encéfalo/metabolismo , Encéfalo/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Poro Nuclear/metabolismo , Proteinopatías TDP-43/metabolismo , Animales , Animales Modificados Genéticamente , Encéfalo/patología , Lesiones Traumáticas del Encéfalo/genética , Lesiones Traumáticas del Encéfalo/patología , Estudios de Casos y Controles , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Células HEK293 , Humanos , Longevidad , Masculino , Glicoproteínas de Membrana/metabolismo , Actividad Motora , Poro Nuclear/genética , Poro Nuclear/patología , Proteínas de Complejo Poro Nuclear/metabolismo , Agregado de Proteínas , Agregación Patológica de Proteínas , Ratas Sprague-Dawley , Proteinopatías TDP-43/genética , Proteinopatías TDP-43/patologíaRESUMEN
Nucleocytoplasmic transport (NCT) decline occurs with aging and neurodegeneration. Here, we investigated the NCT pathway in models of amyotrophic lateral sclerosis-fused in sarcoma (ALS-FUS). Expression of ALS-FUS led to a reduction in NCT and nucleoporin (Nup) density within the nuclear membrane of human neurons. FUS and Nups were found to interact independently of RNA in cells and to alter the phase-separation properties of each other in vitro. FUS-Nup interactions were not localized to nuclear pores, but were enriched in the nucleus of control neurons versus the cytoplasm of mutant neurons. Our data indicate that the effect of ALS-linked mutations on the cytoplasmic mislocalization of FUS, rather than on the physiochemical properties of the protein itself, underlie our reported NCT defects. An aberrant interaction between mutant FUS and Nups is underscored by studies in Drosophila, whereby reduced Nup expression rescued multiple toxic FUS-induced phenotypes, including abnormal nuclear membrane morphology in neurons.
Asunto(s)
Transporte Activo de Núcleo Celular/fisiología , Neuronas/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Animales Modificados Genéticamente , Drosophila , Humanos , Mutación , Proteína FUS de Unión a ARN/genéticaRESUMEN
GEMIN5, an RNA-binding protein is essential for assembly of the survival motor neuron (SMN) protein complex and facilitates the formation of small nuclear ribonucleoproteins (snRNPs), the building blocks of spliceosomes. Here, we have identified 30 affected individuals from 22 unrelated families presenting with developmental delay, hypotonia, and cerebellar ataxia harboring biallelic variants in the GEMIN5 gene. Mutations in GEMIN5 perturb the subcellular distribution, stability, and expression of GEMIN5 protein and its interacting partners in patient iPSC-derived neurons, suggesting a potential loss-of-function mechanism. GEMIN5 mutations result in disruption of snRNP complex assembly formation in patient iPSC neurons. Furthermore, knock down of rigor mortis, the fly homolog of human GEMIN5, leads to developmental defects, motor dysfunction, and a reduced lifespan. Interestingly, we observed that GEMIN5 variants disrupt a distinct set of transcripts and pathways as compared to SMA patient neurons, suggesting different molecular pathomechanisms. These findings collectively provide evidence that pathogenic variants in GEMIN5 perturb physiological functions and result in a neurodevelopmental delay and ataxia syndrome.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/genética , Células Madre Pluripotentes Inducidas/metabolismo , Trastornos del Neurodesarrollo/metabolismo , Neuronas/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Proteínas del Complejo SMN/genética , Alelos , Secuencia de Aminoácidos , Animales , Preescolar , Discapacidades del Desarrollo/genética , Drosophila/genética , Drosophila/crecimiento & desarrollo , Femenino , Técnicas de Silenciamiento del Gen , Ontología de Genes , Células HEK293 , Humanos , Mutación con Pérdida de Función , Masculino , Hipotonía Muscular/genética , Disinergia Cerebelosa Mioclónica/genética , Trastornos del Neurodesarrollo/diagnóstico por imagen , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/fisiopatología , Linaje , Polimorfismo de Nucleótido Simple , RNA-Seq , Ribonucleoproteínas Nucleares Pequeñas/genética , Rigor Mortis/genética , Proteínas del Complejo SMN/metabolismoRESUMEN
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.
Asunto(s)
Esclerosis Amiotrófica Lateral/patología , Ribonucleoproteína Heterogénea-Nuclear Grupo M/metabolismo , Degeneración Nerviosa/metabolismo , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteínas de Unión al ARN/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Animales Modificados Genéticamente , Drosophila , Proteínas de Drosophila/metabolismo , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Humanos , Ratones , Degeneración Nerviosa/patología , Motivos de Unión al ARN/fisiologíaRESUMEN
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.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Proteína C9orf72/genética , Neuronas Motoras/metabolismo , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas de Unión al ARN/genética , ARN/metabolismo , Anciano , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Animales Modificados Genéticamente , Proteína C9orf72/metabolismo , Expansión de las Repeticiones de ADN , Drosophila , Femenino , Demencia Frontotemporal/genética , Demencia Frontotemporal/metabolismo , Demencia Frontotemporal/patología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Persona de Mediana Edad , Neuronas Motoras/patología , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
Proper transport of the Parkinson's disease (PD) protein, α-synuclein (α-syn), is thought to be crucial for its localization and function at the synapse. Previous work has shown that defects in long distance transport within narrow caliber axons occur early in PD, but how such defects contribute to PD is unknown. Here we test the hypothesis that the NAC region is involved in facilitating proper transport of α-syn within axons via its association with membranes. Excess α-syn or fPD mutant α-synA53T accumulates within larval axons perturbing the transport of synaptic proteins. These α-syn expressing larvae also show synaptic morphological and larval locomotion defects, which correlate with the extent of α-syn-mediated axonal accumulations. Strikingly, deletion of the NAC region (α-synΔ71-82) prevented α-syn accumulations and axonal blockages, and reduced its synaptic localization due to decreased axonal entry and axonal transport of α-syn, due to less α-syn bound to membranes. Intriguingly, co-expression α-synΔ71-82 with full-length α-syn rescued α-syn accumulations and synaptic morphological defects, and decreased the ratio of the insoluble higher molecular weight (HMW)/soluble low molecular weight (LMW) α-syn, indicating that this region is perhaps important for the dimerization of α-syn on membranes. Together, our observations suggest that under physiological conditions, α-syn associates with membranes via the NAC region, and that too much α-syn perturbs axonal transport via aggregate formation, instigating synaptic and behavioral defects seen in PD.