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1.
Int J Mol Sci ; 22(5)2021 Feb 28.
Artículo en Inglés | MEDLINE | ID: mdl-33670886

RESUMEN

Deficient intracellular transport is a common pathological hallmark of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Mutations in the fused-in-sarcoma (FUS) gene are one of the most common genetic causes for familial ALS. Motor neurons carrying a mutation in the nuclear localization sequence of FUS (P525L) show impaired axonal transport of several organelles, suggesting that mislocalized cytoplasmic FUS might directly interfere with the transport machinery. To test this hypothesis, we studied the effect of FUS on kinesin-1 motility in vitro. Using a modified microtubule gliding motility assay on surfaces coated with kinesin-1 motor proteins, we showed that neither recombinant wildtype and P525L FUS variants nor lysates from isogenic ALS-patient-specific iPSC-derived spinal motor neurons expressing those FUS variants significantly affected gliding velocities. We hence conclude that during ALS pathogenesis the initial negative effect of FUS (P525L) on axonal transport is an indirect nature and requires additional factors or mechanisms.


Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Transporte Axonal , Microtúbulos/metabolismo , Neuronas Motoras/metabolismo , Mutación , Proteína FUS de Unión a ARN/genética , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/fisiopatología , Línea Celular , Humanos , Cinesina , Neuronas Motoras/fisiología , Proteína FUS de Unión a ARN/metabolismo
2.
Nat Commun ; 12(1): 1809, 2021 03 22.
Artículo en Inglés | MEDLINE | ID: mdl-33753744

RESUMEN

Dynamic membraneless compartments formed by protein condensates have multifunctional roles in cellular biology. Tools that inducibly trigger condensate formation have been useful for exploring their cellular function, however, there are few tools that provide inducible control over condensate disruption. To address this need we developed DisCo (Disassembly of Condensates), which relies on the use of chemical dimerizers to inducibly recruit a ligand to the condensate-forming protein, triggering condensate dissociation. We demonstrate use of DisCo to disrupt condensates of FUS, associated with amyotrophic lateral sclerosis, and to prevent formation of polyglutamine-containing huntingtin condensates, associated with Huntington's disease. In addition, we combined DisCo with a tool to induce condensates with light, CRY2olig, achieving bidirectional control of condensate formation and disassembly using orthogonal inputs of light and rapamycin. Our results demonstrate a method to manipulate condensate states that will have broad utility, enabling better understanding of the biological role of condensates in health and disease.


Asunto(s)
Proteínas Fluorescentes Verdes/química , Ensayos Analíticos de Alto Rendimiento/métodos , Multimerización de Proteína , Proteínas/química , Animales , Células COS , Chlorocebus aethiops , Transferencia Resonante de Energía de Fluorescencia , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Microscopía Fluorescente/métodos , Proteínas/genética , Proteínas/metabolismo , Proteína FUS de Unión a ARN/química , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo
3.
Elife ; 92020 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-33319748

RESUMEN

The AAA+ protein disaggregase, Hsp104, increases fitness under stress by reversing stress-induced protein aggregation. Natural Hsp104 variants might exist with enhanced, selective activity against neurodegenerative disease substrates. However, natural Hsp104 variation remains largely unexplored. Here, we screened a cross-kingdom collection of Hsp104 homologs in yeast proteotoxicity models. Prokaryotic ClpG reduced TDP-43, FUS, and α-synuclein toxicity, whereas prokaryotic ClpB and hyperactive variants were ineffective. We uncovered therapeutic genetic variation among eukaryotic Hsp104 homologs that specifically antagonized TDP-43 condensation and toxicity in yeast and TDP-43 aggregation in human cells. We also uncovered distinct eukaryotic Hsp104 homologs that selectively antagonized α-synuclein condensation and toxicity in yeast and dopaminergic neurodegeneration in C. elegans. Surprisingly, this therapeutic variation did not manifest as enhanced disaggregase activity, but rather as increased passive inhibition of aggregation of specific substrates. By exploring natural tuning of this passive Hsp104 activity, we elucidated enhanced, substrate-specific agents that counter proteotoxicity underlying neurodegeneration.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Agregación Patológica de Proteínas/patología , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , alfa-Sinucleína/metabolismo , Animales , Caenorhabditis elegans , Línea Celular , Endopeptidasa Clp/genética , Endopeptidasa Clp/metabolismo , Escherichia coli , Variación Genética/genética , Células HEK293 , Humanos , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/patología , Pliegue de Proteína , Deficiencias en la Proteostasis/genética , Deficiencias en la Proteostasis/patología , Proteína FUS de Unión a ARN/metabolismo , Saccharomyces cerevisiae
4.
Nat Commun ; 11(1): 6341, 2020 12 11.
Artículo en Inglés | MEDLINE | ID: mdl-33311468

RESUMEN

Mutations in the RNA-binding protein Fused in Sarcoma (FUS) cause early-onset amyotrophic lateral sclerosis (ALS). However, a detailed understanding of central RNA targets of FUS and their implications for disease remain elusive. Here, we use a unique blend of crosslinking and immunoprecipitation (CLIP) and NMR spectroscopy to identify and characterise physiological and pathological RNA targets of FUS. We find that U1 snRNA is the primary RNA target of FUS via its interaction with stem-loop 3 and provide atomic details of this RNA-mediated mode of interaction with the U1 snRNP. Furthermore, we show that ALS-associated FUS aberrantly contacts U1 snRNA at the Sm site with its zinc finger and traps snRNP biogenesis intermediates in human and murine motor neurons. Altogether, we present molecular insights into a FUS toxic gain-of-function involving direct and aberrant RNA-binding and strengthen the link between two motor neuron diseases, ALS and spinal muscular atrophy (SMA).


Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , ARN Nuclear Pequeño/metabolismo , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Esclerosis Amiotrófica Lateral/genética , Animales , Línea Celular , Predisposición Genética a la Enfermedad/genética , Humanos , Ratones , Ratones Noqueados , Modelos Moleculares , Neuronas Motoras/metabolismo , Mutación , Dominios y Motivos de Interacción de Proteínas , ARN Nuclear Pequeño/química , Proteína FUS de Unión a ARN/química , Ribonucleoproteína Nuclear Pequeña U1/química
5.
Nat Commun ; 11(1): 5735, 2020 11 12.
Artículo en Inglés | MEDLINE | ID: mdl-33184287

RESUMEN

Protein domains without the usual distribution of amino acids, called low complexity (LC) domains, can be prone to self-assembly into amyloid-like fibrils. Self-assembly of LC domains that are nearly devoid of hydrophobic residues, such as the 214-residue LC domain of the RNA-binding protein FUS, is particularly intriguing from the biophysical perspective and is biomedically relevant due to its occurrence within neurons in amyotrophic lateral sclerosis, frontotemporal dementia, and other neurodegenerative diseases. We report a high-resolution molecular structural model for fibrils formed by the C-terminal half of the FUS LC domain (FUS-LC-C, residues 111-214), based on a density map with 2.62 Å resolution from cryo-electron microscopy (cryo-EM). In the FUS-LC-C fibril core, residues 112-150 adopt U-shaped conformations and form two subunits with in-register, parallel cross-ß structures, arranged with quasi-21 symmetry. All-atom molecular dynamics simulations indicate that the FUS-LC-C fibril core is stabilized by a plethora of hydrogen bonds involving sidechains of Gln, Asn, Ser, and Tyr residues, both along and transverse to the fibril growth direction, including diverse sidechain-to-backbone, sidechain-to-sidechain, and sidechain-to-water interactions. Nuclear magnetic resonance measurements additionally show that portions of disordered residues 151-214 remain highly dynamic in FUS-LC-C fibrils and that fibrils formed by the N-terminal half of the FUS LC domain (FUS-LC-N, residues 2-108) have the same core structure as fibrils formed by the full-length LC domain. These results contribute to our understanding of the molecular structural basis for amyloid formation by FUS and by LC domains in general.


Asunto(s)
Amiloide/química , Amiloide/metabolismo , Proteína FUS de Unión a ARN/química , Proteína FUS de Unión a ARN/metabolismo , Amiloide/genética , Amiloide/ultraestructura , Microscopía por Crioelectrón , Humanos , Enlace de Hidrógeno , Espectroscopía de Resonancia Magnética , Simulación de Dinámica Molecular , Estructura Molecular , Conformación Proteica , Dominios Proteicos , Dominios y Motivos de Interacción de Proteínas , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/ultraestructura , Análisis de Secuencia de Proteína
6.
Genes Dev ; 34(11-12): 785-805, 2020 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-32381627

RESUMEN

Dysregulation of the DNA/RNA-binding protein FUS causes certain subtypes of ALS/FTD by largely unknown mechanisms. Recent evidence has shown that FUS toxic gain of function due either to mutations or to increased expression can disrupt critical cellular processes, including mitochondrial functions. Here, we demonstrate that in human cells overexpressing wild-type FUS or expressing mutant derivatives, the protein associates with multiple mRNAs, and these are enriched in mRNAs encoding mitochondrial respiratory chain components. Notably, this sequestration leads to reduced levels of the encoded proteins, which is sufficient to bring about disorganized mitochondrial networks, reduced aerobic respiration and increased reactive oxygen species. We further show that mutant FUS associates with mitochondria and with mRNAs encoded by the mitochondrial genome. Importantly, similar results were also observed in fibroblasts derived from ALS patients with FUS mutations. Finally, we demonstrate that FUS loss of function does not underlie the observed mitochondrial dysfunction, and also provides a mechanism for the preferential sequestration of the respiratory chain complex mRNAs by FUS that does not involve sequence-specific binding. Together, our data reveal that respiratory chain complex mRNA sequestration underlies the mitochondrial defects characteristic of ALS/FTD and contributes to the FUS toxic gain of function linked to this disease spectrum.


Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/fisiopatología , Regulación de la Expresión Génica/genética , Mitocondrias/patología , ARN Mensajero/metabolismo , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo , Línea Celular , Respiración de la Célula/genética , Células Cultivadas , Transporte de Electrón/genética , Genoma Mitocondrial , Humanos , Mitocondrias/genética , Mutación , Agregación Patológica de Proteínas/genética , Unión Proteica/genética
7.
J Neuropathol Exp Neurol ; 79(4): 370-377, 2020 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-32142134

RESUMEN

Alterations of RNA metabolism caused by mutations in RNA-binding protein genes, such as transactivating DNA-binding protein-43 (TDP-43) and fused in sarcoma (FUS), have been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). Unlike the accumulation of TDP43, which is accepted as a pathological hall mark of sporadic ALS (sALS), FUS pathology in sALS is still under debate. Although immunoreactive inclusions of FUS have been detected in sALS patients previously, the technical limitation of signal detection, including the necessity of specific antigen retrieval, restricts our understanding of FUS-associated ALS pathology. In this study, we applied a novel detection method using a conventional antigen retrieval technique with Sudan Black B treatment to identify FUS-positive inclusions in sALS patients. We classified pathological motor neurons into 5 different categories according to the different aggregation characteristics of FUS and TDP-43. Although the granular type was more dominant for inclusions with TDP-43, the skein-like type was more often observed in FUS-positive inclusions, suggesting that these 2 proteins undergo independent aggregation processes. Moreover, neurons harboring FUS-positive inclusions demonstrated substantially reduced expression levels of dynactin-1, a retrograde motor protein, indicating that perturbation of nucleocytoplasmic transport is associated with the formation of cytoplasmic inclusions of FUS in sALS.


Asunto(s)
Esclerosis Amiotrófica Lateral/patología , Proteínas de Unión al ADN/metabolismo , Cuerpos de Inclusión/patología , Neuronas Motoras/patología , Proteína FUS de Unión a ARN/metabolismo , Anciano , Anciano de 80 o más Años , Esclerosis Amiotrófica Lateral/metabolismo , Complejo Dinactina/metabolismo , Femenino , Células HEK293 , Humanos , Cuerpos de Inclusión/metabolismo , Masculino , Persona de Mediana Edad , Neuronas Motoras/metabolismo
8.
Nat Rev Neurol ; 16(4): 213-228, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-32203398

RESUMEN

Frontotemporal dementia (FTD) encompasses a spectrum of clinical syndromes characterized by progressive executive, behavioural and language dysfunction. The various FTD spectrum disorders are associated with brain accumulation of different proteins: tau, the transactive response DNA binding protein of 43 kDa (TDP43), or fused in sarcoma (FUS) protein, Ewing sarcoma protein and TATA-binding protein-associated factor 15 (TAF15) (collectively known as FET proteins). Approximately 60% of patients with FTD have autosomal dominant mutations in C9orf72, GRN or MAPT genes. Currently available treatments are symptomatic and provide limited benefit. However, the increased understanding of FTD pathogenesis is driving the development of potential disease-modifying therapies. Most of these drugs target pathological tau - this category includes tau phosphorylation inhibitors, tau aggregation inhibitors, active and passive anti-tau immunotherapies, and MAPT-targeted antisense oligonucleotides. Some of these therapeutic approaches are being tested in phase II clinical trials. Pharmacological approaches that target the effects of GRN and C9orf72 mutations are also in development. Key results of large clinical trials will be available in a few years. However, clinical trials in FTD pose several challenges, and the development of specific brain imaging and molecular biomarkers could facilitate the recruitment of clinically homogenous groups to improve the chances of positive clinical trial results.


Asunto(s)
Anticuerpos/uso terapéutico , Afasia Progresiva Primaria/tratamiento farmacológico , Desarrollo de Medicamentos , Demencia Frontotemporal/tratamiento farmacológico , Moduladores de Tubulina/uso terapéutico , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Afasia Progresiva Primaria/genética , Afasia Progresiva Primaria/metabolismo , Proteína C9orf72/genética , Proteínas de Unión al ADN/metabolismo , Demencia Frontotemporal/genética , Demencia Frontotemporal/metabolismo , Humanos , Inmunización Pasiva , Inmunoterapia Activa , Terapia Molecular Dirigida , Progranulinas/genética , Proteína EWS de Unión a ARN/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Parálisis Supranuclear Progresiva/tratamiento farmacológico , Parálisis Supranuclear Progresiva/genética , Parálisis Supranuclear Progresiva/metabolismo , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismo
9.
Sci Rep ; 10(1): 2629, 2020 02 14.
Artículo en Inglés | MEDLINE | ID: mdl-32060318

RESUMEN

Translocated in liposarcoma (TLS)/fused in sarcoma (FUS) is a multitasking DNA/RNA binding protein implicated in cancer and neurodegenerative diseases. Upon DNA damage, TLS is recruited to the upstream region of the cyclin D1 gene (CCND1) through binding to the promotor associated non-coding RNA (pncRNA) that is transcribed from and tethered at the upstream region. Binding to pncRNA is hypothesized to cause the conformational change of TLS that enables its inhibitive interaction with histone acetyltransferases and resultant repression of CCND1 expression, although no experimental proof has been obtained. Here, the closed-to-open conformational change of TLS on binding pncRNA was implied by fluorescence resonance energy transfer. A small fragment (31 nucleotides) of the full-length pncRNA (602 nucleotides) was shown to be sufficient for the conformational change of TLS. Dissection of pncRNA identified the G-rich RNA sequence that is critical for the conformational change. The length of RNA was also revealed to be critical for the conformational change. Furthermore, it was demonstrated that the conformational change of TLS is caused by another target DNA and RNA, telomeric DNA and telomeric repeat-containing RNA. The conformational change of TLS on binding target RNA/DNA is suggested to be essential for biological functions.


Asunto(s)
ARN no Traducido/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Secuencia de Bases , Sitios de Unión , Transferencia Resonante de Energía de Fluorescencia , Humanos , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas , Unión Proteica , Conformación Proteica , ARN no Traducido/química , Proteína FUS de Unión a ARN/química
11.
In Vitro Cell Dev Biol Anim ; 56(1): 15-23, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31925650

RESUMEN

Invasive mucinous adenocarcinoma of the lung (IMA), a mucinous variant of lung adenocarcinoma, is strongly linked with a worse prognosis. Therefore, a deeper understanding about its molecular mechanism may conduce to a promising IMA therapy. Long non-coding RNAs (lncRNAs) have recently caught great attention for their crucial roles in diverse diseases regarding tumor initiation and progression. However, the potential role of the lncRNA HOXC-AS3 IMA is not well established. Hence, the purpose of present study is to manifest HOXC-AS3-regulated inner mechanism in IMA development. It revealed that HOXC-AS3 was highly expressed in IMA cells. Additionally, it was identified that the significant down-regulation of HOXC-AS3 obstructed cell proliferation and migration in IMA. As far as mechanism is concerned, it found that HOXC-AS3 recruited FUS to stabilize FOXM1 mRNA, accelerating IMA progression. Taken together, these data suggested that HOXC-AS3 may be recognized as a novel therapeutic target for patients with IMA or at least offer new views for molecular therapy.


Asunto(s)
Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/patología , Adenocarcinoma Mucinoso/genética , Adenocarcinoma Mucinoso/patología , Progresión de la Enfermedad , Proteína Forkhead Box M1/metabolismo , ARN Largo no Codificante/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Apoptosis/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Proteína Forkhead Box M1/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Invasividad Neoplásica , Unión Proteica , Estabilidad del ARN , ARN Largo no Codificante/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo
12.
Mol Cell Biol ; 40(4)2020 01 30.
Artículo en Inglés | MEDLINE | ID: mdl-31767634

RESUMEN

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron degenerative disease. TDP-43 (TAR DNA-binding protein 43) and FUS (fused in sarcoma) are aggregation-prone RNA-binding proteins that in ALS can mislocalize to the cytoplasm of affected motor neuron cells, often forming cytoplasmic aggregates in the process. Such mislocalization and aggregation are implicated in ALS pathology, though the mechanism(s) of TDP-43 and FUS cytoplasmic toxicity remains unclear. Recently, we determined that the endocytic function aids the turnover (i.e., protein degradation) of TDP-43 and reduces TDP-43 toxicity. Here, we identified that Cdc48 and Ubx3, a Cdc48 cofactor implicated in endocytic function, regulates the turnover and toxicity of TDP-43 and FUS expressed in Saccharomyces cerevisiae Cdc48 physically interacts and colocalizes with TDP-43, as does VCP, in ALS patient tissue. In yeast, FUS toxicity also depends strongly on endocytic function but not on autophagy under normal conditions. FUS expression also impairs endocytic function, as previously observed with TDP-43. Taken together, our data identify a role for Cdc48/VCP and endocytic function in regulating TDP-43 and FUS toxicity and turnover. Furthermore, endocytic dysfunction may be a common defect affecting the cytoplasmic clearance of ALS aggregation-prone proteins and may represent a novel therapeutic target of promise.


Asunto(s)
Esclerosis Amiotrófica Lateral/patología , Proteínas de Unión al ADN/metabolismo , Endocitosis/fisiología , Proteína FUS de Unión a ARN/metabolismo , Proteína que Contiene Valosina/metabolismo , Esclerosis Amiotrófica Lateral/genética , Línea Celular , Células HEK293 , Humanos , Agregación Patológica de Proteínas/patología , Proteolisis , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína que Contiene Valosina/genética
13.
Insect Mol Biol ; 29(1): 56-65, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31290213

RESUMEN

Fused in sarcoma (FUS) is a DNA/RNA-binding protein associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration. The exact molecular mechanisms by which FUS results in neurotoxicity have not yet been fully elucidated. Here, we found that parkin is a genetic suppressor of defective phenotypes induced by exogenous human wild type FUS in Drosophila. Although parkin overexpression did not modulate the FUS protein expression level, the locomotive defects in FUS-expressing larvae and adult flies were rescued by parkin expression. We found that FUS expression in muscle tissues resulted in a reduction of the levels and assembly of mitochondrial complex I and III subunits, as well as decreased ATP. Remarkably, expression of parkin suppressed these mitochondrial dysfunctions. Our results indicate parkin as a neuroprotective regulator of FUS-induced proteinopathy by recovering the protein levels of mitochondrial complexes I and III. Our findings on parkin-mediated neuroprotection may expand our understanding of FUS-induced ALS pathogenesis.


Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Drosophila/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Adenosina Trifosfato/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Animales Modificados Genéticamente , Modelos Animales de Enfermedad , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Larva , Masculino , Mitocondrias/genética , Mitocondrias/metabolismo , Mitocondrias/patología , Músculos/metabolismo , Músculos/patología , Proteína FUS de Unión a ARN/genética , Ubiquitina-Proteína Ligasas/genética
14.
Nat Commun ; 10(1): 5583, 2019 12 06.
Artículo en Inglés | MEDLINE | ID: mdl-31811140

RESUMEN

Mutations in fused in sarcoma (FUS) lead to amyotrophic lateral sclerosis (ALS) with varying ages of onset, progression and severity. This suggests that unknown genetic factors contribute to disease pathogenesis. Here we show the identification of muscleblind as a novel modifier of FUS-mediated neurodegeneration in vivo. Muscleblind regulates cytoplasmic mislocalization of mutant FUS and subsequent accumulation in stress granules, dendritic morphology and toxicity in mammalian neuronal and human iPSC-derived neurons. Interestingly, genetic modulation of endogenous muscleblind was sufficient to restore survival motor neuron (SMN) protein localization in neurons expressing pathogenic mutations in FUS, suggesting a potential mode of suppression of FUS toxicity. Upregulation of SMN suppressed FUS toxicity in Drosophila and primary cortical neurons, indicating a link between FUS and SMN. Our data provide in vivo evidence that muscleblind is a dominant modifier of FUS-mediated neurodegeneration by regulating FUS-mediated ALS pathogenesis.


Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas Nucleares/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Proteínas del Complejo SMN/metabolismo , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Esclerosis Amiotrófica Lateral/genética , Animales , Citoplasma/metabolismo , Gránulos Citoplasmáticos/metabolismo , Drosophila/genética , Drosophila/metabolismo , Femenino , Células HEK293 , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Neuronas Motoras/metabolismo , Mutación , Fenotipo , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/toxicidad , Proteínas del Complejo SMN/genética , Factores de Transcripción/metabolismo
15.
Mol Brain ; 12(1): 103, 2019 12 04.
Artículo en Inglés | MEDLINE | ID: mdl-31801573

RESUMEN

Amyotrophic lateral sclerosis (ALS) is a degenerative motor neuron disease that has been linked to defective DNA repair. Many familial ALS patients harbor autosomal dominant mutations in the gene encoding the RNA/DNA binding protein 'fused in sarcoma' (FUS) commonly inducing its cytoplasmic mislocalization. Recent reports from our group and others demonstrate a role of FUS in maintaining genome integrity and the DNA damage response (DDR). FUS interacts with many DDR proteins and may regulate their recruitment at damage sites. Given the role of FUS in RNA transactions, here we explore whether FUS also regulates the expression of DDR factors. We performed RT2 PCR arrays for DNA repair and DDR signaling pathways in CRISPR/Cas9 FUS knockout (KO) and shRNA mediated FUS knockdown (KD) cells, which revealed significant (> 2-fold) downregulation of BRCA1, DNA ligase 4, MSH complex and RAD23B. Importantly, similar perturbations in these factors were also consistent in motor neurons differentiated from an ALS patient-derived induced pluripotent stem cell (iPSC) line with a FUS-P525L mutation, as well as in postmortem spinal cord tissue of sporadic ALS patients with FUS pathology. BRCA1 depletion has been linked to neuronal DNA double-strand breaks (DSBs) accumulation and cognitive defects. The ubiquitin receptor RAD23 functions both in nucleotide excision repair and proteasomal protein clearance pathway and is thus linked to neurodegeneration. Together, our study suggests that the FUS pathology perturbs DDR signaling via both its direct role and the effect on the expression of DDR genes. This underscors an intricate connections between FUS, genome instability, and neurodegeneration.


Asunto(s)
Daño del ADN , Enfermedad de la Neurona Motora/metabolismo , Enfermedad de la Neurona Motora/patología , Proteína FUS de Unión a ARN/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal , Daño del ADN/genética , Reparación del ADN/genética , Regulación de la Expresión Génica , Células HEK293 , Humanos , Enfermedad de la Neurona Motora/genética , Neuronas Motoras/metabolismo , Neuronas Motoras/patología
16.
Biochemistry (Mosc) ; 84(10): 1166-1176, 2019 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-31694512

RESUMEN

The aim of this study was to evaluate changes in the content of sphingoid bases - sphingosine (SPH), sphinganine, and sphingosine-1-phosphate (SPH-1-P) - and in expression of genes encoding enzymes involved in their metabolism in the brain structures (hippocampus, cortex, and cerebellum) and spinal cord of transgenic FUS(1-359) mice. FUS(1-359) mice are characterized by motor impairments and can be used as a model of amyotrophic lateral sclerosis (ALS). Lipids from the mouse brain structures and spinal cord after 2, 3, and 4 months of disease development were analyzed by chromatography/mass spectrometry, while changes in the expression of the SPHK1, SPHK2, SGPP2, SGPL1, ASAH1, and ASAH2 genes were assayed using RNA sequencing. The levels of SPH and sphinganine (i.e., sphingoid bases with pronounced pro-apoptotic properties) were dramatically increased in the spinal cord at the terminal stage of the disease. The ratio of the anti-apoptotic SPH-1-P to SPH and sphinganine sharply reduced, indicating massive apoptosis of spinal cord cells. Significant changes in the content of SPH and SPH-1-P and in the expression of genes related to their metabolism were found at the terminal ALS stage in the spinal cord. Expression of the SGPL gene (SPH-1-P lyase) was strongly activated, while expression of the SGPP2 (SPH-1-P phosphatase) gene was reduced. Elucidation of mechanisms for the regulation of sphingolipid metabolism in ALS will help to identify molecular targets for the new-generation drugs.


Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Proteína FUS de Unión a ARN/metabolismo , Esfingolípidos/metabolismo , Médula Espinal/metabolismo , Animales , Ratones , Ratones Transgénicos , Esfingolípidos/química
17.
Eur Rev Med Pharmacol Sci ; 23(20): 8771-8778, 2019 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-31696463

RESUMEN

OBJECTIVE: Recently, the vital role of circular RNAs (circRNAs) in human diseases has attracted much attention. The aim of this research was to verify the potential role of circRNA_0000285 in the development of cervical cancer (CC). PATIENTS AND METHODS: CircRNA_0000285 expression in both CC cells and tissue samples was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Functional experiments were performed, including cell counting kit-8 (CCK-8) assay, cell cycle assay and transwell assay. Meanwhile, the underlying mechanism was explored through qRT-PCR and Western blot assay, respectively. In addition, the function of circRNA_0000285 was identified in vivo. RESULTS: CircRNA_0000285 expression level was significantly higher in CC samples than that of corresponding normal tissues. Moreover, the growth and migration abilities of CC cells were significantly inhibited after circRNA_0000285 was knocked down in vitro. Furthermore, the expression of FUS was remarkably down-regulated after knockdown of circRNA_0000285. Subsequent results indicated that the expression level of FUS was positively correlated with the expression of circRNA_0000285 in CC tissues. In addition, the knockdown of circRNA_0000285 significantly inhibited the formation and metastasis of CC in nude mice. CONCLUSIONS: CircRNA_0000285 could enhance the proliferation and metastasis of CC by up-regulating FUS, which might be a potential therapeutic target for CC treatment.


Asunto(s)
ARN Circular/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Neoplasias del Cuello Uterino/patología , Animales , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Femenino , Humanos , Ratones , Ratones Desnudos , Metástasis de la Neoplasia , Interferencia de ARN , ARN Circular/antagonistas & inhibidores , ARN Circular/genética , ARN Interferente Pequeño/metabolismo , ARN Interferente Pequeño/uso terapéutico , Proteína FUS de Unión a ARN/genética , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto
18.
Neuropathology ; 39(5): 358-367, 2019 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-31599037

RESUMEN

Huntington's disease (HD) is an inherited neurodegenerative disease caused by a polyglutamine repeat expansion in the huntingtin protein. Immunohistochemical studies using the 1C2 antibody for polyglutamine expansion have detected characteristic intranuclear inclusions (INIs) in affected neurons in HD. Further, in vitro and mouse models of HD have shown that the INIs recruit several proteins relating to RNA splicing and translation. In the present study, we immunohistochemically investigated the association of INIs with various heterogeneous nuclear ribonucleoproteins in the cerebral cortex of four autopsy cases of HD. Fused in sarcoma (FUS) was colocalized with 1C2-positive nuclear inclusions in all examined cases. Localization of poly (rC)-binding protein 1 (PCBP1) in 1C2-positive nuclear inclusions was also observed. Double immunofluorescence revealed complete or partial loss of the normal, diffuse nuclear distribution of FUS or PCBP1 in neurons with 1C2-positive nuclear inclusions. This maldistribution of FUS in cortical neurons suggests a severe disturbance of messenger RNA processing, which may be a common pathogenetic mechanism of FUS-related familial amyotrophic lateral sclerosis.


Asunto(s)
Corteza Cerebral/patología , Proteínas de Unión al ADN/metabolismo , Enfermedad de Huntington/metabolismo , Cuerpos de Inclusión Intranucleares/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Anciano , Femenino , Humanos , Proteína Huntingtina , Enfermedad de Huntington/patología , Cuerpos de Inclusión Intranucleares/patología , Masculino , Persona de Mediana Edad , Péptidos , Transporte de Proteínas/fisiología , Procesamiento Postranscripcional del ARN/fisiología
19.
Nat Neurosci ; 22(11): 1793-1805, 2019 11.
Artículo en Inglés | MEDLINE | ID: mdl-31591561

RESUMEN

Neuromuscular junction (NMJ) disruption is an early pathogenic event in amyotrophic lateral sclerosis (ALS). Yet, direct links between NMJ pathways and ALS-associated genes such as FUS, whose heterozygous mutations cause aggressive forms of ALS, remain elusive. In a knock-in Fus-ALS mouse model, we identified postsynaptic NMJ defects in newborn homozygous mutants that were attributable to mutant FUS toxicity in skeletal muscle. Adult heterozygous knock-in mice displayed smaller neuromuscular endplates that denervated before motor neuron loss, which is consistent with 'dying-back' neuronopathy. FUS was enriched in subsynaptic myonuclei, and this innervation-dependent enrichment was distorted in FUS-ALS. Mechanistically, FUS collaborates with the ETS transcription factor ERM to stimulate transcription of acetylcholine receptor genes. Co-cultures of induced pluripotent stem cell-derived motor neurons and myotubes from patients with FUS-ALS revealed endplate maturation defects due to intrinsic FUS toxicity in both motor neurons and myotubes. Thus, FUS regulates acetylcholine receptor gene expression in subsynaptic myonuclei, and muscle-intrinsic toxicity of ALS mutant FUS may contribute to dying-back motor neuronopathy.


Asunto(s)
Esclerosis Amiotrófica Lateral/fisiopatología , Regulación de la Expresión Génica/fisiología , Degeneración Nerviosa/fisiopatología , Unión Neuromuscular/metabolismo , Proteína FUS de Unión a ARN/fisiología , Adulto , Esclerosis Amiotrófica Lateral/patología , Animales , Células Cultivadas , Femenino , Técnicas de Sustitución del Gen , Humanos , Masculino , Ratones , Ratones Noqueados , Neuronas Motoras/patología , Fibras Musculares Esqueléticas/patología , Unión Neuromuscular/patología , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo , Receptores Colinérgicos/metabolismo , Adulto Joven
20.
Cell Death Dis ; 10(11): 792, 2019 10 17.
Artículo en Inglés | MEDLINE | ID: mdl-31624242

RESUMEN

Prostate cancer (PCa) is one of the major men's malignancies with high mortality worldwide. Circular RNAs (circRNAs) have been shown to serve as essential regulators in human cancers. CircRNA can exert their functions by cooperating with their host genes. In the present study, microarray analysis revealed an upregulated mRNA in PCa samples. X-linked inhibitor of apoptosis protein (XIAP), a key regulator in the progression of human cancers. Through bioinformatics analysis, we determined that XIAP is a host gene for circRNA0005276. Therefore, this study focused on the interaction between circ0005276 and XIAP as well as their functions in PCa progression. The upregulation of XIAP and circ0005276 was determined in PCa tissues and cell lines. Moreover, we confirmed the positive regulation of circ0005276 on XIAP expression. Functionally, we validated that circ0005276 and XIAP promoted cell proliferation, migration and epithelial-mesenchymal transition. Mechanistically, we verified that circ0005276 interacted with FUS binding protein (FUS) so as to activate the transcription of XIAP. Rescue assays were conducted to determine the crucial role of XIAP in circ0005276 and FUS-mediated PCa cellular processes. Collectively, our study revealed the mechanism and function of circ0005276 and its host gene XIAP in PCa progression.


Asunto(s)
Neoplasias de la Próstata/genética , ARN Circular/metabolismo , Proteína FUS de Unión a ARN/genética , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Animales , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Movimiento Celular/fisiología , Progresión de la Enfermedad , Xenoinjertos , Humanos , Masculino , Ratones , Ratones Desnudos , Células PC-3 , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , ARN Circular/biosíntesis , ARN Circular/genética , Proteína FUS de Unión a ARN/metabolismo , Activación Transcripcional , Regulación hacia Arriba , Proteína Inhibidora de la Apoptosis Ligada a X/biosíntesis , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo
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