RESUMEN
Dipeptide repeat proteins are a major pathogenic feature of C9orf72 amyotrophic lateral sclerosis (C9ALS)/frontotemporal dementia (FTD) pathology, but their physiological impact has yet to be fully determined. Here we generated C9orf72 dipeptide repeat knock-in mouse models characterized by expression of 400 codon-optimized polyGR or polyPR repeats, and heterozygous C9orf72 reduction. (GR)400 and (PR)400 knock-in mice recapitulate key features of C9ALS/FTD, including cortical neuronal hyperexcitability, age-dependent spinal motor neuron loss and progressive motor dysfunction. Quantitative proteomics revealed an increase in extracellular matrix (ECM) proteins in (GR)400 and (PR)400 spinal cord, with the collagen COL6A1 the most increased protein. TGF-ß1 was one of the top predicted regulators of this ECM signature and polyGR expression in human induced pluripotent stem cell neurons was sufficient to induce TGF-ß1 followed by COL6A1. Knockdown of TGF-ß1 or COL6A1 orthologues in polyGR model Drosophila exacerbated neurodegeneration, while expression of TGF-ß1 or COL6A1 in induced pluripotent stem cell-derived motor neurons of patients with C9ALS/FTD protected against glutamate-induced cell death. Altogether, our findings reveal a neuroprotective and conserved ECM signature in C9ALS/FTD.
Asunto(s)
Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Células Madre Pluripotentes Inducidas , Animales , Humanos , Ratones , Demencia Frontotemporal/patología , Esclerosis Amiotrófica Lateral/metabolismo , Factor de Crecimiento Transformador beta1 , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Neuronas Motoras/metabolismo , Drosophila , Matriz Extracelular/metabolismo , Dipéptidos/metabolismo , Expansión de las Repeticiones de ADN/genéticaRESUMEN
An intronic GGGGCC repeat expansion in C9orf72 is a common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. The repeats are transcribed in both sense and antisense directions to generate distinct dipeptide repeat proteins, of which poly(GA), poly(GR), and poly(PR) have been implicated in contributing to neurodegeneration. Poly(PR) binding to RNA may contribute to toxicity, but analysis of poly(PR)-RNA binding on a transcriptome-wide scale has not yet been carried out. We therefore performed crosslinking and immunoprecipitation (CLIP) analysis in human cells to identify the RNA binding sites of poly(PR). We found that poly(PR) binds to nearly 600 RNAs, with the sequence GAAGA enriched at the binding sites. In vitro experiments showed that poly(GAAGA) RNA binds poly(PR) with higher affinity than control RNA and induces the phase separation of poly(PR) into condensates. These data indicate that poly(PR) preferentially binds to poly(GAAGA)-containing RNAs, which may have physiological consequences.
Asunto(s)
Perfilación de la Expresión Génica , Transcriptoma , Humanos , Transcriptoma/genética , Proteína C9orf72/genética , Poli A , Dipéptidos , ARN/genéticaRESUMEN
Mutations in the RNA-binding protein FUS cause amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disease. FUS plays a role in numerous aspects of RNA metabolism, including mRNA splicing. However, the impact of ALS-causative mutations on splicing has not been fully characterized, as most disease models have been based on overexpressing mutant FUS, which will alter RNA processing due to FUS autoregulation. We and others have recently created knockin models that overcome the overexpression problem, and have generated high depth RNA-sequencing on FUS mutants in parallel to FUS knockout, allowing us to compare mutation-induced changes to genuine loss of function. We find that FUS-ALS mutations induce a widespread loss of function on expression and splicing. Specifically, we find that mutant FUS directly alters intron retention levels in RNA-binding proteins. Moreover, we identify an intron retention event in FUS itself that is associated with its autoregulation. Altered FUS levels have been linked to disease, and we show here that this novel autoregulation mechanism is altered by FUS mutations. Crucially, we also observe this phenomenon in other genetic forms of ALS, including those caused by TDP-43, VCP and SOD1 mutations, supporting the concept that multiple ALS genes interact in a regulatory network.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Homeostasis/genética , Proteína FUS de Unión a ARN/genética , Animales , Citoplasma/genética , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/genética , Humanos , Intrones/genética , Mutación con Pérdida de Función , Ratones , Ratones Noqueados , Mutación/genética , Empalme del ARN/genética , Superóxido Dismutasa-1/genética , Proteína que Contiene Valosina/genéticaRESUMEN
Mutations in the endosome-associated protein CHMP2B cause frontotemporal dementia and lead to lysosomal storage pathology in neurons. We here report that physiological levels of mutant CHMP2B causes reduced numbers and significantly impaired trafficking of endolysosomes within neuronal dendrites, accompanied by increased dendritic branching. Mechanistically, this is due to the stable incorporation of mutant CHMP2B onto neuronal endolysosomes, which we show renders them unable to traffic within dendrites. This defect is due to the inability of mutant CHMP2B to recruit the ATPase VPS4, which is required for release of CHMP2B from endosomal membranes. Strikingly, both impaired trafficking and the increased dendritic branching were rescued by treatment with antisense oligonucleotides targeting the well validated frontotemporal dementia risk factor TMEM106B, which encodes an endolysosomal protein. This indicates that reducing TMEM106B levels can restore endosomal health in frontotemporal dementia. As TMEM106B is a risk factor for frontotemporal dementia caused by both C9orf72 and progranulin mutations, and antisense oligonucleotides are showing promise as therapeutics for neurodegenerative diseases, our data suggests a potential new strategy for treating the wide range of frontotemporal dementias associated with endolysosomal dysfunction.
Asunto(s)
Dendritas/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Endosomas/metabolismo , Demencia Frontotemporal/metabolismo , Lisosomas/metabolismo , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/metabolismo , Animales , Encéfalo/metabolismo , Células Cultivadas , Femenino , Técnicas de Silenciamiento del Gen , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Plasticidad NeuronalRESUMEN
Spinal and bulbar muscular atrophy (SBMA) is a neuromuscular disease characterized by the loss of lower motor neurons. SBMA is caused by expansions of a polyglutamine tract in the gene coding for androgen receptor (AR). Expression of polyglutamine-expanded AR causes damage to motor neurons and skeletal muscle cells. Here we investigated the effect of ß-agonist stimulation in SBMA myotube cells derived from mice and patients, and in knock-in mice. We show that treatment of myotubes expressing polyglutamine-expanded AR with the ß-agonist clenbuterol increases their size. Clenbuterol activated the phosphatidylinositol-3-kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) pathway and decreased the accumulation of polyglutamine-expanded AR. Treatment of SBMA knock-in mice with clenbuterol, which was started at disease onset, ameliorated motor function and extended survival. Clenbuterol improved muscle pathology, attenuated the glycolytic-to-oxidative metabolic alterations occurring in SBMA muscles and induced hypertrophy of both glycolytic and oxidative fibers. These results indicate that ß-agonist stimulation is a novel therapeutic strategy for SBMA.
Asunto(s)
Agonistas Adrenérgicos beta/farmacología , Clenbuterol/farmacología , Fibras Musculares Esqueléticas/efectos de los fármacos , Trastornos Musculares Atróficos/tratamiento farmacológico , Receptores Androgénicos/genética , Transducción de Señal , Animales , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones , Ratones Transgénicos , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/metabolismo , Trastornos Musculares Atróficos/metabolismo , Trastornos Musculares Atróficos/patología , Péptidos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Expansión de Repetición de TrinucleótidoRESUMEN
Frontotemporal dementia (FTD)-causing mutations in the CHMP2B gene lead to the generation of mutant C-terminally truncated CHMP2B. We report that transgenic mice expressing endogenous levels of mutant CHMP2B developed late-onset brain volume loss associated with frank neuronal loss and FTD-like changes in social behaviour. These data are the first to show neurodegeneration in mice expressing mutant CHMP2B and indicate that our mouse model is able to recapitulate neurodegenerative changes observed in FTD. Neuroinflammation has been increasingly implicated in neurodegeneration, including FTD. Therefore, we investigated neuroinflammation in our CHMP2B mutant mice. We observed very early microglial proliferation that develops into a clear pro-inflammatory phenotype at late stages. Importantly, we also observed a similar inflammatory profile in CHMP2B patient frontal cortex. Aberrant microglial function has also been implicated in FTD caused by GRN, MAPT and C9orf72 mutations. The presence of early microglial changes in our CHMP2B mutant mice indicates neuroinflammation may be a contributing factor to the neurodegeneration observed in FTD.
Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Proteínas del Tejido Nervioso/genética , Neuronas/patología , Enfermedades de la Lengua/genética , Enfermedades de la Lengua/metabolismo , Animales , Demencia/genética , Modelos Animales de Enfermedad , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Demencia Frontotemporal/genética , Demencia Frontotemporal/inmunología , Demencia Frontotemporal/patología , Humanos , Ratones , Ratones Transgénicos , Mutación , Proteínas del Tejido Nervioso/metabolismo , Neuronas/fisiología , Enfermedades de la Lengua/patologíaRESUMEN
Spinal and bulbar muscular atrophy (SBMA) is a neuromuscular disease caused by the expansion of a polyglutamine tract in the androgen receptor (AR). The mechanism by which expansion of polyglutamine in AR causes muscle atrophy is unknown. Here, we investigated pathological pathways underlying muscle atrophy in SBMA knock-in mice and patients. We show that glycolytic muscles were more severely affected than oxidative muscles in SBMA knock-in mice. Muscle atrophy was associated with early-onset, progressive glycolytic-to-oxidative fiber-type switch. Whole genome microarray and untargeted lipidomic analyses revealed enhanced lipid metabolism and impaired glycolysis selectively in muscle. These metabolic changes occurred before denervation and were associated with a concurrent enhancement of mechanistic target of rapamycin (mTOR) signaling, which induced peroxisome proliferator-activated receptor γ coactivator 1 alpha (PGC1α) expression. At later stages of disease, we detected mitochondrial membrane depolarization, enhanced transcription factor EB (TFEB) expression and autophagy, and mTOR-induced protein synthesis. Several of these abnormalities were detected in the muscle of SBMA patients. Feeding knock-in mice a high-fat diet (HFD) restored mTOR activation, decreased the expression of PGC1α, TFEB, and genes involved in oxidative metabolism, reduced mitochondrial abnormalities, ameliorated muscle pathology, and extended survival. These findings show early-onset and intrinsic metabolic alterations in SBMA muscle and link lipid/glucose metabolism to pathogenesis. Moreover, our results highlight an HFD regime as a promising approach to support SBMA patients.
Asunto(s)
Dieta Alta en Grasa/efectos adversos , Glucólisis , Músculo Esquelético/metabolismo , Trastornos Musculares Atróficos/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Animales , Atrofia/metabolismo , Atrofia/patología , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Glucólisis/fisiología , Humanos , Metabolismo de los Lípidos/fisiología , Masculino , Potencial de la Membrana Mitocondrial/fisiología , Ratones Transgénicos , Músculo Esquelético/patología , Trastornos Musculares Atróficos/patología , Oxidación-Reducción , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Distribución Aleatoria , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Transducción de SeñalRESUMEN
Spinal and bulbar muscular atrophy (SBMA) is characterized by loss of motoneurons and sensory neurons, accompanied by atrophy of muscle cells. SBMA is due to an androgen receptor containing a polyglutamine tract (ARpolyQ) that misfolds and aggregates, thereby perturbing the protein quality control (PQC) system. Using SBMA AR113Q mice we analyzed proteotoxic stress-induced alterations of HSPB8-mediated PQC machinery promoting clearance of misfolded proteins by autophagy. In muscle of symptomatic AR113Q male mice, we found expression upregulation of Pax-7, myogenin, E2-ubiquitin ligase UBE2Q1 and acetylcholine receptor (AchR), but not of MyoD, and of two E3-ligases (MuRF-1 and Cullin3). TGFß1 and PGC-1α were also robustly upregulated. We also found a dramatic perturbation of the autophagic response, with upregulation of most autophagic markers (Beclin-1, ATG10, p62/SQSTM1, LC3) and of the HSPB8-mediated PQC response. Both HSPB8 and its co-chaperone BAG3 were robustly upregulated together with other specific HSPB8 interactors (HSPB2 and HSPB3). Notably, the BAG3:BAG1 ratio increased in muscle suggesting preferential misfolded proteins routing to autophagy rather than to proteasome. Thus, mutant ARpolyQ induces a potent autophagic response in muscle cells. Alteration in HSPB8-based PQC machinery may represent muscle-specific biomarkers useful to assess SBMA progression in mice and patients in response to pharmacological treatments.
Asunto(s)
Autofagia/genética , Proteínas de Choque Térmico HSP27/genética , Trastornos Musculares Atróficos/genética , Receptores Androgénicos/genética , Animales , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Técnicas de Sustitución del Gen , Proteínas de Choque Térmico HSP27/biosíntesis , Humanos , Ratones , Trastornos Musculares Atróficos/patología , Pliegue de Proteína , Receptores Androgénicos/metabolismo , Ubiquitina/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Polyglutamine expansion in androgen receptor (AR) is responsible for spinobulbar muscular atrophy (SBMA) that leads to selective loss of lower motor neurons. Using SBMA as a model, we explored the relationship between protein structure/function and neurodegeneration in polyglutamine diseases. We show here that protein arginine methyltransferase 6 (PRMT6) is a specific co-activator of normal and mutant AR and that the interaction of PRMT6 with AR is significantly enhanced in the AR mutant. AR and PRMT6 interaction occurs through the PRMT6 steroid receptor interaction motif, LXXLL, and the AR activating function 2 surface. AR transactivation requires PRMT6 catalytic activity and involves methylation of arginine residues at Akt consensus site motifs, which is mutually exclusive with serine phosphorylation by Akt. The enhanced interaction of PRMT6 and mutant AR leads to neurodegeneration in cell and fly models of SBMA. These findings demonstrate a direct role of arginine methylation in polyglutamine disease pathogenesis.
Asunto(s)
Proteínas de Drosophila/genética , Trastornos Musculares Atróficos/enzimología , Péptidos/genética , Proteína-Arginina N-Metiltransferasas/metabolismo , ARN Mensajero/análisis , Receptores Androgénicos/metabolismo , Animales , Células COS , Chlorocebus aethiops , Drosophila , Proteínas de Drosophila/metabolismo , Células HEK293 , Humanos , Ratones , Trastornos Musculares Atróficos/genética , Trastornos Musculares Atróficos/metabolismo , Proteínas Nucleares/metabolismo , Células PC12 , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Androgénicos/genéticaRESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective loss of upper and lower motor neurons and skeletal muscle atrophy. Epidemiologic and experimental evidence suggest the involvement of androgens in ALS pathogenesis, but the mechanism through which androgens modify the ALS phenotype is unknown. Here, we show that androgen ablation by surgical castration extends survival and disease duration of a transgenic mouse model of ALS expressing mutant human SOD1 (hSOD1-G93A). Furthermore, long-term treatment of orchiectomized hSOD1-G93A mice with nandrolone decanoate (ND), an anabolic androgenic steroid, worsened disease manifestations. ND treatment induced muscle fiber hypertrophy but caused motor neuron death. ND negatively affected survival, thereby dissociating skeletal muscle pathology from life span in this ALS mouse model. Interestingly, orchiectomy decreased androgen receptor levels in the spinal cord and muscle, whereas ND treatment had the opposite effect. Notably, stimulation with ND promoted the recruitment of endogenous androgen receptor into biochemical complexes that were insoluble in sodium dodecyl sulfate, a finding consistent with protein aggregation. Overall, our results shed light on the role of androgens as modifiers of ALS pathogenesis via dysregulation of androgen receptor homeostasis.
Asunto(s)
Esclerosis Amiotrófica Lateral/etiología , Esclerosis Amiotrófica Lateral/genética , Andrógenos/fisiología , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/patología , Músculo Esquelético/metabolismo , Receptores Androgénicos/metabolismo , Superóxido Dismutasa , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Anabolizantes/efectos adversos , Animales , Muerte Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Humanos , Hipertrofia , Masculino , Ratones , Ratones Transgénicos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Nandrolona/efectos adversos , Nandrolona/análogos & derivados , Nandrolona Decanoato , Orquiectomía , Médula Espinal/metabolismo , Superóxido Dismutasa-1RESUMEN
Amyotrophic lateral sclerosis (ALS) is a late onset and progressive motor neuron disease. Mutations in the gene coding for fused in sarcoma/translocated in liposarcoma (FUS) are responsible for some cases of both familial and sporadic forms of ALS. The mechanism through which mutations of FUS result in motor neuron degeneration and loss is not known. FUS belongs to the family of TET proteins, which are regulated at the post-translational level by arginine methylation. Here, we investigated the impact of arginine methylation in the pathogenesis of FUS-related ALS. We found that wild type FUS (FUS-WT) specifically interacts with protein arginine methyltransferases 1 and 8 (PRMT1 and PRMT8) and undergoes asymmetric dimethylation in cultured cells. ALS-causing FUS mutants retained the ability to interact with both PRMT1 and PRMT8 and undergo asymmetric dimethylation similar to FUS-WT. Importantly, PRMT1 and PRMT8 localized to mutant FUS-positive inclusion bodies. Pharmacologic inhibition of PRMT1 and PRMT8 activity reduced both the nuclear and cytoplasmic accumulation of FUS-WT and ALS-associated FUS mutants in motor neuron-derived cells and in cells obtained from an ALS patient carrying the R518G mutation. Genetic ablation of the fly homologue of human PRMT1 (DART1) exacerbated the neurodegeneration induced by overexpression of FUS-WT and R521H FUS mutant in a Drosophila model of FUS-related ALS. These results support a role for arginine methylation in the pathogenesis of FUS-related ALS.