RESUMO
Neuromuscular junctions (NMJs) ensure communication between motor neurons (MNs) and muscle; however, in MN disorders, such as amyotrophic lateral sclerosis (ALS), NMJs degenerate resulting in muscle atrophy. The aim of this study was to establish a versatile and reproducible in vitro model of a human motor unit to investigate the effects of ALS-causing mutations. Therefore, we generated a co-culture of human induced pluripotent stem cell (iPSC)-derived MNs and human primary mesoangioblast-derived myotubes in microfluidic devices. A chemotactic and volumetric gradient facilitated the growth of MN neurites through microgrooves resulting in the interaction with myotubes and the formation of NMJs. We observed that ALS-causing FUS mutations resulted in reduced neurite outgrowth as well as an impaired neurite regrowth upon axotomy. NMJ numbers were likewise reduced in the FUS-ALS model. Interestingly, the selective HDAC6 inhibitor, Tubastatin A, improved the neurite outgrowth, regrowth, and NMJ morphology, prompting HDAC6 inhibition as a potential therapeutic strategy for ALS.
Assuntos
Desacetilase 6 de Histona/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Dispositivos Lab-On-A-Chip , Mutação , Junção Neuromuscular/genética , Junção Neuromuscular/fisiopatologia , Proteína FUS de Ligação a RNA/genética , Agrina/metabolismo , Esclerose Lateral Amiotrófica/etiologia , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Biomarcadores , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Técnicas de Cocultura , Imunofluorescência , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Laminina/metabolismo , Técnicas Analíticas Microfluídicas , Neurônios Motores/citologia , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Junção Neuromuscular/efeitos dos fármacos , Crescimento Neuronal/efeitos dos fármacosRESUMO
Energy metabolism has been repeatedly linked to amyotrophic lateral sclerosis (ALS). Yet, motor neuron (MN) metabolism remains poorly studied and it is unknown if ALS MNs differ metabolically from healthy MNs. To address this question, we first performed a metabolic characterization of induced pluripotent stem cells (iPSCs) versus iPSC-derived MNs and subsequently compared MNs from ALS patients carrying FUS mutations to their CRISPR/Cas9-corrected counterparts. We discovered that human iPSCs undergo a lactate oxidation-fuelled prooxidative metabolic switch when they differentiate into functional MNs. Simultaneously, they rewire metabolic routes to import pyruvate into the TCA cycle in an energy substrate specific way. By comparing patient-derived MNs and their isogenic controls, we show that ALS-causing mutations in FUS did not affect glycolytic or mitochondrial energy metabolism of human MNs in vitro. These data show that metabolic dysfunction is not the underlying cause of the ALS-related phenotypes previously observed in these MNs.
Assuntos
Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Diferenciação Celular , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Mutação/genética , Proteína FUS de Ligação a RNA/genética , Estudos de Casos e Controles , Respiração Celular , Glucose/metabolismo , Glicólise , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Ácido Láctico/metabolismo , Análise do Fluxo Metabólico , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Neurônios Motores/ultraestrutura , Proteína FUS de Ligação a RNA/metabolismoRESUMO
Genome damage and defective repair are etiologically linked to neurodegeneration. However, the specific mechanisms involved remain enigmatic. Here, we identify defects in DNA nick ligation and oxidative damage repair in a subset of amyotrophic lateral sclerosis (ALS) patients. These defects are caused by mutations in the RNA/DNA-binding protein FUS. In healthy neurons, FUS protects the genome by facilitating PARP1-dependent recruitment of XRCC1/DNA Ligase IIIα (LigIII) to oxidized genome sites and activating LigIII via direct interaction. We discover that loss of nuclear FUS caused DNA nick ligation defects in motor neurons due to reduced recruitment of XRCC1/LigIII to DNA strand breaks. Moreover, DNA ligation defects in ALS patient-derived iPSC lines carrying FUS mutations and in motor neurons generated therefrom are rescued by CRISPR/Cas9-mediated correction of mutation. Our findings uncovered a pathway of defective DNA ligation in FUS-linked ALS and suggest that LigIII-targeted therapies may prevent or slow down disease progression.
Assuntos
Esclerose Lateral Amiotrófica/genética , Dano ao DNA/genética , Reparo do DNA/genética , DNA/metabolismo , Mutação/genética , Estresse Oxidativo , Proteína FUS de Ligação a RNA/genética , Esclerose Lateral Amiotrófica/patologia , Sistemas CRISPR-Cas/genética , Linhagem Celular , DNA Ligases/metabolismo , Técnicas de Inativação de Genes , Genes Dominantes , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Modelos Biológicos , Poli(ADP-Ribose) Polimerases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/metabolismoRESUMO
Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disorder due to selective loss of motor neurons (MNs). Mutations in the fused in sarcoma (FUS) gene can cause both juvenile and late onset ALS. We generated and characterized induced pluripotent stem cells (iPSCs) from ALS patients with different FUS mutations, as well as from healthy controls. Patient-derived MNs show typical cytoplasmic FUS pathology, hypoexcitability, as well as progressive axonal transport defects. Axonal transport defects are rescued by CRISPR/Cas9-mediated genetic correction of the FUS mutation in patient-derived iPSCs. Moreover, these defects are reproduced by expressing mutant FUS in human embryonic stem cells (hESCs), whereas knockdown of endogenous FUS has no effect, confirming that these pathological changes are mutant FUS dependent. Pharmacological inhibition as well as genetic silencing of histone deacetylase 6 (HDAC6) increase α-tubulin acetylation, endoplasmic reticulum (ER)-mitochondrial overlay, and restore the axonal transport defects in patient-derived MNs.Amyotrophic lateral sclerosis (ALS) leads to selective loss of motor neurons. Using motor neurons derived from induced pluripotent stem cells from patients with ALS and FUS mutations, the authors demonstrate that axonal transport deficits that are observed in these cells can be rescued by HDAC6 inhibition.
Assuntos
Esclerose Lateral Amiotrófica/genética , Transporte Axonal , Desacetilase 6 de Histona/metabolismo , Neurônios Motores/metabolismo , Proteína FUS de Ligação a RNA/genética , Adolescente , Adulto , Idoso , Sistemas CRISPR-Cas , Feminino , Desacetilase 6 de Histona/antagonistas & inibidores , Humanos , Ácidos Hidroxâmicos , Indóis , Células-Tronco Pluripotentes Induzidas , Masculino , Mutação Puntual , Cultura Primária de Células , PirimidinasRESUMO
Skeletal muscle tissue is a rare site of tumor metastasis but is the main target of the degenerative processes occurring in cancer-associated cachexia syndrome. Beneficial effects of physical activity in counteracting cancer-related muscle wasting have been described in the last decades. Recently it has been shown that, in tumor xeno-transplanted mouse models, physical activity is able to directly affect tumor growth by modulating inflammatory responses in the tumor mass microenvironment. Here, we investigated the effect of physical activity on tumor cell growth in colon carcinoma C26 cells injected tibialis anterior muscles of BALB/c mice. Histological analyses revealed that 4 days of voluntary wheel running significantly counteracts tumor cell growth in C26-injected muscles compared to the non-injected sedentary controls. Since striated skeletal muscle tissue is the site of voluntary contraction, our results confirm that physical activity can also directly counteract tumor cell growth in a metabolically active tissue that is usually not a target for metastasis.