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1.
Nature ; 585(7824): 251-255, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32848248

RESUMO

Mutation of C9orf72 is the most prevalent defect associated with amyotrophic lateral sclerosis and frontotemporal degeneration1. Together with hexanucleotide-repeat expansion2,3, haploinsufficiency of C9orf72 contributes to neuronal dysfunction4-6. Here we determine the structure of the C9orf72-SMCR8-WDR41 complex by cryo-electron microscopy. C9orf72 and SMCR8 both contain longin and DENN (differentially expressed in normal and neoplastic cells) domains7, and WDR41 is a ß-propeller protein that binds to SMCR8 such that the whole structure resembles an eye slip hook. Contacts between WDR41 and the DENN domain of SMCR8 drive the lysosomal localization of the complex in conditions of amino acid starvation. The structure suggested that C9orf72-SMCR8 is a GTPase-activating protein (GAP), and we found that C9orf72-SMCR8-WDR41 acts as a GAP for the ARF family of small GTPases. These data shed light on the function of C9orf72 in normal physiology, and in amyotrophic lateral sclerosis and frontotemporal degeneration.


Assuntos
Esclerose Amiotrófica Lateral/genética , Proteínas Relacionadas à Autofagia/química , Proteína C9orf72/química , Proteína C9orf72/genética , Proteínas de Transporte/química , Microscopia Crioeletrônica , Demência Frontotemporal/genética , Haploinsuficiência , Complexos Multiproteicos/química , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Esclerose Amiotrófica Lateral/metabolismo , Proteínas Relacionadas à Autofagia/deficiência , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/ultraestrutura , Proteína C9orf72/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte/ultraestrutura , Demência Frontotemporal/metabolismo , Humanos , Lisossomos/metabolismo , Modelos Moleculares , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , Domínios Proteicos
2.
Nat Commun ; 11(1): 3848, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737286

RESUMO

Amyotrophic Lateral Sclerosis (ALS) is a fatal disease characterized by the degeneration of upper and lower motor neurons (MNs). We find a significant reduction of the retromer complex subunit VPS35 in iPSCs-derived MNs from ALS patients, in MNs from ALS post mortem explants and in MNs from SOD1G93A mice. Being the retromer involved in trafficking of hydrolases, a pathological hallmark in ALS, we design, synthesize and characterize an array of retromer stabilizers based on bis-guanylhydrazones connected by a 1,3-phenyl ring linker. We select compound 2a as a potent and bioavailable interactor of VPS35-VPS29. Indeed, while increasing retromer stability in ALS mice, compound 2a attenuates locomotion impairment and increases MNs survival. Moreover, compound 2a increases VPS35 in iPSCs-derived MNs and shows brain bioavailability. Our results clearly suggest the retromer as a valuable druggable target in ALS.


Assuntos
Esclerose Amiotrófica Lateral/tratamento farmacológico , Hidrazonas/farmacologia , Neurônios Motores/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Proteínas de Transporte Vesicular/genética , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Diferenciação Celular , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Hidrazonas/síntese química , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Locomoção/efeitos dos fármacos , Locomoção/fisiologia , Masculino , Camundongos , Camundongos Transgênicos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Neuroproteção/efeitos dos fármacos , Fármacos Neuroprotetores/síntese química , Ligação Proteica/efeitos dos fármacos , Multimerização Proteica , Relação Estrutura-Atividade , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Proteínas de Transporte Vesicular/metabolismo
3.
Proc Natl Acad Sci U S A ; 117(31): 18591-18599, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32690681

RESUMO

Repeat associated non-AUG (RAN) translation is found in a growing number of microsatellite expansion diseases, but the mechanisms remain unclear. We show that RAN translation is highly regulated by the double-stranded RNA-dependent protein kinase (PKR). In cells, structured CAG, CCUG, CAGG, and G4C2 expansion RNAs activate PKR, which leads to increased levels of multiple RAN proteins. Blocking PKR using PKR-K296R, the TAR RNA binding protein or PKR-KO cells, reduces RAN protein levels. p-PKR is elevated in C9orf72 ALS/FTD human and mouse brains, and inhibiting PKR in C9orf72 BAC transgenic mice using AAV-PKR-K296R or the Food and Drug Administration (FDA)-approved drug metformin, decreases RAN proteins, and improves behavior and pathology. In summary, targeting PKR, including by use of metformin, is a promising therapeutic approach for C9orf72 ALS/FTD and other expansion diseases.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Proteína C9orf72 , Metformina/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , eIF-2 Quinase , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Modelos Animais de Doenças , Demência Frontotemporal/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Repetições de Microssatélites/genética , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo
4.
Nat Commun ; 11(1): 3753, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32719333

RESUMO

Reactive astrocytes have been implicated in the pathogenesis of neurodegenerative diseases, including a non-cell autonomous effect on motor neuron survival in ALS. We previously defined a mechanism by which microglia release three factors, IL-1α, TNFα, and C1q, to induce neurotoxic astrocytes. Here we report that knocking out these three factors markedly extends survival in the SOD1G93A ALS mouse model, providing evidence for gliosis as a potential ALS therapeutic target.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Astrócitos/metabolismo , Complemento C1q/metabolismo , Progressão da Doença , Interleucina-1alfa/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Complemento C3/metabolismo , Modelos Animais de Doenças , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia , Superóxido Dismutase-1/metabolismo
5.
Mol Cell ; 79(3): 443-458.e7, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32649883

RESUMO

Despite the prominent role of TDP-43 in neurodegeneration, its physiological and pathological functions are not fully understood. Here, we report an unexpected role of TDP-43 in the formation of dynamic, reversible, liquid droplet-like nuclear bodies (NBs) in response to stress. Formation of NBs alleviates TDP-43-mediated cytotoxicity in mammalian cells and fly neurons. Super-resolution microscopy reveals distinct functions of the two RRMs in TDP-43 NB formation. TDP-43 NBs are partially colocalized with nuclear paraspeckles, whose scaffolding lncRNA NEAT1 is dramatically upregulated in stressed neurons. Moreover, increase of NEAT1 promotes TDP-43 liquid-liquid phase separation (LLPS) in vitro. Finally, we discover that the ALS-associated mutation D169G impairs the NEAT1-mediated TDP-43 LLPS and NB assembly, causing excessive cytoplasmic translocation of TDP-43 to form stress granules, which become phosphorylated TDP-43 cytoplasmic foci upon prolonged stress. Together, our findings suggest a stress-mitigating role and mechanism of TDP-43 NBs, whose dysfunction may be involved in ALS pathogenesis.


Assuntos
Esclerose Amiotrófica Lateral/genética , Proteínas de Ligação a DNA/genética , Corpos de Inclusão Intranuclear/metabolismo , Neurônios/metabolismo , RNA Longo não Codificante/genética , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Animais Geneticamente Modificados , Arsenitos/farmacologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Córtex Cerebral/ultraestrutura , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/ultraestrutura , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Drosophila melanogaster , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Corpos de Inclusão Intranuclear/efeitos dos fármacos , Corpos de Inclusão Intranuclear/ultraestrutura , Camundongos , Mutação , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Cultura Primária de Células , Transporte Proteico/efeitos dos fármacos , RNA Longo não Codificante/metabolismo , Transdução de Sinais , Estresse Fisiológico
6.
Proc Natl Acad Sci U S A ; 117(26): 15230-15241, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32513711

RESUMO

Mutations in UBQLN2 cause amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and other neurodegenerations. However, the mechanism by which the UBQLN2 mutations cause disease remains unclear. Alterations in proteins involved in autophagy are prominent in neuronal tissue of human ALS UBQLN2 patients and in a transgenic P497S UBQLN2 mouse model of ALS/FTD, suggesting a pathogenic link. Here, we show UBQLN2 functions in autophagy and that ALS/FTD mutant proteins compromise this function. Inactivation of UBQLN2 expression in HeLa cells reduced autophagic flux and autophagosome acidification. The defect in acidification was rescued by reexpression of wild type (WT) UBQLN2 but not by any of the five different UBQLN2 ALS/FTD mutants tested. Proteomic analysis and immunoblot studies revealed P497S mutant mice and UBQLN2 knockout HeLa and NSC34 cells have reduced expression of ATP6v1g1, a critical subunit of the vacuolar ATPase (V-ATPase) pump. Knockout of UBQLN2 expression in HeLa cells decreased turnover of ATP6v1g1, while overexpression of WT UBQLN2 increased biogenesis of ATP6v1g1 compared with P497S mutant UBQLN2 protein. In vitro interaction studies showed that ATP6v1g1 binds more strongly to WT UBQLN2 than to ALS/FTD mutant UBQLN2 proteins. Intriguingly, overexpression of ATP6v1g1 in UBQLN2 knockout HeLa cells increased autophagosome acidification, suggesting a therapeutic approach to overcome the acidification defect. Taken together, our findings suggest that UBQLN2 mutations drive pathogenesis through a dominant-negative loss-of-function mechanism in autophagy and that UBQLN2 functions as an important regulator of the expression and stability of ATP6v1g1. These findings may have important implications for devising therapies to treat UBQLN2-linked ALS/FTD.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Esclerose Amiotrófica Lateral/genética , Autofagossomos/fisiologia , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia/genética , Demência/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Proteínas Relacionadas à Autofagia/genética , Biomarcadores/metabolismo , Linhagem Celular , Demência/metabolismo , Demência/patologia , Predisposição Genética para Doença , Humanos , Concentração de Íons de Hidrogênio , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Ligação Proteica , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Regulação para Cima , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo
7.
Neuron ; 106(6): 899-911, 2020 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-32553207

RESUMO

In recent years, the nuclear pore complex (NPC) has emerged as a key player in genome regulation and cellular homeostasis. New discoveries have revealed that the NPC has multiple cellular functions besides mediating the molecular exchange between the nucleus and the cytoplasm. In this review, we discuss non-transport aspects of the NPC focusing on the NPC-genome interaction, the extreme longevity of the NPC proteins, and NPC dysfunction in age-related diseases. The examples summarized herein demonstrate that the NPC, which first evolved to enable the biochemical communication between the nucleus and the cytoplasm, now doubles as the gatekeeper of cellular identity and aging.


Assuntos
Envelhecimento/metabolismo , Esclerose Amiotrófica Lateral/metabolismo , Diferenciação Celular , Regulação da Expressão Gênica , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , Transporte Ativo do Núcleo Celular , Envelhecimento/genética , Senilidade Prematura/genética , Senilidade Prematura/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Esclerose Amiotrófica Lateral/genética , Animais , Proteína de Ligação a CREB/metabolismo , Senescência Celular , Genoma , Humanos , Doença de Huntington/genética , Doença de Huntington/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Poro Nuclear/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética
9.
Adv Exp Med Biol ; 1241: 195-217, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32383122

RESUMO

Amyotrophic lateral sclerosis (ALS) is one of the most common neurodegenerative diseases, characterized by inevitable progressive paralysis. To date, only two disease modifying therapeutic options are available for the patients with ALS, although they show very modest effect on disease course. The main reason of failure in the field of pharmacological correction of ALS is inability to untangle complex relationships taking place during ALS initiation and progression. Traditional methods of research, based on morphology or transgenic animal models studying provided lots of information about ALS throughout the years. However, translation of these results to humans was unsuccessful due to incomplete recapitulation of molecular pathology and overall inadequacy of the models used in the research.In this review we summarize current knowledge regarding ALS molecular pathology with depiction of novel methods applied recently for the studies. Furthermore we describe present and potential treatment strategies that are based on the recent findings in ALS disease mechanisms.


Assuntos
Esclerose Amiotrófica Lateral/tratamento farmacológico , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Pesquisa Biomédica , Progressão da Doença , Humanos
10.
Neuron ; 107(1): 95-111.e6, 2020 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-32380032

RESUMO

Progressive synapse loss is an inevitable and insidious part of age-related neurodegenerative disease. Typically, synapse loss precedes symptoms of cognitive and motor decline. This suggests the existence of compensatory mechanisms that can temporarily counteract the effects of ongoing neurodegeneration. Here, we demonstrate that presynaptic homeostatic plasticity (PHP) is induced at degenerating neuromuscular junctions, mediated by an evolutionarily conserved activity of presynaptic ENaC channels in both Drosophila and mouse. To assess the consequence of eliminating PHP in a mouse model of ALS-like degeneration, we generated a motoneuron-specific deletion of Scnn1a, encoding the ENaC channel alpha subunit. We show that Scnn1a is essential for PHP without adversely affecting baseline neural function or lifespan. However, Scnn1a knockout in a degeneration-causing mutant background accelerated motoneuron loss and disease progression to twice the rate observed in littermate controls with intact PHP. We propose a model of neuroprotective homeostatic plasticity, extending organismal lifespan and health span.


Assuntos
Canais Epiteliais de Sódio/metabolismo , Homeostase/fisiologia , Plasticidade Neuronal/fisiologia , Neuroproteção/fisiologia , Terminações Pré-Sinápticas/metabolismo , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Modelos Animais de Doenças , Progressão da Doença , Drosophila melanogaster , Camundongos , Camundongos Knockout , Junção Neuromuscular/metabolismo
11.
Muscle Nerve ; 62(2): 272-283, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32369618

RESUMO

BACKGROUND: The exact mechanisms underlying neuroinflammation and how they contribute to amyotrophic lateral sclerosis (ALS) pathogenesis remain unclear. One possibility is the secretion of neurotoxic factors, such as lipocalin-2 (LCN2), that lead to neuronal death. METHODS: LCN2 levels were measured in human postmortem tissue using Western blot, quantitative real time polymerase chain reaction, and immunofluorescence, and in plasma by enzyme-linked immunosorbent assay. SH-SY5Y cells were used to test the pro-inflammatory effects of LCN2. RESULTS: LCN2 is increased in ALS postmortem motor cortex, spinal cord, and plasma. Furthermore, we identified several LCN2 variants in ALS patients that may contribute to disease pathogenesis. Lastly, while LCN2 treatment caused cell death and increased pro-inflammatory markers, treatment with an anti-LCN2 antibody prevented these responses in vitro. CONCLUSIONS: LCN2 upregulation in ALS postmortem samples and plasma may be an upstream event for triggering neuroinflammation and neuronal death.


Assuntos
Esclerose Amiotrófica Lateral/genética , Inflamação/metabolismo , Lipocalina-2/genética , Córtex Motor/metabolismo , Medula Espinal/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/fisiopatologia , Western Blotting , Estudos de Casos e Controles , Morte Celular , Linhagem Celular Tumoral , Citocinas/efeitos dos fármacos , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Imunofluorescência , Humanos , Técnicas In Vitro , Lipocalina-2/antagonistas & inibidores , Lipocalina-2/metabolismo , Lipocalina-2/farmacologia , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Reação em Cadeia da Polimerase em Tempo Real
12.
Adv Exp Med Biol ; 1195: 179-187, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32468476

RESUMO

Amyotrophic lateral sclerosis (ALS) is a rare, neurodegenerative disease that affects the human motor system. ALS is a highly heterogeneous disease, depending on several causative factors. The heterogeneity of the disease is also reflected in the variation of the symptoms in ALS patients. The worldwide annual incidence of ALS is about 2.08 per 100,000 with uniform rates in Caucasian populations and lower rates in African, Asian, and Hispanic populations, while the number of individuals with ALS is expected to grow significantly between 2015 and 2040 with an estimated increase of 69% (Chio et al. 2013a; Arthur et al. 2016).


Assuntos
Esclerose Amiotrófica Lateral/diagnóstico , Esclerose Amiotrófica Lateral/metabolismo , Biomarcadores/análise , Esclerose Amiotrófica Lateral/epidemiologia , Biomarcadores/metabolismo , Grupos de Populações Continentais/estatística & dados numéricos , Humanos , Incidência
13.
Adv Exp Med Biol ; 1195: 205-212, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32468479

RESUMO

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease involving progressive and selective loss of motor neurons, muscle weakness, paralysis and death. The pathogenesis of ALS is not clearly understood, while reliable prognostic markers have not been identified to detect symptoms at earlier time points. The rapid development of microarray technology offers great potential for simultaneous analysis of the transcriptional expression of thousands of genes, aiming to determine novel candidate targets for efficient treatment. Additionally, metabolomics, as a high-throughput approach, is gaining significant attention in ALS research providing an opportunity to develop predictive biomarkers that may be utilized as indicators of clinical symptoms of ALS. In this review, recent evidences from gene expression profiling studies in ALS are illustrated in order to examine molecular signatures related to the disease's pathogenesis and potential discovery of therapeutic targets. Moreover, potent challenges are presented regarding the utilization of the metabolomics approach as a diagnostic tool in context with distinctive biomarkers' identification.


Assuntos
Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/metabolismo , Metabolômica , Transcriptoma , Perfilação da Expressão Gênica , Humanos
14.
PLoS Comput Biol ; 16(4): e1007773, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32294079

RESUMO

Evolutionarily conserved mechanisms maintain homeostasis of essential elements, and are believed to be highly time-variant. However, current approaches measure elemental biomarkers at a few discrete time-points, ignoring complex higher-order dynamical features. To study dynamical properties of elemental homeostasis, we apply laser ablation inductively-coupled plasma mass spectrometry (LA-ICP-MS) to tooth samples to generate 500 temporally sequential measurements of elemental concentrations from birth to 10 years. We applied dynamical system and Information Theory-based analyses to reveal the longest-known attractor system in mammalian biology underlying the metabolism of nutrient elements, and identify distinct and consistent transitions between stable and unstable states throughout development. Extending these dynamical features to disease prediction, we find that attractor topography of nutrient metabolism is altered in amyotrophic lateral sclerosis (ALS), as early as childhood, suggesting these pathways are involved in disease risk. Mechanistic analysis was undertaken in a transgenic mouse model of ALS, where we find similar marked disruptions in elemental attractor systems as in humans. Our results demonstrate the application of a phenomological analysis of dynamical systems underlying elemental metabolism, and emphasize the utility of these measures in characterizing risk of disease.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Cobre/análise , Dente/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Biomarcadores/metabolismo , Criança , Pré-Escolar , Biologia Computacional , Cobre/sangue , Cobre/urina , Feminino , Homeostase , Humanos , Lactente , Masculino , Espectrometria de Massas , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Curva ROC , Risco , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo
15.
Nat Commun ; 11(1): 1773, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286313

RESUMO

In amyotrophic lateral sclerosis (ALS), immune cells and glia contribute to motor neuron (MN) degeneration. We report the presence of NK cells in post-mortem ALS motor cortex and spinal cord tissues, and the expression of NKG2D ligands on MNs. Using a mouse model of familial-ALS, hSOD1G93A, we demonstrate NK cell accumulation in the motor cortex and spinal cord, with an early CCL2-dependent peak. NK cell depletion reduces the pace of MN degeneration, delays motor impairment and increases survival. This is confirmed in another ALS mouse model, TDP43A315T. NK cells are neurotoxic to hSOD1G93A MNs which express NKG2D ligands, while IFNγ produced by NK cells instructs microglia toward an inflammatory phenotype, and impairs FOXP3+/Treg cell infiltration in the spinal cord of hSOD1G93A mice. Together, these data suggest a role of NK cells in determining the onset and progression of MN degeneration in ALS, and in modulating Treg recruitment and microglia phenotype.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Células Matadoras Naturais/metabolismo , Neurônios Motores/metabolismo , Adulto , Idoso , Esclerose Amiotrófica Lateral/imunologia , Esclerose Amiotrófica Lateral/patologia , Animais , Modelos Animais de Doenças , Progressão da Doença , Feminino , Humanos , Células Matadoras Naturais/imunologia , Masculino , Camundongos , Pessoa de Meia-Idade , Córtex Motor/imunologia , Córtex Motor/metabolismo , Córtex Motor/patologia , Neurônios Motores/imunologia , Neurônios Motores/patologia , Receptor 1 Desencadeador da Citotoxicidade Natural/genética , Receptor 1 Desencadeador da Citotoxicidade Natural/metabolismo , Medula Espinal/imunologia , Medula Espinal/metabolismo , Medula Espinal/patologia
16.
Nat Neurosci ; 23(5): 615-624, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32284607

RESUMO

Hexanucleotide expansions in C9orf72, which encodes a predicted guanine exchange factor, are the most frequent genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Although repeat expansion has been established to generate toxic products, mRNAs encoding the C9ORF72 protein are also reduced in affected individuals. In this study, we tested how C9ORF72 protein levels affected repeat-mediated toxicity. In somatic transgenic mice expressing 66 GGGGCC repeats, inactivation of one or both endogenous C9orf72 alleles provoked or accelerated, respectively, early death. In mice expressing a C9orf72 transgene with 450 repeats that did not encode the C9ORF72 protein, inactivation of one or both endogenous C9orf72 alleles exacerbated cognitive deficits, hippocampal neuron loss, glial activation and accumulation of dipeptide-repeat proteins from translation of repeat-containing RNAs. Reduced C9ORF72 was shown to suppress repeat-mediated elevation in autophagy. These efforts support a disease mechanism in ALS/FTD resulting from reduced C9ORF72, which can lead to autophagy deficits, synergizing with repeat-dependent gain of toxicity.


Assuntos
Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Demência Frontotemporal/patologia , Animais , Expansão das Repetições de DNA/genética , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
17.
Hum Genet ; 139(10): 1233-1246, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32277284

RESUMO

Approximately 3% of the human genome is composed of short tandem repeat (STR) DNA sequence known as microsatellites, which can be found in both coding and non-coding regions. When associated with genic regions, expansion of microsatellite repeats beyond a critical threshold causes dozens of neurological repeat expansion disorders. To better understand the molecular pathology of repeat expansion disorders, precise cloning of microsatellite repeat sequence and expansion size is highly valuable. Unfortunately, cloning repeat expansions is often challenging and presents a significant bottleneck to practical investigation. Here, we describe a clear method for seamless and systematic cloning of practically any microsatellite repeat expansion. We use cloning and expansion of GGGGCC repeats, which are the leading genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), as an example. We employ a recursive directional ligation (RDL) technique to build multiple GGGGCC repeat-containing vectors. We describe methods to validate repeat expansion cloning, including diagnostic restriction digestion, PCR across the repeat, and next-generation long-read MinION nanopore sequencing. Validated cloning of microsatellite repeats beyond the critical expansion threshold can facilitate step-by-step characterization of disease mechanisms at the cellular and molecular level.


Assuntos
Esclerose Amiotrófica Lateral/genética , Proteína C9orf72/genética , Clonagem Molecular/métodos , Expansão das Repetições de DNA , Demência Frontotemporal/genética , Repetições de Microssatélites , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Sequência de Bases , Proteína C9orf72/metabolismo , Enzimas de Restrição do DNA/química , Escherichia coli/genética , Escherichia coli/metabolismo , Demência Frontotemporal/metabolismo , Demência Frontotemporal/patologia , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Genoma Humano , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Reação em Cadeia da Polimerase/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
18.
Proc Natl Acad Sci U S A ; 117(11): 5883-5894, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32132204

RESUMO

Liquid-liquid phase separation (LLPS) is involved in the formation of membraneless organelles (MLOs) associated with RNA processing. The RNA-binding protein TDP-43 is present in several MLOs, undergoes LLPS, and has been linked to the pathogenesis of amyotrophic lateral sclerosis (ALS). While some ALS-associated mutations in TDP-43 disrupt self-interaction and function, here we show that designed single mutations can enhance TDP-43 assembly and function via modulating helical structure. Using molecular simulation and NMR spectroscopy, we observe large structural changes upon dimerization of TDP-43. Two conserved glycine residues (G335 and G338) are potent inhibitors of helical extension and helix-helix interaction, which are removed in part by variants at these positions, including the ALS-associated G335D. Substitution to helix-enhancing alanine at either of these positions dramatically enhances phase separation in vitro and decreases fluidity of phase-separated TDP-43 reporter compartments in cells. Furthermore, G335A increases TDP-43 splicing function in a minigene assay. Therefore, the TDP-43 helical region serves as a short but uniquely tunable module where application of biophysical principles can precisely control assembly and function in cellular and synthetic biology applications of LLPS.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Conformação Proteica em alfa-Hélice , Esclerose Amiotrófica Lateral/genética , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Humanos , Espectroscopia de Ressonância Magnética , Simulação de Acoplamento Molecular , Mutação , Conformação Proteica , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína , Proteínas de Ligação a RNA/metabolismo
19.
Brain ; 143(3): 783-799, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-32185393

RESUMO

Frontotemporal dementia and amyotrophic lateral sclerosis are clinically and pathologically overlapping disorders with shared genetic causes. We previously identified a disease locus on chromosome 16p12.1-q12.2 with genome-wide significant linkage in a large European Australian family with autosomal dominant inheritance of frontotemporal dementia and amyotrophic lateral sclerosis and no mutation in known amyotrophic lateral sclerosis or dementia genes. Here we demonstrate the segregation of a novel missense variant in CYLD (c.2155A>G, p.M719V) within the linkage region as the genetic cause of disease in this family. Immunohistochemical analysis of brain tissue from two CYLD p.M719V mutation carriers showed widespread glial CYLD immunoreactivity. Primary mouse neurons transfected with CYLDM719V exhibited increased cytoplasmic localization of TDP-43 and shortened axons. CYLD encodes a lysine 63 deubiquitinase and CYLD cutaneous syndrome, a skin tumour disorder, is caused by mutations that lead to reduced deubiquitinase activity. In contrast with CYLD cutaneous syndrome-causative mutations, CYLDM719V exhibited significantly increased lysine 63 deubiquitinase activity relative to the wild-type enzyme (paired Wilcoxon signed-rank test P = 0.005). Overexpression of CYLDM719V in HEK293 cells led to more potent inhibition of the cell signalling molecule NF-κB and impairment of autophagosome fusion to lysosomes, a key process in autophagy. Although CYLD mutations appear to be rare, CYLD's interaction with at least three other proteins encoded by frontotemporal dementia and/or amyotrophic lateral sclerosis genes (TBK1, OPTN and SQSTM1) suggests that it may play a central role in the pathogenesis of these disorders. Mutations in several frontotemporal dementia and amyotrophic lateral sclerosis genes, including TBK1, OPTN and SQSTM1, result in a loss of autophagy function. We show here that increased CYLD activity also reduces autophagy function, highlighting the importance of autophagy regulation in the pathogenesis of frontotemporal dementia and amyotrophic lateral sclerosis.


Assuntos
Esclerose Amiotrófica Lateral/genética , Enzima Desubiquitinante CYLD/genética , Enzima Desubiquitinante CYLD/fisiologia , Demência Frontotemporal/genética , Predisposição Genética para Doença/genética , Esclerose Amiotrófica Lateral/metabolismo , Animais , Autofagossomos/metabolismo , Autofagossomos/fisiologia , Axônios/patologia , Encéfalo/metabolismo , Proteínas de Ligação a DNA , Enzima Desubiquitinante CYLD/metabolismo , Enzimas Desubiquitinantes/metabolismo , Demência Frontotemporal/metabolismo , Camundongos , Mutação de Sentido Incorreto/genética , NF-kappa B/antagonistas & inibidores , Cultura Primária de Células , Transfecção
20.
Nat Rev Neurol ; 16(4): 213-228, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32203398

RESUMO

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.


Assuntos
Anticorpos/uso terapêutico , Afasia Primária Progressiva/tratamento farmacológico , Desenvolvimento de Medicamentos , Demência Frontotemporal/tratamento farmacológico , Moduladores de Tubulina/uso terapêutico , Esclerose Amiotrófica Lateral/tratamento farmacológico , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/metabolismo , Afasia Primária Progressiva/genética , Afasia Primária Progressiva/metabolismo , Proteína C9orf72/genética , Proteínas de Ligação a DNA/metabolismo , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Humanos , Imunização Passiva , Imunoterapia Ativa , Terapia de Alvo Molecular , Progranulinas/genética , Proteína EWS de Ligação a RNA/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Paralisia Supranuclear Progressiva/tratamento farmacológico , Paralisia Supranuclear Progressiva/genética , Paralisia Supranuclear Progressiva/metabolismo , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismo
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