RESUMO
Amyotrophic lateral sclerosis (ALS) is a fatal, severely debilitating and rapidly progressing disorder affecting motor neurons in the brain, brainstem, and spinal cord. Unfortunately, there are few effective treatments, thus there remains a critical need to find novel interventions that can mitigate against its effects. Whilst the aetiology of ALS remains unclear, ageing is the major risk factor. Ageing is a slowly progressive process marked by functional decline of an organism over its lifespan. However, it remains unclear how ageing promotes the risk of ALS. At the molecular and cellular level there are specific hallmarks characteristic of normal ageing. These hallmarks are highly inter-related and overlap significantly with each other. Moreover, whilst ageing is a normal process, there are striking similarities at the molecular level between these factors and neurodegeneration in ALS. Nine ageing hallmarks were originally proposed: genomic instability, loss of telomeres, senescence, epigenetic modifications, dysregulated nutrient sensing, loss of proteostasis, mitochondrial dysfunction, stem cell exhaustion, and altered inter-cellular communication. However, these were recently (2023) expanded to include dysregulation of autophagy, inflammation and dysbiosis. Hence, given the latest updates to these hallmarks, and their close association to disease processes in ALS, a new examination of their relationship to pathophysiology is warranted. In this review, we describe possible mechanisms by which normal ageing impacts on neurodegenerative mechanisms implicated in ALS, and new therapeutic interventions that may arise from this.
Assuntos
Esclerose Lateral Amiotrófica , Humanos , Esclerose Lateral Amiotrófica/genética , Longevidade , Autofagia/genética , EncéfaloRESUMO
The endoplasmic reticulum (ER) and mitochondria connect at multiple contact sites to form a unique cellular compartment, termed the 'mitochondria-associated ER membranes' (MAMs). MAMs are hubs for signalling pathways that regulate cellular homeostasis and survival, metabolism, and sensitivity to apoptosis. MAMs are therefore involved in vital cellular functions, but they are dysregulated in several human diseases. Whilst MAM dysfunction is increasingly implicated in the pathogenesis of neurodegenerative diseases, its role in amyotrophic lateral sclerosis (ALS) is poorly understood. However, in ALS both ER and mitochondrial dysfunction are well documented pathophysiological events. Moreover, alterations to lipid metabolism in neurons regulate processes linked to neurodegenerative diseases, and a link between dysfunction of lipid metabolism and ALS has also been proposed. In this review we discuss the structural and functional relevance of MAMs in ALS and how targeting MAM could be therapeutically beneficial in this disorder.
Assuntos
Esclerose Lateral Amiotrófica/genética , Mitocôndrias/genética , Membranas Mitocondriais/metabolismo , Distrofias Musculares/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Apoptose/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Humanos , Mitocôndrias/metabolismo , Membranas Mitocondriais/patologia , Distrofias Musculares/metabolismo , Distrofias Musculares/patologiaRESUMO
BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterised by the loss of upper and lower motor neurons in the brain and spinal cord. ALS and frontotemporal dementia (FTD) are overlapping diseases with shared pathological features. Affected neurons of people with ALS and FTD typically contain ubiquitin-immunoreactive inclusions, of which TDP-43 (Tar DNA-binding protein of 43 kDa) is a major component. However, what triggers the formation of these abnormal TDP-43 inclusions is unclear. Previously, we identified CCNF mutations in cohorts of familial and sporadic cases of ALS and FTD. CCNF encodes cyclin F, the substrate-binding component of a multiprotein E3 ubiquitin ligase complex that ubiquitylates and subsequently directs a set of protein substrates for proteasomal degradation. Here, we explored the relationship between cyclin F and TDP-43. METHODS: We used a series of complementary biochemical approaches including immunoprecipitations, in vitro ubiquitylation assays, immunofluorescence imaging and immunocytochemistry. Unpaired student t-tests were used to determine statistical significance of the results. RESULTS: In this study, we demonstrate that that the SCFcyclin F complex directly mediates the poly-ubiquitylation of TDP-43. Importantly, we demonstrate that cyclin F bearing the pathogenic ALS/FTD mutation, S621G, leads to aberrant ubiquitylation of TDP-43 as well as the accumulation of K48-ubiquitylated TDP-43 in neuron-like cells. Furthermore, we demonstrate that a patient carrying the ALS/FTD cyclin FS195R mutation displayed skein-like cytoplasmic TDP-43 aggregates, implying abnormal TDP-43 degradation in a CCNF mutation bearing patient. CONCLUSION: In summary, this study reports a direct ubiquitylation mechanism for TDP-43, revealing important insights into the regulation of cyclin F-mediated TDP-43 turnover and clues towards understanding the molecular origins of the ubiquitylated TDP-43 inclusions that are the hallmark pathological feature in ALS and FTD.
Assuntos
Esclerose Lateral Amiotrófica , Demência Frontotemporal , Doenças Neurodegenerativas , Esclerose Lateral Amiotrófica/metabolismo , Ciclinas/genética , Ciclinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Demência Frontotemporal/genética , Demência Frontotemporal/patologia , Humanos , Neurônios Motores/patologia , Doenças Neurodegenerativas/patologia , UbiquitinaçãoRESUMO
Human cells are exposed to numerous exogenous and endogenous insults every day. Unlike other molecules, DNA cannot be replaced by resynthesis, hence damage to DNA can have major consequences for the cell. The DNA damage response contains overlapping signalling networks that repair DNA and hence maintain genomic integrity, and aberrant DNA damage responses are increasingly described in neurodegenerative diseases. Furthermore, DNA repair declines during aging, which is the biggest risk factor for these conditions. If unrepaired, the accumulation of DNA damage results in death to eliminate cells with defective genomes. This is particularly important for postmitotic neurons because they have a limited capacity to proliferate, thus they must be maintained for life. Neuronal death is thus an important process in neurodegenerative disorders. In addition, the inability of neurons to divide renders them susceptible to senescence or re-entry to the cell cycle. The field of cell death has expanded significantly in recent years, and many new mechanisms have been described in various cell types, including neurons. Several of these mechanisms are linked to DNA damage. In this review, we provide an overview of the cell death pathways induced by DNA damage that are relevant to neurons and discuss the possible involvement of these mechanisms in neurodegenerative conditions.
Assuntos
Doenças Neurodegenerativas , DNA/metabolismo , Dano ao DNA , Reparo do DNA , Humanos , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismoRESUMO
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease commonly treated with riluzole, a small molecule that may act via modulation of glutamatergic neurotransmission. However, riluzole only modestly extends lifespan for people living with ALS, and its precise mechanisms of action remain unclear. Most ALS cases are characterised by accumulation of cytoplasmic TAR DNA binding protein of 43 kDa (TDP-43), and understanding the effects of riluzole in models that closely recapitulate TDP-43 pathology may provide insights for development of improved therapeutics. We therefore investigated the effects of riluzole in female transgenic mice that inducibly express nuclear localisation sequence (NLS)-deficient human TDP-43 in neurons (NEFH-tTA/tetO-hTDP-43ΔNLS, 'rNLS8', mice). Riluzole treatment from the first day of hTDP-43ΔNLS expression did not alter disease onset, weight loss or performance on multiple motor behavioural tasks. Riluzole treatment also did not alter TDP-43 protein levels, solubility or phosphorylation. Although we identified a significant decrease in GluA2 and GluA3 proteins in the cortex of rNLS8 mice, riluzole did not ameliorate this disease-associated molecular phenotype. Likewise, riluzole did not alter the disease-associated atrophy of hindlimb muscle in rNLS8 mice. Finally, riluzole treatment beginning after disease onset in rNLS8 mice similarly had no effect on progression of late-stage disease or animal survival. Together, we demonstrate specific glutamatergic receptor alterations and muscle fibre-type changes reminiscent of ALS in female rNLS8 mice, but riluzole had no effect on these or any other disease phenotypes. Future targeting of pathways related to accumulation of TDP-43 pathology may be needed to develop better treatments for ALS.
Assuntos
Esclerose Lateral Amiotrófica , Doenças Neurodegenerativas , Esclerose Lateral Amiotrófica/tratamento farmacológico , Animais , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Transgênicos , Riluzol/farmacologia , Riluzol/uso terapêuticoRESUMO
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 Lateral Amiotrófica/genética , Enzima Desubiquitinante CYLD/genética , Enzima Desubiquitinante CYLD/fisiologia , Demência Frontotemporal/genética , Predisposição Genética para Doença/genética , Esclerose Lateral Amiotrófica/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 , TransfecçãoRESUMO
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative diseases that are related genetically and pathologically. Mutations in the UBQLN2 gene, encoding the ubiquitin-like protein ubiquilin2, are associated with familial ALS/FTD, but the pathophysiological mechanisms remain unclear. Here, we demonstrate that ALS/FTD UBQLN2 mutants P497H and P506T inhibit protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus in neuronal cells. In addition, we observed that Sec31-positive ER exit sites are clustered in UBQLN2T487I patient spinal cord tissues. Both the ER-Golgi intermediate (ERGIC) compartment and the Golgi become disorganised and fragmented. This activates ER stress and inhibits ER-associated degradation. Hence, this study highlights perturbations in secretory protein trafficking and ER homeostasis as pathogenic mechanisms associated with ALS/FTD-associated forms of UBQLN2.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Proteínas Relacionadas à Autofagia/genética , Células Cultivadas , Estresse do Retículo Endoplasmático , Demência Frontotemporal/metabolismo , Demência Frontotemporal/patologia , Humanos , Camundongos , Mutagênese Sítio-Dirigida , Neurônios/citologia , Neurônios/metabolismo , Transporte ProteicoRESUMO
Microglial NLRP3 inflammasome activation is emerging as a key contributor to neuroinflammation during neurodegeneration. Pathogenic protein aggregates such as ß-amyloid and α-synuclein trigger microglial NLRP3 activation, leading to caspase-1 activation and IL-1ß secretion. Both caspase-1 and IL-1ß contribute to disease progression in the mouse SOD1G93A model of amyotrophic lateral sclerosis (ALS), suggesting a role for microglial NLRP3. Prior studies, however, suggested SOD1G93A mice microglia do not express NLRP3, and SOD1G93A protein generated IL-1ß in microglia independent to NLRP3. Here, we demonstrate using Nlrp3-GFP gene knock-in mice that microglia express NLRP3 in SOD1G93A mice. We show that both aggregated and soluble SOD1G93A activates inflammasome in primary mouse microglia leading caspase-1 and IL-1ß cleavage, ASC speck formation, and the secretion of IL-1ß in a dose- and time-dependent manner. Importantly, SOD1G93A was unable to induce IL-1ß secretion from microglia deficient for Nlrp3, or pretreated with the specific NLRP3 inhibitor MCC950, confirming NLRP3 as the key inflammasome complex mediating SOD1-induced microglial IL-1ß secretion. Microglial NLRP3 upregulation was also observed in the TDP-43Q331K ALS mouse model, and TDP-43 wild-type and mutant proteins could also activate microglial inflammasomes in a NLRP3-dependent manner. Mechanistically, we identified the generation of reactive oxygen species and ATP as key events required for SOD1G93A -mediated NLRP3 activation. Taken together, our data demonstrate that ALS microglia express NLRP3, and that pathological ALS proteins activate the microglial NLRP3 inflammasome. NLRP3 inhibition may therefore be a potential therapeutic approach to arrest microglial neuroinflammation and ALS disease progression.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Inflamassomos/metabolismo , Microglia/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Modelos Animais de Doenças , Progressão da Doença , Humanos , Camundongos Transgênicos , Superóxido Dismutase-1/genéticaRESUMO
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder and mutations in superoxide dismutase 1 (SOD1) account for 20% of familial ALS cases. The aetiology of ALS remains unclear, but protein misfolding, endoplasmic reticulum (ER) stress and neuronal apoptosis are implicated. We previously established that protein disulphide isomerase (PDIA1) is protective against ER stress and apoptosis in neuronal cells expressing mutant SOD1, and recently mutations in PDIA1 and related PDI family member endoplasmic reticulum protein 57 (ERp57/PDIA3), were associated with ALS. Here, we examined whether ERp57 is also protective against mutant SOD1 or whether distinct specificity exists amongst individual PDI family members. Neuronal cells co-expressing SOD1 and ERp57 were examined for inclusion formation, ER stress, ubiquitin proteasome system (UPS) dysfunction and apoptosis. Over-expression of ERp57 inhibited inclusion formation, ER stress, UPS dysfunction and apoptosis, whereas silencing of ERp57 expression enhanced mutant SOD1 inclusion formation, ER stress and toxicity, indicating a protective role for ERp57 against SOD1 misfolding. ERp57 also inhibited the formation of mutant SOD1 inclusions and apoptosis in primary cortical neurons, thus confirming results obtained from cell lines. ERp57 partially co-localized with TAR DNA-binding protein-43 (TDP-43)-positive inclusions in spinal cords from sporadic ALS patients, thus linking ERp57 to protein misfolding in human sporadic disease. Our results therefore imply that ERp57 has a protective role against pathological events induced by mutant SOD1 and they link ERp57 to the misfolding of TDP-43. This study therefore has implications for the design of novel therapeutics based on the activities of the PDI family of proteins.
Assuntos
Esclerose Lateral Amiotrófica/genética , Proteínas de Ligação a DNA/genética , Estresse do Retículo Endoplasmático/genética , Neurônios/metabolismo , Isomerases de Dissulfetos de Proteínas/genética , Superóxido Dismutase-1/genética , Idoso , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Apoptose , Linhagem Celular , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Mutação , Neurônios/patologia , Cultura Primária de Células , Isomerases de Dissulfetos de Proteínas/antagonistas & inibidores , Isomerases de Dissulfetos de Proteínas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Medula Espinal/metabolismo , Medula Espinal/patologia , Superóxido Dismutase-1/metabolismoRESUMO
OBJECTIVE: Since the first report of CHCHD10 gene mutations in amyotrophiclateral sclerosis (ALS)/frontotemporaldementia (FTD) patients, genetic variation in CHCHD10 has been inconsistently linked to disease. A pathological assessment of the CHCHD10 protein in patient neuronal tissue also remains to be reported. We sought to characterise the genetic and pathological contribution of CHCHD10 to ALS/FTD in Australia. METHODS: Whole-exome and whole-genome sequencing data from 81 familial and 635 sporadic ALS, and 108 sporadic FTD cases, were assessed for genetic variation in CHCHD10. CHCHD10 protein expression was characterised by immunohistochemistry, immunofluorescence and western blotting in control, ALS and/or FTD postmortem tissues and further in a transgenic mouse model of TAR DNA-binding protein 43 (TDP-43) pathology. RESULTS: No causal, novel or disease-associated variants in CHCHD10 were identified in Australian ALS and/or FTD patients. In human brain and spinal cord tissues, CHCHD10 was specifically expressed in neurons. A significant decrease in CHCHD10 protein level was observed in ALS patient spinal cord and FTD patient frontal cortex. In a TDP-43 mouse model with a regulatable nuclear localisation signal (rNLS TDP-43 mouse), CHCHD10 protein levels were unaltered at disease onset and early in disease, but were significantly decreased in cortex in mid-stage disease. CONCLUSIONS: Genetic variation in CHCHD10 is not a common cause of ALS/FTD in Australia. However, we showed that in humans, CHCHD10 may play a neuron-specific role and a loss of CHCHD10 function may be linked to ALS and/or FTD. Our data from the rNLS TDP-43 transgenic mice suggest that a decrease in CHCHD10 levels is a late event in aberrant TDP-43-induced ALS/FTD pathogenesis.
Assuntos
Esclerose Lateral Amiotrófica/genética , Demência Frontotemporal/genética , Proteínas Mitocondriais/genética , Idoso , Esclerose Lateral Amiotrófica/imunologia , Esclerose Lateral Amiotrófica/patologia , Animais , Austrália , Western Blotting , Encéfalo/patologia , Feminino , Imunofluorescência , Demência Frontotemporal/imunologia , Demência Frontotemporal/patologia , Variação Genética/genética , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Córtex Motor/patologia , Medula Espinal/patologia , Sequenciamento do Exoma , Sequenciamento Completo do GenomaRESUMO
Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disease affecting motor neurons. Hexanucleotide (GGGGCC) repeat expansions in a non-coding region of C9orf72 are the major cause of familial ALS and frontotemporal dementia (FTD) worldwide. The C9orf72 repeat expansion undergoes repeat-associated non-ATG (RAN) translation to produce five dipeptide repeat proteins (DRPs), including poly(GR) and poly(PR). Whilst it remains unclear how mutations in C9orf72 lead to neurodegeneration in ALS/FTD, dysfunction to the nucleolus and R loop formation are implicated as pathogenic mechanisms. These events can damage DNA and hence genome integrity. Cells activate the DNA damage response (DDR) with the aim of repairing this damage. However, if the damage cannot be repaired, apoptosis is triggered. In lumbar motor neurons from C9orf72-positive ALS patients, we demonstrate significant up-regulation of markers of the DDR compared to controls: phosphorylated histone 2AX (γ-H2AX), phosphorylated ataxia telangiectasia mutated (p-ATM), cleaved poly (ADP-Ribose) polymerase 1 (PARP-1) and tumour suppressor p53-binding protein (53BP1). Similarly, significant up-regulation of γ-H2AX and p-ATM was detected in neuronal cells expressing poly(GR)100 and poly(PR)100 compared to controls, revealing that DNA damage is triggered by the DRPs. Nucleophosmin (NPM1) is a histone chaperone induced during the DDR, which interacts with APE1 to enhance DNA repair. We also demonstrate that more NPM1 precipitates with APE1 in C9orf72 patients compared to controls. Furthermore, overexpression of NPM1 inhibits apoptosis in cells expressing poly(GR)100 and poly(PR)100. This study therefore demonstrates that DNA damage is activated by the C9orf72 repeat expansion in ALS.
Assuntos
Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Reparo do DNA/genética , Idoso , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Sequência de Bases/genética , Nucléolo Celular/metabolismo , Dano ao DNA , Expansão das Repetições de DNA/genética , Dipeptídeos/genética , Feminino , Demência Frontotemporal/genética , Humanos , Masculino , Pessoa de Meia-Idade , Neurônios Motores/metabolismo , Mutação , Proteínas Nucleares/metabolismo , Nucleofosmina , Proteínas/genética , Regulação para CimaRESUMO
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders that have common molecular and pathogenic characteristics, such as aberrant accumulation and ubiquitylation of TDP-43; however, the mechanisms that drive this process remain poorly understood. We have recently identified CCNF mutations in familial and sporadic ALS and FTD patients. CCNF encodes cyclin F, a component of an E3 ubiquitin-protein ligase (SCFcyclin F) complex that is responsible for ubiquitylating proteins for degradation by the ubiquitin-proteasome system. In this study, we examined the ALS/FTD-causing p.Ser621Gly (p.S621G) mutation in cyclin F and its effect upon downstream Lys48-specific ubiquitylation in transfected Neuro-2A and SH-SY5Y cells. Expression of mutant cyclin FS621G caused increased Lys48-specific ubiquitylation of proteins in neuronal cells compared to cyclin FWT. Proteomic analysis of immunoprecipitated Lys48-ubiquitylated proteins from mutant cyclin FS621G-expressing cells identified proteins that clustered within the autophagy pathway, including sequestosome-1 (p62/SQSTM1), heat shock proteins, and chaperonin complex components. Examination of autophagy markers p62, LC3, and lysosome-associated membrane protein 2 (Lamp2) in cells expressing mutant cyclin FS621G revealed defects in the autophagy pathway specifically resulting in impairment in autophagosomal-lysosome fusion. This finding highlights a potential mechanism by which cyclin F interacts with p62, the receptor responsible for transporting ubiquitylated substrates for autophagic degradation. These findings demonstrate that ALS/FTD-causing mutant cyclin FS621G disrupts Lys48-specific ubiquitylation, leading to accumulation of substrates and defects in the autophagic machinery. This study also demonstrates that a single missense mutation in cyclin F causes hyper-ubiquitylation of proteins that can indirectly impair the autophagy degradation pathway, which is implicated in ALS pathogenesis.
Assuntos
Esclerose Lateral Amiotrófica/genética , Autofagia/genética , Ciclinas/genética , Demência Frontotemporal/genética , Ubiquitinação/genética , Esclerose Lateral Amiotrófica/complicações , Células Cultivadas , Demência Frontotemporal/complicações , Células HEK293 , Humanos , Lisina/metabolismo , Mutação de Sentido Incorreto/fisiologiaRESUMO
Astrocytes contribute to the death of motor neurons via non-cell autonomous mechanisms of injury in amyotrophic lateral sclerosis (ALS). Since mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1) underlie the neuropathology of some forms of familial ALS, we explored how expression of mutant SOD1 protein A4V SOD1-EGFP affected the biology of secondary murine astrocytes. A4V SOD1-EGFP expressing astrocytes (72 h after transfection) displayed decreased mitochondrial activity (~45%) and L-glutamate transport (~25%), relative to cells expressing wild-type SOD1-EGFP. A4V SOD1-EGFP altered F-actin and Hoechst staining, indicative of cytoskeletal and nuclear changes, and altered GM130 labelling suggesting fragmentation of Golgi apparatus. SOD1 inclusion formation shifted from discrete to "punctate" over 72 h with A4V SOD1-EGFP more rapidly producing inclusions than G85R SOD1-EGFP, and forming more punctate aggregates. A4V, not wild-type SOD1-EGFP, exerted a substantial, time-dependent effect on GFAP expression, and ~60% of astrocytes became stellate and hypertrophic at 72 h. Spreading toxicity was inferred since at 72 h ~80% of bystander cells exhibited hypertrophy and stellation. This evidence favours mutant SOD1-containing astrocytes releasing destructive species that alter the biology of adjacent astrocytes. This panoply of mutant SOD1-induced destructive events favours recruitment of astrocytes to non-cell autonomous injury in ALS.
Assuntos
Esclerose Lateral Amiotrófica/genética , Astrócitos/efeitos dos fármacos , Mitocôndrias/metabolismo , Neurônios Motores/citologia , Superóxido Dismutase-1/genética , Animais , Astrócitos/metabolismo , Camundongos Endogâmicos C57BL , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismoRESUMO
Amyotrophic lateral sclerosis (ALS) is a fatal, rapidly progressing neurodegenerative disease affecting motor neurons, and frontotemporal dementia (FTD) is a behavioural disorder resulting in early-onset dementia. Hexanucleotide (G4C2) repeat expansions in the gene encoding chromosome 9 open reading frame 72 (C9orf72) are the major cause of familial forms of both ALS (~40%) and FTD (~20%) worldwide. The C9orf72 repeat expansion is known to form abnormal nuclei acid structures, such as hairpins, G-quadruplexes, and R-loops, which are increasingly associated with human diseases involving microsatellite repeats. These configurations form during normal cellular processes, but if they persist they also damage DNA, and hence are a serious threat to genome integrity. It is unclear how the repeat expansion in C9orf72 causes ALS, but recent evidence implicates DNA damage in neurodegeneration. This may arise from abnormal nucleic acid structures, the greatly expanded C9orf72 RNA, or by repeat-associated non-ATG (RAN) translation, which generates toxic dipeptide repeat proteins. In this review, we detail recent advances implicating DNA damage in C9orf72-ALS. Furthermore, we also discuss increasing evidence that targeting these aberrant C9orf72 confirmations may have therapeutic value for ALS, thus revealing new avenues for drug discovery for this disorder.
Assuntos
Esclerose Lateral Amiotrófica/genética , Proteína C9orf72/genética , Dano ao DNA , Expansão das Repetições de DNA , DNA/química , DNA/genética , Predisposição Genética para Doença , Humanos , Conformação de Ácido NucleicoRESUMO
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder primarily affecting motor neurons. Mutations in optineurin cause a small proportion of familial ALS cases, and wild-type (WT) optineurin is misfolded and forms inclusions in sporadic ALS patient motor neurons. However, it is unknown how optineurin mutation or misfolding leads to ALS. Optineurin acts an adaptor protein connecting the molecular motor myosin VI to secretory vesicles and autophagosomes. Here, we demonstrate that ALS-linked mutations p.Q398X and p.E478G disrupt the association of optineurin with myosin VI, leading to an abnormal diffuse cytoplasmic distribution, inhibition of secretory protein trafficking, endoplasmic reticulum (ER) stress and Golgi fragmentation in motor neuron-like NSC-34 cells. We also provide further insight into the role of optineurin as an autophagy receptor. WT optineurin associated with lysosomes and promoted autophagosome fusion to lysosomes in neuronal cells, implying that it mediates trafficking of lysosomes during autophagy in association with myosin VI. However, either expression of ALS mutant optineurin or small interfering RNA-mediated knockdown of endogenous optineurin blocked lysosome fusion to autophagosomes, resulting in autophagosome accumulation. Together these results indicate that ALS-linked mutations in optineurin disrupt myosin VI-mediated intracellular trafficking processes. In addition, in control human patient tissues, optineurin displayed its normal vesicular localization, but in sporadic ALS patient tissues, vesicles were present in a significantly decreased proportion of motor neurons. Optineurin binding to myosin VI was also decreased in tissue lysates from sporadic ALS spinal cords. This study therefore links several previously described pathological mechanisms in ALS, including defects in autophagy, fragmentation of the Golgi and induction of ER stress, to disruption of optineurin function. These findings also indicate that optineurin-myosin VI dysfunction is a common feature of both sporadic and familial ALS.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Proteínas do Olho/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Fator de Transcrição TFIIIA/metabolismo , Esclerose Lateral Amiotrófica/genética , Animais , Proteínas de Ciclo Celular , Células Cultivadas , Estresse do Retículo Endoplasmático , Proteínas do Olho/genética , Humanos , Proteínas de Membrana Transportadoras , Camundongos , Neurônios Motores/metabolismo , Mutação de Sentido Incorreto , Cadeias Pesadas de Miosina/genética , Ligação Proteica , Transporte Proteico , Medula Espinal/citologia , Medula Espinal/metabolismo , Fator de Transcrição TFIIIA/genéticaRESUMO
Cytosolic accumulation of TAR DNA binding protein 43 (TDP-43) is a major neuropathological feature of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). However, the mechanisms involved in TDP-43 accumulation remain largely unknown. Previously, we reported that inhibitors of cyclin-dependent kinases (CDKs) prevented cytosolic stress granule accumulation of TDP-43, correlating with depletion of heterogeneous ribonucleoprotein (hnRNP) K from stress granules. In the present study, we further investigated the relationship between TDP-43 and hnRNP K and their control by CDKs. Inhibition of CDK2 abrogated the accumulation of TDP-43 into stress granules. Phosphorylated CDK2 co-localized with accumulated TDP-43 and phosphorylated hnRNP K in stress granules. Inhibition of CDK2 phosphorylation blocked phosphorylation of hnRNP K, preventing its incorporation into stress granules. Due to interaction between hnRNP K with TDP-43, the loss of hnRNP K from stress granules prevented accumulation of TDP-43. Mutation of Ser216 and Ser284 phosphorylation sites on hnRNP K inhibited hnRNP K- and TDP-43-positive stress granule formation in transfected cells. The interaction between hnRNP K and TDP-43 was further confirmed by the loss of TDP-43 accumulation following siRNA-mediated inhibition of hnRNP K expression. A substantial decrease of CDK2 and hnRNP K expression in spinal cord motor neurons in ALS patients demonstrates a potential key role for these proteins in ALS and TDP-43 accumulation, indicating that further investigation of the association between hnRNP K and TDP-43 is warranted. Understanding how kinase activity modulates TDP-43 accumulation may provide new pharmacological targets for disease intervention.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Esclerose Lateral Amiotrófica/genética , Animais , Citosol/metabolismo , Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/genética , Humanos , Camundongos , Mutação de Sentido Incorreto , FosforilaçãoRESUMO
Intronic expansion of a hexanucleotide GGGGCC repeat in the chromosome 9 open reading frame 72 (C9ORF72) gene is the major cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. However, the cellular function of the C9ORF72 protein remains unknown. Here, we demonstrate that C9ORF72 regulates endosomal trafficking. C9ORF72 colocalized with Rab proteins implicated in autophagy and endocytic transport: Rab1, Rab5, Rab7 and Rab11 in neuronal cell lines, primary cortical neurons and human spinal cord motor neurons, consistent with previous predictions that C9ORF72 bears Rab guanine exchange factor activity. Consistent with this notion, C9ORF72 was present in the extracellular space and as cytoplasmic vesicles. Depletion of C9ORF72 using siRNA inhibited transport of Shiga toxin from the plasma membrane to Golgi apparatus, internalization of TrkB receptor and altered the ratio of autophagosome marker light chain 3 (LC3) II:LC3I, indicating that C9ORF72 regulates endocytosis and autophagy. C9ORF72 also colocalized with ubiquilin-2 and LC3-positive vesicles, and co-migrated with lysosome-stained vesicles in neuronal cell lines, providing further evidence that C9ORF72 regulates autophagy. Investigation of proteins interacting with C9ORF72 using mass spectrometry identified other proteins implicated in ALS; ubiquilin-2 and heterogeneous nuclear ribonucleoproteins, hnRNPA2/B1 and hnRNPA1, and actin. Treatment of cells overexpressing C9ORF72 with proteasome inhibitors induced the formation of stress granules positive for hnRNPA1 and hnRNPA2/B1. Immunohistochemistry of C9ORF72 ALS patient motor neurons revealed increased colocalization between C9ORF72 and Rab7 and Rab11 compared with controls, suggesting possible dysregulation of trafficking in patients bearing the C9ORF72 repeat expansion. Hence, this study identifies a role for C9ORF72 in Rab-mediated cellular trafficking.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Endossomos/metabolismo , Demência Frontotemporal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Esclerose Lateral Amiotrófica/genética , Animais , Proteínas Relacionadas à Autofagia , Transporte Biológico , Proteína C9orf72 , Demência Frontotemporal/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Humanos , Espectrometria de Massas , Camundongos , Proteínas/genética , Proteínas/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
Cellular studies have been undertaken on a nonamer peptide nucleic acid (PNA) sequence, which binds to mRNA encoding superoxide dismutase 1, and a series of peptide nucleic acids conjugated to synthetic lipophilic vitamin analogs including a recently prepared menadione (vitamin K) analog. Reduction of both mutant superoxide dismutase 1 inclusion formation and endoplasmic reticulum stress, two of the key cellular pathological hallmarks in amyotrophic lateral sclerosis, by two of the prepared PNA oligomers is reported for the first time.