RESUMEN
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.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Endosomas/metabolismo , Demencia Frontotemporal/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Esclerosis Amiotrófica Lateral/genética , Animales , Proteínas Relacionadas con la Autofagia , Transporte Biológico , Proteína C9orf72 , Demencia Frontotemporal/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Humanos , Espectrometría de Masas , Ratones , Proteínas/genética , Proteínas/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión a GTP rab7RESUMEN
Fused in sarcoma (FUS) is mutated in both sporadic amyotrophic lateral sclerosis (ALS) and familial ALS patients. The mechanisms underlying neurodegeneration are not fully understood, but FUS redistributes from the nucleus to the cytoplasm in affected motor neurons, where it triggers endoplasmic reticulum (ER) stress. Ataxin-2 is a polyglutamine protein which normally contains 22 repeats, but expanded repeats (>34) are found in Spinocerebellar Ataxia type 2. Recently ataxin-2 with intermediate length repeats (27-33) was found to increase the risk of ALS. Here we show that ataxin-2 with an ALS-linked intermediate length repeat (Q31) is a potent modifier of FUS pathology in cellular disease models. Translocation of FUS to the cytoplasm and ER stress were significantly enhanced by co-expression of mutant FUS with ataxin-2 Q31. Ataxin-2 also co-localized with FUS in sporadic and FUS-linked familial ALS patient motor neurons, co-precipitated with FUS in ALS spinal cord lysates, and co-localized with FUS in the ER-Golgi compartments in neuronal cell lines. Fragmentation of the Golgi apparatus is linked to neurodegeneration in ALS and here we show that Golgi fragmentation is induced in cells expressing mutant FUS. Moreover, Golgi fragmentation was enhanced, and the early stages of apoptosis were triggered, when ataxin-2 Q31 was co-expressed with mutant FUS. These findings describe new cellular mechanisms linking ALS with ataxin-2 intermediate length polyQ expansions and provide further evidence linking disruption to ER-Golgi compartments and FUS pathology in ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Péptidos/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Esclerosis Amiotrófica Lateral/patología , Animales , Apoptosis , Ataxinas , Estudios de Casos y Controles , Línea Celular , Niño , Citoplasma/metabolismo , Gránulos Citoplasmáticos/metabolismo , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico , Femenino , Aparato de Golgi/metabolismo , Humanos , Cuerpos de Inclusión/metabolismo , Masculino , Ratones , Mitocondrias/metabolismo , Mutación Missense , Proteínas del Tejido Nervioso/genética , Péptidos/genética , Unión Proteica , Transporte de Proteínas , Proteína FUS de Unión a ARN/genética , Médula Espinal/metabolismo , Médula Espinal/patología , Expansión de Repetición de Trinucleótido , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Several diverse proteins are linked genetically/pathologically to neurodegeneration in amyotrophic lateral sclerosis (ALS) including SOD1, TDP-43 and FUS. Using a variety of cellular and biochemical techniques, we demonstrate that ALS-associated mutant TDP-43, FUS and SOD1 inhibit protein transport between the endoplasmic reticulum (ER) and Golgi apparatus in neuronal cells. ER-Golgi transport was also inhibited in embryonic cortical and motor neurons obtained from a widely used animal model (SOD1(G93A) mice), validating this mechanism as an early event in disease. Each protein inhibited transport by distinct mechanisms, but each process was dependent on Rab1. Mutant TDP-43 and mutant FUS both inhibited the incorporation of secretory protein cargo into COPII vesicles as they bud from the ER, and inhibited transport from ER to the ER-Golgi intermediate (ERGIC) compartment. TDP-43 was detected on the cytoplasmic face of the ER membrane, whereas FUS was present within the ER, suggesting that transport is inhibited from the cytoplasm by mutant TDP-43, and from the ER by mutant FUS. In contrast, mutant SOD1 destabilised microtubules and inhibited transport from the ERGIC compartment to Golgi, but not from ER to ERGIC. Rab1 performs multiple roles in ER-Golgi transport, and over-expression of Rab1 restored ER-Golgi transport, and prevented ER stress, mSOD1 inclusion formation and induction of apoptosis, in cells expressing mutant TDP-43, FUS or SOD1. Rab1 also co-localised extensively with mutant TDP-43, FUS and SOD1 in neuronal cells, and Rab1 formed inclusions in motor neurons of spinal cords from sporadic ALS patients, which were positive for ubiquitinated TDP-43, implying that Rab1 is misfolded and dysfunctional in sporadic disease. These results demonstrate that ALS-mutant forms of TDP-43, FUS, and SOD1 all perturb protein transport in the early secretory pathway, between ER and Golgi compartments. These data also imply that restoring Rab1-mediated ER-Golgi transport is a novel therapeutic target in ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Proteínas de Unión al GTP rab1/metabolismo , Anciano , Anciano de 80 o más Años , Esclerosis Amiotrófica Lateral/patología , Animales , Transporte Biológico/fisiología , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Vesículas Cubiertas por Proteínas de Revestimiento/patología , Línea Celular Tumoral , Citoplasma/metabolismo , Citoplasma/patología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Retículo Endoplásmico/patología , Femenino , Aparato de Golgi/patología , Humanos , Masculino , Ratones , Persona de Mediana Edad , Mutación , Neuronas/metabolismo , Neuronas/patología , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo , Médula Espinal/metabolismo , Médula Espinal/patología , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1RESUMEN
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder affecting motor neurons in the brain, brainstem and spinal cord, which is characterized by motor dysfunction, muscle dystrophy and progressive paralysis. Both inherited and sporadic forms of ALS share common pathological features, however, the initial trigger of neurodegeneration remains unknown. Motor neurons are uniquely targeted by ubiquitously expressed proteins in ALS but the reason for this selectively vulnerability is unclear. However motor neurons have unique characteristics such as very long axons, large cell bodies and high energetic metabolism, therefore placing high demands on cellular transport processes. Defects in cellular trafficking are now widely reported in ALS, including dysfunction to the molecular motors dynein and kinesin. Abnormalities to dynein in particular are linked to ALS, and defects in dynein-mediated axonal transport processes have been reported as one of the earliest pathologies in transgenic SOD1 mice. Furthermore, dynein is very highly expressed in neurons and neurons are particularly sensitive to dynein dysfunction. Hence, unravelling cellular transport processes mediated by molecular motor proteins may help shed light on motor neuron loss in ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Proteínas Motoras Moleculares/metabolismo , Esclerosis Amiotrófica Lateral/genética , Animales , Transporte Axonal , Humanos , Proteínas Motoras Moleculares/química , Proteínas Motoras Moleculares/genética , Pliegue de ProteínaRESUMEN
Mutations in Cu, Zn-superoxide dismutase 1 (SOD1) are associated with degeneration of motor neurons in the disease, familial amyotrophic lateral sclerosis. Intracellular protein inclusions containing mutant SOD1 (mSOD1) are associated with disease but it is unclear whether they are neuroprotective or cytotoxic. We report here that the formation of mSOD1 inclusions in a motor neuron-like cell line (NSC-34) strongly correlates with apoptosis via the mitochondrial death pathway. Applying confocal microscopic analyses, we observed changes in nuclear morphology and activation of caspase 3 specifically in cells expressing mSOD1 A4V or G85R inclusions. Furthermore, markers of mitochondrial apoptosis (activation and recruitment of Bax, and cytochrome c redistribution) were observed in 30% of cells bearing mSOD1 inclusions but not in cells expressing dispersed SOD1. In the presence of additional apoptotic challenges (staurosporine, etoposide, and hydrogen peroxide), cells bearing mSOD1 inclusions were susceptible to further apoptosis suggesting they were in a pro-apoptotic state, thus confirming that inclusions are linked to toxicity. Surprisingly, cells displaying dispersed SOD1 [both wildtype (WT) and mutant] were protected against apoptosis upstream of mitochondrial apoptotic signaling, induced by all agents tested. This protection against apoptosis was unrelated to SOD1 enzymatic activity because the G85R that lacks enzymatic function protected cells similarly to both WT SOD1 and A4V that possesses WT-like activity. These findings demonstrate new aspects of SOD1 in relation to cellular viability; specifically, mSOD1 can be either neuroprotective or cytotoxic depending on its aggregation state.
Asunto(s)
Esclerosis Amiotrófica Lateral/patología , Apoptosis/fisiología , Cuerpos de Inclusión/patología , Mitocondrias/fisiología , Transducción de Señal/fisiología , Superóxido Dismutasa/genética , Esclerosis Amiotrófica Lateral/genética , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Citocromos c/metabolismo , Humanos , Inmunohistoquímica , Ratones , Microscopía Confocal , Neuronas Motoras/fisiología , Mutación , Fármacos Neuroprotectores/farmacología , Superóxido Dismutasa-1 , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that results from the death of upper and lower motor neurons. Due to a lack of effective treatment, it is imperative to understand the underlying mechanisms and processes involved in disease progression. Regulations in cellular reduction/oxidation (redox) processes are being increasingly implicated in disease. Here we discuss the possible involvement of redox dysregulation in the pathophysiology of ALS, either as a cause of cellular abnormalities or a consequence. We focus on its possible role in oxidative stress, protein misfolding, glutamate excitotoxicity, lipid peroxidation and cholesterol esterification, mitochondrial dysfunction, impaired axonal transport and neurofilament aggregation, autophagic stress, and endoplasmic reticulum (ER) stress. We also speculate that an ER chaperone protein disulphide isomerase (PDI) could play a key role in this dysregulation. PDI is essential for normal protein folding by oxidation and reduction of disulphide bonds, and hence any disruption to this process may have consequences for motor neurons. Addressing the mechanism underlying redox regulation and dysregulation may therefore help to unravel the molecular mechanism involved in ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Autofagia , Transporte Axonal , Colesterol/metabolismo , Estrés del Retículo Endoplásmico , Ácido Glutámico/metabolismo , Humanos , Peroxidación de Lípido , Mitocondrias/metabolismo , Oxidación-Reducción , Estrés Oxidativo , Proteína Disulfuro Isomerasas/metabolismoRESUMEN
In amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration, TAR DNA binding protein 43 (TDP-43) accumulates in the cytoplasm of affected neurons and glia, where it associates with stress granules (SGs) and forms large inclusions. SGs form in response to cellular stress, including endoplasmic reticulum (ER) stress, which is induced in both familial and sporadic forms of ALS. Here we demonstrate that pharmacological induction of ER stress causes TDP-43 to accumulate in the cytoplasm, where TDP-43 also associates with SGs. Furthermore, treatment with salubrinal, an inhibitor of dephosphorylation of eukaryotic initiation factor 2-α, a key modulator of ER stress, potentiates ER stress-mediated SG formation. Inclusions of C-terminal fragment TDP-43, reminiscent of disease-pathology, form in close association with ER and Golgi compartments, further indicating the involvement of ER dysfunction in TDP-43-associated disease. Consistent with this notion, over-expression of ALS-linked mutant TDP-43, and to a lesser extent wildtype TDP-43, triggers several ER stress pathways in neuroblastoma cells. Similarly, we found an interaction between the ER chaperone protein disulphide isomerase and TDP-43 in transfected cell lysates and in the spinal cords of mutant A315T TDP-43 transgenic mice. This study provides evidence for ER stress as a pathogenic pathway in TDP-43-mediated disease.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Citoplasma/metabolismo , Proteínas de Unión al ADN/metabolismo , Estrés del Retículo Endoplásmico , Transporte Activo de Núcleo Celular/efectos de los fármacos , Esclerosis Amiotrófica Lateral/genética , Animales , Línea Celular , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Cinamatos/farmacología , Citoplasma/efectos de los fármacos , Proteínas de Unión al ADN/genética , Estrés del Retículo Endoplásmico/efectos de los fármacos , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/metabolismo , Humanos , Ratones , Mutación , Proteína Disulfuro Isomerasas/metabolismo , Médula Espinal/metabolismo , Tiourea/análogos & derivados , Tiourea/farmacologíaRESUMEN
Endoplasmic reticulum (ER) stress is an important pathway to cell death in amyotrophic lateral sclerosis (ALS). We previously demonstrated that ER stress is linked to neurotoxicity associated with formation of inclusions of mutant Cu,Zn-superoxide dismutase 1 (SOD1). Cells bearing mutant inclusions undergo mitochondrial apoptotic signalling. Here, we demonstrate that the BH3-only protein, Bim, is a direct link between ER stress and mitochondrial apoptosis. In the murine neuroblastoma cell line, Neuro2a, bearing mutant SOD1 inclusions, indicators of both ER stress and apoptosis are expressed. Bim knockdown by siRNA significantly reduced nuclear apoptotic features in these inclusion-bearing cells (but did not affect the proportion of cells overall that bear inclusions). Further, both Bax recruitment to mitochondria and cytochrome c redistribution were also decreased under Bim-depletion conditions. However, upregulation of CHOP, a marker of ER stress, was not reduced by Bim knockdown. Significantly, knockdown of CHOP by siRNA reduced the extent of apoptosis in cells bearing mutant SOD1 inclusions. These sequential links between ER stress, CHOP upregulation, and Bim activation of mitochondrial apoptotic signalling indicate a clear pathway to cell death mediated by mutant SOD1.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Proteínas Reguladoras de la Apoptosis/fisiología , Apoptosis/fisiología , Estrés del Retículo Endoplásmico/fisiología , Proteínas de la Membrana/fisiología , Proteínas Proto-Oncogénicas/fisiología , Superóxido Dismutasa/genética , Factor de Transcripción Activador 6/metabolismo , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Proteína 11 Similar a Bcl2 , Línea Celular Tumoral , Citocromos c/metabolismo , Regulación hacia Abajo , Humanos , Ratones , Mitocondrias/metabolismo , Neuroblastoma/metabolismo , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1 , Factor de Transcripción CHOP/metabolismo , Regulación hacia Arriba , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Mutations in the gene encoding fused in sarcoma (FUS) are linked to amyotrophic lateral sclerosis (ALS), but the mechanisms by which these mutants trigger neurodegeneration remain unknown. Endoplasmic reticulum (ER) stress is increasingly recognized as an important and early pathway to motor neuron death in ALS. FUS is normally located in the nucleus but in ALS, FUS redistributes to the cytoplasm and forms inclusions. In this study, we investigated whether FUS induces ER stress in a motor neuron like cell line (NSC-34). We demonstrate that ER stress is triggered in cells expressing mutant FUS, and this is closely associated with redistribution of mutant FUS to the cytoplasm. Mutant FUS also colocalized with protein disulfide-isomerase (PDI), an important ER chaperone, in NSC-34 cells and PDI was colocalized with FUS inclusions in human ALS lumbar spinal cords, in both sporadic ALS and mutant FUS-linked familial ALS tissues. These findings implicate ER stress in the pathophysiology of FUS, and provide evidence for common pathogenic pathways in ALS linked to the ER.