RESUMO
A major unanswered question in biology and medicine is the mechanism by which the product of the apolipoprotein E ε4 allele, the lipid-binding protein apolipoprotein E4 (ApoE4), plays a pivotal role in processes as disparate as Alzheimer's disease (AD; in which it is the single most important genetic risk factor), atherosclerotic cardiovascular disease, Lewy body dementia, hominid evolution, and inflammation. Using a combination of neural cell lines, skin fibroblasts from AD patients, and ApoE targeted replacement mouse brains, we show in the present report that ApoE4 undergoes nuclear translocation, binds double-stranded DNA with high affinity (low nanomolar), and functions as a transcription factor. Using chromatin immunoprecipitation and high-throughput DNA sequencing, our results indicate that the ApoE4 DNA binding sites include â¼1700 gene promoter regions. The genes associated with these promoters provide new insight into the mechanism by which AD risk is conferred by ApoE4, because they include genes associated with trophic support, programmed cell death, microtubule disassembly, synaptic function, aging, and insulin resistance, all processes that have been implicated in AD pathogenesis. Significance statement: This study shows for the first time that apolipoprotein E4 binds DNA with high affinity and that its binding sites include 1700 promoter regions that include genes associated with neurotrophins, programmed cell death, synaptic function, sirtuins and aging, and insulin resistance, all processes that have been implicated in Alzheimer's disease pathogenesis.
Assuntos
Apolipoproteína E4/genética , Apolipoproteína E4/metabolismo , Neurônios/fisiologia , Transcrição Gênica/fisiologia , Idoso , Animais , Sequência de Bases , Encéfalo/fisiologia , Linhagem Celular Tumoral , Feminino , Fibroblastos/fisiologia , Humanos , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Ligação Proteica/fisiologiaRESUMO
The canonical pathogenesis of Alzheimer's disease links the expression of apolipoprotein E ε4 allele (ApoE) to amyloid precursor protein (APP) processing and Aß peptide accumulation by a set of mechanisms that is incompletely defined. The development of a simple system that focuses not on a single variable but on multiple factors and pathways would be valuable both for dissecting the underlying mechanisms and for identifying candidate therapeutics. Here we show that, although both ApoE3 and ApoE4 associate with APP with nanomolar affinities, only ApoE4 significantly (i) reduces the ratio of soluble amyloid precursor protein alpha (sAPPα) to Aß; (ii) reduces Sirtuin T1 (SirT1) expression, resulting in markedly differing ratios of neuroprotective SirT1 to neurotoxic SirT2; (iii) triggers Tau phosphorylation and APP phosphorylation; and (iv) induces programmed cell death. We describe a subset of drug candidates that interferes with the APP-ApoE interaction and returns the parameters noted above to normal. Our data support the hypothesis that neuronal connectivity, as reflected in the ratios of critical mediators such as sAPPα:Aß, SirT1:SirT2, APP:phosphorylated (p)-APP, and Tau:p-Tau, is programmatically altered by ApoE4 and offer a simple system for the identification of program mediators and therapeutic candidates.
Assuntos
Doença de Alzheimer/fisiopatologia , Apolipoproteína E4/metabolismo , Regulação da Expressão Gênica/fisiologia , Sirtuínas/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Análise de Variância , Apolipoproteína E3/metabolismo , Western Blotting , Descoberta de Drogas , Humanos , Imunoprecipitação , Fosforilação , Reação em Cadeia da Polimerase em Tempo Real , Ressonância de Plasmônio de Superfície , Proteínas tau/metabolismoRESUMO
This study describes the first proteomic analysis of paraptosis--a non-apoptotic form of programmed cell death. As with apoptosis, the first description of paraptosis was based on morphological criteria. Since there are no known markers for paraptosis, the purpose of this study was to dissect changes in the proteome profile occurring during paraptosis. Using one- and two-dimensional SDS-PAGE, Western analysis, and mass spectrometry, we show that during paraptosis, alterations occur mainly in cytoskeletal proteins, signal transduction proteins, mitochondrial proteins, and some metabolic proteins. We also report the identification of: (1) a paraptosis inhibitor, phosphatidylethanolamine binding protein (PEBP-1), and (2) a candidate mediator of paraptosis, prohibitin. Identification of specific paraptotic changes will ultimately lead to tools to detect this type of programmed cell death in in vivo systems and allow for its further characterization.
Assuntos
Apoptose/fisiologia , Apoptose/genética , Western Blotting , Linhagem Celular , Biologia Computacional , Bases de Dados de Proteínas , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Humanos , Imuno-Histoquímica , Proteína de Ligação a Fosfatidiletanolamina/genética , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Proibitinas , Proteômica , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Espectrometria de Massas em TandemRESUMO
The microenvironment of cancerous cells includes endoplasmic reticulum (ER) stress the resistance to which is required for the survival and growth of tumors. Acute ER stress triggers the induction of a family of ER stress proteins that promotes survival and/or growth of the cancer cells, and also confers resistance to radiation and chemotherapy. Prolonged or severe ER stress, however, may ultimately overwhelm the cellular protective mechanisms, triggering cell death through specific programmed cell death (pcd) pathways. Thus, downregulation of the protective stress proteins may offer a new therapeutic approach to cancer treatment. In this regard, recent reports have demonstrated the roles of the phytochemical curcumin in the inhibition of proteasomal activity and triggering the accumulation of cytosolic Ca(2+) by inhibiting the Ca(2+)-ATPase pump, both of which enhance ER stress. Using a mouse melanoma cell line, we investigated the possibility that curcumin may trigger ER stress leading to programmed cell death. Our studies demonstrate that curcumin triggers ER stress and the activation of specific cell death pathways that feature caspase cleavage and activation, p23 cleavage, and downregulation of the anti-apoptotic Mcl-1 protein.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Curcumina/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/patologia , Animais , Apoptose/fisiologia , Caspases/metabolismo , Linhagem Celular Tumoral , Quelantes/farmacologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Retículo Endoplasmático/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Oxirredutases Intramoleculares/metabolismo , Melanoma Experimental/metabolismo , Camundongos , Poli(ADP-Ribose) Polimerases/metabolismo , Prostaglandina-E Sintases , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Fator de Transcrição CHOP/metabolismoRESUMO
Alzheimer's disease (AD) is characterized by neuronal and synaptic loss. One process that could contribute to this loss is the intracellular caspase cleavage of the amyloid precursor protein (APP) resulting in release of the toxic C-terminal 31-amino acid peptide APP-C31 along with the production of APPΔC31, full-length APP minus the C-terminal 31 amino acids. We previously found that a mutation in APP that prevents this caspase cleavage ameliorated synaptic loss and cognitive impairment in a murine AD model. Thus, inhibition of this cleavage is a reasonable target for new therapeutic development. In order to identify small molecules that inhibit the generation of APP-C31, we first used an APPΔC31 cleavage site-specific antibody to develop an AlphaLISA to screen several chemical compound libraries for the level of N-terminal fragment production. This antibody was also used to develop an ELISA for validation studies. In both high throughput screening (HTS) and validation testing, the ability of compounds to inhibit simvastatin- (HTS) or cerivastatin- (validation studies) induced caspase cleavage at the APP-D720 cleavage site was determined in Chinese hamster ovary (CHO) cells stably transfected with wildtype (wt) human APP (CHO-7W). Several compounds, as well as control pan-caspase inhibitor Q-VD-OPh, inhibited APPΔC31 production (measured fragment) and rescued cell death in a dose-dependent manner. The effective compounds fell into several classes including SERCA inhibitors, inhibitors of Wnt signaling, and calcium channel antagonists. Further studies are underway to evaluate the efficacy of lead compounds - identified here using cells and tissues expressing wt human APP - in mouse models of AD expressing mutated human APP, as well as to identify additional compounds and determine the mechanisms by which they exert their effects.
RESUMO
Recent studies have shown that inoculation of susceptible mice with amyloid-ß (Aß) peptides accelerates Aß deposition in the brain, supporting the idea that Aß may be self-amplifying; however, the exact mechanism is not understood. Here we provide evidence that Aß may self-amplify, in part, by inhibiting α-secretase ADAM10 (a disintegrin and metalloprotease) cleavage of full-length Aß precursor protein (FL AßPP) and therefore allow greater ß-secretase processing, and that Aß itself is a substrate for ADAM10. Exposure of primary neuronal cultures from PDAßPP mice to exogenous rat Aß1- 40 resulted in increased de novo human Aß1-42 production and exposure of cells to Aß decreased production of ADAM10 cleavage product soluble AßPPα (sAßPPα). In a cell-free assay, Aß decreased ADAM10 cleavage of the chimeric substrate MBP-AßPPC125 and Aß itself was apparently cleaved by the enzyme. The axonal guidance and trophic factor netrin-1, however, reduced the Aß1- 40-induced Aß1-42 increase, increased sAßPPα, and reversed the Aß-induced sAßPPα decrease in vitro. In vivo, induction of netrin-1 expression in PDAßPPSwe/Ind transgenic mice resulted in reductions in both Aß1-42 and Aß1- 40, and ICV delivery of netrin-1 to PDAßPPSwe/Ind mice increased sAßPPα, decreased Aß, and improved working memory. Finally, to support further study of netrin-1's potential as a therapeutic for Alzheimer's disease, pilot gene therapy studies were performed and a netrin mimetic peptide synthesized and tested that, like netrin, can increase sAßPPα and decrease Aß1-42in vitro. Taken together, these data provide mechanistic insights into Aß self-amplification and the ability of netrin-1 to disrupt it.
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/terapia , Precursor de Proteína beta-Amiloide/metabolismo , Terapia Genética/métodos , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteína ADAM10/metabolismo , Doença de Alzheimer/psicologia , Animais , Biomimética , Linhagem Celular Tumoral , Cognição/fisiologia , Dependovirus/genética , Modelos Animais de Doenças , Vetores Genéticos , Humanos , Memória de Curto Prazo/fisiologia , Camundongos Transgênicos , Netrina-1 , Projetos Piloto , Ratos , Reconhecimento Psicológico/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Proteolytic cleavage of the amyloid-ß protein precursor (AßPP) by the enzyme BACE1 (BACE) is the initial step in production of amyloid-ß peptide (Aß), and as such has been a major target of Alzheimer's disease (AD) drug discovery efforts. Overproduction of Aß results in neuronal cell death and accumulation of amyloid plaques in AD and in traumatic brain injury, and is also associated with stroke due to cerebral amyloid angiopathy. Herein we report for the first time that sAßPPα, the product of the cleavage of AßPP by α-secretase, is a potent endogenous direct inhibitor of the BACE enzyme, and that its inhibition is likely by an allosteric mechanism. Furthermore, using small-angle X-ray scattering, we show that sAßPPß, which is identical to sAßPPα except for a 16-amino acid truncation at the carboxy terminus, adopts a completely different structure than sAßPPα and does not inhibit BACE. Our data thus reveal a novel mechanistic role played by sAßPPα in regulating overproduction of Aß and restoring neuronal homeostasis and neuroprotection. Identification of sAßPPα as a direct BACE inhibitor may lead to design of new therapeutics targeting pathologies associated with overproduction of Aß.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Precursor de Proteína beta-Amiloide/metabolismo , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Fragmentos de Peptídeos/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/genética , Humanos , Imunoprecipitação , Modelos Moleculares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Conformação Proteica , Proteínas Recombinantes/metabolismo , Espalhamento a Baixo Ângulo , Espectrometria de Fluorescência , Difração de Raios XRESUMO
Tropisetron was identified in a screen for candidates that increase the ratio of the trophic, neurite-extending peptide sAPPα to the anti-trophic, neurite-retractive peptide Aß, thus reversing this imbalance in Alzheimer's disease (AD). We describe here a hierarchical screening approach to identify such drug candidates, moving from cell lines to primary mouse hippocampal neuronal cultures to in vivo studies. By screening a clinical compound library in the primary assay using CHO-7W cells stably transfected with human APPwt, we identified tropisetron as a candidate that consistently increased sAPPα. Secondary assay testing in neuronal cultures from J20 (PDAPP, huAPP(Swe/Ind)) mice showed that tropisetron consistently increased the sAPPα/Aß 1-42 ratio. In in vivo studies in J20 mice, tropisetron improved the sAPPα/Aß ratio along with spatial and working memory in mice, and was effective both during the symptomatic, pre-plaque phase (5-6 months) and in the late plaque phase (14 months). This ameliorative effect occurred at a dose of 0.5mg/kg/d (mkd), translating to a human-equivalent dose of 5mg/day, the current dose for treatment of postoperative nausea and vomiting (PONV). Although tropisetron is a 5-HT3 receptor antagonist and an α7nAChR partial agonist, we found that it also binds to the ectodomain of APP. Direct comparison of tropisetron to the current AD therapeutics memantine (Namenda) and donepezil (Aricept), using similar doses for each, revealed that tropisetron induced greater improvements in memory and the sAPPα/Aß1-42 ratio. The improvements observed with tropisetron in the J20 AD mouse model, and its known safety profile, suggest that it may be suitable for transition to human trials as a candidate therapeutic for mild cognitive impairment (MCI) and AD, and therefore it has been approved for testing in clinical trials beginning in 2014.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Precursor de Proteína beta-Amiloide/efeitos dos fármacos , Cognição/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Indóis/farmacologia , Indóis/uso terapêutico , Administração Oral , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Células CHO , Cricetulus , Modelos Animais de Doenças , Hipocampo/metabolismo , Indóis/administração & dosagem , Camundongos , Camundongos Endogâmicos C57BL , TropizetronaRESUMO
An unbiased screen for compounds that block amyloid-ß protein precursor (AßPP) caspase cleavage identified ADDN-1351, which reduced AßPP-C31 by 90%. Target identification studies showed that ADDN-1351 is a TrkA inhibitor, and, in complementary studies, TrkA overexpression increased AßPP-C31 and cell death. TrkA was shown to interact with AßPP and suppress AßPP-mediated transcriptional activation. Moreover, treatment of PDAPP transgenic mice with the known TrkA inhibitor GW441756 increased sAßPPα and the sAßPPα to Aß ratio. These results suggest TrkA inhibition-rather than NGF activation-as a novel therapeutic approach, and raise the possibility that such an approach may counteract the hyperactive signaling resulting from the accumulation of active NGF-TrkA complexes due to reduced retrograde transport. The results also suggest that one component of an optimal therapy for Alzheimer's disease may be a TrkA inhibitor.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Receptor trkA/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Benzamidas/farmacologia , Células CHO , Morte Celular/efeitos dos fármacos , Cricetulus , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Camundongos , Camundongos Transgênicos , Mutação/genética , Fator de Crescimento Neural/metabolismo , Fator de Crescimento Neural/farmacologia , Inibidores de Proteínas Quinases/química , Pirazóis/farmacologia , Receptor trkA/antagonistas & inibidores , Receptor trkA/genética , TransfecçãoRESUMO
The presence of misfolded proteins elicits cellular responses including an endoplasmic reticulum (ER) stress response that may protect cells against the toxic buildup of misfolded proteins. Accumulation of these proteins in excessive amounts, however, overwhelms the "cellular quality control" system and impairs the protective mechanisms designed to promote correct folding and degrade misfolded proteins, ultimately leading to organelle dysfunction and cell death. Studies from multiple laboratories have identified the roles of several ER stress-induced cell death modulators and effectors. Earlier, we reported the role of the small co-chaperone protein p23 in preventing ER stress-induced cell death. p23 undergoes caspase-dependent cleavage to yield a 19-kD product (p19), and mutation of this caspase cleavage site not only blocks the formation of the 19-kD product but also attenuates the ER stress-induced cell death process triggered by various stressors. Thus, a critical question is whether p23 and/or p19 could serve as an in vivo marker for neurodegenerative diseases featuring misfolded proteins and cellular stress. In the present study, we used an antibody that recognizes both p23 and p19 as well as a specific neo-epitope antibody that detects only the p19 fragment. These antibodies were used to detect the presence of both these proteins in cells, primary neurons, brain samples from a mouse model of Alzheimer's disease (AD), and fixed human AD brain samples. While patients with severe AD did display a consistent reduction in p23 levels, our inability to observe p19 in mouse or human AD brain samples suggests that the usefulness of the p23 neo-epitope antibody is restricted to cells and primary neurons undergoing cellular stress.
Assuntos
Estresse do Retículo Endoplasmático/fisiologia , Oxirredutases Intramoleculares/fisiologia , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Apoptose/fisiologia , Encéfalo/metabolismo , Encéfalo/patologia , Hipóxia Celular/fisiologia , Células Cultivadas/metabolismo , Citosol/química , Modelos Animais de Doenças , Epitopos/imunologia , Feminino , Fibroblastos/metabolismo , Células HEK293/metabolismo , Humanos , Oxirredutases Intramoleculares/análise , Oxirredutases Intramoleculares/biossíntese , Oxirredutases Intramoleculares/imunologia , Masculino , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Dados de Sequência Molecular , Neurônios/metabolismo , Prostaglandina-E Sintases , Proteínas Recombinantes de Fusão/fisiologia , Tapsigargina/farmacologia , TransfecçãoRESUMO
Alzheimer's disease-associated amyloid-ß (Aß) peptide is neurotoxic as an oligomer, but not as a monomer, by an unknown mechanism. We showed previously that Aß interacts with the amyloid-ß precursor protein (AßPP), leading to caspase cleavage and cell death induction. To characterize this structure and interaction further, we purified the extracellular domain of AßPP695 (eAßPP) and its complex with Aß oligomers (AßOs) of varying sizes, and then performed small angle X-ray scattering (SAXS). In the absence of any Aß, eAßPP was a compact homodimer with a tight association between the E1 and E2 domains. Dimeric Aß oligomers induced monomerization of eAßPP while larger oligomers also bound eAßPP but preserved the homodimer. Efficient binding of the larger oligomers correlated with the presence of prefibrillar oligomers, suggesting that the eAßPP binding is limited to a conformational subset of Aß oligomers. Both forms of Aß bound to eAßPP at the Aß-cognate region and induced dissociation of the E1 and E2 domains. Our data provide the first structural evidence for Aß-AßPP binding and suggest a mechanism for differential modulation of AßPP processing and cell death signaling by Aß dimers versus conformationally-specific larger oligomers.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Precursor de Proteína beta-Amiloide/efeitos dos fármacos , Precursor de Proteína beta-Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/química , Humanos , Modelos Moleculares , Peso Molecular , Fragmentos de Peptídeos , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/fisiologiaRESUMO
Previously, we identified valosin-containing protein (VCP) as a mediator of ER stress-induced cell death. Mutations in the VCP gene including R93, R155, and R191 have been described that manifest clinically as hereditary inclusion body myopathy with Paget's disease of bone and frontotemporal dementia. In addition, other studies have demonstrated that as a consequence of a mutation generated in the second ATP binding domain of VCP (K524A), cells accumulated large cytoplasmic vacuoles and underwent programmed cell death. In order to better understand the biochemical and molecular consequences of the clinically relevant VCP mutations as well as the genetically engineered ATPase-inactive mutant K524A and any relationship these may have to ER stress-induced cell death, we introduced analogous mutations separately and together into the human VCP gene and evaluated their effect on proteasome activity, Huntingtin protein aggregation and ER stress-induced cell death. Our results indicate that the VCP K524A mutant and the triple mutant VCP R93C-R155C-K524A block protein degradation, trigger Huntingtin aggregate formation, and render cells highly susceptible to ER stress-induced cell death as compared to VCPWT or other VCP mutants.
Assuntos
Adenosina Trifosfatases/genética , Proteínas de Ciclo Celular/genética , Mutação , Degeneração Neural/patologia , Fenótipo , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Morte Celular/genética , Retículo Endoplasmático/metabolismo , Inibidores Enzimáticos/metabolismo , Células HEK293 , Humanos , Proteína Huntingtina , Degeneração Neural/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Tapsigargina/metabolismo , Ubiquitina/metabolismo , Proteína com ValosinaRESUMO
Resveratrol, a naturally occurring polyphenol, exhibits antioxidant, antiaging, and anticancer activity. Resveratrol has also been shown to inhibit tumor initiation, promotion, and progression in a variety of cell culture systems. Earlier, we showed that paraquat, a bipyridyl herbicide, triggers endoplasmic reticulum stress, cell dysfunction, and dopaminergic cell death. Due to its antioxidant activity, we assessed the ability of resveratrol to rescue cells from the toxic effects of paraquat. While resveratrol did not have any protective effect at low concentrations, it triggered endoplasmic reticulum (ER) stress-induced cell death at higher concentrations (50-250 microM). The present study was carried out to determine the mechanism by which resveratrol triggers ER stress and cell death in dopaminergic N27 cells. Our studies demonstrate that resveratrol triggers ER stress and cell dysfunction, caspase activation, p23 cleavage and inhibition of proteasomal activity in dopaminergic N27 cells. While over expression of uncleavable p23 was associated with decreased cell death, downregulation of p23 protein expression by siRNA resulted in enhancement of ER stress-induced cell death triggered by resveratrol indicating a protective role for the small co-chaperone p23 in dopaminergic cell death.
Assuntos
Antioxidantes/farmacologia , Morte Celular/efeitos dos fármacos , Dopamina/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Estresse Oxidativo , Estilbenos/farmacologia , Animais , Caspases/metabolismo , Linhagem Celular , Cinamatos/farmacologia , Retículo Endoplasmático/metabolismo , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Interferência de RNA , Ratos , Resveratrol , Tioureia/análogos & derivados , Tioureia/farmacologiaRESUMO
Parkinson's disease (PD) features oxidative stress and accumulation of misfolded (unfolded, alternatively folded, or mutant) proteins with associated loss of dopaminergic neurons. Oxidative stress and the accumulated misfolded proteins elicit cellular responses that include an endoplasmic reticulum (ER) stress response that may protect cells against the toxic buildup of misfolded proteins. Chronic ER stress and accumulation of misfolded proteins in excessive amounts, however, overwhelm the cellular 'quality control' system and impair the protective mechanisms designed to promote correct folding and degrade faulty proteins, ultimately leading to organelle dysfunction and neuronal cell death. Paraquat belongs to a class of bipyridyl herbicides and triggers oxidative stress and dopaminergic cell death. Epidemiological studies suggest an increased risk for developing PD following chronic exposure to paraquat. The present study was carried out to determine the role of paraquat in triggering cellular stress particularly ER stress and to elucidate the pathways that couple ER stress to dopaminergic cell death. We demonstrate that paraquat triggers ER stress, cell dysfunction, and dopaminergic cell death. p23, a small co-chaperone protein, is cleaved during ER stress-induced cell death triggered by paraquat and blockage of the caspase cleavage site of p23 was associated with decreased cell death. Paraquat also inhibits proteasomal activity that may further trigger accumulation of misfolded proteins resulting in ER stress. Our results indicate a protective role for p23 in PD-related programmed cell death. The data also underscore the involvement of ER, caspases, and the proteasomal system in ER stress-induced cell death process.
Assuntos
Apoptose/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Oxirredutases Intramoleculares/metabolismo , Chaperonas Moleculares/metabolismo , Neurônios/efeitos dos fármacos , Paraquat/toxicidade , Estresse Fisiológico/efeitos dos fármacos , Animais , Apoptose/fisiologia , Caspases/efeitos dos fármacos , Caspases/metabolismo , Domínio Catalítico/efeitos dos fármacos , Domínio Catalítico/fisiologia , Linhagem Celular Transformada , Citoproteção/fisiologia , Dopamina/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Herbicidas/toxicidade , Neurônios/metabolismo , Neurônios/patologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Doença de Parkinson/etiologia , Doença de Parkinson/metabolismo , Doença de Parkinson/fisiopatologia , Prostaglandina-E Sintases , Dobramento de Proteína/efeitos dos fármacos , Ratos , Estresse Fisiológico/fisiologia , Substância Negra/efeitos dos fármacos , Substância Negra/metabolismo , Substância Negra/fisiopatologiaRESUMO
Programmed cell death (pcd) may take the form of apoptosis or of nonapoptotic pcd. Whereas cysteine aspartyl-specific proteases (caspases) mediate apoptosis, the mediators of nonapoptotic cell death programs are much less well characterized. Here we report that alternative, nonapoptotic pcd induced by the neurokinin-1 receptor (NK(1)R) activated by its ligand Substance P, is mediated by a MAPK phosphorylation cascade recruited by the scaffold protein arrestin 2. The activation of the protein kinases Raf-1, MEK2, and ERK2 is essential for this form of nonapoptotic pcd, leading to the phosphorylation of the orphan nuclear receptor Nur77. NK(1)R-mediated cell death was inhibited by a dominant negative form of arrestin 2, Raf-1, or Nur77, by MEK1/2-specific inhibitors, and by RNA interference directed against ERK2 or MEK2 but not ERK1 or MEK1 and against Nur77. The MAPK pathway is also activated in neurons in primary culture undergoing NK(1)R-mediated death, since the MEK inhibitor PD98059 inhibited Substance P-induced death in primary striatal neurons. These results suggest that Nur77, which is regulated by a MAPK pathway activated via arrestin 2, modulates NK(1)R-mediated nonapoptotic pcd.
Assuntos
Arrestinas/fisiologia , Morte Celular , Proteínas de Ligação a DNA/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Fosfoproteínas/fisiologia , Receptores da Neurocinina-1/fisiologia , Substância P/metabolismo , Fatores de Transcrição/fisiologia , Animais , Apoptose , Western Blotting , Cálcio/química , Cálcio/metabolismo , Linhagem Celular , Corpo Estriado/embriologia , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Genes Dominantes , Humanos , Ligantes , MAP Quinase Quinase 2 , Sistema de Sinalização das MAP Quinases , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Neurônios/metabolismo , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares , Análise de Sequência com Séries de Oligonucleotídeos , Plasmídeos/metabolismo , Proteínas Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Interferência de RNA , Ratos , Ratos Sprague-Dawley , Receptores Citoplasmáticos e Nucleares , Receptores de Esteroides , Transdução de Sinais , Fatores de Tempo , TransfecçãoRESUMO
Alterations in Ca2+ homeostasis and accumulation of misfolded proteins in the endoplasmic reticulum (ER) cause ER stress that ultimately leads to programmed cell death. Recent studies have shown that ER stress triggers programmed cell death via an alternative intrinsic pathway of apoptosis that, unlike the intrinsic pathway described previously, is independent of Apaf-1 and cytochrome c. In the present work, we have used a set of complementary approaches, including two-dimensional gel electrophoresis coupled with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry and nano-liquid chromatography-electrospray ionization mass spectrometry with tandem mass spectrometry, RNA interference, co-immunoprecipitation, immunodepletion of candidate proteins, and reconstitution studies, to identify mediators of the ER stress-induced cell death pathway. Our data identify two molecules, valosin-containing protein and apoptosis-linked gene-2 (ALG-2), that appear to play a role in mediating ER stress-induced cell death.