RESUMEN
Down syndrome (DS) is characterized by the trisomy of chromosome 21 and by cognitive deficits that have been related to neuronal morphological alterations in humans, as well as in animal models. The gene encoding for amyloid precursor protein (APP) is present in autosome 21, and its overexpression in DS has been linked to neuronal dysfunction, cognitive deficit, and Alzheimer's disease-like dementia. In particular, the neuronal ability to extend processes and branching is affected. Current evidence suggests that APP could also regulate neurite growth through its role in the actin cytoskeleton, in part by influencing p21-activated kinase (PAK) activity. The latter effect is carried out by an increased abundance of the caspase cleavage-released carboxy-terminal C31 fragment. In this work, using a neuronal cell line named CTb, which derived from the cerebral cortex of a trisomy 16 mouse, an animal model of human DS, we observed an overexpression of APP, elevated caspase activity, augmented cleavage of the C-terminal fragment of APP, and increased PAK1 phosphorylation. Morphometric analyses showed that inhibition of PAK1 activity with FRAX486 increased the average length of the neurites, the number of crossings per Sholl ring, the formation of new processes, and stimulated the loss of processes. Considering our results, we propose that PAK hyperphosphorylation impairs neurite outgrowth and remodeling in the cellular model of DS, and therefore we suggest that PAK1 may be a potential pharmacological target.
Asunto(s)
Síndrome de Down , Ratones , Humanos , Animales , Síndrome de Down/tratamiento farmacológico , Síndrome de Down/genética , Trisomía , Neuronas/metabolismo , Neuritas/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Proyección Neuronal , Caspasas/metabolismoRESUMEN
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.
RESUMEN
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.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/terapia , Precursor de Proteína beta-Amiloide/metabolismo , Terapia Genética/métodos , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteína ADAM10/metabolismo , Enfermedad de Alzheimer/psicología , Animales , Biomimética , Línea Celular Tumoral , Cognición/fisiología , Dependovirus/genética , Modelos Animales de Enfermedad , Vectores Genéticos , Humanos , Memoria a Corto Plazo/fisiología , Ratones Transgénicos , Netrina-1 , Proyectos Piloto , Ratas , Reconocimiento en Psicología/fisiología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
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.
Asunto(s)
Apolipoproteína E4/genética , Apolipoproteína E4/metabolismo , Neuronas/fisiología , Transcripción Genética/fisiología , Anciano , Animales , Secuencia de Bases , Encéfalo/fisiología , Línea Celular Tumoral , Femenino , Fibroblastos/fisiología , Humanos , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Persona de Mediana Edad , Datos de Secuencia Molecular , Unión Proteica/fisiologíaRESUMEN
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.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Precursor de Proteína beta-Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Fragmentos de Péptidos/metabolismo , Regulación Alostérica , Secuencia de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/genética , Humanos , Inmunoprecipitación , Modelos Moleculares , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Conformación Proteica , Proteínas Recombinantes/metabolismo , Dispersión del Ángulo Pequeño , Espectrometría de Fluorescencia , Difracción de Rayos XRESUMEN
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.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Inhibidores de Proteínas Quinasas/uso terapéutico , Receptor trkA/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Benzamidas/farmacología , Células CHO , Muerte Celular/efectos de los fármacos , Cricetulus , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Células HEK293 , Humanos , Ratones , Ratones Transgénicos , Mutación/genética , Factor de Crecimiento Nervioso/metabolismo , Factor de Crecimiento Nervioso/farmacología , Inhibidores de Proteínas Quinasas/química , Pirazoles/farmacología , Receptor trkA/antagonistas & inhibidores , Receptor trkA/genética , TransfecciónRESUMEN
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.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Precursor de Proteína beta-Amiloide/efectos de los fármacos , Cognición/efectos de los fármacos , Hipocampo/efectos de los fármacos , Indoles/farmacología , Indoles/uso terapéutico , Administración Oral , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Células CHO , Cricetulus , Modelos Animales de Enfermedad , Hipocampo/metabolismo , Indoles/administración & dosificación , Ratones , Ratones Endogámicos C57BL , TropisetrónRESUMEN
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.
Asunto(s)
Enfermedad de Alzheimer/fisiopatología , Apolipoproteína E4/metabolismo , Regulación de la Expresión Génica/fisiología , Sirtuinas/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Análisis de Varianza , Apolipoproteína E3/metabolismo , Western Blotting , Descubrimiento de Drogas , Humanos , Inmunoprecipitación , Fosforilación , Reacción en Cadena en Tiempo Real de la Polimerasa , Resonancia por Plasmón de Superficie , Proteínas tau/metabolismoRESUMEN
A systematic approach was used to identify AßPP-selective BACE inhibitors (ASBI) and to evaluate their in vivo ability to modulate AßPP processing selectively. We identified a bioflavonoid nutritional supplement as a molecular lead that acts as an ASBI in cell models, and show that increasing brain levels of this bioflavonoid through a pro-drug approach leads to reduction of Aß42 in an Alzheimer's disease mouse model. ASBIs represent a novel class of candidate therapeutic agents for Alzheimer's disease.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Flavonoides/farmacología , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animales , Ácido Aspártico Endopeptidasas , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Línea Celular Transformada , Cricetulus , Relación Dosis-Respuesta a Droga , Flavonoides/química , Flavonoides/farmacocinética , Humanos , Ratones , Neurregulina-1/metabolismo , Unión Proteica , Rutina/química , Rutina/farmacología , Resonancia por Plasmón de Superficie , Factores de Tiempo , TransfecciónRESUMEN
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.
Asunto(s)
Estrés del Retículo Endoplásmico/fisiología , Oxidorreductasas Intramoleculares/fisiología , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Apoptosis/fisiología , Encéfalo/metabolismo , Encéfalo/patología , Hipoxia de la Célula/fisiología , Células Cultivadas/metabolismo , Citosol/química , Modelos Animales de Enfermedad , Epítopos/inmunología , Femenino , Fibroblastos/metabolismo , Células HEK293/metabolismo , Humanos , Oxidorreductasas Intramoleculares/análisis , Oxidorreductasas Intramoleculares/biosíntesis , Oxidorreductasas Intramoleculares/inmunología , Masculino , Ratones , Ratones Transgénicos , Microscopía Fluorescente , Datos de Secuencia Molecular , Neuronas/metabolismo , Prostaglandina-E Sintasas , Proteínas Recombinantes de Fusión/fisiología , Tapsigargina/farmacología , TransfecciónRESUMEN
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.
Asunto(s)
Péptidos beta-Amiloides/farmacología , Precursor de Proteína beta-Amiloide/efectos de los fármacos , Precursor de Proteína beta-Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/química , Humanos , Modelos Moleculares , Peso Molecular , Fragmentos de Péptidos , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína/efectos de los fármacos , Estructura Terciaria de Proteína/fisiologíaRESUMEN
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.
Asunto(s)
Adenosina Trifosfatasas/genética , Proteínas de Ciclo Celular/genética , Mutación , Degeneración Nerviosa/patología , Fenotipo , Adenosina Trifosfatasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Muerte Celular/genética , Retículo Endoplásmico/metabolismo , Inhibidores Enzimáticos/metabolismo , Células HEK293 , Humanos , Proteína Huntingtina , Degeneración Nerviosa/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Tapsigargina/metabolismo , Ubiquitina/metabolismo , Proteína que Contiene ValosinaRESUMEN
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.
Asunto(s)
Apoptosis/fisiología , Apoptosis/genética , Western Blotting , Línea Celular , Biología Computacional , Bases de Datos de Proteínas , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Humanos , Inmunohistoquímica , Proteínas de Unión a Fosfatidiletanolamina/genética , Proteínas de Unión a Fosfatidiletanolamina/metabolismo , Prohibitinas , Proteómica , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Espectrometría de Masas en TándemRESUMEN
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.
Asunto(s)
Antioxidantes/farmacología , Muerte Celular/efectos de los fármacos , Dopamina/metabolismo , Retículo Endoplásmico/efectos de los fármacos , Estrés Oxidativo , Estilbenos/farmacología , Animales , Caspasas/metabolismo , Línea Celular , Cinamatos/farmacología , Retículo Endoplásmico/metabolismo , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Interferencia de ARN , Ratas , Resveratrol , Tiourea/análogos & derivados , Tiourea/farmacologíaRESUMEN
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.
Asunto(s)
Apoptosis/efectos de los fármacos , Retículo Endoplásmico/efectos de los fármacos , Oxidorreductasas Intramoleculares/metabolismo , Chaperonas Moleculares/metabolismo , Neuronas/efectos de los fármacos , Paraquat/toxicidad , Estrés Fisiológico/efectos de los fármacos , Animales , Apoptosis/fisiología , Caspasas/efectos de los fármacos , Caspasas/metabolismo , Dominio Catalítico/efectos de los fármacos , Dominio Catalítico/fisiología , Línea Celular Transformada , Citoprotección/fisiología , Dopamina/metabolismo , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/patología , Herbicidas/toxicidad , Neuronas/metabolismo , Neuronas/patología , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Enfermedad de Parkinson/etiología , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/fisiopatología , Prostaglandina-E Sintasas , Pliegue de Proteína/efectos de los fármacos , Ratas , Estrés Fisiológico/fisiología , Sustancia Negra/efectos de los fármacos , Sustancia Negra/metabolismo , Sustancia Negra/fisiopatologíaRESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Curcumina/farmacología , Retículo Endoplásmico/efectos de los fármacos , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/patología , Animales , Apoptosis/fisiología , Caspasas/metabolismo , Línea Celular Tumoral , Quelantes/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Retículo Endoplásmico/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Oxidorreductasas Intramoleculares/metabolismo , Melanoma Experimental/metabolismo , Ratones , Poli(ADP-Ribosa) Polimerasas/metabolismo , Prostaglandina-E Sintasas , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Factor de Transcripción CHOP/metabolismoRESUMEN
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.
Asunto(s)
Arrestinas/fisiología , Muerte Celular , Proteínas de Unión al ADN/fisiología , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Fosfoproteínas/fisiología , Receptores de Neuroquinina-1/fisiología , Sustancia P/metabolismo , Factores de Transcripción/fisiología , Animales , Apoptosis , Western Blotting , Calcio/química , Calcio/metabolismo , Línea Celular , Cuerpo Estriado/embriología , AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Proteínas de Unión al GTP/metabolismo , Genes Dominantes , Humanos , Ligandos , MAP Quinasa Quinasa 2 , Sistema de Señalización de MAP Quinasas , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Modelos Biológicos , Neuronas/metabolismo , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Análisis de Secuencia por Matrices de Oligonucleótidos , Plásmidos/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Interferencia de ARN , Ratas , Ratas Sprague-Dawley , Receptores Citoplasmáticos y Nucleares , Receptores de Esteroides , Transducción de Señal , Factores de Tiempo , TransfecciónRESUMEN
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.