RESUMO
Histone deacetylase 2 (HDAC2) is a major HDAC protein in the adult brain and has been shown to regulate many neuronal genes. The aberrant expression of HDAC2 and subsequent dysregulation of neuronal gene expression is implicated in neurodegeneration and brain aging. Human induced pluripotent stem cell-derived neurons (hiPSC-Ns) are widely used models for studying neurodegenerative disease mechanisms, but the role of HDAC2 in hiPSC-N differentiation and maturation has not been explored. In this study, we show that levels of HDAC2 progressively decrease as hiPSCs are differentiated towards neurons. This suppression of HDAC2 inversely corresponds to an increase in neuron-specific isoforms of Endophilin-B1, a multifunctional protein involved in mitochondrial dynamics. Expression of neuron-specific isoforms of Endophilin-B1 is accompanied by concomitant expression of a neuron-specific alternative splicing factor, SRRM4. Manipulation of HDAC2 and Endophilin-B1 using lentiviral approaches shows that the knock-down of HDAC2 or the overexpression of a neuron-specific Endophilin-B1 isoform promotes mitochondrial elongation and protects against cytotoxic stress in hiPSC-Ns, while HDAC2 knock-down specifically influences genes regulating mitochondrial dynamics and synaptogenesis. Furthermore, HDAC2 knock-down promotes enhanced mitochondrial respiration and reduces levels of neurotoxic amyloid beta peptides. Collectively, our study demonstrates a role for HDAC2 in hiPSC-neuronal differentiation, highlights neuron-specific isoforms of Endophilin-B1 as a marker of differentiating hiPSC-Ns and demonstrates that HDAC2 regulates key neuronal and mitochondrial pathways in hiPSC-Ns.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Histona Desacetilase 2/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/fisiologia , Neurônios/metabolismo , Neurônios/fisiologia , Aciltransferases/metabolismo , Biomarcadores/metabolismo , Encéfalo/metabolismo , Encéfalo/fisiologia , Diferenciação Celular/fisiologia , Células Cultivadas , Humanos , Mitocôndrias/fisiologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Isoformas de Proteínas/metabolismoRESUMO
Endophilin-B1, also known as Bax-interacting factor 1 (Bif-1, and encoded by SH3GLB1), is a multifunctional protein involved in apoptosis, autophagy and mitochondrial function. We recently described a unique neuroprotective role for neuron-specific alternatively spliced isoforms of endophilin-B1. To examine whether endophilin-B1-mediated neuroprotection could be a novel therapeutic target for Alzheimer's disease we used a double mutant amyloid precursor protein and presenilin 1 (APPswe/PSEN1dE9) mouse model of Alzheimer's disease and observed that expression of neuron-specific endophilin-B1 isoforms declined with disease progression. To determine if this reduction in endophilin-B1 has a functional role in Alzheimer's disease pathogenesis, we crossed endophilin-B1(-/-) mice with APPswe/PSEN1dE9 mice. Deletion of endophilin-B1 accelerated disease onset and progression in 6-month-old APPswe/PSEN1dE9/endophilin-B1(-/-) mice, which showed more plaques, astrogliosis, synaptic degeneration, cognitive impairment and mortality than APPswe/PSEN1dE9 mice. In mouse primary cortical neuron cultures, overexpression of neuron-specific endophilin-B1 isoforms protected against amyloid-ß-induced apoptosis and mitochondrial dysfunction. Additionally, protein and mRNA levels of neuron-specific endophilin-B1 isoforms were also selectively decreased in the cerebral cortex and in the synaptic compartment of patients with Alzheimer's disease. Flow sorting of synaptosomes from patients with Alzheimer's disease demonstrated a negative correlation between amyloid-ß and endophilin-B1 levels. The importance of endophilin-B1 in neuronal function was further underscored by the development of synaptic degeneration and cognitive and motor impairment in endophilin-B1(-/-) mice by 12 months. Our findings suggest that endophilin-B1 is a key mediator of a feed-forward mechanism of Alzheimer's disease pathogenesis where amyloid-ß reduces neuron-specific endophilin-B1, which in turn enhances amyloid-ß accumulation and neuronal vulnerability to stress.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Doença de Alzheimer/metabolismo , Neurônios/patologia , Idoso de 80 Anos ou mais , Doença de Alzheimer/patologia , Animais , Western Blotting , Células Cultivadas , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Imunofluorescência , Humanos , Immunoblotting , Masculino , Aprendizagem em Labirinto , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sinaptossomos/metabolismo , Sinaptossomos/patologiaRESUMO
Bax-interacting factor 1 (Bif-1, also known as endophilin B1) is a multifunctional protein involved in the regulation of apoptosis, mitochondrial morphology, and autophagy. Previous studies in non-neuronal cells have shown that Bif-1 is proapoptotic and promotes mitochondrial fragmentation. However, the role of Bif-1 in postmitotic neurons has not been investigated. In contrast to non-neuronal cells, we now report that in neurons Bif-1 promotes viability and mitochondrial elongation. In mouse primary cortical neurons, Bif-1 knockdown exacerbated apoptosis induced by the DNA-damaging agent camptothecin. Neurons from Bif-1-deficient mice contained fragmented mitochondria and Bif-1 knockdown in wild-type neurons also resulted in fragmented mitochondria which were more depolarized, suggesting mitochondrial dysfunction. During ischemic stroke, Bif-1 expression was downregulated in the penumbra of wild-type mice. Consistent with Bif-1 being required for neuronal viability, Bif-1-deficient mice developed larger infarcts and an exaggerated astrogliosis response following ischemic stroke. Together, these data suggest that, in contrast to non-neuronal cells, Bif-1 is essential for the maintenance of mitochondrial morphology and function in neurons, and that loss of Bif-1 renders neurons more susceptible to apoptotic stress. These unique actions may relate to the presence of longer, neuron-specific Bif-1 isoforms, because only these forms of Bif-1 were able to rescue deficiencies caused by Bif-1 suppression. This finding not only demonstrates an unexpected role for Bif-1 in the nervous system but this work also establishes Bif-1 as a potential therapeutic target for the treatment of neurological diseases, especially degenerative disorders characterized by alterations in mitochondrial dynamics.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose/fisiologia , Mitocôndrias/ultraestrutura , Neurônios/metabolismo , Animais , Sobrevivência Celular , Modelos Animais de Doenças , Imunofluorescência , Immunoblotting , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Mitocôndrias/metabolismo , Neurônios/ultraestrutura , Isoformas de Proteínas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologiaRESUMO
The p53 tumor suppressor plays a central role in dictating cell survival and death as a cellular sensor for a myriad of stresses including DNA damage, oxidative and nutritional stress, ischemia and disruption of nucleolar function. Activation of p53-dependent apoptosis leads to mitochondrial apoptotic changes via the intrinsic and extrinsic pathways triggering cell death execution most notably by release of cytochrome c and activation of the caspase cascade. Although it was previously believed that p53 induces apoptotic mitochondrial changes exclusively through transcription-dependent mechanisms, recent studies suggest that p53 also regulates apoptosis via a transcription-independent action at the mitochondria. Recent evidence further suggests that p53 can regulate necrotic cell death and autophagic activity including mitophagy. An increasing number of cytosolic and mitochondrial proteins involved in mitochondrial metabolism and respiration are regulated by p53, which influences mitochondrial ROS production as well. Cellular redox homeostasis is also directly regulated by p53 through modified expression of pro- and anti-oxidant proteins. Proper regulation of mitochondrial size and shape through fission and fusion assures optimal mitochondrial bioenergetic function while enabling adequate mitochondrial transport to accommodate local energy demands unique to neuronal architecture. Abnormal regulation of mitochondrial dynamics has been increasingly implicated in neurodegeneration, where elevated levels of p53 may have a direct contribution as the expression of some fission/fusion proteins are directly regulated by p53. Thus, p53 may have a much wider influence on mitochondrial integrity and function than one would expect from its well-established ability to transcriptionally induce mitochondrial apoptosis. However, much of the evidence demonstrating that p53 can influence mitochondria through nuclear, cytosolic or intra-mitochondrial sites of action has yet to be confirmed in neurons. Nonetheless, as mitochondria are essential for supporting normal neuronal functions and in initiating/propagating cell death signaling, it appears certain that the mitochondria-related functions of p53 will have broader implications than previously thought in acute and progressive neurological conditions, providing new therapeutic targets for treatment.
Assuntos
Mitocôndrias/metabolismo , Neurônios/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose , Autofagia , Humanos , Dinâmica MitocondrialRESUMO
Maintaining proper mitochondrial length is essential for normal mitochondrial function in neurons. Mitochondrial fragmentation has been associated with neuronal cell death caused by a variety of experimental toxic stressors. Despite the fact that oxidative stress is a hallmark of neurodegenerative conditions and aging and the resulting activation of p53 is believed to contribute to the neuropathology, little is still known regarding changes in mitochondrial morphology in p53-dependent neuronal death. Therefore, we specifically addressed the relationship between genotoxic stress, p53 activation, and the regulation of mitochondrial morphology in neurons. In cultured postnatal mouse cortical neurons, treatment with the DNA-damaging agent camptothecin (CPT) resulted in elongated mitochondria, in contrast to fragmented mitochondria observed upon staurosporine and glutamate treatment. In fibroblasts, however, CPT resulted in fragmented mitochondria. CPT treatment in neurons suppressed expression of the mitochondrial fission protein Drp1 and the E3 ubiquitin ligase parkin. The presence of elongated mitochondria and the declines in Drp1 and parkin expression occurred before the commitment point for apoptosis. The CPT-induced changes in Drp1 and parkin were not observed in p53-deficient neurons, while p53 overexpression alone was sufficient to reduce the expression of the two proteins. Elevating Drp1 or parkin expression before CPT treatment enhanced neuronal viability and restored a normal pattern of mitochondrial morphology. The present findings demonstrate that genotoxic stress in neurons results in elongated mitochondria in contrast to fission induced by other forms of stress, and p53-dependent declines in Drp1 and parkin levels contribute to altered mitochondrial morphology and cell death.
Assuntos
Dano ao DNA/fisiologia , GTP Fosfo-Hidrolases/biossíntese , Proteínas Associadas aos Microtúbulos/biossíntese , Mitocôndrias/patologia , Proteínas Mitocondriais/biossíntese , Neurônios/patologia , Ubiquitina-Proteína Ligases/biossíntese , Animais , Morte Celular/fisiologia , Células Cultivadas , Dinaminas , Imunofluorescência , GTP Fosfo-Hidrolases/genética , Humanos , Immunoblotting , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Proteínas Mitocondriais/genética , Neurônios/metabolismo , Estresse Oxidativo/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
p53 plays an essential role in mediating apoptotic responses to cellular stress, especially DNA damage. In a kainic acid (KA)-induced seizure model in mice, hippocampal CA1 pyramidal cells undergo delayed neuronal death at day 3-4 following systemic KA administration. We previously demonstrated that CA1 neurons in p53(-/-) animals are protected from such apoptotic neuronal loss. However, extensive morphological damage associated with DNA strand breaks in CA1 neurons was found in a fraction of p53(-/-) animals at earlier time points (8 h to 2 days). No comparable acute damage was observed in wild-type animals. Stereological counting confirmed that there was no significant loss of CA1 pyramidal cells in p53(-/-) animals at 7 days post-KA injection. These results suggest that seizure-induced DNA strand breaks are accumulated to a greater extent but do not lead to apoptosis in the absence of p53. In wild-type animals, therefore, p53 appears to stimulate DNA repair and also mediate apoptosis in CA1 neurons in this excitotoxicity model. These results also reflect remarkable plasticity of neurons in recovery from injury.
Assuntos
Região CA1 Hipocampal/patologia , Dano ao DNA/fisiologia , Agonistas de Aminoácidos Excitatórios/toxicidade , Ácido Caínico/toxicidade , Células Piramidais/patologia , Proteína Supressora de Tumor p53/genética , Animais , Apoptose/efeitos dos fármacos , Contagem de Células , Sobrevivência Celular , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Feminino , Masculino , Camundongos , Camundongos Knockout , Perfusão , Gravidez , Convulsões/induzido quimicamente , Convulsões/genética , Convulsões/patologiaRESUMO
BACKGROUND: Early-onset familial Alzheimer disease (EOFAD) is caused by heterozygous variants in the presenilin 1 (PSEN1), presenilin 2 (PSEN2), and APP genes. Decades after their discovery, the mechanisms by which these genes cause Alzheimer's disease (AD) or promote AD progression are not fully understood. While it is established that presenilin (PS) enzymatic activity produces amyloid-ß (Aß), PSs also regulate numerous other cellular functions, some of which intersect with known pathogenic drivers of neurodegeneration. Accumulating evidence suggests that microglia, resident innate immune cells in the central nervous system, play a key role in AD neurodegeneration. OBJECTIVE: Previous work has identified a regulatory role for PS2 in microglia. We hypothesized that PSEN2 variants lead to dysregulated microglia, which could further contribute to disease acceleration. To mimic the genotype of EOFAD patients, we created a transgenic mouse expressing PSEN2 N141I on a mouse background expressing one wildtype PS2 and two PS1 alleles. RESULTS: Microglial expression of PSEN2 N141I resulted in impaired γ-secretase activity as well as exaggerated inflammatory cytokine release, NFκB activity, and Aß internalization. In vivo, PS2 N141I mice showed enhanced IL-6 and TREM2 expression in brain as well as reduced branch number and length, an indication of "activated" morphology, in the absence of inflammatory stimuli. LPS intraperitoneal injection resulted in higher inflammatory gene expression in PS2 N141I mouse brain relative to controls. CONCLUSION: Our findings demonstrate that PSEN2 N141I heterozygosity is associated with disrupted innate immune homeostasis, suggesting EOFAD variants may promote disease progression through non-neuronal cells beyond canonical dysregulated Aß production.
Assuntos
Doença de Alzheimer/genética , Variação Genética/genética , Heterozigoto , Microglia/fisiologia , Fenótipo , Presenilina-2/genética , Doença de Alzheimer/patologia , Animais , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/patologiaRESUMO
Histone deacetylases (HDACs) catalyze acetyl group removal from histone proteins, leading to altered chromatin structure and gene expression. HDAC2 is highly expressed in adult brain, and HDAC2 levels are elevated in Alzheimer's disease (AD) brain. We previously reported that neuron-specific splice isoforms of Endophilin-B1 (Endo-B1) promote neuronal survival, but are reduced in human AD brain and mouse models of AD and stroke. Here, we demonstrate that HDAC2 suppresses Endo-B1 expression. HDAC2 knockdown or knockout enhances expression of Endo-B1. Conversely, HDAC2 overexpression decreases Endo-B1 expression. We also demonstrate that neurons exposed to beta-amyloid increase HDAC2 and reduce histone H3 acetylation while HDAC2 knockdown prevents Aß induced loss of histone H3 acetylation, mitochondrial dysfunction, caspase-3 activation, and neuronal death. The protective effect of HDAC2 knockdown was abrogated by Endo-B1 shRNA and in Endo-B1-null neurons, suggesting that HDAC2-induced neurotoxicity is mediated through suppression of Endo-B1. HDAC2 overexpression also modulates neuronal expression of mitofusin2 (Mfn2) and mitochondrial fission factor (MFF), recapitulating the pattern of change observed in AD. HDAC2 knockout mice demonstrate reduced injury in the middle cerebral artery occlusion with reperfusion (MCAO/R) model of cerebral ischemia demonstrating enhanced neuronal survival, minimized loss of Endo-B1, and normalized expression of Mfn2. These findings support the hypothesis that HDAC2 represses Endo-B1, sensitizing neurons to mitochondrial dysfunction and cell death in stroke and AD.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Histona Desacetilase 2/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/toxicidade , Animais , Encéfalo/metabolismo , Isquemia Encefálica/metabolismo , Modelos Animais de Doenças , GTP Fosfo-Hidrolases/metabolismo , Regulação da Expressão Gênica/genética , Histona Desacetilase 2/genética , Histona Desacetilase 2/metabolismo , Histona Desacetilases/genética , Histonas/genética , Isquemia/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/fisiologia , Acidente Vascular Cerebral/fisiopatologiaRESUMO
Recent studies in non-neuronal cells have shown that the tumor suppressor p53 can promote cell death through a transcription-independent mechanism involving its direct action with a subset of Bcl-2 family member proteins in the cytosol and at the mitochondria. In cultured cortical neurons, however, we could not find evidence supporting a significant contribution of the cytosolic/mitochondrial p53 pathway, and available evidence instead corroborated the requirement for the transcriptional activity of p53. When directly targeted to the cytosol/mitochondria, wild-type p53 lost its apoptosis-inducing activity in neurons but not in non-neuronal cells. The N-terminal p53 fragment (transactivation and proline-rich domains), which induces apoptosis in non-neuronal cells via the cytosolic/mitochondrial pathway, displayed no apoptogenic activity in neurons. In neuronal apoptosis induced by camptothecin or an MDM2 (murine double minute 2) inhibitor, nutlin-3, endogenous p53 protein did not accumulate in the cytosol/mitochondria, and transcriptional inhibition after p53 induction effectively blocked cell death. In addition, overexpression of a dominant-negative form of p53 (R273H) completely suppressed induction of proapoptotic p53 target genes and cell death. PUMA (p53-upregulated modulator of apoptosis) was one such gene induced by camptothecin, and its overexpression was sufficient to induce Bax (Bcl-2-associated X protein)-dependent neuronal death, whereas Noxa was not apoptogenic. These results collectively demonstrate that, in contrast to non-neuronal cells, the apoptotic activity of p53 in postnatal cortical neurons does not rely on its direct action at the cytosol/mitochondria but is exclusively mediated through its transcription-dependent functions. The uniqueness of p53-mediated apoptotic signaling in postnatal cortical neurons was further illustrated by the dispensable function of the proline-rich domain of p53.
Assuntos
Apoptose/fisiologia , Citosol/metabolismo , Mitocôndrias/metabolismo , Neurônios/metabolismo , Ativação Transcricional/fisiologia , Proteína Supressora de Tumor p53/biossíntese , Proteínas Supressoras de Tumor/metabolismo , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose , Camptotecina/farmacologia , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/fisiologia , Citosol/efeitos dos fármacos , Humanos , Camundongos , Camundongos Transgênicos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Células NIH 3T3 , Neurônios/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/fisiologiaRESUMO
BACKGROUND: In epithelial ovarian cancer (EOC), fertility-sparing surgery (FSS) has mainly been chosen for stage IA disease. The purpose of this study was to clarify the clinical outcome of patients with clear-cell carcinoma of the ovary (CCC) who would usually undergo radical surgery. CASES: After a central pathological review and search of the medical records from multiple institutions between 1988 and 2005, a total of 10 CCC patients treated with FSS were retrospectively evaluated in the current study. The mean age was 35.9 years (range: 32-39 years). The median follow-up time was 35.4 months (range: 21.7-153.2 months). The stage was IA in 4 patients, and IC in 6 patients [IC(b) in 5 patients, and IC(2) in one]. Nine patients received adjuvant chemotherapy. Nine patients were alive and one patient with stage IC(2) died of the disease at a follow-up time of 36.8 months. Five pregnancies were observed in 4 patients. CONCLUSIONS: Although there is no worldwide criterion for FSS in CCC patients at present, it seems that, in selected patients, this surgical approach could be adopted. This should be investigated by additional studies in a larger series.
Assuntos
Adenocarcinoma de Células Claras/cirurgia , Fertilidade , Neoplasias Ovarianas/cirurgia , Adenocarcinoma de Células Claras/patologia , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Quimioterapia Adjuvante , Cisplatino/administração & dosagem , Feminino , Seguimentos , Procedimentos Cirúrgicos em Ginecologia/métodos , Humanos , Estadiamento de Neoplasias , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologiaRESUMO
Spinocerebellar ataxia (SCA) type 7 is an inherited neurodegenerative disorder caused by expansion of a polyglutamine tract within the ataxin-7 protein. To determine the molecular basis of polyglutamine neurotoxicity in this and other related disorders, we produced SCA7 transgenic mice that express ataxin-7 with 24 or 92 glutamines in all neurons of the CNS, except for Purkinje cells. Transgenic mice expressing ataxin-7 with 92 glutamines (92Q) developed a dramatic neurological phenotype presenting as a gait ataxia and culminating in premature death. Despite the absence of expression of polyglutamine-expanded ataxin-7 in Purkinje cells, we documented severe Purkinje cell degeneration in 92Q SCA7 transgenic mice. We also detected an N-terminal truncation fragment of ataxin-7 in transgenic mice and in SCA7 patient material with both anti-ataxin-7 and anti-polyglutamine specific antibodies. The appearance of truncated ataxin-7 in nuclear aggregates correlates with the onset of a disease phenotype in the SCA7 mice, suggesting that nuclear localization and proteolytic cleavage may be important features of SCA7 pathogenesis. The non-cell-autonomous nature of the Purkinje cell degeneration in our SCA7 mouse model indicates that polyglutamine-induced dysfunction in adjacent or connecting cell types contributes to the neurodegeneration.
Assuntos
Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Peptídeos/genética , Células de Purkinje/patologia , Degenerações Espinocerebelares/etiologia , Animais , Ataxina-7 , Núcleo Celular/patologia , Marcha Atáxica/etiologia , Marcha Atáxica/metabolismo , Marcha Atáxica/patologia , Corpos de Inclusão/patologia , Cinética , Camundongos , Camundongos Transgênicos , Mutação , Proteínas do Tecido Nervoso/fisiologia , Degenerações Espinocerebelares/metabolismo , Degenerações Espinocerebelares/patologiaRESUMO
Technical developments in the field of proteomics are poised to generate advances in our understanding of protein structure, function, and organization in complex signaling and regulatory networks. Improvements in mass spectrometry instrumentation, the implementation of protein arrays, and the development of robust informatics software are providing sensitive, high-throughput technologies for large-scale identification and quantitation of protein expression, protein modifications, subcellular localization, protein function, and protein-protein interactions. These advances have significant implications for understanding how cellular proteomes are regulated in health and disease.
Assuntos
Proteômica , Animais , Perfilação da Expressão Gênica/métodos , Genoma , Humanos , Proteoma/análise , Proteoma/química , Proteoma/metabolismoRESUMO
The continuing improvement and refinement of proteomic and bioinformatic tools has made it possible to obtain increasing amounts of structural and functional information about proteins on a global scale. The emerging field of neuroproteomics promises to provide powerful strategies for further characterizing neuronal dysfunction and cell loss associated with neurodegenerative diseases. Neuroproteomic studies have thus far revealed relatively comprehensive quantitative changes and post-translational modifications (mostly oxidative damage) of high abundance proteins, confirming deficits in energy production, protein degradation, antioxidant protein function, and cytoskeletal regulation associated with neurodegenerative diseases such as Alzheimer and Parkinson disease. The identification of changes in low-abundance proteins and characterization of their functions based on protein-protein interactions still await further development of proteomic methodologies and more dedicated application of these technologies by neuroscientists. Once accomplished, however, the resulting information will certainly provide a truly comprehensive view of neurodegeneration-associated changes in protein expression, facilitating the identification of novel biomarkers for the early detection of neurodegenerative diseases and new targets for therapeutic intervention.
Assuntos
Doenças Neurodegenerativas/genética , Proteômica , Doença de Alzheimer/genética , Animais , Síndrome de Down/genética , Glutamina/genética , Glutamina/fisiologia , Humanos , Doença dos Neurônios Motores/genética , Doença de Parkinson/genética , Traumatismos do Sistema Nervoso/genética , Proteínas tau/genéticaRESUMO
Neuronal viability is maintained through a complex interacting network of signaling pathways that can be perturbed in response to a multitude of cellular stresses. A shift in the balance of signaling pathways after stress or in response to pathology can have drastic consequences for the function or the fate of a neuron. There is significant evidence that acutely injured and degenerating neurons may die by an active mechanism of cell death. This process involves the activation of discrete signaling pathways that ultimately compromise mitochondrial structure, energy metabolism and nuclear integrity. In this review we examine recent evidence pertaining to the presence and activation of anti- and pro-cell death regulatory pathways in nervous system injury and degeneration.
Assuntos
Morte Celular/fisiologia , Sobrevivência Celular/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Neurônios/fisiologia , Calpaína/metabolismo , Inibidores de Caspase , Caspases/metabolismo , Núcleo Celular/metabolismo , Dano ao DNA , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Mitocôndrias/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptor de Fator de Crescimento Neural , Receptores de Fator de Crescimento Neural/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Mitochondria exist as dynamic networks that are constantly remodeled through the opposing actions of fusion and fission proteins. Changes in the expression of these proteins alter mitochondrial shape and size, and may promote or inhibit the propagation of apoptotic signals. Using mitochondrially targeted EGFP or DsRed2 to identify mitochondria, we observed a short, distinctly tubular mitochondrial morphology in postnatal cortical neurons in culture and in retinal ganglion cells in vivo, whereas longer, highly interconnected mitochondrial networks were detected in cortical astrocytes in vitro and non-neuronal cells in the retina in vivo. Differential expression patterns of fusion and fission proteins, in part, appear to determine these morphological differences as neurons expressed markedly high levels of Drp1 and OPA1 proteins compared to non-neuronal cells. This finding was corroborated using optic tissue samples. Moreover, cortical neurons expressed several splice variants of Drp1 including a neuron-specific isoform which incorporates exon 3. Knockdown or dominant-negative interference of endogenous Drp1 significantly increased mitochondrial length in both neurons and non-neuronal cells, but caused cell death only in cortical neurons. Conversely, depletion of the fusion protein, Mfn2, but not Mfn1, caused extensive mitochondrial fission and cell death. Thus, Drp1 and Mfn2 in normal cortical neurons not only regulate mitochondrial morphology, but are also required for cell survival. The present findings point to unique patterns of Drp1 expression and selective vulnerability to reduced levels of Drp1 expression/activity in neurons, and demonstrate that the regulation of mitochondrial dynamics must be tightly regulated in neurons.
Assuntos
Córtex Cerebral/citologia , GTP Fosfo-Hidrolases/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Neurônios/metabolismo , Animais , Sobrevivência Celular , Células Cultivadas , Dinaminas , Imunofluorescência , Células HeLa , Hipocampo/citologia , Humanos , Immunoblotting , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/fisiologia , Neurônios/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Loss of p53 function is a common event in a variety of human cancers including tumors of glial origin. Using an in vitro mouse model of malignant astrocyte transformation, three cleavable isotope coded affinity tag (cICAT) experiments were performed comparing cultured wild-type astrocytes and two p53(-/-) astrocyte cultures before and after malignant transformation. We identified and quantitated an average of 1366 proteins per experiment and demonstrated that the protein quantitation ratios in each individual cICAT experiment correlated well to ratios determined in the other two studies. These data were further supported by microarray analysis which also correlated to changes in protein expression. The results showed significant changes in protein expression in association with malignant transformation. Proteins overexpressed in malignant astrocytes were typically involved in ribosome biogenesis/protein synthesis and DNA replication, while underexpressed proteins were generally associated with the regulation of cell cycle checkpoint control, tumor suppression, and apoptosis. Among the significantly up-regulated proteins and transcripts in malignant mouse astrocytes were members of the minichromosome maintenance (MCM) family. Western blot analysis verified increased expression of MCM proteins in malignant human astrocytoma cell lines, which had not previously been described. These results demonstrate the usefulness of the cICAT approach for comparing differences in protein expression profiles between normal and malignant cells.
RESUMO
Bak is generally recognized as a multidomain, pro-apoptotic member of the Bcl-2 family. Bak and Bax are functionally redundant in non-neuronal cells and represent a mitochondrial convergence point for cell death signaling pathways. This functional redundancy, however, may not exist in neurons in which the single deletion of Bax is sufficient to confer protection against a variety of cytotoxic insults. In the present study, we demonstrate that postnatal cortical and cerebellar granule neurons exclusively express an alternatively spliced, BH3 domain-only form of Bak (N-Bak), whereas astrocytes express only the full-length, multidomain form. Overexpression of N-Bak promotes Bax translocation in HeLa cells and induces neuronal cell death in cortical, hippocampal, and cerebellar granule neurons in a Bax-dependent manner. N-Bak interacts with Bcl-XL but not BAX, suggesting an indirect mechanism for promoting Bax translocation to the mitochondria. N-Bak message and protein levels are elevated in cortical neurons in response to DNA damage, and subsequent induction of neuronal death is significantly delayed by expressing a full-length Bak antisense plasmid. These results demonstrate that postnatal neurons solely express a BH3 domain-only form of Bak, which contributes to DNA damage-induced neuronal apoptosis. The absence of full-length Bak expression explains the near exclusive requirement for Bax in neuronal apoptosis.
Assuntos
Apoptose/fisiologia , Proteínas de Membrana/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Animais , Apoptose/efeitos dos fármacos , Camptotecina/farmacologia , Dano ao DNA , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/deficiência , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2 , Proteína X Associada a bcl-2RESUMO
The p53 tumor suppressor gene is a sequence-specific transcription factor that activates the expression of genes engaged in promoting growth arrest or cell death in response to multiple forms of cellular stress. p53 expression is elevated in damaged neurons in acute models of injury such as ischemia and epilepsy and in brain tissue samples derived from animal models and patients with chronic neurodegenerative diseases. p53 deficiency or p53 inhibition protects neurons from a wide variety of acute toxic insults. Signal transduction pathways associated with p53-induced neuronal cell death are being characterized, suggesting that intervention may prove effective in maintaining neuronal viability and restoring function following neural injury and disease.
Assuntos
Apoptose/fisiologia , Neurônios/metabolismo , Proteína Supressora de Tumor p53/fisiologia , Animais , Humanos , Neurônios/citologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
Proteomics is a field of study directed toward providing a comprehensive view of the characteristics and activity of every cellular protein. Rapid innovations in the core technologies required to characterize proteins on a global scale are poised to bring about a comprehensive understanding of how dynamic changes in protein expression, post-translational modification, and function affect complex signaling and regulatory networks. These advances have significant implications for understanding the multitude of pathways that govern behavior and cognition and the response of the nervous system to injury and disease.