RESUMEN
The observation that aging is regulated by microRNAs (miRNA) and at the same time represents the greatest risk factor for Alzheimer's disease (AD), prompted us to examine the circulating miRNA network in AD beyond aging. We here show that plasma miRNAs in aging are downregulated and predicted to be preferentially targeted to the extracellular vesicle (EV) content. In AD, miRNAs are further downregulated, display altered proportions of motifs relevant to their loading into EVs and secretion propensity, and are forecast to be found exclusively in EVs. The circulating miRNA network in AD, therefore, reflects pathological exacerbation of the aging process whereby physiological suppression of AD pathology by miRNAs becomes insufficient.
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Enfermedad de Alzheimer , Vesículas Extracelulares , MicroARNs , Humanos , MicroARNs/genética , Enfermedad de Alzheimer/genética , Envejecimiento/genéticaRESUMEN
MicroRNAs (miRNAs) regulate the translational potential of their mRNA targets and control many cellular processes. The key step in canonical miRNA biogenesis is the cleavage of the primary transcripts by the nuclear RNase III enzyme Drosha. Emerging evidence suggests that the miRNA biogenic cascade is tightly controlled. However, little is known whether Drosha is regulated. Here, we show that Drosha is targeted by stress. Under stress, p38 MAPK directly phosphorylates Drosha at its N terminus. This reduces its interaction with DiGeorge syndrome critical region gene 8 and promotes its nuclear export and degradation by calpain. This regulatory mechanism mediates stress-induced inhibition of Drosha function. Reduction of Drosha sensitizes cells to stress and increases death. In contrast, increase in Drosha attenuates stress-induced death. These findings reveal a critical regulatory mechanism by which stress engages p38 MAPK pathway to destabilize Drosha and inhibit Drosha-mediated cellular survival.
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Ribonucleasa III/fisiología , Estrés Fisiológico , Proteínas Quinasas p38 Activadas por Mitógenos/fisiología , Transporte Activo de Núcleo Celular , Supervivencia Celular , Células HEK293 , Humanos , Fosforilación , Proteolisis , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/fisiología , Ribonucleasa III/genética , Ribonucleasa III/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Cancer stem cells (CSCs) are a source of tumour recurrence in patients with nasopharyngeal carcinoma (NPC); however, the function of microRNA-124 (miR-124) in NPC CSCs has not been clearly defined. In this study, we investigated the role of miR-124 in NPC CSCs. qRT-PCR was performed to measure miR-124 expression in NPC tissues and cell lines and the effects of miR-124 on stem-like properties and radiosensitivity of NPC cells measured. Luciferase reporter assays and rescue experiments were used to investigate the interaction of miR-124 with the 3'UTR of junctional adhesion molecule A (JAMA). Finally, we examined the effects of miR-124 in an animal model and clinical samples. Down-regulation of miR-124 was detected in cancer tissues and was inversely associated with tumour stage and lymph node metastasis. Overexpression of miR-124 inhibited stemness properties and enhanced radiosensitivity of NPC cells in vitro and in vivo via targeting JAMA. Up-regulation of miR-124 was correlated with superior overall survival of patients with NPC. Our study demonstrates that miR-124 can inhibit stem-like properties and enhance radiosensitivity by directly targeting JAMA in NPC. These findings provide novel insights into the molecular mechanisms underlying therapy failure in NPC.
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Molécula A de Adhesión de Unión/genética , MicroARNs/genética , Carcinoma Nasofaríngeo/genética , Neoplasias Nasofaríngeas/genética , Tolerancia a Radiación/genética , Regiones no Traducidas 3'/genética , Animales , Línea Celular Tumoral , Regulación hacia Abajo/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Metástasis Linfática/genética , Ratones , Ratones Endogámicos BALB C , Recurrencia Local de Neoplasia/genética , Células Madre Neoplásicas/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
Amyloid ß (Aß) oligomers may be a real culprit in the pathogenesis of Alzheimer's disease (AD); therefore, the elimination of these toxic oligomers may be of great significance for AD therapy. Autophagy is the catabolic process by which lysosomes degrade cytosolic components, and heat shock cognate 70 kDa protein (Hsc70) binds to proteins with their KFERQ-like motifs [also known as chaperone-mediated autophagy (CMA) motifs] and carries them to lysosomes through CMA or late endosomes through endosomal microautophagy (eMI) for degradation. In this study, our strategy is to make the pathological Aß become one selective and suitable substrate for CMA and eMI (termed as Hsc70-based autophagy) by tagging its oligomers with multiple CMA motifs. First, we design and synthesize Aß oligomer binding peptides with three CMA motifs. Second, we determine that the peptide can help Aß oligomers enter endosomes and lysosomes, which can be further enhanced by ketone. More importantly, we find that the peptide can dramatically reduce Aß oligomers in induced pluripotent stem cell (iPSC) cortical neurons derived from AD patient fibroblasts and protect primary cultured cortical neurons against the Aß oligomer-induced neurotoxicity. In conclusion, we demonstrate that the peptide targeting Hsc70-based autophagy can effectively eliminate Aß oligomers and have superior neuroprotective activity.
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Péptidos beta-Amiloides/antagonistas & inhibidores , Autofagia Mediada por Chaperones/efectos de los fármacos , Proteínas del Choque Térmico HSC70/metabolismo , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Péptidos/farmacología , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/terapia , Secuencias de Aminoácidos , Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/farmacología , Animales , Diferenciación Celular , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Proteínas del Choque Térmico HSC70/genética , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Terapia Molecular Dirigida , Neuronas/metabolismo , Neuronas/patología , Fármacos Neuroprotectores/síntesis química , Péptidos/síntesis química , Cultivo Primario de Células , Unión Proteica , Proteolisis , Ratas , Ratas Long-EvansRESUMEN
BACKGROUND: Microglia have been implicated in the pathogenesis of radiation-induced brain injury (RIBI), which severely influences the quality of life during long-term survival. Recently, irradiated microglia were speculated to present an aging-like phenotype. Long noncoding RNAs (lncRNAs) have been recognized to regulate a wide spectrum of biological processes, including senescence; however, their potential role in irradiated microglia remains largely uncharacterized. METHODS: We used bioinformatics and experimental methods to identify and analyze the senescence phenotype of irradiated microglia. Western blotting, enzyme-linked immunosorbent assays, immunofluorescence, and quantitative real-time reverse transcription-polymerase chain reaction were performed to clarify the relationship between the radiation-induced differentially expressed lncRNAs (RILs) and the distinctive molecular features of senescence in irradiated microglia. RESULTS: We found that the senescence of microglia could be induced using ionizing radiation (IR). A mutual regulation mode existed between RILs and three main features of the senescence phenotype in irradiated microglia: inflammation, the DNA damage response (DDR), and metabolism. Specifically, for inflammation, the expression of two selected RILs (ENSMUST00000190863 and ENSMUST00000130679) was dependent on the major inflammatory signaling pathways of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK). The two RILs modulated the activation of NF-κB/MAPK signaling and subsequent inflammatory cytokine secretion. For the DDR, differential severity of DNA damage altered the expression profiles of RILs. The selected RIL, ENSMUST00000130679, promoted the DDR. For metabolism, blockade of sterol regulatory element-binding protein-mediated lipogenesis attenuated the fold-change of several RILs induced by IR. CONCLUSIONS: Our findings revealed that certain RILs interacted with senescence in irradiated microglia. RILs actively participated in the regulation of senescence features, suggesting that RILs could be promising intervention targets to treat RIBI.
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Senescencia Celular/efectos de la radiación , Microglía/metabolismo , ARN Largo no Codificante/metabolismo , Transducción de Señal/efectos de la radiación , Animales , Línea Celular , Ratones , Microglía/efectos de la radiación , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , ARN Largo no Codificante/genética , Radiación IonizanteRESUMEN
Protein homeostasis or proteostasis is critical for proper cellular function and survival. It relies on the balance between protein synthesis and degradation. Lysosomes play an important role in degrading and recycling intracellular components via autophagy. Among the three types of lysosome-based autophagy pathways, chaperone-mediated autophagy (CMA) selectively degrades cellular proteins with KFERQ-like motif by unique machinery. During the past several years, significant advances have been made in our understanding of how CMA itself is modulated and what physiological and pathological processes it may be involved in. One particularly exciting discovery is how other cellular stress organelles such as ER signal to CMA. As more proteins are identified as CMA substrates, CMA function has been associated with an increasing number of important cellular processes, organelles, and diseases, including neurodegenerative diseases. Here we will summarize the recent advances in CMA biology, highlight ER stress-induced CMA, and discuss the role of CMA in diseases.
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Autofagia/fisiología , Chaperonas Moleculares/metabolismo , Animales , Estrés del Retículo Endoplásmico/fisiología , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Enfermedades Neurodegenerativas/tratamiento farmacológico , Enfermedades Neurodegenerativas/metabolismoRESUMEN
Sevoflurane, a typical inhaled anesthetic, is widely used in patients of all ages during surgery. The negative effects, such as inducing cell death and damaging spatial memory, of sevoflurane on neurodevelopment have raised increasing concerns in recent years. However, the molecular mechanism remains unclear. This study focused on the crucial role of endoplasmic reticulum (ER) stress in sevoflurane-induced hippocampal injury. Three-week-old rats were exposed to sevoflurane or control air for 5 h with or without ER stress inhibitor (4-phenylbutyric acid, 4-PBA) injection. The hippocampus was harvested to measure the ER stress sensors by western immunoblotting. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling staining was used to detect cell apoptosis and electrophysiology was used to measure the intrinsic excitability of neurons in hippocampus. We measured learning and memory ability by Morris water maze tests 5 weeks after sevoflurane exposure. Interestingly, persistent sevoflurane exposure significantly increased the levels of ER stress sensors in hippocampus. But it resulted in different effects in CA1 and dentate gyrus. Greatly increased caspase-12-mediated apoptotic cells, which were proved to be the neural stem cells, were detected in the dentate gyrus. Meanwhile, CA1 pyramidal neurons exhibited significantly reduced intrinsic excitability. Furthermore, the administration of ER stress inhibitor attenuated the above mentioned detrimental effects evidently and prevented the following relevant learning and memory deficits. In conclusion, sevoflurane-mediated ER stress performs distinct effects on the different subfields of the immature hippocampus and inhibiting ER stress during sevoflurane anesthesia will be a potential method to prevent the following learning and memory deficits in adulthood.
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Estrés del Retículo Endoplásmico/efectos de los fármacos , Hipocampo/efectos de los fármacos , Aprendizaje por Laberinto/efectos de los fármacos , Éteres Metílicos/farmacología , Memoria Espacial/efectos de los fármacos , Anestésicos por Inhalación/farmacología , Animales , Animales Recién Nacidos , Muerte Celular/efectos de los fármacos , Hipocampo/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fenilbutiratos/farmacología , SevofluranoRESUMEN
Rictor, an essential component of mTOR complex 2 (mTORC2), plays a pivotal role in regulating mTOR signaling and other biological functions. Posttranslational regulation of rictor (e.g. via degradation) and its underlying mechanism are largely undefined and thus are the focus of this study. Chemical inhibition of the proteasome increased rictor ubiquitination and levels. Consistently, inhibition of FBXW7 with various genetic means including knockdown, knock-out, and enforced expression of a dominant-negative mutant inhibited rictor ubiquitination and increased rictor levels, whereas enforced expression of FBXW7 decreased rictor stability and levels. Moreover, we detected an interaction between FBXW7 and rictor. Hence, rictor is degraded through an FBXW7-mediated ubiquitination/proteasome mechanism. We show that this process is dependent on glycogen synthase kinase 3 (GSK3): GSK3 was associated with rictor and directly phosphorylated the Thr-1695 site in a putative CDC4 phospho-degron motif of rictor; mutation of this site impaired the interaction between rictor and FBXW7, decreased rictor ubiquitination, and increased rictor stability. Finally, enforced activation of Akt enhanced rictor levels and increased mTORC2 activity as evidenced by increased formation of mTORC2 and elevated phosphorylation of Akt, SGK1, and PKCα. Hence we suggest that PI3K/Akt signaling may positively regulate mTORC2 signaling, likely through suppressing GSK3-dependent rictor degradation.
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Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/metabolismo , Regulación Neoplásica de la Expresión Génica , Regulación de la Expresión Génica , Glucógeno Sintasa Quinasa 3/metabolismo , Complejos Multiproteicos/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Secuencias de Aminoácidos , Sitios de Unión , Línea Celular Tumoral , Proteína 7 que Contiene Repeticiones F-Box-WD , Células HEK293 , Humanos , Diana Mecanicista del Complejo 2 de la Rapamicina , Mutación , Fosforilación , Plásmidos/metabolismo , Unión Proteica , ARN Interferente Pequeño/metabolismo , Proteína Asociada al mTOR Insensible a la Rapamicina , Transducción de Señal , Ubiquitina/metabolismoRESUMEN
We have earlier reported the critical nature of calpain-CDK5-MEF2 signaling in governing dopaminergic neuronal loss in vivo. CDK5 mediates phosphorylation of the neuronal survival factor myocyte enhancer factor 2 (MEF2) leading to its inactivation and loss. However, the downstream factors that mediate MEF2-regulated survival are unknown. Presently, we define Nur77 as one such critical downstream survival effector. Following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment in vivo, Nur77 expression in the nigrostriatal region is dramatically reduced. This loss is attenuated by expression of MEF2. Importantly, MEF2 constitutively binds to the Nur77 promoter in neurons under basal conditions. This binding is lost following 1-methyl-4-phenylpyridinium treatment. Nur77 deficiency results in significant sensitization to dopaminergic loss following 1-methyl-4-phenylpyridinium/MPTP treatment, in vitro and in vivo. Furthermore, Nur77-deficient MPTP-treated mice displayed significantly reduced levels of dopamine and 3,4-Dihydroxyphenylacetic acid in the striatum as well as elevated post synaptic FosB activity, indicative of increased nigrostriatal damage when compared with WT MPTP-treated controls. Importantly, this sensitization in Nur77-deficient mice was rescued with ectopic Nur77 expression in the nigrostriatal system. These results indicate that the inactivation of Nur77, induced by loss of MEF2 activity, plays a critical role in nigrostriatal degeneration in vivo.
Asunto(s)
1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacología , Neuronas Dopaminérgicas/citología , Regulación de la Expresión Génica , Factores Reguladores Miogénicos/metabolismo , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Ácido 3,4-Dihidroxifenilacético/farmacología , Animales , Encéfalo/efectos de los fármacos , Encéfalo/patología , Calpaína/metabolismo , Muerte Celular , Quinasa 5 Dependiente de la Ciclina/metabolismo , Neuronas Dopaminérgicas/metabolismo , Factores de Transcripción MEF2 , Masculino , Ratones , Ratones Noqueados , Neurotoxinas/química , ARN Interferente Pequeño/metabolismo , Sustancia Negra/efectos de los fármacos , Sustancia Negra/metabolismoRESUMEN
Many clinical studies indicate a significant decrease of peripheral T cells in Parkinson's disease (PD). There is currently no mechanistic explanation for this important observation. Here, we found that small extracellular vesicles (sEVs) derived from in vitro and in vivo PD models suppressed IL-4 and INF-γ production from both purified CD4+ and CD8+ T cells and inhibited their activation and proliferation. Furthermore, neuronal-enriched sEVs (NEEVs) isolated from plasma of A53T-syn mice and culture media of human dopaminergic neurons carrying A53T-syn mutation also suppressed Th1 and Th2 differentiation of naive CD4+ T cells. Mechanistically, the suppressed phenotype induced by NEEVs was associated with altered programmed death ligand 1 (PD-L1) level in T cells. Blocking PD-L1 with an anti-PD-L1 antibody or a small molecule inhibitor BMS-1166 reversed T cell suppression. Our study provides the basis for exploring peripheral T cells in PD pathogenesis and as biomarkers or therapeutic targets for the disease.
RESUMEN
Synaptic and extrasynaptic NMDA receptors (NMDARs) appear to play opposite roles in neuronal survival and death. Here we report the new findings on the dysregulation of survival factor, myocyte enhancer factor 2D (MEF2D), by extrasynaptic NMDARs. Excitotoxicity led to the NMDAR-dependent degradation of MEF2D protein and inhibition of its transactivation activity in mature cortical neurons. The activation of extrasynaptic NMDARs alone was sufficient for degradation of MEF2D. Calpain directly cleaved MEF2D in vitro and blocking this protease activity greatly attenuated NMDAR signaled degradation of MEF2D in neurons. Consistently, inhibition of calpain protected cortical neurons from NMDA-induced excitotoxicity. Furthermore, knockdown of MEF2D sensitized neurons to NMDA-induced excitotoxicity, which was not protected by calpain inhibition. Collectively, these findings suggest that dysregulation of MEF2D by calpain may mediate excitotoxicity via an extrasynaptic NMDAR-dependent manner.
Asunto(s)
Calpaína/metabolismo , Neurotoxinas/toxicidad , Proteolisis/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Factores de Transcripción/metabolismo , Animales , Muerte Celular/efectos de los fármacos , Hipoxia de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Glucosa/deficiencia , Factores de Transcripción MEF2 , N-Metilaspartato/toxicidad , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/enzimología , Neuronas/metabolismo , Ratas , Transducción de Señal/efectos de los fármacos , Sinapsis/enzimologíaRESUMEN
Parkinson disease (PD) is characterized by the selective demise of dopaminergic (DA) neurons in the substantial nigra pars compacta. Dysregulation of transcriptional factor myocyte enhancer factor 2D (MEF2D) has been implicated in the pathogenic process in in vivo and in vitro models of PD. Here, we identified a small molecule bis(3)-cognitin (B3C) as a potent activator of MEF2D. We showed that B3C attenuated the toxic effects of neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)) by activating MEF2D via multiple mechanisms. B3C significantly reduced MPP(+)-induced oxidative stress and potentiated Akt to down-regulate the activity of MEF2 inhibitor glycogen synthase kinase 3ß (GSK3ß) in a DA neuronal cell line SN4741. Furthermore, B3C effectively rescued MEF2D from MPP(+)-induced decline in both nucleic and mitochondrial compartments. B3C offered SN4741 cells potent protection against MPP(+)-induced apoptosis via MEF2D. Interestingly, B3C also protected SN4741 cells from wild type or mutant A53T α-synuclein-induced cytotoxicity. Using the in vivo PD model of C57BL/6 mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP), we showed that B3C maintained redox homeostasis, promoted Akt function activity, and restored MEF2D level in midbrain neurons. Moreover, B3C greatly prevented the loss of tyrosine hydroxylase signal in substantial nigra pars compacta DA neurons and ameliorated behavioral impairments in mice treated with MPTP. Collectedly, our studies identified B3C as a potent neuroprotective agent whose effectiveness relies on its ability to effectively up-regulate MEF2D in DA neurons against toxic stress in models of PD in vitro and in vivo.
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Apoptosis/efectos de los fármacos , Neuronas Dopaminérgicas/metabolismo , Factores Reguladores Miogénicos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Fármacos Neuroprotectores/farmacología , Estrés Oxidativo/efectos de los fármacos , Trastornos Parkinsonianos/tratamiento farmacológico , Tacrina/análogos & derivados , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina , 1-Metil-4-fenilpiridinio/efectos adversos , 1-Metil-4-fenilpiridinio/farmacología , Animales , Conducta Animal/efectos de los fármacos , Línea Celular , Dopaminérgicos/efectos adversos , Dopaminérgicos/farmacología , Neuronas Dopaminérgicas/patología , Herbicidas/efectos adversos , Herbicidas/farmacología , Factores de Transcripción MEF2 , Masculino , Ratones , Trastornos Parkinsonianos/metabolismo , Trastornos Parkinsonianos/patología , Sustancia Negra/metabolismo , Sustancia Negra/patología , Tacrina/farmacología , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
TFEB (transcription factor EB) regulates multiple genes involved in the process of macroautophagy/autophagy and plays a critical role in lifespan determination. However, the detailed mechanisms that regulate TFEB activity are not fully clear. In this study, we identified a role for HSP90AA1 in modulating TFEB. HSP90AA1 was phosphorylated by CDK5 at Ser 595 under basal condition. This phosphorylation inhibited HSP90AA1, disrupted its binding to TFEB, and impeded TFEB's nuclear localization and subsequent autophagy induction. Pro-autophagy signaling attenuated CDK5 activity and enhanced TFEB function in an HSP90AA1-dependent manner. Inhibition of HSP90AA1 function or decrease in its expression significantly attenuated TFEB's nuclear localization and transcriptional function following autophagy induction. HSP90AA1-mediated regulation of a TFEB ortholog was involved in the extended lifespan of Caenorhabditis elegans in the absence of its food source bacteria. Collectively, these findings reveal that this regulatory process plays an important role in modulation of TFEB, autophagy, and longevity.Abbreviations : AL: autolysosome; AP: autophagosome; ATG: autophagy related; BafA1: bafilomycin A1; CDK5: cyclin-dependent kinase 5; CDK5R1: cyclin dependent kinase 5 regulatory subunit 1; CR: calorie restriction; FUDR: 5-fluorodeoxyuridine; HSP90AA1: heat shock protein 90 alpha family class A member 1; MAP1LC3: microtubule associated protein 1 light chain 3; NB: novobiocin sodium; SQSTM1: sequestosome 1; TFEB: transcription factor EB; WT: wild type.
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Autofagia , Longevidad , Animales , Autofagia/genética , Núcleo Celular/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Autofagosomas/metabolismo , Transducción de Señal/genética , Chaperonas Moleculares/metabolismo , Caenorhabditis elegans/metabolismo , Lisosomas/metabolismoRESUMEN
Neuroprotective strategies, including free radical scavengers, ion channel modulators, and anti-inflammatory agents, have been extensively explored in the last 2 decades for the treatment of neurological diseases. Unfortunately, none of the neuroprotectants has been proved effective in clinical trails. In the current study, we demonstrated that methylene blue (MB) functions as an alternative electron carrier, which accepts electrons from NADH and transfers them to cytochrome c and bypasses complex I/III blockage. A de novo synthesized MB derivative, with the redox center disabled by N-acetylation, had no effect on mitochondrial complex activities. MB increases cellular oxygen consumption rates and reduces anaerobic glycolysis in cultured neuronal cells. MB is protective against various insults in vitro at low nanomolar concentrations. Our data indicate that MB has a unique mechanism and is fundamentally different from traditional antioxidants. We examined the effects of MB in two animal models of neurological diseases. MB dramatically attenuates behavioral, neurochemical, and neuropathological impairment in a Parkinson disease model. Rotenone caused severe dopamine depletion in the striatum, which was almost completely rescued by MB. MB rescued the effects of rotenone on mitochondrial complex I-III inhibition and free radical overproduction. Rotenone induced a severe loss of nigral dopaminergic neurons, which was dramatically attenuated by MB. In addition, MB significantly reduced cerebral ischemia reperfusion damage in a transient focal cerebral ischemia model. The present study indicates that rerouting mitochondrial electron transfer by MB or similar molecules provides a novel strategy for neuroprotection against both chronic and acute neurological diseases involving mitochondrial dysfunction.
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Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Azul de Metileno/farmacología , Mitocondrias/enzimología , Fármacos Neuroprotectores/farmacología , Enfermedad de Parkinson Secundaria/tratamiento farmacológico , Animales , Línea Celular , Proteínas del Complejo de Cadena de Transporte de Electrón/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Glucólisis/efectos de los fármacos , Masculino , Neuronas/enzimología , Neuronas/patología , Consumo de Oxígeno/efectos de los fármacos , Enfermedad de Parkinson Secundaria/inducido químicamente , Enfermedad de Parkinson Secundaria/enzimología , Enfermedad de Parkinson Secundaria/patología , Enfermedad de Parkinson Secundaria/fisiopatología , Ratas , Ratas Sprague-Dawley , Rotenona/efectos adversos , Rotenona/farmacología , Sustancia Negra/enzimología , Sustancia Negra/patología , Sustancia Negra/fisiopatología , Desacopladores/efectos adversos , Desacopladores/farmacologíaRESUMEN
The myocyte enhancer factor 2A-D (MEF2) proteins are members of the MCM1-agamous-deficiens-serum response factor family of transcription factors. Various MEF2 isoform proteins are enriched in neurons and exhibit distinct patterns of expression in different regions of the brain. In neurons, MEF2 functions as a converging factor to regulate many neuronal functions including survival. MEF2 activities are tightly controlled in neurons in response to stress. Whether stress signal may differentially regulate MEF2s remains largely unknown. In this work, we showed that MEF2A, but not MEF2C or MEF2D, was modified by ubiquitination in dopaminergic neuronal cell line SN4741 cells. MEF2A was ubiquitinated at its N'-terminus, and the ubiquitination of MEF2A was first detectable in the nuclear compartment and later in the cytoplasm. Ubiquitination of MEF2A correlated with reduced DNA-binding activity and transcriptional activity. More importantly, disturbing the degradation of ubiquitinated MEF2A through proteasome pathway by neurotoxins known to induce Parkinson's disease features in model animals caused accumulation of ubiquitinated MEF2A, reduced MEF2 activity, and impaired cellular viability. Our work thus provides the first evidence to demonstrate an isoforms-specific regulation of MEF2s by ubiquitination-proteasome pathway in dopaminergic neuronal cell by neurotoxins, suggesting that stress signal and cellular context-dependent dysregulation of MEF2s may underlie the loss of neuronal viability.
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Neuronas Dopaminérgicas/metabolismo , Factores Reguladores Miogénicos/metabolismo , Estrés Fisiológico/fisiología , Ubiquitinación/fisiología , Animales , Línea Celular , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/fisiología , Factores de Transcripción MEF2 , Ratones , Factores Reguladores Miogénicos/fisiología , Neurotoxinas/toxicidad , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiología , Estrés Fisiológico/efectos de los fármacos , Ubiquitinación/efectos de los fármacosRESUMEN
Degradation of dysfunctional intracellular components in the lysosome system can occur through three different pathways, i.e., macroautophagy, microautophagy and chaperone-mediated autophagy (CMA). In this review, we focus on CMA, a type of autophagy distinct from the other two autophagic pathways owing to its selectivity, saturability and competitivity by which a subset of long-lived cytosolic soluble proteins are directly delivered into the lysosomal lumen via specific receptors. CMA participates in quality control to maintain normal cell functions by clearing "old" proteins and provides energy to cells under nutritional stress. Deregulation of CMA has recently been shown to underlie some diseases, especially neurodegenerative disorders for which the decline with age in the activity of CMA may become a major aggravating factor. Therefore, targeting aberrant alteration in CMA under pathological conditions could serve as a potential therapeutic strategy for treating related diseases.
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Autofagia/fisiología , Chaperonas Moleculares/fisiología , Secuencias de Aminoácidos , Sitios de Unión , Senescencia Celular , Proteínas del Choque Térmico HSC70/fisiología , Humanos , Lisosomas/fisiología , Enfermedades Neurodegenerativas/patología , Estructura Terciaria de Proteína , Transporte de ProteínasRESUMEN
Accumulation of oxidative stress is highly intertwined with aging process and contributes to aging-related diseases, such as neurodegenerative diseases. Deciphering the molecular machinery that regulates oxidative stress is fundamental to further uncovering the pathogenesis of these diseases. Chaperone-mediated autophagy (CMA), a highly selective lysosome-dependent degradation process, has been proven to be an important maintainer of cellular homeostasis through multiple mechanisms, one of which is the attenuation of oxidative stress. However, the specific mechanisms underlying this antioxidative action of CMA are not fully understood. In this study, we found that CMA directly degrades Kelch-like ECH-associated protein 1 (Keap1), an adaptor of E3 ligase complex that promotes the degradation of nuclear factor erythroid 2-related factor 2 (Nrf2), which is a master transcriptional regulator in antioxidative response. Activated CMA induced by prolonged oxidative stress led to an increase in Nrf2 level by effectively degrading Keap1, contributing to Nrf2 nuclear translocation and the expression of multiple downstream antioxidative genes. Meanwhile, together with previous study showing that Nrf2 can also transcriptionally regulate LAMP2A, the rate-limiting factor of CMA process, we reveal a feed-forward loop between CMA and Nrf2. Our study identifies CMA as a previously unrecognized regulator of Keap1-Nrf2 pathway and reinforces the antioxidative role of CMA.
Asunto(s)
Autofagia Mediada por Chaperones , Factor 2 Relacionado con NF-E2 , Antioxidantes/farmacología , Autofagia , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Estrés OxidativoRESUMEN
Isoform A of phosphatidylinositol 3-kinase enhancer (PIKE-A) is a newly identified prooncogenic factor that has been implicated in cancer cell growth. How PIKE-A activity is regulated in response to growth signal is poorly understood. Here, we demonstrate that cyclin dependent kinase 5 (Cdk5), a protein known to function mainly in postmitotic neurons, directly phosphorylates PIKE-A at Ser-279 in its GTPase domain in glioblastoma cells. This phosphorylation event stimulates PIKE-A GTPase activity and the activity of its downstream effector Akt. Growth signal activates Cdk5 and results in a Cdk5-dependent accumulation of phosphorylated PIKE-A and activation of Akt in the nucleus. Furthermore, PIKE-A phosphorylation and Cdk5 are increased in human glioblastoma specimens. Phosphorylation of PIKE-A by Cdk5 mediates growth factor-induced migration and invasion of human glioblastoma cells. Together, these findings identify PIKE as the first Cdk5 target in cancer cells, revealing a previously undescribed regulatory mechanism that mediates growth signal-induced activation of PIKE-A/Akt and tumor invasion.
Asunto(s)
Neoplasias del Sistema Nervioso Central/patología , Quinasa 5 Dependiente de la Ciclina/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Glioblastoma/patología , Transporte Activo de Núcleo Celular , Línea Celular Tumoral , Movimiento Celular , Neoplasias del Sistema Nervioso Central/enzimología , Glioblastoma/enzimología , Humanos , Invasividad Neoplásica , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina/metabolismoRESUMEN
As a highly dynamic organelle, mitochondria undergo constant fission and fusion to change their morphology and function, coping with various stress conditions. Loss of the balance between fission and fusion leads to impaired mitochondria function, which plays a critical role in the pathogenesis of Parkinson disease (PD). Yet the mechanisms behind mitochondria dynamics regulation remain to be fully illustrated. Chaperone-mediated autophagy (CMA) is a lysosome-dependent process that selectively degrades proteins to maintain cellular proteostasis. In this study, we demonstrated that MARCHF5, an E3 ubiquitin ligase required for mitochondria fission, is a CMA substrate. MARCHF5 interacted with key CMA regulators and was degraded by lysosomes. Severe oxidative stress compromised CMA activity and stabilized MARCHF5, which facilitated DNM1L translocation and led to excessive fission. Increase of CMA activity promoted MARCHF5 turnover, attenuated DNM1L translocation, and reduced mitochondria fragmentation, which alleviated mitochondrial dysfunction under oxidative stress. Furthermore, we showed that conditional expression of LAMP2A, the key CMA regulator, in dopaminergic (DA) neurons helped maintain mitochondria morphology and protected DA neuronal viability in a rodent PD model. Our work uncovers a critical role of CMA in maintaining proper mitochondria dynamics, and loss of this regulatory control may occur in PD and underlie its pathogenic process.Abbreviations: CMA: chaperone-mediated autophagy; DA: dopaminergic; DNM1L: dynamin 1 like; FCCP: carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; HSPA8: heat shock protein family A (Hsp70) member 8; LAMP2A: lysosomal associated membrane protein 2A; MARCHF5: membrane-associated ring-CH-type finger 5; MMP: mitochondria membrane potential; OCR: oxygen consumption rate; 6-OHDA: 6-hydroxydopamine; PD: Parkinson disease; SNc: substantia nigra pars compacta; TEM: transmission electron microscopy; TH: tyrosine hydroxylase; TMRE: tetramethylrhodamine ethyl ester perchlorate; WT: wild type.
Asunto(s)
Autofagia Mediada por Chaperones , Enfermedad de Parkinson , Autofagia , Humanos , Dinámicas Mitocondriales , Enfermedad de Parkinson/metabolismo , Ubiquitina-Proteína LigasasRESUMEN
MicroRNAs (miRNAs) are small noncoding RNAs ubiquitously expressed in the brain and regulate gene expression at the post-transcriptional level. The nuclear RNase III enzyme Drosha initiates the maturation process of miRNAs in the nucleus. Strong evidence suggests that dysregulation of miRNAs is involved in many neurological disorders including Alzheimer's disease (AD). Dysfunction of miRNA biogenesis components may be involved in the processes of those diseases. However, the role of Drosha in AD remains unknown. By using immunohistochemistry, biochemistry, and subcellular fractionation methods, we show here that the level of Drosha protein was significantly lower in the postmortem brain of human AD patients as well as in the transgenic rat model of AD. Interestingly, Drosha level was specifically reduced in neurons of the cortex and hippocampus but not in the cerebellum in the AD brain samples. In primary cortical neurons, amyloid-beta (Aß) oligomers caused a p38 MAPK-dependent phosphorylation of Drosha, leading to its redistribution from the nucleus to the cytoplasm and a decrease in its level. This loss of Drosha function preceded Aß-induced neuronal death. Importantly, inhibition of p38 MAPK activity or overexpression of Drosha protected neurons from Aß oligomers-induced apoptosis. Taken together, these results establish a role for p38 MAPK-Drosha pathway in modulating neuronal viability under Aß oligomers stress condition and implicate loss of Drosha as a key molecular change in the pathogenesis of AD.