RESUMO
We previously reported that the cell cycle-related cyclin-dependent kinase 4-retinoblastoma (RB) transcriptional corepressor pathway is essential for stroke-induced cell death both in vitro and in vivo However, how this signaling pathway induces cell death is unclear. Previously, we found that the cyclin-dependent kinase 4 pathway activates the pro-apoptotic transcriptional co-regulator Cited2 in vitro after DNA damage. In the present study, we report that Cited2 protein expression is also dramatically increased following stroke/ischemic insult. Critically, utilizing conditional knockout mice, we show that Cited2 is required for neuronal cell death, both in culture and in mice after ischemic insult. Importantly, determining the mechanism by which Cited2 levels are regulated, we found that E2F transcription factor (E2F) family members participate in Cited2 regulation. First, E2F1 expression induced Cited2 transcription, and E2F1 deficiency reduced Cited2 expression. Moreover, determining the potential E2F-binding regions on the Cited2 gene regulatory sequence by ChIP analysis, we provide evidence that E2F1/4 proteins bind to this DNA region. A luciferase reporter assay to probe the functional outcomes of this interaction revealed that E2F1 activates and E2F4 inhibits Cited2 transcription. Moreover, we identified the functional binding motif for E2F1 in the Cited2 gene promoter by demonstrating that mutation of this site dramatically reduces E2F1-mediated Cited2 transcription. Finally, E2F1 and E2F4 regulated Cited2 expression in neurons after stroke-related insults. Taken together, these results indicate that the E2F-Cited2 regulatory pathway is critically involved in stroke injury.
Assuntos
Fator de Transcrição E2F1/metabolismo , Fator de Transcrição E2F4/metabolismo , Regulação da Expressão Gênica , Neurônios/metabolismo , Proteínas Repressoras/biossíntese , Acidente Vascular Cerebral/metabolismo , Transativadores/biossíntese , Motivos de Aminoácidos , Animais , Morte Celular , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F4/genética , Camundongos , Camundongos Transgênicos , Neurônios/patologia , Proteínas Repressoras/genética , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/patologia , Transativadores/genéticaRESUMO
Loss of function mutations in the PTEN-induced putative kinase 1 (Pink1) gene have been linked with an autosomal recessive familial form of early onset Parkinson's disease (PD). However, the underlying mechanism(s) responsible for degeneration remains elusive. Presently, using co-immunoprecipitation in HEK (Human embryonic kidney) 293 cells, we show that Pink1 endogenously interacts with FK506-binding protein 51 (FKBP51 or FKBP5), FKBP5 and directly phosphorylates FKBP5 at Serine in an in vitro kinase assay. Both FKBP5 and Pink1 have been previously associated with protein kinase B (AKT) regulation. We provide evidence using primary cortical cultured neurons from Pink1-deficient mice that Pink1 increases AKT phosphorylation at Serine 473 (Ser473) challenged by 1-methyl-4-phenylpyridinium (MPP+ ) and that over-expression of FKBP5 using an adeno-associated virus delivery system negatively regulates AKT phosphorylation at Ser473 in murine-cultured cortical neurons. Interestingly, FKBP5 over-expression promotes death in response to MPP+ in the absence of Pink1. Conversely, shRNA-mediated knockdown of FKBP5 in cultured cortical neurons is protective and this effect is reversed with inhibition of AKT signaling. In addition, shRNA down-regulation of PH domain leucine-rich repeat protein phosphatase (PHLPP) in Pink1 WT neurons increases neuronal survival, while down-regulation of PHLPP in Pink1 KO rescues neuronal death in response to MPP+ . Finally, using co-immunoprecipitation, we show that FKBP5 interacts with the kinase AKT and phosphatase PHLPP. This interaction is increased in the absence of Pink1, both in Mouse Embryonic Fibroblasts (MEF) and in mouse brain tissue. Expression of kinase dead Pink1 (K219M) enhances FKBP5 interaction with both AKT and PHLPP. Overall, our results suggest a testable model by which Pink1 could regulate AKT through phosphorylation of FKBP5 and interaction of AKT with PHLPP. Our results suggest a potential mechanism by which PINK1-FKBP5 pathway contributes to neuronal death in PD. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.
Assuntos
Neurônios/metabolismo , Proteínas Quinases/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , 1-Metil-4-fenilpiridínio/toxicidade , Animais , Morte Celular/efeitos dos fármacos , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Neurônios/efeitos dos fármacos , Neurotoxinas/farmacologia , Doença de Parkinson/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologiaRESUMO
The glycosylation of nucleocytoplasmic proteins with O-linked N-acetylglucosamine residues (O-GlcNAc) is conserved among metazoans and is particularly abundant within brain. O-GlcNAc is involved in diverse cellular processes ranging from the regulation of gene expression to stress response. Moreover, O-GlcNAc is implicated in various diseases including cancers, diabetes, cardiac dysfunction, and neurodegenerative diseases. Pharmacological inhibition of O-GlcNAcase (OGA), the sole enzyme that removes O-GlcNAc, reproducibly slows neurodegeneration in various Alzheimer's disease (AD) mouse models manifesting either tau or amyloid pathology. These data have stimulated interest in the possibility of using OGA-selective inhibitors as pharmaceuticals to alter the progression of AD. The mechanisms mediating the neuroprotective effects of OGA inhibitors, however, remain poorly understood. Here we show, using a range of methods in neuroblastoma N2a cells, in primary rat neurons, and in mouse brain, that selective OGA inhibitors stimulate autophagy through an mTOR-independent pathway without obvious toxicity. Additionally, OGA inhibition significantly decreased the levels of toxic protein species associated with AD pathogenesis in the JNPL3 tauopathy mouse model as well as the 3×Tg-AD mouse model. These results strongly suggest that OGA inhibitors act within brain through a mechanism involving enhancement of autophagy, which aids the brain in combatting the accumulation of toxic protein species. Our study supports OGA inhibition being a feasible therapeutic strategy for hindering the progression of AD and other neurodegenerative diseases. Moreover, these data suggest more targeted strategies to stimulate autophagy in an mTOR-independent manner may be found within the O-GlcNAc pathway. These findings should aid the advancement of OGA inhibitors within the clinic.
Assuntos
Autofagia/efeitos dos fármacos , Serina-Treonina Quinases TOR/farmacologia , Doença de Alzheimer/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Modelos Animais de Doenças , Humanos , Neurônios/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Tauopatias/tratamento farmacológico , Tauopatias/metabolismo , Proteínas tau/efeitos dos fármacos , Proteínas tau/metabolismoRESUMO
Inappropriate activation of cell cycle proteins, in particular cyclin D/Cdk4, is implicated in neuronal death induced by various pathologic stresses, including DNA damage and ischemia. Key targets of Cdk4 in proliferating cells include members of the E2F transcription factors, which mediate the expression of cell cycle proteins as well as death-inducing genes. However, the presence of multiple E2F family members complicates our understanding of their role in death. We focused on whether E2F4, an E2F member believed to exhibit crucial control over the maintenance of a differentiated state of neurons, may be critical in ischemic neuronal death. We observed that, in contrast to E2F1 and E2F3, which sensitize to death, E2F4 plays a crucial protective role in neuronal death evoked by DNA damage, hypoxia, and global ischemic insult both in vitro and in vivo. E2F4 occupies promoter regions of proapoptotic factors, such as B-Myb, under basal conditions. Following stress exposure, E2F4-p130 complexes are lost rapidly along with the presence of E2F4 at E2F-containing B-Myb promoter sites. In contrast, the presence of E2F1 at B-Myb sites increases with stress. Furthermore, B-Myb and C-Myb expression increases with ischemic insult. Taken together, we propose a model by which E2F4 plays a protective role in neurons from ischemic insult by forming repressive complexes that prevent prodeath factors such as Myb from being expressed.
Assuntos
Fator de Transcrição E2F4/metabolismo , Hipóxia-Isquemia Encefálica/metabolismo , Neurônios/citologia , Proteína p130 Retinoblastoma-Like/metabolismo , Animais , Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Morte Celular , Fator de Transcrição E2F4/genética , Humanos , Hipóxia-Isquemia Encefálica/genética , Hipóxia-Isquemia Encefálica/fisiopatologia , Masculino , Camundongos Knockout , Neurônios/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Ratos Wistar , Proteína p130 Retinoblastoma-Like/genética , Transativadores/genética , Transativadores/metabolismoRESUMO
Mutations in the mitochondrial PTEN-induced kinase 1 (Pink1) gene have been linked to Parkinson disease (PD). Recent reports including our own indicated that ectopic Pink1 expression is protective against toxic insult in vitro, suggesting a potential role for endogenous Pink1 in mediating survival. However, the role of endogenous Pink1 in survival, particularly in vivo, is unclear. To address this critical question, we examined whether down-regulation of Pink1 affects dopaminergic neuron loss following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the adult mouse. Two model systems were utilized: virally delivered shRNA-mediated knockdown of Pink1 and germ line-deficient mice. In both instances, loss of Pink1 generated significant sensitivity to damage induced by systemic MPTP treatment. This sensitivity was associated with greater loss of dopaminergic neurons in the Substantia Nigra pars compacta and terminal dopamine fiber density in the striatum region. Importantly, we also show that viral mediated expression of two other recessive PD-linked familial genes, DJ-1 and Parkin, can protect dopaminergic neurons even in the absence of Pink1. This evidence not only provides strong evidence for the role of endogenous Pink1 in neuronal survival, but also supports a role of DJ-1 and Parkin acting parallel or downstream of endogenous Pink1 to mediate survival in a mammalian in vivo context.
Assuntos
Neurônios Dopaminérgicos/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Intoxicação por MPTP/genética , Proteínas Oncogênicas/genética , Proteínas Quinases/genética , Ubiquitina-Proteína Ligases/genética , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologia , Adenoviridae/genética , Animais , Neurônios Dopaminérgicos/efeitos dos fármacos , Técnicas de Transferência de Genes , Genes Recessivos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Intoxicação por MPTP/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Degeneração Neural/induzido quimicamente , Degeneração Neural/genética , Degeneração Neural/metabolismo , Neurotoxinas/farmacologia , Proteínas Oncogênicas/metabolismo , Proteína Desglicase DJ-1 , Proteínas Quinases/metabolismo , RNA Interferente Pequeno/genética , Estresse Fisiológico/fisiologia , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Extracellular ATP is elevated by transient ischemia and is a potent signaling molecule in the central nervous system. ATP promotes neuron survival from serum starvation by activating P2Y purinergic receptors. ATP also activates IL-6 production and phosphorylation of Stat3 that promotes neuron survival. The transcription cofactor LMO4 is a positive mediator of IL-6/Stat3 signaling. Here, we found that LMO4 and the pro-survival factor cIAP2 (cellular inhibitor of apoptosis protein 2) are rapidly upregulated in neurons exposed to elevated extracellular ATP. Blocking LMO4 upregulation using siRNA in F11 cells blunted cIAP2 upregulation and abolished the early protective effect of ATP. Similar results were obtained using primary cortical neurons from LMO4 null mice, suggesting that LMO4 is required for ATP to protect neurons from hypoxia-induced apoptosis. Whereas increased Stat3 phosphorylation occurs after LMO4 and cIAP2 induction, the rapid upregulated phosphorylation of ERK and CREB may account for increased LMO4 and cIAP2 by ATP. ATP signaling through ERK and CREB activated LMO4 promoters and ERK activation increased LMO4 protein stability in F11 cells. Taken together, our studies reveal that LMO4 is a rapidly induced downstream effector of ATP signaling that promotes neuron survival from hypoxia.
Assuntos
Trifosfato de Adenosina/metabolismo , Sobrevivência Celular , Proteínas de Homeodomínio/metabolismo , Hipóxia , Neurônios/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Proteínas com Domínio LIM , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/citologia , Regiões Promotoras Genéticas , Ratos , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Fatores de Transcrição/genéticaRESUMO
LIM only domain protein 4 (LMO4) interacts with many signaling and transcription factors to regulate cellular proliferation, differentiation and plasticity. In Drosophila, mutations in the 3' untranslated region (UTR) of the homologue dLMO cause a gain of function by increasing mRNA stability. LMO4 3'UTR contains several AU-rich elements (ARE) and is highly conserved among vertebrates, suggesting that RNA destabilizing mechanisms are evolutionarily conserved. Here, we found that extracellular ATP stabilized LMO4 mRNA in F11 cells. The LMO4 3'UTR added to a luciferase reporter markedly reduced reporter activity under basal conditions, but increased activity with ATP treatment. Two ARE motifs were characterized in the LMO4 3'UTR. ATP increased binding of HuD protein to ARE1. ARE1 conferred ATP and HuD-dependent mRNA stabilization. In contrast, sequences flanking ARE2 bound CUGBP1 and ATP destabilized this complex. Thus, our results suggest that ATP modulates recruitment of RNA-binding proteins to the 3'UTR to stabilize LMO4 mRNA.