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1.
EMBO Rep ; 25(3): 1256-1281, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38429579

RESUMO

The plant homeodomain zinc-finger protein, PHF6, is a transcriptional regulator, and PHF6 germline mutations cause the X-linked intellectual disability (XLID) Börjeson-Forssman-Lehmann syndrome (BFLS). The mechanisms by which PHF6 regulates transcription and how its mutations cause BFLS remain poorly characterized. Here, we show genome-wide binding of PHF6 in the developing cortex in the vicinity of genes involved in central nervous system development and neurogenesis. Characterization of BFLS mice harbouring PHF6 patient mutations reveals an increase in embryonic neural stem cell (eNSC) self-renewal and a reduction of neural progenitors. We identify a panel of Ephrin receptors (EphRs) as direct transcriptional targets of PHF6. Mechanistically, we show that PHF6 regulation of EphR is impaired in BFLS mice and in conditional Phf6 knock-out mice. Knockdown of EphR-A phenocopies the PHF6 loss-of-function defects in altering eNSCs, and its forced expression rescues defects of BFLS mice-derived eNSCs. Our data indicate that PHF6 directly promotes Ephrin receptor expression to control eNSC behaviour in the developing brain, and that this pathway is impaired in BFLS.


Assuntos
Epilepsia , Face/anormalidades , Dedos/anormalidades , Transtornos do Crescimento , Hipogonadismo , Deficiência Intelectual , Deficiência Intelectual Ligada ao Cromossomo X , Obesidade , Humanos , Camundongos , Animais , Deficiência Intelectual/genética , Proteínas Repressoras , Deficiência Intelectual Ligada ao Cromossomo X/genética , Deficiência Intelectual Ligada ao Cromossomo X/metabolismo , Epilepsia/genética , Epilepsia/metabolismo , Fatores de Transcrição
2.
BMC Biol ; 21(1): 240, 2023 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-37907898

RESUMO

BACKGROUND: PFTK1/Eip63E is a member of the cyclin-dependent kinases (CDKs) family and plays an important role in normal cell cycle progression. Eip63E expresses primarily in postnatal and adult nervous system in Drosophila melanogaster but its role in CNS development remains unknown. We sought to understand the function of Eip63E in the CNS by studying the fly ventral nerve cord during development. RESULTS: Our results demonstrate that Eip63E regulates axogenesis in neurons and its deficiency leads to neuronal defects. Functional interaction studies performed using the same system identify an interaction between Eip63E and the small GTPase Rho1. Furthermore, deficiency of Eip63E homolog in mice, PFTK1, in a newly generated PFTK1 knockout mice results in increased axonal outgrowth confirming that the developmental defects observed in the fly model are due to defects in axogenesis. Importantly, RhoA phosphorylation and activity are affected by PFTK1 in primary neuronal cultures. We report that GDP-bound inactive RhoA is a substrate of PFTK1 and PFTK1 phosphorylation is required for RhoA activity. CONCLUSIONS: In conclusion, our work establishes an unreported neuronal role of PFTK1 in axon development mediated by phosphorylation and activation of GDP-bound RhoA. The results presented add to our understanding of the role of Cdks in the maintenance of RhoA-mediated axon growth and its impact on CNS development and axonal regeneration.


Assuntos
Quinases Ciclina-Dependentes , Drosophila melanogaster , Animais , Camundongos , Ciclo Celular , Quinases Ciclina-Dependentes/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Neurônios/metabolismo , Fosforilação , Proteína rhoA de Ligação ao GTP/metabolismo
3.
BMC Biol ; 20(1): 115, 2022 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-35581583

RESUMO

BACKGROUND: Activated Cdk5 regulates a number of processes during nervous system formation, including neuronal differentiation, growth cone stabilization, and axonal growth. Cdk5 phosphorylates its downstream substrates located in axonal growth cones, where the highly expressed c-Jun N-terminal kinase (JNK)-interacting protein1 (JIP1) has been implicated as another important regulator of axonal growth. In addition, stringent control of the level of intracellular domain of Notch1 (Notch1-IC) plays a regulatory role in axonal outgrowth during neuronal differentiation. However, whether Cdk5-JIP1-Notch1 cooperate to regulate axonal outgrowth, and the mechanism of such joint contribution to this pathway, is presently unknown, and here we explore their potential interaction. RESULTS: Our interactome screen identified JIP1 as an interactor of p35, a Cdk5 activator, and we sought to explore the relationship between Cdk5 and JIP1 on the regulation of axonal outgrowth. We demonstrate that JIP1 phosphorylated by Cdk5 at Thr205 enhances axonal outgrowth and a phosphomimic JIP1 rescues the axonal outgrowth defects in JIP1-/- and p35-/- neurons. Axonal outgrowth defects caused by the specific increase of Notch1 in JIP1-/- neurons are rescued by Numb-mediated inhibition of Notch1. Finally, we demonstrate that Cdk5 phosphorylation of JIP1 further amplifies the phosphorylation status of yet another Cdk5 substrate E3-ubiquitin ligase Itch, resulting in increased Notch1 ubiquitination. CONCLUSIONS: Our findings identify a potentially critical signaling axis involving Cdk5-JIP1-Itch-Notch1, which plays an important role in the regulation of CNS development. Future investigation into the way this pathway integrates with additional pathways regulating axonal growth will further our knowledge of normal central nervous system development and pathological conditions.


Assuntos
Neurônios , Transdução de Sinais , Células Cultivadas , Neurônios/metabolismo , Fosforilação , Transdução de Sinais/fisiologia
4.
J Biol Chem ; 294(21): 8617-8629, 2019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-30967472

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/genética
5.
J Neurochem ; 150(3): 312-329, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30734931

RESUMO

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/fisiologia
6.
J Biol Chem ; 290(51): 30441-52, 2015 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-26538564

RESUMO

Emerging evidence has demonstrated a growing genetic component in Parkinson disease (PD). For instance, loss-of-function mutations in PINK1 or PARKIN can cause autosomal recessive PD. Recently, PINK1 and PARKIN have been implicated in the same signaling pathway to regulate mitochondrial clearance through recruitment of PARKIN by stabilization of PINK1 on the outer membrane of depolarized mitochondria. The precise mechanisms that govern this process remain enigmatic. In this study, we identify Bcl2-associated athanogene 2 (BAG2) as a factor that promotes mitophagy. BAG2 inhibits PINK1 degradation by blocking the ubiquitination pathway. Stabilization of PINK1 by BAG2 triggers PARKIN-mediated mitophagy and protects neurons against 1-methyl-4-phenylpyridinium-induced oxidative stress in an in vitro cell model of PD. Collectively, our findings support the notion that BAG2 is an upstream regulator of the PINK1/PARKIN signaling pathway.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Mitocôndrias/metabolismo , Chaperonas Moleculares/metabolismo , Neurônios/metabolismo , Proteínas Quinases/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Sobrevivência Celular , Camundongos , Camundongos Mutantes , Mitocôndrias/genética , Membranas Mitocondriais/metabolismo , Mitofagia/genética , Chaperonas Moleculares/genética , Proteínas Quinases/genética , Estabilidade Proteica , Transporte Proteico , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
7.
J Neurosci ; 34(23): 8043-50, 2014 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-24899725

RESUMO

DJ-1 (PARK7) is a gene linked to autosomal recessive Parkinson disease (PD). We showed previously that DJ-1 loss sensitizes neurons in models of PD and stroke. However, the biochemical mechanisms underlying this protective role are not completely clear. Here, we identify Von Hippel Lindau (VHL) protein as a critical DJ-1-interacting protein. We provide evidence that DJ-1 negatively regulates VHL ubiquitination activity of the α-subunit of hypoxia-inducible factor-1 (HIF-1α) by inhibiting HIF-VHL interaction. Consistent with this observation, DJ-1 deficiency leads to lowered HIF-1α levels in models of both hypoxia and oxidative stress, two stresses known to stabilize HIF-1α. We also demonstrate that HIF-1α accumulation rescues DJ-1-deficient neurons against 1-methyl-4-phenylpyridinium-induced toxicity. Interestingly, lymphoblast cells extracted from DJ-1-related PD patients show impaired HIF-1α stabilization when compared with normal individuals, indicating that the DJ-1-VHL link may also be relevant to a human context. Together, our findings delineate a model by which DJ-1 mediates neuronal survival by regulation of the VHL-HIF-1α pathway.


Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neurônios/metabolismo , Proteínas Oncogênicas/metabolismo , Transdução de Sinais/fisiologia , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologia , Animais , Células Cultivadas , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Masculino , Camundongos , Camundongos Knockout , Neuroblastoma/patologia , Neurônios/efeitos dos fármacos , Neurotoxinas/farmacologia , Proteínas Oncogênicas/deficiência , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Doença de Parkinson/patologia , Peroxirredoxinas , Proteína Desglicase DJ-1 , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Proteína Supressora de Tumor Von Hippel-Lindau/genética
8.
Proc Natl Acad Sci U S A ; 109(39): 15918-23, 2012 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-23019375

RESUMO

DJ-1 mutations cause autosomal recessive early-onset Parkinson disease (PD). We report a model of PD pathology: the DJ1-C57 mouse. A subset of DJ-1-nullizygous mice, when fully backcrossed to a C57BL/6 [corrected] background, display dramatic early-onset unilateral loss of dopaminergic (DA) neurons in their substantia nigra pars compacta, progressing to bilateral degeneration of the nigrostriatal axis with aging. In addition, these mice exhibit age-dependent bilateral degeneration at the locus ceruleus nucleus and display mild motor behavior deficits at aged time points. These findings effectively recapitulate the early stages of PD. Therefore, the DJ1-C57 mouse provides a tool to study the preclinical aspects of neurodegeneration. Importantly, by exome sequencing, we identify candidate modifying genes that segregate with the phenotype, providing potentially critical clues into how certain genes may influence the penetrance of DJ-1-related degeneration in mice.


Assuntos
Neurônios Dopaminérgicos/patologia , Peptídeos e Proteínas de Sinalização Intracelular , Locus Cerúleo/patologia , Proteínas do Tecido Nervoso , Proteínas Oncogênicas , Doença de Parkinson/genética , Doença de Parkinson/patologia , Substância Negra/patologia , Animais , Modelos Animais de Doenças , Neurônios Dopaminérgicos/metabolismo , Humanos , Locus Cerúleo/metabolismo , Camundongos , Camundongos Knockout , Doença de Parkinson/metabolismo , Substância Negra/metabolismo
9.
Front Cell Dev Biol ; 12: 1371568, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38606319

RESUMO

The mammalian brain, especially the cerebral cortex, has evolved to increase in size and complexity. The proper development of the cerebral cortex requires the coordination of several events, such as differentiation and migration, that are essential for forming a precise six-layered structure. We have previously reported that Cdk5-mediated phosphorylation of JIP1 at T205 modulates axonal out-growth. However, the spatiotemporal expression patterns and functions of these three genes (Cdk5, Cdk5r1 or p35, and Mapk8ip1 or JIP1) in distinct cell types during cortical development remain unclear. In this study, we analyzed single-cell RNA-sequencing data of mouse embryonic cortex and discovered that Cdk5, p35, and JIP1 are dynamically expressed in intermediate progenitors (IPs). Pseudotime analysis revealed that the expression of these three genes was concomitantly upregulated in IPs during neuronal migration and differentiation. By manipulating the expression of JIP1 and phospho-mimetic JIP1 using in utero electroporation, we showed that phosphorylated JIP1 at T205 affected the temporal migration of neurons.

10.
PLoS Genet ; 6(4): e1000914, 2010 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-20421934

RESUMO

LRRK2 plays an important role in Parkinson's disease (PD), but its biological functions are largely unknown. Here, we cloned the homolog of human LRRK2, characterized its expression, and investigated its biological functions in zebrafish. The blockage of zebrafish LRRK2 (zLRRK2) protein by morpholinos caused embryonic lethality and severe developmental defects such as growth retardation and loss of neurons. In contrast, the deletion of the WD40 domain of zLRRK2 by morpholinos targeting splicing did not induce severe embryonic developmental defects; rather it caused Parkinsonism-like phenotypes, including loss of dopaminergic neurons in diencephalon and locomotion defects. These neurodegenerative and locomotion defects could be rescued by over-expressing zLRRK2 or hLRRK2 mRNA. The administration of L-dopa could also rescue the locomotion defects, but not the neurodegeneration. Taken together, our results demonstrate that zLRRK2 is an ortholog of hLRRK2 and that the deletion of WD40 domain of zLRRK2 provides a disease model for PD.


Assuntos
Neurônios/metabolismo , Transtornos Parkinsonianos/genética , Proteínas Serina-Treonina Quinases/genética , Deleção de Sequência , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Embrião não Mamífero/metabolismo , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Mutação , Degeneração Neural/genética , Transtornos Parkinsonianos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Proteínas de Peixe-Zebra/metabolismo
11.
Neuron ; 55(1): 37-52, 2007 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-17610816

RESUMO

We reported previously that calpain-mediated Cdk5 activation is critical for mitochondrial toxin-induced dopaminergic death. Here, we report a target that mediates this loss. Prx2, an antioxidant enzyme, binds Cdk5/p35. Prx2 is phosphorylated at T89 in neurons treated with MPP+ and/or MPTP in animals in a calpain/Cdk5/p35-dependent manner. This phosphorylation reduces Prx2 peroxidase activity. Consistent with this, p35-/- neurons show reduced oxidative stress upon MPP+ treatment. Expression of Prx2 and Prx2T89A, but not the phosphorylation mimic Prx2T89E, protects cultured and adult neurons following mitochondrial insult. Finally, downregulation of Prx2 increases oxidative stress and sensitivity to MPP+. We propose a mechanistic model by which mitochondrial toxin leads to calpain-mediated Cdk5 activation, reduced Prx2 activity, and decreased capacity to eliminate ROS. Importantly, increased Prx2 phosphorylation also occurs in nigral neurons from postmortem tissue from Parkinson's disease patients when compared to control, suggesting the relevance of this pathway in the human condition.


Assuntos
Quinase 5 Dependente de Ciclina/fisiologia , Proteínas de Homeodomínio/fisiologia , Intoxicação por MPTP/metabolismo , Doença de Parkinson Secundária/metabolismo , Adenoviridae/genética , Sequência de Aminoácidos , Animais , Western Blotting , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/enzimologia , Córtex Cerebral/metabolismo , Técnicas de Transferência de Genes , Proteínas de Homeodomínio/metabolismo , Imuno-Histoquímica , Imunoprecipitação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Neurônios/enzimologia , Neurônios/metabolismo , Doença de Parkinson Secundária/induzido quimicamente , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Substância Negra/citologia , Substância Negra/enzimologia
12.
J Neurosci ; 29(40): 12497-505, 2009 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-19812325

RESUMO

Recent evidence suggests that abnormal activation of cyclin-dependent kinase 5 (cdk5) is a critical prodeath signal in stroke. However, the mechanism(s) by which cdk5 promotes death is unclear. Complicating the role of cdk5 are the observations that cdk5 can exist in multiple cellular regions and possess both prosurvival and prodeath characteristics. In particular, the critical role of cytoplasmic or nuclear cdk5 in neuronal jury, in vivo, is unclear. Therefore, we determined where cdk5 was activated in models of ischemia and how manipulation of cdk5 in differing compartments may affect neuronal death. Here, we show a critical function for cytoplasmic cdk5 in both focal and global models of stroke, in vivo. Cdk5 is activated in the cytoplasm and expression of DNcdk5 localized to the cytoplasm is protective. Importantly, we also demonstrate the antioxidant enzyme Prx2 (peroxiredoxin 2) as a critical cytoplasmic target of cdk5. In contrast, the role of cdk5 in the nucleus is context-dependent. Following focal ischemia, nuclear cdk5 is activated and functionally relevant while there is no evidence for such activation following global ischemia. Importantly, myocyte enhancer factor 2D (MEF2D), a previously described nuclear target of cdk5 in vitro, is also phosphorylated by cdk5 following focal ischemia. In addition, MEF2D expression in this paradigm ameliorates death. Together, our results address the critical issue of cdk5 activity compartmentalization, as well as define critical substrates for both cytoplasmic and nuclear cdk5 activity in adult models of stroke.


Assuntos
Isquemia Encefálica/metabolismo , Quinase 5 Dependente de Ciclina/metabolismo , Citoplasma/metabolismo , Peroxirredoxinas/metabolismo , Animais , Isquemia Encefálica/etiologia , Morte Celular/fisiologia , Células Cultivadas , Cerebelo/metabolismo , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Ratos , Ratos Wistar , Acidente Vascular Cerebral/complicações
13.
J Neurochem ; 112(2): 497-510, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19895669

RESUMO

DNA damage is a critical component of neuronal death underlying neurodegenerative diseases and injury. Neuronal death evoked by DNA damage is characterized by inappropriate activation of multiple cell cycle components. However, the mechanism regulating this activation is not fully understood. We demonstrated previously that the cell division cycle (Cdc) 25A phosphatase mediates the activation of cyclin-dependent kinases and neuronal death evoked by the DNA damaging agent camptothecin. We also showed that Cdc25A activation is blocked by constitutive checkpoint kinase 1 activity under basal conditions in neurons. Presently, we report that an additional factor is central to regulation of Cdc25A phosphatase in neuronal death. In a gene array screen, we first identified Pim-1 as a potential factor up-regulated following DNA damage. We confirmed the up-regulation of Pim-1 transcript, protein and kinase activity following DNA damage. This induction of Pim-1 is regulated by the nuclear factor kappa beta (NF-kappaB) pathway as Pim-1 expression and activity are significantly blocked by siRNA-mediated knockdown of NF-kappaB or NF-kappaB pharmacological inhibitors. Importantly, Pim-1 activity is critical for neuronal death in this paradigm and its deficiency blocks camptothecin-mediated neuronal death. It does so by activating Cdc25A with consequent activation of cyclin D1-associated kinases. Taken together, our results demonstrate that Pim-1 kinase plays a central role in DNA damage-evoked neuronal death by regulating aberrant neuronal cell cycle activation.


Assuntos
Ciclo Celular/fisiologia , Dano ao DNA/fisiologia , Neurônios/fisiologia , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Camptotecina/farmacologia , Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Linhagem Celular Transformada , Córtex Cerebral/citologia , Imunoprecipitação da Cromatina/métodos , Dano ao DNA/efeitos dos fármacos , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Proteínas de Fluorescência Verde/genética , Humanos , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , Neurônios/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-pim-1/deficiência , Proteínas Proto-Oncogênicas c-pim-1/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Estaurosporina/farmacologia , Isótopos de Enxofre/metabolismo , Fatores de Tempo , Transfecção/métodos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologia , Fosfatases cdc25/metabolismo
14.
Aging (Albany NY) ; 13(1): 77-88, 2020 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-33291077

RESUMO

Pink1, Parkin and Fbxo7, three autosomal recessive familial genes of Parkinson's disease (PD), have been implicated in mitophagy pathways for quality control and clearance of damaged mitochondria, but the interplay of these three genes still remains unclear. Here we present that Fbxo7 and Pink1 play a reciprocal role in the regulation of their protein levels. Regardless of the genotypes of Fbxo7, the wild type and the PD familial mutants of Fbxo7 stabilize the processed form of Pink1, supporting the prior study that none of the PD familial mutations in Fbxo7 have an effect on the interaction with Pink1. On the other hand, the interaction of Fbxo7 with Bag2 further facilitates its capability to stabilize Pink1. Intriguingly, the stabilization of Fbxo7 by Pink1 is specifically observed in substantial nigra pars compacta but striatum and cerebral cortex. Taken together, our findings support the notion that Fbxo7 as a scaffold protein has a chaperon activity in the stabilization of proteins.


Assuntos
Encéfalo/metabolismo , Proteínas F-Box/metabolismo , Doença de Parkinson/metabolismo , Proteínas Quinases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Córtex Cerebral/metabolismo , Corpo Estriado/metabolismo , Proteínas F-Box/genética , Técnicas de Introdução de Genes , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Chaperonas Moleculares/metabolismo , Mutação , Doença de Parkinson/genética , Parte Compacta da Substância Negra/metabolismo , Complexo de Endopeptidases do Proteassoma , Proteínas Quinases/genética , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Proteólise , Ubiquitinação
15.
Cell Death Dis ; 10(2): 135, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30755590

RESUMO

The unfolded protein response (UPR) triggered by endoplasmic reticulum (ER) stress is a feature of many neurodegenerative diseases including Alzheimer's disease, Huntington's disease and Parkinson's disease (PD). Although the vast majority of PD is sporadic, mutations in a number of genes including PARK7 which encodes the protein DJ-1 have been linked to early-onset, familial PD. In this regard, both PD of sporadic and genetic origins exhibit markers of ER stress-induced UPR. However, the relationship between pathogenic mutations in PARK7 and ER stress-induced UPR in PD pathogenesis remains unclear. In most contexts, DJ-1 has been shown to protect against neuronal injury. However, we find that DJ-1 deficiency ameliorates death in the context of acute ER stress in vitro and in vivo. DJ-1 loss decreases protein and transcript levels of ATF4, a transcription factor critical to the ER response and reduces the levels of CHOP and BiP, its downstream effectors. The converse is observed with DJ-1 over-expression. Importantly, we find that over-expression of wild-type and PD-associated mutant form of PARK7L166P, enhances ER stress-induced neuronal death by regulating ATF4 transcription and translation. Our results demonstrate a previously unreported role for wild-type and mutant DJ-1 in the regulation of UPR and provides a potential link to PD pathogenesis.


Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Morte Celular/genética , Estresse do Retículo Endoplasmático/genética , Proteína Desglicase DJ-1/metabolismo , Resposta a Proteínas não Dobradas , Regulação para Cima , Fator 4 Ativador da Transcrição/genética , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Fibroblastos , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Estresse Oxidativo/genética , Doença de Parkinson/metabolismo , Proteína Desglicase DJ-1/genética , RNA Mensageiro/metabolismo
16.
J Neurosci ; 26(34): 8819-28, 2006 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-16928871

RESUMO

Cell cycle regulators appear to play a paradoxical role in neuronal death. We have shown previously that cyclin-dependent kinases (CDKs), along with their downstream effectors, Rb (retinoblastoma) and E2F/DP1 (E2 promoter binding factor/deleted in polyposis 1), regulate neuronal death evoked by the DNA damaging agent camptothecin. However, the mechanism by which CDKs are activated in this model is unclear. The cell division cycle 25A (Cdc25A) phosphatase is a critical regulator of cell cycle CDKs in proliferating cells. In cortical neurons, we presently show that expression of Cdc25A promotes death even in the absence of DNA damage. Importantly, Cdc25A activity is rapidly increased during DNA damage treatment. Inhibition of Cdc25A blocks death and reduces cyclin D1-associated kinase activity and Rb phosphorylation. This indicates that endogenous Cdc25A activity is important for regulation of cell cycle-mediated neuronal death. We also examined how Cdc25A activity is regulated after DNA damage. Cultured embryonic cortical neurons have a significant basal activity of checkpoint kinase 1 (Chk1), a kinase that regulates cell cycle arrest. During camptothecin treatment of neurons, this activity is rapidly downregulated with a concomitant increase in Cdc25A activity. Importantly, expression of wild-type Chk1, but not kinase-dead Chk1, inhibits the camptothecin-induced increase in Cdc25A activity. In addition, Chk1 expression also promotes survival in the presence of the DNA-damaging agent. Together, our data suggest that a Chk1/Cdc25A activity participates in activation of a cell cycle pathway-mediated death signal in neurons. These data also define how a proliferative signal may be abnormally activated in a postmitotic environment.


Assuntos
Apoptose/fisiologia , Camptotecina/farmacologia , Ciclo Celular/fisiologia , Neurônios/citologia , Neurônios/metabolismo , Proteínas Quinases/metabolismo , Fosfatases cdc25/metabolismo , Animais , Células Cultivadas , Quinase 1 do Ponto de Checagem , Quinases Ciclina-Dependentes/metabolismo , Dano ao DNA , Camundongos , Camundongos Endogâmicos , Neurônios/efeitos dos fármacos , Proteínas Quinases/fisiologia , Interferência de RNA , Fosfatases cdc25/antagonistas & inibidores , Fosfatases cdc25/genética
17.
Cell Signal ; 18(10): 1572-83, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16446076

RESUMO

Studies have suggested that cholesterol imbalance in the brain might be related to the development of neurological disorders such as Alzheimer's disease and Niemann-Pick disease type C. Previously, we have reported that U18666A, a cholesterol transport-inhibiting agent, leads to apoptosis and intracellular cholesterol accumulation in primary cortical neurons. In this study, we examined the effects of U18666A-mediated neuronal apoptosis, and found that chronic exposure to U18666A led to the activation of caspases and calpains and hyperphosphorylation of tau. Tau hyperphosphorylation is regulated by several kinases that phosphorylate specific sites of tau in vitro. Surprisingly, the kinase activity of cyclin-dependent kinase 5 decreased in U18666A-treated cortical neurons whereas its protein level remained unchanged. The amount of glycogen synthase kinase 3 and mitogen-activated protein kinases were found to decrease in their phosphorylated states by Western blot analysis. Gene transcription was further studied using microarray analysis. Genes encoding for kinases and phosphatases were differentially expressed with most up-regulated and some down-regulated in expression upon U18666A treatment. The activation of cysteine proteases and cholesterol accumulation with tauopathies may provide clues to the cellular mechanism of the inhibition of cholesterol transport-mediated cell death in neurodegenerative diseases.


Assuntos
Androstenos/farmacologia , Apoptose/efeitos dos fármacos , Calpaína/metabolismo , Caspases/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neurônios/efeitos dos fármacos , Animais , Células Cultivadas , Quinase 5 Dependente de Ciclina/metabolismo , Citoesqueleto/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/metabolismo , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/enzimologia , Fosfoproteínas Fosfatases/metabolismo , Fosforilação/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas tau/metabolismo
18.
Nat Commun ; 8(1): 1399, 2017 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-29123128

RESUMO

Mutations in PTEN-induced kinase 1 (PINK1) result in a recessive familial form of Parkinson's disease (PD). PINK1 loss is associated with mitochondrial Ca2+ mishandling, mitochondrial dysfunction, as well as increased neuronal vulnerability. Here we demonstrate that PINK1 directly interacts with and phosphorylates LETM1 at Thr192 in vitro. Phosphorylated LETM1 or the phospho-mimetic LETM1-T192E increase calcium release in artificial liposomes and facilitates calcium transport in intact mitochondria. Expression of LETM1-T192E but not LETM1-wild type (WT) rescues mitochondrial calcium mishandling in PINK1-deficient neurons. Expression of both LETM1-WT and LETM1-T192E protects neurons against MPP+-MPTP-induced neuronal death in PINK1 WT neurons, whereas only LETM1-T192E protects neurons under conditions of PINK1 loss. Our findings delineate a mechanism by which PINK1 regulates mitochondrial Ca2+ level through LETM1 and suggest a model by which PINK1 loss leads to deficient phosphorylation of LETM1 and impaired mitochondrial Ca2+ transport..


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Mitocôndrias/patologia , Doença de Parkinson/patologia , Proteínas Quinases/genética , Animais , Células Cultivadas , Transporte de Íons/fisiologia , Lipossomos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Neurônios/patologia , Doença de Parkinson/genética , Fosforilação
19.
PLoS One ; 9(9): e106601, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25210784

RESUMO

Loss-of-function mutations in DJ-1 (PARK7) gene account for about 1% of all familial Parkinson's disease (PD). While its physiological function(s) are not completely clear, DJ-1 protects neurons against oxidative stress in both in vitro and in vivo models of PD. The molecular mechanism(s) through which DJ-1 alleviates oxidative stress-mediated damage remains elusive. In this study, we identified Paraoxonase-2 (PON2) as an interacting target of DJ-1. PON2 activity is elevated in response to oxidative stress and DJ-1 is crucial for this response. Importantly, we showed that PON2 deficiency hypersensitizes neurons to oxidative stress induced by MPP+ (1-methyl-4-phenylpyridinium). Conversely, over-expression of PON2 protects neurons in this death paradigm. Interestingly, PON2 effectively rescues DJ-1 deficiency-mediated hypersensitivity to oxidative stress. Taken together, our data suggest a model by which DJ-1 exerts its antioxidant activities, at least partly through regulation of PON2.


Assuntos
Antioxidantes/metabolismo , Arildialquilfosfatase/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Proteínas Oncogênicas/biossíntese , Doença de Parkinson/genética , Animais , Apoptose/genética , Arildialquilfosfatase/genética , Sobrevivência Celular , Regulação da Expressão Gênica , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Neurônios/metabolismo , Neurônios/patologia , Proteínas Oncogênicas/genética , Estresse Oxidativo , Doença de Parkinson/patologia , Proteína Desglicase DJ-1
20.
Nat Cell Biol ; 12(6): 563-71, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20473298

RESUMO

Accumulating evidence suggests that deregulated cyclin-dependent kinase 5 (Cdk5) plays a critical part in neuronal death. However, the pathogenic targets of Cdk5 are not fully defined. Here we demonstrate that the Cdk5 activator p35 interacts directly with apurinic/apyrimidinic endonuclease 1 (Ape1), a protein crucial for base excision repair (BER) following DNA damage. Cdk5 complexes phosphorylate Ape1 at Thr 232 and thereby reduces its apurinic/apyrimidinic (AP) endonuclease activity. Ape1 phosphorylation is dependent on Cdk5 in in vitro and in vivo. The reduced endonuclease activity of phosphorylated Ape1 results in accumulation of DNA damage and contributes to neuronal death. Overexpression of Ape1(WT) and Ape1(T232A), but not the phosphorylation mimic Ape1(T232E), protects neurons against MPP(+)/MPTP. Loss of Ape1 sensitizes neurons to death. Importantly, increased phosphorylated Ape1 was also observed in post-mortem brain tissue from patients with Parkinson's and Alzheimer's diseases, suggesting a potential link between Ape1 phosphorylation and the pathogenesis of neurodegenerative diseases.


Assuntos
Quinase 5 Dependente de Ciclina/metabolismo , Dano ao DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Endonucleases/metabolismo , Neurônios/metabolismo , Animais , Quinase 5 Dependente de Ciclina/fisiologia , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/fisiologia , Humanos , Masculino , Camundongos , Camundongos Knockout , Fosforilação
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