The Role Of Differentiated Embryonic Chondrocyte-expressed Gene 1 (DEC1) In Parkinson's Disease (PD)

Parkinson's disease(PD),the second most common neurodegenerative disorder after Alzheimer's disease(AD),is pathologically characterized by selective loss of midbrain dopaminergic neurons in the substantianigra pars compacta(SNpc).The etiology of PD is that the genetic and microenvironmental factors may mutually cooperate to promote dopaminergic neuronal death.The microenvironmental factors have been associated with oxidative stress,endoplasmic reticulum(ER)stress,neuroinflammation,mitochondrial dysfunction,loss of calcium homeostasis and neuron apoptosis.Recently,the major progress of dopaminergic neuronal death has been made in the genetic and signaling networks that control the generation of midbrain dopaminergic neurons.The studies have revealed the crucial role of several transcription factors in midbrain dopaminergic neurons development.Human differentiated embryonic chondrocyte gene 1(DEC1),also called mouse stimulated with retinoic acid 13(STRA13)and rat enhancer of split and hairy related protein 2(SHARP2),is one of the basic helix-loop-helix(bHLH)transcription factors.It is widely expressed in normal tissues and is associated with cell differentiation,proliferation,biological rhythm,apoptosis and lipid metabolism.DEC1 is related to neuronal differentiation and neurons mature in the central nervous system(CNS).For example,overexpression of STRA13(DEC1)promotes neuronal differentiation in P19 cells,and SHARP1 and SHARP2(DEC1)are expressed in the subregions of CNS that have been associated with adult plasticity.However,the role of DEC1 in dopaminergic neurons is totally unknown.In this study,we reported that DEC1 decreased in SNpc using MPTP mice model of PD.In vitro,we found that DEC1 expression decreased in MPP+-induced apoptosis in SH-SY5Y cells.Next,we explore the role and the mechanisms of DEC1 on MPP+-induced apoptosis.In the second part of our study,we used wild type(DEC1+/+)mice and DEC1 knockout(DEC1-/-)mice with different ages(6-month and 4-month)to explore the role of DEC1 on dopaminergic neurons in SNpc.We found that DEC1 deficiency in 6-month mice led to motor dysfunction compared to the corresponding wild type mice.Furthermore,we found there was obvious damages selectively in SNpc neurons in 6-month DEC1-/-mice.Our study suggests that DEC1 is a novel and potent candidate target in PD.The article is divided into two parts:Part ?:The role and mechanisms of DEC1 in MPTP/MPP+-induced neurontoxityPart ?:The influence and mechanisms of DEC1 deficiency on dopaminergic neurons in SNpcPart I:The role and mechanisms of DEC1 in MPTP/MPP+-induced neurontoxityPurpose:We aimed to investigate the effect of DEC1 on MPTP/MPP+-induced apoptosis.Methods:The method is devided into two parts.1.Adult mice were injected with MPTP or saline for 5 days and subsequently received behavior tests.After that,we measured TH and Stral3 expression in SNpc by immunofluorescence.Western blot analysis was used to detect protein levels of DEC1,TH,DAT,apoptosis-related proteins(cleaved caspase 3,caspase 3,Bax,Bel-2)and PI3K/Akt pathway(PI3Kp110?,the p-Akt(Ser473)/Akt,p-GSK-3?(Ser9)/GSK-3? and ?-catenin).2.SH-SY5Y cells treated with MPP+ were used as cell model in vitro.1)We measured DEC1,TH,DAT,apoptosis-related proteins and PI3K/Akt pathway related proteins in SH-SY5Y cells treated with different concentrations of MPP+.2)We modulated DEC1 by overexpression and shRNA and used TUNEL staining to detect apoptosis in MPP+-induced neurotoxcity.3)Analysis of apoptosis-related proteins was performed by Western blot in MPP+-induced apoptosis when cells were modulated with overexpression of DEC1.4)Administration of drugs to inhibit or activate PI3K/Akt pathway were used to explore the relationship of DEC1 and PI3K/Akt pathway in MPP+-induced apoptosis.5)We modulate DEC1 expression by overexpression experiment to study whether DEC1 affect PI3K/Akt in SH-SY5Y cells treated with MPP+.Results:1.In vivo,DEC1 expression decreased in MPTP-injected mice in SNpc;2.The activity of caspase 3 increased and the levels of PI3K/Akt pathway proteins(PI3Kp110?,the p-Akt(Ser473)/Akt,p-GSK-3?(Ser9)/GSK-3?,?-catenin)decreased in MPTP-treated mice;3..In vitro,DEC1 downregulated in MPP+-induced apoptosis which was in accord with the results in vivo.4.Overexpression of DEC1 relieved MPP+-induced toxicity and knockdown of DEC1 aggravated apoptosis in MPP+-induce toxicity;5.Overexpression of DEC1 abolished the increased activity of caspase 3 and the decrease of TH and DAT;6.We found DEC1 was associated with PI3K/Akt pathway by using inhibitor and activator of PI3K;7.Overexpression of DEC1 selectively abolished the decrease of proteins in PI3K/Akt/GSK3?/?-catenin pathway.Conclusion:DEC1 plays a protective role in MPP+-induced apoptosis via PI3K/Akt pathway.Part ?:The influence and mechanisms of DEC1 deficiency on dopaminergic neurons in SNpcPurpose:On the basis of the first part of our results,we investigated the role and mechanisms of DEC1 deficiency on dopaminergic neurons.Methods:1)We used wild type(DEC1+/+)mice and DEC1 knockout(DEC1-/-)mice with different ages(4-month and 6-month)to detect motor ability in open-field test,beam walking test and rotated test and to assess the learning and memory ability in Morris water maze.2)Immunohistochemistry was used to measure TH+ cells in SNpc in 4-month or 6-month wild type(DEC1+/+)mice and DEC1 knockout(DEC1-/-)mice.After that we used Western blot to analyse TH and DAT protein levels in 4-month or 6-month wild type(DEC1+/+)mice and DEC1 knockout(DEC1-/-)mice.3)Nissle staining was used to detect neuron of mibrain and hippocampus in 4-month or 6-month wild type(DEC1+/+)mice and DEC1 knockout(DEC1-/-)mice.4)TUNEL assay was used to measure apoptosis in SNpc in widtype and knockout mice of both ages.Apoptosis-ralated proteins were detected by Western blot in widtype and knockout mice of both ages.5)PI3K/Akt pathway related protein levels were measured in widtype and knockout mice by Western blot.Results:(1)6-month DEC1 knockout mice showed motor deficiency compared to the corresponding wild type mice;(2)There was no significant difference between widtype(DEC1+/+)mice and DEC1 knockout(DEC1-/-)mice in both ages on learning and memory ability in Morris water maze;(3)6-month knockout mice showed decrease of TH+ cells selectively in SNpc;(4)The apoptotic cells in SNpc were mainly related to the increased caspase 3 activity via PI3K/Akt pathway.Conclusion:DEC1 plays a protective role on dopaminergic neurons and it may decreases caspase 3 activity via PI3K/Akt pathway.