The Roles And Mechanisms Of Phosphodiesterase 4 In Neurodegeneration Of Dopaminergic Neurons

Objective:Parkinson's disease(PD)is a neurodegenerative disease caused by dopaminergic neuronal lesions in the substantia nigra pars compacta(SNpc)and striatum.Although the etiology of PD is not completely clear,mitochondrial dysfunction and dopaminergic neuronal lesion apoptosis caused by enhanced cerebral oxidative stress are considered to be one of the main factors for the occurrence and development of PD.Therefore,neuroprotectants that improve mitochondrial dysfunction and block the dopaminergic neuron damage induced by oxidative stress may help to prevent the progression of PD.Motor function is regulated by direct or indirect pathways through dopaminergic neurons transmitted to the striatum,and cyclic Adenosine Monophosphate(cAMP)mediates dopamine receptor signaling in the striatum,while phosphodiesterase 4(PDE4)regulates cAMP.The role of PDE4 in the apoptosis of dopaminergic neurons and its mechanism have not yet been clarified.Therefore,this paper explored the roles and mechanisms of PDE4 in the neurodegeneration of dopaminergic neurons based on the in vivo and in vitro model of PD.Methods:(1)The cell models of dopaminergic neuron degeneration were constructed by using MPP+(500?M)to treat SH-SY5Y cells,and using MPP+(500?M)to treat human dopaminergic neurons and primary neurons.The cell model also included the overexpression of ?-synucleinaA53T mutant in SH-SY5Y cells.The expression of apoptosis-related proteins were detected after knockdown of PDE4B or PDE4D.(2)PDE4 inhibitors FCPR16(25 ?M)and roflupram(20?M)treated SH-SY5Y cells stimulated by MPP+(500 ?M)to detect cell viability,lactate dehydrogenase(LDH)activity,apoptosis rate,mitochondrial membrane potential(MMP),oxidative stress response and the expression of apoptosis-related proteins.The changes of cAMP-response element binding protein(CREB)-related pathway proteins and their effects on cell function were detected.Lyso-Tracker Red staining and CYTO-ID staining were carried out,and the changes of the activation of AMP-activated protein kinase(AMPK),LC3-II protein and p62 protein were measured.(3)The behaviors of mice were detected by pole test,rotarod test and gait experiment.Nissl staining,tyrosine hydroxylase(TH)immunohistochemistry,and TH immunofluorescence assay were used to detect neuroprotective effects.The expression of Peroxisome proliferator-activated receptor-gamma coactivator(PGC)-1? and the phosphorylation of CREB were detected by Western blot and immunofluorescence techniques.Results:(1)The effects of knockdown PDE4B or PDE4D on modeling SH-SY5Y cells include the improvement of cell viability and MMP,as well as the reduction of LDH activity and reactive oxygen species level.Knockdown of PDE4B or PDE4D may also increase the ratio of Bcl-2/Bax protein and reduce the expression of cleaved caspase-3.(2)PDE4 inhibitors FCPR16 or ROF play the role in MPP+-treated SH-SY5Y cells and primary neurons,including the improvement of cell viability and MMP,the decrease of LDH activity,apoptosis rate and reactive oxygen species level,and the improvement of mitochondrial function.FCPR16 or ROF processes MPP+-treated SH-SY5Y cells to promote the phosphorylation of CREB and activate its associated signaling pathways.(3)FCPR16 inhibits PDE4 and plays the role in MPP+-treated SH-SY5Y cells,including increased lysosomal numbers,autophagic flux,and the expression of LC3-?,promoting the activation of AMPK.(4)The effects of ROF or rolipram on MPTP-induced mice include shortening the time required to climb the pole,increasing the running time on the roller,improving gait,increasing the number of Nissl positive cells,upregulating the expression of PGC-1? and TH,and promoting the phosphorylation of the CREB.Conclusion:Inhibition of PDE4 had anti-apoptotic effects on model cells induced by MPP+or overexpressed ?-synucleinA53T mutants,improved the motor deficiency of MPTP model mice,and played a neuroprotective role on dopaminergic neurons by activating the cAMP/EPAC/Akt signaling pathway,CREB/PGC-1? signaling pathway and AMPK-dependent autophagy pathway.