The Role Of Endogenous Ghrelin Receptor In The Degeneration Of Nigral Dopaminergic Neurons In MPTP-induced Parkinson’s Disease Mice

Parkinson’s disease(PD)is the second prevalent neurodegenerative disorder,which is pathologically charactized by a selective loss of dopaminergic neurons in the substantia nigra(SN)and the subsequent dopamine(DA)depletion in the striatum(Str).The pathological hallmark of PD is the intracellular inclusions,termed Lewy bodies(LBs),which are mainly composed of the aggregated alpha-synuclein,resulting in the imbalance of direct and indirect pathway in the basal ganglia.This disorder is characterized symptomatically by bradykinesia,resting tremor and rigidity.Multiple factors might be involved in the pathogenesis of PD,such as genetic factors,environmental factors,aging,oxidative stress,inflammation and apoptosis.However,up to now,its etiology is largely unknown.Ghrelin,a novel brain-gut peptide,has been identified as an endogenous ligand for the growth hormone(GH)secretagogue receptor(GHS-R).There are two subtypes of GHS-R,GHS-R1 a and GHS-R1 b,in which GHS-R1 a is its fully functional receptor.GHS-R1 a was expressed both in the peripheral tissues and the central nervous system.In the central nervous system,GHS-R1 a is mainly distributed in the hypothalamus,pituitary,hippocampus,midbrain,SN and some other parts.Several lines of evidence suggest that the effects of ghrelin are not only restricted to feeding behavior,growth hormone secretion and energy homeostasis,but also concerned with neuroprotective effects on some brain regions.Our previous studies have shown that ghrelin could enhance the excitability of nigral dopaminergic neurons,resulting in increased DA release in the striatum by activation of GHS-R1 a.We also observed the neuroprotective effects of ghrelin on dopaminergic neurons against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-induced neurotoxicity,which was mediated by inhibition of the apoptosis and attenuation of the oxidative stress.In the presence of ghrelin,exogenous GHS-R1 a over-expression in the SN could protect dopaminergic neurons against MPTP-induced neurotoxicity.All the above results demonstrated that the neuroprotective effects of ghrelin in PD were mediated by its activation of GHS-R1 a.However,recent studies indicate that GHS-R1 a could also undergo dimerization with a variety of G-protein coupled receptors to form heterologous dimers,such as the dopamine type 1 receptor(D1R),dopamine type 2 receptor(D2R),melanocortin-3 receptor(MC3R),serotonin type 2C receptor(5-HT2C),and cannabinoid type 1 receptor(CB1),which play important roles in the physiological functions of the cells through activation of signal transduction pathwayswithout ghrelin binding.However,the role of endogenous GHS-R1 a in the nigral dopaminergic neurons is unclear.In order to explore the role of endogenous ghrelin receptor in the degeneration of nigral dopaminergic neurons in MPTP-induced Parkinson’s disease mice,we choose Ghsr+/-male and WT male mice,which were injected with MPTP and NS.The mice were divided into four groups,WT-NS group,WT-MPTP group,Ghsr+/--NS group and Ghsr+/--MPTP group.The contents of DA in the Str were measured by high performance liquid chromatography(HPLC).The numbers of the tyrosine hydroxylase(TH)-positive neurons and microglia in the SN were counted by immunofluorescent technique.The protein levles of Bcl-2,Bax,TH,SOD1 and IL-6 in the SN were detected by western blots.The results were as follows:1.After 1-week,3-week and 5-week MPTP treatment,the number of TH positive neurons in the SN of WT mice was decreased by 16%,34% and 48%,respectively(P<0.05).The protein levels of TH were decreased by 26%,51% and 44%,correspondingly(P<0.05).For the Ghsr+/-mice,the number of TH positive neurons in the SN was reduced by 40%,50% and 58%,respectively(P<0.05),and the protein levels of TH was decreased by 42%,61% and 74%(P<0.05).Meanwhile,compared with WTMPTP group,the number of TH positive neurons in the SN of the Ghsr+/--MPTP group was reduced by 33%,26% and 30%,respectively,and the levels of TH protein were reduced by 21%,40% and 59%,respectively(P<0.05).2.After 5-week MPTP treatment,for the WT mice,the contents of DA in the Str were decreased from 13.81±0.27 ng/mg to 6.65±0.67 ng/mg(P<0.01).For the Ghsr+/-mice,the contents of DA in the Str were reduced from 11.67±2.25 ng/mg to 3.81±0.49 ng/mg(P<0.001).Simultaneously,the contents of DA in the Str of Ghsr+/--MPTP group were significantly decreased when compared with that of WT-MPTP group(P<0.01).3.For the WT mice,the number of microglia in the SN was increased by 66% after 5-week MPTP treatment(P<0.001).For the Ghsr+/-mice,after 3-week and 5-week MPTP treatment,the number of microglia in the SN was increased by 60% and 107%,respectively(P<0.001,P<0.001).Compared with that of WT-MPTP group,the number of microglia in the SN of Ghsr+/--MPTP group was increased by 31% after 5-week MPTP injection(P<0.001).4.After 5-week MPTP treatment,the protein levels of IL-6 in WT mice were increased,but no significant difference was observed(P>0.05),and the protein levels of IL-6 in Ghsr+/-mice were increased by 65%(P<0.05).Meanwhile,compared with that of WT-MPTP group,the protein levels of IL-6 in the SN of Ghsr+/--MPTP group were increased by 23%(P<0.05).5.After 3-week and 5-week MPTP treatment,the protein levels of SOD1 in the SN of WT mice were decreased by 29% and 36%(P<0.05).In the Ghsr+/-mice,the protein levels of SOD1 was significantly decreased by 42% and 63%(P<0.05,P<0.01).After 5-week MPTP treatment,compared with that of WT-MPTP group,the protein levels of SOD1 in the SN of Ghsr+/--MPTP group were decreased by 42%(P<0.05).6.After 5-week MPTP treatment,the Bcl-2/Bax ratio in the SN was decreased by 50%(P<0.01).Meanwhile,the Bcl-2/Bax ratio in the SN of Ghsr+/--MPTP group was decreased by 34% compared with that of WT-MPTP group(P<0.05)In summary,the present data showed that endogenous GHS-R1 a knockdown could dramatically aggravate the degeneration of nigral dopaminergic neurons,including decreases in the number of TH-positive cells and TH expression in the early stages of MPTP-induced injury.The probable mechanisms of these effects might be mediated by activation of microglial,increased IL-6 expression,decreased SOD1 levels,enhanced Bcl-2 expression and reduced Bax expression.This study elucidates the important role of endogenous GHS-R1 a on the nigral dopaminergic neurons,and of which deficiency could make the dopaminergic neurons more susceptible to the neurotoxicity of MPTP.