Neuroprotective Effect And Mechanism Of Rapamycin In A Mouse Model Of Parkinson's Disease

Parkinson's disease(PD)is a common chronic neurodegenerative disease in elderly and is characterized by the progressive degeneration of dopaminergic(DA)neurons in the pars compacta of substantia nigra(SNpc)and the formation of Lewy bodies.Glutamate excitotoxicity also plays an important role in the pathogenesis of PD.In the normal condition,glutamate is released into the synaptic cleft and participates in the excitatory neurotransmission via binding on the glutamate receptors located on the postsynaptic neurons.While dysfunctional glutamate metabolism contributes to the movement disorders and cognitive impairment in PD.Excessive glutamate in the synaptic cleft over-stimulates the ionotropic and metabotropic glutamate receptors in the postsynaptic membrane and mediates the excitotoxicity.Through over-activating the N-methyl-D-aspartate(NMDA)receptor,glutamate can induce intracellular Ca2+ overload,production of reactive oxygen species and reactive nitrogen radicals,result in mitochondrial dysfunction,and thus lead to the neuronal death;through over-activating a-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid(AMPA)receptor and kainic acid(KA)receptor,glutamate can induce Na+ influx and acute osmotic swelling of nerve cells and mediate neuronal death.Excitatory amino acid transporters(EAATs)play a predominant role in clearance of extracellular excessive glutamate.Five mammalian EAATs have been characterized:GLAST(glutamate/aspartate transporter,also called EAAT1),GLT-1(glutamate transporter-1,also called EAAT2),EAAC1(excitatory amino acid carrier-1,also called EAAT3),EAAT4 and EAAT5.Among these five isoforms,GLT-1 in astrocytes is responsible for taking up nearly 90%of glutamate.Thus far,evidences have shown that decreased expression of GLT-1 was closely related to the pathogenesis of amyotrophic lateral sclerosis(ALS).In the past decades,multiple expression patterns of glutamate transporters in PD models were reported.Among these studies,impairment of glutamate transporters has been shown in PD models constructed by 6-hydroxydopamine(6-OHDA),1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)and 1-methyl-4-phenylpyridinium(MPP?).However,there had been no direct evidence showing that impaired glutamate transporter was related to the pathogenesis of PD,until Assous et al.used glutamate transporter substrate inhibitor l-trans-pyrrolidine-2,4-dicarboxylate(PDC)in the substantia nigra and striatum to succeed mimic several PD features,including neurons death and axons dystrophy,and the motor disorder when DA neurons loss exceeded 50%.Our recent study showed that MPP+ decreased GLT-1 expression in astrocytes,also partially indicating the correlation between the glutamate transporter level and PD.We also found that ceftriaxone could improve the membrane expression of GLT-1 in MPP+-treated astrocytes and attenuate the apoptosis of astrocytes via suppression of nuclear factor-kappa B(NF-?B)/JNK/c-Jun signal pathway.However,the regulatory mechanism of glutamate transporters in PD has not been completely elucidated.Rapamycin,a lipophilic macrolide antibiotic,is widely used in clinical practice as an immunosuppressant.Rapamycin induces autophagy by inactivating the mammalian target of rapamycin(mTOR)and hence blocking downstream calcium-dependent and non-dependent mTOR signaling pathways,as well as binding intracellularly to the FK506 binding protein.Studies have showed that rapamycin is able to provide neuronal protection in a series of experimental models of neurodegenerative diseases.Moreover,rapamycin is also reported to slow aging process and extend mammalian lifespan.In the previous studies,rapamycin showed potential neuroprotection in PD by inducing clearance of misfolded proteins via autophagy and protecting DA neurons from death via blockade translation of RTP801/REDD1/Ddit4 and phosphorylation of the survival kinase Akt.Rapamycin was also reported to protect DA neurons against rotenone-induced cell death.Besides,rapamycin was shown to stimulate the neurite outgrowth in PC 12 cells,a cellular model of the DA neurons.Study also indicated that PINK1,a familial PD-linked gene.could activate the mammalian target of rapamycin complex 2(mTORC2),induce phosphorylation of Akt and thus exert its neuroprotective effect in SH-SY5Y cells in vitro.Thus rapamycin is a promising drug in treating PD,however,its detailed neuroprotective mechanism needs to be explored.Previous evidences indicated that rapamycin treatment could increase GLT-1 expression in astrocytes and such regulatory mechanism was involved in the PI3K/Akt/mTOR and mTOR/Akt/NF-?B pathways.Yet there is still no evidence to show whether rapamycin regulates the expression of glutamate transporters and thus performs therapeutic effects in PD models.Recently,a number of studies have demonstrated that immune response plays an important role in the pathogenesis of PD and neuroinflammation becomes a novel therapeutic target in the treatment of neurodegenerative disease.MTOR inhibitor RAD001 was shown to promote the immune function and ameliorate immunosenescence in elderly.However,whether rapamycin could modulate the immune response in PD and the related regulatory mechanism are still not fully understood.In this paper,we demonstrated that the neuroprotective effects of rapamycin in PD mice.Firstly,PD mice showed significant movement disorders as well as the striatum and substantia nigra tyrosine hydroxylase(tyrosine hydroxylase,TH)expression was significantly reduced.Compared with MPTP-treated group,rapamycin extended the suspension time of PD mice and shorten their pole-climbing time,showed rapamycin can improve ataxia of the PD mouse model.Rapamycin significantly increased the number of PD mouse substantia nigra and striatum TH positive neurons.Besides,Rapamycin significantly reduces the accumulationa-synuclein in the substantia nigra of the PD mouse.TUNEL experiments have shown that rapamycin can significantly reduce the PD mouse cell death in the substantia nigra.Therefore,we showed that rapamycin have neuroprotective effects in the PD mice.In this study,we found that in MPP?-treated astrocytes,4 hours rapamycin treatment can significantly increase the expression of GLT-1 total protein and membrane proteins.Rapamycin pretreatment can significantly increase glutamate uptake to MPP?-treated astrocytes,but the expression of GLAST did not change significantly.In the following experiments,we treated cells 4 hours with rapamycin.In vitro,rapamycin increases the expression and function of GLT-1.Then we studied the effect of rapamycin for glutamate transporters in PD mice.We found that rapamycin can increase substantia nigra GLAST and striatum GLT 1 expression at the total protein level in PD mouse.In addition,Rapamycin can also increase the expression of GLT-1 and GLAST cell membrane protein in the substantia nigra of PD mouse,and the expression of GLT-1 cytoplasm protein.It also showed rapamycin can increase the expression of GLT-1 by Immunohistochemistry experiment.Consistent with Glutamate transporter expression on the cell membrane,rapamycin also increased synaptic body glutamate uptake in PD mouse substantia nigra.These results showed that rapamycin can promote the expression and function of glutamate transporter in PD mouse.In order to explore the mechanism of rapamycin-induced upregulation of glutamate transporters,we examined the related signal pathway involved in regulating glutamate transporters upon treatment of rapamycin in MPTP-treated mice.The immunofluoresence double staining results showed that the interaction between glutamate transporters and ubiquitin was increased in MPTP-treated mice,suggesting glutamate transporters were ubiquitinated in midbrain in PD mice.We found that rapamcyin decreased the ubiquitination of glutamate transporters in MPTP-treated mice.Previous studies reported that ubiquitin ligase E3 Nedd4-2 mediated the ubiquitination of glutamate transporters and promoted their degradation,and our recent work found that Nedd4-2 mediated the ubiquitination of glutamate transporters.Here we examined the effect of rapamycin on the Nedd4-2-mediated ubiquitination of glutamate transporters.We found that rapamycin did not change the expression of phosphorylated Nedd4-2,an activated state of this E3 ligase in MPTP-treated mice.However,the expression of phosphorylated Nedd4-2 was obviously increased in the cytoplasm in MPTP-treated mice,and rapamycin significantly decreased the expression of phosphorylated Nedd4-2 in the cytoplasm,suggesting that rapamycin possibly suppressed the process which Nedd4-2 submit the ubiquitin to the substrate for degradation.To verify this hypothesis,we examined the Nedd4-2-mediated ubiquitination of glutamate transporters via immunofluorescence assay.Consistent with our recent unpublished data,the interaction between p-Nedd4-2 and glutamate transporters(GLT-1 and GLAST)was increased in MPTP-treated mice.Rapamycin decreased the interaction between p-Nedd4-2 and GLT-1,GLAST in MPTP-treated mice,suggesting rapamycin decreased the ubiquitination of glutamate transporters mediated by Nedd4-2 in PD model.Our results suggest that rapamycin decreases the activity of Nedd4-2,attenuates the Nedd4-2-mediated ubiquitination of glutamate transporters,and thus increases glutamate transporters expression and function in PD model.we found that rapamycin treatment significantly increased IL-6 expression in astrocytes treated with MPP+ for 24 h.Moreover,pretreatment of rapamycin activated the JAK2/STAT3 pathway in astrocytes treated with MPP+ for 24 h.Our ELISA results also revealed that IL-6 secretion was increased in both rapamycin treatment group in MPP?-treated astrocytes.Thus in the following experiments,we treated astrocytes with MPP+ for 24 h.Microglia is also a predominant source for the production of IL-6 and IL-6 secreted from microglia contributes to the inflammatory reaction in the pathogenesis of PD.Here we examined the effect of rapamycin on the IL-6/JAK2/STAT3 pathway in MPP?·treated microglial BV2 cells,which is a widely used model of microglia.We found that rapamycin did not change IL-6 expression in MPP?-treated microglial BV2 cells.Furthermore,rapamycin decreased the expression of phosphorylated JAK2 and STAT3 in MPP?-treated microglial BV2 cells,suggesting rapamycin suppressed the JAK2/STAT3 pathway in MPP+-treated microglial BV2 cells.Consistent with previous evidence,MPP+ elevated the production of IL-6,and interestingly,our ELISA results revealed that IL-6 secretion was decreased in both rapamycin treatment group and MPP+ treatment microglial BV2 cells.Previous study has reported that MPP+ could decrease the IL-6 mRNA expression in PC 12 cells and MPP+ cause cell death in PC12 cells partially by changing IL-6 expression.Thus we examine the effect of rapamycin on the IL-6/JAK2/STAT3 pathway in MPP?-treated PC12 cells.Because PC 12 cells could produce dopamine and possess the dopamine transporters,they have been used as a dopaminergic cellular model.In present work,we found that IL-6 protein expression was decreased in MPP+-treated PC 12 cells,however,rapamycin showed no obvious effects on the IL-6 expression in MPP?-treated PC 12 cells.Moreover,activity of JAK2/STAT3 pathway was reduced in MPP?-treated PC 12 cells compared with control,and rapamycin also showed no obvious effects on the activity of JAK2/STAT3 pathway in MPP?-treated PC 12 cells.These results suggest that rapamycin showed no obvious effects on the IL-6/JAK2/STAT3 pathway in MPP?-treated PC 12 cells.In addition,rapamycin decreased the expression of IL-6 receptor in these three cell types upon MPP+ treatment,these in vitro results indicated that rapamycin promoted the IL-6 secretion from astrocytes and activated IL-6/JAK2/STAT3 pathway,moreover,rapamycin suppressed the IL-6 secreted from microglial BV2 cells and the downstream JAK2/STAT3 pathway.We further explored whether this phenomenon exist in PD mice.Firstly,we examined the IL-6 production in serum,and we found that rapamycin significantly promoted IL-6 production in serum compared with control and single MPTP-treated mice.Then we found that rapamycin treatment increased the IL-6 expression at the protein in the midbrain rather than striatum in MPTP-treated mice.However,rapamycin treatment showed no obvious changes on the downstream JAK2/STAT3 pathway in midbrain and striatum in MPTP-treated mice.Since IL-6 expression was increased in astrocytes in vitro and IL-6 was reported to protect dopamine neurons from the neurotoxin.Here we examined the IL-6 expression in astrocytes and neurons in vivo via double staining,and we found that rapamycin treatment increased the IL-6 expression in astrocytes compared with control and single MPTP-treated mice.However,IL-6 expression in neurons showed no significant changes among these four groups.We further explored the upstream pathway in regulating IL-6 secretion and expression and we also examined the downstream effector molecules of STAT3.Here our in vitro results indicated that rapamycin treatment increased the expression of phosphorylated Akt,NF-?B in MPP?-treated astrocytes,and in vivo results indicated that rapamycin treatment increased the expression of phosphorylated NF-?B in MPTP-treated mice.Rapamycin treatment also decreased the expression of phosphorylated S6 which is the downstream effector of mTORCl in MPP?-treated astrocytes and in the midbrain in MPTP-treated mice,suggesting mTORCl was suppressed in our in vitro and in vivo models.Using the inhibitors of Akt,NF-?B(MK-2206,BAY 11-7082),we found that the expression of IL-6 showed no obvious changes among rapamycin?MPP?,MPP?+rapamycin,and MPP?-treated astrocytes by application of MK-2206,in addition,MK-2206 suppressed the secretion of IL-6 in MPP?+rapamyein group rather than rapamycin+MPP+ group,in MPP?-treated astrocytes.These results suggest Akt is partly responsible for regulating IL-6 secretion and expression in astrocytes.Moreover,we also found that the expression of IL-6 and downstream JAK2/STAT3 pathway showed no significant changes among rapamycin+MPP?,MPP??rapamycin,and MPP?-treated astrocytes by application of BAY 11-7082.BAY 11-7082 significantly decreased the IL-6 secretion in the supernatant of rapamycin?MPP+ and MPP?+rapamycin groups in MPP?-treated astrocytes.Our results suggest that mTOR/Akt/NF-?B is mainly responsible for the IL-6 secretion and expression in astrocytes upon rapamycin application in MPP?-treated astrocytes.In the meanwhile,we examined the downstream effector molecules of STAT3.Since we have found that rapamycin can reduce the neuronal death in SN and then we focus on the anti-apoptotic protein family.Here we demonstrated that rapamycin increased phosphorylated bcl-xl expression in vltro and in vivo in MPP+-treated astrocytes and MPTP-treated mice.Then we used NF-?B BAY 11-7082 to treat the astrocytes in these four groups,and we found that the expression of phosphorylated JAK2 and STAT3 showed no obvious changes among the rapamycin?MPP?,MPP?+rapamycin,and MPP?-treated astrocytes,Furthermore,the expression of phosphorylated bcl-2 and bcl-xl also no significant changes among these three groups by application of BAY 11-7082.showedThus our results suggest that mTOR/Akt/NF-?B are mainly responsible for the IL-6 secretion and expression in astrocytes upon rapamycin application in MPP?-treated astrocytes,moreover,rapamycin activates the IL-6/JAK2/STAT3 pathway to promote the anti-apoptotic protein(bcl-xl)expression in astrocytes.As mTOR/Akt/NF-?B also regulates the expression of glutamate transporters,here we also used the Akt and NF-?B inhibitors to examine whether this pathway is involved in regulating glutamate transporters expression.Inhibitor of Akt,MK-2206 and inhibitor of NF-?B,BAY 11-7082 decreased the GLT-1 expression at the membrane and total level in the rapmycin+MPP+ group,compared with single MPP+-treated astrocytes.MK-2206 and BAY 11-7082 also decreased the glutamate uptake in the rapmycin+MPP+ group,in MPP?-treated astrocytes.These results suggest that mTOR/Akt/NF-?B also partly participate in regulating the expression and function of glutamate transporters by application of rapamycin in MPP+-treated astrocytes.Taken together,we reported that rapamycin treatment improved the glutamate transporters expression and function in MPP?-treated astrocytes and in a MPTP mouse model of PD,and this regulatory mechanism was partially involved in the decreased activity of ubiquitin ligase E3 Nedd4-2(neuronal precursor cell expressed developmentally down-regulated 4-2)in the cytoplasm by application of rapamycin.We found that rapamycin decreased the interaction between Nedd4-2 and glutamate transporters,reduced the ubiquitination of glutamate transporters mediated by Nedd4-2 and thus increased glutamate transporters expression and function.In addition,we also found that rapamycin increased interleukin-6(IL-6)expression in astrocytes and promoted astrocyte-derived IL-6 secretion in vitro and in vivo.Rapamycin activated the IL-6/Janus kinase 2(JAK2)/signal transducer and activator of transcription 3(STAT3)pathway via astroglial autocrine.We also demonstrated that through activation of JAK2/STAT3 pathway,rapamycin increased the expression of anti-apoptotic protein bcl-xl and decreased the astrocytes death in PD model.Furthermore,we reported that mTOR-Akt-NF-?B cascade was responsible for the upregulated glutamate transporters and IL-6.We also found that rapamycin attenuated the immune inflammation in the in vitro and in vivo models of PD possibly via suppressing inflammatory cytokines production of microglia.Taken together,our studies indicate that rapamycin could be a potential drug in treatment of PD and our findings explore novel neuroprotective effects of rapamycin in PD.