Mechanism And Regulatory Strategies Of Change Of Glutamate Transporters Expression In Parkinson's Disease

Parkinson's disease(PD)is a common neurodegenerative disease,which is mainly appeared in the middle aged and elderly people.Its' clinical symptoms mainly contain rest tremor,bradykinesia,hypermyotonia and postural balance disorders.In recent years,the non-motor symptoms of PD gradually attracted people's attention,and these symptoms include sweating,constipation and other autonomic dysfunction,also olfactory loss,impaired visual-spatial ability,memory loss and other cognitive disorders,anxiety,depression,hallucinations,paranoia,dementia and other mental disorders.Currently the pathogenesis of PD is recognized as the degeneration and death of dopamine(DA)neurons in the substantia nigra and the formation of Lewy bodies.Meanwhile,excitotoxicity,environmental factors,calcium overload,mitochondrial dysfunction,excessive oxidative stress,aging and neural stem cell degeneration also contribute to the pathogenesis of PD.Based on the pathogenesis of PD,delaying dopaminergic degeneration and supplying exogenous dopamine are the basic principles,and the application of levodopa is still the "gold standard" in treating PD.Levodopa could cross the blood brain barrier,turn into the dopamine under the effect of dopamine decarboxylase and function as a replacement therapy.However,some side effects appeared in the long-term usage,which contain orthostatic hypotension,arrhythmia,delirium,hallucinations and other mental symptoms,even contain dyskinesia,deterioration phenomenon in the end dose,"on-off" phenomenon and other complications.Transplantation of neural stem cell and gene therapy are emerging treatments in recent years,but because of the high costs and risks,they could not be used in the clinical practice yet.Among the pathogenesis of PD,glutamate excitotoxicity is the hot research topic.Glutamate transporters decrease the excitotoxicity via uptake the excessive glutamate in the synaptic cleft.Glutamate is the most important excitatory neurotransmitter in the mammalian central nervous system.It is released to the synaptic cleft and taken into cells by the glutamate transporters located on the membrane of neurons and astrocytes.Eukaryotes high affinity glutamate transporters(excitatory amino acid transporters,EAATs)contain GLAST(EAAT1),GLT-1(EAAT2),EAAC1(EAAT3),EAAT4 and EAAT5.Glutamate transporters transport the glutamate via the Na+ and K+electrochemical gradient formed by Na+-K-ATP enzyme.When glutamate transporters transport one molecule glutamate,it accompanies by the symport of three Na+ and one H+ into the cells and antiporter of one K+ outside.In 2004,Gouaux's laboratory successfully indicated the crystal structure of prokaryotes aspartate transporter Pyrococcus horikoshii GltPh,which is the homologue of the glutamate transporter.Studies have shown that GltPh is a trimer consisting of three monomers,wherein each monomer has a substrate transport channel,and each monomer is an independent functional unit.GltPph consists of eight transmembrane domain(TM)TM1-TM8 and two opposite hairpin structure(helical hairpin,HP)HP 1,HP2.The crystal structure studies of Gltph in the different transport phases show that HP1,HP2 and TM7,TM8 compose the substrate binding pocket and it accompanies by the reciprocal motion of various segments in the different transport phases.It has been reported that HP1 plays an important role in the transport phases,due to it functions as the intracellular gate of transporters and a part of the substrate binding pocket.TM2 has been thought as a part of the trimer interface,and its location is not changed in the transport cycle.However,the positional relationship between HP1 and TM2 in the transport cycle is not yet reported.Studies have shown that upregulation of EAATs could weaken excitotoxic damage to the nervous system.Some groups showed that ceftriaxone could increase the expression of GLT-1 via activation of NF-kB.Also,ceftriaxone could decrease the death of dopamine neurons in the substantia nigra and striatum and ceftriaxone also could improve the movement disorder and the cognitive abilities in PD mice.In addition,studies also showed that ceftriaxone could reduce the side effects of levodopa via increasing the expression of glutamate transporters and decreasing the concentration of glutamate in the synaptic cleft.These results show that ceftriaxone is a prospective drug in treatment of PD.However,the mechnism of antiapoptotic effect by ceftriaxone in PD models is stil unclear.Therefore,in this study,we focus on the mechanism of glutamate transporters and their regulatory mechanism in the pathogenesis of PD.We want to improve the progress of PD via upregulation of EAATs.Our work is divided into four sections as below.Part ?.Relative movement between hairpin loop 1 and transmembrane domain 2 of the glutamate transporter EAAT1 in substrate transportAs reported previously,the spatial relationship between HP1 and TM2 in EAAT1 during the transport cycle remains unknown.In this study,we used chemical cross-linking of introduced cysteine pair(V96C and S366C)in a cysteine-less version of EAAT1 to assess the proximity of TM2 and HP1.Here we show that inhibition of transport by Copper(?)(1,10-Phenanthroline)3(CuPh)and cadmium ion(Cd2+)were observed in the V96C/S366C mutant.Glutamate or potassium significantly protected against the inhibition of transport activity of V96C/S366C by CuPh(P<0.01),while TBOA(D,L-threo-?-benzyloxyaspartate)potentiated the inhibition of transport activity of V96C/S366C by CuPh(P<0.01).We also checked the kinetic parameters of V96C/S366C in different tansport phases.CuPh had no significant effect on the Km in different substrates or inhibtor(F value:0.125;P value:0.998).However,CuPh significantly decreased the Vmax in different substrates or inhibtor(F value:21.317;P value:<0.001).We analyzed the spatial relationship between Val-96 of TM2 and Ser-366 of HP1 in different transport phases.V96 of TM2 and S366 of HP1 are far apart in the inward-facing state of glutamate transporters,while V96 of TM2 and S366 of HP1 are close to each other in the outward-facing state of glutamate transporters.In addition,we found that during the transport process of EAAT1,HP1 moves downward in the inward-facing state,and thus HP1 is far apart from TM2.In this part,we report the spatial relationship between HP1 and TM2 in the EAAT1 transport cycle and we provide the theoretical basis for studying the transport mechanism of glutamate transporters.Reveal of the EAATs transport mechanism could significantly guide the study of mechanism and regulatory strategies of glutamate transporters expression in PD.Part ?.Ceftriaxone protects astrocytes from MPP+ via suppression of NF-kB/JNK/c-Jun signalingAs stated previously,glutamate transporters could rapidly reuptake the glutamate released from the presynaptic neurons and prevent the excitotoxicity effectively.In 2005,the antibiotic ceftriaxone was firstly demonstrated to play a neuroprotective effect by increasing the expression of glutamate transporter GLT-1.However,whether ceftriaxone could play a protective effect in Parkinson's disease via upregulation of glutamate transporters and the regulatory mechanism are unknown.We created the cellular model by using the neurotoxin MPP+ in astrocytes and we confirmed the effect of MPP+ by the transport activity of glutamate transporters and cell viability experiments.MPP+ decreased the transport activity and viability(F value:11.107;P value:0.001)and(F value:37.372;P value:<0.001;F value:39.283;P value:<0.001).We observe the protective effects of different concentrations of ceftriaxone and found that 100 ?M ceftriaxone could significantly improve the transport activity in MPP+-treated astrocytes(P<0.05).Altinough ceftriaxone increased the MPP+-treated astrocytes viability,there showed no significance between these two groups(P>0.05).Then we examined the effects of ceftriaxone on the expression of glutamate transporter GLT-1 in the membrane and total protein levels in MPP+-treated astrocytes via imunofluorescence and Western blotting.We found that ceftriaxone could improve the expression of GLT-1 in the normal astrocytes,while it had no significant effect on the GLT-1 expression in the cell membrane.Ceftriaxone could promote the expression of GLT-1 in the membrane in MPP+-treated astrocytes(P<0.05).We studied the NF-kB signaling pathway by ceftriaxone.Here we found that single treatment with ceftriaxone or MPP+ could increase the expression of NF-kB,while co-treatment with ceftriaxone and MPP+ could decrease the expression of NF-kB.Application of NF-kB inhibitor BAY 11-7082 could significantly increase the transport activity of glutamate transporters and the expression of GLT-1 in MPP+-treated astrocytes.While BAY 11-7082 showed no significant effects on the transport activity of glutamate transporters and the expression of GLT-1 in single treatment of ceftriaxone in astroyctes.These results suggest us that on the one hand ceftriaxone could inhibit the NF-kB pathway and exert a neuroprotective effect in MPP+-treated astrocytes.However,on the other hand,ceftriaxone upregulated the expression of GLT-1 maybe not involved in the NF-kB pathway.We also examined the JNK/c-Jun pathway,we found that ceftriaxone could inhibit the JNK/c-Jun pathway in MPP+-treated astrocytes and BAY 11-7082 could also suppress the JNK/c-Jun pathway in MPP+-treated astrocytes,which indicates that NF-kB is the upstream regulatory pathway of JNK/c-Jun pathway.BAY 11-7082 or SP600125 increased the transport activity of glutamate transporters,GLT-1 expression in the membrane and the cell vitality in MPP+-treated astrocytes.Thus we could speculate that ceftriaxone could inhibit the NF-kB/JNK/c-Jun pathway,reduce the apoptosis of astrocytes and show the neuroprotective effect in MPP+-treated astrocytes.In this part,we indicate the regulatory mechanism of GLT-1 by ceftriaxone and we also provide the theoretical basis for application of ceftriaxone in treatment of PD.Part ?.Regulation of glutamate transporters expression by ubiquitin ligase E3 Nedd4-2 in MPP+-treated astrocytes and PD miceAccording to the results in the Part two,we found that the expression of EAATs in the membrane plays an important role in the normal transport activity,while the expression of EAATs between the cell membrane and cytoplasm is in dynamic equilibrium.Ubiquitination regulates the dynamic circulation between the cell membrane and cytoplasm.So in this part,we mainly focus on the regulation of EAATs by ubiquitin ligase E3.Previous studies have reported that the ubiquitin ligase E3 play an important role in the pathogenesis of PD.Nedd4-2(Neuronal precursor cell Expressed Developmentally Down-regulated 4-2)forms the complexes with EAATs and regulates the expression of EAATs.Downregulation of Nedd4-2 could increase the expression of EAATs and reduce the excitotoxic damage to the nervous system.However,whether Nedd4-2 mediates the ubiquitination of glutamate transporters in PD and whether downregulation of Nedd4-2 could promote the expression of glutamate transporter and exert neuroprotective effect are still unknown.In the present work,we created the PD mice models.In the three days after the MPTP injection,one day after the created PD mice models and three days after the created PD mice models,we isolate the midbrain,striatum and cerebral cortex from certain number of mice,and we examine the expression of GLT-1,GLAST,p-Nedd4-2 and Nedd4-2.Western blotting results show that the protein expression of GLT-1 is reduced in the midbrain and striatum in three days after the created PD mice models compared with control,(midbrain:t value:5.336;P value:0.013)and(striatum:t value:4.087;P value:0.015)and the protein expression of GLAST is reduced in the striatum in three days after the created PD mice models compared with control(t value:4.254;P value:0.041).Q-PCR results show that the mRNA expression of GLT-1 and GLAST is reduced in the striatum and cerebral cortex in three days after the created PD mice models compared with control(striatum GLT-1:t value:4.242;P value:0.013),(striatum GLAST:t value:4.165;P value:0.014),(cerebral cortex GLT-1:t value:4.434;P value:0.047),(cerebral cortex GLAST:t value:4.065;P value:0.021).It is worth noting that the mRNA expression of GLT-1 is increased in the midbrain in three days after the created PD mice models compared with control(P<0.05).We speculate that some post-transcriptional factors participate in the regulation of GLT-1.Then we examined the interaction between Nedd4-2 and EAATs and the ubiquitination of EAATs in MPP+-treated astrocytes and PD mice via co-immunoprecipitation and immunofluorescence.Using Nedd4-2 or GLT-1 as the capture antibody,we found the interaction between Nedd4-2 and GLT-1 in MPP+-treated astrocytes.While the interaction between Nedd4-2 and GLAST showed no obvious difference in MPP+-treated and control astrocytes.Using GLT-1 or Ub as the capture antibody,we found the ubiquitination of GLT-1 in MPP+-treated astrocytes rather than GLAST.We found the interaction between Nedd4-2 and GLT-1 or GLAST in the midbrain in PD mice models.Using Ub as the capture antibody,we found the ubiquitination of GLT-1 and GLAST in the midbrain,and the ubiquitination of GLAST is much more obvious(P<0.05).The interaction between Nedd4-2 and GLT-1 only exists in the normal condition in the striatum,and the interaction between Nedd4-2 and GLAST showed no obvious difference in PD mice and control.These results showed that interaction between Nedd4-2 and EAATs and the ubiquitination of EAATs in MPP+-treated astrocytes and PD mice.So we want to know whether downregulation of Nedd4-2 could promote the expression of glutamate transporter and exert neuroprotective effect.In this work,we designed and synthesized three small interfering RNA(siRNA)sequences against Nedd4-2,we examined the transfection efficiency and interference efficiency,and we dealt the astrocytes with siNedd4-2 and MPP+.We found that siNedd4-2 could increase the expression of GLT-1 in the membrane and total level,and furthermore,siNedd4-2 could improve the glutamate uptake in MPP+-treated astrocytes(P<0.05).In this part,we indicated the interaction between Nedd4-2 and EAATs and the ubiquitination of EAATs in MPP+-treated astrocytes and PD mice,and we found that siNedd4-2 could increase the expression of GLT-1 and the glutamate uptake in MPP+-treated astrocytes.Nedd4-2 mediates the ubiquitination of GLT-1 and induces the excitotoxicity.Downregulation of Nedd4-2 may be a promising therapy in treatment of PD.Part IV Research on the neuroprotective effects and mechanism of rapamycin in PDAccording to the results in the Part two and three,we found the decreased expression and function of EAATs in MPP+-treated astrocytes and PD mice and the upregulation of EAATs exerts the neuroprotective effect.It has been reported that rapamycin,which was used in immunosuppressive therapy,could promote the expression of EAATs via the mTOR/Akt/NF-KB signal pathway.However,the effect of rapamycin on the expression and function of EAATs in PD was unknown.In addition,rapamycin shows multiple neuroprotective effects via mTOR pathway.So in this part,we examined the effects of rapamycin on the expression and function of glutamate transporters in MPP+-treated astrocytes and in PD mice.We also examined the effects of rapamycin on the expression of interlukin-6(IL-6)in MPP+-treated astrocytes and in the serum and midbrain of PD mice.According to the methods mentioned in the text,we treated the PD mice with rapamycin and we found that rapamycin improved the movement disorder of PD mice.(suspension test:P<0.01;pole-climbing test:P<0.05).We also found that rapamycin increased the expression of tyrosine hydroxylase in the midbrain and striatum(P<0.05)and decreased the apoptosis of neurons(P<0.01)and the expression of a-synuclein in the midbrain(P<0.01).These results suggest that rapamycin is neuroprotective in the PD models.Furthermore,we found rapamycin increased the expressions of IL-6,p-JAK2,p-STAT3 and GLT-1 in MPP+-treated astrocytes.Meanwhile,rapamycin increased the expression of IL-6 in the serum and midbrain,and also increased the expression of GLT-1 in the midbrain in the PD mice.(IL-6 expression in the serum:P<0.01;IL-6 expression in the midbrain:P<0.05;GLT-1 expression in the midbrain:P<0.01).Besides,rapamycin significantly reduced the serum expression of IFN-? in PD mice(P<0.01).We speculate that on the one hand,rapamycin reduces the glutamate excitotoxicity on the dopamine neurons via upregulation of glutamate transporter,on the other hand,rapamycin exerts neuroprotective effects via upregulation of IL-6 and activation of JAK2/STAT3 signaling pathway.In addition,rapamycin could also protect the central nervous system by inhibiting the inflammatory response.In this part,we found the neuroprotective effects of rapamycin on PD mice.Rapamycin increased the expression of GLT-1 in the MPP+-treated astrocytes and in the midbrain of PD mice.In addition,rapamycin increased the expression of IL-6 in the MPP+-treated astrocytes and in the serum and midbrain of PD mice.Moreover,rapamycin decreased the expression of IFN-y in the serum of PD mice.Our study provides theoretical and experimental basis for the reveal of neuroprotective effects of rapamycin in Parkinson's disease.In summary,we revealed the spatial relationship between HP 1 and TM2 in the EAAT1 transport cycle.We reported that ceftriaxone increased the expression of GLT-1,enhanced the transport activity of glutamate transporters and promoted primary astrocyte viability in MPP+-treated astrocytes via suppression of NF-kB/JNK/c-Jun signaling.We also found the interaction between Nedd4-2 and EAATs and the ubiquitination of EAATs in MPP+-treated astrocytes and PD mice,and we found that siNedd4-2 could increase the expression of GLT-1 and the glutamate uptake in MPP+-treated astrocytes.In addition,we found the neuroprotective effects of rapamycin on PD mice.Furthermore,rapamycin increased the expression of GLT-1 in the MPP+-treated astrocytes and in the midbrain of PD mice.Meanwhile,rapamycin increased the expression of IL-6 in the MPP+-treated astrocytes and in the serum and midbrain of PD mice.Also rapamycin reduced the serum expression of IFN-y in PD mice.Our studies provide theoretical and experimental basis for the transport mechanism of glutamate transporters and their regulatory mechanism in PD.