The Role Of Glutamate Transporter-1(GLT-1) In Parkinson’s Disease-like Changes From The Depression Model And The Mechanisms Underlying It

Objectives:Clinically retrospective data suggest that the incidence of Parkinson’s disease(PD)is higher in patients with depression than those without.However,the key mechanisms in promoting the development of PD from depression,and the series of changes happening as well as characteristics appearing during this process are all unclear.The establishment of transformed animal model from depression to PD will help to address the interaction between depression and PD,and find out targets for preventing the process of transformation.The aim of this study is to investigate the changes and characteristics of depression transforming into PD by establishing a natural transformation model and screen out a potential molecule that changed with the transformation.Then,to explore the role of this molecule in the pathogenesis of PD and the mechanisms underlying it by directly regulating the expression of the molecule.In short,we hope our work could ultimately provide a new idea for the prevention and treatment of PD(especially PD associated with depression).Methods:1)Using maternal separation(MS)to induce early life stress-related depression.Male pups from SD rats were randomly assigned to the control(Ctrl)or MS group on postnatal day(PND)1.Pups for the MS group were separated from their mothers for 3 hours every day from PND 1 to 21,and pups in the Ctrl group were not separated.Rats in two groups were not given any other treatment except being fed normally until 60th week after MS.At the timepoint of 9th,36th and 60th week,behavioral test(sucrose preference test,buried food pellet test,open field test and automated gait analysis),neuroimaging(microPET/CT),western blotting,high performance liquid chromatography(HPLC),immunohistochemical staining,and transcriptome(RNA sequencing)were performed to evaluate the following indicators:depression-like behavior,olfactory function,motor performance,biochemical indexes and gene expression profile.2)Kyoto encyclopedia of genes and genomes(KEGG)pathway analysis and gene ontology(GO)analysis were used to analyze the functional classification of the differentially expressed genes(DEGs)from RNA sequencing data.The target molecule for further study was selected out via proteinprotein interaction(PPI)network analysis in combination with literature review.Moreover,the target molecule was verified by real-time quantitative PCR(qRT-PCR)and western blotting.3)Whether changes of the target molecule in the subacute PD mice with intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)and PD rats with bilateral substantia nigra stereotactic injection of 6-hydroxydopamine(6-OHDA)were consistent with the result in the natural outcome of MS-induced depression model was also validated by behavior tests and western blotting.4)After the down-regulated molecule glutamate transporter-1(GLT-1)was screened out,8-week-old male SD rats were randomly divided into the control group(Scramble group)and the Glt-1 knockdown group(Glt-1 KD group)according to its distribution and functional characteristics in brain with unilateral two-point stereotactic injection of recombinant adeno-associated virus(rAAV)in the right striatum.The Glt-1 KD group used rAAV containing shRNA sequence with GFAP promotor that could interfere GLT-1 expression in astrocytes,while the Scramble group used empty vector rAAV with GFAP promotor parallelly.After 6 weeks of rAAV injection,behavior(rotarod test,open field test,automated gait analysis,sucrose preference test and buried food pellet test),neuroimaging(micro-PET/CT),and histochemistry(western blotting and immunohistochemical staining)were used to evaluate the motor performance,depressionlike behavior and olfactory function,as well as brain biochemical indexes of dopamine system in the two groups.5)Primary astrocytes were derived from newborn C57BL/6 mice of 1-3-days age,and primary mesencephalic neurons were derived from E16-18 pups from C57BL/6 mice.Small-interfering RNAs(siRNAs)targeting Glt-1 sequence was designed and employed to knock down the expression of GLT-1 in primary astrocytes.Concurrently,the negative control(NC)RNAs without interference effect was used in the control group.The primary astrocytes with Glt-1 interference(Glt-1 KD group)or not(NC group)were co-cultured with primary mesencephalic neurons in a Transwell cells co-culture system with or without ceftriaxone treatment.After 72 hours of co-culture,the supernatants from the coculture system of two groups were collected to detect the concentration of glutamate by ultra high performance liquid chromatography with triple quadrupole mass spectrometer(UHPLC-TQ-MS).The cellular morphology of tyrosine hydroxylase(TH)positive neurons from the primary mesencephalic neurons in two groups was observed by immunofluorescence staining.The changes of calcium signal in primary midbrain neurons in two groups were detected by calcium imaging and western blotting.Results:1)Rats in MS group showed stable depression symptom,whereas they exhibited impaired olfactory ability,reduced distance of locomotion,and longer stance and brake time of gait during walking with aging.The results of micro-PET/CT and western blotting indicated that the dopamine system in striatum of SD rats was impaired after MS.The decrease of striatal dopamine level calculated from the result of HPLC was about 33%.Consistently,TH immune-staining positive neurons of MS rats in the substantia nigra pars compacta(SNpc)showed about 20%of cell loss.2)At 60 weeks,we found that extensive DEGs in the striatum of MS rats significantly enriched in the pathway of dopaminergic synapse and the biological process of locomotion and neuromuscular process controlling balance by transcriptome sequencing.Solute carrier family 1 member 2(Slc1a2)gene was conditionally screened out from DEGs combined with literature review and PPI network analysis.qRT-PCR and western blotting experiments confirmed that Slc1a2 gene transcription and its encoded protein GLT-1 expression were all decreased in the striatum of SD rats in MS group relative to Ctrl group.This was consistent with the result of transcriptome sequencing analysis in 60-week-old SD rats with depression.3)Expression of GLT-1 in the striatum of the subacute PD mice with intraperitoneal injection of MPTP and PD rats with bilateral substantia nigra stereotactic injection of 6-OHDA were also diminished.Additionally,MPTP treated mice showed longer immobility time in tail suspension test than normal saline-treated mice;6-OHDA treated rats showed less sucrose intake than normal saline-treated rats.4)After 6 weeks of rAAV injection in vivo,SD rats in Glt-1 KD group showed poorer motor performance(less time on the rod and shorter total distance in locomotion),abnormal gait(longer average stance time and average brake time on the right rear limb),and depression-like feature(less sucrose intake)compared with the SD rats in Scramble group.However,there was no statistic difference in the time taken to find the buried pellet between two groups in the buried food pellet test.This suggested that there was no olfactory dysfunction of SD rats in Glt-1 KD group.The result of microPET/CT indicated the radiation signal of dopamine transporter in the Glt-1-knockdown side of striatum was weaker than that in their own un-rAAV-injected side and Scramble group.Western blotting result showed the expression of TH protein in the Glt-1-knockdown side of striatum was reduced compared to un-rAAV-injected side as well as the Scramble group.Furthermore,the number of TH positive neurons in the Glt-1-knockdown side of the SNpc was also reduced compared to the Scramble group and their own un-rAAV-injected side.5)The morphology of TH positive neurons from primary mesencephalic neurons in Glt-1 KD group displayed decreased or shortened neurites compared with that of NC group after immunofluorescence staining in vitro.Moreover,the GLT-1 agonist ceftriaxone could reverse the abnormal morphology.The glutamate content detected by UHPLC-TQ-MS in supernatants from Glt-1 KD group was higher than that from NC group.Calcium imaging indicated that the 340 nm/380 nm ratio of Glt-1 KD group was increased compared with that of NC group.Western blotting result showed that the expression of p-CaMKII(phosphorylated Ca2+/calmodulin-dependent protein kinase Ⅱ)protein in Glt-1 KD group was also increased relative to NC group.Conclusions:1)Reduced expression of GLT-1 in the striatum plays an important role in age-relate PD-like changes in depressed rats induced by MS,and knockdown of astrocytic Glt-1 in the striatum could cause PD-like behavior and pathological changes in SD rats,which are also accompanied with depression-like behavior.2)The deficiency of GLT-1 in astrocytes can cause morphological damage to dopaminergic neurons,and the abnormal calcium signal induced by glutamate accumulation after Glt-1 knockdown may contribute to the damage.3)GLT-1 may be a potential target for the prevention and treatment of PD(especially PD associated with depression).