The Mechanism Of Astrocytic JWA Regulates Injury Of Dopaminergic Neurons Induced By Exposure Of Environmental Toxicants

Parkinson's disease(PD),originally described by James Parkinson in 1817,is the second most common neurodegenerative disorder after Alzheimer's disease.The prevalence of PD is approximately 0.2%on average in the general population,but it rises steeply with increasing age' reaching more than 4%at 85 years of age.As regards its clinical manifestations,motor(resting tremor,rigidity,brady-and hypokinesia and postural instability)and non-motor(cognitive,psychiatric,autonomic,olfactory and sleeping disturbances)symptomscan be distinguished.The symptoms develop in consequence of the loss of brain stem catecholaminergic,and especially mesencephalic dopaminergic(DA)neurons in the substantianigra pars compacta,with a resultant disturbance of DA signalling in the innervate brain areas,and mainly in the dorsal striatum.The treatment of PD is mainly on compensatory strategy and is hard to stop the process of the disease completely.Therefore,the research on molecular mechanism of PD will provide scientific basis for effective treatment strategy.Astrocytes,along with other cells of the glial lineage such as oligodendrocytes and microglia,are believed to be structural cells,and the main function of which is to hold neurons together.However,astrocytes serve many housekeeping functions,including maintenance of the extra cellular environment and stabilization of cell-cell communications in the central nervous system(CNS).The function of astrocytes in regulating cerebral blood flow and maintaining synaptic function is becoming increasingly recognized as being of paramount importance in the maintenance of the neuronal environment.Astrocytes are also central to the maintenance of neuronal metabolism and neurotransmitter synthesis.Understanding these functions has allowed a refocusing on the role of astrocytes in neurodegenerative diseases,which has led to astrocyte-specific analyses with potential for drug discovery.Our group has been focusing on the functions of JWA gene in cancers and neural networks for a long time,and it is showed that JWA affects neurons uptake of glutamate in vitro and participates in the regulation of ERK and NF-?B signaling pathway.The previous studies have shown that the genes are affected in neuronal specific JWA knockout mice mainly enriched in ribosome,oxidative stress and oxidative phosphorylation and neurodegenerative diseases related signal pathways.Suggesting that JWA may play an important role in development of CNS deseases.AIMS:To investigate the mechanism of astrocytic JWA in regulation of dopaminergic neurons under the exposure of environmental toxin praquate and 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP).METHODS:The astrocytic specific JWA knockout mouse(JWA CKO)was generated by use of JWA exon2 Loxp floxed mouse and followed by GFAP-Cre mouse mediated recombination.The chronic MPTP/p PD model was created according to the routine strategy.Briefly,JWA CKO and WT mice were injected subcutaneously with 20 mg/kg MPTP in saline and 1 h later followed by intraperitoneally with 250 mg/kg probenecid in DMSO for 10 times with intervals of 3.5 days.The PD like mice were then used to investigate the effects of astrocytic JWA deficiency on behavior,monoamine neurotransmitter,dopaminergic neurons survival.We further explored the molecular mechanisms how JWA involved in MPTP induced neurodegeneration in mice.Western bloting,immunofluorescence and realtime-PCR was used to detect the related biomarkers in vivo and in vitro and then verified in primary astrocytes.Moreover,we cultured primary astrocytes from JWA CKO and JWA WT mice and tested the activity of conditioned media(CM)on neurons after MPP+stimulation.In addition,to evaluate whether astrocytic JWA knockout involved in environmental toxin paraquat induced CNS injury and the potential mechanisms,JWA CKO mice were received a total of 10 injections of paraquat at a dose of 7 mg/kg at 2-day intervals.RESULTS:1.After chronic MPTP/p administration,C57BL/6 mice displayed a-46%reduction in the number of the TH positive cells in the midbrain.In comparison with MPTP/p-untreated mice,MPTP/p treatment decreased-42%JWA protein expression.2.JWA CKO mice had a lower basal levels of striatal dopamine and its metabolites content.After MPTP/p treatment,DA and DOPAC content was decreased by 46%and 41%in the JWA CKO mice compared with JWA WT mice.The deletion of JWA gene in astrocytes had no significant effect on the release level of amino acids.JWA CKO mice did not significantly alter locomotion ability in the absence of MPTP.However,chronic MPTP/p administration in JWA CKO mice dramatically shortened time by 45%spent on rotarod performance,reduced activity by 42%,and increased the total time by 48%in pole test compared with the JWA WT mice3.The number of TH-positive cells in JWA CKO mice was decreased by 25%in comparison with JWA WT mice.The number of TH-positive cells was reduced?37%in MPTP/p-treated JWA CKO mice compared to the WT control mice.The glial fibrillary acidic protein(GFAP)-positive astrocytes were examined in substantia nigra.Deletion of astrocytic JWA showed an obvious increased in the number of GFAP-positive cells as 2.1-fold as JWA WT mice.In comparison with untreated JWA WT mice,the number of activated GFAP-positive cells was increased approximately 3.5-fold in MPTP/p-treated JWA CKO mice.The expression of GLT-1 was greatly reduced in MPTP/p-treated JWA CKO mice compared with untreated JWA WT mice.However,analysis of expression patterns of GLAST,another glial glutamate transporter,indicated almost no difference between JWA CKO mice and JWA WT mice.In comparison with untreated MPP+groups,GLT-1 expression levels were significantly decreased in MPP+groups in C8D1A cell line.4.Knocking down or overexpressing JWA in C8D1A cells 2 days downregulated or elevated GLT-1 expression compared with the vector control.At the same time,there was a significantly decrease of ERK and Akt phosphorylation in JWA knocking down C8D1A cells.In contrast,JWA overexpression greatly increased ERK and Akt phosphorylation.Similarly,knocking down or overexpressing JWA significantly downregulated or elevated glutamate uptake activity compared with the vector control in C8D1A cells.Knocking down JWA greatly reduced the GLT-1 mRNA levels by 45%,whereas significantly increased by 2.1-fold in C8D1A cells with overexpression of JWA.However,GLAST expression was not affected by JWA in C8D1A cells.5.Knocking down or overexpressing JWA significantly downregulated or elevated p-CREB compared with the vector control in C8D1A cells,however,the p-P65 expression was negatively regulated by JWA.Inhibitions of the ERK or Akt pathway abolished JWA-induced p-CREB and GLT-1 expressions.Consistent with protein results,the inhibitors significantly decreased glutamate uptake activity after 4 h incubation in C8D1A.The conditional media(CM)from JWA CKO astrocytes were less protective of neurons against MPP+compared with CM from JWA WT astrocytes.6.Knocking down or overexpressing JWA significantly downregulated or elevated GLT-1 protein and mRNA expression levels compared with the vector control in primary mouse astrocytes.Consistent with previous results,JWA overexpression in primary astrocytes led to 1.63-fold increases of glutamate uptake activity,whereas it led to a decrease by 44%.The expression of GFAP was significantly elevated in primary astrocytes from JWA CKO mice compared with primary astrocytes from JWA WT mice7.The astrocytes were obviously activated and showing with short and thick processes in striatum(STR),substantia nigra(SN),hypothalamus(HY),periaqueductal gray(PAG)and hippocampus(HP)of JWA CKO mice.The activated astrocytes indicated significantly increased expressions of STAT3 at basal and phosphorylation;GFAP expression was also obviously increased in these activated astrocytes.In contrast,the unacticvated astrocytes were showing with thin processes and denoting as resting phenotype in relative brain regions of JWA WT mice.These were further confirmed in C8D1A cells,and indicated that knocking down JWA activated expressions of STAT3 and GFAP,whereas overexpressing JWA suppressed expressions of STAT3 and GFAP.8.Astrocytic JWA deficiency accelerated dopaminergic neuron degeneration,motor dysfunction,reduced dopamine levels and the expression of GLT-1 in paraquat-treated JWA CKO mice compared with JWA WT mice.9.Overexpression of JWA increased the survival in MPP+treated C8D1A cells while low expression of JWA increased the toxicity of MPP+in C8D1A cells.The content of GSH was increased 45%in JWA overexpressed C8D1A cells while decreased 48%in JWA knocked down C8D1A cells.The mRNA expressions of Nrf2(increased 33%)and its down stream molecules such as NQO1(increased 35%),HO-1(increased 167%),GCLC(increasedl12%)and GCLM(increased 41%)were all upregulated upon JWA overexperssion.In contrast,the expression of Nrf2(decreased 49%)and its down stream molecules NQO1(decreased 19%),HO-1(decreased 61%),GCLC(decreased 25%)were all downregulated upon JWA knocking down.Moreover,si-Nrf2 reversed the regulation of JWA on the down stream molecules Nrf2.However,no obvious effects were observed on the expressions of nerve nutrition factors BDNF and GDNF upon overexpressing or knocking down JWA in C8D1A cells.CONCLUTION:In the present study our data indicated at first time that JWA gene deletion in astrocytes increased the sensitivity of mice to MPTP and Paraquat exposure in both in vivo and in vitro models.We also found that JWA activated CREB expression through MAPK/ERK and PI3K/Akt signaling pathways to regulate GLT-1 and eventually led changes of glutamate content in extracellular space.Our findings may provide new insights for the mechanisms of PD pathogenesis and with potential traslational signifcance to develop JWA based new strategy for PD therapy.