Inhibition Of Glutamine Synthetase Aggravates Neural Impairment In Mouse Model Of Parkinson's Disease Induced By MPTP

Parkinson's disease(PD)is the second most common neurodegenerative disorder,after Alzheimer's disease.The pathological features of PD are the progressive loss of dopaminergic neurons in the substantia nigra pars compacta(SNc)and severe decrease in dopamine(DA)level in the striatum,accompanied by the deposition of Lewy bodies(LB).The clinical symptoms include bradykinesia,resting tremor,rigidity,and postural instability.Many factors contribute to the development of PD,including environmental factors,genetic factors,immune dysfunction,neuroinflammation,excessive oxidative stress,mitochondrial dysfunction,excitotoxicity,etc.,but its precise aetiology remains obscure.Levodopa is widely recognized as the most effective therapy for PD,but long-term treatment is associated with adverse reactions such as movement disorders.Therefore,it is ugent to explore the pathogenesis of PD and to develop new strategies for PD.Astrocytes have been viewed as the most abundant,the largest and most widely distributed cells in the mammalian brain,and mammalian brain neuronal circuits are built in networks of astrocytes.Astrocytes not only participate in the formation of blood-brain barrier,but also can secrete a variety of neurotrophic factors.Astrocytes can sense neuronal inputs through transporters,receptors and membrane ion channels and respond by releasing a variety of neurotransmitters such as y-aminobutyric acid(GABA),D-tryptophan,glutamate.In recent years,astrocyte has become a new target for the treatment of neurodegenerative diseases such as PD.Glutamate is the most important neurotransmitter in the central nervous system,but when its content increases pathologically,it will contribute to the death of neurons.This pathological process exists in many neurological disorders,such as PD,epilepsy,muscle atrophy lateral sclerosis and stroke.Therefore,maintaining glutamate homoeostasis may be a new neuronal protection strategy to treat PD.Glutamine synthetase(GS)is specifically expressed in astrocytes.Glutamate is released into synaptic clefts when glutamatergic neurons are activated.Glutamate uptake into strocytes is mainly through glutamate transporter-1(GLT1)and glutamate-aspartate transporter(GLAST).After uptake into astrocytes,glutamate may be converted to glutamine via glutamine synthetase.Glutamine can subsequently be transferred from astrocytes to neurons and re-converted to glutamate.GABAergic neurons generate glutamate which subsequently converts to GABA by glutamate decarboxylase.GABA is removed from the extrasynaptic space predominantly by transporters in the presynaptic neurons and to a lesser extent into astrocytes.Reuptake of extracellular GABA into the presynaptic interneurons is performed mainly by the GABA transporter GAT1.Astrocytes are also involved in GABA reuptake that escapes the synapse and this is primarily via GAT3.In the first part of our work,we used wild type(WT)mice to induce subacute 1-methyl,4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)PD mouse model.We explored the time course between the expressions of GLT1,GLAST,GS,GAT3,DAT and dopaminergic neurons loss.In the second part of our work,we investigated the effects of glutamine synthetase inhibitor on MPTP-induced Parkinson-like symptoms.We found that in MPTP induced Parkinson's model of mice,changes in astrocyte functional markers were earlier than dopaminergic neurons loss.Inhibition of glutamine synthetase had a detrimental effect on MPTP induced Parkinson-like symptoms,aggravated dopaminergic neurons loss and broke striatal amino acid neurotransmitters balance.Our work revals the inhibtion of GS promotes the process of PD and suggests that regulation of GS expression and the maintenance of neurotransmitters homoeostasis is a prising new strategies for PD treatment.Part I Expressions of astrocyte functional markers in MPTP mouse model of Parkinson's diseaseAIM:To elucidate the time course between the expressions of astrocyte functional markers and dopaminergic neurons loss in MPTP mouse model of PD.METHODS:C57BL/6J male mice received MPTP or saline injections.MPTP was injected once daily(20mg/kg i.H.)for 2 days and for 6 consecutive days.The rotarod test,locomotor activity test and pole test were performed to evaluate the coordination and movement 6 days after the final injection of MPTP or saline.Tyrosine hydroxylase(TH),glial fibrillary acidic protein(GFAP)and ionized calcium binding adnexin 1(Iba-1)immunohistochemical staining were taken in the substantia nigra and striatum to observe the activation of astrocytes and microglia.The levels of monoamine neurotransmitters and amino acids neurotransmitters in the striatum were measured by high performance liquid chromatography(HPLC).Western blot was used to detect the changes in astrocyte functional markers in the midbrain and striatum.RESULTS:1)Behavior tests results showed that compared with the control group,mice with MPTP injection for 2 days showed no difference in climbing time,rod stay time and exercise speed while mice with MPTP injection for 6 days induced the coordination ability of movement dysfunction,but the speed of movement did not change.2)Histopathological analysis showed that TH-positive neurons in the substantia nigra pars compacta in mice with MPTP injection for 2 days were less a bit than those in the control group.The TH-positive neurons were significantly lost in mice with MPTP injection for 6 days.HPLC showed that DA?DOPAC?HVA were lower in mice with MPTP injection for 2 days than those in the control group,mice with MPTP injection for 6 days further reduced the content.Amino acids neurotransmitters showed no change.3)Mice with MPTP injection for 2 days,astrocytes had been activated significantlycompared with MPTP group,but microglia activation was not significant.Mice with MPTP injection for 2 days,astrocytes and microglia were significantly activated and proliferated.4)Western Blot showed that GLT1 was down-regulated,the expressions of GS and GAT3 was up-regulated in the midbrain and striatum in mice with MPTP inj ection for 2 days compared with the control group.Mice with MPTP injection for 6 days,GLT1 expression was down-regulated.GS expression was further up-regulated,aquaporin-4(AQP4)and DAT expression was down-regulated.The expression of GLAST had no significant change in the midbrain,but was down-regulated in the striatum.The expression of GAT3 was still at high level.CONCLUTIONS:1)The changes of astrocytes functional markers were earlier than dopaminergic neurons loss.2)The changes of astrocytes functional markers may compensate the homeostasis of glutamate and GABA in MPTP-induced parkinsonism.Part II Inhibiting glutamine synthetase aggravates neural impairment in MPTP mouse model of Parkinson's diseaseAIM:To investigate whether inhibiting glutamine synthetase affecting amino acid neurotransmitter balance aggravates MPTP-induced parkinsonism.METHODS:C57BL/6J male mice received MPTP or saline injections.MPTP was injected once daily(20 mg/kg i.H.)for 6 consecutive days.Mice were intraperitoneally injected with glutamine synthetase inhibitor methionine sulfoximine(MSO)for 2 weeks(0.1 ml/10 g,qod).Six days after the last MPTP injection,behavioral tests were performed to evaluate the coordination and movement of mice.TH,GFAP and Iba-1 immunohistochemical staining were taken in the substantia nigra and striatum to observe glial activation and dopaminergic neuronal loss.The levels of monoamine neurotransmitters and amino acids neurotransmittersin the striatum were measured by HPLC.Western blot was used to detect the changes of astrocyte functional markers in the midbrain and striatum.RESULTS:1)MSO aggravated motor dysfunction compared with MPTP group,including prolonged the time of rod climbing,shortened the residence time of rod,decreased the coordination ability of movement and the speed of movement.2)MSO aggravated progressive loss of dopaminergic neurons in the midbrain.MSO disrupted the balance among glutamate,GABA and glutamine.Compared with MPTP group,the contents of DOPAC,HVA and DA in the striatum were further decreased in MSO+MPTP group.The glutamine content was decreased and resulted in the decline of excitatory neurotransmitter glutamate level and the rise of the inhibitory neurotransmitter y-aminobutyric acid level.3)MSOpromoted the glial activation in the midbrain and striatum.4)Western Blot showed that the expression of GS was down-regulated in the midbrain and striatum in MSO and MSO+MPTP group compared with the control group.The expression of GLT1 was up-regulated and the expression of GAT3 was down-regulated in the midbrain and striatumin MSO group compared with the control group.The expression of GLAST was up-regulated in the midbrain but not in striatum.The expressions of DAT in the midbrain and striatumin MSO+MPTP group were further down-regulated compared with MPTP group.CONCLUTIONS:1)Up-regulation of GS and GAT3 expression may compensate the homeostasis of glutamate and GABA in MPTP-induced parkinsonism.2)MSO,as a glutamine synthetase inhibitor,contributed to destruction of striatum amino acid neurotransmitter balance,increased loss of TH neurons and decreased striatum DA level and ultimately exacerbated movement dysfunction.The major contributions of the present study lie in:1.The changes of astrocytes functional markers protein expressions were earlier than dopaminergic neurons loss.There was no significant difference in the number of dopaminergic neurons in mice with MPTP injection for 2 days while astrocytes in SNr,SNc and striatum which mainly receive dopamine fiber from SNc were significantly activated.The changes of astrocytes functional markers protein expressions were earlier than dopaminergic neurons loss.2.Inhibition of glutamine synthetase aggravates MPTP-induced parkinsonism.MSO inhibited glutamine synthetase which aggravated progressive loss of dopaminergic neurons in the midbrain in MPTP mouse model.The contents of DOPAC,HVA and DA in the striatum were further reduced,and the balance of amino acid neurotransmitters was destroyed in MPTP mouse model.