Mechanisms Underlying The Neuroprotection Of Group â…¡ And â…¢ Metabotropic Glutamate Receptors

Neuroprotection is the intervention strategy to delay or prevent theprogress of disease, and it is suggested to be the most prospectivetreatment for acute or chronic diseases of central neural system. It hasbeen demonstrated that many mechanisms are implicated in the processof neural damage, including apoptosis, excitotoxicitity, neuralinflammation, abnormality of neurogenesis and so on. Accordingly, variesof neuroprotective agents have been developed, such as antagonists ofexcitory amino acid receptors, blockers of Ca²⁺ channels, scavengers offree radicals and so on. Unfortunately, although most of them exertprotective effects during laboratory research, they have little therapeuticaleffects in clinic. Otherwise, some drugs generate serious adversereactions. Therefore, searching the new targets of neuroprotection anddeveloping agents of exact neuroprotection are the focus in neurosciencereseach.So far, there have been several theories implicated in themechanisms of neural damage, among of which, theory of glutamateexcitotoxicity is the one of great importance. Glutamate accumulation inthe synaptic spaces causes excitotoxic damages to the adjacent neurons.Clearance of extracellular glutamate is mainly accomplished by glutamatetransporters on astrocytes. Uptake inhibition leads to the enhancement ofextracellular glutamate levels with subsequent neurotoxic effects throughactivating glutamate receptors. Thus, astrocytic glutamate transport plays a major role in regulating glutamatergic transmission and maintainingextracellular glutamate concentrations below neurotoxic levels.Additionally, neuroinflammation mediated by microglia has beenimplicated in the pathogenesis and progress of neuronal damage. Byreleasing toxic molecules including tumor necrosis factor (TNF-α), or byinjuring neighboring cells and further activating more microglia,activated microglia may form vicious self-perpelling neuronaldegeneration cycle. Furthermore, recent studies have shown thatneurogenesis is reduced in the subventricular zone (SVZ), subgranularzone (SGZ) in the process of neural system diseases. Therefore, inducingendogenous neural stem cells to proliferate could also be one of theavailable regenerative interventions for neural damages.Recently, mounting evidence has indicated that activation ofmetabotropic glutamate receptors (mGluRs) exerted neuroprotection.mGluRs are G-protein-coupled-receptors extensively localizing in thecentral nervous system. The mGluR family includes at least eightsubtypes classified into three groups on the basis of signal transductionmechanisms, pharmacological properties, and gene sequence homology;groupâ… includes mGluR1 and mGluR5, groupâ…¡includes mGluR2 andmGluR3, and groupâ…¢includes mGluR4, 6, 7, 8. It has been reported thatagonists of groupâ…¡orâ…¢mGluRs have neuroprotective effects. Despitethe direct neuroprotective roles, activation of groupâ…¡orâ…¢mGluRs ofglia also exerts neuroprotection. In addition, mGluRs are expressed incertain 'neurogenetic regions' including SVZ and SGZ, and agonists ofgroupâ…¡andâ…¢mGluRs can promote proliferation of neural progenitorcells. Therefore, groupâ…¡andâ…¢mGluRs may be the promising targetsfor neuroprotection.The present research aimed to study the neuroprotective effects ofagonists of groupâ…¡andâ…¢mGluRs against MPP⁺-induced damage inPC12 cells. Also, further experiments were undertaken to explore the regulation of glutamate uptake of C6 glioma cells, inflammativecytokines released by microglia, proliferation of neural stem cells. Theresults demonstrated the neuroprotective roles of groupâ…¡and groupâ…¢mGluRs as well as the relative mechanisms of the neuroprotection,suggesting groupâ…¡andâ…¢mGluRs to be the promising targets for theneuroprotection.AIM: To determine whether activation of groupâ…¡orâ…¢mGluRsprotects PC12 cells aginst MPP⁺-induced damage, and to explore therelative mechanisms of the protective effect.METHODS: The viability and apoptosis of PC12 cells weredetermined by LDH level and Hoechst33342 labeling respectively. Theglutamate uptake into C6 glioma cells was investigated by uptake of[³H]-D,L-glutamate. The TNF-αreleased by microglia is detected byELISA method, and [³H]-TdR was used to measure the proliferation ofneural stem cells. The expression of mGluRs in microglia was analyzedby RT-PCR and immunocytochemistry.RESULTS: 1) Activation of groupâ…¡orâ…¢mGluRs with selectiveagonists APDC or L-AP4 inhibited the damage and apoptosis of PC12cells induced by MPP⁺. 2) LPS (4, 6μg/mL)inhibited glutamate uptakesignificantly compared to the control group without effects on theapoptosis and viability of C6 glioma cells. Pretreatment of C6 gliomacells with groupâ…¡andâ…¢mGluRs agonists DCG-â…£and L-AP4 reversedLPS-induced glutamate uptake inhibition. These recovery effects wereabolished by their antagonists APICA and MSOP respectively. 3)Microglia expressed groupâ…¡andâ…¢mGluRs both in mRNA and proteinlevels; MPP⁺ enhanced the level of TNF-α, activation of groupâ…¢mGluRs inhibited the release of TNF-αinduced by MPP⁺, whileactivation of groupâ…¡mGluRs further increased the level of TNF-α. 4)Activation of groupâ…¡mGluRs increased the proliferation of neural stem cells isolated from SVZ of newbom mouse, while activation of groupâ…¢mGluRs with L-AP4 had no effect on proliferation.CONCLUSION: Activation of groupâ…¡orâ…¢mGluRs protectedPC12 cells against the neuronal damages induced by MPP⁺. Further studyshowed that agonists of groupâ…¡orâ…¢mGluRs enhanced glutamateuptake, decreased release of inflammation cytokines, and promotedproliferation of neural stem cells. The results suggested that groupâ…¡andâ…¢mGluRs have prospects to be the potent targets of neuroprotection.The major contributions of the present study lie in:1. Systemly studying the neuroprotection of agonists of groupâ…¡andâ…¢mGluRs, as well as the relative mechanisms; providing essentialacademic and experimental foundation for developing the mGluRs to bethe potent targets for development of neuroprotective agents.2. Activation of groupâ…¡mGluRs with APDC could promote theproliferation of neural stem cells isolated from SVZ of newborn mouse,providing new views for demonstrating the neuropharmacologicalprofiles of groupâ…¡mGluRs.