Metformin-mediated Neuroprotective Roles In MPTP/p-induced Mouse Model Of Parkinson’s Disease

Parkinson’s disease (PD), the second most common neurodegenerative disorder,is characterized by the progressive loss of dopaminergic (DA) neurons in thesubstantia nigra compacta (SNc) and aggregation of Lewy bodies in neurons. Themajor clinical features include rigidity, bradykinesia, postural instability and restingtremor. Although aging, oxidative stress and neuronal inflammation have beenrecognized to play crucial roles in the pathogenesis of PD, the precise aetiologyremains obscure. Clinically, L-DOPA, a precursor of DA, is the first-class drug totreat PD, and even in today it is considered to be the “gold standard” treatment for PD.Although L-DOPA ameliorates the symptoms of PD but fails to prevent DA neuronsdegeneration. Thus, it is crucial to develop effective therapetutic drugs that canreverse or alleviate DA neurons degeneration process in PD patients.Autophagy is an essential function for organism and mediates the degradation ofmisfolded/damaged protein and dysfunctional organelles. Emerging evidencessuggest that dysregulation of autophagy plays an important role in neuron death in PD.On one hand, it reduces α-synuclein degradation under autophagy dysregulationcondition, and exacerbates the accumulation of α-synuclein. On the other hand,autophagy dysregulation could not effectively remove dysfunctional mitochondria, which generate an abundance of ROS. And ROS, in turn, causes oxidative stressand increases cellular toxicity. It reported that rapamycin, a classic autophagyinducer, could prevent the loss of dopaminergic neuron in MPTP PD mice. Thisindicates that induction of autophagy is beneficial for DA neuron survival in PD.AMP-activated protein kinase (AMPK) is a serine–threonine heterotrimeric kinasewhich acts as a sensor of cellular energy status. AMPK is switched on by anincrease of AMP/ATP ratio, which leads to the phosphorylation of AMPK at Thr172by AMPK kinases. AMPK activation affects many pathways, generally causingconservation and generation of ATP. It reported that activation or overexpression ofAMPK alleviated DA neuron injury suffered by rotenone or MPP+. These implicateregulation of AMPK may become a new neuroprotective strategy for PD.Metformin, a biguanide family membr commonly used in treatment for type2diabetes. Metformin also is a AMPK agonist, which can accelerate AMPKphosphorylation at Thr172site of α subunit, and induce autophagy. Futhermore,metformin can regulate the system metabolic status, inhibit system metabolicinflammation, and improve insulin sensitivity. Emerging evidences show that PD isa metabolic disease, and metabolic inflammation and insulin resistance in brain playimportant roles in PD pathology. So we surmise that metformin may be beneficialfor PD therapy.Astrocytes, as the largest number of cells in brain, plays important roles not onlyin neurotrophy and support, but also in the maintainance for barin energy metabolism.Astrocytses also can release inflammatory factors, and mediate neuroinflmmation.So, the disturbance of astrocytes functions becomes more and more important in PDpathology. To explore the role of metformin for PD therapy, we established themouse model of PD by MPTP/p (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine plusprobenecid) chronic injection. Furthermore, to explore the neuroprotective mechanism of metformin, we established SHSY-5Y celluar model induced by MPP+.And we cultured midbrain primary astrocytes to investigate the mechanism ofmetformin for neuroinflammation inhibition. We demonstrated that metforminexerted its neuroprotective role in PD via enhancement of AMPK induced autophagyand inhibition of neuroinflammation.AIM: To investigate the protective roles and mechanisms of metformin inMPTP-induced Parkinson’s disease model.METHODS:1. The protocol of chronic MPTP mice PD model: mice were treated with20mg/kg MPTP in saline subcutaneously, and1h later were injected250mg/kgprobenecid in DMSO intraperitoneally every3.5d over a period of5weeks.2.Rotarod test was tested on6th day after the last injection of MPTP/p. Mice wereplaced in a separate compartment on the rod and tested at20rpm.3. Blood glucosewas quantified by glucose kit by the glucose oxidase reaction method.4. Monoamine transmitters (DA, DOPAC, HVA) of striatum were detected byHPLC-ECD method.5. Immunohistochemistry was carried out to determine thenumber of TH, a-synuclein and MAC-1positive cells.6. pAMPK, LC3, p62, p65,NLRP3inflammasome and IL-1B were semi-quantified by western blotting.7. mRNA of IL-6, TNF-a, IL-4, IL-10and TGF-B were detected by real-time PCR.8. MTT assay and LDH measurement were applied to examine SH-SY5Y cellsviability and injury.9. Cellular ROS was detected by H2DCF-DA.10. Mitochon-dria membrane potential was analysed by flow cytometric analyses. RESULTS:1. Metformin attenuated DA neuron injury and midbrain neuroinflamm-ation in MPTP/p-treated miceMetformin increased the number of TH-positive neurons by25%in SNc (P <0.05) and DA by115%compared with MPTP/p-treated mice (P <0.05). Metforminextended the latency of MPTP/p-treated mice in rotarod test (P <0.05). MPTP/pinjections for5weeks did not impair blood glucose (P>0.05). Metformin did notinfluence blood glucose (P>0.05). Metformin inhibited microglia activation, p65nuclear translocation, NLRP3inflammasome activation. Metformin reduced IL-1βformation, TNF-α and IL-6mRNA expression, increased IL-4mRNA levels (P <0.05).2. Metformin induced autophagy and decreased α-synuclein accumulationin MPTP/p-treated miceMetformin activated AMPK, induced LC3-II expression, reduced p62andα-synuclein expression (P <0.05).3. Metformin prevented SH-SY5Y cells damage suffered by MPP+viaAMPK-autophagy pathwayMetformin attenuated SH-SY5Y cells damage induced by MPP+(P <0.05), butit was blocked by AMPK inhibitor Compound C (CC) and autophagy inhibitor3MA(P <0.05). Metformin promoted dysfunctional mitochondria clearance and inhibitsROS generation in SH-SY5Y cells (P <0.05), and these effects were also blocked byCC and3MA (P <0.05).4. Metformin inhibited midbrain primary astrocyte inflammation reactioninduced by MPP+via AMPK-autophagy pathwayMetformin accelerated AMPK phosphorylation, increased LC3-II expression,decreased autophagy substrate p62level (P <0.05), and decresased ROS in primary astrocyte induced by MPP+(P <0.05). metformin inhibited IKK phosphorylationand p65neuclear translocation induced by MPP+(P <0.05). Metformin inhibitedNLRP3inflammasome activation, and decreased IL-1β production and TNF-α、IL-6mRNA levels in primary astrocyte, which were induced by MPP+(P <0.05), andthese effects were blocked by CC and3MA (P <0.05).CONCLUSION:Metformin is beneficial for PD therapy. And the protective effect is partiallymediated by AMPK-autophagy enhancement and inhibition of neuroinflammation.