The Regulation Of Notch Signaling Pathway On The Function Of Mouse Microglia

Neurodegenerative diseases are one of the killers to threat human health,and more elder people, more patients who had one of the neurodegenerativediseases. Parkinson's disease (PD) is one of the most common neurodegenerativedisorders. In developed countries, it affects approximately 1% of population agedover 60. But we know very little about the causes and mechanisms of the diseases.Activated microglia-induced localized neuroinflammation has been provedparticularly important in the initiation and progress of the disease, and moderateintervention of the activation process of microglia could possibly attenuate theprogression of neurodenegerative diseases such as PD. Activated microgliaparticipate in neuroinflammatory responses in a polarized manner: M1-likemicroglia produce neurotoxic molecules to promote neuroinflammation, whereasM2-like microglia suppress the neuroinflammation to protect neurons.Notch signaling pathway is related to the activation process of microglia. Itis highly conserved in evolution, and plays an important role in regulatingcell-fate decision during embryonic and postnatal development through theinteraction between adjacent cells. Notch signaling pathway plays a decisive role in cell fate differentiation in many different systems, such as the nervous system.And Notch signaling pathway regulates the function of many kinds of immunecells. It is reported that Notch signaling pathway has significant relevance on thedevelopment and function of the microglia.But the insufficient evidences resulted in the polarization of N9 microglialcell and the relationship between the Notch pathway and the microglialpolarization was still poorly revealed.A murine microglial cell line N9 is obtained by immortalization of E13mouse embryonic cultures with the 3RV retrovirus carrying an activated v-myconcogene that is similar to primary microglia in producing substantial amounts ofNO and various cytokines after stimulation.ObjectsObjects:A murine microglial cell line N9 was employed:(1) to verify the polarization of microglia, and to unveil reversibility of thepolarized activation;(2) to confirm the expression of Notch signal-associated molecules inmicroglia;(3) to investigate the effect of Notch signaling on proliferation of microglia;(4) to observe the role of Notch signaling in the determination of M1 versusM2 polarization in microglia activation.Methods:(1) Use LPS or IL4 to stimulate N9 microglia respectively firstly, and thensequentially use LPS/IL4 or IL4/LPS to treated N9 microglia; and the levels ofIL-6 and TNF-αin the culture medium were assayed by using different ELISAkits, the total nitrite was measured based on the Griess reaction, and the polarizedmarker genes were examined using quantitative PCR analysis. (2) The Notch-signal associated molecules were examined usingquantitative PCR analysis.(3) Divide N9 microglia into three groups: the blank control, theDMSO-treated control and the GSI-treated experimental group; the growth curveof microglia was determined by MTT.(4) Incubate N9 microglia with GSI continuously, and/or withdraw the GSImidway for a while, and then Use LPS or IL4 to stimulate N9 microgliarespectively; and the levels of IL-6 and TNF-αin the culture medium wereassayed by using different ELISA kits, the total nitrite was measured based on theGriess reaction, and the polarized marker genes were examined using quantitativePCR analysis.ResultsResults:(1) When N9 cells were stimulated with LPS, the expression of IL-6, TNF-α,NO and iNOS were enhanced remarkably, while IL4 stimulation upregulated theexpression of Arg, Ym1, CCR2, and Mmr. And the expression of M1 markersIL-6, TNF-αand NO were maintained upon secondary stimulation with IL4, butM2 markers Ym1 and CCR2 failed to upregulate; on the other hand, the Ym1 andCCR2 induced by IL4 pretreatment were reversed by the secondary treatmentwith LPS, while the expression of IL-6, TNF-αand NO was upregulated.(2) The expression of Notch receptor (Notch1), ligands (Jagged2, Dll-1,Dll-4) and downstream target genes (Hes1, Hes5) were detected on microglialcell line N9.(3) Compared to the control, there was no difference on the proliferation ofmicroglia with GSI treated.(4) Blocking the Notch signaling pathway with GSI, and stimulating M1activation with LPS, or M2 activation with IL4, the expression of IL-6, TNF-α and NO were downregulated, while the expression of M2 molecules includingArg, Ym1, CCR2, and Mmr were enhanced. And after withdrawing GSI from theculture medium, LPS failed to upregulate the expression levels of M1 markersIL-6, TNF-αand NO, while IL4 induced an exacerbated expression of Arg andYm1.ConclusionsConclusions:(1) Microglia presents obvious polarization, and the LPS-induced M1polarization did not shift to M2 upon a secondary IL4 stimulation, but theIL4-induced M2 polarization did shift to M1 upon a secondary LPS stimulation.(2) Notch signal-associated molecules are expressed in microglia, andNotch pathway can not affect the proliferation of microglia in vitro.(3) Notch signaling modulates M1 versus M2 polarization of microglia, andthe Notch signal-modulated microglial polarization appears to be irreversible.