Microglia Polarization Regulated By Notch Signaling Impact Neurons In Parkinson’s Disease Model

Parkinson’s disease (Parkinson’s disease, PD) is the second largest neurodegenerativediseases more often in the elderly population. The prevalence is about1-2%aged over65，and with the development of the aging society, the incidence is still gradually increased. Atpresent，the etiology and pathological mechanisms of PD are not fully understood.，but theneuroinflammation caused by toxic effects is one of the important mechanisms of nervedegeneration. Microglia, reside immune cells of the central nervous system（CNS）, are themain participants of the inflammatory process. Wherefore, Moderately intervenemicroglial activation may delay the progress of PD pathology. As a result of the differentsurrounding microenvironment, microglia exhibits two different types of polarizedphenotype，classic activation M1-like can promotes inflammation reaction and bringsabout neurotoxicity，choose activated M2-like is anti-inflammatory and can repair damage.The Notch signaling pathway achieves the communication, cytosolic signal transduction and nuclear transcription by the interaction between the adjacent cells. Then,the Notch signaling controls cell’s proliferation, differentiation, apoptosis, migration andadhesion. It is very conservative in evolution and plays an important role in the regulationof the body’s growth and development. The Notch signaling pathway is significant inregulating the development and function of macrophages. Similarly, it involved in theregulation of microglial activation reaction in different environmental stimuli conditions.At present, experimental studies have confirmed the performance of primarymicroglia, MMGT12and BV2cells lines in different stimuli. Regarding this reaction andthe role of Notch signaling in polarization response, we have studied using N9microgliacell lines. In brain of PD mouse and Patient，however, there is no clear reports about whichphenotypes activated microglia showed and what role played, as well as whether the Notchsignaling’s regulation affected them. In the experiment, we observed the phenotypicchanges of microglia based on mouse model of PD, and observed the polarizationresponse though blocking microglial Notch signaling in vivo and in vitro, then its impacton the surrounding tissue.Objective:（1） Make the Parkinson Disease model in C57/BL wild mouse and observe dopamineneuronal apoptosis in the brain SNc and microglia polarization changes;（2） Test and verify the primary microglia polarization responses and observe thechange of the polarization response after Notch signaling is blocked, underdifferent stimulating environment;（3） Make the Parkinson Disease model in Notch signaling knockout mouse, observethe changes in number of dopamine neurons after blocking microglial Notchsignaling in vivo.In this study, by blocking Notch signaling in vivo and vitro, observe that Notchsignaling impact polarization response of microglia. In order to provide preliminarytheoretical basis for reducing the loss of neurons in PD and other neurodegenerativediseases, then slowing disease progression by interference Notch signaling activity. Methods:（1） Intraperitoneal injected of MPTP to production model of PD, observe the generalbehavior performance.Open-field test and forced swimming test detected thebehavioral changes in mice. Making frozen sections of mice brain tissue,detected change of the microglia polarization and the number of dopamineneurons by immunofluorescence;（2） On the basis of primary cultured mixed glial cells, refine microglia use the shockSeparation, identified the purity by flow cytometry. ELISA to detect TNF-α/IL6content in the culture supernatant and real-time PCR method to detect theexpression of M1and M2marker molecule expression levels;（3） Clipped the mouse tail and extracted the mouse genome, after amplificated byPCR, identified the genotype of Notch signaling knockout mice using agarosegel electrophoresis. Select the mice that Notch signaling is knocked out and thecontrol mice，produced PD model, detected the change in the number ofdopamine neurons by immunofluorescence on the brain tissue frozen section.Result:（1） After injected with MPTP，PD model mice showed activity weakened, shortnessof breath, arched back and limbs were stiff, unsteady gait, vertical tail andtremors, seizures, but the mice of control group was not evident. The totaldistance of the model mice was reduced compared with the mice of control group,but no significant difference (P>0.05)in open field test activities, but its centralactivity was significantly shorter (P <0.05)than the normal control group; theimmobility time in the water of the model mice was significantly prolonged (P<0.05) in forced swimming experiment.TH-positive neurons in the brain SNc ofmodel group mice decreased to approximately87%（P＜0.01）of that of thecontrol group; M1microglia increased approximately54%（P＜0.05）of that ofthe control group, and the number of M2microglia was less than the controlgroup, but no significant difference; （2） Microglia, obtained by the shocking,it’s purity was96.33%; after LPS stimulation,the IL-6/TNF-а content in culture supernatant increased（P＜0.05）,the expressionof iNOS/CD86increased; But in IL4treatment group, these were not differencecompared with the blank control group. In contrary, the MMR, YM1and Arg1expression was elevated（P＜0.05）in IL4group. After blocking Notch signal usingGSI, IL-6decreased（P＜0.05）but TNF-а didn’t change in LPS stimulation groupculture supernatant and iNOS, CD86expression reduced（P＜0.05）; yet, the MMRYM1and Arg1expression tended to increase in IL-4treatment groups;（3） In Notch signal knock pups, the number and the morphology of microglia didn’tsignificantly change in areas of the brain such as cortex, hippocampus, midbrainand cerebellum area. Made PD model in the adult Lyz2Cre;RBP-J flox-/-mouseand Lyz2CreRBP-J flox+/-mice, the number of survival neurons in knockoutmice was more than that of the control mice（P＜0.05）.Conclusion:（1） The changes of ethology and histopathology on the PD model mice showing themodel is practical. In PD brain, the M1and M2polarization phenotypes ofMicroglia are both present, the role of M1polarization is greater than M2type;（2） Notch signal regulated the polarization phenotypes that microglia performanceunder the different micro-environmental stimuli, blocking Notch signal, M1polarization weakened but M2polarization Strengthen;（3） Knocking Notch signal didn’t affect the microglial congenital development. Afterblocking the Notch signaling of microglia in vivo, the number of survival neuronsof brain SNc Increased.