Activated Microglial Cells Induce Dopaminergic Neuronal Apoptosis Through ProNGF-JNK-jun Signaling Pathway

Study purpose:Parkinson’s disease (PD) is a neurodegenerative disease that is characterized by a lossof dopaminergic neuron in the substantia nigra (SN). Its pathogenesis mechanism is animportant basic and clinical question. Recent studies have revealed that brain glial cellscan produce neurotrophin (neurotrophin, NT) precursors and involve in brain developmentor damage process. For instance, pro-nerve growth factor (proNGF) can bind its receptorp75NTR and sortilin with high affinity and induce neuronal apoptosis, so proNGF mayplay a critical role in this process.Many studies have shown that glial cells can regulate growth, survival and functionalof dopaminergic neuron in SN. The activated glial cells can generate a variety ofneurotrophin precursors such as proNGF, but an important issue needs to be clarifiedabout their biological activity and implication in neurodegenerative diseases. In thisproject, we intend to focus on the activated microglial cells and proNGF, to explorewhether proNGF could be produced and released from microglial cells, whether proNGFhas biological function, and what signaling pathways are involved in proNGF-inducingapoptosis of dopaminergic neuron. We expect to elucidate a potential role of proNGF inneurotoxic or inflammatory response, which may provide new experimental evidences forunderstanding PD pathogenesis mechanisms. Material and Methods:1) LPS-lesioned animal model was prepared by stereotaxic injection of LPS into SN ofadult rats.2) Immunohistochemistry, double immunofluorescence and laser scanning confocalmicroscopy were performed to show the localization and expression change of proNGF,CD11b and iba1in the dopaminergic neuron and microglia in SN.3) Cell culture of N9/BV2cells was performed to study the expression of proNGF,proBDNF, proGDNF, MMP-9in microglia cells.4) Western blot was performed to show the expression of proNGF、p75NTR and sortilinin SH-SY5Ycells.5) TUNEL, Hoechst/PI and CCK-8were performed to display and analyze SH-SY5Yapoptosis.6) JNK, c-jun signaling pathways were analyzed for apoptosis mechanisms.Main results:1. LPS stimulated activation of N9/BV2microglial cells, upregulation of proNGFsynthesis and extracellular release1) By cell culture of N9/BV2cells and LPS model, LPS stimulated activation ofmicroglial cells with iba1-positivity and amoeba-like cell morphology.2) By western blot and immunocytochemistry, LPS induced up-regulation of proNGF inN9and BV2microglial cells.3) By western blot, extracellular release of proNGF from N9microglial cells was observedafter LPS stimulation.2. Administration of proNGF could induce apoptosis of SH-SY5Y dopaminergic cells,indicating that proNGF might exhibit a biological function1) Cell culture of SH-SY5Y dopaminergic neuron cells was prepared for neurotoxicityexperiment.2) TUNEL, Hoechst/PI and caspase-3method, administration of proNGF inducedapoptosis of SH-SY5Y cells, but did not show significant effect on cell viability. 3) Animal study showed that LPS injection also induced microglial activation,proNGFupregulation and dopaminergic neuron degeneration.3. Administration of proNGF increased expression and phosphorylation of JNK,c-jun, indicating that JNK/C-JUN signaling might mediate proNGF biological effectin SH-SY5Y cells1) Administration of proNGF increased expression and phosphorylation of JNK and c-junin cultured SH-SY5Y cells, suggesting that biological effect of proNGF might be mediatedthrough activation of JNK-c-jun signaling pathway.2) Specific signaling inhibitor experiment was performed to study possible mechanism inLPS-induced microglial activation and proNGF synthesis. We assigned a comparison ofcontrol group, LPS group, LPS+IWR1(Wnt signaling pathway inhibitor) group andLPS+GSI (Notch signaling pathway inhibitor) group. Our pilot experimental evidenceindicated that Wnt and Notch signaling pathway might be involved in activation ofmiroglial cells and proNGF synthesis process, which should need further extensiveinvestigation.Summary and conclusionMajor finding of our study is that LPS could stimulate activation of microglial cells,upregulation and release of proNGF, and then induce apoptosis of dopaminergic neuronpossibly through proNGF-JNK-JUN signaling pathway. The results of this study have thusprovided new evidence for role of proNGF in neuroinflammation or neurotoxic effect ondopaminergic neurons, as well as understanding of pathogenesis mechanisms ofneurodegenerative diseases and potential therapeutic application.