The Role Of Notch Signaling In Microglia Activation

Microglia, the first description dates back to the late 19th century are the latest identified major cell types besides neurons astrocytes, oligodendrocytes in the central nervous system (CNS). Microglial cells are ubiquitous in the CNS and account for 5%-20% of all glia cells. The description about the source of microglial precursors had been a debated issue and focus of many investigators in the past few decades. In animals such as rodents and in humans two different pools of myeloid cells are thought to be the precursors of microglia. The two different pools of myeloid cells include progenitors invading the early embryonic brain from the yolk sacand monocytes invading the brain during late embryonic or early postnatal period. It indicated the monocytic and macrophagic nature of microglia. Microglia plays important role in the CNS including physiological processes and also in pathological conditions, such as neurodegenerative diseases、CNS injury and tumor formation.Two major forms of microglial cells known as amoeboid and ramified microglia have been recognized and they may be reflective of different stages of development or activation. In the mature central nervous system, resting microglia are ramified with small cell bodies and processes to actively survey their environment. The primary role of the resting microglia is therefore to maintain cellular, synaptic, and myelin homeostasis both during development and normal function of the CNS. The resting microglia could also secret growth factors which promote neuron survival and therefore regulate endogenous immune system. In the developing brain, microglia exist as the amoeboid microglial cells (AMCs). In pathological conditions, resting microglia will be activated and becoming AMCs characterized by a round or oval cell body bearing pseudopodia and filopodia-like processes, the cytoplasm shows conspicuous vacuoles and a large number of lysosomes which are the primary immune cells of the CNS dealing with invasions by infectious agents, tumors and removal of cellular debris and are endowed with potentiality in antigen presentation. The macrophagic role of microglia in clearing the cellular debris under pathological conditions of the CNS has been demonstrated by many studies.Pathological factors including Lipopolysaccharides (LPS), adenosine triphosphate (ATP), β-amyloid precursor protein (P-APP), hypoxia and pro-inflammatoryfactorscould activate microglia and induced the inflammation process. Besides being involved inphagocytosis, microglial cells are also involved in immune response of brain as well as have other functional roles. Firstly, in response to the prompt recognition of a variety of inflammatory and infectious stimuli, microglia express a wide array of surface receptors which include the pattern recognition receptors, complement receptors and cytokine receptors. Besides, microglia also secrete cytokines, including interleukin 1β(IL-1β)、interleukin 6 (IL-6)、nterleukin 8(IL-8)、tumor necrosis factor-a (TNF-α)、transforming growth factor-β(TGF-β)、prostain、reactive oxygen species (ROS)、nitric oxide (NO) and glutamate. These are key regulators of immune responses in inflamed CNS and lead to apoptosis of neuron or oligodendrocytes. These inflammatorymediatorscould also activate astrocyte and promote astrocyte inflammatory factors release which enhanced microglia activation feedbackedly. Secondly, AMCs could also secrete chemotaxin and the receptors which mediated microglia migration to the site of injury and proliferate at the meantime thus exacerbating the inflammation process. Taken together, the over activated microglia could lead to neural injury and disease progression through the behaviors above. Therapy targeted on microglia activation inhibition has becoming prospective in the treatment of CNS diseases, therefore clarifying the various mechanisms controlling microglial activation is the first step in the suppression of neuro inflammation. Although pathways such as NF-κB and MAPK in activated microglia in inflammation have been well investigated, the mechanisms regulating microglial maturation are dubious. In this connection, clarifying the microglia activation mechanism is the premise and key point in the current study.Notch signaling pathway is one of the most conserved pathways with versatility in function but simplicity in molecular design. It can determine the cell fate through regulating cell-cell interaction of neighbor cells thus playing a fundamental role in various developmental processes and the pathogenesis of several human cancers and disorders. Four different Notch receptors Notch-1, Notch-2, Notch-3 and Notch-4 and many ligands including five classic Notch ligands Jagged 1 (JAG1), JAG2, and Delta-like proteins (Deltal,3, and 4) have been identified in mammals. Ligand-activated Notch receptors are cleaved at the Notchintracellular domain (NICD). The NICD translocates to the nucleus where it binds the recombining binding protein suppressor of hairless (RBP-jκ) transcription factor and initiates transcription of Notch target genes. In the central nervous system (CNS), Notch signaling is prominent among those pathways known to regulate neural development and pathogenesis of CNS disorders. The distinct function of Notch in CNS is manifested by its ubiquitous expression in different CNS cells, including neural stem cells, neurons, oligodendrocytes, astrocytes and microglia. Manyfindings have revealed important roles for the Notch signaling pathway in immune cells including T cell、B cells and macrophages.Especially, Notch signaling plays a crucial role in macrophage activation and M1/M2 polarization. Considering the macrophagic nature of microglia, we proposed that Notch signaling participate in microglia activation and play roles in neuro inflammation.It is well documented that hypoxia is linked to different neurodevelopmental and traumaticdiseases. The developing brain is highly vulnerable to oxygen deprivation or hypoxia. Hypoxia in the developing brain will mainly damage the periventricular white matter (PWM) which is named periventricular white matter diseases (PWMD) or worsely periventricular Leukomalacia (PLV) and lead to neonatal mortality and long-term neurodevelopmental deficits. Neuroinflammation, characterized by microglial activation has been reported to play an important role in the hypoxic injuries in the neonatal brain. In view of this, we have used in this study in vitro hypoxia models of both primary cultures of microglia and BV-2 cell line and an in vivo experimental rodent model with postnatal hypoxic exposure to investigate the role and mechanism of Notch signaling in neuro inflammation in the hypoxic developing brain. We first observe Notch signaling expression in neonatal brain after hypoxia exposure to investigate the expression and changes of Notch signaling in microglia after hypoxia exposurein vivo. In addition, primary cultured microglia and microglia cell line BV-2 cells were used to observe the expression of Notch signaling in primary microglia and BV-2 cells following hypoxia exposure in vitro. N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-phenyglycine t-butyl ester (DAPT), a y-secretase inhibitor, which can efficiently block the y-secretase complex was applied to investigate the inflammatory factor expression and release in microglia. Then, we investigate the effect and the mechanism of Notch signaling inhibition on proliferation and migration of BV-2 cells following LPS stimulation. Finally, we explore the regulation of mechanism Notch signaling on microglia activation. The results are as following:Part Ⅰ Expression and effect of Notch signaling in microglia following hypoxia exposure.In neonatal mice after hypoxic brain injury, Notch signaling ligand Delta-1 and receptor Notch-1 immunoexpression was markedly increased on microglia in the corpus callusm. NICD which represent the activation of Notch signaling was noticeably increased in lectin-labeled microglia after hypoxia compared with the control. Next, qPCR and immunostaining was adopted to investigate the mRNA expression and immunofluorescence intensity of Notch signaling elements in primary cultured microglia. The results show that Notch-1 and Delta-1 mRNA expression and immunofluorescence intensity was significantly elevated after hypoxia, NICD immunofluorescence intensity was increased after hypoxia and RBP-Jκ and Hes-1 mRNA expression was also significantly increased. The results suggest the expression and activation of Notch signaling in microglia following hypoxia exposure was enhanced. In addition, DAPT was used to block Notch signaling activation in microglia. Results show that 10μm DAPT could effectively blocked Notch signaling activation. Western blot analysis results show thatNotch signaling blockagecould significantly suppressed mRNA expression of most inflammatory mediators. Thirdly, expression of Notch signaling and the effect of Notchsignalingblockage in production of inflammatory mediators in microglia cell line BV-2 cells were investigated. The expression of important proteins in Notch signaling was investigated by qPCR and Western blot in BV-2 cells. The results show enhanced expression of Notch-1, Delta-1, NICD, RBP-Jκ and Hes-1 after hypoxia exposure in BV-2 cells. Next, y-secretase inhibitor DAPT was adopted to block Notch signaling in BV-2 cells. Results show that 10μm DAPT could effectively blocked Notch signaling activation in BV-2 cells. Western blot analysis results show that Notch signaling blockage could significantly suppressed protein expression of inflammatory mediators as well as nitric oxide (NO) release as shown by Griess assay. NF-κB signaling was the most important and well studied pathway that mediates inflmammatory factors release. We found that Notch signaling blockage could suppress NF-κB signaling activation both in BV-2 cells and in microglia of neonatal rats following hypoxia brain injury. These results suggest that activation of microglia induced by hypoxia exposure was dependent on Notch signaling. Notch signaling participate the hypoxia induced microglia activation.Part Ⅱ Role of Notch signaling in microglia proliferation and migrationWe next investigate the effect of Notch signaling on microglia proliferation and migration using the classic microglia activation model by LPS stimulation. First, microglia proliferation was investigated. BrdU incorporation andflow cytometery results show that Notch blockage will lead to a significant reduction in the percentage of proliferative microglia cells and cell cycle arrest in G1 stage. Further we found that Cyclin Dl protein expression was decreased and both p27kip1 mRNA and protein expression level was enhanced significantly after Notch signaling blockage in BV-2 cells. Chromatin immunoprecipitation assay (CHIP) results show that both RBP-Jκ and Hes-1 can negatively regulate p27kip1 expression by binding to the promoter region of p27kip1 as transcription factors. Next, transwellassay results show that BV-2 cells migration induced by LPS was significantly suppressed by Notch signaling blockage. Furthermore, Rhodamine phalloidin was used to investigate the distribution of F-actin in BV-2 cells and the results show that F-actin redistribution to the cells processes in activated microglia was blocked by DAPT treatment. These results suggest that Notch signaling could affect microglia proliferation and migration.Part Ⅲ The mechanism of Notch regulates microglia activationWe next investigate the mechanism of Notch signaling on controlling microglia activation. Western blot analysis results show that after Notch signaling blockage, activation of signaling including NF-κB, p38 MAPK, and JNK was significantly suppressed. Importantly, TAK1 phosphorylation was decreased. Western blot analysis using a K63 specific ubiquitination antibody shows that TAK1 ubitiquitination was decreased. Next, qPCR, Western blot and immunofluorescence results show that mRNA and protein expression of CYLD (Cylindromatosis) was significantly increased after Notch signaling blockage. Finally, chromatin immunoprecipitation assay (CHIP) results show that Hes-1 can negatively regulate CYLD expression by binding to the promoter region of CYLD as a transcription factor. These results suggest that Notch signaling may affect NF-κB, p38 MAPK, and JNK signaling activation by controlling CYLD expression, thus influencing microglia activation.Conclusion:We found in this study that Notch signaling in microglia of neonatal rats following hypoxia exposure was increased in vivo. In vitroresults show that Notch signaling could regulate microglia mediated expression of inflammatory mediators, microglia proliferation and migration. Blockage Notch signaling could suppress microglia mediated neuroinflammation. In conclusion, the study shows that Notch signaling could regulate many properties of activated microglia. In this connection, regulation of Notch signaling expression and activation in microglia may therefore present as a novel therapeutic target for the treatment of various pathological states that involve hypoxia in the CNS.