| The adult central nerve system (central nervous system, CNS) in mammals shows a limited capacity for anatomical and functional recovery after nerve injury. Exception to this rule is the olfactory system. Because olfactory receptor neurons (olfactory receptor neurons, ORNs) can be continuously replaced throughout life, both in response to injury and as part of normal turnover, the system become a good model for the study on neurgenesis. Insight into the development of CNS and the repair of CNS after injury will help to understand the limited capacity for neural regeneration after adult CNS injury and look for new therapeutic strategies for SCI. However, little is known about the regeneration mechanism of olfactory, the reason of ORNs turnover, how OECs migrate from olfactory epithelium to olfactory bulb, how the axons of ORNs enter into and extend within the CNS at the olfactory bulb, how interact between OECs and olfactory nerve, and how the axons of ORNs sort in the olfactory bulb during olfactory neurogenesis. Our studies are focused on the underlying mechanism of olfactory neurogenesis.Here, a series of experimental methods were used to explore the underlying mechanism of olfactory neurogenesis. Our studies are intended to further understand the roles of OECs on olfactory development, olfactory plasticity and nerve regeneration after injuring, and looking for new therapeutic strategies for neural development abnormality, nervous system neoplasms, nervous recessive diseases and CNS injury. The results of our studies are as follows:1. Purinergic signaling inhibits OECs migrationAdult mammalian nerve regeneration and functional recovery is extremely difficult. In the central nervous system, the ORNs is very special, they can turnover for life, which is related with olfactory ensheathing cells (olfactory ensheathing cells, OECs) that the existence of special kind of glial cells closely . Currently, the role of olfactory ensheathing cells in neurogenesis is very limited known. In the first part of our studies we focused on the roles of purinergic signaling released by olfactory receptor nervers (ORNs) on olfactory ensheathing cells (OECs) during olfactory neurogenesis.Olfactory ensheathing cells (OECs) are a unique type of glia cell that exist in olfactory system. They share the phenotypes of both Schwann cells and astrocytes, and are the only type of glia cells which can be find both in the central nervous system (olfactory bulb) and the peripheral nervous system (olfactory epithelium). During development, they originate from the olfactory placode, wrap olfactory receptor neuron axon bundles and help them to extend through the cribriform plate into the olfactory bulb.Studies have shown that the migration of olfactory ensheathing cells play a critical role on olfactory system development. Recently, with the insight into the mechanism of nervous development, injury and signal transmission, neuroglial cell evolve as the focus of research in the neuroscience. The migrating ability of OECs, which closely affect neuron migration, axon growth, regrowth and target, was thought to be essential for neural development and regeneration after injury. However, the specific mechanism remains elusive.Purins as a kind of signal molecular play an important role in the signal transduction of cells. Physiological and pathological sources of extracellular ATP have been clearly elucidated.In the olfactory system, ATP may be released from synaptic vesicles in olfactory nerves. Purinergic signaling have been reported that it take part in the regulation of proliferation and migration of glial cells. It is now recognized that purinergic signaling can be released by neurons effect the migration as 'host damage tissue"signaling. It also has been reported that the purinergic signal can induced the neuroprotective by activating the expression of heat shock protein in olfactory neuron injury in OE. However, it remains elusive whether purinergic signaling can regulate the migration of OECs in adult OE neurogenesis.We examined the effects of purinergic signaling on OECs migration. Initially, the "scratch" migration assay, Boyden chamber assay showed that purinergic signaling inhibits OECs migration in vitro. We used immunohistochemical staining and RT-PCR method further, found that OECs express the P2Y1 and P2Y2 receptors not others. Furthermore, purinergic signaling inhibitors can reverse the above phenomenon.Together, we report, to our knowledge for the first time, that purinergic signaling inhibits OECs migration via P2Y1 and P2Y2 receptors. In this research, we investigate a new mechanism of the regulation of cell migration, and deepened the interaction between olfactory ensheathing cells and olfactory receptor neurons, which makes understanding of neural development and regeneration deeply. And the mechanism provides important theoretical basis for the search for central nervous system diseases and injuries of effective treatment strategies and therapeutic targets to provide new breakthrough.2. Effects of HDACs on olfactory neurogenesisOlfactory epithelium neurogenesis process and the controlling mechanisms are the important issues of olfactory developmental biology. Olfactory receptor neurons (ORNs) are primary neurons in olfactory system, which is the only neurons that can direct contact with the peripheral environment in vertebrate.ORNs is very important for the normal olfactory function. So mammalian ORNs are in regeneration for life. Olfactory epithelium neurogenesis is an extremely complex process. ORNs are subject to appropriate regulation, but the regulation of this important process is not clear in each stage of development.The acetylation and deacetylation of core histones of chromatin are among the most important histone modifications and are essential for many biological processes, including proliferation, differentiation and gene silencing. Histone deacetylases,HDACs, which regulate the the process of histone acetylation modification, play an important role in the process involved in neural stem cell proliferation, fate decisions, differentiation and other processes. The research of MacDonald shows the expression of HDACs in olfactory stem cells and ORNs。HDACs may involve in the neurogenesis of olfactory. However, little is known about the effect of HDACs on olfactory neurogenesis.Here, we report that histone deacetylases (HDACs) is required for the olfactory neurogenesis. We found that VPA, the inhibitor of HDACs, can affect the thickness of the olfactory epithelium and cell number. HDACs involves in the process of neurogenesis in the olfactory epithelium. HDAC inhibitors can cause the cells of olfactory to cease proliferating in vitro and in vivo. In order to explore the effect of HDACs on the mature of olfactory receptor neurons, we used in vivo models and western blotting methods, found in the mouse olfactory epithelium the number of OMP-positive neurons in the reduction and reduction in the expression of OMP after treatment VPA. These results strongly suggest, HDACs with the regulation of the progenitor cells of olfactory epithelial proliferation and the mature of olfactory receptor neurons. HDACs activity was inhibited will lead to inhibit the progenitor cells of olfactory epithelial proliferation and the mature of olfactory receptor neurons was inhibited, thus affecting the normal form of the olfactory epithelium, resulting in its thickness reduced, the total number of cells decreased and the number of mature ORNs decreased by Notch2 signal pathway. Taken together, we demonstrate, for the first time, that HDACs, a regulater in the process of histone acetylation modification, can affect the olfactory neurogenesis.Primary findings are summarized as follow:1. In the adult olfactory neurogenisis, purinergic signaling inhibits migration of OECs by P2Y1 and P2Y2 receptors.2. HDACs can inhibit the progenitor cells of olfactory epithelial proliferation and the mature of olfactory receptor neurons, involving in the process of olfactory neurogenesis through by Notch2 signal pathway.
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