| BackgroundIn adult mammalian brain,there are two main neurogenic areas,subventricular zone and sub-granular zone,which are termed neural niches.Neural stem cells stay in quiescent state,and maintain the stem cell pool.At the same time,some of them are constantly activated,and differentiate into neurons,astrocytes and oligodendrocytes,maintain the homeostatic state of the brain,participate learning and memory,damage repair and other physiological and pathological processes.Taking the subventricular zone of rodents as an example,the stem cells in the neural niche are continuously activated into neural progenitor cells during the development of neurogenesis.They leave the neural niche and migrate along the rostral side to the olfactory bulb to form new neurons integrated into the neural network.With the increase of age,the endogenous neural activity in the neurogenic niche gradually weakens,correspondingly,the homeostasis and damage repair functions decrease,and the incidence of neurodegeneration and brain tumor increases.Therefore,neural stem cells play an important role in maintaining the health of mammalian nervous system.How to regulate the stemness and differentiation potential of neural stem cells and maintain brain homeostasis are important scientific questions.At present,there are three main strategies for stem cell therapy: the first is cell rescue by using neurotrophic factors or cell-based methods;the second is cell replacement therapy by using exogenous and in vitro cultured cell transplantation;the third is cell replacement by using endogenous neural precursor cells or strategies involving direct reprogramming of resident cells in the brain.However,different treatment strategies also have different problems to be solved.In the strategy of using neurotrophic factor to rescue cells,neurotrophic factor is difficult to be effectively delivered to neuron targets.Exogenous cell transplantation may result in abnormal neuronal connections and epilepsy.In the face of the above problems,we focus on the regulation of quiescence/activation of endogenous neural stem cells.Adult mammalian brain still retains the function of neurogenesis.If we can reasonably and effectively regulate endogenous cells to repair and ameliorate nervous system diseases,we can avoid the risks and limitations of neurotrophic factor therapy and exogenous cell transplantation therapy,and find new route and targets for the treatment of nervous system diseases.The key of endogenous cell therapy is to find the key regulatory molecules of neural stem cell stemness and neurogenesis.Ttyh1 is a transmembrane protein,which was identified by Campbell in 1993 in a region of fli(flightless)locus.Previous studies have suggested that Ttyh1 acts as a chloride channel.However,our previous experiments showed that Ttyh1 may play key regulatory roles in the stemness,proliferation and differentiation of neural stem cells.In order to further study the regulatory effect and mechanism of ttyh1 on neural stem cells,we designed the following studies.Objective1.To analyze the expression and distribution characteristics of Ttyh1 in each stage of NSCs;2.To reveal the regulatory effects of Ttyh1 on the stemness,proliferation and differentiation of NSCs;3.To clarify the molecular mechanism of the regulation by Ttyh1 on NSCs.Methods1.Use the single-cell sequencing data in the existing database to analyze the distribution of Ttyh1 at various stages of neural stem cells through bioinformatics;2.Construct Ttyh1 reporter mice,induced by tamoxifen,and analyze the expression and distribution of Ttyh1;3.Transfect neural stem cells cultured in vitro with lentivirus,knock down the expression of Ttyh1,and observe the changes in growth and differentiation of neural stem cells;4.Utilize Ttyh1 KO mice,and analyze and compare the effects of Ttyh1 on the stemness and differentiation of neural stem cells at different ages by immunofluorescence staining on frozen sections of adult and old mouse brains;5.Perform transcriptome sequencing on neural stem cells transfected with sh Ttyh1 lentivirus to analyze the mechanism of the regulation by Ttyh1 on neural stem cells6.Use active NFATc3 overexpression lentivirus to transfect Ttyh1 knockdown cells to rescue and verify the downstream role of NFATc3 in the regulation by Ttyh1 on neural stem cells.Results1.Analysis of single-cell sequencing data shows that Ttyh1 is mainly distributed in quiescent neural stem cells and astrocytes,and its expression gradually decreases after quiescent neural stem cells are activated.2.Immunofluorescence staining of frozen sections of Ttyh1 reporter mouse brain showed that Ttyh1 was mainly distributed on striatal side of the lateral ventricle in the SVZ area,and mainly concentrated in the sub-granular zone in the SGZ area,and it could be co-labeled with Sox2 and partially co-labeled with GFAP.Co-labeling rarely occurs with other progenitor cell markers EGFR,DCX and ependymal cell marker CD24;3.Using lentivirus to knock down the expression of Ttyh1 in neural stem cells cultured in vitro,it was found that the growth morphology of neural stem cells changed,appeared adherent growth,and protruded neurites.The results of immunofluorescence staining showed that after knocking down Ttyh1.The proportion of Sox2 positive stem cells and DCX positive neuroblasts decreased,while GFAP and O4 positive glia cells increased,Map2 positive neuron cells decreased,indicating that more cells differentiated toward glial cells.4.Immunofluorescence staining of the brain sections of adult Ttyh1 KO mice showed that the neural stem cells of adult KO mice exhibited enhanced proliferation,and more progenitors migrated from the SVZ area,differentiated into neuroblasts and other progeny cells.The proliferation and neurogenesis of stem cells in the SGZ have also been enhanced;5.Immunofluorescence staining of the brain sections of old Ttyh1 KO mice showed that the number of stem cells in the neural niches decreased significantly,the neural stem cells were exhausted,compared with the control mice.And there were pathological cavities in the brains of the old Ttyh1 KO mice;6.Transcriptome sequencing results showed that a large number of gene expressions changed after Ttyh1 knockdown,and the clustering results showed significant changes in the calcium signal pathway;7.Western blot and q PCR results showed that the expression of calcium ion pathway related molecules decreased after Ttyh1 knockdown,and the downstream transcription factor NFATc3 was significantly down-regulated;8.The rescue experiment showed that the overexpression of NFATc3 overexpression by lentivirus can partially rescue the phenotypic changes of neural stem cells caused by Ttyh1 knockdown.Conclusions1.Ttyh1 is mainly distributed in the quiescent neural stem cells and astrocytes in the neural niche,and the expression of Ttyh1 gradually decreases after the neural stem cells are activated;2.After knocking down Ttyh1 in neural stem cells cultured in vitro,the morphology of neural stem cells changes,the proportion of stem cells is reduced,and the stem cells are differentiated.The existence of Ttyh1 can maintain the stemness of neural stem cells;3.After knocking out Ttyh1,the neurogenesis of adult mice is enhanced,and more neural stem cells are activated to differentiate into progeny cells and participate in the construction of neural networks;4.After knocking out Ttyh1,the stem cells in the neural niches of old mice were significantly reduced,the stem cell pool was exhausted,and pathological cavities appeared in the brain at the same time;5.Ttyh1 regulates the expression of Cyclin D1 and P21 through the downstream transcription factor NFATc3 of the calcium signal pathway to regulate the cell cycle of neural stem cells. |
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