Autophagy Mediates The Proliferation And Mechanism Of Neural Stem Cells Under Hypoxia

Neural stem cells are one type of stem cells that have the capacity of self-renewal,proliferation and differentiation into neuron,astrocytes and oligodendrocytes.NSCs require somewhat microenvironments,including the celluar and non-celluar components,to sustain the potential of self-renewal,proliferation and the multi-directive differentiation,which is so called stem cell niche in the fields of stem cells.Thus,different stem cells have their own unique microenvironment.Hypoxia is the common microenvironment or niche.It has been reported that the concentration of oxygen in the central nervous system is about 1~8 percentage or lower.Consequently,NSCs survive in a relatively hypoxic microenvironment.In addition,it has been documented that an obvious proliferating state was found in NSCs cultured in vitro under the conditions of 2.5~5% O2 Hypoxia,thus,is necessary for the proliferation and stemness of NSCs.However,the mechanisms by which hypoxia promotes the proliferation of NSCs have not been clearly yet.Autophagy is an intracellular lysosomal degradation path way.The classic autophagic degradation pathway is a process that the unnecessary or damaged proteins and organelles are recognized and encapsulated by a double isolated membrane structure,which then infuses with lysosome to form the autolysosome and the contents are digested by the proteolytic enzymes releasing from the lysosome.Hence autophagy has been recognized an important way of cell quality control and metabolic regulation.Moreover,there is increasing researches support the viewpoint that autophagy is critical adaptive mechanism,by which cells cope with the external stresses such as hungry,hypoxia,and so on.Recent studies have shown that autophagy has an important role in keeping the stemness of stem cells like NSCs,and the autophagic activity in NSCs was significantly higher than that of terminally differentiated cells.Therefore,we suppose that hypoxic environment may have an impact on the autophagic activity of NSCs.Hypoxia is one of classical stresses and can induce the occurrance of autophagy through the regulation of several signal pathways,which including(1)the hypoxia inducible factor dependent pathway and(2)the hypoxia inducible factor non-dependent pathway.(1)HIF-1 is a transcript factor that protects the cell survival from environment via sensing the oxygen concentration and activates the transcription of BINIP3 or REDD1 which is involved in the autophagy initiation.Once transcriptional activation by HIF-1,BNIP3 promotes the release the Beclin1 that provokes autophagy.REDD1 induces autophagy through suppressing the activity of m TOR through via TSC1/TSC2-Rheb pathway.(2)HIF-1 non-dependent pathway: hypoxia activates the AMPK,which on the one hand inhibits the activity of m TOR via TSC1/TSC2-Rheb pathway,on the other hand,AMPK directly activates ULK1 that initiates the process of autophagy.Accordingly,the occurrence of autophagy induced by hypoxia is a complicated signal transduction pathway involving multiple molecules.In the present study,we investigated(1)the relationship between proliferation and autophagy in NSCs under 3% O2 conditions;(2)the mechanism of autophagy in NSCs regulated by hypoxia.In this study,we isolated the midbrain tissue from the embryonic rat fetal on day 14.5 to establish the NSCs culture system in vitro.After that,we adopted the 3% O2 as the hypoxia model to observed the relationship between the proliferation and the changes of autophagy in NSCs,and further elucidate the function of autophagy in proliferation of NSCs and the underlying mechanisms.The main results as follows:1 Autophagy mediates the proliferation of NSCs in hypoxiaUsing 20% O2(normoxia)and 3% O2(hypoxia),we first investigated proliferation and autophagy of NSCs under hypoxia,and then determined the role of autophagy in the hypoxia-promoted proliferation of NSCs.1.1 The effects of hypoxia on the proliferation of NSCsWe had detected the relative numbers of NSCs and measured diameter directly by MTT assay in order to assess the effects of two types oxygen models on the proliferation of in different timepoints(24 h,48 h,72 h).The results have showed that hypoxia can significantly promote the proliferation of NSCs,and the number of neurosphere and the diameter of the neurosphere has increased with the prolongation of the hypoxia time.1.2 Hypoxia promotes the autophagic activity of NSCsWe have detected the changes of autophagic activity of NSCs by Western Blot at different timepoints of normoxia and hypoxia condition.The results have demonstrated that hypoxia can facilitate the autophagic activity of NSCs.The expression of autophagy marker LC3 had increased significantly,and a time-effects was found between the length of hypoxia time and the expression of LC3.In addition,once the autophagy being blocked by the Baf A1,the amount of LC3 was accumulated obviously.1.3 The relationship between proliferation and autophagy in NSCs under hypoxiaWe had detected the ability of NSCs to propagate after the block or suppression of autophagy by treatment with 3-MA or Baf A1.The results have showed that the capacity of NSCs to reproduce has suppressed markedly,and the numbers and diameter of neurosphere have decreased significantly.The results from MTT and Ed U assay have further evidenced that block or inhibition of autophagy can clearly repress the reproduction of NCSs under hypoxia condition.Based on the above results we have concluded that hypoxia can clearly promote the proliferation of NSCs and sustain a higher autophagic activity.In contrast,the effects of autophagy on the reproduction has been abolished after the block or inhibition of autophagy in NSCs.Thus,we ensure that it is autophagy that mediate the propagation of NSCs under hypoxia condition.2 The potential mechanisms of regulation of autophagy and proliferation in NSCs by hypoxiaTo clarify how hypoxia regulates autophagic activities and promotes proliferation of NSCs,we firstly detected the effects of HIF-1 on autophagy and proliferation of NSCs under hypoxia.2.1 HIF-1 is necessary for autophagy and proliferation of NSCs under hypoxiaThe relationship between the expression of HIF-1 and the proliferation of NSCs was detected by Western Blot at different time points in normoxic or hypoxic conditions.The results showed that the expression of HIF-1 was positively correlated with the autophagic activity and the expression of proliferating cell nuclear antigen PCNA under hypoxia.We further enforced overexpression of HIF-1 or treated cells with PHD inhibitor FG-4592,and then observed that the autophagic activity and the expression of PCNA in NSCs were significantly increased.On the contrary,the autophagic activity and the expression of PCNA in NSCs were decreased when HIF-1? was knocked down with si RNA prior to hypoxia exposure.2.2 HIF-1 mediates autophagy of NSCs via regulating of m TOR under hypoxiam TOR is an important negative regulator of autophagy.We found that the expression of HIF-1? was negatively correlated with the activity of m TOR,that is,the higher expression of HIF-1 and the lower activity of m TOR.Meanwhile,the expression of LC3-II increased with time.Moreover,the expression of HIF-1 and its target gene BNIP3 showed the highly positive correlation.We also observed the increased activity of m TOR but the decreased proliferation after knockdown of HIF-1 with si RNA in NSCs.In addition,the expressions of LC3-II and PCNA were significantly increased after HIF-1? was enforced overexpression with plasmids under normoxia,while the activities of m TOR and its substrate p70S6 K were inhibited.These results suggest that hypoxia may regulate the autophagic activity of NSCs through the HIF-1-m TOR pathway.2.3 BNIP3 mediates the regulation of HIF-1 on m TOR and involved in the regulation of autophagy and proliferation of NSCs under hypoxiaIt has been reported that BNIP3 modulates autophagy via the Rheb-m TOR pathway.Therefore,we speculate that BNIP3 may be involved in the regulation of HIF-1 on m TOR.After BNIP3 was enforced overexpression with plasmid in normoxia,we observed that the activities of m TOR and its substrate p70S6 K were clearly inhibited,while the expressions of LC3-II and PCNA were significantly increased.Next,we knocked down the expression of BNIP3 with si RNA prior to hypoxia exposure.The results showed that the activities of m TOR and its substrate p70S6 K were increased,while the expressions of LC3-II and PCNA were decreased.In conclusion,in the present study,we found that hypoxia(3% O2)could significantly promote the proliferation and autophagic activity of NSCs,and we confirmed that the higher autophagic activity in NSCs mediated the regulation of hypoxia on NSCs proliferation.Most importantly,we revealed a new mechanism for the first time that the HIF-1-BNIP3-m TOR pathway is required for autophagy and proliferation of NSCs under hypoxia.This study will provide a new insight for the expansion NSCs in vitro and their clinical applications.High altitude brain injury is the category brain disorders consisting of high altitude headache,acute mountain sickness and high altitude cerebral edema,which characterized by a spectrum of non-specific symptoms,occurring to the unacclimated people when they arriving at high altitude areas.With the increase in altitude,the incidence of all of these three high altitude brain injuries has increasingly significantly.In the late years,with the growing needs of economic development,geological disasters,military activities and the plateau tourism,more and more people will arrive into plateau.However,the high altitude brain injury has posed great threat to the people's health.Consequently,it is of great importance to developing the drugs of preventing and treating the high altitude brain injury.The methylene blue is medicine that clinically applied to cure the methemoglobin hemoglobin as well as the useful diagnostic tool of locating certain tissue and organ in surgery.In addition,recent researches have found that methylene blue has an neuroprotection on ischemia brain injury,traumatic brain injury and Alzheimer disease and so on,suggesting methylene blue may as a potential and promising compound applied for various clinical illness.Therefore,uncovering the mechanism underlying methylene blue will contribute to clinical treatment of various neurological disorders.It is found that methylene blue can sustain the function of mitochondrial electron transport chain via the redox,thus reducing the free radicals and maintaining the homeostasis.Also,there is research demonstrated that methylene blue can exert the neuroprotection through inducing the autophagy.Our previous study had showed that methylene blue has a neuroprotection on the ischemia brain injury by inducing the mitophagy.Autophagy is an important regulatory system for the quality control of intracellular proteins and organelles.Autophagy is normal a necessary mechanism for central nervous system to maintain neuronal survival.Many autophagy regulatory molecules regulate intracellular autophagy level according to various stimulus inside and outside the cell,and maintain cell homeostasis.Autophagic deficiency in cells is closely related to the development of a variety of diseases such as cardiovascular diseases and acute and chronic nervous system diseases.In the present study,we used lipopolysaccharide(LPS,0.5 mg/kg)combined with hypobaric hypoxia(6000 m,6 h)to establish a high altitude brain injury model,to evaluate the protective effect of MB on high altitude hypoxia brain injury and the potential mechanism underlying it.The main results are as follows:1 MB decrease the increased brain water content induced by LPS combined with hypobaric hypoxiaWe have adopted LPS(0.5 mg/kg)combined with hypobaric hypoxia(6000 m,6 h)to establish a high altitude brain injury model,to determine the protective effect of MB on high altitude brain injury by measuring the brain water content.The results have showed that LPS(0.5 mg/kg)combined with hypobaric hypoxia(6000 m,6 h)can significantly increase the brain water content in mice.All of different doses of MB could decrease the brain water content,of which the dose of 0.5 mg/kg MB can significantly reduce the brain water content resulting from LPS combining with hypobaric hypoxia exposure.We thus chose the dose of 0.5 mg/kg MB for subsequent assays.2 MB enhance the spatial memory of mice after exposed to LPS combined with hypobaric hypoxiaWe applied the morris water maze assay to detect the effects of LPS combined with hypobaric hypoxia on spatial memory in mice,and to evaluate the protective effects of MB on it.The results had showed that:(1)all of mice has an intact ability to learn and memory so that the time they needed to search for the target has gradually shorten.(2)The spatial memory of mice significantly decreased after exposed to LPS combined with hypobaric hypoxia.The entries they crossed the platform and distance they moved in the target quadrant has markedly decreased,and it also have a trend in that the time first to the target and spent in the target quadrant has separately increased and decreased,but there was no see a statistically significant change;the average speed they moved was significantly reduced as well;(3)MB can clearly improve the decreased spatial memory caused by the LPS combined with the hypobaric hypoxia.Compared to control,the entries the mice crossed the platform and the distance the mice moved in the target quadrant has increased obviously.Though the less time the mice arrived in the target,and the longer time the mice spent in the target quadrant,but all has no statistically significant change.In addition,MB also can enhance the spatial memory under normoxia condition,but failed to change the physical ability.3 The effects of MB on the cellularity of mice brain after exposed to LPS combined with hypobaric hypoxiaWe have detected the changes of cellularity of mice brain by the staining after exposed to LPS(0.5 mg/kg)combined with hypobaric hypoxia(6000 m,6 h),and also evaluated the protective effect of MB.The results have showed that LPS combined with hypobaric hypoxia exposure did not cause obvious structural damage in the cortex and dentate gyrus of mice,so the protective effect of MB was not seen.4 MB enhanced the autophagic activity in the mice brain after exposed to LPS combined with hypobaric hypoxiaWe have further examined autophagic activity in the cortex of mice brain and investigated the effects of MB on it.The results have showed that LPS(0.5 mg/kg)combined with hypobaric hypoxia(6000 m,6 h)could suppress the autophagic activity in the cortex of mice brain.The expression of autophagy maker such as Beclin1 and LC3 II/I has decreased significantly,while MB pretreatment can significantly restore the decreased autophagic activity resulting from the LPS and hypobaric hypoxia exposure.In addition,MB can also significantly increase the autophagy activity in the cortex under normoxia condition.In summary,the present study has demonstrated that LPS(0.5 mg/kg)combined with hypobaric hypoxia(6000 m,6 h)can cause the increase of brain water content in mice,result in the decline in spatial memory and repress the autophagic activity.But pretreatment with a dose of 0.5 mg/kg MB can significantly reduce the increased brain water content and enhance the spatial memory,the mechanism underlying which may be associated with the effects of MB on autophagy in the mice brain.The study provides new evidence for the treatment of hypoxic brain damage at high altitude and enhancement of memory by MB.