Epigenetic Mechanisms Underlying NNOS-PSD-95 Dissociation Promoting Neurogenesis After Stroke

Stroke has become a major public health problem.The absence of effective therapies increases the need for new therapeutic targets.The mammalian brain has the ability to repair itself in order to regain lost function.Promoting regeneration repair will provide a new treatment strategy for stroke,including neurogenesis and dendrite remodeling.According to the experimental finding,the interaction between neuronal nitric oxide synthase?nNOS?and post-synaptic density protein-95?PSD-95?negatively regulates the regeneration repair after stroke in rats.nNOS-PSD-95decoupling in neurons promotes the differentiation of neural stem cells?NSCs?to neurons,promotes the migration of newborn cells to the ischemic injury area,and enhances the growth of neurite of newborn neurons and the formation of dendritic spines of mature neurons after stroke.More interestingly,nNOS-PSD-95 decoupling can improve post-stroke motor function in the recovery phase,which is achieved by intracerebral regeneration repair in rats.Thus,the role of nNOS-PSD-95 decoupling in promoting neurogenesis after stroke has been clearly identified,but the underlying mechanism remains unclear.According to literature reports,epigenetics regulates the differentiation fate of NSCs.Histone acetylation and deacetylation are important components of the epigenetic mechanism.Histone acetyltransfeases?HATs?and histone deacetylases?HDACs?are involved in gene activation and inhibition respectively.Furthermore,histone deacetylase-2?HDAC2?,which is selectively expressed in the neuronal lineage cells,plays an important role in the neurogenesis and differentiation of NSCs in adult neurogenesis.Thus,we hypothesized that HDAC2 might play a critical role in the process of nNOS-PSD-95 dissociation promoting neurogenesis after stroke.In addition,neurogenic differentiation?NeuroD?is a neuron-specific transcription factor.In NSCs,HDACs silences expression of key neurogenetic transcription factors,such as NeuroD.Therefore,NeuroD might be a key downstream effector molecule in the process of HDAC2 regulating neurogenesis.Early studies showed that Best1 expression was significantly up-regulated in the peri-infarct area after stroke.Inhibition of HDACs can down-regulate the expression of Best1 after cerebral ischemia.In other words,epigenetics can positively regulate the expression of Best1 after cerebral ischemia.Therefore,Best1 may play an important role in the process of nNOS-PSD-95 dissociation promoting functional recovery by inhibiting HDAC2 expression.Therefore,this paper studied the epigenetic mechanism of nNOS-PSD-95dissociation promoting neurogenesis after stroke:In chapter 1,the rat's middle cerebral artery embolism?MCAO?model,and the oxygen glucose deprivation?OGD?model in NSCs or the co-cultures by NSCs with neurons,were used in vitro and in vivo experiments to study whether nNOS-PSD-95 dissociation promoted NSCs differentiation to neurons after stroke by affecting HDAC2.In chapter 2,we further explore whether the downstream mechanism of HDAC2 is related to the increased expression of NeuroD mediated by histone acetylation,a key transcription factor for neuronal differentiation,in the process of nNOS-PSD-95 dissociation promoting neurogenesis after stroke.In chapter 3,the photothrombotic stroke model and the OGD model of astrocytes,were used in vitro and in vivo experiments,to study the effects of Best1 on motor function after cerebral ischemia and the mechanism.Chapter 1 To explore how the decoupling of nNOS-PSD-95 in neurons after stroke affects the differentiation fate of NSCs,we first focused on HDAC2.To specificallyregulateHDAC2,lentivirusescontainingHDAC2shRNA?LV-HDAC2-shRNA?and adenoviruses selectively expressing HDAC2?Ad-HDAC2?were designed.The results showed that LV-HDAC2-shRNA-infected NSCs had an increased ability to differentiate into neurons,while Ad-HDAC2-infected NSCs showed the opposite effect.Microinjection of Ad-HDAC2 into dentate gyrus?DG?significantly reduced the number of BrdU⁺/DCX⁺cells.Thus,HDAC2 negatively regulates the differentiation fate of NSCs to neurons.Ischemia leads to an increase in nNOS-PSD-95 interaction and activation of nNOS,thereby increasing the release of NO;so what a role does NO play in the negative regulation of nNOS-PSD-95 coupling in neurogenesis?First,we used NO donor and scavenger in the OGD treated co-cultures of NSCs and neurons,proving that NO also has negative regulatory effect on NSCs differentiation to neurons.Therefore,we hypothesized that neuron-derived NO may regulate HDAC2 to influence the differentiation fate of NSCs.We used NO donor to mimic the neuron-derived NO,and found that the nitrosylation level of HDAC2 in NSCs remained unchanged,but the protein expression and enzyme activity of HDAC2 were significantly up-regulated,and the opposite result was obtained by NO scavenger,that is,NO could indeed up-regulate the expression of HDAC2 in NSCs.Then we detected the protein level and enzyme activity of HDAC2 in the OGD treated co-cultures of NSCs with neurons and the OGD treated NSCs.It was found that only in the OGD treated co-cultures of NSCs and neurons,the protein level and enzyme activity of HDAC2 were up-regulated and could be reversed by NO scavenger.Therefore,the expression of HDAC2 in NSCs was up-regulated after OGD,and NO from neurons played a key role.In other words,if the coupling of nNOS-PSD-95 is removed after stroke,the release of NO in neurons can be reduced,thus inhibiting the HDAC2 in NSCs.Subsequently,we infected NSCs with LV-HDAC2-shRNA,then cocultured it with neurons,and performed OGD.The results showed that?-?-tubulin⁺newborn cells and newborn neurons with multineurites decreased after OGD,and knock-down of HDAC2 in the NSCs reversed these results.It is suggested that up-regulated HDAC2 hinders the differentiation of NSCs into neurons after stroke,and inhibiting the up-regulation of HDAC2 is beneficial to neurogenesis after stroke.So is the effect of nNOS-PSD-95 decoupling on neurogenesis after stroke dependent on the inhibition of HDAC2?ZL006 was given in a co-culture system with HDAC2 over-expression in NSCs to reduce the coupling of neuronal nNOS-PSD-95after OGD,then observing the differentiation of NSCs into neurons.Experimental results showed that ZL006 reversed the neuronal differentiation reduction and neurite growth injury caused by OGD,while the over-expression of HDAC2 cancelled the effect of ZL006.In conclusion,nNOS-PSD-95 decoupling promotes the differentiation fate of NSCs into neurons after stroke by inhibiting HDAC2.Chapter 2 Histone acetylation is crucial for neurogenesis,and NeuroD is a neuron-specific transcription factor.Therefore,we focused on NeuroD in order to elucidate the downstream mechanism of nNOS-PSD-95 association blocking NSCs differentiation into neurons through HDAC2 after stroke.First,we show significant reductions in the expression of acetylated histone H4?acety-H4?and NeuroD when NSCs are treated with NO donors,and NO scavenger can reverse this effect of NO donors.Meanwhile,the HDAC2 knockdown induced by HDAC2 inhibitor TSA and LV-HDAC2-shRNA can also counteract the regulation of NO donor on the expression levels of acety-H4 and NeuroD.That is,NO inhibits histone acetylation by up-regulating of HDAC2,thereby inhibiting NeuroD expression in NSCs.To confirm the above experimental results,we performed OGD operation in the co-culture system with HDAC2 knockdown in NSCs to detect the level of histone acetylation and the expression of NeuroD.As expected,OGD reduced the level of histone acetylation and NeuroD expression in NSCs.Similarly,knockdown of HDAC2 reverses the effect of OGD.It is suggested that nNOS-PSD-95 decoupling promotes NeuroD expression in NSCs by inhibiting HDAC2 to increase histone acetylation level.To sum up,nNOS-PSD-95 dissociation after stroke leads to the reduction of neuron-derived NO,and its role in up-regulating of HDAC2 expression and activity in NSCs is inhibited,so as to increase the level of histone acetylation and realize the epigenetic regulation of NeuroD,a key transcription factor for neuronal differentiation.Chapter 3 Does Best1 play an important role in the process of nNOS-PSD-95dissociation promoting functional recovery by inhibiting HDAC2 expression?In order to solve this problem,Best1 is mainly studied in this chapter for its effect on motor function after cerebral ischemia.First,we found that Best1 expression in the peri-infarct area was significantly increased on day 7 and day 10 after cerebral ischemia.To specifically knockdown Best1,we constructed recombinant adenovirus AAV-Best1-shRNA-GFP,and found that interference of Best1 expression was beneficial to the improvement of motor function after cerebral ischemia.The mutation of Best1 inhibits the function of its ion channel,so we designed the mutation virus AAV-Best1^W93C-3flag-GFP,and the experimental results showed that inhibition of the function of Best1 ion channel was beneficial to the recovery of motor function after cerebral ischemia.Astrocytes in the peri-infarct area will be activated,one of the main sign of the activation is increased expression of glial fibrillary acidic protein?GFAP?,so we tested the GFAP expression in the peri-infarct area.The experimental results showed that GFAP was continuously highly expressed on day 3,7 and 10,and co-labelling of Best1 and GFAP in the peri-infarct area were significantly increased on day 10 after cerebral ischemia,suggesting that Best1 in astrocytes may play an important role in the function recovery in the delayed period of cerebral ischemia.Therefore,we cultured astrocytes in vitro,and found that GFAP and Best1 were highly expressed in48 and 72 h after OGD.Next,it was found that GFAP expression was inhibited by interfering Best1 expression.It has been reported that Best1,which is redistributed by activated astrocytes,can mediate the release of gamma-aminobutyric acid?GABA?,while reducing GABA tonic after stroke is conducive to functional recovery.Combined with the above experimental results,we speculated that the interference of Best1 can ultimately improve the motor function after cerebral ischemia,by inhibiting activated astrocytes,thereby inhibiting Best1 expression and its ion channel function,reducing GABA release.It has been reported that the activation of protease-activated receptor 1?PAR1?can promote the Best1 release of glutamate,GABA or other glial transmitters in astrocytes.Monoamine oxidase B?MAOB?mediated the synthesis of tonic GABA in astrocytes.Therefore,in this chapter,we tested the PAR1 and MAOB expression in the peri-infarct area,the results showed that MAOB was continuously highly expressed on day 3,7 and 10,and PAR1 was highly expressed on day7 after cerebral ischemia.It is suggested that astrocytes may synthesis more GABA and the Best1 can release more GABA in the peri-infarct area after cerebral ischemia.