NNOS From NSCs Regulates Neurogenesis Via HDAC2

Now many reports have established that nNOS-derived NO exerts a negativeregulator on the adult neurogenesis in normal brains. We also in vitro demonstratedthat neurons-derived nNOS negatively regulates neural stem cells (NSCs)proliferation and neuronal differentiation. Besides, we observed that repression ofnNOS from NSCs decreases progenitor proliferation, reduces neuronal differentiationand increases cell apoptosis, thus proving nNOS from NSCs as a positive regulator.However, there is no sufficient data to conclude that nNOS from NSCs is necessaryfor neuronal differentiation, and we do not know whether the reduction of neuronaldifferentiation is only relevant to neuronal survival.It has become apparent that cellular gene transcription plays a critical role inNSC differentiation, and a decrease of histone acetylation by histone deacetylases(HDACs) yields a condensed chromatin structure and thus transcriptional silencing.Among all HDACs, HDAC1and HDAC2are clearly implicated in NSCdifferentiation. Besides, as NSCs commit to the neuronal lineage, expression ofHDAC2is upregulated but that of HDAC1is undetectable. So we mainly study therole of HDAC2in neuronal differentiation, and observe whether HDAC2contributesto nNOS inhibition-mediated depression of neurogenesis.Here, we investigated the following two questions:(1) whether nNOS fromNSCs is necessary for neuronal differentiation;(2) whether HDAC2contributes tonNOS inhibition-mediated depression of neurogenesis. Chapter1. nNOS from NSCs is necessary for neuronal differentiationTo study the role of nNOS from NSCs in neuronal differentiation, we designed thefollowing experiments. First, we cultured embryonic neurospheres from nNOS-/-andWT mice, seeded the single cell suspensions to form monolayer-cultured NSCs, andthen differentiated them for four days. Immunofluorescent results showed thatalthough the knockout of nNOS has much weak effect on the percentage of astrocytes(GFAP+), it substantially reduces neuronal differentiation (β-Ⅲ-tubulin+)(nNOS+/+20.39±1.20%，nNOS-/-11.21±2.69%), indicating that gene deletion of nNOS fromNSCs inhibits neuronal differentiation.The reduction of neuronal differentiation could be due to some combination ofattenuated neuronal survival, decreased proliferation of progenitors, and/or inhibitionof instructive differentiation from NSCs to a neuronal lineage. Now that we hadproved the negative effect of nNOS gene deletion on NSC proliferation, to determinewhether either of the remaining two mechanisms has a role in decreased neuronaldifferentiation when nNOS from NSCs is knocked out, we treated NSCs with nNOSinhibitor L-VNIO (100μM) during the later two days of differentiation. Under theconditions, L-VNIO did not inhibit the proliferation of progenitors, but remarkablyreduced the percentage of neurons at day4after differentiation (Control20.83±0.31%，L-VNIO6.23±0.40%), and significantly increased the apoptotic ratio of cells(PI+)(Control3.48±0.07%，L-VNIO4.91±0.15%). If all cells that die are neurons,that is, L-VNIO notably induces the apoptosis of neurons, and L-VNIO still lowersthe conversion rate of progenitors to neurons, so we could say that L-VNIO inhibitsprogenitors to adopt a neuronal fate. To address this question, we used a mathematicaldescription of neurogenesis: n_j+1=n_j+β_j(N_j-n_j)-δ_jnn_j; PI+_j+1-PI+_j=δ_jN_j, and weobserved that L-VNIO leads to the notably lower value of β_j(_j=4) as δ_jnn_j=δ_jN_j(Control0.426±0.05，L-VNIO0.136±0.06), indicating that repression of nNOS from NSCs negatively regulates neuronal fate during differentiation.To explain the effect of nNOS from NSCs on neuronal differentiation by variousmeans, we treated progenitors with50mM KCl for24h during the first day ofdifferentiation to increase nNOS expression. Results showed that KCl increased thepercentage of neurons, and the effect was abolished when nNOS was knocked out. Inaddition, KCl induced cellular apoptosis and a decrease of total cell number, so weobserved the differences in the number of neurons between control and KCl, and theresult of neuronal number was consistent with that of neuronal percentage. So, nNOSup-regulation promotes neuronal differentiation.Chapter2. nNOS regulates NSCs proliferation and neuronal differentiation viaHDAC2nNOS from NSCs has been proved to be necessary for progenitor proliferation andneuronal differentiation, and HDAC2probably contributes to the process, so we firstobserved that whether nNOS regulated the HDAC2expression and activity.Embryonic neurospheres treated with100μM L-VNIO for24hours exhibited asignificantly decreased HDAC2level. To further confirm the role of nNOS repressionin the regulation of HDAC2, we cultured embryonic NSCs from null mutant micelacking nNOS gene. Consistent with treating with L-VNIO, nNOS gene deletionresulted in a marked decrease in HDAC2expression. In addition,100μM L-VNIOadded into neurospheres for3h had no effect on HDAC2activity. To examine theeffects of nNOS inhibition on HDAC2during differentiation, we treated adherentcells with100μM L-VNIO during the later two days of NSC differentiation. Theresults of Western Blot analysis and HDAC2activity assay showed that L-VNIO didnot inhibit HDAC2expression during differentiation, but significantly increasedHDAC2activity. So, nNOS repression induces the down regulation of HDAC2 expression and has no effect on HDAC2activity in neurospheres, however, nNOSinhibition enhances HDAC2activity and do not have a notable effect on HDAC2expression during NSC differentiation.Now that nNOS affects HDAC2, so do nNOS regulate NSC proliferation andneuronal differentiation via HDAC2? To resolve the question, we first observed therole of HDAC2in neurogenesis. On one hand, we infected single-cell suspensions ofneural stem cells with HDAC2shRNA lentiviral particles (LV-HDAC2shRNA)recommended for the inhibition of HDAC2expression and Control shRNA lentiviralparticles (LV-Control shRNA), and four days later, we dissociated neurospheres tosingle-cell suspensions and counted the number of cells on a hemocytometer. Theresult of cell counting showed that NSCs infected with LV-HDAC2shRNA displayedmarkedly reduced rate of proliferation. In addition, NSCs infected with lentiviralparticles were plated on PORN/LAM-coated coverslips at2×104cells/cm2andcultured as a monolayer for24hours, and then10μM BrdU was added to label thedividing cells for2hours. As expected, inhibition of HDAC2expression decreasedthe percentage of BrdU incorporation (LV-Control shRNA41.67±1.44%, LV-HDAC2shRNA33.27±1.74%). After four days of differentiation, the immunofluorescence ofβ-Ⅲ-Tubulin exhibited the positive effect of HDAC2interference on neuronaldifferentiation, indicating that HDAC2negatively regulates neuronal differentiation.On the other hand, we infected NSCs with HDAC2over expression adeno-associatedvirus (AD-HDAC2) and AD-Null, and the results were that HDAC2over expressiondid not apparently affect neurospheres formation quantified by cell counting, butnotably inhibited neuronal differentiation. The findings collectively indicate thatHDAC2is necessary for NSC proliferation, and acts as a negative regulator ondifferentiation from neural stem cells to neurons.Finally, to explore whether HDAC2contributes to the effects of nNOS from embryonic and adult NSCs on neurogenesis, we observed that whether HDAC2overexpression could restore L-VNIO-induced decrease of neurospheres formation by cellcounting, and whether HDAC2interference could reverse L-VNIO-induced inhibitionof neuronal differentiation by immunofluorescence. Thus, we conclude that nNOSrepression decreases NSC proliferation and neuronal differentiation via the downregulation of HDAC2expression in NSCs and the enhancement of HDAC2activityduring differentiation respectively.