Regulation Of Tanscription Factor Peroxisome Proliferator Activated Receptor γ's Activity In Neurons By Histone Deacetylase 4

Neurons are the basic structural and functional units of the nervous system. Thus neuronal death is considered as a common pathological feature of various disorders of the nervous system.However, the molecular mechanisms of neuronal death remain vague.Owing to the lack of effective therapies, it is important for us to better understand the molecular mechanisms of neuronal death and find new therapeutical strategies.Among several possible mechanisms, people pay more attention to ones in which transcription factors involved.Usually several pathways are involved spatially and temporally in the pathophysiology of nervous system disorders. Transcription factors are able to regulate several genes simultaneously. This pleiotropic effect could be useful to solve the problems.The nuclear receptor peroxisome proliferator-activated receptor y (PPARy) is a ligand-activated transcription factor. PPARy initially characterized as the master regulator for the development of adipose cells.While it has been identified in various tissues later, including breast, colon, lung, brain and so on. In adipose, activity of PPARγis modulated by protein kinases, such as mitogen activated protein kinase (MAPK), or transcription co-repressors, such as HDAC1 and HDAC3,that ultimately block adipocyte differentiation. Recent studies showed that PPARy signaling plays a crucial role in neuronal survival and many agonists of PPARy implicate a neuroprotective effect.Furthermore, conditional neuron-specific PPARy knock-out mice endured more brain damage in response to middle cerebral artery occlusion. Though PPARy activation is known to reduce damage to brain in distinct models of brain disease, the mechanism how extracellular stress regulates PPARy activity in neurons is presently elusive.Histone deacetylases (HDACs) catalyze the deacetylation of histone proteins at Lys (K) residues, cause compactation of the chromatin. So HDACs often function as a component of the transcriptional repressor complex to silence gene expression. Histone acetylation and deacetylation are involved in the epigenetic remodeling of the gene transcription and a wide range of neurologic and psychiatric disorders.The histone acetylation level decreased in the ischemic brain tissues and neurodegenerative animal models, also HDAC inhibitors could improve neuronal survival in both cultured cells and animal models of brain diseases. These evidences indicate that HDAC may regulate neuronal survival.However, there are kinds of HDAC in the brain, different HDAC plays different roles in cell survival and differentiation.Previous studies found that HDAC4, a member of classⅡa HDACs, could regulate neuronal survival.HDAC4 is predominantly in cytoplasm in neurons, it can shuttle between the nucleus and cytoplasm in response to specific signaling events.Under conditions of activity-dependent neuronal death or neurodegenerative disorder protein ataxin-1-induced neuronal death, HDAC4 translocates to the nucleus, and there it promotes cell death through binding and inhibiting the transcription factor myocyte enhancer factor 2 (MEF2).Although these studies indicate that MEF2 is an important mediator of HDAC4-regulated neuronal death, whether any other transcription factors are targeted by HDAC4 in neuronal death is still unclear.In this study, we cultured cerebral cortical neurons and established a cell culture model of oxidative stress. We use this model to test the neuroprotective role of PPARy under oxidative stress and the possible regulation mechanism of HDAC4.At the first, we established the cell culture model.After 7 days, cortical neurons are divided into two groups:control group and H2O2 group. H2O2 group treated with 36μM H2O2 for 24h. DAPI staining showed that the% of apoptotic cells of control group and H2O2 group were 17.05±2.83% and 55.35±4.51%.Compared with control group, more neurons died in H2O2 group, the difference had statistical significance (P<0.01).Next, we use luciferase reporter gene assay to test the activity of PPARy under oxidative stress. Primary neurons were transiently co-transfected (D6) with PPREx3-TK-luc using calcium phosphate transfection.After 12h, neurons were divided into three groups:control group, without any treatment;H2O2 group, cells were treated with 1/1000 DMSO 30 min before H2O2(36μM);TSA group, pretreated with 1μM TSA (HDAC inhibitor) 30 min before H2O2 (36μM).Cell lysates were analyzed for luciferase according after 4h.% of PPARy reporter activity of them were 100±0.06%,55.89±2.50% and 91.05±7.20%.The difference between H2O2 group and control group had statistical significance (P<0.001).Oxidative stress could decrease PPARy activity significantly, TSA could block this decrease (P<0.01). The results indicated that the decrease of PPARy activity in cortical neurons under oxidative stress might be HDAC dependent.To confirm whether the inhibition of PPARy activity by H2O2 is attributed to decrease of PPARy protein in cells, we determined PPARy protein level in whole cell lysates and nuclear fractions by Western Blotting.We observed PPARγexpression level at 30 min, 1h,2h,4h,8h after H2O2 treatment. Whole cell lysates and nuclear fractions western blotting result showed that H2O2 didn't change the expression level of PPARy significantly.Previous studies confirmed that HDAC4 translocates from cytoplasm to nucleus in cerebellar granule neurons under low concentrations of potassium or upon glutamate treatment and represses transcription factors (MEF2 and CREB).To test if HDAC4 shuttles from cytoplasm to nucleus in cortical neurons upon H2O2 treatment, we determined HDAC4 localization by immunofluorescence and Western Blotting. Cortical neurons were treated with 36μM for different time periods as indicated before.The total level of HDAC4 in whole cell lysates didn't change significantly upon H2O2 treatment. The nuclear and cytosolic fractions immunoblots showed that H2O2 induced decrease of HDAC4 in cytoplasm, while a consistent and significant increase of HDAC4 was shown in nucleus.Immunofluorescence staining also showed increased nucleus density of HDAC4 in neurons.These data indicated that H2O2 treatment resulted in an increase of HDAC4 in nucleus.Because HDAC4 possesses histone deacetylase activity which represses transcription, we tested whether HDAC4 inhibited transcriptional activity of exogenous and endogenous PPARγ. Cultured HEK293 cells were divided into four groups:control group% of PPARy reporter activity was extremely low(3.18±0.41%) because HEK 293 cells didn't express PPARy protein. Overexpreesed PPARy group's activity was 100±7.25%;co-transfected with HDAC4 group was 27.00±1.21%; HDAC4 and its inhibitor TSA treatment group was 103.57±9.53%.HDAC4 can inhibit transcriptional activity of exogenous PPARy significantly (P<0.001).HDAC inhibitor TSA can attenuate HDAC4's blocking by repressing its deacetylase activity(P<0.05).Next we tested whether HDAC4 inhibited PPARy activity in cortical neurons. Similarly, HDAC4 group PPARy activity was 29.30±11.04%, compared with control group(100±0.06%), the decrease had statistical significance (P<0.05).By introducing specific HDAC4 microRNA into neurons, we further test whether the repression of PPARy activity under oxidative stress is due to increased nuclear HDAC4.The knocking down effect of HDAC4 microRNA was proved in HEK 293 cells. Then we transfected neurons with HDAC4 microRNA or control vectors.After H2O2 treatment, we found that control group PPARy activity was 55.41±4.86%, control microRNA group was 63.48±5.25%,HDAC4 microRNA group was 84.87±6.07%.The increased PPARγactivity in HDAC4 microRNA group, compared with control one, had statistical significance (P<0.01).The results confirmed our previous inference.Next, we tested whether HDAC4 interacts with PPARy directly. HEK293 cells expressing Flag tagged HDAC4 were lysed, pulled down with GST-PPARγand Western blotted with HDAC4 specific antibody.The results showed that HDAC4 could bind PPARy in vitro. To identify the region of HDAC4 that interacts with PPARγ, we performed the similar experiments by using N terminal(1-1950 bp) or C terminal(1708-3255 bp) HDAC4 fused to GST. The results showed that N terminus of HDAC4 is sufficient for the interaction with PPARy.To further prove the interaction between the N-terminus of HDAC4 and PPARy is direct, both GST-HDAC4-N'and His-tagged PPARy were performed GST-pull-down assay.The results suggested that indeed the two proteins can associate directly.Then we use Co-IP to test whether HDAC4 could associate with PPARγin HEK 293 cells.The results demonstrated that HDAC4 and PPARy might associated with each other in vivo.Previous studies showed that nuclear localized HDAC4 promotes neuronal apoptosis and this process is dependent on HDAC4's transcription repressing activity. In contrast, overexpressing transcription factor PPARy makes neurons more resistant to stress-induced apoptosis. Thus, the inhibition of PPARy transcription activity by HDAC4 in response to oxidative stress suggests that it may suppress the pro-survival effect of PPARy. We tested whether PPARy may attenuate HDAC4-induced neuronal apoptosis by survival assays.The percentage of dead neurons in PPARy group was 14.42±0.60%, HDAC4 group was 37.31±1.47%;PPARy and HDAC4 co-transfect group was 17.91±0.98%.The results showed that PPARy overexpression can reduce HDAC4 induced apoptosis (P<0.001).To determine the role of this regulation in oxidative stress-induced neuronal apoptosis, we knocked down the expression of HDAC4 by its specific miRNA.We transfected neurons with HDAC4 miRNA or control miRNA. The control group of control miRNA didn't treat anything, the percentage of dead neurons was 10.25±1.39%;the percentage of control miRNA group treated with H2O2 was 55.52±1.19%;the percentage of HDAC4 miRNA control group was 9.31±0.77%; the percentage of HDAC4 miRNA group treated with H2O2 was 33.16±0.79%. The results indicated that knocking down HDAC4 can reduce the number of apoptotic neurons triggered by H2O2.The difference between control miRNA treated with H2O2 and HDAC4 miRNA one had statistical significance (P<0. 001).It is reported that neuronal PPARy deficiency increases susceptibility to brain damage after cerebral ischemia. To check the role of PPARy in oxidative stress-induced neuronal apoptosis, we knocked down PPARy by specific miRNA.The knocking down effect of HDAC4 microRNA was proved in HEK 293 cells.Then we transfected neurons with PPARy miRNA 1,PPARγmiRNA2 and control miRNA.The control group of control miRNA didn't treat anything, the percentage of dead neurons was 13.14±0.75%;the percentage of control miRNA group treated with H2O2 was 22.07±0.80%;the percentage of PPARy miRNA1 H2O2 group was 47.71±2.15%;the percentage of PPARy miRNA2 H2O2 group was 47.00±0.58%.The difference between control miRNA treated with H2O2 and PPARy miRNA1 (P<0.01)/PPARγmiRNA2 (P<0.001)one had statistical significance.The results indicated that knocking down PPARγcan increase the number of apoptotic neurons triggered by H2O2.At last, we tested whether overexpressing PPARy could promote the resistance to oxidative stress.We transfected neurons with Flag-PPARy and control vector. The control group of GFP didn't treat anything, the percentage of dead neurons was 12.37±1.00%; the percentage of GFP group treated with H2O2 was 56.24±0.99%;the percentage of PPARγcontrol group was 10.63±0.59%;the percentage of PPARy H2O2 group was 20.92±2.42%;The difference between GFP group treated with H2O2 and PPARy one had statistical significance (P<0.001).The results indicated that PPARy plays an important protective function in neuronal survival.To sum up, HDAC4 could inhibit the activity of PPARy under oxidative stress, which leads to the ultimate neuronal death;PPARy deficiency increases susceptibility to the damage of oxidative stress;PPARγoverexpression protects cortical neurons from H2O2 and HDAC4-induced cell death.