| Transcription factors are important DNA binding protein and play a very important role in cell reprogramming together with chromatin regulators and epigenetic regulators, controlling the cellular fate such as differentiation, aging and survival-death. These progresses are tightly related to the cytothesis and tumorigenesis. Because of the importance of transcription factors in cell life activities, there is importantly practical significance and value in this field research.Peroxisome proliferator-activated receptor-y (PPARy) is a kind of ligand-activated nuclear transcription factor, and belongs to the member of the Peroxisome proliferator-activated receptors (PPARs). The other two subunits are PPARa and PPARβ/δ. PPARy was initially found in the fat cells, mainly involved in the regulation of sugar and lipid metabolism, enhanced insulin sensitivity, maintaining the body metabolic balance. Recently, the researchers find PPARγalso distributes in the spleen of rat, human macrophage and the bone marrow precursor cells, participating in sup-pressing inflammatory cytokines. For the past few years, the scientists discover PPARy even expresses in embryonic brain and neural stem cells, playing important roles in proliferation and differentiation of neural stem cells. More interestingly, the researchers find the expression level of PPARy in adulthood brain was low, however, the expression increased obviously after central nervous system damage. The phenomenon suggests that PPARy maybe play important roles in nervous system development and growth, in addition, the repair of damage. There are a large number of studies suggest that PPARy participates in promoting neural stem cells and neural progenitor cells proliferation, differentiation, migration and neurite outgrowth. In fact, recent studies have shown that PPARy has remarkable neuroprotective effect in spinal cord injury, cerebral ischaemia and neurodegenerative diseases such as alzheimer’s disease, Parkinson’s and Huntington’s disease.The mechanisms of the neuroprotective effect of PPARy include correcting metabolic disorders by regulating the activity of TNF-a (tumor necrosis factor), IL-6(interleukin-6), IDE (insulin-degrading enzyme) and so on to improve insulin sensitivity to increase glucose uptake and reduce lipolysis, suppressing oxidative stress reaction by suppressing the activity of NADPH(nicotinamide adenine dinucleotide phosphate) and promoting the activity of catalase and SOD(superoxide dismutase) that had the role of antioxidation, inflammatory response by suppression activity of NF-B (nuclear factor-kappa B), COX-2(cyclooxygenase-2) and iNOS (inducible nitric oxide synthase) and apoptosis by promoting the PI-3K/PDK-1/Akt pathway to phosphorylate Bad, a apoptosis-promoting protein, to keep the mitochondrial membrane potential stable. In addition, studies have shown that PPARy agonist could promote neural precursor cells proliferation in spinal cord injury model, but the detail of the mechanism is still unknown.The scientists not only research the molecule involved in PPARy pathway signaling, but also study the origin of PPARy. Because of the low level expression of PPARy in neurons, they always consider the PPARy having neuroprotective role comes from microglia. But some studies find PPARy in neurons also has neuroprotective effect, neuron-specific PPARy knock-out-mice caused these mice to experience significantly more brain damage and oxidative stress in response to middle cerebral artery occlusion. However, the mechanism is not clearly.Recent studies shows that PPARy not only has neuroprotective function, but also participates in the neural regulation. PPARy stimulation inhibits physiological responses to psychological stress by reducing the level of HPA axis and sympathetic nervous system. PPARy in hypothalamus has role in regulating food intake behavior and energy homeostasis.Based on the current status, we are not only committed to exploring the molecular mechanism of PPARy in the neuroprotective effect, but also try to find out new potential PPARy functions.The molecular mechanism research of PPARy neuro-protective effects mainly concentrates on finding the genes that promote neuron growth,proliferation,differentiation, growth, neurogenesis and have direct relationship to PPARy. In addition, PPARy new potential features are mainly focused on the genes closely associated with neuronal signal transmission.First of all, we use chip technology for high-throughput screening these genes which are mediated by PPARy. Two adult male C57BL/6J mice are taken and received an intraperitoneal injection of rosiglitazone in10%DMSO at12mg/kg body weight or the same volume saline solution containing10%DMSO for12h, instead, respectively. Meanwhile, conditional neuron-specific PPARy knock-out-mice (N-PPARy-KO) are also taken in this experiment but have no any treatment. Their genotype are PPARγfl/flCre-/-and PPARγfl/flCre+/-, respectively. The differences of gene expression are analyzed by chip technology as well as real-time quantitative PCR, to verify the results of chip technology. The target genes related to promoting neuron growth, proliferation, differentiation, growth, neurogenesis and modulating neuronal signal transmission, were chosen according to the results of SAS system analysis of SBC (Shanghai Biotechnology Corporation). They are IRS-4(insulin substrate-4) which is closely related to cell growth and proliferation,5-HTr2C (serotonin receptor2C) and GABRA6(y-aminobutyric acid receptor subunit a6) which are important neurotransmitter receptors and play important roles in mediating neuron information transfer.Next, we verify the relationship of the expression level changes in IRS-4,5-HTr2C and GABRA6with PPARy activation in primary cortical neuronal culture from E17-18Sprague Dawley rat. In vitro, cortical neurons from E17-18Sprague Dawley rats are exposed to10μM PPARγ agonist rosiglitazone after1week of plating, for Oh,1h,2h,4h,8h or24h, respectively. The expression of IRS-4mRNA,5-HTr2C mRNA and GABRA6mRNA are analyzed by RT-PCR (reverse transcriptase PCR) or RT-QPCR (real-time quantitative PCR). RT-PCR result shows that these is a rising trend in GABRA6mRNA expression at1h and2h, but not significant.The expression of GABRA6mRNA turns back to baseline at4h and8h, and then up-regulates to2.6times of the control group at24h(P=0.037). RT-QPCR results show significant increase in mRNA levels for IRS-4in4h group (4h group to Oh group ratio=1.47, P=0.007) and24h group (24h group to Oh group ratio=2, P=0.019) relative to (3-actin mRNA. However, there are no significant differences in5-HTr2C mRNA levels among each group. The results show that PPARy agonist could suppress GABRA6mRNA expression, while promote IRS-4mRNA expression. But we still have no sufficient evidence to rule out the possibility of the effects of PPARy on5-HTr2C.Then, we verify the association of the expression level change in IRS-4and5-HTr2C with PPARy activation in vivo. In addition, the study also brings in another three genes. They are Syt2(synaptotagmin2), a Ca2+sensor for fast neurotransmitter release in forebrain synapses, Foxc2(forkhead box C2), an important transcript factor that mainly regulates the genes related energy homeostasis and angiogenesis and its mutation very closely to obesity, congenital malformation and tumorigenesis, Check1(checkpoint kinase1) an important cell cycle checkpoints, that maintaining genome stability, has important role in anti-apoptosis and its mutation has close relationship to tumorigenesis. Firstly, adult male C57BL/6J mice are divided into placebo group and agonist group randomly. There are3to5mice in placebo group and3to4mice in agonist group. Agonist group receive an intraperitoneal injection of rosiglitazone in10%DMSO at12mg/kg body weight, while placebo group receive the same volume saline solution containing10%DMSO for12h, instead. Total RNA and protein are isolated from cortex and hippocampus and mRNA expression levels of above genes are measured by RT-QPCR, meanwhile protein levels of5-HTr2C are measured by Western blots method. RT-QPCR results show the expression of IRS-4,5-HTr2C and Checkl mRNA significantly increase by40%,63%and28%in cortex in agonist group, respectively. And Sty2mRNA expression has a51.5%up-regulation in hippocampus in agonist group, while there is no significant difference in cortex. Foxc2mRNA expression in cortex and hippocampus significantly reduces by41%and51.5%, respectively. Western blots results show a25.8%up-regulation in protein level for5-HTr2C in cortex in agonist group, however, we do not observe significant change in hippocampus. Because of restriction to IRS-4antibody source, IRS-4antibody is often humanized antibody, while there is almost no murine antibody in the market. So we could not obtain the Western blots data in cortex and hippocampus tissue in this experiment. Foxc2and Syt2further verification and analysis are still under the way, the protein levels have not yet been detected. The experiment in vivo show that active PPARy maybe promotes the expression of IRS-4,5-HTr2C, Chekl in cortex and Syt2in hippocampus, while inhibites the expression of Foxc2in cortex and hippocampus.Finally, we also use the conditional neuron-specific PPARy knock-out-mice to verify the effects of neuronal PPARy deficiency on the expression of IRS-4,5-HTr2c, Foxc2, Syt2and Check1. The genotyping for determining Nestin-Cre transgenic, PPARy-LoxP and BKO (brain PPARy knockout) mice is performed by PCR method. The expression of recombinant PPARy in BKO mice is tested with RT-PCR. The expression of PPARy in various tissues including cortex, hippocampus, live, muscle and spend is detected with RT-QPCR. Transgenic mice are divided into control group and knockout group, according to their genotype. Expression of total RNA of above genes isolated from cortex and hippocampus are measured by RT-QPCR, meanwhile protein levels of5-HTr2C isolated from cortex and hippocampus aremeasured by Western blots method.Genotyping result shows control mice only have bands at230bp, while knockout mice have bands at230bp and100bp, it means that genotype of control mice is PPARγfl/flCre-/-, while knockout mice is PPARγfl/flCre+/-. RT-QPCR result shows all tissues from control and knockout mice only have bands at700bp, other than cortex and hippocampus from knockout mice that have bands at300bp and400bp. As we known, a full PPARy band is at700bp and two recombinant PPARy bands are at400bp and300bp. The300bp band has the exact sequence predicted from the deletion of the loxP-flanked region. The400bp product is identical to the300bp product, except that an additional100bp of intronic sequence from the intron three of PPARy is spliced into the transcript. So it means that recombinant PPARy specially expresses in knockout mice brain tissues. RT-QPCR result shows significant decrease in mRNA level for PPARγ in cortex and hippocampus in knockout mice (knockout group to control group ratio=0.01, P<0.001). The PPARγ expression in knockout mice brain tissues is too low to detect. Even though, there are significant difference in muscle (knockout group to control group ratio=0.70, P=0.05) and spleen (knockout group to control group ratio=1.6, P=0.01), this change could be compared to brain tissue in knockout mice. However, there is no significant difference in live between control mice and knockout mice. These results demonstrate that we gain the neuron-specific PPARγ knock-out-mice successfully.RT-QPCR results show in cortex these is significant decrease in mRNA level for IRS-4(knockout group to control group ratio=0.86, P=0.04),5-HTr2C (knockout group to control group ratio=0.55, P=0.037) and Chek1mRNA (knockout group to control group ratio=0.76, P=0.001), while there was no significant difference in Foxc2mRNA、Syt2mRNA. In hippocampus, these is significant increase in mRNA level for Foxc2mRNA (knockout group to control group ratio=3.68, P=0.044) and Chek1mRNA (knockout group to control group ratio=1.22, P=0.001), and decrease in Syt2mRNA (knockout group to control group ratio=0.67, P=0.023), while there was no significant difference in IRS-4and5-HTr2C. We did not find significant difference for5-HTr2C protein in cortex or hippocampus between control mice and knockout mice. These results show PPARγ agonist promotes the expression of IRS-4,5-HTr2C, Chek1in cortex and Syt2in hippocampus, while suppresses the expression of Foxc2in hippocampus by PPARγ-dependent pathway.In order to clarify whether PPARy directly modulates the expression of IRS-4,5-HTr2C, Foxc2, Syt2and Check1, we divide six to ten adult BKO mice into placebo group and agonist group.Each group have three to five mice, agonist group receive an intraperitoneal injection of rosiglitazone in10%DMSO at12mg/kg body weight, while placebo group receive the same volume saline solution containing10%DMSO for12h, instead. Total RNA and protein are isolated from cortex and hippocampus and mRNA expression levels of above genes are measured by RT-QPCR. RT-QPCR results show that in cortex these is significant increase in mRNA level for IRS-4(knockout group to control group ratio=1.40, P=0.016) and decrease in Foxc2mRNA (knockout group to control group ratio=0.675, P=0.013) and Syt2mRNA (knockout group to control group ratio=0.39, P<0.001), while there was no significant difference in5-HTr2C and Checkl. In hippocampus, these is significant decrease in mRNA level for5-HTr2C (knockout group to control group ratio=0.72, P=0.014) and Foxc2(knockout group to control group ratio=0.20, P<0.001), while there was no significant difference in IRS-4, Syt2and Checkl. The experiment demonstrates PPARy agonist promotes5-HTr2C mRNA, Chekl mRNA in cortex and Syt2mRNA in hippocampus by PPARy-dependent pathway completely, while mediates the expression of IRS-4in cortex and Foxc2in hippocampus by PPARy-dependent pathway partially.In conclusion, PPARy has effects on the expression of GABRA6, IRS-4,5-HTr2C, Foxc2, Syt2and Checkl. PPARy reduces the expression of GABRA6and Foxc2mRNA, however, promoting the expression of IRS-4,5-HTr2C and Checkl in cortex, in addition, Syt2in hippocampus. The effects of PPARy on candidate genes maybe have the characteristics of tissue-selectivity. Most importantly, PPARy promotes5-HTr2C mRNA, Chekl mRNA in cortex and Syt2mRNA in hippocampus by PPARy-dependent completely, while mediates the expression of IRS-4in cortex and Foxc2in hippocampus by PPARy-dependent partially. |
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