谷氨酸对神经元PPARgamma的调节作用及机制研究

Glutamate (Glu) neurotoxicity is invloved in a variety of diseases of nerve system, but the underlying mechanism of Glu neurotoxicity is far from clear. Our previous studies have shown that 1) peroxisome proliferator-activated receptor γ (PPARγ) is important in self-defense of the brain against injury, and especially PPARγ in neurons plays an essential protective function; 2) Glu decreases PPARγ protein level but increased PPARγ phosphorylation in primary cortical neurons. Accordingly, the proposed study is going to focus on the effects of glutamate on the regulation of neuronal PPARγ and the underlying mechanism. We hope to understand the neurotoxicity of Glu in the view of negative regulation of PPARγ. ..In primary cortical neurons, the following experiments will be carried out. 1) We investigate the neuroprotection of PPARγ in Glu-induced neuronal injury by the addition of PPARγ ligand alone or in combination with PPARγ gene knockout or PPARγ antagonist. 2) Glu-induced reduction in PPARγ activities will be detected by the observation of PPARγ-DNA binding activity, translocation, target genes expression, and transcriptional activity, respectively. 3) To determine the decrease in PPARγ expression is caused by ubiquitin-proteasome system-mediated PPARγ degradation, western blotting and immunoprecipitation assay will be used to detect PPARγ protein expression, turnover, and ubiquitin-PPARγ conjugation after inhibiton of protein synthesis by cycloheximide with or without proteasome inhibitor. The involvement of ERK1/2 signaling in Glu-induced PPARγ phosphorylation will be determined firstly by screening through a variety of candidate protein kinases, then by measurement of total ERK1/2, phosphor-ERK1/2 (active form), and phospho-PPARγ in neurons treated with Glu alone and in combination with ERK1/2 activation inhibitor, or with phosphorylation-deficient mutant of PPARγ (S112A). In this section, total PPARγ, ubiquitin-PPARγ conjugation, and PPARγ turnover will also be detected to show whether ERK1/2-mediated PPARγ phosphorylation will influence the decay of PPARγ by increasing the targeting of PPARγ to the proteasome system. 4) The effect of antioxidant on Glu-induced PPARγ regulation or the reconfirmation of above PPARγ changes in Glu-treated immature primary cortical neurons (the excellent model for oxidative glutamate toxicity) are both applied to determine whether Glu-regulated PPARγ is mediated by Glu excitotoxicity or oxidative glutamate toxicity. In addition, to confirm the above in vitro study, an in vivo model of glutamate toxic injury will be established by injection of glutamate to the cerebral cortex of rats. The effects of Glu on PPARγ of isolated cortical neurons will be observed...The proposed study is believed to provide insight into the understanding of Glu-induced neurotoxicity and the potential therapeutic implications based on PPARγ regulation in the brain.

谷氨酸毒性作用参与多种神经系统疾病，但其作用机制尚不明确。申请人已发表的成果及前期研究表明：1.PPARγ是脑组织重要的自我保护机制，特别是神经元PPARγ在其中具有重要作用；2.谷氨酸降低神经元PPARγ表达、增加其磷酸化。据此，本研究将对谷氨酸调节PPARγ及其机制进行探索，以期从负性调节PPARγ的角度揭示谷氨酸的损伤机制。主要研究内容包括：利用神经元谷氨酸损伤模型，1）证明神经元表达的PPARγ可抑制谷氨酸损伤；2）谷氨酸负性调节PPARγ表达和活性；3）谷氨酸通过ERK1/2诱导PPARγ磷酸化而使其失活，通过泛素蛋白酶体系统降解PPARγ而使其表达下调，且磷酸化促进其降解；4）明确谷氨酸作用的性质（氧化毒性亦或兴奋毒性）。在此基础上，构建谷氨酸脑内损伤模型，进一步体内验证谷氨酸对神经元PPARγ的调节作用。本研究将为探索谷氨酸损伤机制、建立基于PPARγ的神经保护策略奠定基础。

谷氨酸毒性作用参与多种神经系统疾病，但其作用机制尚不明确。转录因子PPARγ是脑组织重要的自我保护机制，特别是神经元PPARγ在其中具有重要作用。前期我们发现谷氨酸降低神经元PPARγ表达、增加其磷酸化，但具体机制不清。据此，本研究对谷氨酸调节PPARγ及其机制进行探索，并确定谷氨酸作用的性质。本研究明确了ERK1/2信号通路和泛素-蛋白酶体系统分别参与了谷氨酸诱导的PPARγ磷酸化和降解作用；证明了ERK1/2信号通路介导的PPARγ磷酸化对泛素-蛋白酶体系统介导PPARγ降解的促进作用；确定了谷氨酸对PPARγ上述调节作用的性质为谷氨酸氧化毒性作用。我们的研究为谷氨酸损伤机制开辟了新的理论系统、为在神经变性性疾病中建立基于PPARγ的神经保护策略提供新的线索和依据。