【作者】 余璐；

【导师】 杨守京；

【作者基本信息】 第四军医大学 ， 病理学与病理生理学， 2010， 博士

【摘要】 研究背景无论是发育过程中还是成年后的视皮质神经元,其结构、功能以及突触数量可随环境发生变化,具有经验依赖的可塑性(Experience- dependent Plasticity)。新近研究发现,神经元兴奋依赖性(Activity-dependent)调节和功能的可塑性与神经元能量代谢紧密偶联(Energy metabolic coupling),这被认为是神经元可塑性的核心环节。神经元兴奋性(Neuronal activity)高度依赖于线粒体氧化磷酸化(Oxdatve phosphorylation system, OXPHOS)产生的三磷酸腺苷(Adenosine triphosphate, ATP)。线粒体功能障碍(Mitochodrial dysfunction)可能是视觉剥夺导致视皮质神经元功能抑制的关键因素。但是,迄今对视皮质神经元兴奋能量偶联的信号机制尚不明确。核呼吸因子(Nuclear respiratory factors, NRFs)是调节线粒体氧化磷酸化和线粒体再生的重要转录因子,通过直接转录调控细胞核DNA(nDNA)编码的呼吸酶链亚单位基因,并通过线粒体转录因子A和B(mtTFA, mtTFB)间接调控线粒体DNA(mtDNA)编码的呼吸酶链亚单位基因,行使其对线粒体呼吸转录调节。我们以往研究发现,核呼吸因子1 (NRF-1)和2 (NRF-2)参与了视皮质神经元线粒体能量代谢调控的偶联过程,其转录和蛋白表达水平与神经元兴奋性呈正相关,且与细胞色素氧化酶(Cytochrome c oxidase, COX)活性是一致的。另一方面,PGC-1α,即过氧化物酶体增生物激活受体γ(Peroxisome proliferators-activated receptor gamma,PPARγ)共激活因子1α(PPARγcoactivator-1-α, PGC-1α),作为重要的转录共激活因子,是线粒体生物合成过程的关键分子。有研究表明,PGC-1α对NRFs的基因表达具有强大的诱导作用,可通过NRFs调节OXPHOS成分的协同表达。因此,PGC-1α能促进NRFs的基因转录,从而协调机体在不同能量供需状态时的线粒体呼吸链功能。然而,视皮质神经元兴奋能量偶联过程中PGC-1α和NRFs的信号调控机制亟待深入。来自动物试验以及临床观察的证据均表明,一磷酸腺苷激活的蛋白激酶(AMP-activated protein kinase, AMPK)被认为是神经系统中调节能量平衡微环境的关键感应及效应分子,但AMPK在视皮质神经元中的作用尚未报道。AMPK是调控线粒体功能,调节细胞能量代谢的关键分子,与PGC-1α关系密切;另有证据显示,AMPK可能是NRFs信号传导通路中重要的上游激活分子。因此,AMPK信号通路可能是视皮质神经元兴奋能量偶联的关键信号途径。本课题拟通过对视神经元兴奋依赖性体内和体外动物模型的研究,采用细胞及分子生物学方法,探讨AMPK,PGC-1α和NRFs信号在视皮质神经元兴奋能量偶联中的作用机制。旨在为阐明AMPK调控下游靶分子参与视觉系统可塑性的信号转导机制提供一些有意义的实验依据,以期通过AMPK-NRFs通路为靶点进行基因治疗从而调节视皮质神经元细胞功能,为弱视及其它神经发育功能不良(全)性疾病的防治提供思路。研究目的本研究的目标是通过在体和离体实验研究,明确内源性AMPK,PGC-1α和NRF-1在视皮质神经元兴奋依赖性转录调节与能量偶联中的作用机制。包括:1.观察视皮质神经元兴奋与线粒体功能之间的关系,明确视皮质神经元的兴奋能量偶联,分析AMPK, PGC-1α和NRF-1在偶联中的作用。2.证明视觉剥夺引起的视皮质神经元损害是由于内源性AMPK,PGC-1α和NRFs系统活性下降进而导致线粒体氧化磷酸化功能下降所致。3.通过在体实验研究,明确能否通过调节AMPK的活性保护视觉剥夺后线粒体的功能,改善视皮质神经元的能量代谢状态。实验结果1.体外培养大鼠视皮质神经元,采用KCl诱导神经元去极化兴奋,可兴奋依赖性上调神经元PGC-1α,NRF-1及mtTFA的mRNA和蛋白表达水平;并且,同时增强线粒体产生ATP的能力。2.视皮质神经元兴奋显著提高细胞内AMPK及其下游激酶ACC的磷酸化水平,提示神经元兴奋可以激活AMPK激酶活性。3.另一方面,AMPK抑制剂Compound C可以有效阻断视皮质神经元兴奋依赖性的PGC-1α,NRF-1,mtTFA的表达水平以及ATP含量。4.与之对应,AMPK的激动剂,白藜芦醇(Resveratrol)或AICAR,可显著增加视皮质神经元内ATP水平,并上调PGC-1α,NRF-1,mtTFA的表达水平。该作用可被Compound C所阻断。5.采用大鼠单眼视觉剥夺(Monocular deprivation, MD)在体模型研究发现,视觉剥夺显著损害对应的视皮质神经元:导致神经元线粒体肿胀,数量减少;神经元内ATP水平显著降低。6.定量检测发现,视觉剥夺显著抑制了对应的视皮质神经元AMPK的激酶活性和磷酸化水平;PGC-1α和NRF-1的表达水平也随之降低。7.更为重要的是,在体给予Resveratrol治疗显著增加神经元线粒体数量,改善线粒体形态;增加神经元内ATP水平;激活AMPK活性,上调PGC-1α和NRF-1的表达,从而可以有效抑制视觉剥夺导致的视皮质神经元损害。结论1.视皮质神经元兴奋与能量调节紧密偶联,而AMPK是兴奋能量偶联的关键分子。2.AMPK介导视皮质神经元PGC-1α,NRF-1及其下游的mtTFA的兴奋依赖性转录调节。3.AMPK对PGC-1α,NRF-1的调控在神经元能量平衡中发挥关键作用,该信号通路功能失调可能是视觉剥夺导致的视皮质兴奋能量偶联障碍的重要分子机制之一。4.本研究首次发现,保护AMPK信号途径可以有效抑制视觉剥夺导致的视皮质神经元线粒体损害,改善视皮质神经元的能量应答,为临床防治弱视及其它相关病提供新的治疗靶点。更多还原

【Abstract】 BackgroundIn development and adult visual cortical neurons, the mitochondrial number, functional capacity, and gene expression are dynamically regulated in accordance with energy demands, namely experience-dependent energy metabolic plasticity. Recent studies show that neuronal activity and functional plasticity are tightly coupled with energy metabolism (energy metabolic coupling). Approximately 90% of adenosine triphosphate (ATP) generated in the brain is synthesized in mitochondria via oxidative phosphorylation (OXPHOS). Thus, brain critically depends upon mitochondrial function and oxygen supply to support its immense energetic demand. However, the signaling mechanisms that couple neuronal activity to energy metabolism are entirely unknown.Nuclear respiratory factors (NRFs), including NRF-1 and NRF-2, are found to act on a number of nuclear genes required for mitochondrial respiratory function. Our previously studies suggest that both NRF-1 and NRF-2 regulate a number of genes required for mitochondrial energy metabolic coupling. The transcription and protein expression of NRF-1 and NRF-2 in visual cortical neurons are positively correlated with neuronal excitability, and consistent with the cytochrome oxidase (COX) activity. Peroxisome proliferator-activated receptorγcoactivator-1-α(PGC-1α), on the other hand, as inducible coregulators of nuclear receptors in the control of cellular energy metabolic pathways, has been adequately represented as a master regulator of mitochondrial biogenesis. PGC-1αhas been shown to coactivate NRFs, which are associated with increased COX activity. Nevertheless, the functional significance of neuronal activity-dependent regulation of PGC-1αand NRFs expression in visual cortical neurons, the signaling pathways that couple neuronal activity to energy metabolism are entirely unknown.AMP-activated protein kinase (AMPK), a metabolic gauge regulating whole-body energy homeostasis, has been reported to regulate mitochondrial biogenesis by sensing the energy state in the cells. Several lines of evidences show that AMPK is a putative upstream activator of NRFs signal transduction pathway. However, the signaling pathway that couples neuronal activity to mitochondrial energy metabolism, particularly whether AMPK mediates activity-dependent regulation of PGC-1αand NRFs for mitochondrial biogenesis and function, has not been examined in visual cortical neurons.AimThe goal of this study was to test our hypothesis that AMPK might be an important mediator of neuronal activity to stimulate mitochondrial biogenesis and function by regulating PGC-1αand NRF-1 gene expression in visual cortical neurons; if so, to detection the functional consequences of these activity-dependent mitochondrial transcriptional regulation. Our study also aims to provide experimental evidences for the treatment of neuronal mitochondrial dysregulation and other related neurodevelopmental dysfunction disease by modulation of AMPK signaling.Results1. Membrane depolarization with 25 mmol/L KCl significantly increases PGC-1α, NRF-1 and mtTFA mRNA and protein level with increased ATP production in primary visual cortical neurons.2. KCl depolarization rapidly increases AMPK phosphorylation in cultured primary visual cortical neurons.3. AMPK inhibition by Compound C, an AMPK inhibitor, completely represses KCl depolarization-induced up-regulation of PGC-1α, NRF-1 and mtTFA mRNA and AMPK ?phosphorylation as well as cellular ATP content.4. AMPK activation by AICAR or resveratrol also markedly increases PGC-1αand NRF-1 mRNA levels as well as cellular ATP content in neuron cultures. All these effects can be completely blocked by an AMPK inhibitor, Compound C.5. Reduction of neuronal activity by one week of MD significantly decreases AMPK phosphorylation and activity, dramatically down-regulates PGC-1αand NRF-1 expression in deprived primary visual cortex.6. Administration of resveratrol in vivo significantly activates AMPK activity and attenuates the effects of MD on mitochondria by significant increase in PGC-1αand NRF-1 levels mitochondria amount, and coupled respiration.Conclusions1. Neuronal activity has a major impact on AMPK signaling, which mediates activity-dependent regulation of mitochondrial energy metabolism by regulating coactivator PGC-1αand its downstream transcription factor NRF-1 and mtTFA in visual cortical neurons. 2. We demonstrated for the first time that pharmacologic interventions, such as resveratrol, that restore AMPK activity in the functional inactivation visual cortices may be a novel means to rescue mitochondrial dysfunction caused by visual deprivation.3. This study provides additional insight into AMPK pathophysiology in the brain, and highlights the importance of AMPK- PGC-1α- NRF-1 networks in activity-dependent regulation of mitochondrial energy metabolism in neurons, which could potentially lead to novel therapeutic targets for the amelioration of neuronal mitochondrial dysfunction and its related disease.更多还原