雄激素改善衰老过程神经元线粒体的功能障碍及TFAM参与的相关机制研究

Mitochondrial dysfunction is an early event associated with aging and age-related neurodegenerative disorders including Parkinson`s disease (PD) and Alzheimer`s disease (AD). Aged men have a greater prevalence of PD than aged women and testosterone deficiency in men increases the incidence of AD, which suggests that testosterone play roles in neurodegenerative diseases. Based on the lower testosterone levels in aged males and our results that orchiectomy to the adults decreases the mitochondrial membrane potential and the activity of mitochondrial complex I, it is presumed that the enhancement of mesodopaminergic activity and alleviation of oxidative damages in the substantia nigra as well as the amelioration of motor behavioral deficits via androgen supplements to aged males in our previous studies are related to the mitochondrial biogenesis induced by testosterone supplements. Due to the essential roles of mitochondrial transcription factor A (TFAM) in the maintenance of mitochondrial DNA and in mitochondrial biogenesis, we hypothesized that androgen would regulate the expression of transcription factors in TFAM signaling pathway to ameliorate the neuronal mitochondrial dysfunction in aging males. In our preliminary experiment, the decreased mitochondrial membrane potential, the reduced ATP levels and the depressed activities of mitochondrial complex I as well as complex V in aged male rats were found and ameliorated by long term supplements of testosterone propionate. To determine the role of TFAM in the effects of androgen on mitochondrial function, further studies on the findings of our preliminary experiment are necessary by analyzing the effects of androgen supplements on mitochondrial DNA-encoded subunits in aging and the influence of testosterone deficiency upon the expression of transcription factors in TFAM signaling pathway. The projects would provide important clues for androgen to improve the aging and age-related neurodegenerative disorders by promoting TFAM expression.

线粒体功能障碍是衰老和衰老相关神经退行性变的早期事件。基于老年雄性睾酮水平降低及我们近期发现成年雄性睾丸切除降低黑质线粒体膜电位、减弱线粒体复合物I活性的事实，推测我们获得的补充雄激素改善老年雄性运动行为缺陷、增强黑质多巴胺能神经元功能活动、减轻氧化损伤的结果与雄激素促进衰老过程中神经元线粒体的生物发生有关；鉴于转录因子TFAM在线粒体生物发生中的作用，推测雄激素通过调控TFAM信号通路相关因子的表达来改善线粒体的功能。课题组经预实验发现补充雄激素显著提高老年雄性大鼠黑质线粒体膜电位、增加ATP含量、增强线粒体复合物I和V的活性。深入研究这一发现，探讨衰老过程雄激素影响哪些mtDNA编码亚基的表达；探寻雄激素缺乏对TFAM信号通路相关因子表达的影响，判断TFAM在雄激素改善衰老过程神经元线粒体功能障碍中的作用，为通过干预TFAM的表达，改善雄激素缺乏所致衰老及衰老相关神经退行性变提供依据。

男性衰老存在脑线粒体功能减弱和睾酮水平降低，虽有研究表明这两种现象相互关联，但尚不清楚1）在衰老过程补充雄激素是否能够改善老年男性脑的线粒体功能；2）雄激素对老年男性线粒体功能的改善是否与线粒体生物发生的改变、特别是与转录因子TFAM有关；3）雄激素缺乏是否直接引起脑线粒体功能的障碍、降低线粒体的生物发生；4）雄激素对老年男性脑线粒体功能的改善是否还有其它影响线粒体功能稳态的因素参与。本项目通过对老年雄性大(小)鼠的研究发现，在衰老过程补充雄激素降低黑质/海马MDA、线粒体H2O2和线粒体3-NT的水平；增加GSH/GSSG的比值和GSH-PX、CAT和Mn-SOD的酶活性；提高线粒体膜电位、线粒体复合物酶活性及ATP含量，表明衰老过程中补充雄激素能够改善脑的线粒体功能。对补充雄激素的老年实验动物进一步的研究观察到线粒体超微结构的改善；CS酶活性、mtDNA拷贝数和线粒体数量的增高；mtDNA编码亚基ND1、COX1和ATP6表达水平的增加；以及线粒体生物发生诱导因子PGC-1α及其效应因子NRF-1/NRF-2和TFAM表达信号的增强，这暗示衰老过程补充雄激素能够促进脑的线粒体生物发生。对成年雄性小鼠行性腺切除可导致黑质线粒体的功能障碍、降低线粒体的生物发生，雄激素替代则逆转因睾丸切除所致线粒体的功能障碍及线粒体生物发生的减弱；表明雄激素与其存在着一定的调控关系。对细胞培养的研究发现，在SH-SY5Y细胞实施TFAM siRNA后，雄激素仍提高细胞的TFAM mRNA和蛋白水平，虽后者与单纯TFAM siRNA处理组相比蛋白水平未达到显著区别，但提示雄激素可能直接影响了TFAM的转录；利用ChIP-qPCR技术在培养的细胞检测到TFAM基因启动子区存在雄激素受体的结合位点，证实了雄激素对线粒体生物发生的直接调控作用。此外，本项目还发现线粒体动力学、线粒体自噬及Nrf2也参与了雄激素对老年雄性脑线粒体功能的改善作用。本课题为雄激素预防或治疗衰老相关神经退行性疾病提供了实验依据，并为针对雄激素缺乏所致衰老及衰老相关神经退行性变的关键分子的药物开发提供了理论依据。