自噬-Sirt3调控轴维持造血干细胞年轻态的作用和机制研究

Autophagy suppresses mitochondrial metabolism to preserve hematopoietic stem cells (HSC) in mice. However, the mechanism by which autophagy regulates hematopoietic aging, in particular in humans, has largely been unexplored. To address this issue, we set to test autophagic activity in human blood samples, and observed that reduction of autophagy in both hematopoietic cells and their stem cells is associated with aged hematopoiesis in human population. Next, using a genetically-modified mouse model that carries atg7 deletion in hematopoietic system, we found that autophagy delays hematopoietic aging by activating the downstream expression of Sirt3, a key mitochondrial protein capable of rejuvenating blood. Furthermore, we found that forced expression of Sirt3 in HSC-enriched cells prevents accelerated hematopoietic aging from autophagy defect. These preliminary data support an autophagy-Sirt3 axis in decelerating hematopoietic stem cell aging. However, the mechanism underlying the direct connection between autophagy and Sirt3 remains unknown. Given the fact that autophagy is essentially a protein degrading process, we hypotheses that autophagy deterioration leads to accumulation of its target protein that inhibits the transcription of Sirt3, whereas activation of autophagy degrades the target protein, thereby causing activation of Sirt3 in transcription. In the proposed project, we intend to further determine the role of autophagy-Sirt3 axis in decelerating hematopoietic aging. More importantly, we plan to identify the putative protein that inhibits Sirt3 transcription and is targeted by autophagy. We will use transcripteomic, proteomic means and analysize ChIP-seq database to address this open question in a set of mouse models with deletion of different autophagy-essential genes in hematopoietic system. The results from mouse system will be validated in human blood samples. Accomplishment of this study is expected to advance our understanding in blood aging, and in a long-run, will improve clinical therapy on hematopoietic diseases associated with aging.

造血干细胞衰老与临床上多种恶性血液病相关。我们和同行分别报道了自噬在造血干细胞抗衰老中的作用，但是自噬抗衰老的分子机制尚不明了。我的预实验结果显示：小鼠造血干细胞的自噬缺陷，可导致抗衰老蛋白Sirt3表达特异性下降；高龄人群中造血干细胞自噬水平显著降低且伴随发生Sirt3表达下降；而且，过表达Sirt3能够挽救自噬缺陷导致的衰老。提示：自噬可能以自噬-Sirt3轴方式对抗造血干细胞衰老。自噬是细胞内的蛋白降解机制，其上调sirt3基因表达是间接调控，可能通过自噬降解负调控Sirt3表达的蛋白因子而实现。本项目将以自噬基因敲除小鼠为模型，运用转录组、蛋白组及数据库联合分析等手段，确证自噬-Sirt3轴的抗衰老作用，并鉴定自噬直接降解的关键靶蛋白分子，以阐明自噬调控小鼠造血干细胞衰老的分子机制，并对关键结果在人血液样本中验证，为未来临床抗血液衰老疗法提供理论依据。

现代社会老龄化问题日益凸显，血液衰老引发众多血液类疾病。血液衰老的源头在于造血干细胞的衰老，通过干预和延缓造血干细胞衰老，将有效预防和治疗衰老性血液病。自噬作为细胞中降解受损、衰老的细胞器和错误折叠的蛋白分子的一种保护性机制，在维持造血干细胞年轻态中发挥至关重要的作用。所以，探明自噬在造血干细胞中发挥抗衰老作用的分子机制，有望为延缓造血干细胞衰老提供可行方案。.本项目运用血液细胞特异性缺失自噬基因的小鼠模型观察到造血干细胞中的自噬发挥维持细胞年轻态的作用。Atg7f/f;Vav-iCre青年小鼠的造血干细胞呈现与90周龄的老龄小鼠造血干细胞相似的衰老状态。进一步观察到抗衰老基因Sirt3在自噬缺失的造血干细胞中呈现年龄依赖的下降，且Sirt3下降发生于转录阶段。利用自噬激活剂、抑制剂以及饥饿方式干预野生型小鼠的造血干细胞的自噬水平，可观察到自噬选择性调控去乙酰化家族成员Sirt3的转录，但在自噬缺基因缺失的造血干细胞中无法观察到类似的变化，提示Sirt3表达水平的变化依赖自噬。在人体造血干细胞中进一步验证了自噬与抗衰老的去乙酰化家族成员Sirt3两者存在协同延缓造血干细胞衰老的作用。从而证明了造血干细胞中，不同的抗衰老途径之间存在功能上的联系，这一发现深化了我们对造血干细胞的衰老和抗衰老机制的理解。