Role of Histone Deacetylase 9 (HDAC9) in adipose tissue aging: mitochondrial function, oxidative stress and senescence

项目摘要 脂肪组织是衰老细胞积累和无菌炎症的主要仓库。虽然脂肪 衰老中的组织衰老与线粒体功能障碍和氧化应激有因果关系， 机制尚未确定。在这里，我们提出了新的假设，组蛋白脱乙酰酶9（HDAC 9），一类 II组蛋白去乙酰化酶，在促进衰老中脂肪组织衰老中起关键作用，通过促进 线粒体功能障碍我们的初步数据表明，HDAC 9表达在脂肪组织中增加， 老年小鼠和人类的组织。此外，HDAC 9基因缺失改善了脂肪组织线粒体 能量消耗和减少脂肪干细胞（ASC）衰老在1岁的饲料喂养的小鼠，而 在体外保护ASCs免于衰老。我们的假设将通过两个具体目标进行检验。目标1将测试 老年脂肪组织中HDAC 9表达增加有助于线粒体 功能障碍和氧化应激通过破坏线粒体动力学。我们的初步数据表明HDAC 9 在老年小鼠的成熟脂肪细胞中强烈上调。因此，我们将采用两种新型鼠标 模型来测试我们的假设：脂肪细胞特异性HDAC 9敲除（A-KO; HDAC 9 flox/flox脂联素-Cre）和 脂肪细胞特异性转基因（A-Tg，HDAC 9 Tg-flox-STOP-flox脂联素-Cre）小鼠。将培养原代ASC 并分化为成熟脂肪细胞。线粒体功能和动力学将在脂肪组织中测量 外植体和成熟脂肪细胞使用Seahorse分析仪，Western印迹，免疫荧光，透射电镜 电子显微镜和共焦成像。目的2将检验HDAC 9基因缺失抑制 脂肪组织衰老的发展，促进健康的脂肪组织衰老，而过度表达 HDAC 9加速脂肪组织老化并增强衰老。我们将量化衰老细胞 通过葡萄糖和胰岛素耐量测试，了解脂肪组织中的累积和代谢健康。ASC培养物 来自全局HDAC 9敲除小鼠的细胞将用于评估对UV诱导的衰老的易感性。此外，本发明还 A-KO和A-Tg小鼠中的衰老细胞积累将通过衰老相关的β-半乳糖苷酶来评估 染色、qRT-PCR和Western印迹。这个项目将使我获得深入的知识，线粒体 生物学，同时发展新的技术技能，包括原代细胞培养，组织染色和成像。最 重要的是，它将使我获得进行严格的，假设驱动的研究的专业知识，从而设置 作为一个物理学家和科学家的职业生涯的舞台。该项目将在尼尔博士的指导下进行 Weintraub和Masuko Ushio-Fukai博士在医学院血管生物学中心的共同指导 在奥古斯塔大学的格鲁吉亚，这有一个成功的博士前和博士后培训的丰富历史。我们 预计这项研究的结果将确定脂肪组织衰老中HDAC 9表达的升高是一个重要的因素。 线粒体功能障碍和衰老的关键介质，并将提供深入了解潜在的治疗 方法来改善脂肪组织健康老化。

PROJECT SUMMARY Adipose tissue is a major depot for senescent cell accumulation and sterile inflammation in aging. While adipose tissue senescence in aging is causally linked to mitochondrial dysfunction and oxidative stress, the underlying mechanisms are undefined. Here, we propose the novel hypothesis that histone deacetylase 9 (HDAC9), a class II histone deacetylase, plays a key role in promoting adipose tissue senescence in aging by promoting mitochondrial dysfunction. Our preliminary data demonstrate that HDAC9 expression is increased in adipose tissues of aged mice and humans. Moreover, HDAC9 gene deletion improves adipose tissue mitochondrial energy expenditure and reduces adipose stem cell (ASC) senescence in 1-year-old chow diet fed mice, while protecting ASCs against senescence in vitro. Our hypothesis will be tested with two specific aims. Aim 1 will test the hypothesis that increased HDAC9 expression in aged adipose tissues contributes to mitochondrial dysfunction and oxidative stress by disrupting mitochondrial dynamics. Our preliminary data suggest that HDAC9 is strongly upregulated in the mature adipocytes of aged mice. Therefore, we will employ two novel mouse models to test our hypothesis: adipocyte-specific HDAC9 knockout (A-KO; HDAC9flox/flox adiponectin-Cre) and adipocyte-specific transgenic (A-Tg, HDAC9Tg-flox-STOP-flox adiponectin-Cre) mice. Primary ASCs will be cultured and differentiated to mature adipocytes. Mitochondrial function and dynamics will be measured in adipose tissue explants and mature adipocytes using the Seahorse analyzer, Western blot, immunofluorescence, transmission electron microscopy and confocal imaging. Aim 2 will test the hypothesis that HDAC9 gene deletion represses the development of adipose tissue senescence and promotes healthy adipose tissue aging, while overexpression of HDAC9 accelerates adipose tissue aging and augments senescence. We will quantify senescent cell accumulation in adipose tissue and metabolic health through glucose and insulin tolerance testing. ASC cultures from global HDAC9 knockout mice will be used to assess susceptibility to UV-induced senescence. Additionally, senescent cell accumulation in A-KO and A-Tg mice will be assessed by senescence-associated β-galactosidase staining, qRT-PCR and Western blot. This project will allow me to acquire deep knowledge of mitochondrial biology while developing new technical skills, including primary cell culture, tissue staining and imaging. Most importantly, it will allow me to gain expertise in conducting rigorous, hypothesis-driven research, thus setting the stage for a career as a physician-scientist. The project will be conducted under the mentorship of Dr. Neal Weintraub and co-mentorship of Dr. Masuko Ushio-Fukai in the Vascular Biology Center at the Medical College of Georgia at Augusta University, which has a rich history of successful pre- and post-doctoral training. We anticipate that findings from this proposal will identify elevated HDAC9 expression in adipose tissue aging as a key mediator of mitochondrial dysfunction and senescence and will provide insight into potential therapeutic approaches to improve adipose tissue health in aging.