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

项目总结 脂肪组织是衰老细胞积累和衰老过程中无菌炎症的主要储存库。虽然肥胖 衰老中的组织衰老与线粒体功能障碍和氧化应激有关，这是潜在的 机制是不确定的。在这里，我们提出了一种新的假设，即组蛋白脱乙酰酶9(HDAC9)，一类 组蛋白脱乙酰基酶在促进脂肪组织衰老过程中起关键作用 线粒体功能障碍。我们的初步数据显示，HDAC9在脂肪组织中的表达增加 衰老的小鼠和人的组织。此外，HDAC9基因缺失改善了脂肪组织线粒体 1岁饲料喂养的小鼠的能量消耗和减少脂肪干细胞(ASC)衰老，而 在体外保护ASCs抗衰老。我们的假设将通过两个具体目标进行检验。AIM 1将测试 老年脂肪组织中HDAC9表达增加与线粒体有关的假说 功能障碍和氧化应激，通过破坏线粒体动力学。我们的初步数据显示，HDAC9 在衰老小鼠的成熟脂肪细胞中强烈上调。因此，我们将使用两个新的鼠标 验证我们假设的模型：脂肪细胞特异性HDAC9基因敲除(A-KO；HDAC9 FLOX/FLOX Adiponectin-CRE)和 脂肪细胞特异性转基因(A-TG，HDAC9Tg-FLOX-STOP-FLOX脂联素-CRE)小鼠。原代ASCs将被培养 分化为成熟的脂肪细胞。将在脂肪组织中测量线粒体的功能和动力学 用海马氏分析仪、免疫印迹、免疫荧光、透射法检测外植体和成熟脂肪细胞 电子显微镜和共聚焦成像。目的2将验证HDAC9基因缺失抑制的假设 脂肪组织衰老的发生和促进脂肪组织的健康衰老，同时过度表达 HDAC9可加速脂肪组织衰老，加速衰老。我们将对衰老细胞进行量化 通过葡萄糖和胰岛素耐量测试，脂肪组织中的蓄积和代谢健康。ASC培养 来自全球HDAC9基因敲除的小鼠将被用来评估对紫外线诱导的衰老的敏感性。另外， 衰老相关β半乳糖苷酶将评估A-KO和A-TG小鼠衰老细胞的积累 染色、定量逆转录聚合酶链式反应和蛋白质印迹。这个项目将使我获得关于线粒体的深入知识 同时开发新的技术技能，包括原代细胞培养、组织染色和成像。多数 重要的是，它将使我在进行严格的、假设驱动的研究方面获得专业知识，从而设定 作为一名内科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 温特劳布和Ushio-Fukai博士在医学院血管生物学中心的共同指导 佐治亚州奥古斯塔大学拥有丰富的博士前和博士后培训成功历史。我们 预期这项提案的发现将确定在脂肪组织老化中HDAC9表达升高是一种 线粒体功能障碍和衰老的关键媒介，将为潜在的治疗提供洞察力 在衰老过程中改善脂肪组织健康的方法。

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.