The Role of Hsf1 in Hematopoietic Stem Cell Aging

项目概要 造血干细胞 (HSC) 在整个生命周期中再生所有血液和免疫细胞。老化 HSC 表现 再生功能减弱、淋巴潜能降低以及与相关的克隆生长 免疫力下降，贫血、骨髓衰竭和血液学的发生率增加 老年人的恶性肿瘤。蛋白质稳态（proteostasis）的调节最近已成为一种 促进 HSC 自我更新所需的基本过程。蛋白质稳态的丧失被认为是其中之一 衰老的标志，但它在多大程度上导致干细胞衰老尚不清楚。一条重要途径 维持蛋白质稳态的是热休克反应，它受到主转录因子的调节 热休克因子 1 (Hsf1)。 Hsf1 诱导热休克蛋白的表达，有助于正确的蛋白质折叠， 贩运和退化。在稳定状态下，Hsf1 通常被隔离在细胞质中，但会易位到 细胞核对细胞和蛋白毒性应激的反应。之前，我们证明了 HSC 经历 离体培养时的细胞应激，Hsf1 激活可以缓解这种应激以维持 HSC 再生 活动。 Hsf1 在年轻和老年 HSC 中高度表达，但在中年衰老过程中被特异性激活。 老年和老年 HSC。衰老是一个与遗传基因积累相关的压力显着的过程 突变、炎症和氧化应激。基于这些初步结果，本文的中心假设 提议认为，Hsf1 激活可促进衰老过程中 HSC 功能和蛋白质稳态。为了检验这个假设， 目标 1 将使用条件性 Hsf1 敲除小鼠检查 Hsf1 在老化 HSC 功能和蛋白质稳态中的作用。 HSC 功能将在竞争性移植测定中进行评估，蛋白质稳态将通过 定量蛋白质合成、蛋白酶体活性、错误折叠蛋白质、未折叠蛋白质和蛋白质聚集体 丰富。我预计老年 Hsf1 缺陷型 HSC 的重建会减少，蛋白质会增加 合成、错误折叠和未折叠的蛋白质以及聚集体。虽然 Hsf1 激活被假设为 Hsf1 异时性激活对于衰老过程中的 HSC 功能很重要，但其机制尚不清楚。 初步 RNA 测序结果显示，转谷氨酰胺酶 2 (Tgm2) 参与 Hsf1 激活， 在老年 HSC 中显着上调。因此，目标 2 将检查与年龄相关的 Hsf1 激活是否取决于 Tgm2 上调。 Hsf1 激活、HSC 功能和蛋白质稳态将在条件 Tgm2 中进行评估 基因敲除小鼠。我预计衰老 HSC 中 Tgm2 的丢失将破坏蛋白质稳态并表现出相关的 由于 Hsf1 激活减少而导致健康和功能下降。总的来说，这些研究将提供更深入的 深入了解干细胞衰老过程中调节蛋白质稳态的机制。这些发现将揭示新的 促进衰老过程中 HSC 健康的治疗目标。

PROJECT SUMMARY Hematopoietic stem cells (HSCs) regenerate all blood and immune cells throughout life. Aging HSCs exhibit diminished regenerative function, reduced lymphoid potential, and clonal outgrowth that is associated with compromised immunity as well as an increased incidence of anemia, bone marrow failure, and hematological malignancies in older adults. The regulation of protein homeostasis (proteostasis) has recently emerged as a fundamental process required to promote HSC self-renewal. Loss of proteostasis is considered one of the hallmarks of aging, but to what extent it contributes to stem cell aging is largely unknown. An essential pathway in maintaining proteostasis is the Heat Shock Response, which is regulated by the master transcription factor Heat shock factor 1 (Hsf1). Hsf1 induces expression of heat shock proteins that aid in proper protein folding, trafficking, and degradation. At steady state, Hsf1 is typically sequestered in the cytoplasm, but translocates into the nucleus in response to cellular and proteotoxic stress. Previously, we demonstrated that HSCs undergo cellular stress when cultured ex vivo and Hsf1 activation can alleviate this stress to maintain HSC regenerative activity. Hsf1 is highly expressed in young and old adult HSCs but is specifically activated during aging in middle- aged and old adult HSCs. Aging is a notably stressful process associated with the accumulation of genetic mutations, inflammation, and oxidative stress. Based on these preliminary results, the central hypothesis of this proposal is that Hsf1 activation promotes HSC function and proteostasis during aging. To test this hypothesis, Aim 1 will examine the role of Hsf1 in aging HSC function and proteostasis using conditional Hsf1 knockout mice. HSC function will be assessed in competitive transplantation assays and proteostasis will be assessed by quantifying protein synthesis, proteasome activity, misfolded protein, unfolded protein, and protein aggregate abundance. I expect that there will be less reconstitution in aged Hsf1-deficient HSCs and more protein synthesis, misfolded and unfolded proteins, and aggregates. While Hsf1 activation is hypothesized to be important for HSC function during aging, the mechanism underlying heterochronic Hsf1 activation is unknown. Preliminary RNA-sequencing results revealed that Transglutaminase 2 (Tgm2), involved in Hsf1 activation, is significantly upregulated in old adult HSCs. Thus, Aim 2 will examine if age-related Hsf1 activation depends on Tgm2 upregulation. Hsf1 activation, HSC function, and proteostasis will be assessed in conditional Tgm2 knockout mice. I expect that loss of Tgm2 in aging HSCs will disrupt proteostasis and exhibit an associated decline in fitness and function due to a decrease in Hsf1 activation. Collectively, these studies will provide deeper insights into mechanisms that regulate proteostasis during stem cell aging. These findings will uncover new therapeutic targets to promote HSC fitness during aging.