The Role of Hsf1 in Hematopoietic Stem Cell Aging

项目总结 造血干细胞(HSCs)在一生中再生所有的血液和免疫细胞。老化的HSCs展览 再生功能减弱，淋巴潜能降低，以及与 免疫功能受损，贫血、骨髓衰竭和血液学发病率增加 老年人的恶性肿瘤。蛋白质稳态的调节最近已经成为一种 促进HSC自我更新所需的基本程序。蛋白平衡的丧失被认为是 衰老的特征，但它在多大程度上导致干细胞衰老在很大程度上是未知的。必经之路 维持蛋白质平衡的是热休克反应，它由主转录因子调节 热休克因子1(HSF1)。HSF1诱导热休克蛋白的表达，有助于蛋白质的正确折叠， 贩卖和堕落。在稳定状态下，HSF1通常隔离在细胞质中，但转位到 对细胞和蛋白毒性应激作出反应的细胞核。在此之前，我们演示了造血干细胞经历 体外培养时的细胞应激和HSF1的激活可以缓解这种应激，以维持HSC的再生 活动。HSF1在青年和老年成人HSCs中高表达，但在中老年HSCs中特异激活。 老年和老年成人HSCs。衰老是一个明显的压力过程，与基因的积累有关 突变、炎症和氧化应激。根据这些初步结果，这一中心假设 有人认为，HSF1的激活促进了HSC的功能和衰老过程中的蛋白平衡。为了检验这一假设， 目的1利用条件性HSF1基因敲除小鼠，研究HSF1在衰老HSC功能和蛋白稳定中的作用。 将在竞争性移植试验中评估HSC的功能，并将通过以下方法评估蛋白稳定性 量化蛋白质合成、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 富足。我预计在HSF1缺乏的老年HSCs中将会有更少的重建和更多的蛋白质 合成，错误折叠和未折叠的蛋白质，以及聚集体。而HSF1的激活被假设为 HSF1在衰老过程中对HSC的功能很重要，但其异时性激活的机制尚不清楚。 初步的RNA测序结果表明，参与HSF1激活的转谷氨酰胺酶2(TGM2)是 在老年成人HSCs中显著上调。因此，Aim 2将检查与年龄相关的HSF1激活是否依赖于 Tgm2上调。HSF1激活、HSC功能和蛋白稳定性将在条件性TGM2中进行评估 基因敲除老鼠。我预计在衰老的HSCs中Tgm2的丢失将扰乱蛋白平衡并表现出相关的 由于HSF1活性降低而导致的体能和功能下降。总的来说，这些研究将提供更深层次的 对干细胞老化过程中蛋白平衡调节机制的洞察。这些发现将揭示新的 在衰老过程中促进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.