Targeting Cellular Senescence and RAGE in Type 2 Diabetes

项目总结 2型糖尿病(T2D)是导致骨骼质量差和骨折的主要危险因素，与早产儿有关 衰老细胞和晚期糖基化终末产物(AGEs)的积累； AGE[RAGE]途径)在多种组织中表达，包括骨骼。直觉上，衰老的细胞和愤怒可以起作用 独立地或通过串扰相互作用对T2D中的骨骼脆性有很大贡献，然而这个概念 没有经过严格的测试。这项提议建立在创新的概念、技术和方法之上 为了验证我们的中心假设，即靶向细胞衰老或RAGE可以改善T2D相关的骨骼 脆弱。为了验证我们的假设，我们将使用新的转基因小鼠和创新技术，包括质量 细胞学以及先进的组织学和分子学工具。骨骼、能量之间的相互作用 新陈代谢和T2D多年来一直是研究的主题，但很少有活体研究严格地 询问衰老细胞或RAGE信号在T2D骨骼功能障碍中的作用。从一个 翻译视角，更好地理解骨骼意志中衰老和愤怒之间的相互作用 产生有影响力的进展，并可能揭示改善T2D加速骨骼老化的新策略。至 为此，在目标1中，我们将识别、定位和表征小鼠的骨骼驻留衰老细胞群 并确定其T2D特异性衰老相关分泌表型(SASP)。在目标2中，使用 携带能够选择性消除p16INK4a+或p21Cip1+衰老细胞的转基因小鼠，我们将 验证T2D建立的小鼠衰老细胞清除将使骨重建正常化的假设 和质量。因此，我们将区分p16INK4a和p21Cip1在介导骨功能障碍中的因果作用。 用我们的全球p16-和p21-ATTAC小鼠品系进行T2D，通过比较系统清除 P16INK4a+与p21Cip1+衰老细胞比较。此外，我们将评估清除衰老的相对影响 使用我们的新型Cre-loxP细胞系-p16-LOX-ATTAC和p21-LOX-ATTAC。全球和骨细胞- 对衰老细胞的特定清除将与使用“抗衰老药物”的药物清除进行比较。 最后，在目标3中，使用我们的新型Cre-loxP小鼠，它通过细胞特异性抑制RAGE信号转导 胞浆结构域缺陷的显性负性RAGE(DN-RAGE)的表达，我们将定义 抑制成骨细胞/骨细胞和髓系/破骨细胞系中的RAGE信号对骨骼脆性的影响 T2D组小鼠。总的来说，这些研究将严格测试细胞衰老和愤怒信号 与T2D相关的骨骼脆性是基础。我们将通过利用我们独特的资源和 专业知识。我们将以令人信服的初步数据和创新方法为基础，包括新颖的 我们期待的分析、转基因和药理学工具将极大地促进我们对 T2D中骨骼功能障碍的基本生物学，导致了新的机械学见解，并有证据- 以治疗方法为基础，促进将临床前发现转化为临床应用。

PROJECT SUMMARY Type 2 diabetes (T2D), a major risk factor for poor bone quality and fractures, is associated with the premature accumulation of senescent cells and advanced glycation endproducts (AGEs; activators of the receptor for AGE [RAGE] pathway) in multiple tissues, including bone. Intuitively, senescent cells and RAGE could act independently or interact via cross-talk to contribute substantially to skeletal fragility in T2D, yet this concept has not been rigorously tested. This proposal is founded on innovative concepts, technology, and approaches to test our central hypothesis that targeting cellular senescence or RAGE can improve T2D-related skeletal fragility. To test our hypothesis, we will use novel transgenic mice and innovative technology, including mass cytometry as well as advanced histological and molecular tools. The interplay among bone, energy metabolism, and T2D has been a topic of research for years, yet few in vivo studies have rigorously interrogated the contributions of senescent cells or RAGE signaling to skeletal dysfunction in T2D. From a translational perspective, better understanding of the cross-talk between senescence and RAGE in bone will yield impactful advances and may reveal novel strategies to ameliorate accelerated skeletal aging in T2D. To this end, in Aim 1 we will identify, locate, and characterize bone-resident senescent cell populations in mice with T2D and define their T2D-specific senescence-associated secretory phenotype (SASP). In Aim 2, using mice harboring transgenes that enable the selective elimination of p16Ink4a+ or p21Cip1+ senescent cells, we will test the hypothesis that senescent cell clearance in mice with established T2D will normalize bone remodeling and quality. Thus, we will distinguish the causal roles of p16Ink4a and p21Cip1 in mediating skeletal dysfunction in T2D using our global p16- and p21-ATTAC mouse strains by comparing the effects of systemic clearance of p16Ink4a+ vs p21Cip1+ senescent cells. In addition, we will assess the relative impact of clearing senescent osteocytes, using our novel Cre-LoxP lines – p16-LOX-ATTAC and p21-LOX-ATTAC. Global and osteocyte- specific clearance of senescent cells will be compared with pharmacological elimination using “senolytics”. Finally, in Aim 3, using our novel Cre-loxP mouse that inhibits RAGE signal transduction via cell-specific cytosolic-domain deficient dominant-negative RAGE (DN-RAGE) expression, we will define the effects of inhibiting RAGE signaling in the osteoblast/osteocyte and myeloid/osteoclast lineages on skeletal fragility in mice with T2D. Collectively, these studies will rigorously test whether cellular senescence and RAGE signaling underlie T2D-related skeletal fragility. We will address these questions by leveraging our unique resources and expertise. We will build upon compelling preliminary data and innovative approaches, including novel analytical, transgenic, and pharmacological tools that we anticipate will significantly advance our understanding of the fundamental biology of skeletal dysfunction in T2D, leading to new mechanistic insights, and evidence- based therapeutic approaches to facilitate the translation of preclinical discoveries to clinical applications.