Reverse Engineering of Cell Senescence

细胞衰老的逆向工程

• 批准号: 10445589
• 负责人: MARC Wallace KIRSCHNER
• 金额: $ 34.66万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2022-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10445589
• 关键词: Address; Affect; Age; Aging; Animals; Biochemical; Cell Aging; Cell Culture Techniques; Cell Cycle; Cell Cycle Arrest; Cell Size; Cell physiology; Cells; Cellular biology; Cessation of life; Coupling; Critical Pathways; Cultured Cells; Dependence; Development; Embryonic Development; Engineering; Etiology; Event; Exposure to; FRAP1 gene; Genes; Growth; Health Benefit; Heterogeneity; Hormones; Hypertrophy; In Vitro; Inflammatory; Insulin; Intention; Ionizing radiation; Kinetics; Knowledge; Laboratories; Learning; Life Extension; Lipids; Liver; Logic; Mass Spectrum Analysis; Measurement; Measures; Medical; Methods; Microscopic; Microscopy; Modification; Molecular; Mus; Normal Cell; Normal tissue morphology; Pathology; Pathway interactions; Pattern; Pharmaceutical Preparations; Pharmacologic Substance; Pharmacology; Phenotype; Phosphoproteins; Phosphorylated Peptide; Phosphorylation; Phosphotransferases; Phylogenetic Analysis; Plant Roots; Play; Process; Protein Kinase; Proteins; Proteome; Research Personnel; Resolution; Role; Signal Transduction; Sirolimus; Site; Stress; Symptoms; Testing; Time; Tissues; aged; cell growth; cell type; density; experimental study; in vivo; instrument; interest; kinase inhibitor; mTOR inhibition; new technology; prevent; programs; protein expression; proteostasis; response; senescence; tool; wound healing

Abstract Cellular senescence is an elusive cell state - it is recognized in embryogenesis, wound healing and aging, where it is not only a symptom, but a major contributor to aging pathology. The striking experiments in the mouse, where eliminating senescent cells bestows broad health benefits and even a reversal of the aging process, drives our interest in understanding and ultimately controlling the transition of normal cells towards senescence. If we understood how cell senescence arises, we would have a chance to find ways to suppress it or reverse it, as well as to develop practical ways of clearing such cells from our bodies. This proposal is to use new tools on the microscopic and on the molecular level to observe the passage of cells from a normal to senescent state and reveal the changes in their molecular circuitry. Among the transitions that cells make is a dramatic increase in cell size. Hypertrophy is known to be driven by mTOR, and inhibited by the drug rapamycin, which is tied to life extension in several phylogenetically diverse species. We focus on hypertrophy to help crack senescence because, as a phenotype, it is in such stark violation of the normal cellular economy, where across cell types size is precisely maintained. Unfortunately, cell size has been one of the hardest phenotypes to study. This situation has changed radically with the development of new forms of microscopy by our group, that directly measure cell dry mass or even directly measure protein and lipid mass separately. Using such instruments, we propose to study with unprecedented temporal and mass resolution how cells, prompted by stress or aging, become hypertrophic and how hypertrophy is connected to other phenotypes of senescence. We will follow these same trajectories with deep quantitative mass spectrometry to correlate protein expression and phosphorylation with size and other senescent markers. We cross-reference some of our findings between in-vitro studies in cell culture and in-vivo studies in young and aged mice. Once we build a baseline description of the process of senescence, the same induction-maturation-death lifecycle of senescent cells will be repeated under perturbation by drugs. We can progress from description to causal analysis using the knowledge that several senolytic drugs are kinase inhibitors. When we perturb senescing cells by a small optimally informative, pre-selected set of poly-specific kinase inhibitors, whose inhibitory activities tile the whole kinome, we can identify key kinases that regulate senescence. Further coupling this to phospho-mass spectrometry will allow us to trace the signaling cascades to specific protein substrates and phosphosites. These observations and pharmacological perturbations can suggest new senolytic strategies and even suggest specific senolytic drugs.

抽象的 细胞衰老是一种难以捉摸的细胞状态——它在胚胎发生、伤口愈合和衰老过程中被识别出来， 它不仅是一种症状，而且是衰老病理学的一个主要因素。引人注目的实验 小鼠，消除衰老细胞可以带来广泛的健康益处，甚至可以逆转衰老 过程，激发了我们理解并最终控制正常细胞向 衰老。如果我们了解细胞衰老是如何发生的，我们就有机会找到抑制细胞衰老的方法 或逆转它，以及开发从我们体内清除此类细胞的实用方法。这个提议是为了 使用微观和分子水平上的新工具来观察细胞从正常细胞的传代 衰老状态并揭示其分子电路的变化。在细胞进行的转变中 是细胞尺寸的急剧增加。已知肥大由 mTOR 驱动，并受药物抑制 雷帕霉素，与多种系统发育不同物种的寿命延长有关。我们专注于肥大 帮助破解衰老，因为作为一种表型，它严重违反了正常的细胞经济， 其中跨细胞类型的大小得到精确维持。不幸的是，细胞大小一直是最困难的问题之一 要研究的表型。随着新型显微镜的发展，这种情况发生了根本性的改变 我们的团队直接测量细胞干质量，甚至直接分别测量蛋白质和脂质质量。 使用此类仪器，我们建议以前所未有的时间和质量分辨率研究细胞如何， 由于压力或衰老而导致肥大，以及肥大如何与其他表型相关 衰老。我们将通过深度定量质谱法遵循这些相同的轨迹来关联 蛋白质表达和磷酸化与大小和其他衰老标记。我们交叉引用了一些 我们在细胞培养体外研究和年轻和老年小鼠体内研究之间的发现。一旦我们建立 衰老过程的基线描述，相同的诱导-成熟-死亡生命周期 衰老细胞会在药物的干扰下重复出现。我们可以从描述发展到因果关系 使用几种 senolytic 药物是激酶抑制剂的知识进行分析。当我们扰乱衰老时 细胞通过一组最佳信息、预先选择的多特异性激酶抑制剂进行抑制，其抑制作用 通过研究整个激酶组的活动，我们可以识别调节衰老的关键激酶。进一步耦合此 磷质谱分析将使我们能够追踪特定蛋白质底物的信号级联反应， 磷酸位点。这些观察结果和药理学扰动可以提出新的 senolytic 策略 甚至建议特定的抗衰老药物。