PROJECT 1 SUMMARY Project 1 (formerly Project 2) builds on the accomplishments of this Project and the Program Project Grant (PPG) as a whole and addresses a number of key unresolved issues relevant to skeletal aging that are also applicable across aging tissues and thus best addressed in the framework of a PPG. First, although both p16Ink4a and p21Cip1 have been identified as key drivers of cellular senescence, the relative contributions of each towards age-related senescence across tissues in vivo remains unclear. In terms of skeletal aging, we have found that clearing p16Ink4a+ senescent cells in the INK-ATTAC mouse model using AP20187 prevents age-related bone loss. With regards to p21Cip1, using single cell proteomic analyses by cytometry by time-of-flight (CyTOF), we have identified age-related increases not only in p16+ but also in p21+ senescent cells in bone and, using scRNAseq analyses, we found that p16Ink4a+ and p21Cip1+ cells express substantially different SASP profiles not only in bone, but also in muscle and brain. Moreover, although genetic models using the ATTAC transgene have been extremely useful in assessing the effects of senescent cell clearance on different tissues with aging, in Aim 1 we propose to use an alternate approach to ask a fundamentally different question: What are the specific roles and interactions of p16Ink4a and p21Cip1 in leading, in the first place, to the formation and propagation of senescent cells across tissues? Thus, in Aim 1, we will utilize common PPG mouse models developed in Core B that have inducible deletions in aged mice of either p16Ink4a or p21Cip1, or both, and evaluate, across bone (Project 1), muscle (Project 2), and brain (Project 3), the resulting phenotypes and senescent cell burden across these tissues. Aim 2 complements this genetic approach by using common PPG pharmacologic mouse models that utilize novel senolytic compounds. These compounds, being developed in Core C, have relative propensities to target senescence driven either by p16 or p21 and provide a unique opportunity, utilizing the senescent cell phenotyping in Core D along with the systems biology approaches in Core A, to integrate the findings from Aims 1 and 2 and define, across tissues and using either genetic or pharmacologic tools, the contributions of p16Ink4a vs. p21Cip1 towards senescence and the SASP across bone, muscle, and brain. Building on the integrative strengths of the PPG, Aims 3 and 4 examine the interactions between senescent osteocytes and muscle and brain aging (Aim 3) and interactions between systemic or bone marrow senescent adipocytes and bone, muscle, and brain aging (Aim 4). In addition to the integration across Projects and Cores, the innovation in the proposed studies lies in the use of novel mouse models developed by us (p16-LOX-ATTAC, SNORKEL) and others (floxed p16Ink4a, floxed p21Cip1, bone marrow adipocyte-specific Cre [BMAd-Cre]); novel analytical tools, including the multiparametric identi...