Project Summary A causal hallmark of brain aging and Alzheimer’s Disease (AD) is loss of proteostasis, the maintenance of proteome health through several processes, including protien degradation/clearance (hereafter, ‘turnover’). Neuronal proteostasis is exceptionally vulnerable during aging and AD as evidenced by the accumulation of dysfunctional or toxic neuronal proteins, such as pTau and Aβ, causal for neuron dysfunction and degeneration. While there is an appreciation for the role of dysregulated proteostasis in brain aging and AD, much remains to be understood about the dynamics of neuronal protein turnover (NPT), consequences of aberrant NPT, and NPT regulators during aging and AD. I hypothesize that NPT is compromised with aging and AD, ultimately worsening age and AD phenotypes, and NPT is at least partially regulated by lysosomal biology and microglia. I am uniquely positioned to address my hypotheses thanks to my establishment of a new in vivo model of bioorthogonal non-canonical amino acid tagging (BONCAT). BONCAT permits the tagging of newly synthesized proteins with an azide-bearing phenylalanine (AzF) in a cell-specific manner. Tagged proteins can be pulled down and analyzed by mass spectrometry (MS). When AzF is provided to BONCAT mice in a pulse- chase administration scheme and mice are sacrificed at different time points in the chase, tagged proteins can be pulled down and their reduction over the chase/time, representative of turnover, can be examined. I have performed this experiment in young and aged neuronal-BONCAT mice, finding >800 relatively longer-lived proteins (RLLPs) with aging, many of which are in pathways of neurodegeneration. Here, I propose to perform an identical experiment in AD models, compare the results to aging, and use an in vitro human transdifferentiated neuron model in conjunction with targeted protein degradation to determine the pathological consequences of select RLLPs common to aging and AD. I will additionally combine BONCAT with a novel lysosome immunoprecipitation (IP) method, LysoTag, to understand the causality of autophagic/lysosomal dysfunction in age/AD NPT aberrations. Lastly, I will apply BONCAT to understand how microglia regulate NPT by (1) examining neuronal protein uptake by microglia and (2) determining how deletion of Trem2, a microglia-specific AD risk factor, alters NPT. Cumulatively, the proposal will address what NPT changes occur with aging/AD, how the changes affect neuronal health and pathology, and how the lysosome and microglia act as intrinsic and extrinsic regulators of NPT. Findings will hold promise to identify new targets to promote healthy brain aging. Through continued training with the K99/R00 award, I will learn new methodologies (MS acquisition/analysis; lysosome IP; targeted protein degradation) and concepts (proteostasis; autophagy) and participate in critical professional development workshops/classes. The research and training proposed herein will empower me t...