Project summary / abstract Given the recent FDA approval of an antibody-based drug that can remove amyloid plaques as measured by positron-emission tomography targeting amyloid (PET-amyloid) (aducanamab1), the FDA approval of the Precivity-AD blood test2, and applications submitted for accelerated approval of lecanemab and donanemab – antibody-based drugs also shown to reduce plaque by PET-amyloid imaging3–5 – there is an urgent need to better understand the natural amyloid-beta (Aβ) turnover in plaques. The overall goal of this proposal is to quantitatively characterize the rate of Aβ turnover within plaques in vivo, in human brain tissue at various stages of Alzheimer's disease (AD) using stable isotope labeling kinetics (SILK). That multiple antibody-based drugs nearly completely remove plaques as measured by PET3–5, taken together with a reduction in the rate of cognitive decline, supports a role for amyloid pathology as critical driver of AD pathogenesis. However, the duration and possible cessation of treatment is partly dependent on whether amyloid plaques continue to grow and turn over. Other drugs (e.g. BACE inhibitors) can stop amyloid plaque growth with minimal reversal of plaque load (~4%/year by PET), suggesting that there is some natural slow turnover of amyloid plaques. Drug trials and clinical use of anti-amyloid therapies thus must be based on accurate models of natural plaque growth. Very recently, microscopic resolution of in vivo metabolic growth of human amyloid plaques was achieved by combining SILK with mass spectrometric-based imaging methods (“iSILK”) to better characterize protein and peptide kinetics within brain parenchyma. Specifically, the Bateman laboratory used nanoscale secondary ion mass spectrometry (NanoSIMS) coupled to SILK to directly image the distribution and rate of protein deposition in plaques at the nanometer level in postmortem tissue from 3 human patients with AD6. However, NanoSIMS imaging fails to specify which molecules contain the detected isotopes. In contrast, matrix-assisted laser desorption / ionization (MALDI) mass spectrometry-based imaging (MALDI-IMS) allows chemically-specific Aβ peptide imaging of pathologic structures in AD mouse models and postmortem brain, which has been pioneered by the Hanrieder lab7–10. Consequently, the group very recently demonstrated MALDI in combination with SILK to follow plaque formation dynamics10. Using postmortem tissue from human patients previously labeled during life by oral ingestion of 15N- labeled spirulina, we will characterize Aβ turnover by MALDI-IMS in brains with a spectrum of AD pathology. These measurements will inform a compartmental model of AD-related protein kinetics starting at the microscopic structure of the plaque and extending throughout the body, a particularly important model in the dawning era of clinically-approved diagnostic biomarkers2, disease-modifying therapies3–5, and the critical need for a precise understanding of the e...