ABSTRACT (Supplement) Humanin (HN), a 24-amino acid mitochondrial-derived peptide from the occipital lobe of postmortem sporadic AD patient brain tissue [1] is a neuroprotective peptide that could protect neurons from Aβ-related and NMDA- induced toxicity [2]. A decrease in the endogenous HN plasma level with age has been reported [3], and decreased HN levels has been linked to cognitive decline during aging. The identification of a SNP (rs2854128) using mitochondrial GWAS in the HN-coding region of the mitochondrial genome from a large sample of older adults further supports the role of HN and its circulating levels with accelerated cognitive decline [4]. The availability of high throughput screening (HTS) formattable assays, allow for screening to identify small molecules that can enhance HN expression and its levels in AD models. In the parent grant we completed screening for small molecules that act as humanin (HN) mimetics and have identified validated hits that increase both the p- gp130 and p-Akt levels in neuronal cells. Further analoging, testing and evaluation of these mimetics are ongoing following the path outlined in the grant. The availability of new assays to assess levels of HN peptide itself in vitro and in tissue has presented the opportunity to also identify small molecules that enhance HN levels. Our preliminary data using this assay shows that we can detect HN levels in SH-SY5Y cells. Hence for the Supplement research we propose is to conduct screening for HN enhancers using the human neuroblastoma SH-SY5Y cells that express and secrete HN [5]. The work we propose in the Supplement is directed toward formatting of the available HN ELISA assay for HTS and screening of a subset of the UCLA compound library to identify compounds that change HN levels in cells. Hits from the screening would be validated and then undergo secondary testing to confirm HN enhancement in SH-SY5Y and iPSC-derived human neurons, as well as in murine neuroblastoma N2a. Validated hits would also be assessed for increasing neuronal p-Akt. Thus, in the supplement we propose an Expansion of Aim1 of the parent grant to include identification of HN enhancers that along with our currently identified HN mimetics would undergo further testing and evaluation as outlined in the parent grant. Hits from the screening would then undergo secondary testing to confirm HN enhancement in SH-SY5Y and iPSC-derived human neurons, as well as in murine neuroblastoma N2a cells. Validated hits would also be assessed using our p-Akt AlphaLISA to confirm biological activity in neurons. Validated HN enhancers would undergo prioritization as part of Expansion of Aim3 of the parent grant by evaluation in in vitro ADME-T assays. Prioritized analogs would undergo pharmacokinetic (PK) and pharmacodynamics (PD) studies to identify brain permeable HN enhancers. Such an HN enhancers identified from the supplement grant would have the potential to be developed as novel therapeutic candidates...