The 24-residue peptide Humanin (HN), containing two Ser residues at positions 7 and 14, protects neuronal cells from insults of various Alzheimer's disease (AD) genes and Ab. It was not known why the rescue function of (S14G)HN is more potent than HN by two to three orders of magnitude. Investigating the possibility that the post-translational modification of Ser14 might play a role, we found that HN with D-Ser at position 14 exerts neuroprotection more potently than HN by two to three orders of magnitude, whereas D-Ser7 substitution does not affect the rescue function of HN. On the other hand, S7A substitution nullified the HN function. Multiple series of experiments indicated that Ser7 is necessary for self-dimerization of HN, which is essential for neuroprotection by this factor. These findings indicate that the rescue function of HN is quantitatively modulated by D-isomerization of Ser14 and Ser7-relevant dimerization, allowing for the construction of a very potent HN derivative that was fully neuroprotective at 10 pM against 25 lM Ab1-43. This study provides important clues to the understanding of the neuroprotective mechanism of HN, as well as to the development of novel AD therapeutics. Keywords: Alzheimer's disease, dimerization, Humanin, neuronal death, neuroprotection, D-serine isomerization. Alzheimer's disease (AD) is the most prevalent neurodegenerative disease associated with progressive dementia. No fundamental therapy for this disease has so far been established. As brain atrophy is the central abnormality in AD, pathological mechanisms leading to neuronal loss must be understood to develop future curative therapy. Three forms of known mutant genes cause familial AD (FAD): amyloid precursor protein (APP) mutants, presenilin (PS)1 mutants, and PS2 mutants (Shastry and Giblin 1999). Multiple groups have found that all examined FAD mutants cause or enhance cell death when they are expressed in neuronal cells (Niikura et al. 2002 for review). In an attempt to find the molecules that suppress neuronal cell death by these AD-related insults, we used 'death-trap' screening, developed by D'Adamio et al. (1997) -an unbiased functional screening of molecules that allow dying cells to survive.We applied this method to V642I-APP-inducible neuronal cells with our unique modification using an expression cDNA library constructed from an occipital lobe of the brain of an autopsy-diagnosed AD patient. As a result of this screening, we identified an ORF cDNA, encoding a novel short peptide MAP-RGFSCLLLLTSEIDLPVKRRA, that suppresses neuronal cell death by various FAD genes [APP mutants (V642I, K595M/N596L, A617G, and L648P), PS1 mutants (M146L, H163R, A246E, L286V, and C410Y), and a PS2 mutant Received February 20, 2003; accepted March 7, 2003. Address correspondence and reprint requests to Ikuo Nishimoto, Departments of Pharmacology and Anatomy, KEIO University School of Medicine, Medical Research Center, 6th Floor, 35 Shinanomachi, Tokyo 160-8582, Japan. E-mail: nisimoto@sc.itc.keio.ac.jp 1 These two ...