Presenilins (PS) form high molecular weight complexes with several other transmembrane proteins, termed NCT, Aph-1 and Pen-2 that are critical for generation of functional gamma-secretase complexes. However, the exact roles of these proteins, particularly for Aph-1 in mammals where two homologous genes exist, in regulation of gamma-secretase complex assembly remain uncertain. Although recent data support the notion that PS, NCT, Aph-1 and Pen-2 comprise the minimal gamma-secretase complex, the precise mechanism whereby these four components are assembled into the final active complex remain undefined. An interesting question is why there exist two mammalian Aph-1 genes, namely Aph-1a and Aph-1b, encoding three Aph-1 homologues called Aph-1aL, Aph-1aS and Aph-1b. Based on our recent find ings that the phenotype of Aph-1a null embryos resemble but not identical to those of Notch1 null or NCT null embryos, we hypothesize that Aph-1a is the principal mammalian Aph-1 homologue in presenilin-dependent gamma-secretase complexes required for embryonic development and that Aph-1 homologues are developmentally regulated. Thus, we plan in Aim 1 to address these issues by generation and characterization of Aph-1a null, Aph-1b null and Aph-1a+Aph-1b null mice. Based on our recent findings that the deletion of Aph-1a significantly reduces the levels of mature and immature NCT coupled with the finding that Aph-1 and NCT physically interact, we hypothesize that Aph-1 and NCT are required to regulate the stability of each other to form a stable precomplex for assembling PS and Pen-2. In such a model, we suggest that the three mammalian Aph-1 homologues (Aph-1aL, Aph-1aS and Aph-1b) define a set of six distinct functional gamma-secretase complexes. To test this model, we will generate and characterize a series of mice harboring different combination of Aph-1a and Aph-1b knockout allele and fibroblasts derived from these mice in Aim 2. Since we showed that Aph-1b null mice are viable and there is reduction in levels of PS and Pen-2 in brains of Aph-1-/- mice, we will test whether deletion of Aph-1b is sufficient to ameliorate Abeta deposition in brains of mutant APP;PS1 mice in Aim 3. Taken together, studies proposed here will address important mechanistic questions regarding physiological roles of mammalian Aph-1 homologues and critically evaluating Aph-1a and Aph-1b as therapeutic targets in efforts to ameliorate Abeta amyloidosis in AD.