Principal Investigator: Philip Wong, Ph.D. [ Bio ]
Alzheimer’s disease (AD), a progressive neurodegenerative disorder of the elderly, is characterized by the deposition of β-amyloid (Aβ) and neurofibrillary tangles in the hippocampus and cerebral cortex. Endoproteolytic cleavages of APP by β-and γ-secretases result in the generation of Aβ peptides that are believed to be neurotoxic. The presenilins (PS1 and PS2), which when mutated cause familial AD, are important for the regulated intramembraneous proteolysis of several proteins, including APP and Notch1. Presenilins are involved in the proteolytic processing of Notch1 and they are critical for Notch1 functions. Recent studies support the view that the presenilins may be the putative γ-secretase.
Nicastrin, a type I transmembrane glycoprotein, forms high molecular weight complexes with presenilins (PS1 and PS2). Studies in C. elegans indicated that aph-2/nicastrin is required for glp-1/notch signaling. In addition, co-immunoprecipitation experiments demonstrated that nicastrin binds to both full-length APP and APP-CTFs and is capable of modulating the secretion of Aβ for HEK293 cells. Similarly, while nicastrin was shown to bind to the membrane-tethered form of Notch, studies indicated that the APP and Notch processing pathways do not compete with one another. These results suggested that nicastrin and presenilins form functional components of a multimeric complex required for the intramembraneous proteolysis of both Notch and APP. Recent studies in nicastrin-deficient Drosophila demonstrate that the loss of nicastrin abolishes the presenilin-dependent intramembraneous proteolysis of Notch. Thus, we hypothesize that nicastrin is required for proteolytic processing and signaling of Notch1 in mammals. We plan in specific aim 1 to determine whether nicastrin is required for presenilin-mediated Notch signaling by generating and characterizing nicastrin-deficient mice. Secondly, because studies indicated that nicastrin plays an important role in APP processing, we hypothesize that nicastrin is required for APP processing in mammals. In specific aim 2, we will test the role of nicastrin in presenilin-mediated γ-secretase cleavage of APP by establishing normal, heterozygous, or homozygous nicastrin knockout ES cells and infect these cells with adenovirus expressing human wild type APP or its variants. Interestingly, while Drosophila mosaic analysis indicated that nicastrin might be required for trafficking of presenilins to the cell surface, RNAi study in Drosophila S2 cells suggested that nicastrin is necessary for the stabilization of presenilins. To clarify this issue, and based on our preliminary observation that the level of PS1 is reduced in brain of nicastrin heterozygous knockout mice, we hypothesize that nicastrin is required for the stabilization of presenilins in mammalian cells. Thus, in specific aim 3 we will determine the mechanism whereby nicastrin influences presenilins to facilitate transmembrane cleavage of Notch and APP by examining the stabilization of presenilins by nicastrin. In addition, we will define the domains of nicastrin that are critical for such stabilization using nicastrin deficient cells.
Taken together, outcomes from our proposed studies will have important implications toward our understanding of the biology of nicastrin and specifically the mechanism whereby nicastrin influences presenilins in facilitating the presenilin-dependent γ-secretase activities on several critical pathways, including Notch and APP. Finally, our proposal is designed to critically evaluate whether nicastrin is a high priority therapeutic target and thus they have important implications for the development of compounds to ameliorate Aβ amyloidosis in AD.
For more information, please contact Phil Wong, PhD, at
(410) 502-5168 or wong@jhmi.edu
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