Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder. Mutations in presenilins 1 and 2 (PS1 and PS2) account for approximately 40% of familial AD (FAD) cases. FAD mutations and genetic deletions of presenilins have been associated with calcium (Ca(2+)) signaling abnormalities. We demonstrate that wild-type presenilins, but not PS1-M146V and PS2-N141I FAD mutants, can form low-conductance divalent-cation-permeable ion channels in planar lipid bilayers. In experiments with PS1/2 double knockout (DKO) mouse embryonic fibroblasts (MEFs), we find that presenilins account for approximately 80% of passive Ca(2+) leak from the endoplasmic reticulum. Deficient Ca(2+) signaling in DKO MEFs can be rescued by expression of wild-type PS1 or PS2 but not by expression of PS1-M146V or PS2-N141I mutants. The ER Ca(2+) leak function of presenilins is independent of their gamma-secretase activity. Our data suggest a Ca(2+) signaling function for presenilins and provide support for the "Ca(2+) hypothesis of AD."

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Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD),

Mutations in presenilins are responsible for approximately 40% of all early-onset familial Alzheimer disease (FAD) cases in which a genetic cause has been identified. In addition, a number of mutations in presenilin-1 (PS1) have been suggested to be associated with the occurrence of frontal temporal dementia (FTD). Presenilins are highly conserved transmembrane proteins that support cleavage of the amyloid precursor protein by γ-secretase. Recently, we discovered that presenilins also function as passive ER Ca 2+ leak channels. Here we used planar lipid bilayer reconstitution assays and Ca 2+ imaging experiments with presenilin-null mouse embryonic fibroblasts to analyze ER Ca 2+ leak function of 6 FAD-linked PS1 mutants and 3 known FTD-associated PS1 mutants. We discovered that L166P, A246E, E273A, G384A, and P436Q FAD mutations in PS1 abolished ER Ca 2+ leak function of PS1. In contrast, A79V FAD mutation or FTD-associated mutations (L113P, G183V, and Rins352) did not appear to affect ER Ca 2+ leak function of PS1 in our experiments. We validated our findings in Ca 2+ imaging experiments with primary fibroblasts obtained from an FAD patient possessing mutant PS1-A246E. Our results indicate that many FAD mutations in presenilins are loss-of-function mutations affecting ER Ca 2+ leak activity. In contrast, none of the FTD-associated mutations affected ER Ca 2+ leak function of PS1, indicating that the observed effects are disease specific. Our observations are consistent with the potential role of disturbed Ca 2+ homeostasis in Alzheimer disease pathogenesis. IntroductionAlzheimer disease (AD) is the most common form of age-related dementia in human beings over the age of 60 years. AD affects about 2% of populations in industrialized countries. The understanding of the molecular processes that lead to the pathogenesis of AD is immensely important in combatting this neurological disease. Most cases of AD are idiopathic and are characterized by late onset (in individuals over 60 years of age). A small fraction of AD cases (familial AD [FAD]) are characterized by an earlier onset and genetic inheritance. Mutations in presenilin-1 (PS1) and PS2 account for about 40% of all known FAD cases in which a genetic cause has been identified (1). Three missense mutations in PS1 have been suggested to be associated with frontal temporal dementia (FTD) (2), a neurological disorder that affects the frontal and temporal lobes of the brain. Presenilins are 50-kD proteins that contain 9 transmembrane domains (3, 4) and reside in the ER membrane (5). The complex of presenilins, that includes aph-1 and pen-2 subunits, is transported to the cell surface and endosomal structures, where it functions as γ-secretase. The γ-secretase cleaves the amyloid precursor protein (APP) and releases the amyloid β-peptide, the principal constituent of the amyloid plaques in the brains of AD patients. Consistent with the role of presenilins as catalytic subunits of γ-secretase (6, 7), FAD mutations in presenilins affect APP processing.

Missense mutations in presenilin 1 (PS1) and presenilin 2 (PS2) proteins are a major cause of familial Alzheimer disease. Presenilins are proteins with nine transmembrane (TM) domains that function as catalytic subunits of the ␥-secretase complex responsible for the cleavage of the amyloid precursor protein and other type I transmembrane proteins. The waterfilled cavity within presenilin is necessary to mediate the intramembrane proteolysis reaction. Consistent with this idea, cysteine-scanning mutagenesis and NMR studies revealed a number of water-accessible residues within TM7 and TM9 of mouse PS1. In addition to ␥-secretase function, presenilins also demonstrate a low conductance endoplasmic reticulum Ca 2؉ leak function, and many familial Alzheimer disease presenilin mutations impair this function. To map the potential Ca 2؉ conductance pore in PS1, we systematically evaluated endoplasmic reticulum Ca 2؉ leak activity supported by a series of cysteine point mutants in TM6, TM7, and TM9 of mouse PS1. The results indicate that TM7 and TM9, but not TM6, could play an important role in forming the conductance pore of PS1. These results are consistent with previous cysteine-scanning mutagenesis and NMR analyses of PS1 and provide further support for our hypothesis that the hydrophilic catalytic cavity of presenilins may also constitute a Ca 2؉ conductance pore.Presenilins belong to the family of aspartic proteases, and they are involved in regulated intramembrane proteolysis, a mechanism that is used to cleave peptide bonds within the lipid bilayer (1-3). Presenilins are 50-kDa proteins that contain nine transmembrane (TM) 5 domains (4, 5) and reside primarily in the endoplasmic reticulum (ER) membrane (6), with the N terminus oriented toward the cytosol and the C terminus toward the ER lumen. The complex of presenilins with nicastrin, APH-1, and PEN-2 subunits is transported to the cell surface and endosomal structures and functions as ␥-secretase, which cleaves the amyloid precursor protein and releases the amyloid ␤-peptide, the principal constituent of the amyloid plaques in the brains of Alzheimer disease patients (7). Following assembly, the presenilin 1 (PS1) and presenilin 2 (PS2) holoproteins undergo endoproteolysis in the cytosolic loop between TM6 and TM7, resulting in the generation of a 35-kDa N-terminal fragment and an 18 -20-kDa C-terminal fragment, which remain associated with each other in the "mature" ␥-secretase complex (8 -15). Mutation of the catalytic residue Asp-257 or Asp-385 in TM6 and TM7, respectively, abolishes the catalytic activity of presenilins as well as binding to transition state inhibitors of ␥-secretase (15-17). Consistent with the role of presenilins as the catalytic subunits of ␥-secretase (7, 15), genetic inactivation of the PS genes or familial Alzheimer disease (FAD) PS mutations affect amyloid precursor protein processing (18). There is increasing evidence that presenilins also have functions outside of ␥-secretase (3, 19). One of these functions is related to Ca 2ϩ sign...