Notch receptors and the amyloid precursor protein are type I membrane proteins that are proteolytically cleaved within their transmembrane domains by a presenilin (PS)-dependent ␥-secretase activity. In both proteins, two peptide bonds are hydrolyzed: one near the inner leaflet and the other in the middle of the transmembrane domain. Under saturating conditions the substrates compete with each other for proteolysis, but not for binding to PS. At least some Alzheimer's disease-causing PS mutations reside in proteins possessing low catalytic activity. We demonstrate (i) that differentially tagged PS molecules coimmunoprecipitate, and (ii) that PS N-terminal fragment dimers exist by using a photoaffinity probe based on a transition state analog ␥-secretase inhibitor. We propose that ␥-secretase contains a PS dimer in its catalytic core, that binding of substrate is at a site separate from the active site, and that substrate is cleaved at the interface of two PS molecules.

Gene knockout studies in mice suggest that presenilin 1 (PS1) is the major c-secretase and that it contributes disproportionately to amyloid b (Ab) peptide generation from b-amyloid precursor protein (APP), whereas PS2 plays a more minor role. Based on this and other observations we hypothesized that familial Alzheimer's disease (FAD) mutations in PS2 would have a dramatic effect on function in order to have an observable effect on Ab levels in the presence of normal PS1 alleles. Only four of the eight reported FAD mutations in PS2 have altered function in vitro suggesting that the other variants represent rare polymorphisms rather than disease-causing mutations. In support of our hypothesis, the four verified PS2 FAD mutations cause substantial changes in the Ab 42/40 ratio, comparable with PS1 mutations that cause very-earlyonset FAD. Most of the PS2 mutations also cause a significant decrease in Ab 40, APP C-terminal fragment (CTF)c and Notch intracellular domain (NICD) production suggesting that they are partial loss of function mutations. PS2 M239V, its PS1 homolog M233V, and other FAD mutations within transmembrane (TM) 5 of PS1 differentially affect CTFc and NICD production suggesting that TM5 of PS are important for c-secretase cleavage of APP but not Notch.

Alzheimer's disease (AD) is the most common form of dementia and is characterized pathologically by the accumulation of b-amyloid (Ab) plaques and neurofibrillary tangles in the brain. Genetic studies of AD first highlighted the importance of the presenilins (PS). Subsequent functional studies have demonstrated that PS form the catalytic subunit of the c-secretase complex that produces the Ab peptide, confirming the central role of PS in AD biology. Here, we review the studies that have characterized PS function in the c-secretase complex in Caenorhabditis elegans, mice and in in vitro cell culture systems, including studies of PS structure, PS interactions with substrates and other c-secretase complex members, and the evidence supporting the hypothesis that PS are aspartyl proteases that are active in intramembranous proteolysis. A thorough knowledge of the mechanism of PS cleavage in the context of the c-secretase complex will further our understanding of the molecular mechanisms that cause AD, and may allow the development of therapeutics that can alter Ab production and modify the risk for AD. Keywords: Alzheimer's disease, amyloid precursor protein, presenilin, presenilinase, c-secretase. Alzheimer's diseaseAlzheimer's disease (AD) is the most common cause of progressive neurodegeneration and is the fourth leading cause of death in the United States (Lendon et al. 1997). Risk for developing AD increases with age such that nearly 30% of 85-year-olds have AD (reviewed in Cummings 2004). AD is characterized pathologically by neuronal cell loss and the accumulation of neurofibrillary tangles (NFT) and senile plaques in the brain. The primary component of neuritic (senile) plaques is the amyloid-b peptide (Ab) (Glenner and Wong 1984a,b;Masters et al. 1985;Gorevic et al. 1986;Selkoe et al. 1986), which is produced by proteolytic processing of amyloid precursor protein (APP) (Fig 1). APP is first cleaved by either the a-or b-secretase activity to form the C83 or C99 fragments, respectively (Esch et al. 1990;Seubert et al. 1993). C83 and C99 are substrates for a second cleavage by the c-secretase activity at the cytoplasmic edge of the transmembrane domain (e-site) to produce a C-terminal fragment (CTFc) that may directly interact with transcription factors in the nucleus ( . c-Secretase is promiscuous and produces Ab peptides that range in length from 37 to 43 residues (Dovey et al. 1993;Wang et al. 1996).Genetic studies in families that have a history of AD (FAD) have identified four genes that contribute to AD: the e4 allele of apolipoprotein E is a risk factor while causative mutations have been demonstrated in the genes encoding b-APP, presenilin-1 and -2 (PS1, PS2). Most mutations in APP and PS increase the ratio of a 42-residue form of Ab et al. 1996). The vast majority of these mutations have been reported in the PS genes (Cruts and Rademakers 2004). As a result, much research has focused on understanding the role of PS in APP metabolism and the molecular mechanisms that cause AD, with the goal o...

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