The pathogenic event common to all forms of Alzheimer's disease is the abnormal accumulation of the amyloid beta-peptide (Abeta). Here we provide strong evidence that intracellular cholesterol compartmentation modulates the generation of Abeta. Using genetic, biochemical and metabolic approaches, we found that cholesteryl-ester levels are directly correlated with Abeta production. Acyl-coenzyme A:cholesterol acyltransferase (ACAT), the enzyme that catalyses the formation of cholesteryl esters, modulates the generation of Abeta through the tight control of the equilibrium between free cholesterol and cholesteryl esters. We also show that pharmacological inhibitors of ACAT, developed for the treatment of atherosclerosis, are potent modulators of Abeta generation, indicating their potential for use in the treatment of Alzheimer's disease.

We studied a novel function of the presenilins (PS1 and PS2) in governing capacitative calcium entry (CCE), a refilling mechanism for depleted intracellular calcium stores. Abrogation of functional PS1, by either knocking out PS1 or expressing inactive PS1, markedly potentiated CCE, suggesting a role for PS1 in the modulation of CCE. In contrast, familial Alzheimer's disease (FAD)-linked mutant PS1 or PS2 significantly attenuated CCE and store depletion-activated currents. While inhibition of CCE selectively increased the amyloidogenic amyloid beta peptide (Abeta42), increased accumulation of the peptide had no effect on CCE. Thus, reduced CCE is most likely an early cellular event leading to increased Abeta42 generation associated with FAD mutant presenilins. Our data indicate that the CCE pathway is a novel therapeutic target for Alzheimer's disease.

The morphological transition of growth cones to synaptic boutons characterizes synaptogenesis. Here we have isolated mutations in immaculate connections (imac; CG8566), a previously uncharacterized Drosophila gene encoding a member of the Kinesin-3 family. Whereas earlier studies in Drosophila implicated Kinesin-1 in transporting synaptic vesicle precursors, we find that Imac is essential for this transport. An unexpected feature of imac mutants is the failure of synaptic boutons to form. Motor neurons lacking imac properly target to muscles but remain within target fields as thin processes, a structure that is distinct from either growth cones or mature terminals. Few active zones form at these endings. We show that the arrest of synaptogenesis is not a secondary consequence of the absence of transmission. Our data thus indicate that Imac transports components required for synaptic maturation and provide insight into presynaptic maturation as a process that can be differentiated from axon outgrowth and targeting.

Perturbed Ca2؉ homeostasis is a common molecular consequence of familial Alzheimer's disease-linked presenilin mutations. We report here the molecular interaction of the large hydrophilic loop region of presenilin 2 (PS2) with sorcin, a penta-EF-hand Ca 2؉ -binding protein that serves as a modulator of the ryanodine receptor intracellular Ca 2؉ channel. The association of endogenous sorcin and PS2 was demonstrated in cultured cells and human brain tissues. Membrane-associated sorcin and a subset of the functional PS2 complexes were co-localized to a novel subcellular fraction that is distinctively positive for calcineurin B. Sorcin was found to interact with PS2 endoproteolytic fragments but not full-length PS2, and the sorcin/PS2 interaction was greatly enhanced by treatment with the Ca 2؉ ionophore A23187. Our findings reveal a molecular link between PS2 and intracellular Ca 2؉ channels (i.e. ryanodine receptor) and substantiate normal and/or pathological roles of PS2 in intracellular Ca 2؉ homeostasis.Nearly half of early-onset familial Alzheimer's disease (FAD) 1 is associated with mutations in genes encoding two homologous proteins, presenilin 1 (PS1) and presenilin 2 (PS2) (1). Recent studies have shown that PS1 (and perhaps PS2) plays an essential role in the ␥-secretase cleavage of amyloid ␤-protein precursor (2-4) and the trafficking/maturation of other select cellular proteins, including Notch and TrkB (5-8). Common molecular consequences of presenilin FAD mutations include the increased production of amyloid ␤-peptide x-42 and increased apoptosis (reviewed in Refs. 9 -11). In addition, FAD mutations in both PS1 and PS2 have been shown to disrupt intracellular Ca 2ϩ homeostasis (12, 13). However, the mechanism by which Ca 2ϩ dyshomeostasis contributes to FAD pathogenesis is still unresolved.Recently, a number of molecules that form complexes with the presenilins have been identified, including ␤-and ␦-catenin (14 -19), p0071 (20), amyloid ␤-protein precursor (21), filamin/ Fh-1 (22), Notch (23), GSK3␤ (24), Rab11 (25), calsenilin (26), calmyrin (27), QM/Jif-1 (28), and Bcl-X L (29). It is currently unclear whether these interactions mediate pathogenesis in presenilin FAD. It is also noteworthy that some of these proteins have been shown to interact either preferentially or exclusively with full-length presenilin over the N-or C-terminal fragments. Since only a subset of presenilin proteins are cleaved to form stable, functional presenilin complexes (30 -35), and the remaining full-length proteins are degraded by the proteasome (32, 36 -38), proteins that interact with the Nand/or C-terminal presenilin fragments are more likely to mediate presenilin function as opposed to maturation of the presenilins. Additionally, many of these presenilin-interacting proteins have been identified and characterized using overexpression in cell systems, whereas only a few have been demonstrated to interact with PS1 and/or PS2 endogenously (e.g. ␤-catenin).Although the N-terminal and loop domains are not conserved betw...

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