Store-operated Ca2+ (SOC) channels regulate many cellular processes, but the underlying molecular components are not well defined. Using an RNA interference (RNAi)-based screen to identify genes that alter thapsigargin (TG)-dependent Ca2+ entry, we discovered a required and conserved role of Stim in SOC influx. RNAi-mediated knockdown of Stim in Drosophila S2 cells significantly reduced TG-dependent Ca2+ entry. Patch-clamp recording revealed nearly complete suppression of the Drosophila Ca2+ release-activated Ca2+ (CRAC) current that has biophysical characteristics similar to CRAC current in human T cells. Similarly, knockdown of the human homologue STIM1 significantly reduced CRAC channel activity in Jurkat T cells. RNAi-mediated knockdown of STIM1 inhibited TG- or agonist-dependent Ca2+ entry in HEK293 or SH-SY5Y cells. Conversely, overexpression of STIM1 in HEK293 cells modestly enhanced TG-induced Ca2+ entry. We propose that STIM1, a ubiquitously expressed protein that is conserved from Drosophila to mammalian cells, plays an essential role in SOC influx and may be a common component of SOC and CRAC channels.

Amyloid precursor protein (APP) is endoproteolytically processed by BACE1 and gamma-secretase to release amyloid peptides (Abeta40 and 42) that aggregate to form senile plaques in the brains of patients with Alzheimer's disease (AD). The C-terminus of Abeta40/42 is generated by gamma-secretase, whose activity is dependent upon presenilin (PS 1 or 2). Missense mutations in PS1 (and PS2) occur in patients with early-onset familial AD (FAD), and previous studies in transgenic mice and cultured cell models demonstrated that FAD-PS1 variants shift the ratio of Abeta40 : 42 to favor Abeta42. One hypothesis to explain this outcome is that mutant PS alters the specificity of gamma-secretase to favor production of Abeta42 at the expense of Abeta40. To test this hypothesis in vivo, we studied Abeta40 and 42 levels in a series of transgenic mice that co-express the Swedish mutation of APP (APPswe) with two FAD-PS1 variants that differentially accelerate amyloid pathology in the brain. We demonstrate a direct correlation between the concentration of Abeta42 and the rate of amyloid deposition. We further show that the shift in Abeta42 : 40 ratios associated with the expression of FAD-PS1 variants is due to a specific elevation in the steady-state levels of Abeta42, while maintaining a constant level of Abeta40. These data suggest that PS1 variants do not simply alter the preferred cleavage site for gamma-secretase, but rather that they have more complex effects on the regulation of gamma-secretase and its access to substrates.

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Summary Alzheimer's disease (AD) is characterized pathologically by the abundance of senile plaques and neurofibrillary tangles in the brain. We synthesized over 1200 novel gamma-secretase modulator (GSM) compounds that reduced Abeta42 levels without inhibiting epsilon-site cleavage of APP and Notch, the generation of the APP and Notch intracellular domains, respectively. These compounds also reduced Abeta40 levels while concomitantly elevating levels of Abeta38 and Abeta37. Immobilization of a potent GSM onto an agarose matrix quantitatively recovered Pen-2 and to a lesser degree PS-1 NTFs from cellular extracts. Moreover, oral administration (once daily) of another potent GSM to Tg 2576 transgenic AD mice displayed dose-responsive lowering of plasma and brain Abeta42; chronic daily administration led to significant reductions in both diffuse and neuritic plaques. These effects were observed in the absence of Notch-related changes (e.g. intestinal proliferation of goblet cells), which are commonly associated with repeated exposure to functional gamma-secretase inhibitors (GSIs).

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