Presenilins are components of the gamma-secretase protein complex that mediates intramembranous cleavage of betaAPP and Notch proteins. A C. elegans genetic screen revealed two genes, aph-1 and pen-2, encoding multipass transmembrane proteins, that interact strongly with sel-12/presenilin and aph-2/nicastrin. Human aph-1 and pen-2 partially rescue the C. elegans mutant phenotypes, demonstrating conserved functions. The human genes must be provided together to rescue the mutant phenotypes, and the inclusion of presenilin-1 improves rescue, suggesting that they interact closely with each other and with presenilin. RNAi-mediated inactivation of aph-1, pen-2, or nicastrin in cultured Drosophila cells reduces gamma-secretase cleavage of betaAPP and Notch substrates and reduces the levels of processed presenilin. aph-1 and pen-2, like nicastrin, are required for the activity and accumulation of gamma-secretase.

Acute, s.c. administration of a ␥-secretase inhibitor, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), to young PDAPP mice dose dependently decreases cortical amyloid-␤ (A␤). The present studies replicated these findings in Tg2576 mice and examined further whether DAPT would reduce cerebrospinal fluid (CSF) A␤ comparably in young (plaque-free) and aged (plaque-bearing) mice. In the first study, vehicle or DAPT (10, 30, or 100 mg/kg s.c.) administered to young Tg2576 mice (6 months old) dose dependently reduced A␤ peptide levels in the cortex as seen previously in the PDAPP mice. Additionally, a dose-dependent decrease in plasma A␤ levels was evident. The same dosing regime was applied next to aged mice (17 months old) to assess A␤ changes in the CSF in addition to plasma and brains. DAPT dose dependently reduced A␤ levels in the CSF and plasma, but not in the brain wherein A␤ levels were 400 to 500 times higher than those in young mice, consistent with a large pool of A␤ extracted from amyloid deposits. In subsequent studies, effects of oral DAPT (100 or 200 mg/kg) were examined concurrently in young and aged mice. DAPT reduced A␤ levels in CSF and plasma to a similar extent at both ages. In contrast, DAPT reduced brain A␤ levels primarily in young mice, with minimal effects in aged mice. These results demonstrate that A␤ levels in CSF and plasma decrease dose dependently after ␥-secretase inhibition, and this response is not affected by amyloid plaque burden. We conclude that CSF and plasma A␤ may offer a clinically applicable, mechanism-based biomarker for inhibitors of A␤ production.

Riluzole was tested in a dose-ranging study for preservation of motor function in a transgenic mouse model of familial ALS. The model is based on expression of mutant human Cu,Zn superoxide dismutase in mouse brain and spinal cord. In contrast with the human ALS trials, in the mouse model, riluzole significantly preserved motor function as assessed by nightly running in a wheel. The effect of riluzole on motor performance was greater earlier in disease than later, suggesting that riluzole may have benefit for "quality-of-life" measures in ALS. Treatment with riluzole was initiated earlier in the transgenic model than in the human ALS trials, which may account for the significantly better outcome.

Nicastrin is a component of the ␥-secretase complex that has been shown to adhere to presenilin-1 (PS1), Notch, and APP. Here we demonstrate that Nicastrindeficient mice showed a phenotype that is indistinguishable from PS1/PS2 double knock-out mice, whereas heterozygotes were healthy and viable. Fibroblasts derived from Nicastrin-deficient embryos were unable to generate amyloid ␤-peptide and failed to release the intracellular domain of APP-or Notch1-Gal4-VP16 fusion proteins. Additionally, C-and N-terminal fragments of PS1 and the C-terminal fragments of PS2 were not detectable in Nicastrin-null fibroblasts, whereas full-length PS1 accumulated in null fibroblasts, indicating that Nicastrin is required for the endoproteolytic processing of presenilins. Interestingly, cells derived from Nicastrin heterozygotes produced relatively higher levels of amyloid ␤-peptide whether the source was endogenous mouse or transfected human APP. These data demonstrate that Nicastrin is essential for the ␥-secretase cleavage of APP and Notch in mammalian cells and that Nicastrin has both positive and negative functions in the regulation of ␥-secretase activity.

Presenilin (PS1 or PS2) is an essential component of the active γ-secretase complex that liberates the Aβ peptides from amyloid precursor protein (APP). PS1 is regarded as an atypical aspartyl protease harboring two essential aspartic acids in the context of the sequence D257LV and D385FI, respectively, rather than the typical DTG...DTG catalytic motif of classical aspartyl proteases. In the present studies, we introduced the sequence DTG in PS1 at and around the catalytic D257 and D385 residues to generate three PS1 mutants: D257TG, D385TG, and the double-mutant D257TG/D385TG. The effects of these changes on the γ-secretase activity in the presence or absence of γ-secretase inhibitors and modulators were investigated. The results showed that PS1 mutants having D385TG robustly enhanced Aβ 42 production compared to the wild type (wt), and were more sensitive than wt to inhibition by a classical aspartyl protease transition state mimic, and fenchylamine, a sulfonamide derivative. Unlike wt PS1 and some of its clinical mutants, all three PS1 artificial mutants decreased cleavage of Notch S3-site, suggesting that these artificial mutations may trigger conformational changes at the substrate docking and catalytic site that cause alteration of substrate specificity and inhibition pattern. Consistent with this notion, we have found that NSAID enzymatic inhibitors of COX, known modulators of the γ-secretase activity, cause PS1 mutants containing D385TG to produce higher levels of both Aβ 38 and Aβ 42 , but to reduce levels of Aβ 39 , showing a pattern of Aβ formation different from that observed with wild type PS1 and its clinical mutants. This study provides an important structural clue for the rational design of drugs to inhibit processing of APP at the γ-site without interfering with Notch processing.

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