No abstract

The loss of synapses and neurons in Alzheimer's disease (AD) is thought to be, at least partly, induced by toxic species formed by the amyloid b-peptide (Ab) [1]. Ab is produced from the amyloid precursor protein (APP) by sequential proteolytic cleavages mediated by b-secretase (BACE) and c-secretase [2]. An initial cleavage by b-secretase produces soluble APP (b-APPs) and a membrane-bound C-terminal fragment (C99) that is cleaved by c-secretase, generating the APP intracellular domain (AICD) and Ab. Two major forms of this amyloidogenic peptide are produced, Ab40 and Ab42, the latter being less abundant but more prone to aggregation [3][4][5] Several lines of evidence suggest that polymerization of the amyloid b-peptide (Ab) into amyloid plaques is a pathogenic event in Alzheimer's disease (AD). Ab is produced from the amyloid precursor protein as the result of sequential proteolytic cleavages by b-secretase and c-secretase, and it has been suggested that these enzymes could be targets for treatment of AD. c-Secretase is an aspartyl protease complex, containing at least four transmembrane proteins. Studies in cell lines have shown that c-secretase is partially localized to lipid rafts, which are detergent-resistant membrane microdomains enriched in cholesterol and sphingolipids. Here, we studied c-secretase in detergent-resistant membranes (DRMs) prepared from human brain. DRMs prepared in the mild detergent CHAPSO and isolated by sucrose gradient centrifugation were enriched in c-secretase components and activity. The DRM fraction was subjected to size-exclusion chromatography in CHAPSO, and all of the c-secretase components and a lipid raft marker were found in the void volume (> 2000 kDa). Co-immunoprecipitation studies further supported the notion that the c-secretase components are associated even at high concentrations of CHAPSO. Preparations from rat brain gave similar results and showed a postmortem time-dependent decline in c-secretase activity, suggesting that DRMs from fresh rat brain may be useful for c-secretase activity studies. Finally, confocal microscopy showed co-localization of c-secretase components and a lipid raft marker in thin sections of human brain. We conclude that the active c-secretase complex is localized to lipid rafts in human brain.

Alzheimer’s disease (AD) is caused by synaptic and neuronal loss in the brain. One of the characteristic hallmarks of AD is senile plaques containing amyloid β-peptide (Aβ). Aβ is produced from amyloid precursor protein (APP) by sequential proteolytic cleavages by β-secretase and γ-secretase, and the polymerization of Aβ into amyloid plaques is thought to be a key pathogenic event in AD. Since γ-secretase mediates the final cleavage that liberates Aβ, γ-secretase has been widely studied as a potential drug target for the treatment of AD. γ-Secretase is a transmembrane protein complex containing presenilin, nicastrin, Aph-1, and Pen-2, which are sufficient for γ-secretase activity. γ-Secretase cleaves >140 substrates, including APP and Notch. Previously, γ-secretase inhibitors (GSIs) were shown to cause side effects in clinical trials due to the inhibition of Notch signaling. Therefore, more specific regulation or modulation of γ-secretase is needed. In recent years, γ-secretase modulators (GSMs) have been developed. To modulate γ-secretase and to understand its complex biology, finding the binding sites of GSIs and GSMs on γ-secretase as well as identifying transiently binding γ-secretase modulatory proteins have been of great interest. In this review, decades of findings on γ-secretase in AD are discussed.

Background:In AD, APP can be processed in lipid rafts, and ␥-secretase-associated proteins (GSAPs) can affect A␤ production. Results: We identify novel GSAPs in detergent-resistant membranes (DRMs). Conclusion: VDAC1 and CNTNAP1 associate with ␥-secretase in DRMs and affect APP processing with less effect on Notch processing. Significance: Novel GSAPs that regulate A␤ production can be used as AD therapeutic targets.

BackgroundA key player in the development of Alzheimer's disease (AD) is the γ-secretase complex consisting of at least four components: presenilin, nicastrin, Aph-1 and Pen-2. γ-Secretase is crucial for the generation of the neurotoxic amyloid β-peptide (Aβ) but also takes part in the processing of many other substrates. In cell lines, active γ-secretase has been found to localize primarily to the Golgi apparatus, endosomes and plasma membranes. However, no thorough studies have been performed to show the subcellular localization of the active γ-secretase in the affected organ of AD, namely the brain.Principal FindingsWe show by subcellular fractionation of rat brain that high γ-secretase activity, as assessed by production of Aβ40, is present in an endosome- and plasma membrane-enriched fraction of an iodixanol gradient. We also prepared crude synaptic vesicles as well as synaptic membranes and both fractions showed high Aβ40 production and contained high amounts of the γ-secretase components. Further purification of the synaptic vesicles verified the presence of the γ-secretase components in these compartments. The localization of an active γ-secretase in synapses and endosomes was confirmed in rat brain sections and neuronal cultures by using a biotinylated γ-secretase inhibitor together with confocal microscopy.SignificanceThe information about the subcellular localization of γ-secretase in brain is important for the understanding of the molecular mechanisms of AD. Furthermore, the identified fractions can be used as sources for highly active γ-secretase.