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Alzheimer disease is associated with extracellular deposits of amyloid ␤-peptides in the brain. Amyloid ␤-peptides are generated by proteolytic processing of the ␤-amyloid precursor protein by ␤-and ␥-secretases. The cleavage by secretases occurs predominantly in postGolgi secretory and endocytic compartments and is influenced by cholesterol, indicating a role of the membrane lipid composition in proteolytic processing of the ␤-amyloid precursor protein. To analyze the role of glycosphingolipids in these processes we inhibited glycosyl ceramide synthase, which catalyzes the first step in glycosphingolipid biosynthesis. The depletion of glycosphingolipids markedly reduced the secretion of endogenous ␤-amyloid precursor protein in different cell types, including human neuroblastoma SH-SY5Y cells. Importantly, secretion of amyloid ␤-peptides was also strongly decreased by inhibition of glycosphingolipid biosynthesis. Conversely, the addition of exogenous brain gangliosides to cultured cells reversed these effects. Biochemical and cell biological experiments demonstrate that the pharmacological reduction of cellular glycosphingolipid levels inhibited maturation and cell surface transport of the ␤-amyloid precursor protein. In the glycosphingolipid-deficient cell line GM95, cellular levels and maturation of ␤-amyloid precursor protein were also significantly reduced as compared with normal B16 cells. Together, these data demonstrate that glycosphingolipids are implicated in the regulation of the subcellular transport of the ␤-amyloid precursor protein in the secretory pathway and its proteolytic processing. Thus, enzymes involved in glycosphingolipid metabolism might represent targets to inhibit the production of amyloid ␤-peptides.

Matriptase-2 is a member of the TTSPs (type II transmembrane serine proteases), an emerging class of cell surface proteases involved in tissue homoeostasis and several human disorders. Matriptase-2 exhibits a domain organization similar to other TTSPs, with a cytoplasmic N-terminus, a transmembrane domain and an extracellular C-terminus containing the non-catalytic stem region and the protease domain. To gain further insight into the biochemical functions of matriptase-2, we characterized the subcellular localization of the monomeric and multimeric form and identified cell surface shedding as a defining point in its proteolytic processing. Using HEK (human embryonic kidney)-293 cells, stably transfected with cDNA encoding human matriptase-2, we demonstrate a cell membrane localization for the inactive single-chain zymogen. Membrane-associated matriptase-2 is highly N-glycosylated and occurs in monomeric, as well as multimeric, forms covalently linked by disulfide bonds. Furthermore, matriptase-2 undergoes shedding into the conditioned medium as an activated two-chain form containing the catalytic domain, which is cleaved at the canonical activation motif, but is linked to a released portion of the stem region via a conserved disulfide bond. Cleavage sites were identified by MS, sequencing and mutational analysis. Interestingly, cell surface shedding and activation of a matriptase-2 variant bearing a mutation at the active-site serine residue is dependent on the catalytic activity of co-expressed or co-incubated wild-type matriptase-2, indicating a transactivation and trans-shedding mechanism.

Presenilins (PSs) are components of the ␥-secretase complex that mediates intramembranous cleavage of type I membrane proteins. We show that ␥-secretase is involved in the regulation of cellular lipoprotein uptake. Loss of ␥-secretase function decreased endocytosis of low-density lipoprotein (LDL) receptor. The decreased uptake of lipoproteins led to upregulation of cellular cholesterol biosynthesis by increased expression of CYP51 and enhanced metabolism of lanosterol. Genetic deletion of PS1 or transgenic expression of PS1 mutants that cause early-onset Alzheimer's disease led to accumulation of ␥-secretase substrates and mistargeting of adaptor proteins that regulate endocytosis of the LDL receptor. Consistent with decreased endocytosis of these receptors, PS1 mutant mice have elevated levels of apolipoprotein E in the brain. Thus, these data demonstrate a functional link between two major genetic factors that cause early-onset and late-onset Alzheimer's disease.

Processing of amyloid precursor protein (APP) is a well acknowledged central pathogenic mechanism in Alzheimer disease. However, influences of age-associated cellular alterations on the biochemistry of APP processing have not been studied in molecular detail so far. Here, we report that processing of endogenous APP is down-regulated during the aging of normal human fibroblasts (IMR-90). The generation of intracellular APP cleavage products C99, C83, and AICD gradually declines with increasing life span and is accompanied by a reduced secretion of soluble APP (sAPP) and sAPP␣. Further, the maturation of APP was reduced in senescent cells, which has been shown to be directly mediated by age-associated increased cellular cholesterol levels. Of the APP processing secretases, protein levels of constituents of the ␥-secretase complex, presenilin-1 (PS1) and nicastrin, were progressively reduced during aging, resulting in a progressive decrease in ␥-secretase enzymatic activity. ADAM10 (a disintegrin and metalloprotease 10) and BACE (␤-site APP-cleaving enzyme) protein levels exhibited no age-associated regulation, but interestingly, BACE enzymatic activity was increased in aged cells. PS1 and BACE are located in detergentresistant membranes (DRMs), well structured membrane microdomains exhibiting high levels of cholesterol, and caveolin-1. Although total levels of both structural components of DRMs were up-regulated in aged cells, their particular DRM association was decreased. This age-dependent membrane modification was associated with an altered distribution of PS1 and BACE between DRM and non-DRM fractions, very likely affecting their APP processing potential. In conclusion, we have found a significant modulation of endogenous APP processing and maturation in human fibroblasts caused by age-associated alterations in cellular biochemistry.Aging is the most prevailing risk factor of Alzheimer disease, even though the biochemical basis of this association is unknown. A significant pathological feature of Alzheimer disease is the appearance of senile plaques that are composed primarily of amyloid ␤ (A␤), 2 a 38 -42-amino-acid peptide derived from proteolytic processing of the ubiquitously expressed amyloid precursor protein (APP) (1). At least three APP processing secretases are identified. ADAM10 (␣-secretase) is involved in non-amyloidogenic processing and cleaves APP within the A␤ domain, whereby release of A␤ is prevented and soluble sAPP␣ is secreted (2). Amyloidogenic processing is driven by BACE (␤-secretase), which cleaves APP at the N-terminal site of the A␤ domain (3). Subsequently, a complex of presenilin, nicastrin, anterior pharynx defective-1 and presenilin enhancer-2 (␥-secretase complex) cleaves the generated C-terminal fragments (CTFs) C83 or C99 at the C-terminal site of the A␤ domain (4 -6). This results in the generation of the APP intracellular domain (AICD), and in the amyloidogenic pathway, A␤ is released (7,8).BACE and the ␥-secretase complex have been well described to be associated to deterge...

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Presenilins (PS) are critical components of the ␥-secretase complex that mediates cleavage of type I membrane proteins including the ␤-amyloid precursor protein to generate the amyloid ␤-peptide. In addition, PS1 interacts with ␤-catenin and facilitates its metabolism. We demonstrate that phosphorylation of serines 353 and 357 by glycogen synthase kinase-3␤ (GSK3␤) induces a structural change of the hydrophilic loop of PS1 that can also be mimicked by substitution of the phosphorylation sites by negatively charged amino acids in vitro and in cultured cells. The structural change of PS1 reduces the interaction with ␤-catenin leading to decreased phosphorylation and ubiquitination of ␤-catenin. The decreased interaction of PS1 with ␤-catenin leads to stabilization of ␤-catenin thereby increasing its nuclear signaling and the transcription of target genes, including c-MYC. Consistent with increased expression of c-myc, a PS1 mutant that mimics phosphorylated PS1 increased cell proliferation as compared with wild-type PS1. These results indicate a regulatory mechanism in which GSK3␤-mediated phosphorylation induces a structural change of the hydrophilic loop of PS1 thereby negatively modulating the formation of a ternary complex between ␤-catenin, PS1, and GSK3␤, which leads to stabilization of ␤-catenin.