Macroautophagy, which is a lysosomal pathway for the turnover of organelles and long-lived proteins, is a key determinant of cell survival and longevity. In this study, we show that neuronal macroautophagy is induced early in Alzheimer's disease (AD) and before β-amyloid (Aβ) deposits extracellularly in the presenilin (PS) 1/Aβ precursor protein (APP) mouse model of β-amyloidosis. Subsequently, autophagosomes and late autophagic vacuoles (AVs) accumulate markedly in dystrophic dendrites, implying an impaired maturation of AVs to lysosomes. Immunolabeling identifies AVs in the brain as a major reservoir of intracellular Aβ. Purified AVs contain APP and β-cleaved APP and are highly enriched in PS1, nicastrin, and PS-dependent γ-secretase activity. Inducing or inhibiting macroautophagy in neuronal and nonneuronal cells by modulating mammalian target of rapamycin kinase elicits parallel changes in AV proliferation and Aβ production. Our results, therefore, link β-amyloidogenic and cell survival pathways through macroautophagy, which is activated and is abnormal in AD.

Oxidizing conditions must be maintained in the endoplasmic reticulum (ER) to allow the formation of disulfide bonds in secretory proteins. Here we report the cloning and characterization of a mammalian gene (ERO1-L) that shares extensive homology with the Saccharomyces cerevisiae ERO1 gene, required in yeast for oxidative protein folding. When expressed in mammalian cells, the product of the human ERO1-L gene colocalizes with ER markers and displays Endo-H-sensitive glycans. In isolated microsomes, ERO1-L behaves as a type II integral membrane protein. ERO1-L is able to complement several phenotypic traits of the yeast thermosensitive mutant ero1-1, including temperature and dithiothreitol sensitivity, and intrachain disulfide bond formation in carboxypeptidase Y. ERO1-L is no longer functional when either one of the highly conserved Cys-394 or Cys-397 is mutated. These results strongly suggest that ERO1-L is involved in oxidative ER protein folding in mammalian cells.

Mitochondria are central in the regulation of cell death. Apart from providing the cell with ATP, mitochondria also harbor several death factors that are released upon apoptotic stimuli. Alterations in mitochondrial functions, increased oxidative stress, and neurons dying by apoptosis have been detected in Alzheimer's disease patients. These findings suggest that mitochondria may trigger the abnormal onset of neuronal cell death in Alzheimer's disease. We previously reported that presenilin 1 (PS1), which is often mutated in familial forms of Alzheimer's disease, is located in mitochondria and hypothesized that presenilin mutations may sensitize cells to apoptotic stimuli at the mitochondrial level. Presenilin forms an active ␥-secretase complex together with Nicastrin (NCT), APH-1, and PEN-2, which among other substrates cleaves the ␤-amyloid precursor protein (␤-APP) generating the amyloid ␤-peptide and the ␤-APP intracellular domain. Here we have identified dual targeting sequences (for endoplasmic reticulum and mitochondria) in NCT and showed expression of NCT in mitochondria by immunoelectron microscopy. We also showed that NCT together with APH-1, PEN-2, and PS1 form a high molecular weight complex located in mitochondria. ␥-Secretase activity in isolated mitochondria was demonstrated using C83 (␣-secretasecleaved C-terminal 83-residue ␤-APP fragment from BD8 cells lacking presenilin and thus ␥-secretase activity) or recombinant C100-Flag (C-terminal 100-residue ␤-APP fragment) as substrates. Both systems generated an APP intracellular domain, and the activity was inhibited by the ␥-secretase inhibitors L-685,458 or Compound E. This novel localization of NCT, PS1, APH-1, and PEN-2 expands the role and importance of ␥-secretase activity to mitochondria.

One characteristic feature of Alzheimer's disease is the deposition of amyloid beta-peptide (Abeta) as amyloid plaques within specific regions of the human brain. Abeta is derived from the amyloid beta-peptide precursor protein (beta-APP) by the intramembranous cleavage activity of gamma-secretase. Studies in cells have revealed that gamma-secretase is a large multimeric membrane-bound protein complex that is functionally dependent on several proteins, including presenilin, nicastrin, Aph-1, and Pen-2. However, the precise biochemical and molecular nature of gamma-secretase is as yet to be fully elucidated, and no investigations have analyzed gamma-secretase in human brain. To address this we have developed a novel in vitro gamma-secretase activity assay using detergent-solubilized cell membranes and a beta-APP-derived fluorescent probe. We report that human brain-derived gamma-secretase activity co-purifies with a high molecular weight protein complex comprising presenilin, nicastrin, Aph-1, and Pen-2. The inhibitor profile and solubility characteristics of brain-derived gamma-secretase are similar to those described in cells, and proteolysis occurs at the Abeta40- and Abeta42-generating cleavage sites. The ability to isolate gamma-secretase from post-mortem human brain may facilitate the identification of brain-specific modulators of beta-APP processing and provide new insights into the biology of this important factor in the pathogenesis of Alzheimer's disease.

Presenilin (PS) provides the catalytic core of the ␥-secretase complex. ␥-Secretase activity leads to generation of the amyloid ␤-peptide, a key event implicated in the pathogenesis of Alzheimer disease. PS has ten hydrophobic regions, which can all theoretically form membrane-spanning domains. Various topology models have been proposed, and the prevalent view holds that PS has an eighttransmembrane (TM) domain organization; however, the precise topology has not been unequivocally determined. Previous topological studies are based on non-functional truncated variants of PS proteins fused to reporter domains, or immunocytochemical staining. In this study, we used a more subtle N-linked glycosylation scanning approach, which allowed us to assess the topology of functional PS1 molecules. Glycosylation acceptor sequences were introduced into full-length human PS1, and the results showed that the first hydrophilic loop is oriented toward the lumen of the endoplasmic reticulum, whereas the N terminus and large hydrophilic loop are in the cytosol. Although this is in accordance with most current models, our data unexpectedly revealed that the C terminus localized to the luminal side of the endoplasmic reticulum. Additional studies on the glycosylation pattern after TM domain deletions, combined with computer-based TM protein topology predictions and biotinylation assays of different PS1 mutants, led us to conclude that PS1 has nine TM domains and that the C terminus locates to the lumen/extracellular space.Most proteases operate in aqueous environments, but ␥-secretase, along with other intramembrane cleaving proteases, is unusual in the sense that it executes proteolysis in the lipid bilayer (1). ␥-Secretase is a high molecular weight protein complex composed of presenilin (PS) 2 1 or 2, nicastrin, Aph-1, and Pen-2 (2-5). Growing evidence suggests that the polytopic PS1 protein is a TM aspartyl protease and that two of its aspartate residues, Asp-257 in hydrophobic domain (HD) 6 and Asp-385 in HD 8, form the catalytic site of the ␥-secretase complex (6 -8). An endoproteolytic cleavage within HD 7 of PS1 generates an N-terminal fragment (NTF) and a C-terminal fragment (CTF) (9). The NTF/ CTF heterodimer is believed to be the biologically active form of PS. Nicastrin, Aph-1, and Pen-2 are all essential cofactors in the ␥-secretase complex, but their precise biochemical functions are not fully known. Nicastrin and Aph-1 have been proposed to form a subcomplex that can stabilize full-length PS, and the final addition of Pen-2 has been suggested to trigger PS endoproteolysis (10) and stabilize the NTF/CTF heterodimer (11). ␥-Secretase cleavage occurs within the lipid bilayer of the membrane through a mechanism referred to as "regulated intramembrane proteolysis" (12). Recently, not only APP, but a large number of other type-1 TM proteins, such as the Notch receptors, CD44 and E-cadherin, have been shown to undergo PS-dependent regulated intramembrane proteolysis (reviewed in Ref. 13). Given that target proteins of ␥-secret...