Interaction with telencephalin and the amyloid precursor protein predicts a ring structure for presenilins.

The carboxyl terminus of presenilin 1 and 2 (PS1 and PS2) binds to the neuron-specific cell adhesion molecule telencephalin (TLN) in the brain. PS1 deficiency results in the abnormal accumulation of TLN in a yet unidentified intracellular compartment. The first transmembrane domain and carboxyl terminus of PS1 form a binding pocket with the transmembrane domain of TLN. Remarkably, APP binds to the same regions via part of its transmembrane domain encompassing the critical residues mutated in familial Alzheimer's disease. Our data surprisingly indicate a spatial dissociation between the binding site and the proposed catalytic site near the critical aspartates in PSs. They provide important experimental evidence to support a ring structure model for PS.

The amyloid precursor protein (APP)-cytoplasmic fragment generated by gamma-secretase is rapidly degraded but distributes partially in a nuclear fraction of neurones in culture.

The gamma-secretase cleavage is the last step in the generation of the beta-amyloid peptide (Abeta) from the amyloid precursor protein (APP). The Abeta precipitates in the amyloid plaques in the brain of Alzheimer's disease patients. The fate of the intracellular APP carboxy-terminal stub generated together with Abeta has been, in contrast, only poorly documented. The analogies between the processing of APP and other transmembrane proteins like SREBP and Notch suggests that this intracellular fragment could have important signalling functions. We demonstrate here that APP-C59 is rapidly degraded (half-life approximately 5 min) when overexpressed in baby hamster kidney cells or primary cultures of neurones by a mechanism that is not inhibited by endosomal/lysosomal or proteasome inhibitors. Furthermore, APP-C59 binds to the DNA binding protein Fe65, although this does not increase the half-life of APP-C59. Finally, we demonstrate that a fraction of APP-C59 becomes redistributed to the nuclear detergent-insoluble pellet, in which the transcription factor SP1 is also present. Overall our results reinforce the analogy between Notch and APP processing, and suggest that the APP intracellular domain, like the Notch intracellular domain, could have a role in signalling events from the plasma membrane to the nucleus.

The discrepancy between presenilin subcellular localization and gamma-secretase processing of amyloid precursor protein.

We investigated the relationship between PS1 and gamma-secretase processing of amyloid precursor protein (APP) in primary cultures of neurons. Increasing the amount of APP at the cell surface or towards endosomes did not significantly affect PS1-dependent gamma-secretase cleavage, although little PS1 is present in those subcellular compartments. In contrast, almost no gamma-secretase processing was observed when holo-APP or APP-C99, a direct substrate for gamma-secretase, were specifically retained in the endoplasmic reticulum (ER) by a double lysine retention motif. Nevertheless, APP-C99-dilysine (KK) colocalized with PS1 in the ER. In contrast, APP-C99 did not colocalize with PS1, but was efficiently processed by PS1-dependent gamma-secretase. APP-C99 resides in a compartment that is negative for ER, intermediate compartment, and Golgi marker proteins. We conclude that gamma-secretase cleavage of APP-C99 occurs in a specialized subcellular compartment where little or no PS1 is detected. This suggests that at least one other factor than PS1, located downstream of the ER, is required for the gamma-cleavage of APP-C99. In agreement, we found that intracellular gamma-secretase processing of APP-C99-KK both at the gamma40 and the gamma42 site could be restored partially after brefeldin A treatment. Our data confirm the "spatial paradox" and raise several questions regarding the PS1 is gamma-secretase hypothesis.

Pathogenic APP mutations near the gamma-secretase cleavage site differentially affect Abeta secretion and APP C-terminal fragment stability.

Release of amyloid beta (Abeta) from the amyloid precursor protein (APP) requires cleavages by beta- and gamma-secretases and plays a crucial role in Alzheimer's disease (AD) pathogenesis. Missense mutations in the APP gene causing familial AD are clustered around the beta-, alpha- and particular gamma-secretase cleavage sites. We systematically compare in primary neurons the effect on APP processing of a series of clinical APP mutations (two of which not characterized before) located in close proximity to the gamma-secretase cleavage site. We confirm and extend previous observations showing that all these mutations (T714I, V715M, V715A, I716V, V717I and V717L) affect gamma-secretase cleavage causing an increased relative ratio of Abeta42 to Abeta40. Taking advantage of these extended series of APP mutations we were able to demonstrate an inverse correlation between these ratios and the age at onset of the disease in the different families. In addition, a subset of mutations caused the accumulation of APP C-terminal fragments indicating that these mutations also influence the stability of APP C-terminal fragments. However, it is unlikely that these fragments contribute significantly to the disease process.

Processing of beta-secretase by furin and other members of the proprotein convertase family.

The amyloid peptide is the main constituent of the amyloid plaques in brain of Alzheimer's disease patients. This peptide is generated from the amyloid precursor protein by two consecutive cleavages. Cleavage at the N terminus is performed by the recently discovered beta-secretase (Bace). This aspartyl protease contains a propeptide that has to be removed to obtain mature Bace. Furin and other members of the furin family of prohormone convertases are involved in this process. Surprisingly, beta-secretase activity, neither at the classical Asp(1) position nor at the Glu(11) position of amyloid precursor protein, seems to be controlled by this maturation step. Furthermore, we show that Glu(11) cleavage is a function of the expression level of Bace, that it depends on the membrane anchorage of Bace, and that Asp(1) cleavage can be followed by Glu(11) cleavage. Our data suggest that pro-Bace could be active as a beta-secretase in the early biosynthetic compartments of the cell and could be involved in the generation of the intracellular pool of the amyloid peptide. We conclude that modulation of the conversion of pro-Bace to mature Bace is not a relevant drug target to treat Alzheimer's disease.

Presenilin 2 deficiency causes a mild pulmonary phenotype and no changes in amyloid precursor protein processing but enhances the embryonic lethal phenotype of presenilin 1 deficiency.

Mutations in the homologous presenilin 1 (PS1) and presenilin 2 (PS2) genes cause the most common and aggressive form of familial Alzheimer's disease. Although PS1 function and dysfunction have been extensively studied, little is known about the function of PS2 in vivo. To delineate the relationships of PS2 and PS1 activities and whether PS2 mutations involve gain or loss of function, we generated PS2 homozygous deficient (-/-) and PS1/PS2 double homozygous deficient mice. In contrast to PS1(-/-) mice, PS2(-/-) mice are viable and fertile and develop only mild pulmonary fibrosis and hemorrhage with age. Absence of PS2 does not detectably alter processing of amyloid precursor protein and has little or no effect on physiologically important apoptotic processes, indicating that Alzheimer's disease-causing mutations in PS2, as in PS1, result in gain of function. Although PS1(+/-) PS2( -/-) mice survive in relatively good health, complete deletion of both PS2 and PS1 genes causes a phenotype closely resembling full Notch-1 deficiency. These results demonstrate in vivo that PS1 and PS2 have partially overlapping functions and that PS1 is essential and PS2 is redundant for normal Notch signaling during mammalian embryological development.

Presenilin 1 controls gamma-secretase processing of amyloid precursor protein in pre-golgi compartments of hippocampal neurons.

Mutations of presenilin 1 (PS1) causing Alzheimer's disease selectively increase the secretion of the amyloidogenic betaA4(1-42), whereas knocking out the gene results in decreased production of both betaA4(1-40) and (1-42) amyloid peptides (De Strooper et al. 1998). Therefore, PS1 function is closely linked to the gamma-secretase processing of the amyloid precursor protein (APP). Given the ongoing controversy on the subcellular localization of PS1, it remains unclear at what level of the secretory and endocytic pathways PS1 exerts its activity on APP and on the APP carboxy-terminal fragments that are the direct substrates for gamma-secretase. Therefore, we have reinvestigated the subcellular localization of endogenously expressed PS1 in neurons in vitro and in vivo using confocal microscopy and fine-tuned subcellular fractionation. We show that uncleaved PS1 holoprotein is recovered in the nuclear envelope fraction, whereas the cleaved PS fragments are found mainly in post-ER membranes including the intermediate compartment (IC). PS1 is concentrated in discrete sec23p- and p58/ERGIC-53-positive patches, suggesting its localization in subdomains involved in ER export. PS1 is not found to significant amounts beyond the cis-Golgi. Surprisingly, we found that APP carboxy-terminal fragments also coenrich in the pre-Golgi membrane fractions, consistent with the idea that these fragments are the real substrates for gamma-secretase. Functional evidence that PS1 exerts its effects on gamma-secretase processing of APP in the ER/IC was obtained using a series of APP trafficking mutants. These mutants were investigated in hippocampal neurons derived from transgenic mice expressing PS1wt or PS1 containing clinical mutations (PS1(M146L) and PS1(L286V)) at physiologically relevant levels. We demonstrate that the APP-London and PS1 mutations have additive effects on the increased secretion of betaA4(1-42) relative to betaA4(1-40), indicating that both mutations operate independently. Overall, our data clearly establish that PS1 controls gamma(42)-secretase activity in pre-Golgi compartments. We discuss models that reconcile this conclusion with the effects of PS1 deficiency on the generation of betaA4(1-40) peptide in the late biosynthetic and endocytic pathways.

Developmental expression of wild-type and mutant presenilin-1 in hippocampal neurons from transgenic mice: evidence for novel species-specific properties of human presenilin-1.

Presenilins 1 (PS1) and 2 (PS2) are multispanning transmembrane proteins associated with familial Alzheimer disease (FAD). They are developmentally regulated, being expressed at highest levels during neuronal differentiation and are sustained at a lower level throughout life. We investigated the distribution and metabolism of endogenous murine PS1 as well as human wild-type (wtPS1) and the familial AD Met146Leu (M146L) mutant presenilins in dissociated cultures of hippocampal neurons derived from control and transgenic mice. We found that the PS1 endoproteolytic fragments and, to a lesser extent, the full-length protein, were expressed as early as day 3 post-plating. Both species increased until the cells were fully differentiated at day 12. Confocal microscopy revealed that presenilin is present in the Golgi and endoplasmic reticulum and, as in punctate, vesicle-like structures within developing neurites and growth cones. Using a human-specific PS1 antibody, we were able to independently examine the distribution of the transgenic protein which, although similar to the endogenous, showed some unique qualities. These included (i) some heterogeneity in the proteolytic fragments of human PS1; (ii) significantly reduced levels of full-length human PS1, possibly as a result of preferential processing; and (iii) a more discrete intracellular distribution of human PS1. Colocalization with organelle-specific proteins revealed that PS1 was located in a diffuse staining pattern in the MAP2-positive dendrites and in a punctate manner in GAP43-positive axons. PS1 showed considerable overlap with GAP43, particularly at the growth cones. Similar patterns of PS1 distribution were detected in cultures derived from transgenic animals expressing human wild-type or mutant presenilins. The studies demonstrate that mutant presenilins are not grossly different in their processing or distribution within cultured neurons, which may represent more physiological models as compared to transfection systems. Our data also suggest that the molecular pathology associated with PS1 mutations results from subtle alterations in presenilin function, which can be further investigated using these transgenic neuronal cell culture models.

A presenilin-1-dependent gamma-secretase-like protease mediates release of Notch intracellular domain.

Signalling through the receptor protein Notch, which is involved in crucial cell-fate decisions during development, requires ligand-induced cleavage of Notch. This cleavage occurs within the predicted transmembrane domain, releasing the Notch intracellular domain (NICD), and is reminiscent of gamma-secretase-mediated cleavage of beta-amyloid precursor protein (APP), a critical event in the pathogenesis of Alzheimer's disease. A deficiency in presenilin-1 (PS1) inhibits processing of APP by gamma-secretase in mammalian cells, and genetic interactions between Notch and PS1 homologues in Caenorhabditis elegans indicate that the presenilins may modulate the Notch signalling pathway. Here we report that, in mammalian cells, PS1 deficiency also reduces the proteolytic release of NICD from a truncated Notch construct, thus identifying the specific biochemical step of the Notch signalling pathway that is affected by PS1. Moreover, several gamma-secretase inhibitors block this same step in Notch processing, indicating that related protease activities are responsible for cleavage within the predicted transmembrane domains of Notch and APP. Thus the targeting of gamma-secretase for the treatment of Alzheimer's disease may risk toxicity caused by reduced Notch signalling.

Deficiency of presenilin-1 inhibits the normal cleavage of amyloid precursor protein.

Point mutations in the presenilin-1 gene (PS1) are a major cause of familial Alzheimer's disease. They result in a selective increase in the production of the amyloidogenic peptide amyloid-beta(1-42) by proteolytic processing of the amyloid precursor protein (APP). Here we investigate whether PS1 is also involved in normal APP processing in neuronal cultures derived from PS1-deficient mouse embryos. Cleavage by alpha- and beta-secretase of the extracellular domain of APP was not affected by the absence of PS1, whereas cleavage by gamma-secretase of the transmembrane domain of APP was prevented, causing carboxyl-terminal fragments of APP to accumulate and a fivefold drop in the production of amyloid peptide. Pulse-chase experiments indicated that PS1 deficiency specifically decreased the turnover of the membrane-associated fragments of APP. As in the regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor, PS1 appears to facilitate a proteolytic activity that cleaves the integral membrane domain of APP. Our results indicate that mutations in PS1 that manifest clinically cause a gain of function and that inhibition of PS1 activity is a potential target for anti-amyloidogenic therapy in Alzheimer's disease.

Phosphorylation, subcellular localization, and membrane orientation of the Alzheimer's disease-associated presenilins.

Presenilins 1 and 2 are unglycosylated proteins with apparent molecular mass of 45 and 50 kDa, respectively, in transfected COS-1 and Chinese hamster ovary cells. They colocalize with proteins from the endoplasmic reticulum and the Golgi apparatus in transfected and untransfected cells. In COS-1 cells low amounts of intact endogeneous presenilin 1 migrating at 45 kDa are detected together with relative larger amounts of presenilin 1 fragments migrating between 18 and 30 kDa. The presenilins have a strong tendency to form aggregates (mass of 100-250 kDa) in SDS-polyacrylamide gel electrophoresis, which can be partially resolved when denatured by SDS at 37 degrees C instead of 95 degrees C. Sulfation, glycosaminoglycan modification, or acylation of the presenilins was not observed, but both proteins are posttranslationally phosphorylated on serine residues. The mutations Ala-246 --> Glu or Cys-410 --> Tyr that cause Alzheimer's disease do not interfere with the biosynthesis or phosphorylation of presenilin 1. Finally, using low concentrations of digitonin to selectively permeabilize the cell membrane but not the endoplasmic reticulum membrane, it is demonstrated that the two major hydrophilic domains of presenilin 1 are oriented to the cytoplasm. The current investigation documents the posttranslational modifications and subcellular localization of the presenilins and indicates that postulated interactions with amyloid precursor protein metabolism should occur in the early compartments of the biosynthetic pathway.

Amyloidogenic processing of human amyloid precursor protein in hippocampal neurons devoid of cathepsin D.

betaA4-Amyloid peptide, the main component of the amyloid plaques in the brain of Alzheimer's disease patients is produced from amyloid precursor protein (APP) by proteolytical processing. Several lines of evidence suggest a direct role for cathepsin D, the major endosomal/lysosomal aspartic endopeptidase, in betaA4-amyloid peptide generation. Here we tested this hypothesis using primary cultures of hippocampal neurons derived from cathepsin D-deficient (knock out) mice and expressing wild-type human APP and two clinical APP variants via recombinant Semliki Forest virus. We demonstrate APP secretory processing, production of carboxyl-terminal amyloid fragments, and secretion of the betaA4-amyloid peptide in the complete absence of cathepsin D. The results rule out cathepsin D as a critical component of alpha-, beta-, or gamma-secretase and therefore as a primary target for drugs aimed at decreasing the betaA4-amyloid peptide burden in Alzheimer's disease.

Exchanging the extracellular domain of amyloid precursor protein for horseradish peroxidase does not interfere with alpha-secretase cleavage of the beta-amyloid region, but randomizes secretion in Madin-Darby canine kidney cells.

Secretory processing and polarized sorting of horseradish peroxidase fused to the amyloid precursor protein transmembrane domain were compared with those of wild-type amyloid precursor protein in COS and polarized Madin-Darby canine kidney (MDCK) cells. The cellular and secreted forms of the chimeric protein were enzymatically active in colorimetric and cytochemical assays after reconstitution with hemin and Ca2+. The peroxidase enzyme was secreted by a proteolytic process, similar to the parent amyloid precursor protein. In polarized MDCK cells, amyloid precursor protein was secreted exclusively in the basolateral compartment, while the peroxidase chimeric protein was secreted in both compartments. The basolateral sorting determinant for secretion must therefore be located in the extracellular domain of amyloid precursor protein. On the other hand, cell surface-associated peroxidase chimeric protein was similar to cell surface-associated wild-type amyloid precursor protein, mainly expressed at the basolateral side. The basolateral cell-surface expression, in contrast to the basolateral secretion, is therefore controlled by determinants in the cytoplasmic domain. Methylamine inhibited and bafilomycin slightly increased the basolateral secretion of both proteins, but both drugs strongly increased apical secretion. The default secretory pathway of COS cells and the basolateral (but not the apical) secretory pathway of MDCK cells are therefore comparably sensitive to methylamine and not to bafilomycin.

Basolateral secretion of amyloid precursor protein in Madin-Darby canine kidney cells is disturbed by alterations of intracellular pH and by introducing a mutation associated with familial Alzheimer's disease.

The analysis of potential sorting signals in amyloid precursor protein (APP) by site-directed mutagenesis and the disturbance of metabolic pathways by drugs is used here to define the parameters that determine polarized secretion of APP in Madin-Darby canine kidney cells. Endogenously produced APP751/770 and APP695 produced from transfected constructs are secreted almost exclusively into the basolateral compartment. The sorting mechanism is highly dependent on intracellular pH as demonstrated by its sensitivity to primary amines and inhibitors of the acidifying vacuolar protein ATPase. The role of potential basolateral sorting signals in the cytoplasmic, transmembrane, and beta A4 amyloid region of APP was investigated. Neither deletion of the endocytosis and putative basolateral sorting signal GY.NPTY nor complete deletion of the cytoplasmic domain causes apical secretion of soluble APP. Further deletion of the transmembrane domain and of the beta A4 amyloid region confirmed that the major basolateral sorting determinant resides in the extracellular domain of APP. Increased beta-secretase cleavage of APP after introduction of the "swedish" double mutation causes apical missorting of about 20% of beta-secretase-cleaved APP. The data underline the complexity of processing and sorting APP in polarized cells and suggest a possible problem of protein sorting in Alzheimer's Disease.