[Frontiers in Bioscience, 5, d72-83, January 1, 2000]

Current Issue

Send correspondence to:

Dr Huaxi Xu,
Fisher Center for Alzheimer Research
and Laboratory of Molecular and Cellular Neuroscience,
The Rockefeller University, New York NY 1002,

Tel/Fax: 212-327-7888,
E-mail: xuh@rockvax.


Alzheimer's disease, b-amyloid precursor protein, Beta-amyloid, Presenilin proteins, Intracellular trafficking, Signal transduction, Phosphorylation, Estrogen, Endoplasmic reticulum, Golgi, trans-Golgi Network, Review


Copyright © Frontiers in Bioscience, 1995

Version 1 (1998)
Version 2 (2000)


Jeffrey P. Greenfield, Rachel S. Gross, Gunnar K. Gouras, and Huaxi Xu

Fisher Center for Alzheimer Research and Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York NY 10021


1. Abstract
2. Introduction
3. Discussion
3.1. bAPP Structure Gives Clues to its Function
3.2. bAPP is Trafficked and Processed Through the Secretory Pathway
3.3. a-Secretase Cleaves bAPP at the Cell Surface
3.4. Alternative bAPP Metabolism Gives Rise to β-Amyloid Peptides
3.5. Ab Peptides may be Generated Intracellularly
3.6. FAD Mutations and the b-secretase
3.7. Role of Presenilin in Ab Generation and AD Pathogenesis
3.8. Signal Transduction Regulates bAPP Trafficking
3.9. Hormonal Regulation of bAPP Metabolism
4. Perspective
5. Acknowledgement
6. References

In molecular neurobiology, perhaps no molecule has been as thoroughly examined as Alzheimer's b-amyloid precursor protein (bAPP). In the years since the cDNA encoding bAPP was cloned, the protein has been the subject of unparalleled scrutiny on all levels. From molecular genetics and cellular biology to neuroanatomy and epidemiology, no scientific discipline has been left unexplored - and with good reason. b-amyloid (Ab) is the main constituent of the amyloidogenic plaques which are a primary pathological hallmark of Alzheimer's disease, and bAPP is the protein from which Ab is cleaved and released. Unraveling the molecular events underlying Ab generation has been, and remains, of paramount importance to scientists in our field. In this review we will trace the progress that has been made in understanding the molecular and cellular basis of bAPP trafficking and processing, or alternatively stated, the molecular basis for Ab generation. Imperative to a complete understanding of Ab generation is the delineation of its subcellular localization and the identification of proteins that play either direct or accessory roles in Ab generation. We will focus on the regulation of bAPP cleavage through diverse signal transduction mechanisms and discuss possible points of therapeutic intercession in what has been postulated to be a seminal molecular step in the cascade of events terminating in the onset of dementia, loss of neurons, and eventual death from Alzheimer's disease.


Alzheimer's disease (AD) is the most common form of dementia in the elderly, accounting for over 50% of the typical, late-onset cases of dementia. AD is characterized clinically by an insidious onset and an inexorable progression of dementia, and pathologically by the abnormal accumulation of neuritic plaques and neurofibrillary tangles in vulnerable brain regions. Plaques consist of deposits of variously sized peptides called b-amyloid (Ab) (1, 2)which are derived through proteolytic processing of the b-amyloid precursor protein (bAPP). The neurofibrillary tangles are composed largely of hyperphosphorylated twisted filaments of a cytoskeletal protein, tau, (3) whose contribution to AD pathogenesis is beyond the scope of this review. Evidence causally linking bAPP to AD was first provided by the discovery of mutations within the bAPP coding sequence that segregated with disease phenotypes in autosomal dominant familial AD (FAD) (4-6). Although documented FAD is rare (<10% of all AD), the characteristic clinicopathological features, amyloid plaques, neurofibrillary tangles, synaptic and neuronal loss, neurotransmitter deficits and dementia are apparently indistinguishable when FAD is compared with typical, common, "non-familial," or sporadic AD.

In addition to mutations within bAPP, there are actually many more reported pedigrees in which early onset FAD segregates with two other genetic loci. Mutations in the presenilin 1 gene on chromosome 14 (7) and the presenilin 2 gene on chromosome 1 (8) cause FAD by altering bAPP metabolism. Specifically, presenilin mutations cause a selective increase in the production of the 42 amino acid form of Ab (Ab42) (9-10). This increase of Ab42 occurs in the plasma, and in media from cultured skin fibroblasts derived from patients carrying these mutations, and can be detected presymptomatically (10). This elevation of Ab42 is significant because Ab42, despite being produced far less abundantly than Ab40, is more highly amyloidogenic than Ab40 (11, 12) and is believed to form the core of the amyloidogenic plaques (13, 14). Cells tranfected with mutant PS1 cDNA secrete higher levels of Ab42 (9, 15); similarly, transgenic mice expressing mutant PS1 show increased levels of Ab42 levels in their brains (16). Furthermore, it was already known that patients with Down's syndrome or trisomy 21 (bAPP is on chromosome 21) invariably develop AD pathology by age 50 and can show Ab42 immunoreactive plaques as early as age 12 (17). This data from humans, transgenic mice and cells transfected with mutations in bAPP and PS1, has strengthened the integrated amyloid cascade hypothesis: diverse molecular anomalies all lead to enhanced production or aggregation of Ab peptides. It is the goal of this review to introduce the various hypotheses regarding the metabolism of bAPP and the generation of Ab, and to suggest how interrupting or delaying this early and invariable event in AD may be therapeutically feasible.


3.1. bAPP Structure Gives Clues to its Function

The partial purification of amyloid peptides from the microvasculature of AD brains by Glenner and Wong (1) began the modern AD research era; within three years, the sequence of this small peptide was used to successfully clone the full-length cDNA of bAPP (18). The deduced amino acid sequence of bAPP predicts a type 1 transmembrane protein encoded by alternatively spliced mRNA resulting in isoform diversity. Isoforms of 751 and 770 amino acids contain a protease inhibitor domain in the extracellular region of the bAPP molecule (19, 20). An isoform of 695 amino acids missing the protease inhibitor domain, however, is the main isoform expressed in neurons. A definitive cell biological role has yet to be ascribed for bAPP and its metabolites, however, physiological roles for bAPP in transmembrane signal transduction (21), calcium metabolism (22) and neuritic outgrowth (23) have been suggested. Potential functional motifs within bAPP identified by the presence of consensus sequences, suggest roles in metal ion binding (24), heparin binding (25), cell adhesion (26), as a receptor for a currently unrecognized ligand, and in regulating cell growth (27). Initial studies of bAPP knockout mice did not reveal an obvious disease phenotype (28), however, more recent examinations have suggested deficits in specific synaptic proteins (29). A family of novel bAPP-like proteins (APLPs) was discovered (30, 31) suggesting that bAPP may be a member of a larger family of related molecules. APLPs lack the Ab domain, however, and therefore cannot serve as precursors to Ab.

3.2. bAPP is Trafficked and Processed Through the Secretory Pathway

bAPP is synthesized and cotranslationally inserted into membranes in the endoplasmic reticulum (ER). Although it has been recently suggested that Ab42 can be generated within a compartment early in the secretory pathway (32), such as the ER or early Golgi, most bAPP molecules exit the ER uncleaved. Once exiting the ER, bAPP molecules are transported to the Golgi apparatus where the majority of bAPP molecules are found under steady state conditions. Within the Golgi apparatus, bAPP is significantly modified by N- and O-glycosylation, tyrosyl sulfation, and sialylation (33, 34). bAPP is also phosphorylated in both the extracellular and intracellular domains (34, 35). In addition, some bAPP molecules are chondroitin-sulfated in their ectodomains (36).

The proteolytic processing events underlying bAPP metabolism have been studied intensely. The first proteolytic cleavage product of bAPP processing to be definitively identified by purification and sequencing was a fragment which results primarily from a cleavage event within the Ab domain. The entire amino-terminal fragment of the bAPP extracellular domain, termed s-bAPP or bAPPs, is released into the medium of cultured cells (26, 34, 37, 38) and into the cerebrospinal fluid, leaving a nonamyloidogenic carboxyl-terminal fragment associated with the cell (39). This pathway was designated the a-secretory pathway after the still unidentified enzymatic activity termed the a-secretase which cleaves the protein with that specificity. Thus one important processing event in the biology of bAPP acts to preclude Ab formation by cleaving bAPP within the Ab domain.

3.3. a-Secretase Cleaves bAPP at the Cell Surface

An increasing number of secreted proteins are now recognized as being derived from integral plasma membrane proteins in which the secretory event is actually post-translational hydrolysis from the cell surface. This shedding or solubilization involves either an endoprotease or a phospholipase. bAPP appears to belong to this large family of proteins including membrane receptors, receptor ligands, ectoenzymes, leukocyte antigens, and cell adhesion molecules in which an enzymatic event at the cell surface may be physiologically relevant (40). It has been demonstrated that a-secretase is most active late within the secretory pathway and at the plasma membrane (41). The a-cleaved bAPP molecule is likely to have a physiological relevance that extends beyond merely preventing Ab formation: both in vitro and in vivo effects of bAPPs on cytoprotection and cell neurotrophy have been documented (27).

Recent evidence has suggested that Tumor Necrosis Factor-a Converting Enzyme (TACE), a member of the ADAM family (A Disintegrin And Metalloprotease family) of proteases, may be involved in regulating a-cleavage of bAPP (42). Inhibiting TACE cleavage affected both bAPP secretion and Ab generation in cultured cells suggesting that TACE or a closely related molecule, could be involved in an a-secretase-like cleavage event. Membrane-bound tumor necrosis factor-a (TNF-a) is also similar to bAPP in that it is a transmembrane protein that can be proteolytically cleaved to release its extracellular domain as soluble TGF-a. Amino acid substitutions around the bAPP a-secretase cleavage site do not alter bAPP cleavage suggesting that a-secretase cleavage relies more upon the distance from the membrane than on the primary amino acid sequence (41). Progressive deletion of the extracellular juxtamembrane amino acids demonstrated that only 11 amino acids of the protein sequence were required to sustain bAPP cleavage at the cell surface (41). The cell regulation of the a-secretase pathway will be discussed later in this review. Simply, however, the activation of Protein Kinase C (PKC), through diverse routes, strongly stimulates a-secretase cleavage. This event can be ascribed either to indirect effects of PKC on the cellular localization of bAPP and the a-secretase or direct effects on the enzymes themselves through altering their state of phosphorylation (43-45).

3.4. Alternative bAPP Metabolism Gives Rise to the β-Amyloid Peptide

Until 1992, Ab was, by general consensus, an abnormal metabolite derived from bAPP. Soluble Ab was not initially detected in brain, cerebrospinal fluid or from cell culture systems due to the limitations imposed by antibody reagent sensitivity. The conformation, aggregation and low endogenous levels of Ab produced by cells also made Ab detection difficult. Most early information regarding bAPP metabolism therefore, was derived from experiments studying the carboxy-terminal fragments of bAPP. High-level overexpression of human bAPP using recombinant vaccinia viruses (46), baculoviruses (47), or stable transfections in association with supraphysiological levels of protein phosphorylation (43) resulted in the accumulation of heterogeneous C-terminal bAPP fragments. These studies supported the hypothesis that an alternative cleavage of bAPP molecules could occur in which C-terminal fragments not cleaved by the a-secretase, but rather containing the complete Ab sequence, remained as potential precursors from which Ab could be further derived. This cleavage activity was designated the b-secretase. Sequencing of these putative b-cleaved C-terminal species confirmed them as potential intermediaries in the pathway to Ab generation (48).

This non a-secretase-mediated cleavage immediately prompted the search for alternative intracellular routes through which bAPP could be trafficked and metabolized into Ab-generating precursors. The existence of an alternative secretory pathway for bAPP was difficult to prove. Several groups began searching for the secretory compartments, within which bAPP was metabolized and Ab generated. A favored hypothesis posited that Ab was generated within an acidic intracellular compartment with a pH permissive for maximal secretase activity. Vesicle neutralizing agents such as chloroquine and ammonium chloride applied to cultured cells facilitated the recovery of full length bAPP and an array of C-terminal fragments (44, 49, 50) - a similar population of fragments as was recovered from purified lysosomes (51).

The possibility that an endosomal-mediated metabolism of bAPP was important in the generation of Ab peptides was further indicated by the discovery of a clatherin-coated vesicle (CCV) targeting motif in the LDL receptor. This NPXY motif, required for proper internalization of the LDL receptor, was also present on the cytoplasmic tail of bAPP (52). The co-purification of bAPP with CCVs (53) suggested the possibility that bAPP may be a receptor for a yet undiscovered or recognized ligand. These experiments helped define a second normal processing pathway for bAPP that does not involve a cleavage event within the Ab domain of bAPP. This work supported the hypothesis that bAPP can be reinternalized from the cell surface and targeted to the endosome/lysosome pathway where it may contribute, in part, to the generation of bAPP-derived fragments containing Ab (54).

Despite progress in elucidating the subcellular compartments within which bAPP processing enzymes are active, until recently, all attempts to discover their identities have failed using traditional mammalian systems. To overcome the difficulties inherent in mammalian gene discovery, several groups attempted to reconstitute bAPP processing in Saccharomyces cerevisiae, an organism with a much simpler, and completely sequenced genome. Combined genetic and biochemical approaches identified several proteolytic processing enzymes in yeast which have been found to be homologous to enzymes that catalyze the same reactions in higher organisms. Using similar approaches, it was demonstrated using yeast strains transfected with human bAPP, that yeast contain enzymes that exhibit a-secretase-like activity (55). This reaction is catalyzed by the glycosyl-phosphatidylinositol-linked yeast aspartyl proteases, MKC7 and YAP3 (56, 57). A more recent study demonstrates the presence of b-secretase-like enzymatic activity in yeast as well (58). Given these findings along with the availability of mutant yeast strains and the well-characterized yeast secretory pathway, yeast may become an attractive system for studying bAPP processing and Ab generation.

3.5. Ab Peptides may be Generated Intracellularly

Hypotheses regarding the abundance of a 4 kD Ab species were based upon the belief that Ab was an abnormal and toxic species - a product restricted to the brains of aged or demented humans. It was believed that a cell would need to be significantly injured for a protease to gain access to the interior of the membrane bilayer to cleave bAPP and generate Ab. This concept was soundly refuted when several groups discovered that a soluble Ab species was detectable in body fluids from various species (59) and in the conditioned medium of cultured cells (60) but was not detectable in the lysates of cultured cells. Cultured cells were shown to release the 4 kD Ab peptide into medium in high picomolar to low nanomolar concentrations (60, 61). In addition to cultures of neuronal-like cell lines, cortical cultures derived from the brains of fetal mice, rats and humans all secrete Ab peptides in quantifiable levels (59, 62) and Ab can be similarly detected from the cerebrospinal fluid of humans and rats (59).

The subcellular processing pathway involved in Ab generation was also debated, with the generation of Ab through either endocytic or exocytic pathways, each rigorously defended. One shared feature of the two pathways is the unquestioned reliance of bAPP passage through an acidic intracellular compartment. In support of this, treatment of bAPP-expressing cells with Bafilomycin A (Baf A), a vacuolar-type H+-ATPase inhibitor, abolished Ab generation (63). Attempts to isolate Ab from the late endosomes/lysosomes failed, leading to the hypothesis that Ab production requires maturation of bAPP through the Golgi and processing in an acidic compartment other than lysosomes (64).

This hypothesis was confirmed when it was demonstrated with the use of a cell-free system that Ab can be generated from the trans-Golgi network (TGN) in the absence of vesicle formation (65). This result is consistent with previous findings because the TGN, like the endosome, provides an ideal acidic environment for maximal secretase activity and Ab generation. b-secretase activity within the late Golgi was confirmed in cells expressing bAPP harboring a pathogenic point mutation (66). The late Golgi has many known cellular functions that support it as a likely site for Ab generation. As previously mentioned, under steady state conditions bAPP is principally localized within the TGN. It has been appreciated that physiologically relevant events, such as prohormone processing, occur here as well (67). Finally, the TGN is a known sorting station for many lysosomal enzymes and plasma membrane proteins. These facts support the notion that if bAPP and relevant secretases are co-localized within the TGN, this late secretory compartment could be a major site of Ab generation. Interestingly, implicating the TGN as an important location for Ab production raises the possibility that the excess production of Ab seen in AD may be due to a defect in the general secretory/processing apparatus of neurons which could occur after many decades of unfaltering function.

bAPP is initially synthesized and cotranslationally inserted into the endoplasmic reticulum (ER) and recent studies have demonstrated ER generation of Ab42 (68-72). Retaining bAPP in the ER using brefeldin A or ER retention signals increases production of Ab42, reinforcing the hypothesis that it is generated within the ER. Ab42 generation was detected in the ER of both primary neurons and cultured human NT2N cells. Using human NT2N cells, a pool of insoluble Ab42 making up over half of the total intracellular Ab42 was extracted with formic acid from the ER (71). These studies, using ELISA assays with C-terminal specific antibodies, could not distinguish between the N-termini of the peptides and differentiate between Ab42 species. A cell-free assay was used to investigate the subcellular compartments within which distinct amyloid species are produced and those from which they are secreted (72). It was determined that Ab40 (Ab1-40 and Abx-40) is generated exclusively in the TGN and secreted in post-TGN secretory vesicles. It was also demonstrated that while the Abx-42 is produced and retained in the ER in an insoluble state, both Ab1-42 and Abx-42 species are generated in the TGN and packaged into secretory vesicles which accounts for the secreted pool of Ab42. The identification of the subcellular compartments in which the distinct species of Ab are produced and from which they are secreted could aid in the search for the proteolytic enzymes responsible for Ab formation.

Several studies have recently been conducted which support the hypothesis that Ab is generated intracellularly. Recent studies demonstrated that an insoluble pool of intracellular Ab was highly stable and accumulated in NT2N cells as a function of aging in culture. These studies showed a 12-fold increase in the amount of intracellular Ab over a seven-week period in culture (71). This substantial increase in intracellular Ab suggests that intracellular Ab accumulation may be more important than previously thought in AD pathogenesis. A recent study showed that human neurons in AD-vulnerable brain regions contain Ab42 immunoreactivity which appears to precede both neurofibrillary tangles and Ab plaque formation (73), suggesting that intracellular Ab42 accumulation may be an early pathogenic event.

3.6. FAD Mutations and the b-secretase

The early discovery of mutations within bAPP lent support to those who believed the accumulation of Ab was a crucial event in AD pathogenesis because it linked, for the first time, mutations in bAPP, with early onset forms of AD. All bAPP mutations studied in transfected or primary cells led to an increase of Ab secretion, particularly of Ab42 (74, 75). Strikingly, Ab levels within the plasma of families harboring these mutations are significantly raised well before AD symptoms manifest (10). For this reason, there is little doubt that these mutations cause AD, in these patients, by providing an enhanced cleavage site upon which the proteolytic secretases can act more quickly or efficiently. Of all the known genetic causes of FAD, however, bAPP missense mutations at or near the sites of endoproteolysis are a relatively rare cause of familial AD. Nevertheless, the belief that FAD and sporadic AD have a similar amyloid-dependent pathogenesis suggested that discovering a secretase, would provide a single therapeutic target for all forms of AD. The recent cloning and characterization of a transmembrane aspartyl protease with b-secretase activity (76, 77) named BACE (for beta-site APP-cleaving enzyme) finally achieved this goal.

Overexpression of BACE increased b-secretase cleavage products, Ab1-42 and Ab11-40/42 (78). BACE was found to be expressed in higher levels in neurons than in glial cells, supporting neurons as the primary source of extracellular Ab. BACE's active site is lumenal which agrees with the topological orientation of the b-secretase cleavage site in bAPP. BACE was maximally active at an acidic pH and was localized within acidic intracellular compartments of the secretory pathway, primarily the Golgi and the endosomes. Whether or not other enzymes with b-secretase activity exist as well is still uncertain. Inhibitors of this characterized aspartyl protease with b-secretase activity could provide a novel approach to treating against AD.

3.7. Role of Presenilin in Ab Generation and AD Pathogenesis

Although bAPP and BACE are integral to AD pathogenesis, mutations in two genes actually cause disease in the majority of known FAD pedigrees: the presenilin 1 (PS1) gene on chromosome 14 (7) and the presenilin 2 (PS2) gene on chromosome 1 (8). These genes encode polytopic membrane proteins with high homology to one another. The functions of these proteins are not well understood, but clues have been provided. Homologues of the PS proteins occur in Caenorhabditis elegans. Mutations in the C. elegans Spe-4 gene lead to deficits in spermatogenesis by disrupting protein trafficking in the Golgi (79), and mutations in the Sel-12 gene can disrupt a crucial intracellular Notch signaling mechanism (80). Notch, a protein involved in regulating cell-fate decisions and development within the nervous system initiates proper signaling pathways after being cleaved within a transmembrane domain. This processing event closely parallels the g-secretase cleavage predicted to occur within the transmembrane domain of bAPP. Interestingly, wild-type PS1 can rescue sel-12 mutants (81), further underscoring their functional similarities. Finally, PS1 knockout mice exhibit developmental deficits similar to those observed in mice in which other components of the Notch signaling pathway have been mutated or knocked out (82). These genetic interactions between Notch and PS1 homologues suggested that PS1 may be involved in the Notch signaling pathways.

While examining the effects of PS1 deficiency on Notch processing, it was demonstrated that the release of the Notch intracellular domain was reduced in neurons derived from PS1 knockout mice (83). It was also determined that g-secretase inhibitors blocked the parallel step in Notch processing, suggesting that a similar proteolytic activity was cleaving within the transmembrane domain of both Notch and bAPP. It was demonstrated that loss-of-function mutations in the Drosophila PS1 gene cause lethal Notch-like phenotypes (84). These recent discoveries that PS1 may play similar roles in both Notch and bAPP processing may lead to a more detailed understanding of the mechanisms behind PS1's involvement in bAPP processing. Unfortunately, it also suggests that targeting the g-secretase activity for AD treatment may cause unwanted side effects by inhibiting important pathways mediated by Notch signaling.

Emphasis has been placed on studying the presenilins because by understanding their functions, we may gain insight into the role that bAPP plays, both as a normally functioning protein and as a disease protein within AD pathogenesis. While the presenilin molecules appear to influence bAPP metabolism, a direct molecular interaction between the two molecules, while proposed (85), has been difficult to convincingly prove (86), keeping elusive the answer of how that influence is achieved on a molecular level. Despite an unclear mechanism, the downstream effects of presenilin mutations on bAPP metabolism have been extensively documented. Plasma from individuals with PS1 mutations provided the first evidence of this link when it was shown that their Ab42 levels were significantly elevated (10). Similarly, brains and fibroblasts from these patients all demonstrate this same significant elevation (10, 87). When transgenic animals, or cultured cells bearing mutated PS genes, were analyzed and compared with animals or cells bearing wild type PS genes, the mutant counterparts all produced a consistent reinforcing result. Expressing mutant PS1 effects bAPP metabolism which results in significantly elevated secretion of Ab42 relative to Ab40 (9, 16). These results provided powerful support for placement of the presenilin proteins within the framework of the amyloid cascade hypothesis. Mutations in the presenilin proteins could now join Down's syndrome and bAPP-linked familial AD as directly causing AD pathogenesis through an increase in Ab generation.

The subcellular localization of PS1 regulation of bAPP processing, has been established using subcellular fractionation studies which suggested that the presence of CTFs was significantly increased in both the ER and the Golgi fractions of fibroblasts from PS1 knockout mice (88). Analysis of these fractions supported results from another study showing that Ab42 was the major species in the ER and that a mutant PS1 strain significantly increased intracellular Ab42 levels (69).

The hypothesis that PS1 can regulate bAPP metabolism received perhaps its most validating experimental evidence from a study examining Ab generation in cells cultured from the hippocampus of PS1 knockout mice and infected with a recombinant Semliki Forest virus encoding human bAPP (89). These cells secreted 80% less Ab (both Ab40 and Ab42) than did control cells which had been infected with the same virus. The authors speculate that PS1 may actually activate the enzyme (g -secretase) which gives rise to the C-terminal cleavage of the Ab peptide. This model has precedence: the SREBP-(Sterol-regulatory element binding protein) cleavage activating protein (SCAP) was demonstrated to facilitate what is suspected to be intramembranous cleavage of SREBP (90)- an event which may closely parallel g-secretases intramembranous cleavage of bAPP.

A recent study demonstrated that mutations in PS1's two conserved transmembrane asparate residues, Asp 257 and Asp 385, significantly reduced Ab generation and enhanced the carboxy-terminal fragments of bAPP, normal substrates of g-secretase (91). These effects, observed in three different cell lines, indicate that these two asparate residues may be necessary for both PS1 endoproteolysis and g-secretase activity. This led the authors to propose that PS1 is either a diaspartyl cofactor for g-secretase or the g-secretase itself, making it an autoactivated intramembranous aspartyl protease.

While that possibility remains intriguing, PS1 may play an alternative role in the sorting or trafficking of bAPP. Recent studies examined the trafficking and metabolism of both bAPP and TrkB in neurons that lacked PS1 (92). It was demonstrated that these neurons did not secrete Ab and had a significantly increased rate of sAPPa secretion. In addition, the accumulation of the carboxyl-terminal fragments (CTFs) derived from bAPP in these PS1 deficient neurons suggested that PS1 may play a role in promoting intramembrane cleavage or degradation of CTFs. It was also observed that the maturation of TrkB and the BDNF-inducible TrkB autophosphorylation are altered by PS1's absence. The combination of these results suggests that PS1 plays an essential role in regulating trafficking and metabolism of proteins involved in bAPP processing. This hypothesis suggests that mutations in PS1 may retard the normal sorting or trafficking of bAPP at an early stage within the secretory pathway. If normal PS1 is a sorting or trafficking molecule responsible for ensuring bAPP's timely and properly directed exit from the ER towards the Golgi, knocking out PS1 may cause bAPP to lag within the ER, resulting in elevated levels of Ab42. Alternatively, this may cause bAPP to enter the anterograde bulk protein trafficking pathway through the Golgi and to the cell surface, resulting in extremely reduced levels of both Ab42 and Ab40 due to the limited exposure of bAPP to the secretases in the ER/Golgi. This hypothesis is exciting because it may not only account for the effects of PS1 mutations or knocking out PS1 entirely, on bAPP metabolism, but it may also account for the lethality of PS1 knockouts. In addition, the retardation of the proper sorting and trafficking of membrane proteins to cell surface during development may aid in explaining the phenotype of mutations in PS1 homologues in C. elegans.

3.8. Signal Transduction Regulates bAPP Trafficking

Although the evidence that the mutated presenilin proteins may cause AD by altering bAPP metabolism is relatively recent, the idea that bAPP metabolism is a highly regulated phenomenon, is not novel. For example, it has been known for a decade that adding phorbol esters to cultured cells leads to a stimulation of the a-secretase pathway, increasing bAPPs secretion and C-terminal fragment generation within the Ab domain, and decreasing secreted Ab. Studies demonstrating this (43-45) provided evidence that the routing of bAPP through the a-secretase pathway was enhanced by protein phosphorylation through activation of protein kinase C (PKC). It was demonstrated that PKC can phosphorylate serine 655 of bAPP, both on synthetic peptides containing this phospho-domain (35), and utilizing in vitro assays using permeabilized PC12 cells (93).

These results were compelling because they suggested that PKC may act to directly phosphorylate bAPP, perhaps in response to direct depolarization of the neuron itself. In support of this hypothesis, it was demonstrated that when muscarinic acetylcholine receptors were transfected into PC12 cells, receptor agonists led to the same increase in bAPPs, but could not elicit that response when staurosporine was present simultaneously (94). Alternatively, it was possible that PKC, although able to directly phosphorylate bAPP, actually exerted its effects on bAPP metabolism by phosphorylating an entirely different target such as the a-secretase or other phospho-proteins. This latter hypothesis revealed itself to be true following the demonstration that PKC can stimulate bAPPs secretion even when the phosphorylation sites on bAPP are themselves mutated, or when the entire cytoplasmic domain has been deleted (95). So although it was clear that PKC played a crucial role in bAPP metabolism, alternative hypotheses were needed to explain PKC's potent anti-amyloidogenic effects.

One mechanistic explanation is that PKC phosphorylates a TGN phosphoprotein resulting in the redistribution of bAPP from the TGN to the cell surface. The subcellular localization of bAPP within the TGN and a-secretase in the plasma membrane support this hypothesis, confirmed in studies in which the formation of bAPP-containing vesicles was reconstituted from the TGN in a cell-free system (93, 96). In these studies it was demonstrated that PKC treatment results in a redistribution of bAPP from the TGN to post-TGN compartments where it can undergo a-secretase processing. Evidence was also provided that protein kinase A (PKA) can similarly reduce Ab formation and stimulate the release of constitutive secretory proteins from the TGN via a mechanism involving enhanced formation of post-TGN transport vesicles (96). Although PKC and PKA converge on the level of formation from the TGN of bAPP-containing vesicles, additional evidence indicates that the regulatory mechanisms involved are distinct; the effects of PKC and PKA are additive, suggestive of independent substrate sites. Recently processing events that underlie bAPP metabolism have been examined in primary rodent neuronal cultures (76). This study showed that neurons generate abundant Ab peptide variants, especially Ab Glut11 peptides, and demonstrated the role of protein phosphatases in regulating Ab generation in neurons.

One alternative hypothesis, which may help explain PKC's effect on a-secretase cleavage, involves the multicatalytic complex of the proteasome. It was demonstrated that proteasome inhibitors can drastically decrease PDBu-stimulated bAPPa secretion and may play a role in the basal secretion levels of bAPPs (97). Unfortunately from a therapeutic standpoint, PKC activation can stimulate bAPP transcription, indirectly increasing Ab formation; therefore, it remains an important goal to definitively identify phosphorylation targets for the kinases if we are to have a realistic hope of therapeutically intervening in AD pathogenesis via stimulation of this anti-amyloidogenic pathway. In addition, calcium, a cofactor for the activation of PKC and tyrosine kinases, may play an important role in the pathogenesis of AD. Calcium's effect on APP processing has been examined by increasing the ion's intracellular levels through upregulating its release from internal storage compartments or through the use of ionophores. Calcium's role in the regulation of bAPP processing was first examined in studies demonstrating that thapsigargin, an inhibitor of calcium reuptake in the ER, increased sbAPPa release and reduced Ab generation (98). This effect was independent of PKC activation suggesting the presence of an alternative pathway.

3.9. Hormone Regulation of bAPP Metabolism

Increasing epidemiological evidence has suggested that post-menopausal women receiving estrogen replacement therapy have both a delayed onset and reduced risk for developing AD (99, 100). The central role of Ab in AD pathogenesis together with the indication that estrogen replacement therapy may prevent AD, suggested to us that one mechanism of action of estrogen in the brain might be to modulate bAPP metabolism and Ab generation. A recent study reported that physiological concentrations of estrogen reduce neuronal Ab generation (101) which is in agreement with prior data demonstrating that estrogen increased secretion of bAPPs (102). Importantly, these changes were not due to changes in bAPP transcription, or to alteration of PS1 levels. These data suggest one compelling mechanism through which estrogen may protect against AD.

The anti-degenerative effects of estrogen have received increased attention in recent years. In addition to its anti-amyloidogenic properties, estrogen may also modulate (i) basal forebrain cholinergic activity and integrity (103), (ii) dendritic plasticity (104), (iii) NMDA receptor density (105), and (iv) neurotrophin signaling (106), as well as play a role in the prevention of oxidative toxicity due to glutamate, free radicals and Ab (107). Although it plays many roles in the brain, the Ab-reducing effect of estrogen strongly supports the notion that one relevant neuropharmacological activity of estrogen is to reduce Ab generation, and that such an activity contributes to the ability of estrogen replacement therapy to protect against AD.

The cell biological basis for estrogen's regulation of Ab formation remains to be elucidated. It has been demonstrated that estrogen can affect the morphology of the trans-Golgi network by enhancing the biogenesis and trafficking of post-TGN constitutive secretory vesicles and granules (108, 109). Since formation of Ab in the TGN and its export from that organelle are regulated by signal transduction pathways, we hypothesize that estrogen promotes the egress of bAPP from the TGN thereby reducing the local concentration of bAPP available as a substrate for Ab production.

The finding that estrogen reduced the generation of Ab peptides raised the question of whether these effects were specific for estrogen or if other estrogen-like steroid molecules would have similar effects on neuronal Ab secretion. It appears that neuronal cultures treated with testosterone show a similar increase in the secretion of the non-amyloidogenic bAPP fragment, sAPPa and decrease in the secretion of Ab (110). Although clinical studies on testosterone supplementation have not been conducted to date, these results may suggest that testosterone may have a therapeutic benefit in protecting against the development of AD in elderly men, similar to estrogen's efficacy in protecting post-menopausal women against AD.

Levels of adrenal glucocorticoid hormones are known to increase with age, especially in individuals with AD (111, 112). However, treating neurons with corticosterone does not effect sbAPPa or Ab secretion (110) suggesting that estrogen's and testosterone's ability to reduce Ab secretion is not characteristic of all steroid hormones. In addition, cholesterol's influence on bAPP metabolism has been studied by several groups. Increasing cholesterol levels in cellular membranes resulted in a decreased secretion of sbAPPa (113, 114). Interestingly, reducing cellular cholesterol using mevalonate can decrease Ab generation in cultured hippocampal neurons (115), while increasing cholesterol levels by direct treatment of cultured neurons leads to an increase in Ab secretion (110). These results, demonstrating the complex influences of estrogen, testosterone and cholesterol on Ab generation, highlight the inherent difficulty in studying the molecular and cellular basis of bAPP metabolism and underscore the challenge we face in trying to intercede in this pathologically crucial event.


The last decade has provided tremendous insight into the molecular mechanisms that are responsible for bAPP metabolism. In particular, several signal transduction pathways have been identified which can regulate bAPP metabolism to reduce Ab generation. A major goal, however, remains the elucidation of compounds that, while effective at reducing Ab generation, are therapeutically safe. Reducing the amyloid burden in the brain, and therefore the progression of AD, may eventually prove analogous to lowering one's cholesterol to prevent heart disease. As scientists, we are close to fully understanding the molecular basis for bAPP metabolism and Ab generation. However, that will fulfill only half our mission; as healers we have much work to do before we can offer AD patients realistic hope that their disease will become anything other than an irreversible decline into dementia.


The authors would like to thank Dr. Gopal Thinakaran of The Johns Hopkins Medical Institutions and Dr. Lars Lannfelt of The Karolinska Institute for their insightful critique and helpful suggestions for improving this manuscript.


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