APP and Amyloid Beta in Alzhiemer's
Intracellular APP processing and A-beta production in Alzheimer's disease
Alzheimer's disease (AD) is a progressive neurodegenerative dementia afflicting 1% of the population over age 65. Characteristic features of the disease include neurofibrillary tangles composed of abnormal tau paired helical filaments, neuronal loss, and alterations in multiple neurotransmitter systems. A most significant pathological feature, however, is an overabundance of diffuse and compact senile plaques in association and limbic areas of the brain. Although these plaques contain multiple proteins, their cores are composed primarily of b-amyloid, a 39-42 amino acid proteolytic fragment derived from the amyloid precursor protein (APP; for review, see 1).
APP is a single-transmembrane protein with a 590-680 aa long extracellular amino terminal domain and an approximately 55aa cytoplasmic tail which contains intracellular trafficking signals (Fig 1). mRNA from the APP gene on chromosome 21 undergoes alternative splicing to yield eight possible isoforms, three of which (the 695, 751 and 770 amino acid isoforms) predominate in the brain (2,3). APP695 is the shortest of the three isoforms and is produced mainly in neurons. Alternatively, APP751, which contains a Kunitz-protease inhibitor (KPI) domain, and APP770, which contains both the KPI domain and an MRC-OX2 antigen domain, are found mostly in non-neuronal glial cells. All three isoforms share the same Ab, transmembrane and intracellular domains and are thus all potentially amyloidogenic. The normal function of APP is currently unknown, although in neurons it has been demonstrated to be localized in synapses where is may play a role in neurite extension or memory (4).
Figure 1. APP and its proteolytic products. Shown is APP770, the largest of the three predominant isoforms found in the brain. Shorter isoforms are produced by alternative splicing of mRNA to remove the OX2 antigen domain (APP751) or both the OX2 and Kunitz protease inhibitor domains (APP695). APP can undergo proteolytic processing via 2 pathways. Cleavage by a-secretase occurs within the Ab domain and generates the large soluble N-terminal APPa and a non-amyloidogenic C-terminal fragment. Further proteolysis of this fragment by g-secretase generates the non-amyloidogenic peptide p3. Alternatively, cleavage of APP by b-secretase occurs at the beginning of the Ab domain and generates a shorter soluble N-terminus, APPb, as well as an amyloidogenic C-terminal fragment (C99). Further cleavage of this C-terminal fragment by g-secretase generates Ab. Cleavage by g-secretase or multiple g-secretases can result in C-terminal heterogeneity of Ab to generate Ab40 and Ab42.
APP is trafficked through the constitutive secretory pathway, where it undergoes post-translational processing including a variety of proteolytic cleavage events. APP can be cleaved by three enzymatic activities termed a-, b-, and g-secretase (Fig 1). a-secretase cleaves APP at amino acid 17 of the Ab domain, thus releasing the large amino-terminal fragment sAPPa for secretion. Since a-secretase cleaves within the Ab domain, this cleavage precludes Ab formation. Rather, the intracellular carboxy-terminal domain of APP generated by a-secretase cleavage is subsequently cleaved by g-secretase within the predicted transmembrane domain to generate a 22-24 residue (~3kD) fragment termed p3 which is non-amyloidogenic (5). Alternatively, APP can be cleaved by b-secretase to define the amino terminus of Ab and to generate the soluble amino-terminal fragment APPb. Subsequent cleavage of the intracellular carboxy-terminal domain of APP by g-secretase yields full-length Ab. Carboxy-terminal cleavage of Ab by g-secretase results in the generation of multiple peptides, the two most common being 40-amino acid Ab (Ab40) and 42-amino acid Ab (Ab42). Ab40 comprises 90-95% of secreted Ab and is the predominant species recovered from cerebrospinal fluid (6). In contrast, less than 10% of secreted Ab is Ab42. Despite the relative paucity of Ab42 production, Ab42 is the predominant species found in plaques and is deposited initially (7), perhaps due to its ability to form insoluble amyloid aggregates more rapidly than Ab40 (8, 9).
Ab has been postulated to be a causal factor in the pathogenesis of AD. The presence of Ab-containing amyloid plaques is necessary for the neuropathological diagnosis of AD, suggesting that these entities may be involved in the etiology of the disease. Supportive evidence for the causal role of Ab in AD can be found in patients with Down's syndrome, who often develop AD-like symptoms and pathology after age 40 (10). Down's syndrome patients produce elevated APP presumably due to an additional copy of chromosome 21 and exhibit florid AD-like amyloid plaques prior to the onset of other AD symptoms, suggesting that amyloid deposition is an initial event (11). Furthermore, alterations in APP processing have been linked to a subset of familial AD patients (FAD) with autosomal dominant mutations in APP (12, 13), presenilin 1 (PS1; 14) and presenilin 2 (PS2; 15). FAD individuals comprise 10% of all AD cases and generally exhibit symptoms of the disease much earlier than sporadic AD patients. For example, a double mutation of amino acids 670 and 671 in APP from Lys-Met to Asn-Lys immediately upstream of the b-cleavage site of Ab ("Swedish" mutation or APPDNL) results in a 5-8 fold increase in the formation of Ab by cells (13, 16, 17, 18). Furthermore, mutations adjacent to the carboxy-terminal g-cleavage site of Ab such as the Val717 mutations specifically increase the production of the more-amyloidogenic Ab42 relative to Ab40 (19). The fact that these alterations are sufficient to cause AD-like pathology is supported by studies which show that transgenic mice overexpressing either APPDNL (20) or APPV717F (21) produce higher levels of Ab prior to the exhibition of other AD pathological features such as abnormal phosphorylation of cytoskeletal tau, microgliosis, reactive astrocytosis, reduced levels of synaptic marker proteins and memory deficits (22, 23, 24).
?FAD-associated mutations in genes other than APP also affect Ab production. The presenilins, multi-transmembrane proteins localized predominantly to the ER and Golgi, play a crucial role in APP processing. APP and PS1 may form a complex in vivo (25) and PS1 is necessary for the production of Ab, as PS1 knockout mice produce less Ab due to impaired g-secretase cleavage of APP (26). Over 40 dominant point mutations in PS1 (chromosome 14) and PS2 (chromosome 1) as well as one splice site mutation in PS1 have been associated with FAD phenotypes to date (27). FAD-linked mutations in the presenilins also provide a link between Ab and AD in that expression of these mutants in cells favors the production of Ab42 (28). Thus, presenilins are involved in the carboxy-terminal cleavage of APP in both normal and pathological states. Since there may be different g-secretases for the generation of Ab40 and Ab42 (29, 30), it is interesting to speculate that presenilin mutations may increase the ratio of Ab42/Ab40 by preferentially encouraging the activity of the 42-specific g-secretase.
The study of Ab has historically focused on extracellular (i.e. secreted) Ab, as these Ab molecules are assumed to be the building blocks of the extracellular plaques in AD. Extracellular Ab may have an intracellular origin prior to secretion, as Ab can be detected endogenously within neuronal cells such as NT2N (31, 32, 33) as well as within other cell types (34, 35). Both Ab40 and Ab42 can be produced intracellularly (31). Furthermore, although much of the intracellularly-generated Ab is enroute to secretion, there is a significant pool of Ab which is not secreted (33, 36, 37). Thus, there appear to be two distinct pools of intracellularly-generated Ab: a pool that is eventually secreted, and a pool that is destined to remain within the cell.
Given the evidence that altered production of Ab may be an initial event in the development of AD, much research has focused on understanding the mechanisms by which APP is processed to generate Ab. The main cleavage pathways appear to be conserved in both neuronal and non-neuronal cells, but the predominant intracellular sites of production and the particular products formed are cell-type dependent. Non-neuronal cells preferentially process APP via a- and g-secretase cleavage to generate APPa and the non-amyloidogenic fragment p3. Thus, non-neuronal cells are not a significant source of Ab under normal conditions. However, although non-neuronal cells predominantly utilize a-secretase, neurons do not rely heavily on this pathway and produce very low levels of p3 (38). Regardless of the cell type, a-secretase cleaves APP constitutively (5) and is thought to occur mainly at the cell surface since APPa cannot be detected intracellularly (38, 39) and cell-surface labeled APP can be recovered as APPa in the medium (40). In addition to constitutive a-secretase activity, a-secretase cleavage can also be regulated in both neurons and non-neuronal cells via activation of glutamate, muscarinic receptors, and protein kinase C (PKC, see 41 for review). PKC-stimulated a-secretase activity requires the activity of tumor necrosis factor-a converting enzyme (TACE), which can cleave APP at the a-cleavage site in vitro. TACE has a broad sequence specificity and appears to cleave a wide range of proteins at extracellular residues near their transmembrane domains (42).
Cleavage by b- and g-secretases yields Ab and is a constitutive event, as Ab can be detected in normal brains in picomolar to nanomolar concentrations (43, 44). APP is trafficked intracellularly through the default secretory pathway and the generation of Ab can occur at several distinct locations along this route. APP produced in the endoplasmic reticulum (ER) transits to the Golgi, where it is post-translationally modified via N-and O-linked glycosylation and tyrosine sulfation before vesicular transport to the cell surface (45). Cell surface APP is then reinternalized into the endosomal/lysosomal system where it may be degraded (46). Ab can be produced in at least three sites along this pathway: the endosomal-lysosomal system, the Golgi apparatus, and the ER (Fig 2).
Figure 2. Three intracellular pathways of Ab production. APP is synthesized in the endoplasmic reticulum (ER) and is trafficked through the Golgi network to the cell surface. From the cell surface it is reinternalized via endocytosis into the endosomal/lysosomal system. Cleavage of APP to form Ab can occur at three sites along this pathway. The endosomal/lysosomal system may contribute minor amounts of secreted Ab, particularly in non-neuronal cells. The trans-Golgi network (TGN) is the major site of intracellular Ab40 production in neurons and in non-neuronal cells engineered to express the FAD APPDNL "Swedish" mutation. In addition, either the TGN or post-Golgi vesicles are responsible for the production of secreted Ab in neurons. Finally, the ER is a site for the production of Ab42 that is destined to remain within the cell. (Figure courtesy of D. Skovronsky)
APP enters the endosomal-lysosomal system following reinternalization from the cell surface mediated by the APP carboxy-terminal NPTY motif (46, 47). Amyloidogenic carboxy-terminal fragments (CTFs) of APP have been demonstrated to exist in the endosomes/lysosomes, where they may serve as a substrate for g-secretase cleavage to generate Ab (47). Indeed, treatment of cells with agents which interfere with the pH of lysosomes such as chloroquine or NH4Cl reduce the secretion of Ab (48, 49). However, Ab itself cannot be purified from lysosomes of radiolabeled cells (49), suggesting that Ab produced in this organelle is rapidly secreted, and more recent studies have shown that blocking the endosomal/lysosomal system has no effect on the production of intracellular Ab in neurons (50). Thus, the endosomal/lysosomal system contributes a small amount of Ab exclusively to the secreted pool. This pathway may be mostly involved in non-neuronal Ab production which has been shown by immunoelectron microscopy to occur near the surface of cells (51).
A second intracellular site of Ab production is within the Golgi apparatus. This pathway was originally identified in non-neuronal cells expressing APP with the Swedish APPDNL mutation. For example, undifferentiated neuro2a (N2a) cells that overexpress wild-type APP cleave it mainly by a-secretase cleavage at the surface of the cell. However, N2a cells that overexpress APP with the Swedish mutation tend to process APP via b-secretase cleavage to form Ab (18, 52). The intracellular localization of this b-cleavage was investigated by blocking APP exit from the ER with the fungal antibiotic brefeldin A (BFA). This abolishes secreted Ab (48) as well as intracellular Ab40, demonstrating that these species are produced downstream of the ER. The specific location was determined by treatment of N2a cells with the ionophore monensin or incubation at 20°C, both of which block protein trafficking past the trans-Golgi network. These treatments reveal that the Golgi is the main site of intracellular Ab40 production. Secreted Ab40 and Ab42 may be produced here as well (52), although this is controversial (53). By correlating the formation of APPb with APP post-translational modifications known to occur at specific points in the Golgi apparatus, it has been demonstrated that b-cleavage can occur as early as the medial Golgi (18). Endogenous b-cleavage in the Golgi was also demonstrated in non-neuronal H4 cells and PC12 cells, although subsequent g-cleavage was not detected (54, 35)
?Although this pathway contributes to the pool of Ab in non-neuronal cells expressing the FAD Swedish APP mutation, Golgi processing also appears to be constitutively active in neurons expressing wild-type APP (39). Expression of APPDNL in non-neuronal cells results in an eight-fold increase in Ab production, whereas expression in neurons results in a very modest, less than two-fold increase in Ab levels, suggesting that this pathway is already active. As further evidence of the importance of this pathway in neurons, immunoelectron microscopy using an Ab40 end-specific antibody has demonstrated Ab40 to be localized predominantly in the trans-Golgi network (51, 56). As Ab40 is the main secreted and intracellular Ab species produced, the Golgi appears to be the main site of Ab production in neurons.
?A third pathway for the production of Ab was recently identified in the endoplasmic reticulum/intermediate compartment (ER/IC; 36, 37, 51). Although treatment of cells with BFA or incubation at 15oC to retain proteins in the ER abolishes Ab secretion, some b-cleavage can still occur in the ER compartment, as amyloid-containing APP CTFs are produced (55) and APPb can be detected intracellularly in neuronal NT2N cells (38). Furthermore, g-cleavage occurs as well, as intracellular Ab can be detected in transfected kidney 293 cells (37), NT2N neurons (33, 36) or a cell-free reconstitution system (56) following treatment with BFA. Moreover, formation of Ab when protein exit from the ER is blocked is not simply due to non-physiological retention of b- and g-secretases in the ER, as identical results were obtained when APP alone was retained in the ER with a carboxy-terminal dilysine retention motif (36). Interestingly, the intracellular Ab produced in the ER is almost exclusively Ab42 (36, 37, 51) and is not destined for secretion. Immunoelectron microscopy studies using an end-specific antibody for Ab42 offer further confirmation that Ab42 can be localized to the ER in neurons (51, 56).
Taken together, these results point to the production of two pools of Ab. The first is a secretable pool which consists preferentially of Ab40 and is generated mainly in the Golgi and/or post-Golgi vesicles in neurons and in the endosomal/lysosomal system in non-neuronal cells. The second is a non-secreted pool consisting of Ab40 generated in the TGN as well as Ab42 generated in the ER/IC. Significantly, relative levels of Ab40 and Ab42 are different between the secreted and intracellular pools. Although absolute levels of secreted Ab are higher than levels of intracellular Ab, secreted Ab has a much lower ratio of Ab42/Ab40 (1:10) than intracellular Ab (1:3, 32, 33, 36, 37, 50). Given that Ab42 is the more amyloidogenic species and may serve as the nidus for amyloid plaques, the higher intracellular ratio of Ab42/40 may be important for AD. Indeed, the FAD V717F mutation has differential effects on secreted and intracellular Ab and elevates intracellular Ab42 following retention of APP in the ER with BFA (37).
Intracellularly-generated Ab may be important in understanding the role of amyloid in senile plaque formation. While senile plaques are large extracellular accumulations of Ab, they likely result from Ab produced intracellularly and secreted by neurons. However, secretion of Ab alone is insufficient to explain the pathogenesis of senile plaques, as CSF levels of Ab in normal or AD patients are too low to initiate fibril formation. Rather, there must be some mechanism for concentrating Ab to form a nidus for a plaque. One possible mechanism may arise if Ab, particularly the more amyloidogenic Ab42, is concentrated intracellularly. The ER-generated Ab42 that is not secreted may increase slowly over time within the cell until it reaches concentrations necessary for fibril formation. This idea is supported by the recent discovery of a large, detergent-insoluble pool of Ab in NT2N cells that can be liberated by extraction with formic acid (33). This pool has a much higher Ab42/40 ratio than the detergent-soluble pool, with Ab42 surpassing Ab40 as the predominant Ab species. The detergent-insoluble Ab42 is generated within the ER/IC and increases with time in culture due to slower overall turnover of Ab42 than Ab40. Moreover, since this Ab accumulates intracellularly in the absence of any FAD mutations it is possible that pathological mutations may accelerate this phenomenon, as mutations in APP and PS1 increase the relative production of Ab42. This may involve intracellular Ab42, since presenilins are localized within the ER where intracellular Ab42 is generated (27) and the APP V717 mutations can increase the relative production of intracellular Ab42 (37).
Since the detergent-insoluble intracellular pool of Ab42 is degraded slowly, it might form an overwhelming amyloid burden within the cell that may lead to the formation of extracellular amyloid plaques. In transgenic mice harboring the V717F FAD APP mutation, Ab fibrils were detected intracellularly, particularly in the vicinity of the rough endoplasmic reticulum (57), and Ab can also be found inside neurons prior to plaque formation in aged macaque brains (58). Neuronal cell death is a prominent feature of AD (59), and dying neurons might rupture and release the accumulated intracellular Ab42 into the surrounding extracellular milieu. Once released, Ab42 could have multiple effects. First, Ab42 might stimulate further production of amyloidogenic APP fragments in neighboring neurons. Bahr et al. (60) demonstrated that exogenous Ab42 added to hippocampal slice cultures was selectively internalized within AD-vulnerable CA1 neurons and induced a buildup of C99, the amyloidogenic precursor to Ab. Thus, a local release of Ab42 could amplify the levels of intracellular Ab in surrounding cells. Secondly, the release of insoluble intracellular Ab42 from dying neurons might form a nidus for the accumulation of secreted Ab into diffuse or senile extracellular plaques. It has been observed that extracellular deposition of Ab plaques is localized within the vicinity of neuronal cell death and often is seen surrounding dead neurons (61). More striking evidence for intracellular Ab serving as a seed for subsequent plaque formation is the identification and amplification of mRNAs from senile plaques. Ginsberg et al (62) recently demonstrated that the majority of mRNAs which can be amplified from immunohistochemically-defined senile plaques in AD hippocampus are of neuronal origin, suggesting the contribution of neuronal cell components in the development of senile plaques.
Although neuronal cell death in AD might occur for multiple reasons unrelated to the buildup of intracellular Ab, the possibility exists that the intracellular Ab itself is causally involved in neuronal death and other features of AD pathology. Ab42 has been shown to have greater toxicity in vitro than Ab40 (63). Thus, accumulation of high levels of intracellular Ab42 as neurons age may eventually prove toxic to the cell. Correlational evidence for the toxicity of intracellular Ab is derived from the observation that neurons with TUNEL-positive DNA damage in AD brains contain intracellular Ab (61). Detectable intracellular Ab is also found in association with characteristic features of AD pathology. In AD brain tissue intracellular Ab is found in the same neurons which contain neurofibrillary tangles (64) and has been shown to overlap with intracellular neurofibrillary tangles when the tissue is treated with 10% formic acid (65). Additionally, consistent with the relative levels of secreted and intracellular Ab described in previous sections, intracellular neurofibrillary tangles are often associated with the more amyloidogenic Ab42 (66), whereas extracellular neurofibrillary tangles are more likely to be associated with Ab40 (67). It has also been demonstrated that an intracellular increase in potentially amyloidogenic fragments is correlated with a decrease in synaptic marker protein staining (60), suggesting that intracellular Ab may be involved in AD-associated synaptic deficits. Furthermore, these effects can be exerted in the absence of or prior to the formation of senile plaques, suggesting the potential involvement of intracellular Ab. For example, Hsia and colleagues recently demonstrated that transgenic mice harboring the FAD APP V717F mutation display morphological and electrophysiological deficits in the CA1 and CA3 regions of the hippocampus several months prior to the development of amyloid plaques (23). These deficits include diminished presynaptic terminals, decreased neuronal number, and impairments in synaptic transmission which do not correlate with the level of plaque burden. Although the presence of extracellular plaques is apparently not necessary, the authors demonstrated that increased Ab levels do play a role. Transgenic mice with both the Swedish and V717F FAD mutations produce very high levels of Ab and have correspondingly more severe deficits in synaptic transmission.
Thus, we propose a theoretical model for the generation of senile plaques as well as possibly other features of AD pathology (Fig 3). Ab42 is generated in the neuronal ER under normal circumstances, and this production may be increased by the presence of FAD mutations in APP or the presenilins. Due to reduced clearance, Ab42 preferentially accumulates inside the cell and becomes insoluble. The insoluble, intracellular Ab42 may eventually achieve a local concentration within neurons sufficient for the formation of fibrils. This process may require decades, thus accounting for the late onset of AD symptoms. Eventually, the neuron may die due to causes related or unrelated to the accumulation of this Ab and thereby release the aggregated Ab42. The now-extracellular Ab42 fibrils are free to complex with secreted Ab from other neurons (or from non-neuronal cells in some rare cases), forming extracellular amyloid plaques. Thus, in this model the extracellular plaque is seeded from intracellular sources. Furthermore, intracellular Ab may have other effects inside the cell, contributing directly or indirectly to the etiology of other features of AD such as synaptic degeneration and hyperphosphorylated PHF tau. Currently, this theory of intracellular Ab42 in the pathogenesis of AD remains hypothetical and clearly requires more experimentation to substantiate or refute it. However, by shifting the focus of early events in AD from outside of the neuron to inside, this model may be useful in illuminating new directions of research as well as identifying new targets to combat the progression of AD.
Figure 3. Theoretical model for an involvement of intracellular Ab42 in the early events leading to AD. Ab42 generated in the ER remains intracellular and accumulates in a detergent-insoluble pool over time. The insoluble amyloid may eventually achieve local concentrations necessary for the formation of amyloid fibrils. This intracellular burden of insoluble and fibrillar amyloid may be involved in the etiology of other intracellular features of AD, such as neurofibrillary tangles and synaptic degeneration. If death of the neuron eventually occurs (due to causes related or unrelated to AD), degeneration of the cell releases the accumulated Ab42 into the extracellular space, where it may interact with secreted Ab to form the extracellular plaque.
1. Selkoe DJ. Cellular and molecular biology of b-amyloid precursor and Alzheimer's disease. In: Prusiner SB, Rosenberg RN, Mauro SD, et al, eds. The molecular and genetic basis of neurological disease. Boston: Butterworth Heinemann Press, 1997:601-602
2. Golde TE, Estus SC, Usiak M, Younkin LH, Younkin SG. Expression of b amyloid protein precursor mRNAs: Recognition of a novel alternatively spliced form and quantitation in Alzheimer's disease using PCR. Neuron 1990;4:253-67
3. Kang J, Muller-Hill B. Differential splicing of Alzheimer's disease amyloid A4 precursor RNA in rat tissues: PreA4-695 is predominantly produced in rat and human brain. Biochem Biophys Res Commun 1990;166:1192-1200
4. Muller U, Cristina N, Li ZW, et al. Behavioral and anatomical deficits in mice homozygous for a modified b-amyloid precursor protein. Cell 1994;79:755-65
5. Sisodia SS, Koo EH, Beyreuther K, Unterbeck A, Price DL. Evidence that b-amyloid protein in Alzheimer's disease is not derived by normal processing. Science 1990;248:492-5
6. Seubert P, Vigo-Pelfry C, Esch F, et al. Isolation and quantification of soluble Alzheimer's b-peptide from biological fluids. Nature 1992;359:325-7
7. Iwatsubo T, Okada A, Suzuki N, Mizusawa H, Nukina N, Ihara Y. Visualization of Ab42(43) and Ab40 in senile plaques with specific Ab monoclonals: evidence that the initially deposited species is Ab42(43). Neuron 1993;13:45-53
8. Jarrett JT, Berger EP, Lansbury PT. The carboxy terminus of b-amyloid protein is critical for the seeding of amyloid formation: Implications for pathogenesis of Alzheimer's disease. Biochemistry 1993;32:4693-7.
9. Jarrett JT, Lansbury PT Jr. Seeding "one-dimensional crystallization" of amyloid: a pathogenic mechanism in Alzheimer's disease and scrapie? Cell 1993; 73:1055-8.
10. Wisniewski KE, Dalton AJ, McLachlan C, Wen GY, Wisniewski HM. Alzheimer's disease in Down's syndrome: clinicopathologic studies. Neurol 1985; 35:957-61
11. Giaccone G, Tagliavini F, Linoli G, et al. Down patients: extracellular preamyloid deposits precede neuritic degeneration and senile plaques. Neurosci Lett 1989;97:232-8
12. Goate A, Chartier-Harlin MC, Mullan M, et al. Segregation of a missense mutation in the amyloid precursor gene with familial Alzheimer's disease. Nature 1991;349:704-6
13. Citron M, Oltersdorf T, Haass C, et al. Mutation of the b-amyloid precursor protein in familial Azlheimer's disease increases b-protein production. Nature 1992;360:672-4
14. Sherrington R, Rogaev EI, Liang Y, et al. Cloning of a gene bearing missense mutations in early-onset familial Alzheimer's disease. Nature 1995;375:754-60
15. Levy-Lehad E, Wijsman EM, Nemens E, et al. A familial Alzheimer's disease locus on chromosome 1. Science 1995;269:970-3
16. Cai XD, Golde TE, Younkin SG. Release of excess amyloid b-protein from a mutant amyloid b-protein precursor. Science 1993;259:514-6
17. Haass C, Lemere CA, Capell A, et al. The Swedish mutation causes early-onset Alzheimer's disease by b-secretase cleavage within the secretory pathway. Nat Med 1995;1:1291-6
18. Thinakaran G, Teplow DB, Siman R, Greenberg B, Sisodia SS. Metabolism of the "Swedish" amyloid precursor protein variant in neuro2a (N2a) cells. Evidence that cleavage at the "b-secretase" site occurs in the Golgi apparatus. J Biol Chem 1996;271:9390-7
19. Suzuki N, Cheung TT, Cai XD, et al. An increased percentage of long amyloid b protein is secreted by familial amyloid b protein precursor (bAPP 717) mutants. Science 1994;264:1336-40
20. Hsiao K, Chapman P, Nilsen S, et al. Correlative memory deficits, Ab elevation, and amyloid plaques in transgenic mice. Science 1996;274:99-102
21. Games D, Adams D, Alessandrini R, et al. Alzheimer-type neuropathology in transgenic mice overexpressing V717F b-amyloid precursor protein. Nature 1995;373:523-7
22. Johnson-Wood K, Lee M, Motter R, et al. Amyloid precursor protein processing and Ab42 deposition in a transgenic mouse model of Alzheimer disease. Proc Natl Acad Sci USA 1997;94:1550-5
23. Hsia AY, Masliah E, McConlogue L, et al. Plaque-independent disruption of neural circuits in Alzheimer's disease mouse models. Proc Natl Acad Sci USA 1999;96:3228-33
24. Chen KS, Masliah E, Grajeda H, et al. Neurodegenerative Alzheimer-like pathology in PDAPP 717V->F transgenic mice. Prog Brain Res 1998;117:327-34
25. Xia W, Zhang J, Perez R, Koo EH, Selkoe DJ. Interactions between amyloid precursor protein and presenilins in mammalian cells: implications for the pathogenesis of Alzheimer's disease. Proc Natl Acad Sci USA 1997;94:8208-13
26. DeStrooper B, Saftig P, Craessaerts K, et al. Deficiency of presenilin-1 inhibits the normal cleavage of amyloid precursor protein. Nature 1998;391:387-90
27. Younkin SG, Tanzi RE, Christen Y. Presenilins and Alzheimer's disease. New York: Springer-Verlag 1998
28. Scheuner D, Eckman C, Jensen M, et al. Secreted amyloid b-protein similar to that in the senile plaques of Alzheimer's disease is increased in vivo by the presenilin 1 and 2 and APP mutations linked to familial Alzheimer's disease. Nat Med 1996;2:864-70
29. Klafki HW, Abramowski D, Swoboda R, Paganetti PA, Staufenbiel M. The carboxyl termini of b amyloid peptides 1-40 and 1-42 are generated by distinct g-secretase activities. J Biol Chem 1996;271:28655-9
30. Skovronsky DM, Doms RW, Lee VM-Y. Evidence that distinct proteases produce intracellular versus secreted Ab. Submitted.
31. Wertkin AM, Turner RS, Pleasure SJ, et al. Human neurons derived from a teratocarcinoma cell line express solely the 695-amino acid amyloid precursor protein and produce intracellular b-amyloid or A4 peptides. Proc Natl Acad Sci USA 1993; 90:9513-7
32. Turner RS, Suzuki N, Chyung ASC, Younkin SG, Lee VM-Y. Amyloid b 40 and b 42 are generated intracellularly in human neurons and their secretion increases with maturation. J Biol Chem 1996;271:8966-70
33. Skovronsky DM, Doms RW, Lee VM-Y. Detection of a novel intraneuronal pool of insoluble amyloid b-protein that accumulates with time in culture. J Cell Bio 1998;141:1031-9
34. Fuller SJ, Storey E, Li QX, Smith AI, Beyreuther K, Masters CL. Intracellular production of bA4 amyloid of Alzheimer's disease: modulation by phosphoramidon and lack of coupling to the secretion of the amyloid precursor protein. Biochemistry 1995;34:8091-98
35. Kuentzel SL, Ali SM, Altman RA, Greenberg BD, Raub TJ. The Alzheimer b-amyloid protein precursor/protease nexin-II is cleaved by secretase in a trans-Golgi secretory compartment in human neuroglioma cells. Biochem J 1993;295:367-78
36. Cook DG, Forman MS, Sung JC et al. Alzheimer Ab (42) is generated in the endoplasmic reticulum/intermediate compartment of NT2N cells. Nat Med 1997;3:1021-3
37. Wild-Bode C, Yamazaki T, Capell A et al. Intracellular generation and accumulation of amyloid b-peptide terminating at amino acid 42. J Biol Chem 1997;272:10685-8
38. Chyung ASC, Greenberg BD, Cook DG, Doms RW, Lee VM-Y. Novel b-secretase cleavage of b-amyloid precursor protein in the endoplasmic reticulum/intermediate compartment of NT2N cells. J Cell Bio 1997;138:671-80
39. Forman MS, Cook DG, Leight S, Doms RW, Lee VM-Y. Differential effects of the Swedish mutant amyloid precursor protein on b-amyloid accumulation and secretion in neurons and non-neuronal cells J Biol Chem 1997;272:32247-53
40. Sisodia SS. b-amyloid precursor protein cleavage by a membrane-bound protease. Proc Natl Acad Sci USA 1992;89:6075-9
41. Rossner S, Ueberham U, Schliebs R, Perez-Polo JR, Bigl V. The regulation of amyloid precursor protein metabolism by cholinergic mechanisms and neurotrophin receptor signaling. Prog Neurobiol 1998;56:541-69
42. Buxbaum JD, Liu KN, Luo Y, et al. Evidence that tumor necrosis factor a converting enzyme is involved in regulated a-secretase cleavage of the Alzheimer amyloid protein precursor. J Biol Chem 1998;273:27765-7
43. Haass C, Schlossmacher MG, Hung AY, et al. Amyloid b-peptide is produced by cultured cells during normal metabolism. Nature 1992;359:322-5
44. Seubert P, Oltersdorf T, Lee MG, et al. Secretion of b-amyloid precursor protein cleaved at the amino terminus of the b-amyloid peptide. Nature 1993;361:260-3
45. Weidemann A, Koenig G, Bunke D, et al. Identification, biogenesis, and localization of precursors of Alzheimer disease A4 amyloid protein. Cell 1989;57:115-26
46. Golde TE, Estus S, Younkin LH, Selkoe DJ, Younkin SG. Processing of the amyloid protein precursor to potentially amyloidogenic derivatives. Science 1992;255:728-30
47. Koo EH, Squazzo S. Evidence that production and release of amyloid b-protein involves the endocytic pathway. J Biol Chem 1994;269:17386-9
48. Shoji M, Golde TE, Ghiso J, et al. Production of the Alzheimer amyloid b protein by normal proteolytic processing. Science 1992;258:126-9
49. Haass C, Hung AV, Schlossmacher MG, Teplow DB, Selkoe DJ. b-amyloid peptide and a 3-kDa fragment are derived by distinct cellular mechanisms. J Biol Chem 1993;268:3021-4
50. Tienari PJ, Ida N, Ikonen E, et al. Intracellular and secreted Alzheimer b-amyloid species are generated by distinct mechanisms in cultured hippocampal neurons. Proc Natl Acad Sci USA 1997;94:4125-30
51. Hartmann T, Bieger SC, Bruhl B, et al. Distinct sites of intracellular production for Alzheimer's disease Ab40/42 amyloid peptides. Nat Med 1997;3:1016-7
52. Xu H, Sweeney D, Wang R, et al. Generation of Alzheimer's b-amyloid protein in the trans-Golgi in the apparent absence of vesicle formation. Proc Natl Acad Sci USA 1997;94:3748-52
53. Martin BL, Schrader-Fischer G, Busciglio J, Duke M, Paganetti P, Yankner BA. Intracellular accumulation of b-amyloid in cells expressing the swedish mutant amyloid precursor protein. J Biol Chem 1995;270:26727-26730
54. Sambamurti K, Shioi J, Anderson JP, Pappolla MA, Robakis NK. Evidence for intracellular cleavage of the Alzheimer's amyloid precursor in PC12 cells. J Neurosci Res 1992;33:319-29
55. Gabudza D, Busciglio J, Chen LB, Masudaira P, Yankner BA. Inhibition of energy metabolism alters the processing of amyloid precursor protein and induces a potentially amyloidogenic derivative. J Biol Chem 1994;249:13623-8
56. Greenfield JP, Tsai J, Gouras GK, et al. Endoplasmic reticulum and trans-Golgi network generate distinct populations of Alzheimer b-amyloid peptides. Proc Natl Acad Sci USA 1999;96:742-7
57. Masliah E, Sisk A, Mallory M, Mucke L, Schenk D, Games D. Comparison of neurodegenerative pathology in transgenic mice overexpressing V717F b-amyloid precursor protein and Alzheimer's disease. J Neurosci 1996;16:5795-811
58. Murphy GM, Forno LS, Higgins L, Scardina JM, Eng LF, Cordell B. Development of a monoclonal antibody specific for the COOH-terminal of b-amyloid 1-42 and its immunohistochemical reactivity in Alzheimer's disease and related disorders. Am J Pathol 1994;144:1082-8
59. Cotman CW, Su JH. Mechanisms of neuronal death in Alzheimer's disease. Brain Pathol 1996;6:493-506.
60. Bahr BA, Hoffman KB, Yang AJ, Hess US, Glabe CG, Lynch G. Amyloid b-protein is internalized selectively by hippocampal field CA1 and causes neurons to accumulate amyloidogenic carboxy-terminal fragments of the amyloid precursor protein. J Comp Neurol 1998; 397:139-47
61. LaFerla FM, Troncoso JC, Strickland DK, Kawas CH, Jay G. Neuronal cell death in Alzheimer's disease correlates with apoE uptake and intracellular Ab stabilization. J Clin Invest 1997;100:310-20
62. Ginsberg SD, Crino PB, Hemby SE, et al. Predominance of neuronal mRNAs in individual Alzheimer's disease senile plaques. Ann Neurol 1999;45:174-81
63. Iverson LL, Mortishire-Smith RJ, Pollack SJ, Shearman MS. The toxicity in vitro of b-amyloid protein. Biochem J 1995;311:1-16
64. Grundke-Iqbal I, Iqbal K, George L, Tung YC, Kim KS, Wisniewski HM. Amyloid protein and neurofibrillary tangles coexist in the same neuron in Alzheimer disease. Proc Natl Acad Sci USA 1989;86:2853-7
65. Perry G, Cras P, Siedlak SL, Tabaton M, Kawai M. b-protein immunoreactivity is found in the majority of neurofibrillary tangles of Alzheimer's disease. Am J Pathol 1992;140:283-90
66. Murphy GM, Forno LS, Higgins L, Scardina JM, Eng LF, Cordell B. Development of a monoclonal antibody specific for the COOH-terminal of b-amyloid 1-42 and its immunohistochemical reactivity in Alzheimer's disease and related disorders. Am J Pathol 1994;144:1082-8
67. Schwab C, Akiyama H, McGeer EG, McGeer PL. Extracellular neurofibrillary tangles are immunopositive for the 40 carboxy-terminal sequence of b-amyloid protein. J Neuropathol Exp Neurol 1998;57:1131-7