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The relationship between amyloid beta and cognitive dysfunction in mouse models of Alzheimer's disease has been evaluated predominantly with the spatial reference memory version of the water maze task. However, as Alzheimer's disease encompasses decline in multiple memory systems, it is important to also utilize non-spatial tasks to fully characterize the role of amyloid on behaviour in animal models. We used the TgCRND8 mouse model of Alzheimer's disease to evaluate the effect of amyloid on spatial reference memory, as well as on the non-spatial task of acquisition of conditioned taste aversion, and on the procedural task of swimming to a visible platform. We demonstrate that 8- to 12-month-old TgCRND8 mice are significantly impaired in all three tasks, and that the levels of soluble amyloid beta are significantly correlated with impairment in spatial reference memory, but not with impairment in conditioned taste aversion or swimming to a visible platform. Insoluble fractions of amyloid, which correspond closely to amyloid plaque burden in the brain, are not associated with any behavioural measure. Our study extends the characterization of the model to stages of advanced amyloid pathology and demonstrates that older TgCRND8 mice are impaired in multiple memory systems, including procedural tasks, which are spared at younger ages. The lack of association between amyloid plaques and memory decline supports clinical findings in Alzheimer's patients.

The Arctic mutation (APP E693G) is unique, since it is located within the amyloid-beta (Abeta) sequence and leads to Alzheimer's disease (AD). Arctic Abeta peptides more easily form Abeta protofibrils in vitro, but little is known about the pathogenic mechanism of the Arctic mutation in vivo. Here, we analyzed APP transgenic mice with both the Swedish and Arctic mutations (tg-APPArcSwe) and transgenic mice with the Swedish mutation alone (tg-APPSwe). Intense intraneuronal Abeta-immunoreactive staining was present in young tg-APPArcSwe mice, but not in tg-APPSwe mice. Intracellular Abeta aggregates in tg-APPArcSwe were strongly stained by antibodies recognizing the N-terminus of Abeta, while those recognizing the C-terminus of Abeta stained weakly. The Abeta aggregates inside neurons increased with age and predated extracellular Abeta deposition in both tg-APPArcSwe and tg-APPSwe mice. Senile plaque deposition was markedly accelerated in tg-APPArcSwe mice, as compared to tg-APPSwe mice. We conclude that the Arctic mutation causes AD by facilitating amyloidosis through early accumulation of intracellular Abeta aggregates in association with a rapid onset of senile plaque deposition.

Deregulation of amyloid precursor protein (APP) processing with increased production of amyloid beta-peptide (Abeta) is considered to be a key pathogenic event in Alzheimer's disease (AD). It has been suggested that stimulation of the muscarinic M(1) receptor subtype affects APP processing and leads to a change in Abeta concentration. To test the hypothesis that treatment with a cholinesterase inhibitor could change the levels of Abeta in plasma, we measured Abeta42 and Abeta40 plasma levels in AD subjects before tacrine treatment and at weeks 2 and 6 of treatment. Treatment with tacrine had no statistically significant effect on plasma Abeta42 and Abeta40 either at 2 weeks or at 6 weeks of administration compared to baseline levels. Plasma Abeta42 and Abeta40 levels showed large subject-to-subject variation but small variation within the same patient over the 3-sample interval. After 2 weeks of treatment with tacrine, there was a strong negative correlation between tacrine concentration and levels of Abeta42 (r = -0.64; p = 0.01) and Abeta40 (r = -0.55; p = 0.04). However, after 6 weeks there was no correlation between plasma concentrations of tacrine and Abeta42 (r = 0.33; p = 0.34) or Abeta40 (r = -0.22; p = 0.54) levels in plasma. After 2 weeks of treatment with an acetylcholinesterase inhibitor, we found a correlation between higher drug concentrations and lower beta-amyloid levels. This might indicate an effect on APP metabolism with an increased alpha-cleavage. But after 6 weeks of drug treatment, there was no obvious drug effect on beta-amyloid concentrations. This finding may indicate that compensatory mechanisms have started at 6 weeks and that no long-term effect on key pathological features in AD is to be expected by an inhibition of acetylcholinesterase., (Copyright 2002 S. Karger AG, Basel)

Several pathogenic Alzheimer's disease (AD) mutations have been described, all of which cause increased amyloid beta-protein (Abeta) levels. Here we present studies of a pathogenic amyloid precursor protein (APP) mutation, located within the Abeta sequence at codon 693 (E693G), that causes AD in a Swedish family. Carriers of this 'Arctic' mutation showed decreased Abeta42 and Abeta40 levels in plasma. Additionally, low levels of Abeta42 were detected in conditioned media from cells transfected with APPE693G. Fibrillization studies demonstrated no difference in fibrillization rate, but Abeta with the Arctic mutation formed protofibrils at a much higher rate and in larger quantities than wild-type (wt) Abeta. The finding of increased protofibril formation and decreased Abeta plasma levels in the Arctic AD may reflect an alternative pathogenic mechanism for AD involving rapid Abeta protofibril formation leading to accelerated buildup of insoluble Abeta intra- and/or extracellularly.

JNPL3 transgenic mice expressing a mutant tau protein, which develop neurofibrillary tangles and progressive motor disturbance, were crossed with Tg2576 transgenic mice expressing mutant beta-amyloid precursor protein (APP), thus modulating the APP-Abeta (beta-amyloid peptide) environment. The resulting double mutant (tau/APP) progeny and the Tg2576 parental strain developed Abeta deposits at the same age; however, relative to JNPL3 mice, the double mutants exhibited neurofibrillary tangle pathology that was substantially enhanced in the limbic system and olfactory cortex. These results indicate that either APP or Abeta influences the formation of neurofibrillary tangles. The interaction between Abeta and tau pathologies in these mice supports the hypothesis that a similar interaction occurs in Alzheimer's disease.

Evidence gathered over the last two decades suggests that beta amyloid (Abeta), the predominant proteinaceous component of senile plaques, plays an early and critical role in the etiology and pathogenesis of Alzheimer's disease (AD). Thus, it is reasonable to hypothesize that compounds capable of reducing the accumulation of Abeta may be of value therapeutically. Additionally, compounds that influence Abeta accumulation may be useful as tools to further dissect the cellular pathways that regulate Abeta production and accumulation. To screen for compounds that affect Abeta levels, we have established high throughput, cell-based assays capable of the sensitive and selective detection of Abeta40 in parallel with the more amyloidogenic form of the peptide, Abeta42. To validate the approach, we examined the effects of several compounds previously identified to influence Abeta accumulation. Analysis of peptide accumulation following treatment with these compounds showed results similar to those previously published. Currently, we are using this assay to screen drugs that have already received FDA approval for the treatment of other diseases and over-the-counter natural product extracts. If compounds such as these can be identified that lower Abeta in the brain, they may represent one of the fastest and most cost effective methods to therapy.

Deposition of beta-amyloid (Abeta) peptides in the brain is an early and invariant feature of all forms of Alzheimer's disease. As with any secreted protein, the extracellular concentration of Abeta is determined not only by its production but also by its catabolism. A major focus of Alzheimer's research has been the elucidation of the mechanisms responsible for the generation of Abeta. Much less, however, is known about the mechanisms responsible for Abeta removal in the brain. In this report, we describe the identification of endothelin-converting enzyme-1 (ECE-1) as a novel Abeta-degrading enzyme. We show that treatment of endogenous ECE-expressing cell lines with the metalloprotease inhibitor phosphoramidon causes a 2-3-fold elevation in extracellular Abeta concentration that appears to be due to inhibition of intracellular Abeta degradation. Furthermore, we show that overexpression of ECE-1 in Chinese hamster ovary cells, which lack endogenous ECE activity, reduces extracellular Abeta concentration by up to 90% and that this effect is completely reversed by treatment of the cells with phosphoramidon. Finally, we show that recombinant soluble ECE-1 is capable of hydrolyzing synthetic Abeta40 and Abeta42 in vitro at multiple sites.

Plasma amyloid beta42 peptide (Abeta42) levels are significantly elevated in all genetic forms of early-onset Alzheimer's disease caused by familial Alzheimer's disease mutations or Down's syndrome. Moreover, recent studies have determined that both plasma Abeta42 and Abeta40 levels are significantly elevated in late-onset Alzheimer's disease (LOAD) patients, their cognitively normal first-degree relatives, and members of typical LOAD families when compared to appropriate controls. To determine the magnitude of the genetic component affecting plasma Abeta levels, we estimated the heritability of plasma Abeta42 and Abeta40 in 15 extended, multigenerational LOAD pedigrees, using a variance components method. Heritability estimates as high as 73 and 54% were found for plasma Abeta42 and Abeta40 levels, respectively. Inclusion of the ApoE epsilon4 dosage as a covariate was not found to have a significant effect on the heritability of these traits. These results suggest that genetic determinants other than ApoE account for a very substantial percentage of the phenotypic variance in plasma Abeta levels. The high heritability and the significant elevation of these traits in LOAD pedigrees suggest that at least some of the genetic determinants of plasma Abeta levels may lead to elevated Abeta and LOAD in these families. Thus, we suggest that plasma Abeta levels are quantitative traits that may be excellent surrogate markers for use in linkage analysis to identify loci that are important in typical LOAD., (Copyright 2001 Wiley-Liss, Inc.)

Alzheimer's disease is characterized by the deposits of the 4-kDa amyloid beta peptide (A beta). The A beta protein precursor (APP) is cleaved by beta-secretase to generate a C-terminal fragment, CTF beta, which in turn is cleaved by gamma-secretase to generate A beta. Alternative cleavage of the APP by alpha-secretase at A beta 16/17 generates the C-terminal fragment, CTFalpha. In addition to A beta, endoproteolytic cleavage of CTF alpha and CTF beta by gamma-secretase should yield a C-terminal fragment of 57-59 residues (CTF gamma). However, CTF gamma has not yet been reported in either brain or cell lysates, presumably due to its instability in vivo. We detected the in vitro generation of A beta as well as an approximately 6-kDa fragment from guinea pig brain membranes. We have provided biochemical and pharmacological evidence that this 6-kDa fragment is the elusive CTF gamma, and we describe an in vitro assay for gamma-secretase activity. The fragment migrates with a synthetic peptide corresponding to the 57-residue CTF gamma fragment. Three compounds previously identified as gamma-secretase inhibitors, pepstatin-A, MG132, and a substrate-based difluoroketone (t-butoxycarbonyl-Val-Ile-(S)-4-amino-3-oxo-2, 2-difluoropentanoyl-Val-Ile-OMe), reduced the yield of CTF gamma, providing additional evidence that the fragment arises from gamma-secretase cleavage. Consistent with reports that presenilins are the elusive gamma-secretases, subcellular fractionation studies showed that presenilin-1, CTF alpha, and CTF beta are enriched in the CTF gamma-generating fractions. The in vitro gamma-secretase assay described here will be useful for the detailed characterization of the enzyme and to screen for gamma-secretase inhibitors.

The abnormal accumulation of the amyloid beta protein (Abeta) has been implicated as an early and critical event in the etiology and pathogenesis of Alzheimer's disease (AD). Compounds that reduce Abeta accumulation may therefore be useful therapeutically. In cell-based screens we detected a significant reduction in Abeta concentration after treatment with the phosphatidylinositol kinase inhibitors wortmannin and LY294002. To determine the effect of this class of compounds on in vivo Abeta accumulation, we administered wortmannin to the Tg2576 mouse model of AD. Oral administration of wortmannin over four months resulted in a significant, non-overlapping 40%-50% reduction in the number of senile plaques, one of the pathological hallmarks of AD. Sandwich ELISA analysis of formic acid extractable Abeta in the brain of treated animals indicates that both Abeta40 and the longer, more amyloidogenic form of the peptide, Abeta42, were significantly reduced. These data provide the first direct evidence that compounds identified by their ability to reduce Abeta concentration in vitro can reduce Abeta accumulation and deposition in the brain, thus establishing a basic paradigm for the identification and evaluation of additional compounds that lower Abeta accumulation.

Plasma Abeta42 (amyloid beta42 peptide) is invariably elevated in early-onset familial Alzheimer's disease (AD), and it is also increased in the first-degree relatives of patients with typical late-onset AD (LOAD). To detect LOAD loci that increase Abeta42, we used plasma Abeta42 as a surrogate trait and performed linkage analysis on extended AD pedigrees identified through a LOAD patient with extremely high plasma Abeta. Here, we report linkage to chromosome 10 with a maximal lod score of 3.93 at 81 centimorgans close to D10S1225. Remarkably, linkage to the same region was obtained independently in a genome-wide screen of LOAD sibling pairs. These results provide strong evidence for a novel LOAD locus on chromosome 10 that acts to increase Abeta.

The amyloid beta protein (Abeta) deposited in the Alzheimer's disease (AD) brain is heterogeneous at both its amino and carboxyl termini. Recent studies of the genetic forms of AD indicate that the aggregation and deposition of Abeta42 may be a common initiating event in all forms of AD. Here, we analyzed the amino termini of the Abeta species deposited in the AD brain, focusing specifically on species with amino-terminal pyroglutamate at position 3 (Abeta3(pE)). Immunocytochemical analysis of AD brains with an antibody specific for Abeta3(pE) confirmed that these species deposit in blood vessels and senile plaques. Using specific sandwich ELISAs, we determined the amounts of Abeta3(pE)-40 and Abeta3(pE)-42(43) in AD brain compared with other forms. This analysis showed that Abeta3(pE)-40 is closely correlated with the extent of Abeta deposition in blood vessels, whereas Abeta3(pE)-42(43) is not. In addition, Abeta3(pE)-42(43) is an important component of the Abeta deposited in senile plaques of the AD brain, constituting approximately 25% of the total Abeta42(43). In vitro comparison of Abeta1-42 and Abeta3(pE)-42 showed that Abeta3(pE)-42 is highly prone to oligomerization. These findings suggest that Abeta3(pE)-42 may be particularly important in AD pathogenesis., (Copyright 2000 Academic Press.)

Prior to the identification of the various abnormal proteins deposited as fibrillar aggregates in the Alzheimer's disease (AD) brain, there was tremendous controversy over the importance of the various lesions with respect to primacy in the pathology of AD. Nevertheless, based on analogy to systemic amyloidosis, many investigators believed that the amyloid deposits in AD played a causal role and that characterization of these deposits would hold the key to understanding this complex disease. Indeed, in retrospect, it was the initial biochemical purifications of the approximately 4 kDa amyloid beta-peptide (Abeta) from amyloid deposits in the mid 1980s that launched a new era of AD research (Glenner and Wong, Biochem. Biophys. Res. Commun. 122 (1984) 1121-1135; Wong et al., Proc. Natl. Acad Sci. USA 82 (1985) 8729 8732; and Masters et al., Proc. Natl. Acad Sci. USA 82 (1985) 4245-4249). Subsequent studies of the biology of Abeta together with genetic studies of AD have all supported the hypothesis that altered Abeta metabolism leading to aggregation plays a causal role in AD. Although there remains controversy as to whether Abeta deposited as classic amyloid or a smaller, aggregated, form causes AD, the relevance of studying the amyloid deposits has certainly been proven. Despite the significant advances in our understanding of the role of Abeta in AD pathogenesis, many important aspects of Abeta biology remain a mystery. This review will highlight those aspects of Abeta biology that have led to our increased understanding of the pathogenesis of AD as well as areas which warrant additional study.

Autosomal dominant mutations in the presenilin 1 (PS1) gene are associated with familial, early-onset Alzheimer's disease. Although the pathogenic mechanism of these mutations is unclear, their common feature is that they lead to an increased concentration of amyloid beta-peptide (Abeta) 42 in the plasma of early-onset patients, in the conditioned media of transfected cells, and in the brains of transgenic mice that overexpress mutant PS1. To address the mechanism(s) by which the pathogenic PS1 mutations increase Abeta42, we constructed human cell lines expressing a doxycyclin (dox)-inducible antisense PS1 RNA and measured its effects on the levels of PS1, amyloid precursor protein (APP), and Abeta. In time course experiments, we observed a statistically significant (p = 0.0038) more than twofold elevation in secreted Abeta42 as early as 12 days after addition of dox. This correlated with an 80% decrease in the 46-kDa PS1 holoprotein and a 30% decrease in the 26-kDa N-terminal fragment (NTF). Furthermore, there was a significant fivefold (p = 0.002) increase in Abeta42 after 14-day dox treatment; this correlated with a >90% decrease in PS1 holoprotein and 60% decrease in NTF. At no time point did we observe significant changes in Abeta40, APP holoprotein, presenilin 2, or tubulin. Ten days after the removal of dox, we observed a return to constitutive levels for Abeta42, PS1 holoprotein, and NTF. These results suggest that in human cell lines, the reduction of normal PS1 activity results in the increased production of Abeta42. Furthermore, our results are consistent with a loss of function or dominant negative mechanism for the pathogenic PS1 mutations.

The Alzheimer's amyloid protein (Abeta) is released from the larger amyloid beta-protein precursor (APP) by unidentified enzymes referred to as beta- and gamma-secretase. beta-Secretase cleaves APP on the amino side of Abeta producing a large secreted derivative (sAPPbeta) and an Abeta-bearing C-terminal derivative that is subsequently cleaved by gamma-secretase to release Abeta. Alternative cleavage of the APP by alpha-secretase at Abeta16/17 releases the secreted derivative sAPPalpha. In yeast, alpha-secretase activity has been attributed to glycosylphosphatidylinositol (GPI)-anchored aspartyl proteases. To examine the role of GPI-anchored proteins, we specifically removed these proteins from the surface of mammalian cells using phosphatidylinositol-specific phospholipase C (PI-PLC). PI-PLC treatment of fetal guinea pig brain cultures substantially reduced the amount of Abeta40 and Abeta42 in the medium but had no effect on sAPPalpha. A mutant CHO cell line (gpi85), which lacks GPI-anchored proteins, secreted lower levels of Abeta40, Abeta42, and sAPPbeta than its parental line (GPI+). When this parental line was treated with PI-PLC, Abeta40, Abeta42, and sAPPbeta decreased to levels similar to those observed in the mutant line, and the mutant line was resistant to these effects of PI-PLC. These findings provide strong evidence that one or more GPI-anchored proteins play an important role in beta-secretase activity and Abeta secretion in mammalian cells. The cell-surface GPI-anchored protein(s) involved in Abeta biogenesis may be excellent therapeutic target(s) in Alzheimer's disease.

gamma-Secretase activity is the final cleavage event that releases the amyloid beta peptide (Abeta) from the beta-secretase cleaved carboxyl-terminal fragment of the amyloid beta protein precursor (APP). No protease responsible for this highly unusual, purportedly intramembranous, cleavage has been definitively identified. We examined the substrate specificity of gamma-secretase by mutating various residues within or adjacent to the transmembrane domain of the APP and then analyzing Abeta production from cells transfected with these mutant APPs by enzyme-linked immunosorbent assay and mass spectrometry. Abeta production was also analyzed from a subset of transmembrane domain APP mutants that showed dramatic shifts in gamma-secretase cleavage in the presence or absence of pepstatin, an inhibitor of gamma-secretase activity. These studies demonstrate that gamma-secretase's cleavage specificity is primarily determined by location of the gamma-secretase cleavage site of APP with respect to the membrane, and that gamma-secretase activity is due to the action of multiple proteases exhibiting both a pepstatin- sensitive activity and a pepstatin-insensitive activity. Given that gamma-secretase is a major therapeutic target in Alzheimer's disease these studies provide important information with respect to the mechanism of Abeta production that will direct efforts to isolate the gamma-secretases and potentially to develop effective therapeutic inhibitors of pathologically relevant gamma-secretase activities.

Numerous mutations in the presenilin (PS) genes cause early onset familial Alzheimer's disease (FAD). Here we characterize the expression of two naturally occurring alternative PS2 transcripts which lack either exons 3 and 4 (PS2 deltaexon3,4) or exons 3, 4, and 8 (PS2 deltaexon3,4,8). These transcripts do not contain the natural initiation codon within exon 3. The transcripts are efficiently translated as N-terminal truncated proteins. These deleted proteins are still able to regulate formation of endogenous PS fragments, indicating that the C-terminal half of the PS2 protein is sufficient for this phenomenon. Although approximately 50% of the PS1 and both PS2 mutations occur within the N-terminal region lacking in the PS2 deltaexon3,4 and PS2 deltaexon3,4,8 proteins, expression of these truncated proteins does not affect pathological generation of amyloid beta-peptide (Abeta). This suggests that point mutations causing AD are gain of function mutations.

Mutations in the amyloid beta precursor protein (APP) gene cosegregate with autosomal dominant Alzheimer disease (AD). Brain pathology of AD is characterized by amyloid deposition in senile plaques and by neurofibrillary tangles. Amyloid deposits in AD brains consist of amyloid beta (A beta), a 4-kDa proteolytic product of APP. In contrast, two other mutations in APP, the Flemish APP692 and Dutch APP693 mutations, are associated with autosomal dominant cerebral hemorrhages due to congophilic amyloid angiopathy (CAA) in the presence or absence of AD pathology, respectively. Both mutations are located within A beta near the constitutive cleavage site. While a common effect of AD-linked mutations is to elevate A beta 42 extracellular concentrations, not much is known about the effect of APP692 and APP693. Here we provide evidence that APP692 and APP693 have a different effect on A beta secretion as determined by cDNA transfection experiments. While APP692 upregulates both A beta 40 and A beta 42 secretion, APP693 does not. These data corroborate with previous findings that increased A beta secretion and particularly of A beta 42, is specific for AD pathology.

Humans harboring missense mutations in the presenilin 1 (PS1) gene undergo progressive cerebral deposition of the 42-residue amyloid beta-protein (A beta 42) at an early age and develop severe Alzheimer's disease. A beta 42 is selectively elevated in the conditioned media of cells expressing mutant but not wild-type PS1, indicating that presenilin mutations alter APP processing. Here we analyze the effects of various PS1 mutant constructs on the cellular production of A beta 42. A construct expressing only the PS1 N-terminal endoproteolytic fragment with the mutation Y115H causes no significant increase in A beta 42, whereas a full-length PS1 construct with the same mutation does. This result suggests that the pathogenic effect of mutant presenilins is produced by the full-length molecule even though only a minor proportion of total PS1 occurs as holoprotein in tissues and cell lines. We demonstrate that the effects of two different PS1 mutations are additive when engineered into the same PS1 molecule. Therefore, two mutations alter gamma-secretase processing of APP more than one and the PS1 mutations described to date do not cause the maximum possible PS1-mediated rise in A beta 42. When a PS1 mutation was expressed in cells carrying the APPV717I mutation, A beta 42 rose dramatically to become the predominant secreted A beta species, an observation of interest for transgenic modeling of AD. Our results are consistent with the hypothesis that presenilin is a major regulator of the proteolytic processing of APP by gamma-secretases.

By using transgenic mice that overexpress human beta-amyloid precursor proteins (APPs) at levels twofold higher than endogenous APPs, following introduction of the human APP gene in a yeast artificial chromosome (YAC), we examined the effects of controlled cortical impact (CCI) brain injury on neuromotor/cognitive dysfunction and the development of Alzheimer's disease (AD)-like neuropathology. Neuropathological analyses included Nissl-staining and immunohistochemistry to detect APPs, beta-amyloid (Abeta), neurofilament proteins, and glial fibrillary acidic protein, whereas Abeta levels were measured in brain homogenates from mice subjected to CCI and control mice by using a sensitive sandwich enzyme-linked immunosorbent assay. Twenty APP-YAC transgenic mice and 17 wild type (WT) littermate controls were anesthetized and subjected to CCI (velocity, 5 m/second; deformation depth, 1 mm). Sham (anesthetized but uninjured) controls (n = 10 APP-YAC; n = 8 WT) also were studied. Motor function was evaluated by using rotarod, inclined-plane, and forelimb/hindlimb flexion tests. The Morris water maze was used to assess memory. Although CCI induced significant motor dysfunction and cognitive deficits, no differences were observed between brain-injured APP-YAC mice and WT mice at 24 hours and 1 week postinjury. By 1 week postinjury, both cortical and hippocampal CA3 neuron loss as well as extensive astrogliosis were observed in all injured animals, suggesting that overexpression of human APPs exhibited no neuroprotective effects. Although AD-like pathology (including amyloid plaques) was not observed in either sham or brain-inj ured animals, a significant decrease in brain concentrations of only Abeta terminating at amino acid 40 (Abeta x-40) was observed following brain injury in APP-YAC mice (P < 0.05 compared with sham control levels). Our data show that the APP-YAC mice do not develop AD-like neuropathology following traumatic brain injury. This may be because this injury does not induce elevated levels of the more amyloidogenic forms of human Abeta (i.e., Abeta x-42/43) in these mice.

The presenilin proteins, localized predominantly to the endoplasmic reticulum, are multi-transmembrane spanning proteins that are processed to form an approximately 27 kD N-terminal fragment and a corresponding 19 kD C-terminal fragment. Mutations in the presenilin 1 and presenilin 2 genes have been identified that cause early onset familial Alzheimer's disease. Analysis of plasma and fibroblasts from patients revealed that these mutations increase the concentration of A beta 42. Cells and transgenic animals containing these mutations both give rise to increases in the extracellular concentration of this peptide providing further evidence for the role of A beta, particularly A beta 42, in Alzheimer's disease. These data provide strong evidence that alterations in A beta concentration are an early and critical event in the pathology of Alzheimer's disease.

We report a novel mutation in the amyloid precursor protein gene (APP I716V) which probably leads to familial early onset Alzheimer's disease with an onset age in the mid 50s. Cells transfected with cDNAs bearing this mutation produce more A beta 1-42(43) than those transfected with wild-type APP and this effect is additive with that of the previously reported APP V717I mutation thus providing a novel approach for further increasing A beta 1-42(43) in model systems.

Five different Alzheimer mutations of the beta-amyloid precursor protein (APP) were expressed in neurons via recombinant herpes simplex virus (HSV) vectors, and the levels of APP metabolites were quantified. The predominant intracellular accumulation product was a C-terminal fragment of APP that co-migrated with the protein product of an HSV recombinant expressing the C-terminal 100 amino acids (C100) of APP, which is known to cause neurodegeneration. Fractionation studies revealed that the C-terminal fragment generated by expression of the Alzheimer mutations, like C100, partitioned into membrane fractions and was particularly enriched in synaptosomes. The processing abnormality caused by expression of the Alzheimer mutations occurs predominantly in neurons. Expression of these mutations or of C100 alone in neurons caused increased secretion of Abeta relative to that of neurons infected with wild type APP recombinant vectors. These data show that expression of APP mutations that cause familial Alzheimer's disease increases the intracellular accumulation of potentially amyloidogenic and neurotoxic C-terminal fragments of APP in neurons.

Mutations in the genes encoding amyloid-beta precursor protein (APP), presenilin 1 (PS1) and presenilin 2 (PS2) are known to cause early-onset, autosomal dominant Alzheimer's disease. Studies of plasma and fibroblasts from subjects with these mutations have established that they all alter amyloid beta-protein (beta APP) processing, which normally leads to the secretion of amyloid-beta protein (relative molecular mass 4,000; M(r) 4K; approximately 90% A beta1-40, approximately 10% A beta1-42(43)), so that the extracellular concentration of A beta42(43) is increased. This increase in A beta42(43) is believed to be the critical change that initiates Alzheimer's disease pathogenesis because A beta42(43) is deposited early and selectively in the senile plaques that are observed in the brains of patients with all forms of the disease. To establish that the presenilin mutations increase the amount of A beta42(43) in the brain and to test whether presenilin mutations act as true (gain of function) dominants, we have now constructed mice expressing wild-type and mutant presenilin genes. Analysis of these mice showed that overexpression of mutant, but not wild-type, PS1 selectively increases brain A beta42(43). These results indicate that the presenilin mutations probably cause Alzheimer's disease through a gain of deleterious function that increases the amount of A beta42(43) in the brain.

Transgenic mice overexpressing the 695-amino acid isoform of human Alzheimer beta-amyloid (Abeta) precursor protein containing a Lys670 --> Asn, Met671 --> Leu mutation had normal learning and memory in spatial reference and alternation tasks at 3 months of age but showed impairment by 9 to 10 months of age. A fivefold increase in Abeta(1-40) and a 14-fold increase in Abeta(1-42/43) accompanied the appearance of these behavioral deficits. Numerous Abeta plaques that stained with Congo red dye were present in cortical and limbic structures of mice with elevated amounts of Abeta. The correlative appearance of behavioral, biochemical, and pathological abnormalities reminiscent of Alzheimer's disease in these transgenic mice suggests new opportunities for exploring the pathophysiology and neurobiology of this disease.

To determine whether the presenilin 1 (PS1), presenilin 2 (PS2) and amyloid beta-protein precursor (APP) mutations linked to familial Alzheimer's disease (FAD) increase the extracellular concentration of amyloid beta-protein (A beta) ending at A beta 42(43) in vivo, we performed a blinded comparison of plasma A beta levels in carriers of these mutations and controls. A beta 1-42(43) was elevated in plasma from subjects with FAD-linked PS1 (P < 0.0001), PS2N1411 (P = 0.009), APPK670N,M671L (P < 0.0001), and APPV7171 (one subject) mutations. A beta ending at A beta 42(43) was also significantly elevated in fibroblast media from subjects with PS1 (P < 0.0001) or PS2 (P = 0.03) mutations. These findings indicate that the FAD-linked mutations may all cause Alzhelmer's disease by increasing the extracellular concentration of A beta 42(43), thereby fostering cerebral deposition of this highly amyloidogenic peptide.

Some forms of familial Alzheimer's disease are caused by mutations in the amyloid beta protein precursor (beta APP), and there is excellent evidence that these mutations foster amyloid deposition by increasing secretion of total amyloid beta protein (A beta) or the highly amyloidogenic A beta 1-42 form. These observations provide a powerful rationale for developing an animal model of AD by generating transgenic mice in which cerebral amyloid deposition is induced by A beta overproduction. To produce substantial A beta in vivo, we generated mice expressing the transgene of signal peptide and 99 residues of carboxyl-terminal fragment (CTF) of beta APP under control of the cytomegalovirus enhancer/chicken beta-actin promoter. The transgenic mRNA was detected in many tissues of these mice, but the levels of transgenic mRNA, CTF, and A beta did not correlate well indicating that tissue-specific posttranslational processing may play an important role in determining the amount of A beta that accumulates in various tissues. A beta was detected biochemically in brain, kidney, and pancreas with the largest amount present in pancreas. In transgenic plasma, there was a marked accumulation of human A beta 1-40 and A beta 1-42(43) to levels over 30-times those observed in normal human plasma. Thus, the transgenic mice produce and secrete considerable A beta. Despite this increase in A beta secretion and the elevated A beta in brain, immunohistochemistry revealed no consistent cerebral A beta deposition. In pancreas, however, intracellular A beta deposits were detected immunohistochemically in acinar cells and interstitial macrophages, some of which showed severe degeneration. In addition, examination of these cells by immunoelectron microscopy revealed many putative amyloid fibrils (7-12 nm) that were stained by anti-A beta antibodies. Overall, our findings indicate that tissue-specific posttranslational processing may play a pivotal role in A beta production and amyloid fibril formation in vivo. By carefully analyzing the changes that occur in the transgenic mice described here as compared to the transgenic line that has recently been shown to form extracellular amyloid plaques in brain, it may be possible to gain considerable insight into the factors that determine the location and amount of A beta that accumulates as amyloid.

Biochemical and immunocytochemical analyses were performed to evaluate the composition of the amyloid beta protein (A beta) deposited in the brains of patients with Alzheimer's disease (AD). To quantitate all A beta s present, cerebral cortex was homogenized in 70% formic acid, and the supernatant was analyzed by sandwich enzyme-linked immunoabsorbent assays specific for various forms of A beta. In 9 of 27 AD brains examined, there was minimal congophilic angiopathy and virtually all A beta (96%) ended at A beta 42(43). The other 18 AD brains contained increasing amounts of A beta ending at A beta 40. From this set, 6 brains with substantial congophilic angiopathy were separately analyzed. In these brains, the amount of A beta ending at A beta 42(43) was much the same as in brains with minimal congophilic angiopathy, but a large amount of A beta ending at A beta 40 (76% of total A beta) was also present. Immunocytochemical analysis with monoclonal antibodies selective for A beta s ending at A beta 42(43) or A beta 40 confirmed that, in brains with minimal congophilic angiopathy, virtually all A beta is A beta ending at A beta 42(43) and showed that this A beta is deposited in senile plaques of all types. In the remaining AD brains, A beta 42(43) was deposited in a similar fashion in plaques, but, in addition, widely varying amounts of A beta ending at A beta 40 were deposited, primarily in blood vessel walls, where some A beta ending at A beta 42(43) was also present. These observations indicate that A beta s ending at A beta 42(43), which are a minor component of the A beta in human cerebrospinal fluid and plasma, are critically important in AD where they deposit selectively in plaques of all kinds.

Normal processing of the amyloid beta protein precursor (beta APP) results in secretion of a soluble 4-kilodalton protein essentially identical to the amyloid beta protein (A beta) that forms insoluble fibrillar deposits in Alzheimer's disease. Human neuroblastoma (M17) cells transfected with constructs expressing wild-type beta APP or the beta APP717 mutants linked to familial Alzheimer's disease were compared by (i) isolation of metabolically labeled 4-kilodalton A beta from conditioned medium, digestion with cyanogen bromide, and analysis of the carboxyl-terminal peptides released, or (ii) analysis of the A beta in conditioned medium with sandwich enzyme-linked immunosorbent assays that discriminate A beta 1-40 from the longer A beta 1-42. Both methods demonstrated that the 4-kilodalton A beta released from wild-type beta APP is primarily but not exclusively A beta 1-40. The beta APP717 mutations, which are located three residues carboxyl to A beta 43, consistently caused a 1.5- to 1.9-fold increase in the percentage of longer A beta generated. Long A beta (for example, A beta 1-42) forms insoluble amyloid fibrils more rapidly than A beta 1-40. Thus, the beta APP717 mutants may cause Alzheimer's disease because they secrete increased amounts of long A beta, thereby fostering amyloid deposition.