Increased amyloid-beta42(43) in brains of mice expressing mutant presenilin 1

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.     

Hypoglycemia enhances the expression of mRNA encoding beta-amyloid precursor protein in rat primary cortical astroglial cells

Deposition of beta-amyloid (A beta) is a characteristic feature of the pathology of Alzheimer's disease (AD). Since glucose metabolism and the consequential ATP production are depressed in the temporal and parietal regions of the cortex in patients with AD, we designed the present study to investigate the possible role of hypometabolism in the pathogenesis of AD. We incubated rat primary cortical astroglial cells for 2 h to 4 days in a media deprived of 95% of its glucose and assessed the expression and alternative splicing of the mRNA that encoding beta-amyloid precursor protein (APP) using RT-PCR. Hypoglycemia caused a time-dependent increase in APP mRNA expression, which reaches a peak level of 173.2% of control expression (P < 0.05) at 24 h of hypoglycemia. Noteworthy, hypoglycemia favors the alternative splicing that includes the exon 7 segment, which encodes a Kunitz-type serine protease inhibitor domain. This study demonstrates that hypoglycemia increases APP mRNA expression in astroglial cells and processing of APP mRNA to a form that may encourage A beta deposits in AD. These data suggest that the observed hypometabolism in AD may contribute to its deposition of A beta in affected brain regions.     

A comprehensive study of the spatiotemporal pattern of beta-amyloid precursor protein mRNA and protein in the rat brain: lack of modulation by exogenously applied nerve growth factor

Nerve growth factor (NGF) is a neurotrophic factor for basal forebrain cholinergic neurons, a population that degenerates and dies in Alzheimer's disease (AD). It has been suggested that NGF be used to treat AD patients. However, in vivo administration of NGF to the developing hamster brain was shown to induce the expression of the beta-amyloid precursor protein (beta APP) gene. The association of alterations in beta APP gene expression with AD-like neuropathological changes and cognitive impairment in animals, and with AD-like neurodegeneration in Down syndrome patients suggests that NGF-mediated increases in beta APP expression could negate or attenuate NGF's neurotrophic activity in AD treatment trials. The present study was undertaken to explore further the influence of NGF on beta APP expression, and to determine which, if any, of the beta APP mRNAs is altered in response to NGF treatment. We first examined the spatiotemporal pattern of beta APP-695 and Kunitz protease inhibitor (KPI)-containing beta APP mRNA expression in the rat brain. Specific oligonucleotide probes were used to show that these mRNAs are present during embryonic development. In addition, we evaluated postnatal expression in nine brain regions and showed that beta APP mRNAs were readily detected in all regions at postnatal day 2. In human brain, the relative levels of beta APP-695 and beta APP-KPI mRNA and their protein are discordant, in that the level of beta APP-695 mRNA is slightly higher than that of beta APP-KPI, but beta APP-KPI protein predominates. In contrast, the several-fold excess of beta APP-695 mRNA relative to beta APP-KPI mRNA in the rat brain was also reflected at the protein level. Surprisingly, administration of exogenous NGF failed to affect rat beta APP mRNA levels either in vitro or during postnatal development in vivo.     

Zyme, a novel and potentially amyloidogenic enzyme cDNA isolated from Alzheimer's disease brain

The deposition of the beta amyloid peptide in neuritic plaques and cerebral blood vessels is a hallmark of Alzheimer's disease (AD) pathology. The major component of the amyloid deposit is a 4.2-kDa polypeptide termed amyloid beta-protein of 39-43 residues, which is derived from processing of a larger amyloid precursor protein (APP). It is hypothesized that a chymotrypsin-like enzyme is involved in the processing of APP. We have discovered a new serine protease from the AD brain by polymerase chain reaction amplification of DNA sequences representing active site homologous regions of chymotrypsin-like enzymes. A cDNA clone was identified as one out of one million that encodes Zyme, a serine protease. Messenger RNA encoding Zyme can be detected in some mammalian species but not in mice, rats, or hamster. Zyme is expressed predominantly in brain, kidney, and salivary gland. Zyme mRNA cannot be detected in fetal brain but is seen in adult brain. The Zyme gene maps to chromosome 19q13.3, a region which shows genetic linkage with late onset familial Alzheimer's disease. When Zyme cDNA is co-expressed with the APP cDNA in 293 (human embryonic kidney) cells, amyloidogenic fragments are detected using C-terminal antibody to APP. These co-transfected cells release an abundance of truncated amyloid beta-protein peptide and shows a reduction of residues 17-42 of Abeta (P3) peptide. Zyme is immunolocalized to perivascular cells in monkey cortex and the AD brain. In addition, Zyme is localized to microglial cells in our AD brain sample. The amyloidogenic potential and localization in brain may indicate a role for this protease in amyloid precursor processing and AD.     

Specific detection of an interleukin 1- and tumour necrosis factor-activated beta-casein kinase in HeLa and KB cells

beta-amyloid (beta A) deposition is a key event in the etiopathogenesis of Alzheimer's disease (AD), contributing to neuronal degeneration and cognitive impairment in AD patients. Both neurotrophic and neurotoxic actions of beta A have been demonstrated in experimental conditions. In order to further characterize the effects of brain beta A deposits on behavioral processes, we evaluated psychomotor activity (PMA), psychomotor coordination (PMC) and learning in a passive avoidance task (PAL) in rats with unilateral or bilateral 2 microliters injections of beta-amyloid (1-28) protein (beta A; 1.5 nmol/microliter) or vehicle (water; W) into the hippocampus, 1 and 4 weeks after neurosurgery. The extent of neuronal loss in the lateral blade of the gyrus dentatus (LBGD) and the area percentage occupied by APP immunoreactivity in neurons of the CA3c subfield of the hippocampus were also measured in animals with unilateral beta A implants. PMA levels were similar in water- and beta A-injected animals 1 and 4 weeks after recovery. As compared to water-injected rats, beta A animals showed reduced PMC values 1 week, but not 4 weeks, after injections. beta A also impaired learning acquisition in a passive avoidance task, reducing the number of avoidances and mean latency per trial at both 1 and 4 weeks postsurgery in rats with unilateral or bilateral beta A implants. The extent of neuronal loss in the LBGD) was not different in rats receiving water or beta A injections. Hippocampal APP expression tended to increase in beta A-implanted rats and showed a negative correlation with cognitive performance at the 4-week period. According to these results it seems that beta A implants into the hippocampus reduce psychomotor coordination performance in a transient manner, with no effect on psychomotor activity, and induce durable learning impairment in rats, and that changes in cognitive performance correlate with histochemical parameters such as APP expression. In conclusion, the present results contribute to a better understanding of beta A-induced behavioral alterations and to the identification of potential molecular mechanisms involved in cognitive dysfunctions in this animal model of neurodegeneration.     

Deletion of an endosomal/lysosomal targeting signal promotes the secretion of Alzheimer's disease amyloid precursor protein (APP)

Alzheimer's disease amyloid precursor protein (APP) generates a beta-amyloid protein (A beta) that is a main component of the senile plaques found in the brains of Alzheimer's disease patients. APP is thought to undergo proteolysis via two different pathways, the amyloidogenic pathway which produces A beta, and the non-amyloidogenic pathway which releases a large N-terminal fragment into the medium. The proteases that mediate these processes remain unidentified. The physiological function of APP is not clear yet. Therefore, the cytoplasmic region of APP has attracted much interest, because this region is highly conserved among species, and members of the amyloid precursor-like protein (APLP) family. Several potentially functional sequences exist in the region, including signal sequences for protein sorting and a G0-protein binding sequence. We constructed two mutants, 695 deltaNPTY and 695 deltaGYEN. They lack potential endosome/lysosome targeting signals, NPTY and GY, in the cytoplasmic domain of APP695, respectively. The mutant APPs had longer half-lives and were secreted more easily into the medium than the wild type, suggesting that these sequences are important for the secretion and metabolism of APP.     

Detection of the membrane-retained carboxy-terminal tail containing polypeptides of the amyloid precursor protein in tissue from Alzheimer's disease brain

A major hallmark of Alzheimer's disease (AD) is the presence of extracellular amyloid plaques consisting primarily of amyloid beta peptide (A beta) which is derived from a larger beta-amyloid precursor protein (APP). APP is processed via secretory and endosomal/lysosomal pathways by a group of proteases called secretases. During the processing of APP, the carboxy-terminal tail fragment has been suggested to remain within the cell. To investigate the fate of this fragment, we generated an antibody specific for a nine amino acid residue, the sequence of which was derived from the carboxy-terminal putative cytoplasmic tail of APP. Computer analysis of the entire APP gene, searching for regions of greatest antigenicity, surface probability, hydrophilicity, and presence of beta turns, indicated that the cytoplasmic tail region is an immunodominant region of APP. The peptide coupled to keyhole limpet hemocyanin protein, produced a very high titer antibody (1:1 x 10(6)). To evaluate the specificity of the antibody, immunoprecipitation of in vitro transcribed and translated DNA encoding the carboxy-terminal amino acids of APP in wheat germ extract was carried out. A single immunoprecipitated band of the correct size was seen by SDS-PAGE. The antibody was also able to specifically detect the accumulation of the stable C-terminal tail containing fragments of APP in neurites of the amygdala and hippocampus regions of the human brain tissue from AD subjects, but did not react with age-matched control normal brain tissue. The localization of the C-terminal tail of APP within the brain tissue of AD patients underscores the likely importance of the C-terminus in the pathogenesis of AD.     

The Cystatin-C gene is not linked to early onset familial Alzheimer's disease

The APP717 mutations discovered in only a few early onset Alzheimer's disease (AD) families have confirmed the genetic heterogeneity of this disorder. To identify the other gene(s) involved in the disease we selected the protease inhibitor, Cystatin-C, as a candidate gene. Cystatin-C is an amyloidogenic protein causing hereditary cerebral haemorrhage with amyloidosis-Icelandic type (HCHWA-I). It is localised with the beta-amyloid peptide in the arterial walls of AD brains. We have analysed the segregation of a polymorphic marker in this gene in 8 early onset AD families. Two early onset families showed clear non-segregation of the marker with the disease. When the 8 families are analysed together (assuming only one other gene is involved), they present exclusion linkage criteria. These data indicate that Cystatin-C is not the site of the defect in 2 families and is not likely to be in the other families analysed. We conclude that the deposition of Cystatin-C in AD is a secondary event in the disease process, and that this gene is not pathogenic in familial AD.     

Carboxyl-terminal fragments of beta-amyloid precursor protein bind to microtubules and the associated protein tau

Alzheimer's disease is a neurodegenerative disorder characterized by protein depositions in intracellular and extracellular spaces in the brain. The intraneuronal deposits are formed by neurofibrillary tangles composed mainly of abnormally phosphorylated tau, a microtubule-associated protein, whereas the major constituent of the amyloid deposited extracellularly in the brain parenchyma and vessel walls is amyloid beta-protein (A beta). The proteolytic processing of the beta-amyloid precursor protein (beta PP) results in the generation of a complex set of carboxyl-terminal peptides that contain A beta. In this study, we have used fusion proteins containing carboxyl-terminal fragments of beta PP to investigate the association of beta PP with cellular components. We demonstrate that specific domains within the carboxyl end of beta PP contain binding sites for cytoskeletal components; one, within residues 1 to 28 of A beta, binds directly to tubulin, and the second one, within sequences carboxyl-terminal to A beta, binds tau and tubulin. We propose that the two neuropathological hallmarks of Alzheimer's disease, A beta deposition and neurofibrillary tangles, represent the residual of a disrupted beta PP-tubulin-tau complex.     

Presenilins and early-onset familial Alzheimer's disease

Thirty-seven missense mutations and a splice-site mutation in the presenilin gene PS1 on chromosome 14 and two missense mutations PS2 on chromosome 1 co-segregate with early-onset familial Alzheimer's disease (AD). The presenilins belong to a family of conserved integral membrane proteins which include Caenorhabditis elegans SPE4 and SEL12 and the rat apoptosis-linked gene, ALG3. This review summarizes the genetics of presenilins in AD and indicators of putative function based on cellular localization and the functions of non-human homologues. Findings to date suggest an important role of presenilins in beta-amyloid (A beta) production: in vitro and in vivo studies have shown that presenilin mutations are associated with relatively increased production of the longer, and highly fibrillogenic A beta 42(43) peptide, and a marked elevation in the number of A beta 42-immunoreactive plaques in the brains of individuals with familial AD who carry PS1 and PS2 mutations. There is growing evidence that the deposition of A beta 42(43) could in some cases be an early and key event in the AD pathogenic cascade. The genetic and molecular biological data discussed in this review describe mechanisms by which presenilin mutations could lead to the development of AD. Also, mutant presenilins may be more proapoptotic. It is argued that the understanding of the processes by which presenilin mutations lead to the development of AD will help in devising a coherent framework for therapeutic strategies.     

The role of A beta 42 in Alzheimer's disease

Our recent studies of plasma, fibroblasts, transfected cells and transgenic mice show that a fundamental effect of the mutations linked to familial Alzheimer's disease (FAD) is to increase the extracellular concentration of A beta 42. This effect of the FAD-linked mutations is likely to be directly related to the pathogenesis of Alzheimer's disease (AD) because A beta 42 is deposited early and selectively in the senile plaques that are an invariant feature of all forms of AD. Thus our results provide strong evidence that the FAD-linked mutations all cause AD by increasing the extracellular concentration of A beta 42 (43), thereby fostering A beta deposition, and they support the hypothesis that cerebral A beta deposition is an essential early event in the pathogenesis of all forms of AD. Interactions between the basal forebrain cholinergic system and A beta that could influence AD pathogenesis are discussed.     

Expression of V642 APP mutant causes cellular apoptosis as Alzheimer trait-linked phenotype

APP is a transmembrane precursor of beta-amyloid. In dominantly inherited familial Alzheimer's disease (FAD), point mutations V6421, V642F and V642G have been discovered in APP695. Here we show that expression of these mutants (FAD-APPs) causes a clone of COS cells to undergo apoptosis associated with DNA fragmentation. Apoptosis by the three FAD-APPs was the highest among all possible V642 mutants; normal APP695 had no effect on apoptosis, suggesting that apoptosis by APP mutants in this system is phenotypically linked to the FAD trait. FAD-APP-induced apoptosis was sensitive to bcl-2 and most probably mediated by heteromeric G proteins. This study presents a model system allowing analysis of the mechanism for FAD-APP-induced cytotoxicity.     

Neurodegeneration and gliosis in transgenic mice overexpressing a carboxy-terminal fragment of Alzheimer amyloid-beta protein precursor

In order to study the properties of beta-amyloid in vivo, we generated a total of 28 transgenic founder mice that harbored the gene for the 17-amino acid signal sequence and the 99-amino acid carboxy-terminal fragment (CTF) of the human amyloid-beta protein precursor (beta APP) linked to the cytomegalovirus enhancer and chicken beta-actin promoter. Two of these founders, termed 0304 and 0809, exhibited decreased behavioral activity with gliosis and neurodegeneration in the hippocampus at 2.5 and 9 months of age. Both mice had also decreased levels of synaptophysin, a presynaptic marker, but no evidence for beta-amyloid deposition in their brains. Neurodegeneration in the hippocampus was transmitted to the offspring of mouse 0304, although the frequency was low (5 of 44 mice examined) and the time of onset of the disorder was rather later than that in the founder mouse. This is probably due to reduced levels of the transgene-derived products in the offspring of mouse 0304. The 0809 line failed to produce its offspring. The other remaining transgenic founders appeared normal and had lesser amounts of the CTF mRNA and protein in their brains than did 0304 and 0809 founders, though some mice died in earlier stages or exhibited hydrocephalus. These findings suggest that overexpression of the CTF of human beta APP has the potential to elicit neurodegeneration in vivo without appreciable production of beta-amyloid fibrils.     

Progress of the molecular genetic analysis for Alzheimer's disease

The pathogenesis of Alzheimer's disease (AD) remains unclear. We ascertained 57 Japanese families with early onset familial Alzheimer's disease (EOFAD; mean onset age < 65 years). Screening the known beta/A4 amyloid precursor protein (APP) mutations in familial AD by a simplified PCR product detection system disclosed only one EOFAD with the APP717Val-Ile mutation (AD1 locus). Four of 6 families showing a positive linkage to AD3 locus had four different mutations on the presenilin 1 gene. These mutations were Val 96 Phe, Ile223Thr, His163Arg and splicing mutation with an AG-AA substitution at the acceptor site of intron 9. The allele frequency of APOE-e4 of late-onset and early-onset AD was significantly higher than that of age-matched controls (p < 0.0001). These results suggest that APOE-e4 (AD2 locus) is a susceptibility gene for AD in the Japanese population, regardless of the age at onset. It is probable that the risk for AD in most subjects is likely to arise from the cumulative effects of environmental factors along with various genetic factors. Advances in molecular biology and molecular genetics have enabled us to more easily understand these genetic factors.     

Transgenic mice expressing Alzheimer amyloid precursor proteins

Nearly a decade after the identification of the Alzheimer amyloid precursor protein (APP) gene several groups of investigators have created transgenic mice expressing APP that simulate some of the prominent behavioral and pathological features of Alzheimer's disease (Quon et al., 1991; Games et al., 1995; Hsiao et al., 1995, 1996; Moechars et al., 1996; Sturchler-Pierrat et al., 1997). These features, which are present to various degrees in different lines of mice, include age-related impairment in learning and memory, neuronal loss, gliosis, neuritic changes, amyloid deposition, and abnormal tau phosphorylation. No mouse model exhibiting every neuropathological feature of Alzheimer's disease exists. Whether an exact simulation of Alzheimer neuropathology is required to understand neural dysfunction in Alzheimer's disease is unclear. Various mouse models of Alzheimer's disease are summarized in this article.     

The vacuolar H(+)-ATPase inhibitor bafilomycin A1 differentially affects proteolytic processing of mutant and wild-type beta-amyloid precursor protein

We analyzed the effect of the vacuolar H(+)-ATPase inhibitor bafilomycin A1 (bafA1) on the processing of beta-amyloid precursor protein (beta APP). In kidney 293 cells stably transfected with the wild-type beta APP cDNA, bafA1 caused a stabilization of mature beta APP and its 10-kDa COOH-terminal fragment. Moreover, it caused a 2-3-fold increase in secretion of soluble APP and amyloid-beta protein (A beta). Interestingly, bafA1 treatment of cells transfected with a mutant beta APP isoform that occurs in a Swedish kindred with familial Alzheimer's disease resulted in a decrease of A beta production and no increase of soluble APP secretion. Identical results were obtained when the effect of bafA1 was analyzed on fibroblasts derived from affected versus unaffected members of the Swedish family. These data demonstrate a differential effect of bafA1 on the production of A beta derived from wild-type or Swedish mutant beta APP. Radiosequencing of A beta derived from bafA1-treated cells expressing wild-type beta APP revealed a marked increase of A beta peptides starting at amino acids phenylalanine 4 and valine -3 and a relative decrease of A beta molecules beginning at the typical NH2 terminus of aspartate 1. Cells transfected with the Swedish mutation and treated with bafA1 did not produce these alternative A beta peptides, so that bafA1 treatment resulted in a decrease of A beta starting at aspartate 1. Our data indicate that multiple proteases are able to cleave A beta at or near its NH2 terminus. Inhibition of the protease cleaving at aspartate 1 by bafA1 and perhaps other similar agents can result in an increase of alternatively cleaved peptides.     

Reversibility of the effect of tacrine on the secretion of the beta-amyloid precursor protein in cultured cells

The amyloid beta-protein (A beta) of Alzheimer's disease is derived from a family of large integral membrane glycoproteins, beta-amyloid precursor proteins (beta APP). Two secretory proteolytic pathways are involved in the metabolism of beta APP. The major pathway involves cleavage within the A beta sequence and generates carboxyl-truncated derivatives of beta APP which are secreted into the conditioned medium of cells. The minor 'amyloidogenic' pathway results in the production of A beta. Here, cell cultures were used to examine the metabolism of beta APP by tacrine, a centrally active cholinesterase inhibitor reported to improve cognitive deficits. Treatment with tacrine in cells resulted in the drastic inhibition of secretion of the major isoforms of beta APP into the medium. The effect of tacrine can be reversed by washing away the drug from the cells. Treatment with tacrine did not change the level of either HSP-70 or LDH. Thus, the inhibitory effect of tacrine on the secretion of beta APP was not due to the permanent damage or loss of cells as normal release of beta APP could be restored when the drug was washed away.     

Modulation of secreted beta-amyloid precursor protein and amyloid beta-peptide in brain by cholesterol

The effects of dietary cholesterol on brain amyloid precursor protein (APP) processing were examined using an APP gene-targeted mouse, genetically humanized in the amyloid beta-peptide (Abeta) domain and expressing the Swedish familial Alzheimer's disease mutations. These mice express endogenous levels of APP holoprotein and abundant human Abeta. Increased dietary cholesterol led to significant reductions in brain levels of secreted APP derivatives, including sAPPalpha, sAPPbeta, Abeta1-40, and Abeta1-42, while having little to no effect on cell-associated species, including full-length APP and the COOH-terminal APP processing derivatives. The changes in levels of sAPP and Abeta in brain all were negatively correlated with serum cholesterol levels and levels of serum and brain apoE. These results demonstrate that secreted APP processing derivatives and Abeta can be modulated in the brain of an animal by diet and provide evidence that cholesterol plays a role in the modulation of APP processing in vivo. APP gene-targeted mice lacking apoE, also have high serum cholesterol levels but do not show alterations in APP processing, suggesting that effects of cholesterol on APP processing require the presence of apoE.     

Presenilin 1 regulates the processing of beta-amyloid precursor protein C-terminal fragments and the generation of amyloid beta-protein in endoplasmic reticulum and Golgi

Progressive cerebral deposition of the amyloid beta-protein (Abeta) is believed to play a pivotal role in the pathogenesis of Alzheimer's disease (AD). The highly amyloidogenic 42-residue form of Abeta (Abeta42) is the first species to be deposited in both sporadic and familial AD. Mutations in two familial AD-linked genes, presenilins 1 (PS1) and 2 (PS2), selectively increase the production of Abeta42 in cultured cells and the brains of transgenic mice, and gene deletion of PS1 shows that it is required for normal gamma-secretase cleavage of the beta-amyloid precursor protein (APP) to generate Abeta. To establish the subcellular localization of the PS1 regulation of APP processing to Abeta, fibroblasts from PS1 wild-type (wt) or knockout (KO) embryos as well as Chinese hamster ovary (CHO) cells stably transfected with wt or mutant PS1 were subjected to subcellular fractionation on discontinuous Iodixanol gradients. APP C-terminal fragments (CTF) were markedly increased in both endoplasmic reticulum- (ER-) and Golgi-rich fractions of fibroblasts from KO mice; moreover, similar increases were documented directly in KO brain tissue. No change in the subcellular distribution of full-length APP was detectable in fibroblasts lacking PS1. In CHO cells, a small portion of APP, principally the N-glycosylated isoform, formed complexes with PS1 in both ER- and Golgi-rich fractions, as detected by coimmunoprecipitation. When the same fractions were analyzed by enzyme-linked immunosorbent assays for Abetatotal and Abeta42, Abeta42 was the major Abeta species in the ER fraction (Abeta42:Abetatotal ratio 0.5-1.0), whereas absolute levels of both Abeta42 and Abeta40 were higher in the Golgi fraction and the Abeta42:Abetatoal ratio was 0.05-0.16 there. Mutant PS1 significantly increased Abeta42 levels in the Golgi fraction. Our results indicate PS1 and APP can interact in the ER and Golgi, where PS1 is required for proper gamma-secretase processing of APP CTFs, and that PS1 mutations augment Abeta42 levels principally in Golgi-like vesicles.