Tissue-specific expression of three types of beta-protein precursor mRNA: enhancement of protease inhibitor-harboring types in Alzheimer's disease brain

Expression of three types of mRNA encoding amyloid beta-protein precursor (APP) in various tissues was analysed, using a ribonuclease protection assay, with special reference to Alzheimer's disease (AD). The total content and the proportion of APP mRNAs were specific to each tissue. Among eight tissues examined, the brain was distinct in that the expression level was highest and APP695 mRNA was expressed in abundance. The ratio of APP770/APP751/APP695 mRNAs was approximately 1:10:20 in the cerebral cortex of control brain. The proportions of APP770 mRNA and APP770-plus-APP751 mRNAs increased up to 2.6- and 1.4-fold, respectively, in various regions of AD brain compared with control. The enhanced expression of protease inhibitor-harboring types (APP770 and APP751) may disturb the balance between biosynthesis and degradation of APPs and ultimately lead to accumulation of beta-protein as amyloid.     

Amyloid precursor protein trafficking, processing, and function

Intracellular trafficking and proteolytic processing of amyloid precursor protein (APP) have been the focus of numerous investigations over the past two decades. APP is the precursor to the amyloid beta-protein (Abeta), the 38-43-amino acid residue peptide that is at the heart of the amyloid cascade hypothesis of Alzheimer disease (AD). Tremendous progress has been made since the initial identification of Abeta as the principal component of brain senile plaques of individuals with AD. Specifically, molecular characterization of the secretases involved in Abeta production has facilitated cell biological investigations on APP processing and advanced efforts to model AD pathogenesis in animal models. This minireview summarizes salient features of APP trafficking and amyloidogenic processing and discusses the putative biological functions of APP.     

Alzheimer's disease and control brain contain soluble derivatives of the amyloid protein precursor that end within the beta amyloid protein region.

The 39-43 amino acid beta amyloid protein (A beta) that deposits as amyloid in the brains of patients with Alzheimer's disease (AD) is encoded as an internal sequence within a larger membrane-associated protein known as the amyloid protein precursor (APP). In cultured cells, the APP is normally cleaved within the A beta to generate a large secreted derivative and a small membrane-associated fragment. Neither of these derivatives can produce amyloid because neither contains the entire A beta. Our study was designed to determine whether the soluble APP derivatives in human brain end within the A beta as described in cell culture or whether AD brain produces potentially amyloidogenic soluble derivatives that contain the entire A beta. We find that both AD and control brain contain nonamyloidogenic soluble derivatives that end at position 15 of the A beta. We have been unable to detect any soluble derivatives that contain the entire A beta in either the AD or control brain.     

Amyloid beta.

Amyloid beta (A beta) is a 39-43 residue amyloidogenic peptide that is deposited into the extracellular amyloid plaques which characterize an Alzheimer's disease (AD) brain. A beta is derived from the amyloid precursor protein (APP) and undergoes a toxic conformational change (gain of toxic function). The length of the A beta peptide dramatically influences its properties with the longer 42 and 43 residue species being more amyloidogenic. The genetics of familial AD (FAD) supports a central role for A beta in AD since mutations in the FAD causing genes APP and the presenilins (PS1 and PS2) increase the formation of A beta 42,43. Considerable activity is directed towards A beta as a therapeutic target. These strategies aim to inhibit A beta synthesis, A beta fibril formation, its toxic actions on cells or promote its clearance from the brain.     

The evolution of A beta peptide burden in the APP23 transgenic mice: implications for A beta deposition in Alzheimer disease.

BACKGROUND: High levels of A beta in the cerebral cortex distinguish demented Alzheimer's disease (AD) from nondemented elderly individuals, suggesting that decreased amyloid-beta (A beta) peptide clearance from the brain is a key precipitating factor in AD. MATERIALS AND METHODS: The levels of A beta in brain and plasma as well as apolipoprotein E (ApoE) in brain were investigated by enzyme-linked immunosorbent assay (ELISA) and Western blotting at various times during the life span of the APP23 transgenic (Tg) and control mice. Histochemistry and immunocytochemistry were used to assess the morphologic characteristics of the brain parenchymal and cerebrovascular amyloid deposits and the intracellular amyloid precursor protein (APP) deposits in the APP23 Tg mice. RESULTS: No significant differences were found in the plasma levels of A beta between the APP23 Tg and control mice from 2-20 months of age. In contrast, soluble A beta levels in the brain were continually elevated, increasing 4-fold at 2 months and 33-fold in the APP23 Tg mice at 20 months of age when compared to the control mice. Soluble A beta42 was about 60% higher than A beta40. In the APP23 Tg mice, insoluble A beta40 remained at basal levels in the brain until 9 months and then rose to 680 microg/g cortex by 20 months. Insoluble A beta40 was negligible in non-Tg mice at all ages. Insoluble A beta42 in APP23 Tg mice rose to 60 microg/g cortex at 20 months, representing 24 times the control A beta42 levels. Elevated levels of ApoE in the brain were observed in the APP23 Tg mice at 2 months of age, becoming substantially higher by 20 months. ApoE colocalized with A beta in the plaques. Beta-amyloid precursor protein (betaAPP) deposits were detected within the neuronal cytoplasm from 4 months of age onward. Amyloid angiopathy in the APP23 Tg mice increased markedly with age, being by far more severe than in the Tg2576 mice. CONCLUSIONS: We suggest that the APP23 Tg mouse may develop an earlier blockage in A beta clearance than the Tg2576 mice, resulting in a more severe accumulation of A beta in the perivascular drainage pathways and in the brain. Both Tg mice reflect decreased A beta elimination and as models for the amyloid cascade they are useful to study AD pathophysiology and therapy.     

Molecular biology of Alzheimer's amyloid—Dutch variant

Hereditary cerebral hemorrhage with amyloidosis, Dutch type (HCHWA-D) (or familial cerebral amyloid angiopathy) and familial Alzheimer's disease (FAD) share several properties. Both are autosomal dominant forms of cerebral amyloidosis characterized by beta-amyloid (A beta) deposition. In HCHWA-D the A beta is predominantly found in blood vessels and in early parenchymal plaques, whereas in AD parenchymal A beta deposits in the form of senile plaques and neurofibrillary tangles are a more prominent finding. Point mutations in the amyloid precursor protein (APP) have recently been described, in both conditions. A G to C transversion at codon 618 (extracellular portion of APP695), producing a single amino acid substitution of glutamine instead of glutamine acid, occurs in HCHWA-D; whereas mutations at codon 642 in the intramembrane region of APP695 (phenylalanine, isoleucine, or glycine instead of valine) are associated with early onset FAD. This suggests that the site of particular mutations in the APP gene and the type of amino acid substitution in the APP holoprotein are more important in determining clinicopathological phenotype and age at which A beta is deposited. Thus FAD and HCHWA-D can be regarded as two sides of the same coin.     

Immunohistochemical investigation of amyloid beta-protein (Abeta) in the brain of aged cats

To clarify the immunohistochemical features of amyloid deposits and cerebral amyloid angiopathy (CAA), the distribution of the amyloid beta-protein subtypes Abeta40, Abeta42, Abeta43 and Abeta precursor protein (APP) were examined in the brains of fourteen aged cats (7.5-21 year-old). Two types of plaques were detected. The first type was characterized by Ass positive antigenic material and detected in the cortical layers of the frontal and parietal lobes of all examined cats. The second type was characterized by diffuse positive immune staining representing diffuse plaques, which were detected only in the very aged cats (17-21 years old) and distributed throughout the cortical layers of the parietal lobes. Vascular amyloid and the amyloid deposits were strongly positive-stained with the antibody Abeta42. APP was exhibited in neurons and axons while the staining was stronger in the very aged cats (17-21 years old). Our findings suggest that the feline forms a spontaneous model for understanding the early changes of normal brain aging and the early stage of amyloid beta-protein deposition.     

Human amyloid-beta causes changes in the levels of endothelial protein kinase C and its alpha isoform in vitro

Amyloid-beta (A(beta)) deposits and neurofibrillary pathology are characteristic features of Alzheimer's disease (AD). The association of A(beta) with cerebral vessels is an intriguing feature of AD. While there is considerable evidence of altered activities of the major isoforms of protein kinase C (PKC) in the vasculature and neurons of AD brains, little is known about the relationship between the Abeta toxicity and the altered PKC levels in cerebral endothelial cells.In this study, cultured brain endothelial cells exposed to A(beta)1-40 revealed a translocation of PKC from the membrane fraction to the cytosol. The content of the isoform PKC(alpha), involved in the regulation of amyloid precursor protein (APP) secretion, was decreased in the membrane-bound fraction of rat endothelial cells and increased in the cytosol after A(beta)1-40 treatment. These data suggest that the accumulation of A(beta) peptide in the cerebral vasculature may play a significant role in the down-regulation of PKC seen in the AD cerebral vasculature.     

Antisera to an amino-terminal peptide detect the amyloid protein precursor of Alzheimer's disease and recognize senile plaques

The cerebral amyloid deposited in Alzheimer's disease (AD) contains a 4.2 kDa beta amyloid polypeptide (beta AP) that is derived from a larger beta amyloid protein precursor (beta APP). Three beta APP mRNAs encoding proteins of 695, 751, and 770 amino acids have previously been identified. In each of these, there is a single membrane-spanning domain close to the carboxyl-terminus of the beta APP, and the 42 amino acid beta AP sequence extends from within the membrane-spanning domain into the large extracellular region of the beta APP. We raised rabbit antisera to a peptide corresponding to amino acids 45-62 near the amino-terminus of the beta APP. We show that these antisera detect the beta APP by demonstrating that they (i) label a set of approximately 120 kDa membrane-associated proteins in human brain previously detected by antisera to the carboxyl-terminus of beta APP and (ii) label a set of approximately 120 kDa membrane-associated proteins that are selectively overexpressed in cells transfected with a full length beta APP expression construct. The beta APP45-62 antisera specifically stain senile plaques in AD brains. This finding, along with the previous demonstration that antisera to the carboxyl-terminus of the beta APP label senile plaques, indicates that both near amino-terminal and carboxyl-terminal domains of the beta APP are present in senile plaques and suggests that proteolytic processing of the full length beta APP molecule into insoluble amyloid fibrils occurs in a highly localized fashion at the sites of amyloid deposition in AD brains.     

The role of amyloid precursor protein processing by BACE1, the beta-secretase, in Alzheimer disease pathophysiology

Amyloid plaques, composed of the amyloid beta-protein (Abeta), are hallmark neuropathological lesions in Alzheimer disease (AD) brain. Abeta fulfills a central role in AD pathogenesis, and reduction of Abeta levels should prove beneficial for AD treatment. Abeta generation is initiated by proteolysis of amyloid precursor protein (APP) by the beta-secretase enzyme BACE1. Bace1 knockout (Bace1(-/-)) mice have validated BACE1 as the authentic beta-secretase in vivo. BACE1 is essential for Abeta generation and represents a suitable drug target for AD therapy, especially because this enzyme is up-regulated in AD. However, although initial data indicated that Bace1(-/-) mice lack an overt phenotype, the BACE1-mediated processing of APP and other substrates may be important for specific biological processes. In this minireview, topics range from the initial identification of BACE1 to the fundamental knowledge gaps that remain in our understanding of this protease. We address pertinent questions such as putative causes of BACE1 elevation in AD and discuss why, nine years since the identification of BACE1, treatments that address the underlying pathological mechanisms of AD are still lacking.     

Glu11 site cleavage and N-terminally truncated A beta production upon BACE overexpression

Amyloid beta peptides (A beta) are generated by the proteolytic processing of the amyloid beta precursor protein (APP). The newly identified beta-site APP-cleaving enzyme (BACE) cleaves APP at Asp1 as well as between Tyr10 and Glu11 of A beta, producing C-terminal fragments (CTFs) C99 and C89, respectively. Subsequent cleavage by gamma-secretase gives rise to A beta 1-40/42 and A beta 11-40/42. Although both full-length and A beta peptides truncated at residue 11 have been identified in amyloid plaques in the AD brain, the relative proportion of these two cleavage products produced by BACE and secreted into the medium by cultured cells is unknown. Using cell lines stably overexpressing BACE, we found that A beta 11-40 and A beta 11-42 are major A beta cleavage products generated by BACE. We further showed that BACE utilizes both full-length APP as well as C99 as substrates for the production of C89, and that A beta 11-40/42 can be generated by sequential cleavage of single APP molecules by BACE and gamma-secretase. Taken together, the abundance of A beta 11-40/42 produced by BACE suggests that their roles in AD pathogenesis may be underestimated.     

The human amyloid-beta precursor protein770 mutation V717F generates peptides longer than amyloid-beta-(40-42) and flocculent amyloid aggregates

One of the familial forms of Alzheimer's disease (AD) encodes the amyloid-beta precursor protein (AbetaPP) substitution mutation V717F. This mutation is relevant to AD research, since it has been utilized to generate transgenic mice models to study AD pathology and therapeutic interventions. Amyloid beta (Abeta) peptides were obtained from the cerebral tissue of three familial AD subjects carrying the AbetaPP V717F mutation. A combination of ultracentrifugation, size-exclusion, and reverse-phase high performance liquid chromatography, tryptic and cyanogen bromide hydrolysis, amino acid analysis, and matrix-assisted laser desorption ionization and surface-enhanced laser desorption ionization mass spectrometry was used to characterize the familial AD mutant Abeta peptides. The AbetaPP V717F mutation, located 4-6 residues beyond the wild-type AbetaPP gamma-secretase cleavage site, yielded longer Abeta peptides with C termini between residues 43 and 54. In the cerebral cortex these peptides aggregated into thin water- and SDS-insoluble amyloid bundles that condensed into flocculent spherical plaques. In the leptomeningeal arteries the amyloid was deposited in moderate amounts and was primarily composed of the shorter and more soluble Abeta species ending at residues 40, 42, and 44. The single V717F mutation in AbetaPP results in distinctive and drastic changes in the length and tertiary structure of Abeta peptides, which appear to be responsible for the earlier clinical manifestations of dementia and death of these patients.     

The 717Val----Ile substitution in amyloid precursor protein is associated with familial Alzheimer's disease regardless of ethnic groups

Alzheimer's disease (AD) is a devastating neurological disorder and the leading cause of dementia among aged individuals. The human amyloid beta protein, which is a cleavage product of amyloid precursor protein (APP), is a major component of the amyloid deposited in the brain of patients with AD. By using PCR direct sequencing of exon 17 (encoding part of the beta protein) of the APP gene, we have found that a Japanese AD patient harbours a C to T substitution, responsible for a valine to isoleucine change at position 717, heterogeneously. The mutation is exactly the same as that found in a Caucasian AD family by Goate et al. (1). Furthermore, the mutation was shown to co-segregate with AD in his family. These results suggest that the Val----Ile change in the APP causes AD, regardless of ethnic background.     

Amino-terminally truncated Abeta peptide species are the main component of cotton wool plaques

Cotton wool plaques (CWPs) are round lesions that lack a central amyloid core. CWPs have been observed in individuals affected by early-onset familial Alzheimer disease (FAD) associated with mutations in the presenilin 1 (PSEN1) gene. Here we present the characterization of the amyloid-beta (Abeta) peptides deposited in the brain of an individual affected by FAD carrying the novel missense (V261I) mutation in the PSEN1 gene. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry was used to determine the Abeta peptide species present in the cerebral and cerebellar cortices, in leptomeningeal vessels, and in CWPs isolated by laser microdissection (LMD). Our results indicate that amino-terminally truncated Abeta peptide species ending at residues 42 and 43 are the main Abeta peptides deposited in brain parenchyma and LMD-CWPs in association with the PSEN1 V261I mutation. Full-length Abeta1-42 and Abeta1-43 peptide species were underrepresented. CWPs were not found to be associated with vessels and did not contain Abeta1-40 peptides, the main component of the vascular deposits. Although Abeta deposits were present mostly in the form of CWPs in the cerebral cortex and as diffuse deposits in the cerebellar cortex, a similar array of amino-terminally truncated Abeta peptide species was seen in both cases. The biochemical data support the concept that parenchymal and vascular amyloid deposits are associated with a different array of Abeta peptide species. The generation and parenchymal deposition of highly insoluble amino-terminally truncated Abeta peptides may play an important role in the pathogenesis of AD and must be taken into consideration in developing new diagnostic and therapeutic strategies.     

Effects of human apolipoprotein E isoforms on the amyloid beta-protein concentration and lipid composition in brain low-density membrane domains

Apolipoprotein E4 (apoE4) encoded by epsilon 4 allele is a strong genetic risk factor for Alzheimer's disease (AD). ApoE4 carriers have accelerated amyloid beta-protein (A beta) deposition in their brains, which may account for their unusual susceptibility to AD. We hypothesized that the accelerated A beta deposition in the brain of apoE4 carriers is mediated through cholesterol-enriched low-density membrane (LDM) domains. Thus, the concentrations of A beta and various lipids in LDM domains were quantified in the brains of homozygous apoE3 and apoE4 knock-in (KI) mice, and in the brains of those mice bred with beta-amyloid precursor protein (APP) transgenic mice (Tg2576). The A beta 40 and A beta 42 concentrations and the A beta 42 proportions in LDM domains did not differ between apoE3 and apoE4 KI mice up to 18 months of age. The A beta 40 concentration in the LDM domains was slightly, but significantly higher in apoE3/APP mice than in apoE4/APP mice. The lipid composition of LDM domains was modulated in an apoE isoform-specific manner, but its significance for A beta deposition remains unknown. These data show that the apoE isoform-specific effects on the A beta concentration in LDM domains do not occur in KI mouse models.     

Interaction of N-terminal acetyltransferase with the cytoplasmic domain of beta-amyloid precursor protein and its effect on A beta secretion

The processing of beta-amyloid precursor protein (APP) generates the amyloid beta-protein (A beta) and contributes to the development of Alzheimer's disease (AD). Elucidating the regulation of APP processing will, therefore, contribute to the understanding of AD. Many APP-binding proteins, such as FE65, X11s, and JNK-interacting proteins (JIPs), bind the motif 681-GYENPTY-687 within the cytoplasmic domain of APP. Here we found that the human homologue of yeast amino-terminal acetyltransferase ARD1 (hARD1) interacts with a novel motif, 658-HGVVEVD-664, in the cytoplasmic domain of APP695. hARD1 expressed its acetyltransferase activity in association with a human subunit homologous to another yeast amino-acetyltransferase, hNAT1. Co-expression of hARD1 and hNAT1 in cells suppressed A beta40 secretion and the suppression correlated with their enzyme activity. These observations suggest that the association of APP with hARD1 and hNAT1 and/or their N-acetyltransferase activity contributes to the regulation of A beta generation.     

Glutamine synthetase, hemoglobin alpha-chain, and macrophage migration inhibitory factor binding to amyloid beta-protein: their identification in rat brain by a novel affinity chromatography and in Alzheimer's disease brain by immunoprecipitation

Proteins binding to amyloid beta-protein (Abeta) may modulate the accumulation of Abeta in Alzheimer's disease (AD) brain. We developed a monomeric Abeta column for isolation of the proteins binding to Abeta from rat brain. By amino acid sequence analysis and immunoreactivity with specific antibodies, we identified three new Abeta-binding proteins, glutamine synthetase, hemoglobin alpha-chain, and macrophage migration inhibitory factor as well as serum albumin, beta-tubulin, and glyceraldehyde-3-phosphate dehydrogenase already identified as proteins bound to amyloid beta-protein precursor. In addition, the retained fraction contained both apolipoprotein E and alpha(1)-antichymotrypsin already known as Abeta binding proteins. Furthermore, we detected the complexes of these new binding proteins with Abeta in a soluble fraction of the cerebral cortex of AD brain by immunoprecipitation. Our results suggest that these binding proteins also associate with Abeta, leading to the clearance or the accumulation of Abeta and the neuronal cell damage in human brain.     

The neuronal adaptor protein X11beta reduces amyloid beta-protein levels and amyloid plaque formation in the brains of transgenic mice

Accumulation of cerebral amyloid beta-protein (Abeta) is believed to be part of the pathogenic process in Alzheimer's disease. Abeta is derived by proteolytic cleavage from a precursor protein, the amyloid precursor protein (APP). APP is a type-1 membrane-spanning protein, and its carboxyl-terminal intracellular domain binds to X11beta, a neuronal adaptor protein. X11beta has been shown to inhibit the production of Abeta in transfected non-neuronal cells in culture. However, whether this is also the case in vivo in the brain and whether X11beta can also inhibit the deposition of Abeta as amyloid plaques is not known. Here we show that transgenic overexpression of X11beta in neurons leads to a decrease in cerebral Abeta levels in transgenic APPswe Tg2576 mice that are a model of the amyloid pathology of Alzheimer's disease. Moreover, overexpression of X11beta retards amyloid plaque formation in these APPswe mice. Our findings suggest that modulation of X11beta function may represent a novel therapeutic approach for preventing the amyloid pathology of Alzheimer's disease.     

Clearing the brain's amyloid cobwebs.

Elevated cerebral levels of amyloid beta-protein occur universally in Alzheimer's disease, yet only a few patients show evidence of increased Abeta production. Therefore, defects in proteases that degrade Abeta could underlie some or many cases of familial and sporadic AD. This previously neglected topic has begun receiving serious attention. Understanding how proteolysis regulates Abeta levels in the cerebral cortex has implications for both the pathogenesis and the treatment of this protean disorder.     

Phenylbutyric acid reduces amyloid plaques and rescues cognitive behavior in AD transgenic mice

Trafficking through the secretory pathway is known to regulate the maturation of the APP-cleaving secretases and APP proteolysis. The coupling of stress signaling and pathological deterioration of the brain in Alzheimer's disease (AD) supports a mechanistic connection between endoplasmic reticulum (ER) stress and neurodegeneration. Consequently, small molecular chaperones, which promote protein folding and minimize ER stress, might be effective in delaying or attenuating the deleterious progression of AD. We tested this hypothesis by treating APPswePS1delta9 AD transgenic mice with the molecular chaperone phenylbutyric acid (PBA) for 14 months at a dose of 1 mg PBA g(-1) of body weight in the drinking water. Phenylbutyric acid treatment increased secretase-mediated APP cleavage, but was not associated with any increase in amyloid biosynthesis. The PBA-treated AD transgenic mice had significantly decreased incidence and size of amyloid plaques throughout the cortex and hippocampus. There was no change in total amyloid levels suggesting that PBA modifies amyloid aggregation or pathogenesis independently of biogenesis. The decrease in amyloid plaques was paralleled by increased memory retention, as PBA treatment facilitated cognitive performance in a spatial memory task in both wild-type and AD transgenic mice. The molecular mechanism underlying the cognitive facilitation of PBA is not clear; however, increased levels of both metabotropic and ionotropic glutamate receptors, as well as ADAM10 and TACE, were observed in the cortex and hippocampus of PBA-treated mice. The data suggest that PBA ameliorates the cognitive and pathological features of AD and supports the investigation of PBA as a therapeutic for AD.