Coexistence of Alzheimer's amyloid precursor protein and amyloid protein in cerebral vessel walls

Polyclonal antibodies to synthetic peptides homologous to amino acid residues 45-62, 597-624, and 676-695 of the predicted sequence of Alzheimer's amyloid precursor protein (APP) were used to investigate the site of origin of APP, and the relationship between APP and amyloid protein in Alzheimer's disease (AD) and hereditary cerebral hemorrhage with amyloidosis, Dutch type (HCHWA-D). Cortical sections as well as homogenates of isolated leptomeningeal and cortical microvessels from three patients with AD, two patients with HCHWA-D, and two nondemented controls were probed. In vessel extracts of both groups of patients and the controls, APP was detected as a set of proteins with electrophoretic mobility of 105 to 135 kilodaltons. In cortical sections of all subjects, APP immunoreactivity was found in leptomeningeal and cortical vessel walls. In patients with AD and HCHWA-D, APP and amyloid fibrils coexisted in the same vessels. Moreover, APP immunoreactivity was found in association with 50% of senile plaques in AD brains, but was not evidenced in parenchymal amyloid deposits in patients with HCHWA-D. These data suggest that the vascular system is a source of APP and that the processing of APP into insoluble fibrils in AD and HCHWA-D may take place in situ.     

RFLP analysis for APP 717 mutations associated with Alzheimer''s disease.

Familial Alzheimer's disease (FAD) has been shown to be associated with three distinct point mutations within the same codon of the amyloid precursor protein (APP) gene. The mutation identified in the Indiana kindred is a G-->T transversion at the first position of the codon for amino acid 717, resulting in a substitution of phenylalanine for valine in the APP protein. Screening of persons at risk for the APP Phe-717 mutation using a variation of the polymerase chain reaction identified nine positives among 34 tested. In addition, DNA from 145 FAD subjects were tested for the three known APP 717 mutations.     

Altered metabolism of familial Alzheimer's disease-linked amyloid precursor protein variants in yeast artificial chromosome transgenic mice

Missense mutations in the beta-amyloid precursor protein gene (APP) co- segregate with a small subset of autosomal dominant familial Alzheimer's disease (FAD) cases wherein deposition of the 39-43 amino acid beta-amyloid (A beta) peptide and neurodegeneration are principal neuropathological hallmarks. To accurately examine the effect of missense mutations on APP metabolism and A beta production in vivo, we have introduced yeast artificial chromosomes (YACs) containing the entire approximately 400 kbp human APP gene encoding APP harboring either the asparagine for lysine and leucine for methionine FAD substitution at codons 670 and 671 (APP(K670N/M671L)), the isoleucine for valine FAD substitution at codon 717 (APP(V7171)) or a combination of both substitutions into transgenic mice. We demonstrate that, relative to YAC transgenic mice expressing wild-type APP, high levels of A beta peptides are detected in the brains of YAC transgenic mice expressing human APP(K670N/M671L) that is associated with a concomitant diminution in the levels of apha-secretase-generated soluble APP derivatives. Moreover, the levels of longer A beta peptides (species terminating at amino acids 42/43) are elevated in YAC transgenic mice expressing human APP(V7171). These mice should prove valuable for detailed analysis of the in vivo effects of the APP FAD mutations in a variety of tissues and throughout aging and for testing therapeutic agents that specifically alter APP metabolism and A beta production.      

Immunoreactivity of the amino-terminal portion of the amyloid-beta precursor protein in the nucleolus

The functioning and metabolic pathway of the amyloid β-precursor protein (APP) have not been fully elucidated. To fill this research gap, this study immunocytochemically investigated the intracellular localization of APP in the neuroblastoma cell line SK-N-SH and in normal primary cells. Using antibodies against the amino-terminal portion of the APP molecule, immunoreactivity was detected not only in the cytoplasm but also in the nucleus and nucleolus. Further analysis revealed the co-localization of amino acids 44-63 of the APP molecule with fibrillarin, a nucleolus marker. These findings indicate that a fraction of APP, including its amino-terminal portion, may be localized in the nucleus as well as in the nucleolus, suggesting an important role of APP in RNA metabolism and other intra-nucleolus functions.     

Tenuigenin treatment decreases secretion of the Alzheimer's disease amyloid beta-protein in cultured cells

Amyloid beta-protein (A beta) is a pivotal pathological factor in Alzheimer's disease (AD). Tenuigenin, extracted from the Chinese herb Polygala tenuifolia, seems to ameliorate the reduction in cholinergic function on rat models induced by A beta. To examine this therapeutic effect, we tested whether Tenuigenin could inhibit secretion of A beta in neuroblastoma cells stably transfected with two amyloid precursor protein (APP) constructs: the APP695 cDNA (SH-SY5Y APP695) and the C-terminal 99 amino acid residues of APP plus the signal peptide (SH-SY5Y SPA4CT). Tenuigenin inhibited the secretion of A beta and the C-terminal 99 amino acids of APP (C99) in SH-SY5Y APP695 cells, but did not change the A beta and C99 levels in SH-SY5Y SPA4CT cells. Fluorescence Resonance Energy Transfer (FRET) assays showed that Tenuigenin inhibited the proteolytic activities of BACE1 (beta-secretase) on its substrate in vitro. In addition, Tenuigenin did not demonstrate any cytotoxic effects, nor did it affect APP mRNA expression, holoAPP synthesis or sAPP alpha secretion. Our data suggest that Tenuigenin can inhibit the secretion of A beta in SH-SY5Y APP 695 cells via BACE1 inhibition. Taken together, these results suggest that Tenuigenin may be worthy of future study as an anti-AD drug.     

Subcellular localization of amyloid precursor protein in senile plaques of Alzheimer''s disease.

The authors have previously shown that amyloid precursor protein (APP) accumulates in neurites present in senile plaques of Alzheimer's disease (AD). In this ultrastructural immunocytochemical study, we describe the subcellular site of APP accumulation. Vibratome sections of glutaraldehyde-paraformaldehyde fixed hippocampi from five cases of AD were pretreated with methanol and immunostained with an antibody raised against recombinant APP 770 by using either indirect immunogold or peroxidase methods. Immunolabeling was localized in cell processes filled with amorphous, irregular-shaped materials, which were identified as dense bodies deformed by postmortem autolysis and methanol treatment, as well as multilamellar membranous bodies. Identification of these bodies was obtained with comparative ultrastructural examination of biopsy and autopsy tissue fixed with and without methanol treatment. These electron-dense organellae were positive for the lysosomal marker, acid phosphatase. At light microscopy, acid phosphatase and APP colocalized to the same cell processes in senile plaques. Many of those cell processes contained abnormal straight or paired helical filaments supporting their neuritic nature. These results suggest that APP accumulates in the lysosomal system of the dystrophic neurites present in senile plaques and are consistent with a neuronal origin of the APP forming the amyloid in senile plaques.     

Role of APP phosphorylation in FE65-dependent gene transactivation mediated by AICD

Consecutive cleavages of Alzheimer's amyloid beta-protein precursor (APP) generate intracellular domain fragment (AICD). Interaction of APP and/or AICD with the adaptor protein FE65 is thought to modulate the metabolism of APP and the function of AICD. Phosphorylation or amino acid substitution of APP and AICD at threonine 668 (Thr668) suppresses their association with FE65. Here, we analyzed the function of APP and AICD phosphorylation in the nuclear translocation of FE65. In brain, AICD was present as phosphorylated and non-phosphorylated forms with non-phosphorylated AICD being dominantly detected in the nucleus. However, a mutant AICD (AICDa), in which Thr668 of AICD was replaced with Ala, was also mostly localized to the nucleus. These observations indicate that phosphorylation of AICD does not regulate the translocation of FE65 and that FE65 does not accompany AICD into the nucleus. APP was known to tether FE65 to the membrane. We found that phosphorylation of APP liberated membrane-bound FE65, which was then translocated into the nucleus where it up-regulated gene transactivation mediated by AICD, which was translocated into the nucleus independently of FE65. Therefore, phosphorylation of APP but not AICD modulates FE65-dependent gene transactivation mediated by AICD through the regulation of FE65 intracellular localization.     

Multiple forms of beta-amyloid peptide precursor RNAs in a single cell type

The longest open reading frames (ORFs) of three different cDNAs ([10, 12, 18, 26], and this report) contain the exact 42 amino acid (aa) sequence of the beta-amyloid peptide (BAP) which is selectively deposited in Alzheimer's diseased (AD) brains. Each of the three cDNAs for the putative amyloid peptide precursor (APP) has been cloned from a different cell type. Using an HL 60 library, we have cloned two of these three APP cDNAs from a single cell type. The sequences of the HL 60 cDNAs are identical to the APP 751 and to the APP 770 forms of APP cDNAs. Northern blots show that oligonucleotide probes drawn from unique regions of the APP 751 and APP 770 cDNAs both hybridize to 4.0 Kilobase (Kb) and to 1.6 Kb APP RNAs from HL 60 cells. In human adult brain, an oligonucleotide probe drawn from the unique region of the APP 751 cDNA hybridizes to a 3.5 Kb APP RNA. However, a DNA probe drawn from the BAP region, which is common to the 695, 751, and 770 forms of APP cDNAs, hybridizes to 3.5, 3.2 and 1.6 Kb APP RNAs. Taken together, these results show that at least two forms of APP RNAs can exist within a single cell type and that the diversity of possible APP RNAs and complexity of their expression may have been underestimated. Thus, in addition to identifying the cells which produce BAP, a new challenge consists of determining which form of forms of APP RNAs and hence APP proteins are associated with BAP deposition in AD and Down syndrome (DS).     

Immunohistochemical colocalization of amyloid precursor protein with cerebrovascular amyloid of Alzheimer''s disease.

Molecular cloning and cDNA sequencing have indicated that the fibril-forming, amyloidogenic beta/A4 peptide of cerebrovasculature and plaque core in AD is encoded as part of a larger precursor, amyloid precursor protein (APP). A panel of antibodies directed against synthetic peptides, which correspond to distinct domains of this putative APP molecule (i.e., amino acid residues 45-62, 587-596, 597-606, 597-638 [beta/A4 peptide], 638-658 and 653-661), were used to probe immunohistochemically serial sections of formalin-fixed, paraffin-embedded Alzheimer's disease (AD) brains for the presence of APP and/or its derivatives. Histochemical staining of adjacent sections with Bielschowsky's silver impregnation and with Congo red or thioflavin S-staining techniques was also done to identify the structures with amyloid deposition. All these antibodies exhibited intense immunoreactivity with amyloidotic cerebral vessels, including meningeal and parenchymal. This observation indicates that the amyloidotic vasculature of AD brain contains, in addition to the fibril-forming beta/A4 protein, nonamyloidogenic APP and/or its derivatives. More importantly, this APP immunoreactivity colocalized with angiopathic amyloid, which is characterized by phenol-resistant, birefringent congophilia. Parallel analyses with a dual SABC/silver impregnation procedure further confirmed that APP and/or its derivatives, including the amyloidogenic beta/A4, colocalized with argentophilic amyloid in the cerebrovasculature of AD.     

Carboxyl-terminus of the amyloid protein precursor and ERbeta are required for estrogenic effect in activating mitogen-activated protein kinase

Estrogen influences the processing of the amyloid beta precursor protein (APP) in the pathogenesis of Alzheimer's disease, and this effect is mediated by estrogen receptors (ERs) in activating mitogen-activated protein kinase (MAPK)-signaling pathway. To test whether the estrogenic effect on both carboxyl-terminal amino acid fragment (C-terminal) of APP (APP-C105)- and ERbeta-mediated MAPK activation in in vitro, two hybrid genes containing each human ERbeta and APP-C105 gene fused to the neuron-specific enolase (NSE) promoter were constructed and were transfected to the neuronal SK-N-MC cells. Western blot shows that the activation of JNK-signaling pathway, but not p38 and ERK, is dependent on ERbeta through estrogen treatment and APP-C105 is also mediated through estrogen in activating MAPK-signaling pathway. The results suggest that ERbeta and APP-C105 derived from APP are necessary for estrogenic effect in activating MAPK-signaling pathway.     

Cloning,sequencing and expression in the dog of the main amyloid precursor protein isoforms and some of the enzymes related with their processing

Alzheimer's disease (AD) is characterized by neuronal loss and the presence of both neurofibrillary tangles and senile plaques in the brain. These plaques arise from the deposition of beta-amyloid (Aβ) peptides (38–43 amino acids), which are generated from enzymatic cleavage of the amyloid precursor protein (APP) by β- and γ-secretases. In the present work, we cloned the principal APP isoforms as well as some enzymes that have been implicated in their amyloidogenic and non-amyloidogenic processing in dogs. Additionally, the main proteases implicated in the degradation of Aβ were also studied. We also investigated the level of expression of these APP isoforms and enzymes in different brain regions and in peripheral tissues. Our data demonstrate that these canine proteins are highly homologous to their human counterparts. In addition, the expression pattern of these proteins in dogs is consistent with previous data reported in human beings. Thus, dogs may be a natural model to study the biology of AD and could also serve as an animal model for Aβ-targeted drugs against this devastating disease.     

Genetic background regulates beta-amyloid precursor protein processing and beta-amyloid deposition in the mouse

Alzheimer's disease (AD) is a multigenic neurodegenerative disorder characterized by distinct neuropathological hallmarks including deposits of the beta-amyloid (A beta) peptide. A beta is a 39- to 43-amino acid peptide derived from the proteolytic processing of the amyloid precursor protein (APP). While increasing evidence suggests that altered APP processing and A beta metabolism is a common feature of AD, the relationship between the levels of A beta and various APP products and the onset of AD remains unclear. We have undertaken a screen to characterize genetic factors that modify APP processing, A beta metabolism and A beta deposition in a genomic-based yeast artificial chromosome (YAC) transgenic mouse model of AD. A mutant human APP YAC transgene was transferred to three inbred mouse strains. Despite similar levels of holo-APP expression in the congenic strains, the levels of APP C-terminal fragments as well as brain and plasma A beta in young animals varied by genetic background. Furthermore, we demonstrate that age-dependent A beta deposition in the APP YAC transgenic model is dramatically altered depending on the congenic strain examined. These studies demonstrate that APP processing, A beta metabolism and A beta deposition are regulated by genetic background and that analysis of these phenotypes in mice should provide new insights into the factors that regulate AD pathogenesis.     

Nonfibrillar diffuse amyloid deposition due to a gamma(42)-secretase site mutation points to an essential role for N-truncated A beta(42) in Alzheimer's disease

Amyloidogenic processing of the amyloid precursor protein (APP) with deposition in brain of the 42 amino acid long amyloid beta-peptide (A beta(42)) is considered central to Alzheimer's disease (AD) pathology. However, it is generally believed that nonfibrillar pre-amyloid A beta(42) deposits have to mature in the presence of A beta(40) into fibrillar amyloid plaques to cause neurodegeneration. Here, we describe an aggressive form of AD caused by a novel missense mutation in APP (T714I) directly involving gamma-secretase cleavages of APP. The mutation had the most drastic effect on A beta(42)/A beta(40) ratio in vitro of approximately 11-fold, simultaneously increasing A beta(42) and decreasing A beta(40) secretion, as measured by matrix-assisted laser disorption ionization time-of-flight mass spectrometry. This coincided in brain with deposition of abundant and predominant nonfibrillar pre-amyloid plaques composed primarily of N-truncated A beta(42) in complete absence of A beta(40). These data indicate that N-truncated A beta(42) as diffuse nonfibrillar plaques has an essential but undermined role in AD pathology. Importantly, inhibiting secretion of full-length A beta(42 )by therapeutic targeting of APP processing should not result in secretion of an equally toxic N-truncated A beta(42).     

Overexpression of APP stimulates basal and constitutive exocytosis in PC12 cells

The mechanisms that underlie the altered neurotransmitter system in Alzheimer's disease (AD) are not well understood. Amyloid precursor protein (APP) is a precursor protein for beta-amyloid, an important trigger protein in the pathogenesis of AD. Duplication of the APP gene as well as APP genes that contain certain mutations has been reported to be associated with familial AD (FAD), and a role of APP in neurotransmission has been suggested recently. This study examines the role of APP in exocytosis in PC12 cells using transfected human growth hormone (hGH) as a reporter for secretion. It was found that overexpression of APP or expression of the Swedish FAD mutation (APPsw) in PC12 cells significantly increased the basal secretion and constitutive secretion of hGH. Expression of an APP phosphorylation-deficient mutant decreased both basal and constitutive secretion relative to the APP wild-type, suggesting a role for APP-Thr668 phosphorylation in secretion in PC12 cells. Overexpression of X11alpha, a protein that stabilizes cellular APP, also increased the basal secretion of hGH but, contrary to APP, decreased the constitutive secretion of hGH, suggesting that basal and constitutive secretion is likely to proceed via distinct pathways and that the increase in the basal secretion of hGH may result from APP-X11alpha interaction. These results demonstrate an unknown role for APP in secretion, and suggest that elevated levels of APP or APP mutation in FAD brains contribute to the altered neurotransmitter pathology of AD through stimulation of basal and constitutive secretion.     

A novel pathway for amyloid precursor protein processing

Amyloid precursor protein (APP) can be proteolytically processed along two pathways, the amyloidogenic that leads to the formation of the 40-42 amino acid long Alzheimer-associated amyloid β (Aβ) peptide and the non-amyloidogenic in which APP is cut in the middle of the Aβ domain thus precluding Aβ formation. Using immunoprecipitation and mass spectrometry we have shown that Aβ is present in cerebrospinal fluid (CSF) as several shorter isoforms in addition to Aβ1-40 and Aβ1-42. To address the question by which processing pathways these shorter isoforms arise, we have developed a cell model that accurately reflects the Aβ isoform pattern in CSF. Using this model, we determined changes in the Aβ isoform pattern induced by α-, β-, and γ-secretase inhibitor treatment. All isoforms longer than and including Aβ1-17 were γ-secretase dependent whereas shorter isoforms were γ-secretase independent. These shorter isoforms, including Aβ1-14 and Aβ1-15, were reduced by treatment with α- and β-secretase inhibitors, which suggests the existence of a third and previously unknown APP processing pathway involving concerted cleavages of APP by α- and β-secretase.     

A novel species-specific RNA related to alternatively spliced amyloid precursor protein mRNAs.

Using an S1 nuclease protection assay, we have identified a novel "variant" Amyloid Precursor Protein (APP) RNA in human brain which is 3-6-fold more abundant than APP-770, but less abundant than APP-751 or APP-695. This variant, referred to as amyloid precursor-related protein 365 (APRP-365), is not detected in mouse and rat brain RNAs. A 1.6 kilo-basepair cDNA clone corresponding to this variant APP RNA predicts the existence of a 365 amino acid protein that is similar to the amino-terminal end of APP-770 but lacks the beta-amyloid peptide and any hydrophobic transmembrane spanning domains. In a modified polymerase chain reaction (PCR), we used amplification of reverse transcribed mRNA to confirm and extend our S1 observations. Together, the features of APRP-365 suggest that the human variant is a soluble protein containing a Kunitz protease inhibitor domain.     

Intracellular Abeta is increased by okadaic acid exposure in transfected neuronal and non-neuronal cell lines.

Intracellular Abeta was examined in both a neuronal cell line (B103) expressing human APP with Swedish mutation and a non-neuronal cell line (Chinese hamster ovary, CHO) expressing wild human APP. Exposure of the APP695sw-transfected B103 cells to okadaic acid for 3 h, Abeta immunostaining was enhanced, as demonstrated by two independent anti-Abeta antibodies. The confocal microscopic study revealed that the immunoreactivity of Abeta was mainly colocalized with a Golgi marker and partially with an ER marker. Quantitative analyses, using Abeta sandwich ELISA, showed significantly increased intracellular Abeta. False positive detection of Abeta by antibody cross-reaction with APP was ruled out by extracting the fraction with formic acid and making it alkaline before subjecting it to ELISA. This procedure resulted in a fraction that contained little APP. Using CHO cells, OA treatment was also shown to be effective in increasing Abeta, as demonstrated by Western blot. The increased full-length APP and decreased APPC99 were also observed. This is the first study to demonstrate that OA treatment significantly increases intracellular Abeta.     

Lack of variation in the nucleotide sequence corresponding to the transmembrane domain of the β-amyloid precursor protein in Alzheimer's disease

The nucleotide sequence corresponding to the APP transmembrane domain and flanking regions of charged amino acids was determined for 91 patients with histologically confirmed Alzheimer's disease, 9 patients with dementias of other etiologies, and 14 controls who had no identifiable brain disease. Twentyeight of the AD patients had a first-degree relative with dementia. No mutations were detected among the 100 demented patients. However, one of the 14 controls exhibited a change in the 3′ base of codon 716 which would not be expected to result in an amino acid substitution at this position. © 1993 Wiley-Liss, Inc.     

Presenilin-1 deficiency impairs glutamate-evoked intracellular calcium responses in neurons

Presenilin 1 (PS1) plays a critical role in cleaving amyloid precursor protein (APP) to produce amyloid-beta (Abeta), the primary proteinaceous component of the senile plaques associated with Alzheimer's disease. In addition to mediating the cleavage of APP and a number of other proteins, a growing body of evidence suggests that PS1 also regulates intracellular endoplasmic reticulum calcium levels. Such findings suggest that PS1 activity may modulate neuronal excitability, as well. To address this issue we examined cytosolic intracellular calcium responses in PS1-deficient neurons stimulated by the excitatory amino acid neurotransmitter, glutamate. We found that glutamate-induced intracellular calcium levels were markedly reduced in neurons lacking PS1 (-/-) compared with heterozygous (+/-) and wild-type (+/+) neurons. To prove that PS1 was sufficient to mediate normal glutamate-induced calcium responses, we used a Semliki-forest virus (SFV) vector to express wild-type PS1 in PS1 knock-out neurons. We found that heterologous PS1 expression restored glutamate-evoked calcium responses in PS1-deficient neurons to levels matching non-infected wild-type cells. PS1-deficient neurons infected with SFV directing expression of beta-galactosidase failed to rescue the wild-type phenotype. These results support the idea that normal PS1 activity regulates neuronal responses to neurotransmitter stimulation.