Comparison of neurodegenerative pathology in transgenic mice overexpressing V717F beta-amyloid precursor protein and Alzheimer's disease

Overexpression of mutated human amyloid precursor protein (hAPP717V-->F) under control of platelet-derived growth factor promoter (PDAPP minigene) in transgenic (tg) mice results in neurodegenerative changes similar to Alzheimer's disease (AD). To clarify the pathology of these mice, we studied images derived from laser scanning confocal and electron microscopy and performed comparisons between PDAPP tg mice and AD. Similar to AD, neuritic plaques in PDAPP tg mouse contained a dense amyloid core surrounded by anti-hAPP- and antineurofilament-immunoreactive dystrophic neurites and astroglial cells. Neurons were found in close proximity to plaques in PDAPP tg mice and, to a lesser extent, in AD. In PDAPP tg mice, and occasionally in AD, neuronal processes contained fine intracellular amyloid fibrils in close proximity to the rough endoplasmic reticulum, coated vesicles, and electron-dense material. Extracellular amyloid fibrils (9-11 nm in diameter) were abundant in PDAPP tg and were strikingly similar to those observed in AD. Dystrophic neurites in plaques of PDAPP tg mouse and AD formed synapses and contained many dense multilaminar bodies and neurofilaments (10 nm). Apoptotic-like figures were present in the tg mice. No paired helical filaments have yet been observed in the heterozygote PDAPP tg mice. In summary, this study shows that PDAPP tg mice develop massive neuritic plaque formation and neuronal degeneration similar to AD. These findings show that overproduction of hAPP717V-->F in tg mice is sufficient to cause not only amyloid deposition, but also many of the complex subcellular degenerative changes associated with AD.     

A possible role for cathepsins D, E, and B in the processing of beta-amyloid precursor protein in Alzheimer's disease

Formation of the 4-kDa peptides, which are essential constituents of the extracellular plaques in Alzheimer's disease, involves the sequential cleavage of the amyloid precursor protein (APP) by beta- and gamma-secretases. The carboxy-terminal 99-amino-acid peptide which is liberated from APP by beta-secretase was used as a potential native substrate of the gamma-secretase(s). With the addition of an initiator Met and a FLAG sequence at the C-terminus (betaAPP100-FLAG), it was expressed in Escherichia coli under the control of the T7 promotor. The preferred site(s) of cleavage in the N-terminal 40-amino-acid beta-amyloid peptide and betaAPP100-FLAG by potential gamma-secretase(s) were rapidly identified using matrix-assisted laser-desorption/ionization time-of-flight mass spectroscopy in addition to peptide mapping followed by protein sequence analysis. Since gamma-secretases seem to be active at acidic pH, three cathepsins (D, E and B) were selected for testing. Studies using different detergents indicated that the cleavage preference of cathepsin D for the betaAPP100-FLAG is highly dependent on the surfactant used to solubilize this substrate. All three cathepsins were found to be capable of catabolizing both beta-amyloid peptides and the betaAPP100-FLAG. As cathepsin D was found to cleave the betaAPP100-FLAG in the vicinity of the C-terminus of the beta-amyloid peptides and cathepsin B has a high carboxypeptidase activity at low pH, the possibility cannot be excluded that cathepsins D and B are involved in the amyloidogenic processing of APP.     

Alzheimer's disease lesions: from morphology to cell biology

Four different approaches to Alzheimer disease changes have been successively applied, and allowed a permanent feed forward-feed back enrichment of knowledge: morphologists described neurofibrillary tangles, senile plaques, amyloid angiopathy; with the help of immunohistochemical and biochemical techniques, they recognised A beta- and tau-associated pathologies; this, in turn, allowed more precise analysis of the lesions, and permitted recognising new ones such as neuropil threads; molecular genetics and molecular biology provided new insights, allowing the discovery of additional pathologic proteins, the relevance of which to physiology and pathology of the nervous system has now to be settled down. The increasingly intricate complex of lesions of Alzheimer syndrome is reviewed. A more comprehensive understanding is urgently needed for initiating efficient therapeutic researches. It will require together continuing a multidisciplinary approach, and a renewal of research in neuropathology.     

Inhibition of platelet activation by the Alzheimer's disease amyloid precursor protein

The amyloid precursor protein (APP) of Alzheimer's disease is abundantly expressed in the platelet alpha-granule where its role remains unclear. This study describes a novel function for APP in regulating human platelet activation. Preincubation of platelet-rich plasma with recombinant secreted APP (sAPP) isoforms dose-dependently inhibited platelet aggregation and secretion induced by ADP or adrenaline. Similarly, sAPP potently inhibited low-dose thrombin-induced activation in washed platelet suspensions, indicating that the activity does not require plasma cofactors. There were no functional differences between sAPP forms with or without the Kunitz protease inhibitor domain or derived from either alpha- or beta-secretase cleavage. In fact, the N-terminal cysteine-rich region of APP (residues 18-194) was as effective as the entire sAPP region in the inhibition of platelet activation. The inhibitory activity of sAPP correlated with a significant reduction in the agonist-induced production of the arachidonic acid (AA) metabolites thromboxane B2 and prostaglandin E2. However, sAPP did not affect AA-induced platelet aggregation or secretion, indicating the enzymatic conversion of AA was not inhibited. The addition of a threshold dose of AA reversed the sAPP-inhibition of agonist-induced platelet activation. This suggests that sAPP decreases the availability of free AA, although the mechanism is not yet known. These data provide evidence that the release of sAPP upon platelet degranulation may result in negative feedback regulation during platelet activation.     

Constitutive and protein kinase C-regulated secretory cleavage of Alzheimer's beta-amyloid precursor protein: different control of early and late events by the proteasome

The physiological processing of the beta-amyloid precursor protein (betaAPP) by a protease called alpha-secretase gives rise to APP alpha, a C-terminally truncated fragment of betaAPP with known neurotrophic and cytoprotective properties. Several lines of evidence indicate that protein kinase C (PKC)-mediated events regulate this physiological pathway. We show here that the proteasome multicatalytic complex modulates the phorbol 12,13-dibutyrate-stimulated APP alpha secretion at several levels in human kidney 293 (HK293) cells. Two blocking agents of the proteasome, namely, Z-IE(Ot-Bu)A-leucinal and lactacystin, elicit a dual effect on PKC-regulated APP alpha secretion by metabolically labeled HK293 cells. Thus, short periods of preincubation (2-5 h) of the cells with the inhibitors trigger a drastic potentiation of APP alpha recovery, whereas long-term treatment of the cells (15-20 h) with the blocking agents leads to an overall decrease in the secretion of APP alpha. Such a dual effect was not observed on constitutive APP alpha secretion and intracellular formation generated by HK293 cells, which both only increase upon inhibitor treatments. Similar effects on the constitutive and PKC-regulated APP alpha secretion were observed with PC12 cells. Altogether, these data suggest distinct mechanisms underlying basal and PKC-regulated APP alpha production, indicating that this multicatalytic complex appears as a key contributor of the alpha-secretase pathway.     

Secreted beta-amyloid precursor protein stimulates mitogen-activated protein kinase and enhances tau phosphorylation

Biological effects related to cell growth, as well as a role in the pathogenesis of Alzheimer disease, have been ascribed to the beta-amyloid precursor protein (beta-APP). Little is known, however, about the intracellular cascades that mediate these effects. We report that the secreted form of beta-APP potently stimulates mitogen-activated protein kinases (MAPKs). Brief exposure of PC-12 pheochromocytoma cells to beta-APP secreted by transfected Chinese hamster ovary cells stimulated the 43-kDa form of MAPK by > 10-fold. Induction of a dominant inhibitory form of ras in a PC12-derived cell line prevented the stimulation of MAPK by secreted beta-APP, demonstrating the dependence of the effect upon p21ras. Because the microtubule-associated protein tau is hyperphosphorylated in Alzheimer disease, we sought and found a 2-fold enhancement in tau phosphorylation associated with the beta-APP-induced MAPK stimulation. In the ras dominant inhibitory cell line, beta-APP failed to enhance phosphorylation of tau. The data presented here provide a link between secreted beta-APP and the phosphorylation state of tau.     

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.     

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.     

The amplifying effect of beta-amyloid on cellular calcium signalling is reduced in Alzheimer's disease

The amplifying effect of beta-amyloid fragment 25-35 (beta A25-35) on the mitogen-induced rise of free intracellular calcium in circulating lymphocytes was strongly reduced in 24 patients with Alzheimer's disease when compared with elderly, non-demented controls. Low beta-amyloid responses were significantly correlated with the presence of the apolipoprotein E epsilon 4 allele, suggesting a dose effect.     

Upregulation of actin gene expression in cells expressing exogenous beta-amyloid precursor protein

We investigated the effects of beta-amyloid peptide precursor (APP) overexpression upon the levels of other mRNAs. Using quantitative slot-blot hybridization and immunoblot analysis we observed that enhanced levels of APP elevated the levels of beta-actin and beta-actin mRNA. Our results also suggest that the cytoplasmic domain of APP is crucial for the elevation in beta-actin gene expression.     

Co-expression of beta-amyloid precursor protein (betaAPP) and apolipoprotein E in cell culture: analysis of betaAPP processing

Apolipoprotein E (ApoE) is the major genetic risk factor for Alzheimer's disease (AD). The ApoE4 allele is associated with earlier disease onset and greater cerebral deposition of the amyloid beta peptide (Abeta), the major constituent of senile (amyloid) plaques. The molecular mechanism underlying these effects of ApoE4 remains unclear; ApoE alleles could have different influences on Abeta production, extracellular aggregation, or clearance. Because the missense mutations on chromosomes 14 and 21 that cause familial forms of AD appear to lead to increased secretion of Abeta, it is important to determine whether ApoE4 has a similar effect. Here, we have examined the effects of all three ApoE alleles on the processing of betaAPP and the secretion of Abeta in intact cells. We established neural (HS683 human glioma) and non-neural (Chinese hamster ovary) cell culture systems that constitutively secrete both ApoE and Abeta at concentrations like those in human cerebrospinal fluid. betaAPP metabolites, generated in the presence of each ApoE allele, were analysed and quantified by two methods: immunoprecipitation and phosphorimaging, and ELISA. We detected no consistent allele-specific effects of ApoE on betaAPP processing in either cell type. Our data suggest that the higher amyloid burden found in AD subjects expressing ApoE4 is not due to increased amyloidogenic processing of betaAPP, in contrast to findings in AD linked to chromosome 14 or 21. These co-expressing cell lines will be useful in the further search for the effects of ApoE on Abeta aggregation or clearance under physiologically relevant conditions.     

Ectodomain phosphorylation of beta-amyloid precursor protein at two distinct cellular locations

The beta-amyloid precursor protein (betaAPP) is a transmembrane protein that is exclusively phosphorylated on serine residues within its ectodomain. To identify the cellular site of betaAPP phosphorylation, we took advantage of an antibody that specifically detects the free C terminus of beta-secretase-cleaved betaAPP containing the Swedish missense mutation (APPssw-beta). This antibody previously established the cellular location of the beta-secretase cleavage of Swedish betaAPP as a post-Golgi secretory compartment (Haass, C., Lemere, C., Capell, A., Citron, M., Seubert, P., Schenk, D., Lannfelt, L., and Selkoe, D. J. (1995) Nature Med. 1, 1291-1296). We have now localized the selective ectodomain phosphorylation of betaAPP to the same compartment. Moreover, the phosphorylation sites of betaAPP were identified at Ser198 and Ser206 of betaAPP695 by tryptic peptide mapping, mass spectrometry, and site-directed mutagenesis. Intracellular phosphorylation of betaAPP was inhibited by Brefeldin A and by incubating cells at 20 degrees C, thus excluding phosphorylation in the endoplasmic reticulum or trans-Golgi network. Ectodomain phosphorylation within a post-Golgi compartment occurred not only with mutant Swedish betaAPP, but also with wild type betaAPP. In addition to phosphorylation within a post-Golgi compartment, betaAPP was also found to undergo phosphorylation at the cell surface by an ectoprotein kinase. Therefore, this study revealed two distinct cellular locations for betaAPP phosphorylation.     

Isolation of the genomic clone of the rhesus monkey beta-amyloid precursor protein

In the evaluation of CTS, electromyography of various myotomes as well as proximal and distal muscles remains quite useful in helping to assess other clinically suspected differentials such as plexopathy, systemic neuropathies, radiculopathies, and proximal nerve entrapments. With regards to T-EMG specifically, it can provide information regarding axonal loss; however, it only crudely estimates relative severity and prognosis. These are better and less painfully determined by nerve conduction studies. The addition of nerve conduction studies to the clinical assessment gives one the ability to diagnose, determine severity and prognosis, and determine best management. The addition of T-EMG in typical cases of CTS causes excess pain and adds little to the information already gained. Therefore the author suggests that T-EMG be reserved for atypical or unusual presentations when additional information is needed such as when nerve conduction studies cannot be performed or when the pathology is potentially aggressive causing significant axonal injury over a short time.     

Involvement of cell surface glycosyl-phosphatidylinositol-linked aspartyl proteases in alpha-secretase-type cleavage and ectodomain solubilization of human Alzheimer beta-amyloid precursor protein in yeast

Human beta-amyloid precursor protein (APP) introduced into yeast undergoes alpha-secretase-type cleavage, suggesting that yeast have alpha-secretase-like protease(s). Here we report that two structurally and functionally related glycosyl-phosphatidylinositol-linked yeast aspartyl proteases, Mkc7p and Yap3p (collectively termed yapsin), are responsible for alpha-secretase-type cleavage of APP expressed in yeast, resulting in release of soluble APP into the extracellular space. Disruption of MKC7 and YAP3 in a vacuolar protease-deficient strain abolished this APP cleavage/release, and APP cleavage/release could be restored by introduction of MKC7 or YAP3 on a single copy plasmid. Purified Mkc7p cleaved an internally quenched fluorogenic APP peptide substrate at the alpha-secretase cleavage site. Measurement of proteolytic activity either in yeast homogenates or on the yeast cell surface revealed that most Mkc7p and Yap3p activities were localized at the cell surface. These results establish a molecular basis for alpha-secretase-type cleavage in yeast and support the generally held concept that alpha-secretase cleavage of APP occurs at the cell surface.     

Memory-enhancing effects of secreted forms of the beta-amyloid precursor protein in normal and amnestic mice

When administered intracerebroventricularly to mice performing various learning tasks involving either short-term or long-term memory, secreted forms of the beta-amyloid precursor protein (APPs751 and APPs695) have potent memory-enhancing effects and block learning deficits induced by scopolamine. The memory-enhancing effects of APPs were observed over a wide range of extremely low doses (0.05-5,000 pg intracerebroventricularly), blocked by anti-APPs antisera, and observed when APPs was administered either after the first training session in a visual discrimination or a lever-press learning task or before the acquisition trial in an object recognition task. APPs had no effect on motor performance or exploratory activity. APPs695 and APPs751 were equally effective in the object recognition task, suggesting that the memory-enhancing effect of APPs does not require the Kunitz protease inhibitor domain. These data suggest an important role for APPss on memory processes.     

Amyloid precursor protein (APP) in the striatum in Alzheimer's disease: an immunohistochemical study

Increasing recognition of diffuse plaques has raised questions about the differences between diffuse and neuritic plaques, particularly in regard to the role of amyloid precursor protein (APP) processing in their formation. To address this issue, corpus striatum (containing almost exclusively diffuse plaques) and cerebral cortex (containing an admixture of plaque types) from patients with Alzheimer's disease (AD) were examined immunohistochemically with antibodies to domain-specific sites of APP (N-terminal, C-terminal, beta A4-related, isoform-specific, and other epitopes). Striatal plaques labeled strongly with beta A4 antibodies as did cortical plaques in AD and the occasional diffuse plaques in cortex from nondemented elderly controls. Weak labeling of some cortical neuritic plaques but not diffuse plaques was observed with antibodies directed against other APP epitopes. Electron microscopy of diffuse plaque-rich striatum in AD cases revealed only rare degenerating neurites without apparent fibrillar amyloid; no changes were noted in the plaque-free striatum of controls. These results suggest that antibodies to beta A4 recognize not only fibrillar amyloid of neuritic plaques but also antigenic determinants of diffuse plaques which lack fibrillar amyloid. Furthermore, the finding that antibodies to non-A4 domains of APP labeled only cortical but not striatal plaques suggests that APP processing mechanisms in cortical and striatal tissues may differ.