Swedish Mutant Amyloid Precursor Protein Research Articles

Stable β-Secretase Activity and Presynaptic Cholinergic Markers During Progressive Central Nervous System Amyloidogenesis in Tg2576 Mice

We examined presynaptic cholinergic markers and beta-secretase activity during progressive central nervous system amyloidogenesis in Tg2576 Alzheimer mice (transgenic for human amyloid precursor protein Swedish mutation; hAPPswe). At 14, 18, and 23 months of age there were no significant differences between wild-type and transgenic mice in four distinct central nervous system cholinergic indices--choline acetyltransferase and acetylcholinesterase activities, and binding to vesicular acetylcholine transporter and Na(+)-dependent high-affinity choline uptake sites. A novel enzyme-linked immunosorbent assay measuring only the secreted human beta-secretase cleavage product (APPsbetaswe) of APPswe also revealed no change with aging in Tg2576 mouse brain. In contrast, transgenic but not wild-type mice exhibited an age-dependent increase in soluble Abeta40 and Abeta42 levels and progressive amyloid deposition in brain. Thus, aging Tg2576 mice exhibited presynaptic cholinergic integrity despite progressively increased soluble Abeta40 and Abeta42 levels and amyloid plaque density in brain. Older Tg2576 mice may best resemble preclinical or early stages of human Alzheimer's disease with preserved presynaptic cholinergic innervation. Homeostatic APPsbetaswe levels with aging suggest that progressive amyloid deposition in brain results not from increased beta-secretase cleavage of APP but from impaired Abeta/amyloid clearance mechanisms.

Alterations in Cerebral Blood Flow and Glucose Utilization in Mice Overexpressing the Amyloid Precursor Protein

We have used quantitative autoradiographic techniques to study the relationship between cerebral blood flow (CBF) and glucose utilization (CGU) in two lines of transgenic mice overexpressing Swedish mutant amyloid precursor protein (APP) and APP-derived Aβ peptides. Mice were studied at an age when there are no amyloid plaques. In the 2123 line, CBF was reduced only in telencephalic regions with no corresponding decrease in CGU. In 2576 transgenics, a line with higher levels of Aβ peptide, both CBF and CGU were reduced throughout the brain. The data indicate that Aβ induces alterations in resting CBF that are either associated with or independent of alterations in CGU and that occur in the absence of amyloid deposition in neuropil of blood vessels. These observations support the hypothesis that cerebrovascular and metabolic abnormalities are early events in the pathogenesis of Alzheimer's disease.

Reduction of trophic support enhances apoptosis in PC12 cells expressing Alzheimer's APP mutation and sensitizes cells to staurosporine-induced cell death.

Mutations in the amyloid precursor protein (APP) gene are known as causative factors in the pathogenesis of early-onset familial Alzheimer's disease (FAD). In this study, the influence of the Swedish double-mutation form of APP (APPsw; KM670/671NL) on apoptosis regulation in PC12 cells was investigated. APPsw-transfected PC12 cells were compared with wild-type APP (APPwt)-expressing and vector-transfected PC12 cells with regard to their susceptibility to cell death induced by the reduction of trophic support or by additional treatment with staurosporine. Expression of APPsw markedly enhanced the level of apoptotic PC12 cells induced by serum reduction. A similar hypersensitivity of APPsw-expressing PC12 cells could be detected after differentiation with nerve growth factor under serum-reduced conditions. Likewise, the expression of APPsw rendered PC12 cells more vulnerable to staurosporine but only under serum-reduced conditions. This APPsw-effect disappeared in high serum-containing medium. Thus, expression of APPsw seems to enhance cellular sensitivity not in general but after the reduction of trophic factors probably by causing oxidative stress. This, in turn, may sensitize cells to secondary apoptotic stimuli. Moreover, the mutation-specific increase in vulnerability to cell death was only seen at the stage of apoptotic nuclei, but not using methods measuring cell death by determining metabolic activity or membrane integrity. Therefore, the expression of APPsw seems to affect specifically apoptotic cell death rather than overall cell death in vitro. Our study further emphasizes the pathogenic role of mutant APP and may provide new insights in the mechanisms underlying the massive neurodegeneration in brain from patients bearing the APPsw mutation.

New Insights on How Metals Disrupt Amyloid β-Aggregation and Their Effects on Amyloid-β Cytotoxicity

Amyloid-beta protein (A beta) aggregates in the brain to form senile plaques. By using thioflavin T, a dye that specifically binds to fibrillar structures, we found that metals such as Zn(II) and Cu(II) normally inhibit amyloid beta-aggregation. Another method for detecting A beta, which does not distinguish the types of aggregates, showed that these metals induce a non-beta-sheeted aggregation, as reported previously. Secondary structural analysis and microscopic studies revealed that metals induced A beta to make non-fibrillar aggregates by disrupting beta-sheet formation. These non-fibrillar A beta aggregates displayed much weaker Congo Red birefringence, and in separate cell culture experiments, were less toxic than self beta-aggregates, as demonstrated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. The toxicity of soluble A beta was enhanced in the presence of Cu(II), which suggests the previously hypothesized role of A beta in generating oxidative stress. Finally, under an acidic condition, similar to that in the inflammation associated with senile plaques, beta-aggregation was robustly facilitated at one specific concentration of Zn(II) in the presence of heparin. However, because a higher concentration of Zn(II) virtually abolished this abnormal phenomenon, and at normal pH any concentrations strongly inhibit beta-aggregation and its associated cytotoxicity, including its anti-oxidative nature we suggest that Zn(II) has an overall protective effect against beta-amyloid toxicity.

Mechanisms of Neuroprotection by a Novel Rescue Factor Humanin from Swedish Mutant Amyloid Precursor Protein

We report a novel gene, designated Humanin (HN) cDNA, that suppresses neuronal cell death by K595N/M596L-APP (NL-APP), a mutant causing familial Alzheimer's disease (FAD), termed Swedish mutant. Transfection of neuronal cells with HN cDNA or treatment with the coding HN polypeptide abrogated cytotoxicity by NL-APP. HN suppressed neurotoxicity by Aβ1-43 in the absence of N2 supplement, but could not inhibit Aβ secretion from NL-APP. HN could also protect neuronal cells from death by NL-APP lacking the 41st and 42nd residues of the Aβ region. Therefore, HN suppressed neuronal cell death by NL-APP not through inhibition of Aβ42 secretion, but with two targets for its inhibitory action: (i) the intracellular toxic mechanism directly triggered by NL-APP and (ii) neurotoxicity by Aβ. HN will contribute to the development of curative therapy of AD, especially as a novel reagent that could mechanistically supplement Aβ-production inhibitors.

Elevated vulnerability to oxidative stress-induced cell death and activation of caspase-3 by the Swedish amyloid precursor protein mutation.

The Swedish double mutation (KM670/671NL) of amyloid precursor protein (APPsw) is associated with early-onset familial Alzheimer's disease (FAD) and results in from three- to sixfold increased beta-amyloid production. The goal of the present study was to elucidate the effects of APPsw on mechanisms of apoptotic cell death. Therefore, PC12 cells were stably transfected with human APPsw. Here we report that the vulnerability of APPsw-bearing PC12 cells to undergo apoptotic cell death was significantly enhanced after exposure to hydrogen peroxide compared to human wild-type APP-bearing cells, empty vector-transfected cells, and parent untransfected cells. In addition, we have analyzed the potential influence of several mechanisms that can interfere with the execution of the apoptotic cell death program: the inhibition of cell death by the use of caspase inhibitors and the reduction of oxidative stress by the use of (+/-)-alpha-tocopherol (vitamin E). Interestingly, oxidative stress-induced cell death was significantly attenuated in APPsw PC12 cells by pretreatment with caspase-3 inhibitors but not with caspase-1 inhibitors. In parallel, caspase-3 activity was markedly elevated in APPsw PC12 after stimulation with hydrogen peroxide for 6 hr, whereas caspase-1 activity was unaltered. In addition, oxidative stress-induced cell death could be reduced after pretreatment of APPsw cells with (+/-)-alpha-tocopherol. The protective potency of (+/-)-alpha-tocopherol was even greater than that of caspase-3 inhibitors. Our findings further emphasize the role of mutations in the amyloid precursor protein in apoptotic cell death and may provide the fundamental basis for further efforts to elucidate the underlying processes caused by FAD-related mutations.

The Protease Inhibitor, MG132, Blocks Maturation of the Amyloid Precursor Protein Swedish Mutant Preventing Cleavage by β-Secretase

Amyloid (Abeta) peptides found aggregated into plaques in Alzheimer's disease are derived from the sequential cleavage of the amyloid precursor protein (APP) first by beta- and then by gamma-secretases. Peptide aldehydes, which inhibit cysteine proteases and proteasomes, reportedly block Abeta peptide secretion by interfering with gamma-secretase cleavage. Using a novel, specific, and sensitive enzyme-linked immunosorbent assay for the beta-secretase-cleaved fragment of the Swedish mutant of APP (APPSw), we determined that the peptide aldehyde, MG132, prevented beta-secretase cleavage. This block in beta-secretase cleavage was not observed with clasto-lactacystin beta-lactone and thus, cannot be attributed to proteasomal inhibition. MG132 inhibition of beta-secretase cleavage was compared with the serine protease inhibitor, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF). AEBSF inhibition of beta-secretase cleavage was immediate and did not affect alpha-secretase cleavage. With MG132, inhibition was delayed and it decreased secretion of alpha-cleaved APPSw as well. Furthermore, MG132 treatment impaired maturation of full-length APPSw. Both inhibited intracellular formation of the beta-cleaved product. These results suggest that peptide aldehydes such as MG132 have multiple effects on the maturation and processing of APP. We conclude that the MG132-induced decrease in beta-secretase cleavage of APPSw is due to a block in maturation. This is sufficient to explain the previously reported peptide aldehyde-induced decrease in Abeta peptide secretion.

Amyloid Precursor Proteins Inhibit Heme Oxygenase Activity and Augment Neurotoxicity in Alzheimer's Disease

Amyloid precursor protein (APP) generates the β-amyloid peptide, postulated to participate in the neurotoxicity of Alzheimer's disease. We report that APP and APLP bind to heme oxygenase (HO), an enzyme whose product, bilirubin, is antioxidant and neuroprotective. The binding of APP inhibits HO activity, and APP with mutations linked to the familial Alzheimer's disease (FAD) provides substantially greater inhibition of HO activity than wild-type APP. Cortical cultures from transgenic mice expressing Swedish mutant APP have greatly reduced bilirubin levels, establishing that mutant APP inhibits HO activity in vivo. Oxidative neurotoxicity is markedly greater in cerebral cortical cultures from APP Swedish mutant transgenic mice than wild-type cultures. These findings indicate that augmented neurotoxicity caused by APP–HO interactions may contribute to neuronal cell death in Alzheimer's disease.

Inhibitors of β-Amyloid Formation Based on the β-Secretase Cleavage Site

A series of inhibitors of β-amyloid formation have been developed based on the β-secretase cleavage site (VNL-DA) of the Swedish mutant Amyloid Precursor Protein. A simple tripeptide aldehyde was found to be the most potent (IC50 = 700 nM) in the series displaying an inhibitory profile which is different from reported inhibitors of β-amyloid formation.

Identification of beta-secretase-like activity using a mass spectrometry-based assay system.

We describe an assay system for the identification of site-specific proteases. The assay is based on a protein substrate that is immobilized on ceramic beads. After incubation with cell homogenates, the beads are washed and digested with endoproteinase Lys-C to liberate a defined set of peptides. The peptide fragments are identified by mass spectrometry. The assay was used to screen for beta-secretase, the protease that cleaves amyloid precursor protein (APP) at the beta-site. Cathepsin D was identified as the enzyme responsible for beta-secretase-like activity in two cell lines. Subsequent analysis of the related aspartic protease, cathepsin E, revealed almost identical cleavage specificity. Both enzymes are efficient in cleaving Swedish mutant APP at the beta-site but show almost no reactivity with wild-type APP. Treatment of cell lines with pepstatin inhibited the production of amyloid peptide (Abeta) when they were transfected with a construct bearing the Swedish APP mutant. However, when the cells were transfected with wild-type APP, the generation of Abeta was increased. This suggests that more than one enzyme is capable of generating Abeta in vivo and that an aspartic protease is involved in the processing of Swedish mutant APP.

CDNA cloning of a novel secreted isoform of the human receptor for advanced glycation end products and characterization of cells co-expressing cell-surface scavenger receptors and Swedish mutant amyloid precursor protein

The receptor for advanced glycation end products (RAGE) has been proposed as a cell surface receptor that binds amyloid-β protein (Aβ), thereby triggering its cytotoxic effects [S.D. Yan, X. Chen, J. Fu, M. Chen, H. Zhu, A. Roher, T. Slattery, L. Zhao, M. Nagashima, J. Morser, A. Migheli, P. Nawroth, D. Stern, A.M. Schmidt, RAGE and amyloid-β peptide neurotoxicity in Alzheimer's disease, Nature 382 (1996) 685–691.]. A cDNA library of human lung was screened for RAGE with an appropriate hybridization probe. In addition to cell surface RAGE, one clone was found which encodes a new version of RAGE, termed hRAGEsec, which lacks the 19 amino acids of the membrane-spanning region and is therefore secreted. Comparison with the genomic sequence revealed that the synthesis of the secreted isoform requires alternative splicing. The deduced protein sequence of the mature hRAGEsec consists of 321 amino acids with a predicted molecular mass of 35.66 kDa. The pattern of expression of hRAGEsec in human brain was analyzed by in situ hybridization histochemistry. The most intense expression of the gene in contrast to cell surface RAGE was detected in hippocampal CA3 pyramidal cells, dentate gyrus granule cells, cortical neurons as well as glial cells in white matter. To investigate the interaction between Aβ and RAGE and another scavenger receptor, SRA, under physiological conditions, they were co-expressed with human βAPP695-SFAD in a human cell and the level of Aβ in the condition medium was assessed by immunoprecipitation and enzyme-linked immunosorbent assay (ELISA) analysis. A nearly 100% reduction of Aβ from the conditioned medium of hRAGE cells and ∼40% reduction from the SRA-cells implied that hRAGE could be a prominent cell surface receptor interacting with Aβ.

Histochemically Reactive Zinc in Plaques of the Swedish Mutant β-Amyloid Precursor Protein Transgenic Mice

Endogenous metals such as zinc may contribute to beta-amyloid (Abeta) aggregation and hence the plaque formation. In the present study, we examined brains of four Swedish mutant amyloid precursor protein (APP) transgenic mice at 12 months of age for histochemically reactive zinc in the plaques. Here, we report that all the Congo red (+) mature plaques contained chelatable zinc, as demonstrated by staining with the zinc-specific fluorescent dye 6-methoxy-8-quinolyl-para-toluenesulfonamide (TSQ). On the other hand, Congo red (-) preamyloid Abeta deposits were not stained with TSQ. Interestingly, although cerebellum contained similar degree of preamyloid Abeta deposits as cerebral cortex, it was completely devoid of Congo red- or TSQ-stained mature plaques. Although zinc from plaques was only slowly and partially removed by a specific zinc remover, dithizone, treatment of brain sections with heparinase-III, which degrades heparan sulfate proteoglycan (HSPG), another major constituent of plaques, greatly fastened the zinc removal with dithizone. The present study has demonstrated the presence of histochemically reactive zinc in plaques, but not preamyloid Abeta deposits, of the Swedish mutant APP transgenic mice. Because preamyloid Abeta deposits fail to develop into congophilic plaques in cerebellum where synaptic vesicle zinc is deficient, the synaptic zinc may be a necessary element in the plaque formation. In holding zinc inside plaques, HSPG may contribute in addition to Abeta.

The Chaperone BiP/GRP78 Binds to Amyloid Precursor Protein and Decreases Aβ40 and Aβ42 Secretion

Recent studies of cellular amyloid precursor protein (APP) metabolism demonstrate a beta-/gamma-secretase pathway resident to the endoplasmic reticulum (ER)/Golgi resulting in intracellular generation of soluble APP (APPsbeta) and Abeta42 peptide. Thus, these intracellular compartments may be key sites of amyloidogenic APP metabolism and Alzheimer's disease pathogenesis. We hypothesized that the ER chaperone immunoglobulin binding protein (BiP/GRP78) binds to and facilitates correct folding of nascent APP. Metabolic labeling and immunoprecipitation of transiently transfected human embryonic kidney 293 cells demonstrated co-precipitation of APP with GRP78, revealing their transient interaction in the ER. Maturation of cellular APP was impaired by this interaction. Furthermore, the levels of APPs, Abeta40, and Abeta42 recovered in conditioned medium were lower compared with cells transfected with APP alone. Co-expression with APP of GRP78 T37G, an ATPase mutant, almost completely blocked cellular APP maturation as well as recovery of APPs, Abeta40, and Abeta42 in conditioned medium. The inhibitory effects of GRP78 and GRP78 T37G on Abeta40 and Abeta42 secretion were magnified by co-expression with the Swedish mutation of APP (K670N/M671L). Collectively, these data suggest a transient and direct interaction of GRP78 with APP in the ER that modulates intracellular APP maturation and processing and may facilitate its correct folding.

The X11α Protein Slows Cellular Amyloid Precursor Protein Processing and Reduces Aβ40 and Aβ42 Secretion

Constitutive amyloid precursor protein (APP) metabolism results in the generation of soluble APP (APPs) and Abeta peptides, including Abeta40 and Abeta42-the major component of amyloid plaques in Alzheimer's disease brain. The phosphotyrosine binding (PTB) domain of X11 binds to a peptide containing a YENPTY motif found in the carboxyl terminus of APP. We have cloned the full-length X11 gene now referred to as X11alpha. Coexpression of X11alpha with APP results in comparatively greater levels of cellular APP and less APPs, Abeta40, and Abeta42 recovered in conditioned medium of transiently transfected HEK 293 cells. These effects are impaired by a single missense mutation of either APP (Y682G within the YENPTY motif) or X11alpha (F608V within the PTB domain), which diminishes their interaction, thus demonstrating specificity. The inhibitory effect of X11alpha on Abeta40 and Abeta42 secretion is amplified by coexpression with the Swedish mutation of APP (K595N/M596L), which promotes its amyloidogenic processing. Pulse-chase analysis demonstrates that X11alpha prolongs the half-life of APP from approximately 2 h to approximately 4 h. The effects of X11alpha on cellular APP and APPs recovery were confirmed in a 293 cell line stably transfected with APP. The specific binding of the PTB domain of X11alpha to the YENPTY motif-containing peptide of APP appears to slow cellular APP processing and thus reduces recovery of its soluble fragments APPs, Abeta40, and Abeta42 in conditioned medium of transfected HEK 293 cells. X11alpha may be involved in APP trafficking and metabolism in neurons and thus may be implicated in amyloidogenesis in normal aging and Alzheimer's disease brain.

Differential Effects of the Swedish Mutant Amyloid Precursor Protein on β-Amyloid Accumulation and Secretion in Neurons and Nonneuronal Cells

Expression of the Swedish DeltaNL mutation in the beta-amyloid precursor protein (APPDeltaNL) dramatically increases Abeta generation in nonneuronal cell lines, although it is unclear whether intracellular levels of beta-amyloid (Abeta) are also elevated after APPDeltaNL expression. Furthermore, the effects of expressing APPDeltaNL in neurons on the production and secretion of Abeta-(1-40) and Abeta-(1-42) are unknown. To address these issues, we examined the generation of both intracellular and secreted Abeta-(1-40) and Abeta-(1-42) in human neuronal NT2N cells, in primary rat astrocytes, and in Chinese hamster ovary cells engineered to express wild-type APP or APPDeltaNL using a recombinant Semliki Forest virus expression system. Expression of APPDeltaNL led to a marked increase in APPbeta and the C-terminal fragment containing the entire Abeta sequence (C99) in all cells tested. However, a dramatic elevation of intracellular and secreted Abeta-(1-40) and Abeta-(1-42) was seen only in astrocytes and Chinese hamster ovary cells. The DeltaNL mutation did not cause a significant increase in intracellular or secreted Abeta-(1-40) or Abeta-(1-42) in NT2N cells. Since NT2N cells expressing APPDeltaNL accumulate much higher levels of C99 than cells expressing wild-type APP, we conclude that the rate-limiting step in Abeta production could be the further processing of C99 by gamma-secretase in these cells. These results show that the Swedish DeltaNL mutation causes nonneuronal cells to process APP via pathways more in common with the metabolism of wild-type APP in neurons.

Intracellular Accumulation of β-Amyloid in Cells Expressing the Swedish Mutant Amyloid Precursor Protein

beta-Amyloid (beta A) is a normal metabolic product of the amyloid precursor protein (APP) that accumulates in senile plaques in Alzheimer's disease. Cells that express the Swedish mutant APP (Sw-APP) associated with early onset Alzheimer's disease overproduce beta A. In this report, we show that expression of Sw-APP gives rise to cell-associated beta A, which is not detected in cells that express wild-type APP. Cell-associated beta A is rapidly generated, is trypsin-resistant, and is not derived from beta A uptake, indicating that it is generated from intracellular processing of Sw-APP. Intracellular and secreted beta A are produced with different kinetics. The generation of intracellular beta A is partially resistant to monensin and a 20 degrees C temperature block but is completely inhibited by brefeldin A, suggesting that it occurs in the Golgi complex. Monensin, brefeldin A, and a 20 degrees C temperature block almost completely inhibit beta A secretion without causing increased cellular retention of beta A, suggesting that secreted beta A is generated in a post-Golgi compartment. These results suggest that the metabolism of Sw-APP gives rise to intracellular and secreted forms of beta A through distinct processing pathways. Pathological conditions may therefore alter both the level and sites of accumulation of beta A. It remains to be determined whether the intracellular form of beta A plays a role in the formation of amyloid plaques.