Neurotoxicity of a Carboxyl-Terminal Fragment of the Alzheimer's Amyloid Precursor Protein

Abstract: We have previously shown that a recombinant carboxyl-terminal 105-amino-acid fragment (CT105) of the amyloid precursor protein (APP) induced strong non-selective inward currents in Xenopus oocytes. Here we investigated the toxic effect of CT105 peptide on the cultured mammalian cells. The CT105 peptide induced a significant lactate dehydrogenase (LDH) release from cultured rat cortical neurons and PC12 cells in a concentration (from 10 µ M )- and time (from 48 h)-dependent manner. The toxic effect of CT105 was more potent than that of any fragments of amyloid β protein (Aβ). However, CT105 peptide did not affect the viability of U251 human glioblastoma cells. In contrast to CT105, Aβ increased LDH release only slightly even at 50 µ M but significantly inhibited 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction at submicromolar concentrations. Among the various neuroprotective drugs tested, only cholesterol, which alters membrane fluidity, could attenuate the cytotoxicity of CT105 significantly. The CT105 peptide formed multiple self-aggregates on solubilization. Pretreatment with a sublethal concentration of CT105 did not significantly alter the susceptibility of cells to hydrogen peroxide and glutamate. Endogenous CT peptides were found not only in the cell lysates but also in the conditioned medium of PC12 cells. These results imply that CT peptide can directly attack the cell membrane probably by making pores or nonselective ion channels, whereas Aβ impairs the intracellular metabolic pathway first. Thus, it is thought that both CT and Aβ, which are formed during the processing of APP, may participate in the neuronal degeneration in Alzheimer's disease by different mechanisms.     

Iron Levels Modulate α-Secretase Cleavage of Amyloid Precursor Protein

Abstract: The amyloid precursor protein (APP) is a membrane-spanning glycoprotein that is the source of βA4 peptides, which aggregate in Alzheimer's disease to form senile plaques. APP is cleaved within the βA4 sequence to release a soluble N-terminal derivative (APP_sol), which has a wide range of trophic and protective functions. In the current study we have examined the hypothesis that iron availability may modulate expression or processing of APP, whose mRNA contains, based on sequence homology, a putative iron response element (IRE). Radiolabeled APP and its catabolites were precipitated from lysates and conditioned medium of stably transfected HEK 293 cells using antibodies selective for C-terminal, βA4, and N-terminal domains. The relative abundance of the different APP catabolites under different conditions of iron availability was determined by quantitative densitometry after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The data show a specific effect on the production of APP_sol. Using standard conditions previously established for IRE studies, it was found that iron chelation reduces APP_sol production, whereas iron level elevation augments it. No changes were observed in levels of immature and mature APP holoprotein or in the C-terminal α-secretase derivative C83, βA4, and p3 peptides. The specificity for modulatory changes in APP_sol suggests that iron acts at the level of α-secretase activity. In addition to its modulatory effects, iron at very high levels was found to inhibit maturation of APP and production of its downstream catabolites without blocking formation of immature APP. The data establish a potential physiological role for iron in controlling the processing of APP. If APP_sol were to function trophically, as suggested by other studies, the current conclusion suggests that changes in iron and iron-regulating proteins in Alzheimer's disease could contribute to neuronal degeneration by decreasing the production of APP_sol.     

Evidence Against a Role for the Kunitz Domain in Amyloidogenic and Secretory Processing of the Amyloid Precursor Protein

Abstract: The effect of the Kunitz proteinase inhibitor (KPI) on potential β-amyloid precursor protein (βPP)-processing activities from control and Alzheimer's disease (AD) brains was examined using fluorogenic substrates designed to mimic the secretory and amyloidogenic cleavages in βPP. In addition, the level of secretion of KPI-containing βPP751 and KPI-lacking βPP695 from transfected cells was examined to assess the effect of the KPI on βPP secretion. βPP751 and βPP695, obtained from conditioned media of transfected cells, had no effect on proteinase activities against the secretory and amyloidogenic substrates in extracts from control and AD brains. At similar concentrations βPP751, but not βPP695, completely inhibited the activity of trypsin against these substrates. Serine proteinase inhibitors had only modest effects on activities from brain, whereas cysteine modification completely inhibited them, indicating that these proteinase activities were not of the serine type. Thus, the results do not support a role for the KPI in the secretion of βPP or in the amyloidogenic cleavage of βPP. The amounts of βPP695 and βPP751 collected from the media of transfected cells after 48 h of growth were similar, indicating an equal rate of secretion. This result suggests that the KPI domain in βPP751 did not inhibit the secretory cleavage in transfected cells.     

Proteasome Contributes to the α-Secretase Pathway of Amyloid Precursor Protein in Human Cells

Abstract: A major histopathological hallmark in Alzheimer's disease consists of the extracellular deposition of the amyloid β-peptide (Aβ) that is proteolytically derived from the β-amyloid precursor protein (βAPP). An alternative, nonamyloidogenic cleavage, elicited by a protease called α-secretase, occurs inside the Aβ sequence and gives rise to APPα, a major secreted C-terminal-truncated form of βAPP. Here, we demonstrate that human embryonic kidney 293 (HK293) cells contain a chymotryptic-like activity that can be ascribed to the proteasome and that selective inhibitors of this enzyme reduce the phorbol 12,13-dibutyrate-sensitive APPα secretion by these cells. Furthermore, we establish that a specific proteasome blocker, lactacystin, also induces increased secretion of Aβ peptide in stably transfected HK293 cells overexpressing wild-type βAPP751. Altogether, this study represents the first identification of a proteolytic activity, namely, the proteasome, contributing likely through yet unknown intracellular relays, to the α-secretase pathway in human cells.     

Processing of Amyloid Precursor Protein in Human Primary Neuron and Astrocyte Cultures

Abstract: Increased production of amyloid β peptide (Aβ) is highly suspected to play a major role in Alzheimer's disease (AD) pathogenesis. Because Aβ deposits in AD senile plaques appear uniquely in the brain and are fairly restricted to humans, we assessed amyloid precursor protein (APP) metabolism in primary cultures of the cell types associated with AD senile plaques: neurons, astrocytes, and microglia. We find that neurons secrete 40% of newly synthesized APP, whereas glia secrete only 10%. Neuronal and astrocytic APP processing generates five C-terminal fragments similar to those observed in human adult brain, of which the most amyloidogenic higher-molecular-weight fragments are more abundant. The level of amyloidogenic 4-kDa Aβ exceeds that of nonamyloidogenic 3-kDa Aβ in both neurons and astrocytes. In contrast, microglia make more of the smallest C-terminal fragment and no detectable Aβ. We conclude that human neurons and astrocytes generate higher levels of amyloidogenic fragments than microglia and favor amyloidogenic processing compared with previously studied culture systems. Therefore, we propose that the higher amyloidogenic processing of APP in neurons and astrocytes, combined with the extended lifespan of individuals, likely promotes AD pathology in aging humans.     

β-Amyloid-Induced Neurotoxicity of a Hybrid Septal Cell Line Associated with Increased Tau Phosphorylation and Expression of β-Amyloid Precursor Protein

Abstract: Recent evidence suggests that β-amyloid peptide (β-AP) may induce tau protein phosphorylation, resulting in loss of microtubule binding capacity and formation of paired helical filaments. The mechanism by which β-AP increases tau phosphorylation, however, is unclear. Using a hybrid septal cell line, SN56, we demonstrate that aggregated β-AP_1–40 treatment caused cell injury. Accompanying the cell injury, the levels of phosphorylated tau as well as total tau were enhanced as detected immunochemically by AT8, PHF-1, Tau-1, and Tau-5 antibodies. Alkaline phosphatase treatment abolished AT8 and PHF-1 immunoreactivity, confirming that the tau phosphorylation sites were at least at Ser^199/202 and Ser³⁹⁶. In association with the increase in tau phosphorylation, the immunoreactivity of cell-associated and secreted β-amyloid precursor protein (β-APP) was markedly elevated. Application of antisense oligonucleotide to β-APP reduced expression of β-APP and immunoreactivity of phosphorylated tau. Control peptide β-AP_1–28 did not produce significant effects on tau phosphorylation, although it slightly increased cell-associated β-APP. These results suggest that βAP_1–40-induced tau phosphorylation may be associated with increased β-APP expression in degenerated neurons.     

Secreted Forms of β-Amyloid Precursor Protein Protect Against Ischemic Brain Injury

Abstract: The β-amyloid precursor protein (βAPP) is the source of the amyloid β-peptide that accumulates in the brain in Alzheimer's disease. A major processing pathway for βAPP involves an enzymatic cleavage within the amyloid β-peptide sequence that liberates secreted forms of βAPP (APP^Ss) into the extracellular milieu. We now report that postischemic administration of these APP^Ss intracerebroventricularly protects neurons in the CA1 region of rat hippocampus against ischemic injury. Treatment with APP^S695 or APP^S751 resulted in increased neuronal survival, and the surviving cells were functional as demonstrated by their ability to synthesize protein. These data provide direct evidence for a neuroprotective action of APP^Ss in vivo.     

Caffeine Stimulates Amyloid β-Peptide Release from β-Amyloid Precursor Protein-Transfected HEK293 Cells

Abstract: Extracellular amyloid β-peptide (Aβ) deposition is a pathological feature of Alzheimer's disease and the aging brain. Intracellular Aβ accumulation is observed in the human muscle disease, inclusion body myositis. Aβ has been reported to be toxic to neurons through disruption of normal calcium homeostasis. The pathogenic role of Aβ in inclusion body myositis is not as clear. Elevation of intracellular calcium following application of calcium ionophore increases the generation of Aβ from its precursor protein (βAPP). A receptor-based mechanism for the increase in Aβ production has not been reported to our knowledge. Here, we use caffeine to stimulate ryanodine receptor (RYR)-regulated intracellular calcium release channels and show that internal calcium stores also participate in the genesis of Aβ. In cultured HEK293 cells transfected with βAPP cDNA, caffeine (5–10 m M ) significantly increased the release of Aβ fourfold compared with control. These actions of caffeine were saturable, modulated by ryanodine, and inhibited by the RYR antagonists ruthenium red and procaine. The calcium reuptake inhibitors thapsigargin and cyclopiazonic acid potentiated caffeine-stimulated Aβ release. NH₄Cl and monensin, agents that alter acidic gradients in intracellular vesicles, abolished both the caffeine and ionophore effects. Immunocytochemical studies showed some correspondence between the distribution patterns of RYR and cellular βAPP immunoreactivities. The relevance of these findings to Alzheimer's disease and inclusion body myositis is discussed.     

Cysteine 144 Is a Key Residue in the Copper Reduction by the β-Amyloid Precursor Protein

The beta-amyloid precursor protein (beta-APP) contains a copper-binding site localized between amino acids 135 and 156 (beta-APP(135-156)). We have employed synthetic beta-APP peptides to characterize their capacities to reduce Cu(II) to Cu(I). Analogues of the wild-type beta-APP(135-156) peptide, containing specific amino acid substitutions, were used to establish which residues are specifically involved in the reduction of copper by beta-APP(135-156). We report here that beta-APP's copper-binding domain reduced Cu(II) to Cu(I). The single-mutant beta-APP(His147-->Ala) and the double-mutant beta-APP(His147-->Ala/His149-->Ala) showed a small decrease in copper reduction in relation to the wild-type peptide and the beta-APP(Cys144-->Ser) mutation abolished it, suggesting that Cys144 is the key amino acid in the oxidoreduction reaction. Our results confirm that soluble beta-APP is involved in the reduction of Cu(II) to Cu(I).     

Amyloid Precursor Protein Metabolism in Primary Cell Cultures of Neurons, Astrocytes, and Microglia

Abstract: Amyloid precursor protein (APP) gives rise by proteolytic processing to the amyloid β peptide (Aβ) found abundantly in cerebral senile plaques of individuals with Alzheimer's disease. APP is highly expressed in the brain. To assess the source of cerebral Aβ, the metabolism of APP was investigated in the major cell types of the newborn rat cerebral cortex by pulse/chase labeling and immunoprecipitation of the APP and APP metabolic fragments. We describe a novel C-terminally truncated APP isoform that appears to be made only in neurons. The synthesis, degradation, and metabolism of APP were quantified by phosphorimaging in neurons, astrocytes, and microglia. The results show that although little APP is metabolized through the amyloidogenic pathways in each of the three cultures, neurons appear to generate more Aβ than astrocytes or microglia.     

Regulation of Secretion of Alzheimer Amyloid Precursor Protein by the Mitogen-Activated Protein Kinase Cascade

Abstract: Activation of protein kinase C (PKC) regulates the processing of Alzheimer amyloid precursor protein (APP) into its soluble form (sAPP) and amyloid β-peptide (Aβ). However, little is known about the intermediate steps between PKC activation and modulation of APP metabolism. Using a specific inhibitor of mitogen-activated protein (MAP) kinase kinase activation (PD 98059), as well as a dominant negative mutant of MAP kinase kinase, we show in various cell lines that stimulation of PKC by phorbol ester rapidly induces sAPP secretion through a mechanism involving activation of the MAP kinase cascade. In PC12-M1 cells, activation of MAP kinase by nerve growth factor was associated with stimulation of sAPP release. Conversely, M1 muscarinic receptor stimulation, which is known to act in part through a PKC-independent pathway, increased sAPP secretion mainly through a MAP kinase-independent pathway. Aβ secretion and its regulation by PKC were not affected by PD 98059, supporting the concept of distinct secretory pathways for Aβ and sAPP formation.     

An Etiological Role of Amyloidogenic Carboxyl-Terminal Fragments of the β-Amyloid Precursor Protein in Alzheimer's Disease

Abstract: Amyloid β protein (Aβ), 39–43 amino acids long, is the principal constituent of the extracellular amyloid deposits in brain that are characteristic of Alzheimer's disease (AD). Several lines of evidence indicate that Aβ may play an important role in the pathogenesis of AD. However, there are several discrepancies between the production of Aβ and the development of the disease. Thus, Aβ may not be the sole active fragment of β-amyloid precursor protein (βAPP) in the neurotoxicity associated with AD. Consequently, the possible effects of other cleaved products of βAPP need to be explored. The recent concentration on other potentially amyloidogenic products of βAPP has produced interesting candidates, the most promising of which are the amyloidogenic carboxyl-terminal (CT) fragments of βAPP. This review discusses a possible etiological role of CT fragments of βAPP in AD.     

Isoform-Specific Modulation by Apolipoprotein E of the Activities of Secreted β-Amyloid Precursor Protein

Abstract: The genes for both the β-amyloid precursor protein and apolipoprotein E (ApoE) have been linked to Alzheimer's disease. This connection suggests the possibility that these proteins interact physically or functionally. To explore this idea, we focused on the neuroprotective activity of secreted amyloid precursor protein (sAPP) and related signal transduction events. After coincubation with ApoE, sAPP exhibited an enhanced [Ca²⁺]_i-lowering activity and enhanced protection against excitotoxicity in rat primary hippocampal neurons. In contrast, the stimulation of phosphoinositide production by sAPP was inhibited by ApoE. Kinetic analyses and coimmunoprecipitation experiments indicated that these actions result from formation of a heteromeric complex between ApoE and sAPP. Furthermore, the ApoE4 isoform, which seems to accelerate the onset of Alzheimer's disease, was less potent than ApoE3 in modifying each activity of sAPP. These data suggest that sAPP-dependent neuroprotective mechanisms would be compromised in individuals expressing ApoE4, a scenario that may contribute to the development of Alzheimer's disease.     

Phorbol Esters but Not the Cholinergic Agonists Oxotremorine-M and Carbachol Increase Release of the Amyloid Precursor Protein in Cultured Rat Cortical Neurons

Abstract: Conventional secretory processing of the amyloid precursor protein is nonamyloidogenic, releasing carboxyl-terminus-truncated amyloid precursor protein derivatives while cleaving the amyloid β-peptide within its sequence. Alternative processing routes are potentially amyloidogenic, yielding the amyloid β-peptide segment intact. In continuous cell lines, secretory processing of the amyloid precursor protein is regulated by both protein kinase C and muscarinic receptor stimulation. However, the first and second messenger systems that regulate amyloid precursor protein release in central neurons are still under investigation. In the present investigation, we examined whether or not first and second messengers of cholinergic neurotransmission increase production of soluble derivatives of the amyloid precursor protein in primary cultures of rat cortical neurons. Activation of protein kinase C by the phorbol esters phorbol 12,13-dibutyrate and phorbol 12-myristate 13-acetate increased production of the soluble form of the amyloid precursor protein dramatically. In contrast, activation of muscarinic receptors by oxotremorine-M or carbachol did not result in a significant increase in amyloid precursor protein release. Similarly, chemically induced depolarization using 35 m M KCI did not alter production of soluble amyloid precursor protein derivatives. Our data suggest that although protein kinase C stimulation plays an important role in regulating release of the amyloid precursor protein, cholinergic neurotransmission does not regulate its release in cultured rat cortical neurons.     

β-Amyloid Precursor Protein Isoforms in Various Rat Brain Regions and During Brain Development

To address the question of the possible functions of different Alzheimer's disease beta-amyloid precursor protein (beta-APP) isoforms in the brain, we studied their expression at different times during postnatal rat brain development and in various regions of the adult rat brain. Polyclonal antibodies directed to two peptide antigens were used. The majority of all beta-APP forms was found to be soluble as revealed by western blot analysis. The highest level of most beta-APP forms was reached in the second postnatal week, which is the time of brain maturation and completion of synaptic connections. Strikingly high concentrations of the Kunitz protease inhibitor-containing beta-APP were present in the adult olfactory bulb, where continuous synaptogenesis occurs in the adult animal. These findings support the idea of an involvement of beta-APPs in the processes of cell differentiation and, probably, in the establishment of synaptic contacts.     

Inhibition of β-Amyloid Production by Activation of Protein Kinase C

The cellular factors regulating the generation of β-amyloid from the amyloid precursor protein (APR) are unknown. Activation of protein kinase C (PKC) by phorbol ester treatment inhibited the generation of the 4-kDa β-amyloid peptide in transfected COS cells, a human glioma cell line, and human cortical astrocytes. An analogue of diacylglycerol, the endogenous cellular activator of PKC, also inhibited the generation of β-amyloid. Activation of PKC increased the level of secreted APP in transfected COS cells but did not significantly affect the level of secreted APP in primary human astrocytes or in the glioma cell line. Cell-associated APP and the secreted APP derivative, but not β-amyloid, were phosphorylated on serine residues. Activation of PKC did not increase the level of APP phosphorylation, suggesting that PKC modulates the proteolytic cleavage of APP indirectly by phosphorylation of other substrates. These results indicate that PKC activation inhibits β-amyloid production by altering APP processing and suggest that β-amyloid production can be regulated by the phospholipase C-diacylglycerol signal transduction pathway.     

Selective Aggregation of Endogenous β-Amyloid Peptide and Soluble Amyloid Precursor Protein in Cerebrospinal Fluid by Zinc

Abstract: Zinc added to buffered solutions of synthetic β-amyloid peptide (Aβ) has been reported to induce accelerated formation of insoluble aggregates. This observation suggests that zinc may play a role in the formation of senile plaques, which contain Aβ, in Alzheimer's disease. To test this hypothesis under conditions more representative of the brain, we investigated the ability of zinc to induce aggregation of Aβ in freshly drawn canine CSF, which contains the same sequence as human Aβ. Aggregates were separated from CSF by ultracentrifugation before and after incubation with zinc and assayed by quantitative western blotting and ELISA. We found that zinc induced the rapid aggregation of endogenous Aβ in CSF, with an EC₅₀ of 120–140 µ M . The reaction was specific, because most (≥95%) CSF protein remained soluble under conditions where most Aβ was insoluble, as assayed by scanning densitometry of Coomassie-stained gels. Staining of the precipitated material resulted in the visualization of punctate regions that were thioflavin positive or birefringent when stained with Congo red, suggesting the formation of amyloid-related structures. These results suggest that zinc could play a role in amyloid deposition, because there is overlap between the regions of the brain where zinc concentrations are highest and regions with the highest amyloid content. It is surprising that zinc induced the aggregation of endogenous soluble APP at lower concentrations than required for Aβ (EC₅₀ 80 µ M ). The possibility that zinc-induced aggregation of APP may precede the deposition of Aβ into plaques is discussed. Investigation of aggregation of Aβ in CSF will aid in assessing the biological relevance of other agents that have been reported to accelerate amyloid formation.     

Human Brain βA4 Amyloid Protein Precursor of Alzheimer''s Disease: Purification and Partial Characterization

The major component of the amyloid deposition that characterizes Alzheimer's disease is the 4-kDa beta A4 protein, which is derived from a much larger amyloid protein precursor (APP). A procedure for the complete purification of APP from human brain is described. The same amino terminal sequence of APP was found in two patients with Alzheimer's disease and one control subject. Two major forms of APP were identified in human brain with apparent molecular masses of 100-110 kDa and 120-130 kDa. Soluble and membrane fractions of brain contained nearly equal amounts of APP in both humans and rats. Immunoprecipitation with carboxyl terminus-directed antibodies indicates that the soluble forms of APP are truncated. Carboxyl terminus truncation of membrane-associated forms of human brain APP was also found to occur during postmortem autolysis. The availability of purified human brain APP will facilitate the investigation of its normal function and the events that lead to its abnormal cleavage in patients with Alzheimer's disease.     

An Alternative Secretase Cleavage Produces Soluble Alzheimer Amyloid Precursor Protein Containing a Potentially Amyloidogenic Sequence

Cell culture studies have shown that the Alzheimer amyloid precursor protein (APP) is secreted after full-length APP is cleaved by a putative secretase at the Lys16-Leu17 bond (secretase cleavage I) of the amyloid peptide sequence. Because this cleavage event is incompatible with amyloid production, it has been assumed that secreted APP cannot serve as a precursor of the amyloid depositions observed in Alzheimer's disease. Here we show that in neuronally differentiated PC12 cells and human kidney 293 cell cultures a portion of the secreted extracytoplasmic APP reacted specifically with both a monoclonal antibody recognizing amyloid protein residues Leu17-Val24 and a polyclonal antiserum directed against amyloid protein residues Ala21-Lys28. Furthermore, this APP failed to react with antisera recognizing the cytoplasmic domain of the full-length protein. These data indicate the presence of an alternative APP secretase cleavage site (secretase cleavage II), C-terminal to the predominant secretase cleavage I. Depending on the exact location of cleavage site II, potentially amyloidogenic secreted APP species may be produced.     

Secreted Form of Amyloid β/A4 Protein Precursor (APP) Binds to Two Distinct APP Binding Sites on Rat B103 Neuron-Like Cells Through Two Different Domains, but Only One Site Is Involved in Neuritotropic Activity

Abstract: Recent studies have identified the Alzheimer's disease amyloid β/A4 protein precursor (APP) as a trophic and/or tropic protein on several types of cells, including fibroblasts, primary culture neurons, PC12 cells, and B103 neuron-like cells. Many trophic proteins bind heparin, and it is believed that the heparin-binding domain is crucial for the trophic activity of these proteins. APP also binds heparin. The current studies were undertaken to examine the hypothesis that the neuritotropic activity of APP requires heparin binding. It was found that APP produced in E. coli bound B103 cells through detergent-extractable molecules. Approximately 50% of the binding sites were heparinase-sensitive, and heparin and heparan sulfate competed for APP binding to these sites. The heparinase-insensitive sites were recognized by a stretch of 17 amino acids of APP (residues 319–335) that contains the neuritotropic activity of APP. A mutant APP with a deletion at this site was capable of binding to the heparinase-sensitive sites, although this molecule was not neuritotropic to B103 neuron-like cells. Therefore, the neuritotropic site and the heparin-binding site are distinct in APP, and the neuritotropic effect of APP is produced through its binding to detergent-extractable and heparinase-insensitive sites.