Restored degradation of the Alzheimer's amyloid-β peptide by targeting amyloid formation

Accumulation of neurotoxic amyloid-β (Aβ) is central to the pathology of Alzheimer's disease (AD). Elucidating the mechanisms of Aβ accumulation will therefore expedite the development of Aβ-targeting AD therapeutics. We examined activity of an Aβ-degrading protease (matrix metalloprotease 2) to investigate whether biochemical factors consistent with conditions in the AD brain contribute to Aβ accumulation by altering Aβ sensitivity to proteolytic degradation. An Aβ amino acid mutation found in familial AD, Aβ interactions with zinc (Zn), and increased Aβ hydrophobicity all strongly prevented Aβ degradation. Consistent to all of these factors is the promotion of specific Aβ aggregates where the protease cleavage site, confirmed by mass spectrometry, is inaccessible within an amyloid structure. These data indicate decreased degradation due to amyloid formation initiates Aβ accumulation by preventing normal protease activity. Zn also prevented Aβ degradation by the proteases neprilysin and insulin degrading enzyme. Treating Zn-induced Aβ amyloid with the metal-protein attenuating compound clioquinol reversed amyloid formation and restored the peptide's sensitivity to degradation by matrix metalloprotease 2. This provides new data indicating that therapeutic compounds designed to modulate Aβ-metal interactions can inhibit Aβ accumulation by restoring the catalytic potential of Aβ-degrading proteases.     

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.     

Amyloid β Protein Primes Cultured Rat Microglial Cells for an Enhanced Phorbol 12-Myristate 13-Acetate-Induced Respiratory Burst Activity

Abstract: Activated microglia, often associated with neuritic amyloid plaques in the Alzheimer's disease brain, are likely to contribute to the progression of the disease process, e.g., by releasing neurotoxic reactive oxygen and/or nitrogen intermediates. In the present study, whether the amyloid β peptide (Aβ), the principal constituent of amyloid plaques, can stimulate microglial respiratory burst activity and/or microglial production of nitric oxide was examined. Using neonatal rat microglial cultures as a model, it was found that neither the spontaneous release of nitric oxide nor the lipopolysaccharide-induced production of nitric oxide was altered in cultures previously incubated with synthetic Aβ(1–40). for 24 h. In addition, no direct stimulatory effect of Aβ(1–40) on the respiratory burst activity was observed. Nevertheless, concomitant with an increase in the number of responsive cells, a profound priming of the phorbol 12-myristate 13-acetate-evoked production of superoxide anion was observed in Aβ(1–40)-treated cultures. Thus, both the maximal rate and the total phorbol 12-myristate 13-acetate-induced production of superoxide appeared to be statistically significantly higher as compared with untreated cultures. It is concluded that, as far as activation of the microglial respiratory burst is concerned, Aβ(1–40) may merely act as a priming rather than a triggering stimulus.     

Proinflammatory cytokine interferon-γ increases induction of indoleamine 2,3-dioxygenase in monocytic cells primed with amyloid β peptide 1–42: implications for the pathogenesis of Alzheimer's disease

Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting enzyme of the kynurenine pathway of tryptophan metabolism, ultimately leading to production of the excitotoxin quinolinic acid (QUIN) by monocytic cells. In the Tg2576 mouse model of Alzheimer's disease, systemic inflammation induced by lipopolysaccharide leads to an increase in IDO expression and QUIN production in microglia surrounding amyloid plaques. We examined whether the IDO over-expression in microglia could be mediated by brain proinflammatory cytokines induced during the peripheral inflammation using THP-1 cells and peripheral blood mononuclear cells (PBMC) as models for microglia. THP-1 cells pre-treated with 5–25 μM amyloid β peptide (Aβ) (1–42) but not with Aβ (1–40) or Aβ (25–35) became an activated state as indicated by their morphological changes and enhanced adhesiveness. IDO expression was only slightly increased in the reactive cells but strongly enhanced following treatment with proinflammatory cytokine interferon-γ (IFN-γ) but not with interleukin-1β, tumor necrosis factor-α, or interleukin-6 at 100 U/mL. The concomitant addition of Aβ (1–42) with IFN-γ was totally ineffective, indicating that Aβ pre-treatment is prerequisite for a high IDO expression. The priming effect of Aβ (1–42) for the IDO induction was also observed for PBMC. These findings suggest that IFN-γ induces IDO over-expression in the primed microglia surrounding amyloid plaques.     

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.     

Congo Red Inhibits Proteoglycan and Serum Amyloid P Binding to Amyloid β Fibrils

Abstract: Various data suggest that Alzheimer's disease results from the accumulation of amyloid β (Aβ) peptide fibrils and the consequent formation of senile plaques in the cognitive regions of the brain. One approach to lowering senile plaque burden in Alzheimer's disease brain is to identify compounds that will increase the degradation of existing amyloid fibrils. Previous studies have shown that proteoglycans and serum amyloid P (SAP), molecules that localize to senile plaques, bind to Aβ fibrils and protect the amyloid peptide from proteolytic breakdown. Therefore, molecules that prevent the binding of SAP and/or proteoglycans to fibrillar Aβ might increase plaque degradation and prove useful in the treatment of Alzheimer's disease. The nature of SAP and proteoglycan binding to Aβ is defined further in the present study. SAP binds to both fibrillar and nonfibrillar forms of Aβ. However, only the former is rendered resistant to proteolysis after SAP association. It is interesting that both SAP and proteoglycan binding to Aβ fibrils can be inhibited by glycosaminoglycans and Congo red. Unexpectedly, Congo red protects fibrillar Aβ from breakdown, suggesting that this compound and other structurally related molecules are unlikely to be suitable for use in the treatment of Alzheimer's disease.     

In Vitro Induction of Apoptosis or Differentiation by Dopamine in an Immortalized Olfactory Neuronal Cell Line

Abstract: Amyloid β-peptide (Aβ) is deposited as insoluble fibrils in the brain parenchyma and cerebral blood vessels in Alzheimer's disease (AD). In addition to neuronal degeneration, cerebral vascular alterations indicative of damage to vascular endothelial cells and disruption of the blood-brain barrier occur in AD. Here we report that Aβ25-35 can impair regulatory functions of endothelial cells (ECs) from porcine pulmonary artery and induce their death. Subtoxic exposures to Aβ25-35 induced albumin transfer across EC monolayers and impaired glucose transport into ECs. Cell death induced by Aβ25-35 was of an apoptotic form, characterized by DNA condensation and fragmentation, and prevented by inhibitors of macromolecular synthesis and endonucleases. The effects of Aβ25-35 were specific because Aβ1-40 also induced apoptosis in ECs with the apoptotic cells localized to the microenvironment of Aβ1-40 aggregates and because astrocytes did not undergo similar changes after exposure to Aβ25-35. Damage and death of ECs induced by Aβ25-35 were attenuated by antioxidants, a calcium channel blocker, and a chelator of intracellular calcium, indicating the involvement of free radicals and dysregulation of calcium homeostasis. The data show that Aβ induces increased permeability of EC monolayers to macromolecules, impairs glucose transport, and induces apoptosis. If similar mechanisms are operative in vivo, then Aβ and other amyloidogenic peptides may be directly involved in vascular EC damage documented in AD and other disorders that involve vascular amyloid accumulation.     

Full-Length and Truncated Alzheimer Amyloid Precursors in Chromaffin Granules: Solubilization of Membrane Amyloid Precursor Is Mediated by an Enzymatic Mechanism

Abstract: The amyloid β peptide (Aβ) of Alzheimer disease is derived from the proteolytic processing of the amyloid precursor proteins (APPs), which are considered type I transmembrane proteins. Here we report that the soluble fraction of isolated adrenal medullary chromaffin granules (CG), a model neuronal secretory vesicle system, contains an antigen that immunochemically and on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was indistinguishable from full-length APP. A truncated APP fragment with intact Aβ sequence was also detected in the soluble fraction of CG. In vitro experiments showed that full-length APP was solubilized from CG membranes at 37°C as a function of pH, with a peak of activity between pH 8.5 and pH 9.0. Solubilization of full-length APP was inhibited by several protease inhibitors, including aprotinin, cystatin, and iodoacetamide, by the divalent cations Ca²⁺ and Zn²⁺, and by preheating of the membranes. These results are consistent with and suggest the involvement of an enzymatic mechanism in the solubilization of potentially amyloidogenic full-length APP. Production of Aβ from a transmembrane APP predicts a proteolytic cleavage within the lipid bilayer, a site relatively inaccessible to proteases. Thus, the detected soluble, potentially amyloidogenic, full-length APP may be a substrate for the proteases producing Aβ. The detection of soluble APP with intact Aβ sequence in secretory vesicles is consistent with the extracellular topology of amyloid depositions.     

Alzheimer's β-Amyloid Peptide 1–42 Induces a Phagocytic Response in Murine Microglia

Abstract: β-Amyloid (Aβ) peptides are a key component of the senile plaques that characterize Alzheimer's disease. Cytokine-producing microglia have been shown to be intimately associated with amyloid deposits and have also been implicated as scavengers responsible for clearing Aβ deposits. However, little is known about the initial activation of these microglia or the effect of Aβ on phagocytosis. Murine BV-2 microglia were used to assess the effect of synthetic Aβ 1–42 on phagocytosis by quantifying uptake of fluorescent microspheres, acetylated low-density lipoproteins, and zymosan particles by flow cytometry. Aβ 1–42 stimulated microglial phagocytosis in a time- and dose-dependent manner. Aβ fibrils produced the greatest potentiation, and once activated, phagocytosis remained elevated after removal of Aβ from the cultures. Aβ-stimulated phagocytosis could be blocked if proteoglycans were first complexed to Aβ fibrils. These data suggest that Aβ fibrils act as an immune signal to stimulate microglial phagocytosis and that extracellular matrix molecules may modify Aβ function.     

Endoplasmic reticulum-localized amyloid beta-peptide is degraded in the cytosol by two distinct degradation pathways

The paradigm of endoplasmic reticulum (ER)-associated degradation (ERAD) holds that misfolded secretory and membrane proteins are translocated back to the cytosol and degraded by the proteasome in a coupled process. Analyzing the degradation of ER-localized amyloid β-peptide (Aβ), we found a divergence from this general model. Cell-free reconstitution of the export in biosynthetically loaded ER-derived brain microsomes showed that the export was mediated by the Sec61p complex and required a cytosolic factor but was independent of ATP. In contrast to the ERAD substrates known so far, the exported Aβ was degraded by both, a proteasome-dependent and a proteasome-independent pathway. RNA interference experiments in Aβ-transfected cells identified the protease of the proteasome-independent pathway as insulin-degrading enzyme (IDE). The IDE-mediated clearance mechanism for ER-localized Aβ represents an as yet unknown type of ERAD which is not entirely dependent on the proteasome.     

Alzheimer Aβ Amyloid Forms an Inhibitory Neuronal Substrate

Abstract: Alzheimer's disease (AD) is identified by the accumulation of amyloid plaques, neurofibrillary degeneration, and the accompanying neuronal loss. AD amyloid assembles into compact fibrous deposits from the amyloid β(Aβ) protein, which is a proteo-lytic fragment of the membrane-associated amyloid precursor protein. To examine the effects of amyloid on neuron growth, a hybrid mouse motoneuron cell line (NSC34) exhibiting spontaneous process formation was exposed to artificial "plaques" created from aggregated synthetic Aβ peptides. These correspond to full-length Aβ residues 1–40 (Aβ1–40), an internal β-sheet region comprising residues 11–28 (Aβ11–28), and a proposed toxic fragment comprising residues 25–35 (Aβ25–35). Fibers were immobilized onto culture dishes, and addition of cells to these in vitro plaques revealed that Aβ was not a permissive substrate for cell adhesion. Neurites in close contact with these deposits displayed abnormal swelling and a tendency to avoid contact with the Aβ fibers. In contrast, Aβ did not affect the adhesion or growth of rat astrocytes, implicating a specific Aβ-neuron relationship. The inhibitory effects were also unique to Aβ as no response was observed to deposits of pancreatic islet amyloid poly-peptide fibers. Considering the importance of cell adhesion in neurite elongation and axonal guidance, the antiadhesive properties of Aβ amyloid plaques found in vivo may contribute to the neuronal loss responsible for the clinical manifestations of AD.     

Androgens regulate neprilysin expression: role in reducing beta-amyloid levels

Age-related testosterone depletion in men is a risk factor for Alzheimer's disease. Prior studies suggest that androgens affect Alzheimer's disease risk by regulating accumulation of β-amyloid protein (Aβ) by an undefined mechanism. In this study, we investigated the role of the Aβ-catabolizing enzyme neprilysin (NEP) in this process. First, we observed that androgens positively regulate neural expression of NEP in adult male rats. Next, we investigated androgen regulatory effects on both NEP expression and Aβ levels using cultured hippocampal neurons and neuronally differentiated rat pheochromocytoma cell 12 with or without androgen receptor (AR). Dihydrotestosterone (DHT) induced a time-dependent increase in NEP expression. DHT also significantly decreased levels of Aβ in AR-expressing cells transfected with amyloid precursor protein, but did not affect levels of either full-length or non-amyloidogenic, soluble amyloid precursor protein. Importantly, the DHT induced decrease of Aβ was blocked by pharmacological inhibition of NEP. The DHT-mediated increase in NEP expression and decrease in Aβ levels were (i) not observed in rat pheochromocytoma cell 12 lacking AR and (ii) blocked in AR-expressing cells by the antagonists, cyproterone acetate and flutamide. Together, these findings suggest that androgen regulation of Aβ involves an AR-dependent mechanism requiring up-regulation of the Aβ catabolizing enzyme NEP.     

High-Resolution Separation of Amyloid β-Peptides: Structural Variants Present in Alzheimer's Disease Amyloid

Abstract: In Alzheimer's disease (AD), one of the cardinal neuropathological signs is deposition of amyloid, primarily consisting of the amyloid β-peptide (Aβ). Structural variants of AD-associated Aβ peptides have been difficult to purify by high-resolution chromatographic techniques. We therefore developed a novel chromatographic protocol, enabling high-resolution reverse-phase liquid chromatography (RPLC) purification of Aβ variants displaying very small structural differences. By using a combination of size-exclusion chromatography and the novel RPLC protocol, Aβ peptides extracted from AD amyloid were purified and subsequently characterized. Structural analysis by microsequencing and electrospray-ionization mass spectrometry revealed that the RPLC system resolved a complex mixture of Aβ variants terminating at either residue 40 or 42. Aβ variants differing by as little as one amino acid residue could be purified rapidly to apparent homogeneity. The resolution of the system was further illustrated by its ability to separate structural isomers of Aβ^1–40. The present chromatography system might provide further insight into the role of N-terminally and posttranslationally modified Aβ variants, because each variant can now be studied individually.     

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.     

Inhibition of glutaminyl cyclase prevents pGlu-Abeta formation after intracortical/hippocampal microinjection in vivo/in situ

Modified amyloid β (Aβ) peptides represent major constituents of the amyloid deposits in Alzheimer's disease and Down's syndrome. In particular, N-terminal pyroglutamate (pGlu) following truncation renders Aβ more stable, increases hydrophobicity and the aggregation velocity. Recent evidence based on in vitro studies suggests that the cyclization of glutamic acid, leading to pGlu-Aβ, is catalyzed by the enzyme glutaminyl cyclase (QC) following limited proteolysis of Aβ at the N-terminus. Here, we studied the pGlu-formation by rat QC in vitro as well as after microinjection of Aβ(1–40) and Aβ(3–40) into the rat cortex in vivo / in situ with and without pharmacological QC inhibition. Significant pGlu-Aβ formation was observed following injection of Aβ(3–40) after 24 h, indicating a catalyzed process. The generation of pGlu-Aβ from Aβ(3–40) was significantly inhibited by intracortical microinjection of a QC inhibitor. The study provides first evidence that generation of pGlu-Aβ is a QC-catalyzed process in vivo . The approach per se offers a strategy for a rapid evaluation of compounds targeting a reduction of pGlu formation at the N-terminus of amyloid peptides.     

The Amyloid β-Protein of Alzheimer's Disease Increases Acetylcholinesterase Expression by Increasing Intracellular Calcium in Embryonal Carcinoma P19 Cells

Abstract: One of the characteristic changes that occurs in Alzheimer's disease is the loss of acetylcholinesterase (AChE) from both cholinergic and noncholinergic neurons of the brain. However, AChE activity is increased around amyloid plaques. This increase in AChE may be of significance for therapeutic strategies using AChE inhibitors. The aim of this study was to examine the effect of amyloid β-protein (Aβ), the major component of amyloid plaques, on AChE expression. Aβ peptides spanning residues 1–40 or 25–35 increased AChE activity in P19 embryonal carcinoma cells. A peptide containing a scrambled Aβ_25–35 sequence did not stimulate AChE expression. To examine the possibility that the increase in AChE expression was mediated by an influx of calcium through voltage-dependent calcium channels (VDCCs), drugs acting on VDCCs were tested for their effects. Inhibitors of L-type VDCCs (diltiazem, nifedipine, and verapamil), but not N- or P- or Q-type VDCCs, resulted in a decrease in AChE expression. Agonists of L-type VDCCs (maitotoxin and S (−)-Bay K 8644) increased AChE expression. As L-type VDCCs are known to be modulated by cyclic AMP-dependent protein kinase, the effect of the adenylate cyclase activator forskolin was also examined. Forskolin stimulated AChE expression, an action that was blocked by the L-type VDCC antagonist nifedipine. The Aβ_25–35-induced increase in AChE expression was mediated by an L-type VDCC, as the effect was also blocked by nifedipine. The results suggest that the increase in AChE expression around amyloid plaques could be due to a disturbance in calcium homeostasis involving the opening of L-type VDCCs.     

Effects of HNE-modification induced by Aβ on neprilysin expression and activity in SH-SY5Y cells

The cerebral accumulation of β-amyloid (Aβ) is a consistent feature of and likely contributor to the development of Alzheimer's disease. In addition to dysregulated production, increasing experimental evidence suggests reduced catabolism also plays an important role in Aβ accumulation. We have previously shown that neprilysin (NEP), the major protease which cleaves Aβ in vivo , is modified by 4-hydroxy-nonenal (HNE) adducts in the brain of Alzheimer's disease patients. To determine if these changes affected Aβ, SH-SY5Y cells were treated with HNE or Aβ, and then NEP mRNA, protein levels, HNE adducted NEP, NEP activity and secreted Aβ levels were determined. Intracellular NEP developed HNE adducts after 24 h of HNE treatment as determined by immunoprecipitation, immunoblotting, and double immunofluorescence staining. HNE-modified NEP showed decreased catalytic activity, which was associated with elevations in Aβ1–40 in SH-SY5Y and H4 APP695wt cells. Incubation of cells with Aβ1–42 also induced HNE adduction of NEP. In an apparent compensatory response, Aβ-treated cells showed increased NEP mRNA and protein expression. Despite elevations in NEP protein, the activity was significantly lower compared with the NEP protein level. This study demonstrates that NEP can be inactivated by HNE-adduction, which is associated with, at least partly, reduced Aβ cleavage and enhanced Aβ accumulation.     

Monoclonal Antibodies Against the Human Metalloprotease EC 3.4.24.15 Label Neurofibrillary Tangles in Alzheimer's Disease Brain

Abstract: Alzheimer's disease is characterized neuropathologically by the presence of neuritic and amyloid plaques, vascular amyloid, and neurofibrillary tangles in specific brain areas. The main constituent of amyloid deposits is amyloid β protein, a 40–42 amino acid proteolytic product of the amyloid β-precursor protein. In our search for proteases that can generate the N-terminus of amyloid β protein (β-secretases), we discovered a thiol-dependent metalloprotease that was identified, by peptide sequencing, as metalloendopeptidase EC 3.4.24.15. In vitro, the metalloprotease cleaves the methionine-aspartic acid bond in a 10 amino acid synthetic peptide, indicating that it could generate the N-terminus of amyloid β protein, and generates amyloidogenic fragments from full-length recombinant amyloid β-precursor protein. Mouse monoclonal antibodies produced against a unique synthetic peptide from the metalloprotease labeled various monkey tissues as detected by western blots and immunohistochemistry. Unexpectedly, two monoclonal antibodies, IVD6 and IIIF3, immunolabeled strongly intracellular neurofibrillary tangles, neurites of senile plaques, and neuropil threads, but not "ghost" tangles or amyloid in sections taken from Alzheimer's disease brain. This finding provides further evidence for the metalloprotease's relevance to Alzheimer's disease pathology, although the connection between tangle staining and the formation of amyloid β protein remains to be elucidated.     

Soluble aggregates of the amyloid-beta protein activate endothelial monolayers for adhesion and subsequent transmigration of monocyte cells

Increasing evidence suggests that the deposition of amyloid plaques, composed primarily of the amyloid-β protein (Aβ), within the cerebrovasculature is a frequent occurrence in Alzheimer's disease and may play a significant role in disease progression. Accordingly, the pathogenic mechanisms by which Aβ can alter vascular function may have therapeutic implications. Despite observations that Aβ elicits a number of physiological responses in endothelial cells, ranging from alteration of protein expression to cell death, the Aβ species accountable for these responses remains unexplored. In the current study, we show that isolated soluble Aβ aggregation intermediates activate human brain microvascular endothelial cells for both adhesion and subsequent transmigration of monocyte cells in the absence of endothelial cell death and monolayer disruption. In contrast, unaggregated Aβ monomer and mature Aβ fibril fail to induce any change in endothelial adhesion or transmigration. Correlations between average Aβ aggregate size and observed increases in adhesion illustrate that smaller soluble aggregates are more potent activators of endothelium. These results support previous studies demonstrating heightened neuronal activity of soluble Aβ aggregates, including Aβ-derived diffusible ligands, oligomers, and protofibrils, and further show that soluble aggregates also selectively exhibit activity in a vascular cell model.     

Opposing Actions of Thrombin and Protease Nexin-1 on Amyloid β-Peptide Toxicity and on Accumulation of Peroxides and Calcium in Hippocampal Neurons

Abstract: Amyloid β-peptide (Aβ) is the principal component of neuritic plaques in the brain in Alzheimer's disease (AD). Recent studies revealed that Aβ can be neurotoxic by a mechanism involving free radical production and loss of cellular ion homeostasis, thus implicating Aβ as a key factor in the pathogenesis of AD. However, other proteins are present in plaques in AD, including the protease thrombin and protease nexin-1 (PN1), a thrombin inhibitor. We therefore tested the hypothesis that thrombin and PN1 modify neuronal vulnerability to Aβ toxicity. In dissociated rat hippocampal cell cultures the toxicity of Aβ was significantly enhanced by coincubation with thrombin, whereas PN1 protected neurons against Aβ toxicity. Aβ induced an increase in levels of intracellular peroxides and calcium. Thrombin enhanced, and PN1 attenuated, the accumulation of peroxides and calcium induced by Aβ. Taken together, these data demonstrate that thrombin and PN1 have opposing effects on neuronal vulnerability to Aβ and suggest that thrombin and PN1 play roles in the pathogenesis of neuronal injury in AD.