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.     

Brain Expression of Apolipoproteins E, J, and A-I in Alzheimer's Disease

Abstract: Inheritance of the ε4 allele of apolipoprotein (apo) E is associated with increased risk of Alzheimer's disease (AD) and with increased β-amyloid peptide (Aβ) deposition in the cortex. Apo E is a member of a family of exchangeable apos, characterized by the presence of amphipathic α-helical segments that allow these molecules to act as surfactants on the surface of lipoprotein particles. Two members of this family, apo E and apo J, have been shown to bind soluble Aβ, and both are associated with senile plaques in the AD cortex. We now have studied the pattern of brain apo expression and found that five members of this class are present: apo A-I, A-IV, D, E, and J. By contrast, apos A-II, B, and C-II were not detectable. Immunohistochemistry revealed that, in addition to apo E and apo J, apo A-I immunostained occasional senile plaques in AD cortex. Immunoblot analysis showed no difference in the relative amounts of any of these apos in tissue homogenates of frontal lobe from AD or control patients. Comparison by APO E genotype showed no differences in the amount of apo E in brain among APO E ε3/3, ε3/4, or ε4/4 individuals; however, a significant decrease in the amount of apo J was associated with the APO E ε4 allele. No differences in apo J levels were detected in CSF samples of AD subjects. We propose that several members of the exchangeable apo family may interact with Aβ deposits in senile plaques through common amphipathic α-helical domains. Competition among these molecules for binding of Aβ or Aβ aggregates may influence the deposition of Aβ in senile plaques.     

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.     

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.     

Acetylcholinesterase Is Increased in the Brains of Transgenic Mice Expressing the C-Terminal Fragment (CT100) of the β-Amyloid Protein Precursor of Alzheimer's Disease

Abstract: Acetylcholinesterase (AChE) expression is markedly affected in Alzheimer's disease (AD). AChE activity is lower in most regions of the AD brain, but it is increased within and around amyloid plaques. We have previously shown that AChE expression in P19 cells is increased by the amyloid β protein (Aβ). The aim of this study was to investigate AChE expression using a transgenic mouse model of Aβ overproduction. The β-actin promoter was used to drive expression of a transgene encoding the 100-amino acid C-terminal fragment of the human amyloid precursor protein (APP CT100). Analysis of extracts from transgenic mice revealed that the human sequences of full-length human APP CT100 and Aβ were overexpressed in the brain. Levels of salt-extractable AChE isoforms were increased in the brains of APP CT100 mice. There was also an increase in amphiphilic monomeric form (G^A₁) of AChE in the APP CT100 mice, whereas other isoforms were not changed. An increase in the proportion of G^A₁ AChE was also detected in samples of frontal cortex from AD patients. Analysis of AChE by lectin binding revealed differences in the glycosylation pattern in APP CT100 mice similar to those observed in frontal cortex samples from AD. The results are consistent with the possibility that changes in AChE isoform levels and glycosylation patterns in the AD brain may be a direct consequence of altered APP metabolism.     

Trafficking of Alzheimer's disease-related membrane proteins and its participation in disease pathogenesis

Alzheimer's disease (AD) is a common neurodegenerative disorder that causes senile dementia. The pathological characteristics are the appearance of neurofibrillary tangles comprising abnormally phosphorylated tau and senile plaques composed of amyloid beta-protein depositions. Amyloid beta-protein precursor (APP) and presenilin (PS) are known to be causative genes of familial AD. Recent analyses have documented that APP functions in the axonal transport of vesicles and PS regulates intracellular protein trafficking. Dystrophic neurites, in which APP and Alcadein accumulate in swollen axons, are also observed in AD brain. These pathological characteristics and the features of AD-related proteins suggest that AD is a disease of the vesicular transport system. Here we review recent progress of research on AD pathogenesis from the viewpoint of membrane trafficking.     

Aβ43 is more frequent than Aβ40 in amyloid plaque cores from Alzheimer disease brains

One hallmark of Alzheimer disease (AD) is the extracellular deposition of the amyloid β-peptide (Aβ) in senile plaques. Two major forms of Aβ are produced, 40 (Aβ40) and 42 (Aβ42) residues long. The most abundant form of Aβ is Aβ40, while Aβ42 is more hydrophobic and more prone to form toxic oligomers and the species of particular importance in early plaque formation. Thus, the length of the hydrophobic C-terminal seems to be very important for the oligomerization and neurotoxicity of the Aβ peptide. Here we investigated which Aβ species are deposited in AD brain. We analyzed plaque cores, prepared from occipital and frontal cortex, from sporadic and familial AD cases and performed a quantitative study using Aβ standard peptides. Cyanogen bromide was used to generate C-terminal Aβ fragments, which were analyzed by HPLC coupled to an electrospray ionisation ion trap mass spectrometer. We found a longer peptide, Aβ43, to be more frequent than Aβ40. No variants longer than Aβ43 could be observed in any of the brains. Immunohistochemistry was performed and was found to be in line with our findings. Aβ1-43 polymerizes rapidly and we suggest that this variant may be of importance for AD.     

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.     

Review on the APP/PS1KI mouse model: intraneuronal Aβ accumulation triggers axonopathy, neuron loss and working memory impairment

Accumulating evidence points to an important role of intraneuronal Aβ as a trigger of the pathological cascade of events leading to neurodegeneration and eventually to Alzheimer's disease (AD) with its typical clinical symptoms, like memory impairment and change in personality. As a new concept, intraneuronal accumulation of Aβ instead of extracellular Aβ deposition has been introduced to be the disease-triggering event in AD. The present review compiles current knowledge on the amyloid precursor protein (APP)/PS1KI mouse model with early and massive intraneuronal Aβ42 accumulation: (1) The APP/PS1KI mouse model exhibits early robust brain and spinal cord axonal degeneration and hippocampal CA1 neuron loss. (2) At the same time-point, a dramatic, age-dependent reduced ability to perform working memory and motor tasks is observed. (3) The APP/PS1KI mice are smaller and show development of a thoracolumbar kyphosis, together with an incremental loss of body weight. (4) Onset of the observed behavioral alterations correlates well with robust axonal degeneration in brain and spinal cord and with abundant hippocampal CA1 neuron loss.     

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.     

Retinoid X receptor-α mediates (R )-flurbiprofen's effect on the levels of Alzheimer's β-amyloid

Alzheimer's disease (AD) is characterized by the formation of extracellular senile plaques in the brain, whose major component is a small peptide called β-amyloid (Aβ). Long-term use of non-steroidal anti-inflammatory drugs (NSAIDs) has been found beneficial for AD and several reports suggest that NSAIDs reduce the generation of Aβ, especially the more amyloidogenic form Aβ42. However, the exact mechanism underlying NSAIDs' effect on AD risk remains largely inconclusive and all clinical trials using NSAIDs for AD treatment show negative results so far. Recent studies have shown that some NSAIDs can bind to certain nuclear receptors, suggesting that nuclear receptors may be involved in NSAID's effect on AD risk. Here we find that ( R )-flurbiprofen, the R -enantiomer of the racemate NSAID flurbiprofen, can significantly reduce Aβ secretion, but at the same time, increases the level of intracellular Aβ. In addition, we find that a nuclear receptor, retinoid X receptor α (RXRα), can regulate Aβ generation and that down-regulation of RXRα significantly increases Aβ secretion. We also show that ( R )-flurbiprofen can interfere with the interaction between RXRα and 9- cis -retinoid acid, and that 9- cis -retinoid acid decreases ( R )-flurbiprofen's reduction of Aβ secretion. Moreover, the modulation effect of ( R )-flurbiprofen on Aβ is abolished upon RXRα down-regulation. Together, these results suggest that RXRα can regulate Aβ generation and is also required for ( R )-flurbiprofen-mediated Aβ generation.     

Differential effects of γ-secretase and BACE1 inhibition on brain Aβ levels in vitro and in vivo

Alzheimer's disease (AD) is hypothesized to result from elevated brain levels of β-amyloid peptide (Aβ) which is the main component of plaques found in AD brains and which cause memory impairment in mice. Therefore, there has been a major focus on the development of inhibitors of the Aβ producing enzymes γ-secretase and β-site amyloid precursor protein-cleaving enzyme 1 (BACE1). In this study, we investigated the Aβ-lowering effects of the BACE1 inhibitor LY2434074 in vitro and in vivo , comparing it to the well characterized γ-secretase inhibitor LY450139. We sampled interstitial fluid Aβ from awake APPswe/PS1dE9 AD mice by in vivo Aβ microdialysis. In addition, we measured levels of endogenous brain Aβ extracted from wildtype C57BL/6 mice. In our in vitro assays both compounds showed similar Aβ-lowering effects. However, while systemic administration of LY450139 resulted in transient reduction of Aβ in both in vivo models, we were unable to show any Aβ-lowering effect by systemic administration of the BACE1 inhibitor LY2434074 despite brain exposure exceeding the in vitro IC₅₀ value several fold. In contrast, significant reduction of 40–50% of interstitial fluid Aβ and wildtype cortical Aβ was observed when infusing LY2434074 directly into the brain by means of reverse microdialysis or by dosing the BACE1 inhibitor to p-glycoprotein (p-gp) mutant mice. The effects seen in p-gp mutant mice and subsequent data from our cell-based p-gp transport assay suggested that LY2434074 is a p-gp substrate. This may partly explain why BACE1 inhibition by LY2434074 has lower in vivo efficacy, with respect to decreased Aβ40 levels, compared with γ-secretase inhibition by LY450139.     

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.     

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.     

Flavonoid-mediated presenilin-1 phosphorylation reduces Alzheimer's disease beta-amyloid production

Glycogen synthase kinase 3 (GSK-3) dysregulation is implicated in the two Alzheimer's disease (AD) pathological hallmarks: β-amyloid plaques and neurofibrillary tangles. GSK-3 inhibitors may abrogate AD pathology by inhibiting amyloidogenic γ-secretase cleavage of amyloid precursor protein (APP). Here, we report that the citrus bioflavonoid luteolin reduces amyloid-β (Aβ) peptide generation in both human 'Swedish' mutant APP transgene-bearing neuron-like cells and primary neurons. We also find that luteolin induces changes consistent with GSK-3 inhibition that ( i ) decrease amyloidogenic γ-secretase APP processing, and ( ii ) promote presenilin-1 (PS1) carboxyl-terminal fragment (CTF) phosphorylation. Importantly, we find GSK-3α activity is essential for both PS1 CTF phosphorylation and PS1-APP interaction. As validation of these findings in vivo , we find that luteolin, when applied to the Tg2576 mouse model of AD, decreases soluble Aβ levels, reduces GSK-3 activity, and disrupts PS1-APP association. In addition, we find that Tg2576 mice treated with diosmin, a glycoside of a flavonoid structurally similar to luteolin, display significantly reduced Aβ pathology. We suggest that GSK-3 inhibition is a viable therapeutic approach for AD by impacting PS1 phosphorylation-dependent regulation of amyloidogenesis.     

Membrane Currents Induced in Xenopus Oocytes by the C-Terminal Fragment of the β-Amyloid Precursor Protein

Abstract: There is mounting evidence that at least some of the neurotoxicity associated with Alzheimer's disease (AD) is due to proteolytic fragments of the β-amyloid precursor protein (βAPP). Most research has focused on the amyloid β protein (Aβ), which has been shown to possess ion channel activity. However, the possible role of other cleaved products of the βAPP is less clear. We have investigated the ability of various products of βAPP to induce membrane ion currents by applying them to Xenopus oocytes, a model system used extensively for investigating electrophysiological aspects of cellular, including neuronal, signalling. We focussed on the 105-amino-acid C-terminal fragment (CT₁₀₅) (containing the full sequence Aβ), which has previously been found to be toxic to cells, although little is known about its mode of action. We have found that CT₁₀₅ is exceedingly potent, with a threshold concentration of 100–200 n M , in inducing nonselective ion currents when applied from either outside or inside the oocyte and is more effective than either βAPP or the Aβ fragments, β_25–35 or β_1–40. The ion channel activity of CT₁₀₅ was concentration dependent and blocked by a monoclonal antibody to Aβ. These results suggest the possible involvement of CT₁₀₅ in inducing the neural toxicity characteristic of AD.     

Aβ aggregation and possible implications in Alzheimer's disease pathogenesis

Amyloid β protein (Aβ) has been associated with Alzheimer's disease (AD) because it is a major component of the extracellular plaque found in AD brains. Increased Aβ levels correlate with the cognitive decline observed in AD. Sporadic AD cases are thought to be chiefly associated with lack of Aβ clearance from the brain, unlike familial AD which shows increased Aβ production. Aβ aggregation leading to deposition is an essential event in AD. However, the factors involved in Aβ aggregation and accumulation in sporadic AD have not been completely characterized. This review summarizes studies that have examined the factors that affect Aβ aggregation and toxicity. By necessity these are studies that are performed with recombinant-derived or chemically synthesized Aβ. The studies therefore are not done in animals but in cell culture, which includes neuronal cells, other mammalian cells and, in some cases, non-mammalian cells that also appear susceptible to Aβ toxicity. An understanding of Aβ oligomerization may lead to better strategies to prevent AD.     

Neurotoxic traffic: uncovering the mechanics of amyloid production in Alzheimer's disease

Alzheimer's disease (AD) is thought by many to result from the accumulation of the neurotoxic amyloid-β (Aβ) peptide in brain parenchyma. The process by which Aβ is proteolytically derived from the larger amyloid precursor protein (APP) has been the focus of much attention in the AD research field over the past decade. Recently, several of the proteins directly involved in the generation of Aβ have been identified and characterized providing a number of viable therapeutic targets for the treatment of AD. However, the cellular mechanisms by which these proteins interact in the proteolytic processing of APP have not been well defined, nor are they readily apparent when one considers what is known about the intracellular localization and trafficking of the various participants. This article will review the underlying cell biology of Aβ production and discuss the mechanistic options for APP processing given the current knowledge of the proteases involved.     

Enhancement of β-Amyloid Peptide Aβ(1–40)-Mediated Neurotoxicity by Glutamine Synthetase

Abstract: The β-amyloid peptide (Aβ), a main constituent in both senile and diffuse plaques in Alzheimer's disease brains, was previously shown to be neurotoxic and to be able to interact with several macromolecular components of brain tissue. Previous investigations carried out in our laboratory demonstrated free radical species formation in aqueous solutions of Aβ(1–40) and its C-end fragment, Aβ(25–35). Toxic forms of Aβ rapidly inactivate the oxidation-sensitive cytosolic enzyme glutamine synthetase (GS). In this regard, we suggested and subsequently demonstrated that Aβ radicals can cause an oxidative damage of cell proteins and lipids resulting in disruption of membrane functions, enzyme inactivation, and cell death. Because GS can be a substrate for Aβ-derived oxidizing species, the present study was conducted to determine if GS could protect against Aβ neurotoxicity. In contrast to this initial hypothesis, we here report that GS significantly enhances the neurotoxic effects of Aβ(1–40). The Aβ-mediated inactivation of GS was found to be accompanied by the loss of immunoreactive GS and the significant increase of Aβ(1–40) neurotoxicity.     

α2-Macroglobulin Attenuates β-Amyloid Peptide 1–40 Fibril Formation and Associated Neurotoxicity of Cultured Fetal Rat Cortical Neurons

Abstract: β-Amyloid peptides (Aβ) are deposited in an aggregated fibrillar form in both diffuse and senile plaques in the brains of patients with Alzheimer's disease. The neurotoxicity of Aβ in cultured neurons is dependent on its aggregation state, but the factors contributing to aggregation and fibril formation are poorly understood. In the present study, we investigated whether α₂-macroglobulin (α₂M), a protein present in neuritic plaques and elevated in Alzheimer's disease brain, is a potential regulatory factor for Aβ fibril formation. Previous studies in our laboratory have shown that α₂M is an Aβ binding protein. We now report that, in contrast to another plaque-associated protein, α₁-antichymotrypsin, α₂M coincubated with Aβ significantly reduces aggregation and fibril formation in vitro. Additionally, cultured fetal rat cortical neurons are less vulnerable to the toxic actions of aged Aβ following pretreatment with α₂M. We postulate that α₂M is able to maintain Aβ in a soluble state, preventing fibril formation and associated neurotoxicity.