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.     

Zn(II)- and Cu(II)-induced non-fibrillar aggregates of amyloid-β (1–42) peptide are transformed to amyloid fibrils, both spontaneously and under the influence of metal chelators

Aggregation of amyloid-β (Aβ) peptides is a central phenomenon in Alzheimer's disease. Zn(II) and Cu(II) have profound effects on Aβ aggregation; however, their impact on amyloidogenesis is unclear. Here we show that Zn(II) and Cu(II) inhibit Aβ₄₂ fibrillization and initiate formation of non-fibrillar Aβ₄₂ aggregates, and that the inhibitory effect of Zn(II) (IC₅₀ = 1.8 μmol/L) is three times stronger than that of Cu(II). Medium and high-affinity metal chelators including metallothioneins prevented metal-induced Aβ₄₂ aggregation. Moreover, their addition to preformed aggregates initiated fast Aβ₄₂ fibrillization. Upon prolonged incubation the metal-induced aggregates also transformed spontaneously into fibrils, that appear to represent the most stable state of Aβ₄₂. H13A and H14A mutations in Aβ₄₂ reduced the inhibitory effect of metal ions, whereas an H6A mutation had no significant impact. We suggest that metal binding by H13 and H14 prevents the formation of a cross-β core structure within region 10–23 of the amyloid fibril. Cu(II)-Aβ₄₂ aggregates were neurotoxic to neurons in vitro only in the presence of ascorbate, whereas monomers and Zn(II)-Aβ₄₂ aggregates were non-toxic. Disturbed metal homeostasis in the vicinity of zinc-enriched neurons might pre-dispose formation of metal-induced Aβ aggregates, subsequent fibrillization of which can lead to amyloid formation. The molecular background underlying metal-chelating therapies for Alzheimer's disease is discussed in this light.     

Pathologic Amyloid β-Protein Cell Surface Fibril Assembly on Cultured Human Cerebrovascular Smooth Muscle Cells

Abstract: Cerebrovascular amyloid β-protein (Aβ) deposition is a key pathological feature of Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D). Aβ_1–40 containing the E22Q HCHWA-D mutation, but not wild-type Aβ_1–40, potently induces several pathologic responses in cultured human cerebrovascular smooth muscle cells, including cellular degeneration and a robust increase in the levels of cellular Aβ precursor. In the present study, we show by several quantitative criteria, including thioflavin T fluorescence binding, circular dichroism spectroscopy, and transmission electron microscopic analysis, that at a concentration of 25 µ M neither HCHWA-D Aβ_1–40 nor wild-type Aβ_1–40 appreciably assembles into β-pleated sheet-containing fibrils in solution over a 6-day incubation period. In contrast, at the same concentrations, HCHWA-D Aβ_1–40, but not wild-type Aβ_1–40, selectively binds and assembles into abundant fibrils on the surfaces of cultured human cerebrovascular smooth muscle cells. The simultaneous addition of an equimolar concentration of the dye Congo red prevents the cell surface fibril assembly of HCHWA-D Aβ_1–40. Moreover, Congo red effectively blocks the key pathologic responses induced by HCHWA-D Aβ_1–40 in these cells. The present findings suggest that the surface of human cerebrovascular smooth muscle cells may selectively orchestrate the assembly of pathogenic Aβ fibrils and that cell surface Aβ fibril formation plays an important role in causing the pathologic responses in these cells.     

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.     

Etazolate, a neuroprotective drug linking GABA(A) receptor pharmacology to amyloid precursor protein processing

Pharmacological modulation of the GABA_A receptor has gained increasing attention as a potential treatment for central processes affected in Alzheimer disease (AD), including neuronal survival and cognition. The proteolytic cleavage of the amyloid precursor protein (APP) through the α-secretase pathway decreases in AD, concurrent with cognitive impairment. This APP cleavage occurs within the β-amyloid peptide (Aβ) sequence, precluding formation of amyloidogenic peptides and leading to the release of the soluble N-terminal APP fragment (sAPPα) which is neurotrophic and procognitive. In this study, we show that at nanomolar-low micromolar concentrations, etazolate, a selective GABA_A receptor modulator, stimulates sAPPα production in rat cortical neurons and in guinea pig brains. Etazolate (20 nM–2 μM) dose-dependently protected rat cortical neurons against Aβ-induced toxicity. The neuroprotective effects of etazolate were fully blocked by GABA_A receptor antagonists indicating that this neuroprotection was due to GABA_A receptor signalling. Baclofen, a GABA_B receptor agonist failed to inhibit the Aβ-induced neuronal death. Furthermore, both pharmacological α-secretase pathway inhibition and sAPPα immunoneutralization approaches prevented etazolate neuroprotection against Aβ, indicating that etazolate exerts its neuroprotective effect via sAPPα induction. Our findings therefore indicate a relationship between GABA_A receptor signalling, the α-secretase pathway and neuroprotection, documenting a new therapeutic approach for AD treatment.     

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.     

Rapid Degeneration of Cultured Human Brain Pericytes by Amyloid β Protein

Abstract: Amyloid β protein (Aβ) deposition in the cerebral arterial and capillary walls is one of the major characteristics of brains from patients with Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D). Vascular Aβ deposition is accompanied by degeneration of smooth muscle cells and pericytes. In this study we found that Aβ_1–40 carrying the "Dutch" mutation (HCHWA-D Aβ_1–40) as well as wild-type Aβ_1–42 induced degeneration of cultured human brain pericytes and human leptomeningeal smooth muscle cells, whereas wild-type Aβ_1–40 and HCHWA-D Aβ_1–42 were inactive. Cultured brain pericytes appeared to be much more vulnerable to Aβ-induced degeneration than leptomeningeal smooth muscle cells, because in brain pericyte cultures cell viability already decreased after 2 days of exposure to HCHWA-D Aβ_1–40, whereas in leptomeningeal smooth muscle cell cultures cell death was prominent only after 4–5 days. Moreover, leptomeningeal smooth muscle cell cultures were better able to recover than brain pericyte cultures after short-term treatment with HCHWA-D Aβ_1–40. Degeneration of either cell type was preceded by an increased production of cellular amyloid precursor protein. Both cell death and amyloid precursor protein production could be inhibited by the amyloid-binding dye Congo red, suggesting that fibril assembly of Aβ is crucial for initiating its destructive effects. These data imply an important role for Aβ in inducing perivascular cell pathology as observed in the cerebral vasculature of patients with Alzheimer's disease or HCHWA-D.     

Mild cholesterol depletion reduces amyloid-β production by impairing APP trafficking to the cell surface

It has been suggested that cellular cholesterol levels can modulate the metabolism of the amyloid precursor protein (APP) but the underlying mechanism remains controversial. In the current study, we investigate in detail the relationship between cholesterol reduction, APP processing and γ-secretase function in cell culture studies. We found that mild membrane cholesterol reduction led to a decrease in Aβ₄₀ and Aβ₄₂ in different cell types. We did not detect changes in APP intracellular domain or Notch intracellular domain generation. Western blot analyses showed a cholesterol-dependent decrease in the APP C-terminal fragments and cell surface APP. Finally, we applied a fluorescence resonance energy transfer (FRET)-based technique to study APP–Presenilin 1 (PS1) interactions and lipid rafts in intact cells. Our data indicate that cholesterol depletion reduces association of APP into lipid rafts and disrupts APP–PS1 interaction. Taken together, our results suggest that mild membrane cholesterol reduction impacts the cleavage of APP upstream of γ-secretase and appears to be mediated by changes in APP trafficking and partitioning into lipid rafts.     

Intracellular Cyclic AMP Inhibits Constitutive and Phorbol Ester-Stimulated Secretory Cleavage of Amyloid Precursor Protein

Abstract: α-Secretase cleaves the full-length Alzheimer's amyloid precursor protein (APP) within the amyloid β peptide sequence, thus precluding amyloid formation. The resultant soluble truncated APP is constitutively secreted. This nonamyloidogenic processing of APP is increased on stimulation of the phospholipase C/protein kinase C pathway by phorbol esters. Here we used C6 cells transfected with APP₇₅₁ to examine whether the α-secretase cleavage is regulated by the adenylate cyclase signal transduction pathway. Forskolin, an activator of adenylate cyclase, inhibited both the constitutive and phorbol ester-stimulated secretion of nexin II (NXII), the secreted product of the α-secretase cleavage of APP₇₅₁. At 1 µ M , forskolin inhibited secretion of NXII by ∼50% without affecting either the intracellular levels of total APP or the secretion of secretory alkaline phosphatase. In contrast, 1,9-dideoxyforskolin, an inactive analogue of forskolin, did not affect secretion of NXII. These results indicated that forskolin specifically inhibited the α-secretase cleavage of APP₇₅₁. Forskolin treatment increased the intracellular concentration of cyclic AMP (cAMP), suggesting that the forskolin effects on APP cleavage may be mediated by cAMP. In support of this suggestion, both dibutyryl cAMP, a cAMP analogue, and isoproterenol, an activator of adenylate cyclase, also inhibited secretion of NXII. These data indicate that forskolin inhibition of the nonamyloidogenic cleavage of APP is mediated by the second messenger cAMP, which together with the protein kinase C signal transduction pathway modulates the secretory cleavage of APP.     

A traditional medicinal herb Paeonia suffruticosa and its active constituent 1,2,3,4,6-penta-O-galloyl-β-d-glucopyranose have potent anti-aggregation effects on Alzheimer's amyloid β proteins in vitro and in vivo

The deposition of amyloid β (Aβ) protein is a consistent pathological hallmark of Alzheimer's disease (AD) brains; therefore, inhibition of Aβ fibril formation and destabilization of pre-formed Aβ fibrils is an attractive therapeutic and preventive strategy in the development of disease-modifying drugs for AD. This study demonstrated that Paeonia suffruticosa , a traditional medicinal herb, not only inhibited fibril formation of both Aβ_1–40 and Aβ_1–42 but it also destabilized pre-formed Aβ fibrils in a concentration-dependent manner. Memory function was examined using the passive-avoidance task followed by measurement of Aβ burden in the brains of Tg2576 transgenic mice. The herb improved long-term memory impairment in the transgenic mice and inhibited the accumulation of Aβ in the brain. Three-dimensional HPLC analysis revealed that a water extract of the herb contained several different chemical compounds including 1,2,3,4,6-penta- O -galloyl-β- d -glucopyranose (PGG). No obvious adverse/toxic were found following treatment with PGG. As was observed with Paeonia suffruticosa , PGG alone inhibited Aβ fibril formation and destabilized pre-formed Aβ fibrils in vitro and in vivo . Our results suggest that both Paeonia suffruticosa and its active constituent PGG have strong inhibitory effects on formation of Aβ fibrils in vitro and in vivo . PGG is likely to be a safe and promising lead compound in the development of disease-modifying drugs to prevent and/or cure AD.     

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.     

In Vitro Phosphorylation of the Cytoplasmic Domain of the Amyloid Precursor Protein by Glycogen Synthase Kinase-3β

Abstract: The two pathological lesions found in the brains of Alzheimer's disease patients, neurofibrillary tangles and neuritic plaques, are likely to be formed through a common pathway. Neurofibrillary tangles are intracellular aggregates of paired helical filaments, the main component of which is hyperphosphorylated forms of the microtubule-associated protein τ. Extracellular neuritic plaques and diffuse and vascular amyloid deposits are aggregates of β-amyloid protein, a 4-kDa protein derived from the amyloid precursor protein (APP). Using conditions in vitro under which two proline-directed protein kinases, glycogen synthase kinase-3β (GSK-3β) and mitogen-activated protein kinase (MAPK), were able to hyperphosphorylate τ, GSK-3β but not MAPK phosphorylated recombinant APP_cyt. The sole site of phosphorylation in APP_cyt by GSK-3β was determined by phosphoamino acid analysis and phosphorylation of APP_cyt mutant peptides to be Thr⁷⁴³ (numbering as for APP₇₇₀). This site was confirmed by endoproteinase Glu-C digestion of APP_cyt and peptide sequencing. The ability of GSK-3β to phosphorylate APP_cyt and τ provides a putative link between the two lesions and indicates a critical role of GSK-3β in the pathogenesis of Alzheimer's disease.     

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.     

Estrogens Attenuate and Corticosterone Exacerbates Excitotoxicity, Oxidative Injury, and Amyloid β-Peptide Toxicity in Hippocampal Neurons

Abstract: Steroid hormones, particularly estrogens and glucocorticoids, may play roles in the pathogenesis of neurodegenerative disorders, but their mechanisms of action are not known. We report that estrogens protect cultured hippocampal neurons against glutamate toxicity, glucose deprivation, FeSO₄ toxicity, and amyloid β-peptide (Aβ) toxicity. The toxicity of each insult was significantly attenuated in cultures pretreated for 2 h with 100 n M -10 µ M 17β-estradiol, estriol, or progesterone. In contrast, corticosterone exacerbated neuronal injury induced by glutamate, FeSO₄, and Aβ. Several other steroids, including testosterone, aldosterone, and vitamin D, had no effect on neuronal vulnerability to the different insults. The protective actions of estrogens and progesterone were not blocked by actinomycin D or cycloheximide. Lipid peroxidation induced by FeSO₄ and Aβ was significantly attenuated in neurons and isolated membranes pretreated with estrogens and progesterone, suggesting that these steroids possess antioxidant activities. Estrogens and progesterone also attenuated Aβ- and glutamate-induced elevation of intracellular free Ca²⁺ concentrations. We conclude that estrogens, progesterone, and corticosterone can directly affect neuronal vulnerability to excitotoxic, metabolic, and oxidative insults, suggesting roles for these steroids in several different neurodegenerative disorders.     

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.     

Amyloid β-Mediated Oxidative and Metabolic Stress in Rat Cortical Neurons: No Direct Evidence for a Role for H2O2 Generation

Abstract: H₂O₂ and free radical-mediated oxidative stresses have been implicated in mediating amyloid β(1–40) [Aβ(1–40)] neurotoxicity to cultured neurons. In this study, we confirm that addition of the H₂O₂-scavenging enzyme catalase protects neurons in culture against Aβ-mediated toxicity; however, it does so by a mechanism that does not involve its ability to scavenge H₂O₂. Aβ-mediated elevation in intracellular H₂O₂ production is suppressed by addition of a potent H₂O₂ scavenger without any significant neuroprotection. Three intracellular biochemical markers of H₂O₂-mediated oxidative stress were unchanged by Aβ treatment: (a) glyceraldehyde-3-phosphate dehydrogenase activity, (b) hexose monophosphate shunt activity, and (c) glucose oxidation via the tricarboxylic acid cycle. Ionspray mass spectra of Aβ in the incubation medium indicated that Aβ itself is an unlikely source of reactive oxygen species. In this study we demonstrate that intracellular ATP concentration is compromised during the first 24-h exposure of neurons to Aβ. Our results challenge a pivotal role for H₂O₂ generation in mediating Aβ toxicity, and we suggest that impairment of energy homeostasis may be a more significant early factor in the neurodegenerative process.     

One-year longitudinal evaluation of sensorimotor functions in APP751SL transgenic mice

Intracerebral amyloid-beta (Aβ) peptide deposition is considered to play a key role in Alzheimer's disease and is designated as a principal therapeutic target. The relationship between brain Aβ levels and clinical deficits remains, however, unclear, both in human patients and in animal models of the disease. The purpose of the present study was to investigate, in a transgenic mouse model of brain amyloidosis, the consequences of Aβ deposition on basic neurological functions using a longitudinal approach. Animals were phenotyped at different ages corresponding to graded neuropathological stages (from no extracellular Aβ deposition to high amyloid loads). Sensory functions were evaluated by assessing visual and olfactory abilities and did not show any effects of the amyloid precursor protein (APP) transgene. Motor functions were assessed using multiple experimental paradigms. Results showed that motor strength was considerably reduced in APP transgenic mice compared with control animals. No deficit was noted in a motor coordination test although APP transgenic mice displayed decreased locomotion on a stationary beam. Hypolocomotion was also observed in the standard open-field test. Measures of anxiety obtained in the elevated plus-maze show some evidence of hyperanxiety in 15-month-old transgenic mice. Some of the neurological impairments showed by APP mice had an early onset and worsened with progressive aging, in parallel to gradual accumulation of Aβ in brain parenchyma. Relationships between neuropathologically assessed amyloid loads and behavioral deficits were further explored, and it was observed that motor strength deficits were correlated with cortical amyloid burden.     

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.     

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.     

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.