Metal Cations Defibrillize the Amyloid Beta-Protein Fibrils

Amyloid beta-protein (A) is the major constituent of amyloid fibrils composing -amyloid plaques and cerebrovascular amyloid in Alzheimer's disease (AD). We studied the effect of metal cations on preformed fibrils of synthetic A by Thioflavin T (ThT) fluorescence spectroscopy and electronmicroscopy (EM) in negative staining. The amount of cross beta-pleated sheet structure of A 1–40 fibrils was found to decrease by metal cations in a concentration-dependent manner as measured by ThT fluorescence spectroscopy. The order of defibrillization of A 1–40 fibrils by metal cations was: Ca²⁺ and Zn²⁺ (IC₅₀ = 100 M) > Mg²⁺ (IC₅₀ = 300 M) > Al³⁺ (IC₅₀ =1.1 mM). EM analysis in negative staining showed that A 1–40 fibrils in the absence of cations were organized in a fine network with a little or no amorphous material. The addition of Ca²⁺, Mg²⁺, and Zn²⁺ to preformed A 1–40 fibrils defibrillized the fibrils or converted them into short rods or to amorphous material. Al³⁺ was less effective, and reduced the fibril network by about 80 % of that in the absence of any metal cation. Studies with A 1–42 showed that this peptide forms more dense network of fibrils as compared to A 1–40. Both ThT fluorescence spectroscopy and EM showed that similar to A 1–40, A 1–42 fibrils are also defibrillized in the presence of millimolar concentrations of Ca²⁺. These studies suggest that metal cations can defibrillize the fibrils of synthetic A.     

Media from Rhabdomyosarcoma and Neuroblastoma Cell Cultures Stimulate in Vitro Aggregation and Fibrillization of Amyloid Beta-Protein

In vitro aggregation and fibrillization of synthetic amyloid beta-protein A 1–40 was assessed in the conditioned media from rhabdomyosarcoma (CRL 1598, HTB 82, HTB 153, CCL 136), adenocarcinoma (CCL 218), neuroblastoma (SY5Y), and COS cells cultured in the absence and presence of 10% heat-inactivated fetal bovine serum (FBS). The aggregation and formation of cross -pleated sheet structures in A was quantitated by Thioflavin T (ThT) fluorescence spectroscopy, while the morphology of A fibrils was examined in negative staining in the electronmicroscope (EM). In cultures supplemented with 10% FBS, the conditioned media from CRL 1598, HTB 82, CCL 218, and SY5Y cell cultures stimulated A aggregation in a time-dependent manner as compared to that of control (serum-containing medium that had not been exposed to cells). The order of stimulation was SY5Y > CRL 1598 HTB 82 > CCL 218, and the stimulation was higher in 2 week cultures than in 1 week cultures. Similar studies using media from HTB 153, CCL 136 and COS cell cultures showed no effect on A 1–40 aggregation. In serum-free cell cultures, only media from SY5Y and CRL 1598 could promote significant aggregation of A 1–40. Negative staining in EM revealed A fibril formation only with conditioned media from SY5Y and CRL 1598 cultured under serum free conditions; no A fibrils were noticed in media from cell cultures supplemented with 10% FBS. We propose that both the SY5Y neuroblastoma cell line and the CRL 1598 rhabdomyosarcoma cell line may serve as experimental models for in vitro studies of extracellular aggregation and fibrillization of A-protein in cell cultures, while rhabdomyosarcoma HTB 82 and adenocarcinoma CCL 218 may be models for study of A aggregation only.     

Amyloid beta-protein interactions with membranes and cholesterol: causes or casualties of Alzheimer's disease

Amyloid beta-protein (Aβ) is thought to be one of the primary factors causing neurodegeneration in Alzheimer's disease (AD). This protein is an amphipathic molecule that perturbs membranes, binds lipids and alters cell function. Several studies have reported that Aβ alters membrane fluidity but the direction of this effect has not been consistently observed and explanations for this lack of consistency are proposed. Cholesterol is a key component of membranes and cholesterol interacts with Aβ in a reciprocal manner. Aβ impacts on cholesterol homeostasis and modification of cholesterol levels alters Aβ expression. In addition, certain cholesterol lowering drugs (statins) appear to reduce the risk of AD in human subjects. However, the role of changes in the total amount of brain cholesterol in AD and the mechanisms of action of statins in lowering the risk of AD are unclear. Here we discuss data on membranes, cholesterol, Aβ and AD, and propose that modification of the transbilayer distribution of cholesterol in contrast to a change in the total amount of cholesterol provides a cooperative environment for Aβ synthesis and accumulation in membranes leading to cell dysfunction including disruption in cholesterol homeostasis.     

Binding of Amyloid Beta-Protein to Intracellular Brain Proteins in Rat and Human

Amyloid beta-protein (A), in its soluble form, is known to bind several circulatory proteins such as apolipoprotein (apo) E, apo J and transthyretin. However, the binding of A to intracellular proteins has not been studied. We have developed an overlay assay to study A binding to intracellular brain proteins. The supernatants from both rat and human brains were found to contain several proteins that bind to A 1–40 and A 1–42. No major difference was observed in the A binding-proteins from brain supernatants of patients with Alzheimer's disease and normal age-matched controls. Binding studies using shorter amyloid beta-peptides and competitive overlay assays showed that the binding site of A to brain proteins resides between 12–28 amino acid sequence of A. The presence of several intracellular A-binding (AB) proteins suggests that these proteins may either protect A from its fibrillization or alternatively promote A polymerization. Identification of these proteins and their binding affinities for A are needed to assess their potential role in the pathogenesis of Alzheimer's disease.     

Lipid Binding to Amyloid β-Peptide Aggregates: Preferential Binding of Cholesterol as Compared with Phosphatidylcholine and Fatty Acids

Abstract: Amyloid β-peptide (Aβ) aggregates are one of the key neuropathological characteristics of Alzheimer's disease. Aβ belongs to a group of proteins that aggregate and form β-sheets, and some of these proteins bind cholesterol and other lipids. The purpose of the experiments reported here was to determine if cholesterol, fatty acids, and phosphatidylcholine (PC) would bind to Aβ_1–40 and if such binding would be dependent on aggregation of Aβ_1–40. Lipid binding was determined using fluorescent-labeled lipids. Incubation of Aβ_1–40 for 0, 1, 3, 6, 21, and 24 h resulted in aggregation of the peptide with formation of dimers, trimers (1–24 h), and polymers (6–24 h) as determined by sodium dodecyl sulfate-gel electrophoresis. No change in the fluorescence of the lipids was observed when lipids were added to Aβ_1–40 that had been incubated for 0, 1, or 3 h. However, the fluorescence intensities of cholesterol, saturated fatty acids, and PC were significantly increased (p < 0.0001) when added to Aβ_1–40 that had been incubated for 6, 21, and 24 h in which Aβ_1–40 polymers were detected. The binding affinity of cholesterol to Aβ_1–40 polymers (K_D of 3.24 ± 0.315 × 10^?9M) was markedly higher as compared with the other lipids (stearic acid, 9.42 ± 0.41 × 10^?8M; PC, 7.07 ± 0.12 × 10^?7M). The results of this study indicate that Aβ_1–40 polymers bind lipids and have a higher affinity for cholesterol than PC or saturated fatty acids. Aggregated Aβ_1–40 may affect lipid transport between cells or remove specific lipids from membranes, and such effects could contribute to neuronal dysfunction.     

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.     

Expression of the Chondroitin Sulfate Proteoglycans of Amyloid Precursor (Appican) and Amyloid Precursor-Like Protein 2

Abstract: The Alzheimer amyloid precursor (APP) protein is a member of a family of glycoproteins that includes the amyloid precursor-like proteins (APLPs). Previously, we showed that in C6 glioma cell cultures, secreted APP nexin II occurs as the core protein of a chondroitin sulfate proteoglycan (CSPG). Here, we report that among seven untransfected cell lines, expression of secreted APP CSPG was restricted to two cell lines of neural origin, namely, C6 glioma and Neuro-2a neuroblastoma (N2a) cells. Addition of dibutyryl cyclic AMP in N2a cultures, a treatment that induces the neuronal phenotype in these cells, resulted in a significant reduction in the amount of the secreted APP CSPG, although secretion of APP was only marginally affected. Growth in the presence of serum increased the size of the secreted APP CSPG, suggesting that the number and/or length of the chondroitin sulfate (CS) chains attached to the core APP varies with growth conditions. Extensive mapping with epitope-specific anti-bodies suggested that a CS chain is attached within or proximal to the Aβ sequence of APP. In contrast to the restricted expression of the APP CSPG, expression of secreted APLP2 CSPGs was observed in all cell lines examined. After chondroitinase treatment, two core proteins of ∼100 and 110 kDa were obtained that reacted with an APLP2-specific antiserum, suggesting that non-transfected cell lines contain at least two endogenous APLP2 CSPGs, probably derived by alternative splicing of the APLP2 KPI domain. The fraction of the APLP2 proteins in the CSPG form was dependent on the particular cell line examined. The proteoglycan nature of APP and APLP2 suggests that addition of the CS glycosaminoglycan chains is important for the implementation of the biological function of these proteins. However, the differential expression of these two proteoglycans suggests that their physiological roles and their possible involvement in Alzheimer's disease may differ.     

Identification of Heparin-Binding Domains in the Amyloid Precursor Protein of Alzheimer's Disease by Deletion Mutagenesis and Peptide Mapping

Abstract: Recent studies have shown that the binding of the amyloid protein precursor (APP) of Alzheimer's disease to heparan sulfate proteoglycans (HSPGs) can modulate a neurite outgrowth-promoting function associated with APP. We used three different approaches to identify heparin-binding domains in APP. First, as heparin-binding domains are likely to be within highly folded regions of proteins, we analyzed the secondary structure of APP using several predictive algorithms. This analysis showed that two regions of APP₆₉₅ contain a high degree of secondary structure, and clusters of basic residues, considered mandatory for heparin binding, were found principally within these regions. To determine which domains of APP bind heparin, deletion mutants of APP₆₉₅ were prepared and analyzed for binding to a heparin affinity column. The results suggested that there must be at least two distinct heparin-binding regions in APP. To identify novel heparin-binding regions, peptides homologous to candidate heparin-binding domains were analyzed for their ability to bind heparin. These experiments suggested that APP contains at least four heparin-binding domains. The presence of more than one heparin-binding domain on APP suggests the possibility that APP may interact with more than one type of glycosaminoglycan.     

Inhibition of β-Amyloid Formation by Haloperidol: A Possible Mechanism for Reduced Frequency of Alzheimer's Disease Pathology in Schizophrenia

Abstract: Several reports have suggested that the frequency of Alzheimer's disease (AD) neuropathology is significantly reduced in elderly individuals with schizophrenia (SZ), and it has been proposed that medications used for treatment of SZ may be responsible. A central event in AD pathology is the formation of β-amyloid (Aβ) peptide, which is derived by enzymatic processing of its precursor protein. Haloperidol, an antipsychotic medication commonly used in the treatment of SZ, can act as an inhibitor of select proteinases; hence, we examined the ability of this compound to inhibit Aβ formation by cultured cells. Haloperidol and, to a lesser extent, droperidol inhibited Aβ in a dose-dependent manner. These results may explain the apparent reduction of AD neuropathological changes in elderly patients with SZ as well as provide a possible mechanism for this difference.     

Aggregation and Metal-Binding Properties of Mutant Forms of the Amyloid Aβ Peptide of Alzheimer's Disease

Abstract: The fibrillogenic properties of Alzheimer's Aβ peptides corresponding to residues 1–40 of the normal human sequence and to two mutant forms containing the replacement Ala²¹ to Gly or Glu²² to Gln were compared. At pH 7.4 and 37°C the Gln²² peptide was found to aggregate and precipitate from solution faster than the normal Aβ, whereas the Gly²¹ peptide aggregated much more slowly. Electron microscopy showed that the aggregates all had fibrillar structures. Circular dichroism spectra of these peptides revealed that aggregation of the normal and Gln²² sequences was associated with spectral changes consistent with a transformation from random coil to β sheet, whereas the spectrum of the Gly²¹ peptide remained almost unchanged during a period in which little or no aggregation occurred. When immobilised by spotting onto nitrocellulose membranes the peptides bound similar amounts of the radioisotope ⁶⁵Zn²⁺. Of several competing metal ions, tested at 20× the concentration of Zn²⁺, Cu²⁺ displaced >95% of the radioactivity from all three peptides and Ni²⁺ produced >50% displacement in each case. Some other metal ions tested caused lesser displacement, but Fe²⁺ and Al³⁺ were without effect. In a saturation binding assay, a value of 3.2 µM was obtained for the binding of Zn²⁺ to Aβ but our data provided no evidence for a reported higher affinity site (107 nM). The results suggest that the neuropathology associated with the Gly²¹ mutation is not due to enhanced fibrillogenic or different metal-binding properties of the peptide and that the binding of zinc to amyloid peptides is not a specific phenomenon.     

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.     

Amyloid peptide Abeta(1-42) binds selectively and with picomolar affinity to alpha7 nicotinic acetylcholine receptors

We have recently reported evidence that a very high affinity interaction between the beta-amyloid peptide Abeta(1-42) and the alpha7 nicotinic acetylcholine receptor (alpha7nAChR) may be a precipitating event in the formation of amyloid plaques in Alzheimer's disease. In the present study, the kinetics for the binding of Abeta(1-42) to alpha7nAChR and alpha4beta2nAChR were determined using the subtype-selective nicotinic receptor ligands [(3)H]methyllycaconitine and [(3)H]cytisine. Synaptic membranes prepared from rat and guinea pig cerebral cortex and hippocampus were used as the source of receptors. Abeta(1-42) bound to the alpha7nAChR with exceptionally high affinity, as indicated by K(i) values of 4.1 and 5.0 pM for rat and guinea pig receptors, respectively. When compared with the alpha7nAChR, the affinity of Abeta(1-42) for the alpha4beta2nAChR was approximately 5,000-fold lower, as indicated by corresponding K(i) values of 30 and 23nM. The results of this study support the concept that an exceptionally high affinity interaction between Abeta(1-42) and alpha7nAChR could serve as a precipitating factor in the formation of amyloid plaques and thereby contribute to the selective degeneration of cholinergic neurons that originate in the basal forebrain and project to the cortex and hippocampus.     

Increased Secretion of the Amino-Terminal Fragment of Amyloid Precursor Protein in Brains of Rats with a Constitutive Up-Regulation of Protein Kinase C

Abstract: Protein kinase C (PKC) activation stimulates release of secreted amyloid precursor protein (APP_s) in several cell lines. To ascertain the role of PKC in regulating APP metabolism in vivo, we used an animal model (methylazoxymethanol-treated rats; MAM rats) in which PKC is permanently hyperactivated in selected brain areas, i.e., cortex and hippocampus. A significant decrease in membrane-bound APP concentration was found in synaptosomes derived from cortex and hippocampus of MAM rats, where PKC is up-regulated, with a concomitant increase in APP_s production in soluble fractions of the same brain areas. In contrast, in a brain area not affected by MAM treatment (i.e., cerebellum), APP secretion is similar in control and MAM rats, indicating that altered metabolism of APP is restricted to only those areas in which the PKC system is up-regulated. In addition, phorbol esters or H-7 modulate APP_s release in hippocampal slices from both control and MAM rats, further supporting an in vivo role for this enzyme in regulating metabolism of mature APP.     

A Monoclonal Antibody That Reacts Immunohistochemically with Amyloid Deposits in the Brain Tissue of Alzheimer Patients Binds to an Epitope Present on Complement Factor 4

The mouse monoclonal antibody SMP has previously been demonstrated to react immunohistochemically with neurofibrillary tangles, argyrophilic plaques, and leptomeningeal vascular amyloid deposits in the brain tissue of individuals dying from pathologically diagnosed Alzheimer's disease. In preliminary studies the antibody was shown, by size exclusion chromatography, to bind to a protein with an apparent molecular mass of 260 kDa present in the CSF and serum of demented individuals. Chromatographic separation of a 40% ammonium sulphate precipitate of CSF and serum yielded immunoreactive fractions that were subjected to 9% sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by western blotting. Probing the nitrocellulose blot with the antibody revealed that the antibody selectively binds to a protein chain with an apparent molecular mass of 100 kDa. By using a combination of affinity chromatography and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, coupled with western blotting, the serum component with which the antibody reacts has been identified as complement factor 4. In addition, the antibody has been shown to react specifically with an epitope on the alpha-chain of this protein.     

Presenilin 1 Mutations Linked to Familial Alzheimer's Disease Increase the Intracellular Levels of Amyloid β-Protein 1–42 and Its N-Terminally Truncated Variant(s) Which Are Generated at Distinct Sites

Abstract: Mutations in the presenilin genes PS1 and PS2 cause the most common form of early-onset familial Alzheimer's disease. The influence of PS1 mutations on the generation of endogenous intracellular amyloid β-protein (Aβ) species was assessed using a highly sensitive immunoblotting technique with inducible mouse neuro-blastoma (Neuro 2a) cell lines expressing the human wild-type (wt) or mutated PS1 (M146L or Δexon 10). The induction of mutated PS1 increased the intracellular levels of two distinct Aβ species ending at residue 42 that were likely to be Aβ1–42 and its N-terminally truncated variant(s) Aβx-42. The induction of mutated PS1 resulted in a higher level of intracellular Aβ1–42 than of intracellular Aβx-42, whereas extracellular levels of Aβ1–42 and Aβx-42 were increased proportionally. In addition, the intracellular generation of these Aβ42 species in wt and mutated PS1 -induced cells was completely blocked by brefeldin A, whereas it exhibited differential sensitivities to monensin: the increased accumulation of intracellular Aβx-42 versus inhibition of intracellular Aβ1–42 generation. These data strongly suggest that Aβx-42 is generated in a proximal Golgi, whereas Aβ1–42 is generated in a distal Golgi and/or a post-Golgi compartment. Thus, it appears that PS1 mutations enhance the degree of 42-specific γ-secretase cleavage that occurs in the normal β-amyloid precursor protein processing pathway (a) in the endoplasmic reticulum or the early Golgi apparatus prior to β-secretase cleavage or (b) in the distinct sites where Aβx-42 and Aβ1–42 are generated.     

Interaction of enantiomeric alkylacyl and diacylglycerophosphocholines with cholesterol in bilayer membranes

The sn-1 and sn-3 isomers of dioleoylglycerophosphocholine form vesicles of the same size as the racemic lipid. Identical permeability coefficients were found for the diffusion of glucose and chloride across bilayer membranes of vesicles consisting of these lipids. Vesicles made of mixtures of enantiomeric or racemic dioleoyllecithin with 30 mol% cholesterol have identical radii. Cholesterol reduces the permeability of bilayers for glucose and chloride irrespective of the steric configuration of the constituent phospholipid. Increasing concentrations of cholesterol (17, 33 and 50 mol%, respectively) broaden the (CH₂)_n signal in the ¹H-NMR-spectra (90 MHz) of unilamellar vesicles containing sn-1, sn-3 or rac alkyloleoylglycerophosphocholine to the same extent. These results indicate that the steric configuration of phospholipids has no gross effect on the arrangement of phospholipids and cholesterol in bilayer membranes.     

Amyloid β Protein Potentiates Ca2+ Influx Through L-Type Voltage-Sensitive Ca2+ Channels: A Possible Involvement of Free Radicals

Abstract: Amyloid β protein (Aβ), the central constituent of senile plaques in Alzheimer's disease (AD) brain, is known to exert toxic effects on cultured neurons. The role of the voltage-sensitive Ca²⁺ channel (VSCC) in β(25–35) neurotoxicity was examined using rat cultured cortical and hippocampal neurons. When L-type VSCCs were blocked by application of nimodipine, β(25–35) neurotoxicity was attenuated, whereas application of ω-conotoxin GVIA (ω-CgTX-GVIA) or ω-agatoxin IVA (ω-Aga-IVA), the blocker for N- or P/Q-type VSCCs, had no effects. Whole-cell patch-clamp studies indicated that the Ca²⁺ current density of β(25–35)-treated neurons is about twofold higher than that of control neurons. Also, β(25–35) increased Ca²⁺ uptake, which was sensitive to nimodipine. The 2',7'-dichlorofluorescin diacetate assay showed the ability of β(25–35) to produce reactive oxygen species. Nimodipine had no effect on the level of free radicals. In contrast, vitamin E, a radical scavenger, reduced the level of free radicals, neurotoxicity, and Ca²⁺ uptake. These results suggest that β(25–35) generates free radicals, which in turn, increase Ca²⁺ influx via the L-type VSCC, thereby inducing neurotoxicity.     

Fate of Cerebrospinal Fluid-Borne Amyloid β-Peptide: Rapid Clearance into Blood and Appreciable Accumulation by Cerebral Arteries

Abstract: In Alzheimer's disease, the neuritic or senile amyloid plaques in hippocampus and association cortex, the diffuse plaques in brain areas such as the cerebellum and sensorimotor cortex, and the amyloid deposits in the walls of pial and parenchymal blood vessels are mainly composed of amyloid β-peptides. In the present study, either soluble 40-residue amyloid β-peptide radiolabeled with ¹²⁵I (I-sAβ) or [¹⁴C]polyethylene glycol ([¹⁴C]-PEG, a reference material) was briefly infused into one lateral ventricle of normal rats. By 3.5 min, 30% of the I-sAβ was cleared from ventricular CSF into blood; another 30% was removed over the next 6.5 min. No [¹⁴C]PEG was lost from the CSF-brain system during the first 5 min, and only 20% was cleared by 10 min. Much of the I-sAβ that reached the subarachnoid space was retained by pial arteries and arterioles. Virtually no I-sAβ was found in brain. The clearance of amyloid β-peptides from the CSF-brain system, reported herein for normal rats, may be reduced in Alzheimer's disease, thus contributing to amyloid deposition in cerebral tissue and blood vessels.     

Amyloid Angiopathy of Alzheimer''s Disease: Amino Acid Composition and Partial Sequence of a 4,200-Dalton Peptide Isolated from Cortical Microvessels

The cardinal lesions of Alzheimer's disease are neurofibrillary tangles, senile neuritic plaques, and vascular amyloid, the latter generally involving cortical arteries and small arterioles. All three lesions are composed of amyloid-like, beta-pleated sheet fibrils. Recently, a 4,200-dalton peptide has been isolated from extraparenchymal meningeal vessels, neuritic plaques, and neurofibrillary tangles. The assumption of N-terminal homogeneity in vascular amyloid has been used as an argument for a neuronal (versus blood) origin of the peptide. However, intracortical microvessels from Alzheimer's disease have not been previously isolated. The present studies describe the isolation of a microvessel fraction from Alzheimer's disease and control fresh autopsy human brain. Alzheimer's disease isolated brain microvessels that were extensively laden with amyloid and control microvessels were solubilized in 90% formic acid and analyzed by urea sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The arteriole fraction from the Alzheimer's subject with extensive amyloid angiopathy contained a unique 4,200-dalton peptide, whereas the arterioles or capillaries isolated from two controls and two Alzheimer's disease subjects without angiopathy did not. This peptide was purified by HPLC and amino acid composition analysis showed the peptide is nearly identical to the 4,200-dalton peptide recently isolated from neuritic plaques or from neurofibrillary tangles. Sequence analysis revealed N-terminal heterogeneity. The N-terminal sequence was: Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr, which is identical to the N-terminal sequence of the 4,200-dalton peptide isolated previously from extraparenchymal meningeal vessels and neuritic plaques.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.