Alzheimer's Beta-amyloid Research Articles

In situ characterization of beta-amyloid in Alzheimer's diseased tissue by synchrotron Fourier transform infrared microspectroscopy

We report the first evidence of the structure of beta-amyloid protein as it exists in situ within a slice of human Alzheimer's diseased brain tissue. Using a Fourier transform infrared microspectroscopic technique, areas of interest can be selected for spectral measurements with regions of potential contamination masked. In so doing, it is possible to obtain infrared spectra only of beta-amyloid and not the surrounding grey matter within which it lies. However, to obtain spectra of high-quality signal-to-noise ratio using a conventional infrared source, we were limited to aperture sizes between 24 microns x 24 microns to 50 microns x 50 microns. Markedly improved high-quality spectra were acquired with infrared radiation provided by a synchrotron light source (National Synchrotron Light Source, Brookhaven National Laboratories), using aperture sizes as small as 12 microns x 12 microns. This allowed spectroscopic mapping of brain tissue regions containing amyloid. We observe that in situ protein of grey matter exist predominantly in an alpha-helical and/or unordered conformation, whereas within amyloid deposits a beta-sheet structure predominates. The hydrogen bonding strength of the beta-structure found in situ is different from that reported in the literature for isolated/chemically synthesized beta-amyloid peptides.

Degradation of Alzheimer's beta-amyloid protein by human cathepsin D.

The aim of the study was to identify and characterize human brain peptidases capable of degrading Alzheimer's beta-amyloid protein. Synthetic beta-amyloid protein (1-40) was rapidly degraded by a human brain soluble fraction, optimum activity occurring at around pH4. Pepstatin totally inhibited the activity showing that an aspartyl protease was responsible. HPLC separation and identification of the degradation products showed that the L34-M35 bond was the primary site of cleavage followed by hydrolysis of the F19-F20 and F20-A21 bonds. The major lysosomal aspartyl protease, cathepsin D, hydrolysed beta-amyloid protein with the same pH profile, inhibitor sensitivity and bond specificity as the activity present in human brain soluble fraction. We suggest that cathepsin D may play an important role in regulating brain concentrations of beta-amyloid protein (1-40).

Glycoprotein 330/megalin: probable role in receptor-mediated transport of apolipoprotein J alone and in a complex with Alzheimer disease amyloid beta at the blood-brain and blood-cerebrospinal fluid barriers.

A soluble form of Alzheimer disease amyloid beta-protein (sA beta) is transported in the blood and cerebrospinal fluid mainly complexed with apolipoprotein J (apoJ). Using a well-characterized in situ perfused guinea pig brain model, we recently obtained preliminary evidence that apoJ facilitates transport of sA beta (1-40)-apoJ complexes across the blood-brain barrier and the blood-cerebrospinal fluid barrier, but the mechanisms remain poorly understood. In the present study, we examined the transport process in greater detail and investigated the possible role of glycoprotein 330 (gp330)/megalin, a receptor for multiple ligands, including apoJ. High-affinity transport systems with a Km of 0.2 and 0.5 nM were demonstrated for apoJ at the blood-brain barrier and the choroid epithelium in vivo, suggesting a specific receptor-mediated mechanism. The sA beta (1-40)-apoJ complex shared the same transport mechanism and exhibited 2.4- to 10.2-fold higher affinity than apoJ itself. Binding to microvessels, transport into brain parenchyma, and choroidal uptake of both apoJ and sA beta (1-40)-apoJ complexes were markedly inhibited (74-99%) in the presence of a monoclonal antibody to gp330/megalin and were virtually abolished by perfusion with the receptor-associated protein, which blocks binding of all known ligands to gp330. Western blot analysis of cerebral microvessels with the monoclonal antibody to gp330 revealed a protein with a mass identical to that in extracts of kidney membranes enriched with gp330/megalin, but in much lower concentration. The findings suggest that gp330/megalin mediates cellular uptake and transport of apoJ and sA beta (1-40)-apoJ complex at the cerebral vascular endothelium and choroid epithelium.

Metabolism of Alzheimer beta-amyloid precursor protein: regulation by protein kinase A in intact cells and in a cell-free system.

Various compounds that affect signal transduction regulate the relative utilization of alternative processing pathways for the beta-amyloid precursor protein (beta APP) in intact cells, increasing the production of nonamyloidogenic soluble beta APP (s beta APP) and decreasing that of amyloidogenic beta-amyloid peptide. In a recent study directed toward elucidating the mechanisms underlying phorbol ester-stimulated s beta APP secretion from cells, it was demonstrated that protein kinase C increases the formation from the trans-Golgi network (TGN) of beta APP-containing secretory vesicles. Here we present evidence that forskolin increases s beta APP production from intact PC12 cells, and protein kinase A stimulates formation from the TGN of beta APP-containing vesicles. Although protein kinase A and protein kinase C converge at the level of formation from the TGN of beta APP-containing vesicles, additional evidence indicates that the regulatory mechanisms involved are distinct.

The production of an amyloidogenic metabolite of the Alzheimer amyloid beta precursor protein (APP) in thyroid cells is stimulated by interleukin 1 beta, but inhibited by interferon gamma.

Thyroid epithelial cells have been shown to have a high APP expression and to produce large amounts of its metabolic derivatives, namely secreted APPs and a potentially amyloidogenic 41-kDa C-terminal fragment. It was the aim of the present study to analyze how APP production and metabolism were regulated in human thyroid cells. The effects of three cytokines, interferon gamma (IFN gamma), interleukin 1beta (IL-1 beta) and transforming growth factor (TGF) beta, were investigated. Cell extracts and supernatants were studied by immunoblotting using specific N- and C-terminal APP antibodies. Quantification was performed by densitometric scanning. We demonstrate that IFN gamma has a strong suppressive effect on the production and metabolism of APP. From a concentration of 30 U/ml upwards it reduces the cellular APP content, decreases the amounts of secreted APPs and inhibits the generation of the 41-kDa amyloidogenic APP fragment. In contrast, IL-1 beta has a stimulatory influence on the generation of the amyloidogenic 41-kDa APP metabolite, but does not affect the cellular holoprotein or APPs. TGFbeta has no significant effect on APP. Our results demonstrate that cytokines can regulate APP production and metabolism in thyroid cells. IFN gamma is the first naturally occurring agent described to inhibit the generation of amyloidogenic APP fragments. It may be of relevance in preventing amyloid deposition during inflammatory processes in the thyroid gland, but may exert a similar protective effect in other non-neuronal and neuronal tissues.

The production of an amyloidogenic metabolite of the Alzheimer amyloid beta precursor protein (APP) in thyroid cells is stimulated by interleukin 1 beta, but inhibited by interferon gamma

The production of an amyloidogenic metabolite of the Alzheimer amyloid beta precursor protein (APP) in thyroid cells is stimulated by interleukin 1 beta, but inhibited by interferon gamma

Zn2+ interaction with Alzheimer amyloid beta protein calcium channels.

The Alzheimer disease 40-residue amyloid beta protein (AbetaP[1-40]) forms cation-selective channels across acidic phospholipid bilayer membranes with spontaneous transitions over a wide range of conductances ranging from 40 to 4000 pS. Zn2+ has been reported to bind to AbetaP[1-40] with high affinity, and it has been implicated in the formation of amyloid plaques. We now report the functional consequences of such Zn2+ binding for the AbetaP[1-40] channel. Provided the AbetaP[1-40] channel is expressed in the low conductance (<400 pS) mode, Zn2+ blocks the open channel in a dose- dependent manner. For AbetaP[1-40] channels in the giant conductance mode (>400 pS), Zn2+ doses in the millimolar range were required to exert substantial blockade. The Zn2+ chelator o-phenanthroline reverses the blockade. We also found that Zn2+ modulates AbetaP[1-40] channel gating and conductance only from one side of the channel. These data are consistent with predictions of our recent molecular modeling studies on AbetaP[1-40] channels indicating asymmetric Zn(2+)-AbetaP[1-40] interactions at the entrance to the pore.

Monoclonal antibodies inhibit in vitro fibrillar aggregation of the Alzheimer beta-amyloid peptide.

The beta-amyloid peptide, the hallmark of Alzheimer disease, forms fibrillar toxic aggregates in brain tissue that can be dissolved only by strong denaturing agents. To study beta-amyloid formation and its inhibition, we prepared immune complexes with two monoclonal antibodies (mAbs), AMY-33 and 6F/3D, raised against beta-amyloid fragments spanning amino acid residues 1-28 and 8-17 of the beta-amyloid peptide chain, respectively. In vitro aggregation of beta-amyloid peptide was induced by incubation for 3 h at 37 degrees C and monitored by ELISA, negative staining electron microscopy, and fluorimetric studies. We found that the mAs prevent the aggregation of beta-amyloid peptide and that the inhibitory effect appears to be related to the localization of the antibody-binding sites and the nature of the aggregating agents. Preparation of mAbs against "aggregating epitopes," defined as sequences related to the sites where protein aggregation is initiated, may lead to the understanding and prevention of protein aggregation. The results of this study may provide a foundation for using mAbs in vivo to prevent the beta-amyloid peptide aggregation that is associated with Alzheimer disease.

589 Melatonin prevents oxidative damage and heat-shock protein induction in cultured cells exposed to the Alzheimer's amyloid beta protein (Aβ)

589 Melatonin prevents oxidative damage and heat-shock protein induction in cultured cells exposed to the Alzheimer's amyloid beta protein (Aβ)

In vitro growth of Alzheimer's disease beta-amyloid plaques displays first-order kinetics.

A salient pathological feature of Alzheimer's disease (AD) is the presence of amyloid plaques in the brains of affected patients. The plaques are predominantly composed of human beta-amyloid peptide (A beta). Although the aggregation of synthetic A beta has been extensively studied, the mechanism of AD plaque growth is poorly understood. In order to address this question, we used an in vitro model of plaque growth to determine if assembly or aggregation of A beta is required for deposition. Labeled A beta at physiological concentrations readily deposited onto both neuritic and diffuse plaques and cerebrovascular amyloid in unfixed AD brain tissue, whereas essentially no deposition was detected in tissue without performed amyloid. Using this in vitro model of plaque growth, the kinetics of A beta deposition onto plaques was examined in two independent but complementary systems. Intact sections of unfixed AD brain cortex (analyzed by autoradiographic densitometry) allowed definitive morphological analysis of the site of deposition, while homogenates of the same tissue (analyzed by radioisotope counting) allowed precise quantitation of deposition over a wide range of conditions. Essentially identical results were obtained for both systems. Growth of preexisting tissue plaques by deposition of A beta was found to follow first-order dependence on A beta concentration and exhibited a pH optimum of 7. In sharp contrast, A beta aggregation in the absence of template follows higher order kinetics and shows a pH optimum of 5. On the basis of criteria of kinetic order, pH dependence, and structure-activity relationships, we conclude that aggregation of A beta (template-independent initial nidus formation) and deposition of A beta (template-dependent subsequent plaque growth) are fundamentally distinct biochemical processes. The process of plaque growth and maturation by A beta deposition may be an important target for therapeutic intervention to block the progression of AD.

331 Apolipoprotein E4 modulate cholinergic activity in Alzheimer's disease and beta-amyloid 1–40 and 25–35 modulate apolipoprotein E metabolism in primary neuronal cell culture

331 Apolipoprotein E4 modulate cholinergic activity in Alzheimer's disease and beta-amyloid 1–40 and 25–35 modulate apolipoprotein E metabolism in primary neuronal cell culture

Activation of K+ channels and suppression of neuronal activity by secreted beta-amyloid-precursor protein.

The Alzheimer's beta-amyloid precursor protein (beta-APP) is widely expressed in neural cells, and in neurons secreted forms of beta-APP (sAPPs) are released from membrane-spanning holo-beta APP in an activity-dependent manner. Secreted APPs can modulate neurite outgrowth, synaptogenesis, synaptic plasticity and cell survival; a signal transduction mechanism of sAPPs may involve modulation of intracellular calcium levels ([Ca2+]i). Here we use whole-cell perforated patch and single-channel patch-clamp analysis of hippocampal neurons to demonstrate that sAPPs suppress action potentials and hyperpolarize neurons by activating high-conductance, charybdotoxin-sensitive K+ channels. Activation of K+ channels by sAPPs was mimicked by a cyclic GMP analogue and sodium nitroprusside and blocked by an antagonist of cGMP-dependent kinase and a phosphatase inhibitor, suggesting that the effect is mediated by cGMP and protein dephosphorylation. Calcium imaging studies indicate that activation of K+ channels mediates the ability of sAPPs to decrease [Ca2+]i. Modulation of neuronal excitability may be a major mechanism by which beta-APP regulates developmental and synaptic plasticity in the nervous system.

Heat shock factor-1 mediates the transcriptional activation of Alzheimer's beta-amyloid precursor protein gene in response to stress.

Stress may be involved in the pathogenesis of Alzheimer's disease. There is a heat shock element located at position -317 bp on the beta-amyloid precursor protein (beta-APP) gene promoter. Recently we demonstrated [4] that stress, in the form of heat shock, ethanol and sodium arsenite treatment, transcriptionally activates the beta-APP gene. In this report we demonstrate that the nuclear factor that mediates this activation is heat shock factor-1.

Differential Effects of a Rab6 Mutant on Secretory Versus Amyloidogenic Processing of Alzheimer's β-Amyloid Precursor Protein

The Ras-related GTP-binding protein, Rab6, is localized in late Golgi compartments where it mediates intra-Golgi vesicular trafficking. Herein we report that coexpression of Alzheimer's beta-amyloid precursor protein (beta APP751) with a dominant-negative Rab6 mutant (Rab6N126I) in human embryonal kidney 293 cells causes an increase in secretion of the soluble amino-terminal exodomain (s-APP alpha) derived from non-amyloidogenic processing of beta-APP751 by alpha-secretase. The effect was specific to Rab6N126I, since the corresponding mutation in Rab8 (i.e. Rab8N121I), which has been implicated in protein transport to the plasma membrane, caused a modest reduction in s-APP alpha secretion. While Rab6N126I stimulated secretion of APP alpha, the accumulation of amyloid beta peptide (A beta) in the medium was either moderately reduced or unaffected. Similar differential effects of Rab6N126I on secretion of s-APP alpha versus A beta were observed in cell cultures that were overproducing A beta after transfection with a plasmid encoding Swedish variant of beta APP751. Moreover, assays of medium from the latter cultures revealed a marked increase in secretion of s-APP alpha relative to s-APP beta (the immediate product derived from cleavage of beta APP by beta-secretase). The results indicate that vesicular transport events controlled by Rab6 occur at or near a critical juncture in the trans-Golgi network where beta APP is sorted into either the constitutive alpha-secretase pathway or the amyloidogenic beta-secretase pathway.

Loss of vessel wall viability in cerebral amyloid angiopathy.

Cerebral amyloid angiopathy (CAA) is a neuropathological feature of Alzeheimer's disease and an important cause of cerebral haemorrhage in the elderly. CAA is characterized by the deposition of Alzheimer amyloid beta protein (A beta) in cerebral and leptomeningeal vessel walls. In order to study the effect of cerebrovascular A beta deposits in vivo, living canine leptomeninges obtained from old dogs affected by CAA were analysed by confocal laser scanning microscopy after immunofluorescence staining for A beta and viability staining with fluorescein diacetate (FDA). Simultaneous detection of the two signals showed a segmental loss of leptomeningeal vessel wall viability at some site of A beta deposition. Many of the non-viable vessels segments were also dilated, suggesting that A beta-induced vascular cell death creates the loci minores resistentiae for the development of cerebral haemorrhage in CAA.

Systemic overexpression of a C-terminal fragment of human amyloid beta-protein precursor causes accumulation of Alzheimer beta-amyloid fibrils in pancreas of transgenic mice.

The deposition of amyloid beta-protein (A beta), derived from amyloid beta-protein precursor (beta APP), is a specific and early event in development of Alzheimer's disease. Transgenic mice carrying the carboxyl-terminus of beta APP gene linked to the cytomegalovirus enhancer/chicken beta-actin promoter sequence were studied. Deposition of amyloid fibrils, composing A beta, was observed in the transgenic pancreas, accompanied with cell degeneration. This result will provide a model to investigate the beta APP processing mechanism in vivo and a clue to generate possible A beta amyloidosis in animal brains.

Dehydroepiandrosterone augments M1-muscarinic receptor-stimulated amyloid precursor protein secretion in desensitized PC12M1 cells.

Epidemiologic studies suggest that the age-related decline in dehydroepiandrosterone (DHEA) levels may be associated with Alzheimer's disease (AD). Cholinergic markers also decline with age, and are associated with AD pathology. Activation of m1AChR-transfected PC12 cells (PC12M1) with cholinergic agonists results in secretion of Alzheimer's beta-amyloid precursor protein (APP) which in turn reduces beta-amyloid production. This study examined whether DHEA affects APP processing in m1AChR-transfected PC12 cells. DHEA treatment did not significantly alter basal or m1AChR-stimulated APP secretion. However, DHEA (0.1 microM) significantly diminished the desensitization of APP secretion in cells exposed to carbachol for 24 h. The effect of DHEA on APP processing is probably not related to up-regulation of m1AChR or increased m1AChR-activated phosphoinositide hydrolysis since these parameters did not change following DHEA treatment. These findings imply a possible involvement of DHEA in APP processing. Thus, the age-associated decline in DHEA levels may contribute to decreased APP secretion and a consecutive increase in beta-amyloid deposition, which in turn may play a role in the development of AD.

Thyroid epithelial cells produce large amounts of the Alzheimer beta-amyloid precursor protein (APP) and generate potentially amyloidogenic APP fragments.

The Alzheimer beta-amyloid precursor protein (APP) is a transmembrane glycoprotein from which the amyloid beta-protein is proteolytically derived. The latter is a hydrophobic peptide that can aggregate and forms the core of the senile plaques found in the brains of patients suffering from Alzheimer's disease (AD). In view of the known association between familial AD and thyroid autoimmune disease, the expression pattern and cellular processing of APP in human thyroid cells were investigated. Cultured thyroid epithelial cells and homogenized thyroid tissue from normal and pathological thyroid samples were analyzed by immunoblotting using specific N- and C-terminal APP antibodies as well as by reverse transcription-polymerase chain reaction in which two sets of oligonucleotide primers were used. The results of these studies demonstrated that APP isoforms 770 and 751 were expressed in fresh thyroid extracts as well as in cultured thyroid epithelial cells, with APP 770 being the predominant form. Compared to other types of cells, such as lymphocytes and fibroblasts, thyroid epithelial cells produced larger amounts of APP. Most of the mature protein was cleaved within the amyloid beta region, as a result of which a large N-terminal APP fragment was released into the culture medium, whereas a C-terminal nonamyloidogenic fragment of 14 kilodaltons (kDa) was retained within the cell. Interestingly, thyroid epithelial cells also contained larger C-terminal APP fragments of 21, 35, and 41 kDa. From the sizes of these fragments it could be deduced that they contained the entire amyloid beta sequence and were thus potentially amyloidogenic. The 41-kDa fragment was unique to thyroid cells. These fragments may be released into the circulation after thyroid cell damage. Increased/altered thyroid APP expression in familiar AD may induce alterations in thyroid epithelial cells and cell damage, and thus explain the frequent occurrence of thyroid autoimmunity in this disease.

The secreted form of the Alzheimer's beta-amyloid precursor protein stimulates a membrane-associated guanylate cyclase.

We previously demonstrated that secreted forms of the Alzheimer's beta-amyloid precursor protein (sAPP) elevate cyclic GMP (cGMP) in primary neuronal cultures and that this effect is responsible for the modulation of neuronal calcium homoeostasis by sAPP. We have investigated further the mechanism by which sAPP elevates cGMP. Inhibition of the formation of nitric oxide or carbon monoxide did not affect the ability of sAPP to lower rapidly intraneuronal calcium levels or elevate cGMP, suggesting that sAPP does not activate a soluble (cytosolic) guanylate cyclase. A dose-dependent stimulation of cGMP formation by sAPP was observed in brain membrane preparations. The stimulation was also dependent on the presence of ATP. These data suggest that sAPP activates a membrane-associated guanylate cyclase, perhaps similar to those present in the receptors for the natriuretic peptides and sperm motility factors.

Regulated Formation of Golgi Secretory Vesicles Containing Alzheimer β-Amyloid Precursor Protein

Phorbol esters, activators of protein kinase C (PKC), regulate the relative utilization of alternative processing pathways for the Alzheimer beta-amyloid precursor protein (beta-APP) in intact cells, increasing the production of nonamyloidogenic soluble beta-APP (s beta-APP) and decreasing that of neurotoxic beta-amyloid (A beta) peptide. The molecular and cellular bases of PKC-regulated beta-APP cleavage are poorly understood. Here we demonstrate in a reconstituted cell-free system that activation of endogenous PKC increases formation from the trans-Golgi network of secretory vesicles containing beta-APP and that this effect can be mimicked by purified PKC. The results demonstrate directly that PKC is involved in regulation of secretory vesicle formation and provide a mechanism by which PKC may reduce the formation of the A beta peptide characteristic of Alzheimer disease.