Alzheimer's Amyloid Beta Protein Research Articles

Intracellular calcium changes in neuronal cells induced by Alzheimer's beta-amyloid protein are blocked by estradiol and cholesterol.

1. The elevation of intracellular Ca2+ levels ([Ca2+]i) in immortalized hypothalamic neurons (GT1-7 cells) after exposure to Alzheimer's beta-amyloid protein (AbetaP[25-35]) was investigated using a multisite fluorometry system. 2. The marked rise in [Ca2+]i appeared after exposure to 5-20-microM AbetaP[25-35]. Analysis of the spatiotemporal patterns of [Ca2+]i changes revealed that the magnitude and the latency of the response to AbetaP in each cell were highly heterogeneous. 3. The preadministration of 17beta-estradiol, 17alpha-estradiol, phloretin and cholesterol, which influence the properties of membranes, such as membrane fluidity or membrane potential, significantly decreased the rise in [Ca2+]i. 4. These findings support the idea that disruption of calcium homeostasis by AbetaP channels may be the molecular basis of the neurotoxicity of AbetaP and of the pathogenesis of Alzheimer's disease. It is also suggested that membrane properties may play key roles in the expression of neurotoxicity.

Prevention of the neurotoxicity of the amyloid beta protein by genipin.

Genipin, which was shown in our previous investigation to have prominent neuritogenic activity in paraneurons such as PC12h cells, was studied to determine whether it could prevent the toxicity of Alzheimer's amyloid beta protein (Abeta) in cultured hippocampal neurons. Increased release of lactate dehydrogenase from hippocampal neurons after 2 d of Abeta25-35 administration was prevented dose dependently by the addition of genipin 20-40 microm. Morphological observations and trypan blue staining of cells confirmed the protection of hippocampal neurons from Abeta toxicity by genipin. Geniposide had less effect in preventing Abeta toxicity.

Electron microscopy and X-ray diffraction studies further confirm the efficacy of PTI-000703 ™ (Cat's claw derivative) as a potent inhibitor of Alzheimer's beta-amyloid protein fibrillogenesis

Electron microscopy and X-ray diffraction studies further confirm the efficacy of PTI-000703 ™ (Cat's claw derivative) as a potent inhibitor of Alzheimer's beta-amyloid protein fibrillogenesis

Alzheimer's β-Amyloid, Human Islet Amylin, and Prion Protein Fragment Evoke Intracellular Free Calcium Elevations by a Common Mechanism in a Hypothalamic GnRH Neuronal Cell Line

A growing number of reports suggest that elevated levels of extracellular Alzheimer's beta-amyloid protein alter the homeostasis of free [Ca(2+)](i) in different cell types of the mammalian brain. In line with these results, we have previously shown that AbetaP[1-40] forms cation-selective channels (Ca(2+) included) across artificial planar bilayers formed from acidic phospholipids and across excised membrane patches from immortalized hypothalamic GnRH neurons (GT1-7 cells), suggesting that the nonregulated Ca(2+)-influx through these spontaneously formed "amyloid channels" may provide a mechanism to explain its toxicity (1). We have now found and report here that the application of AbetaP[1-40] to GT1-7 neurons consistently elevates [Ca(2+)](i) levels. We also found that human islet amylin and the prion protein fragment (PrP106-126), peptides that acquire beta-pleated sheet conformation in water solutions and have been reported to form ion channels across planar bilayer membranes, also increase cytosolic free calcium in GT1-7 neurons. Searching for protective agents, we found that soluble cholesterol, known to decrease the fluidity of the cell membrane, inhibits AbetaP[1-40]-evoked [Ca(2+)](i) rise. These results suggest that unregulated Ca(2+) entry across amyloid channels may be a common mechanism causing cell death, not only in diseases of the third age, including Alzheimer's disease and type 2 diabetes mellitus, but also in prion-induced diseases.

Influence of lipoproteins on microglial degradation of Alzheimer's amyloid beta-protein.

Amyloid beta-protein (Abeta), the major component of plaques in Alzheimer's disease, is a small hydrophobic protein that is carried on apolipoprotein E (ApoE)- and ApoJ-containing lipoprotein particles in plasma and cerebrospinal fluid (CSF). Microglia, the scavenger cells of the CNS, take up and degrade Abeta via lipoprotein receptors including scavenger receptors A and B, and possibly via other receptors. Lipoproteins, ApoE, and ApoJ influence the uptake and degradation of Abeta in vitro and in vivo. Differences in ApoE-E4, -E3, and -E2 isoforms with respect to Abeta binding to lipoproteins and delivery to cells, including microglia, may contribute to the increased risk of Alzheimer's disease for people with an APOE4 genotype and to risk reduction with APOE2.

L-glutamate suppresses amyloid beta-protein-induced stellation of cultured rat cortical astrocytes.

Alzheimer's amyloid beta-protein (Abeta) has been reported to potentiate glutamate toxicity in neurons, but very little is known about interaction between Abeta and glutamate in astrocytes. Therefore, in the present study, we investigated the effects of Abeta and glutamate on morphology of astrocytes. Cultured rat cortical astrocytes exhibited polygonal morphology in the absence of stimulation and differentiated into process-bearing stellate cells following exposure to Abeta (20 microM). L-Glutamate (30-1,000 microM) had no effect on astrocyte morphology in the absence of stimulation but strongly suppressed Abeta-induced stellation. The suppressive effect of L-glutamate on Abeta-induced stellation was not mimicked by glutamate receptor agonists and not blocked by glutamate receptor antagonists. In contrast, the suppressive effect of L-glutamate was mimicked by D- and L-aspartate and transportable glutamate uptake inhibitors. These results suggest that Abeta-induced astrocyte stellation is suppressed by a mechanism related to glutamate transporters.

Alzheimer's amyloid β interaction with normal human plasma high density lipoprotein: association with apolipoprotein and lipids

We report studies of the interaction of Alzheimer's amyloid beta protein (Aβ) with normal human plasma high density lipoprotein (HDL), aiming to clarify to which lipoprotein (LP) structural constituent (apolipoprotein or lipid) soluble Aβ is primarily bound. Purified HDLs were incubated with biotinylated Aβ1–40 followed by LP repurification by size exclusion (SE) HPLC. SDS–PAGE, immunoblot and N-terminal sequence analysis of the biotin–Aβ positive protein bands revealed that Aβ is bound to many apolipoproteins of the HDL, mainly apoA-I, apoA-II, apoE and apoJ. On the other hand, reconstituted, protein-free HDL lipid particles also bind Aβ peptide and inhibit its aggregation, as intact HDL does. This was assessed by SE–HPLC, SDS–PAGE, immunoblot analysis, ultrastructural electron microscopy and Congo Red staining for β amyloid fibrils. Our data imply that Aβ binding to lipids may play an important role in maintaining the peptide in solution and thus be particularly relevant to Aβ normal and pathologic biochemistry and physiology.

Crystal structures of bovine chymotrypsin and trypsin complexed to the inhibitor domain of Alzheimer's amyloid beta-protein precursor (APPI) and basic pancreatic trypsin inhibitor (BPTI): engineering of inhibitors with altered specificities.

The crystal structures of the inhibitor domain of Alzheimer's amyloid beta-protein precursor (APPI) complexed to bovine chymotrypsin (C-APPI) and trypsin (T-APPI) and basic pancreatic trypsin inhibitor (BPTI) bound to chymotrypsin (C-BPTI) have been solved and analyzed at 2.1 A, 1.8 A, and 2.6 A resolution, respectively. APPI and BPTI belong to the Kunitz family of inhibitors, which is characterized by a distinctive tertiary fold with three conserved disulfide bonds. At the specificity-determining site of these inhibitors (P1), residue 15(I)4 is an arginine in APPI and a lysine in BPTI, residue types that are counter to the chymotryptic hydrophobic specificity. In the chymotrypsin complexes, the Arg and Lys P1 side chains of the inhibitors adopt conformations that bend away from the bottom of the binding pocket to interact productively with elements of the binding pocket other than those observed for specificity-matched P1 side chains. The stereochemistry of the nucleophilic hydroxyl of Ser 195 in chymotrypsin relative to the scissile P1 bond of the inhibitors is identical to that observed for these groups in the trypsin-APPI complex, where Arg 15(I) is an optimal side chain for tryptic specificity. To further evaluate the diversity of sequences that can be accommodated by one of these inhibitors, APPI, we used phage display to randomly mutate residues 11, 13, 15, 17, and 19, which are major binding determinants. Inhibitors variants were selected that bound to either trypsin or chymotrypsin. As expected, trypsin specificity was principally directed by having a basic side chain at P1 (position 15); however, the P1 residues that were selected for chymotrypsin binding were His and Asn, rather than the expected large hydrophobic types. This can be rationalized by modeling these hydrophilic side chains to have similar H-bonding interactions to those observed in the structures of the described complexes. The specificity, or lack thereof, for the other individual subsites is discussed in the context of the "allowed" residues determined from a phage display mutagenesis selection experiment.

Pharmacological modulation of Alzheimer's beta-amyloid precursor protein levels in the CSF of rats with forebrain cholinergic system lesions.

Abnormal deposition and accumulation of Alzheimer's amyloid beta-protein (A beta) and degeneration of forebrain cholinergic neurons are among the principal features of Alzheimer's disease. Studies in rat model systems have shown that forebrain cholinergic deficits are accompanied by induction of cortical beta-amyloid precursor protein (beta-APP) mRNAs and increased levels of secreted beta-APP in the CSF. The studies reported here determined whether the CSF levels of secreted beta-APP could be altered pharmacologically. In different experiments, rats with lesions of the forebrain cholinergic system received injections of vehicle, a muscarinic receptor antagonist scopolamine, or one of two cholinesterase inhibitors - diisopropyl phosphorofluoridate (DFP) or phenserine. Scopolamine was administered to determine whether the levels of beta-APP in the CSF could be increased by anticholinergic agents. The cholinesterase inhibitors were administered to determine whether the forebrain cholinergic system lesion-induced increases in CSF beta-APP could be reduced by cholinergic augmentation. Scopolamine administration led to a significant increase in the CSF levels of secreted beta-APP in sham-lesioned rats. Phenserine, a novel, reversible acetyl-selective cholinesterase inhibitor, significantly decreased the levels of secreted beta-APP in the CSF of forebrain cholinergic system-lesioned rats whereas DFP, a relatively non-specific cholinesterase inhibitor, failed to affect CSF levels of secreted beta-APP. These results suggest that the levels of secreted beta-APP in the CSF can be pharmacologically modulated but that this modulation is dependent upon the status of the forebrain cholinergic system and the pharmacological properties of the drugs used to influence it.

Nanomolar amyloid beta protein-induced inhibition of cellular redox activity in cultured astrocytes.

It has been previously reported that Alzheimer's amyloid beta protein (A beta) induces reactive astrocytosis in culture. In the present study, we found that A beta potently inhibits cellular redox activity of cultured astrocytes, as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay. The following comparative studies revealed several differences between these two actions of A beta on astrocytes. First, A beta-induced reactive morphological change was suppressed by the presence of serum or thrombin, and A beta inhibition of cellular redox activity was observed in either the presence or the absence of serum. Second, micromolar concentrations (10 microM or more) were required for A beta to induce reactive astrocytosis, whereas nanomolar concentrations (0.1-100 nM) were sufficient to inhibit cellular redox activity. Third, the effect of micromolar A beta was virtually irreversible, but nanomolar A beta-induced inhibition of cellular redox activity was reversed by washing out A beta. Furthermore, as it has been reported that A beta neurotoxicity is mediated by reactive oxygen species, we also examined if similar mechanisms are involved in astrocytic response to A beta. However, neither A beta-induced morphological change nor inhibition of redox activity was blocked by antioxidants, suggesting that these effects are not caused by oxidative stress.

Generation of Alzheimer beta-amyloid protein in the trans-Golgi network in the apparent absence of vesicle formation.

beta-amyloid protein (A beta) formation was reconstituted in permeabilized neuroblastoma cells expressing human Alzheimer beta-amyloid precursor protein (beta APP) harboring the Swedish double mutation associated with familial early-onset Alzheimer disease. Permeabilized cells were prepared following metabolic labeling and incubation at 20 degrees C, a temperature that allows beta APP to accumulate in the trans-Golgi network (TGN) without concomitant A beta formation. Subsequent incubation at 37 degrees C led to the generation of A beta. A beta production in the TGN persisted even under conditions in which formation of nascent post-TGN vesicles was inhibited by addition of guanosine 5'-O-(3-thiotriphosphate), a nonhydrolyzable GTP analogue, or by omission of cytosol. These and other results indicate that vesicle budding and trafficking may not be required for proteolytic metabolism of beta APP to A beta, a process that includes "gamma-secretase" cleavage within the beta APP transmembrane domain.

Degradation of Alzheimer's beta-amyloid protein by human and rat brain peptidases: involvement of insulin-degrading enzyme.

We examined the degradation of Alzheimer's beta-amyloid protein (1-40) by soluble and synaptic membrane fractions from post mortem human and fresh rat brain using HPLC. Most of the activity at neutral pH was in the soluble fraction. The activity was thiol and metal dependent, with a similar inhibition profile to insulin-degrading enzyme. Immunoprecipitation of insulin-degrading enzyme from the human soluble fraction using a monoclonal antibody removed over 85% of the beta-amyloid protein degrading activity. Thus insulin-degrading enzyme is the main soluble beta-amyloid degrading enzyme at neutral pH in human brain. The highest beta-amyloid protein degrading activity in the soluble fractions occurred between pH 4-5, and this activity was inhibited by pepstatin, implicating an aspartyl protease. Synaptic membranes had much lower beta-amyloid protein degrading activity than the soluble fraction. EDTA (2mM) caused over 85% inhibition of the degrading activity but inhibitors of endopeptidases -24.11, -24.15, -24.16, angiotensin converting enzyme, aminopeptidases, and carboxypeptidases had little or no effect.

Ion channel formation of Alzheimer's beta amyloid protein and neurodegeneration

Ion channel formation of Alzheimer's beta amyloid protein and neurodegeneration

105 Ion channel formation of Alzheimer's beta amyloid protein and neurodegeneration

105 Ion channel formation of Alzheimer's beta amyloid protein and neurodegeneration

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).

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.

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)

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.

A new compound (AZ36041) promotes the survival of the neurons and reduces neurotoxicity of Alzheimer's beta-amyloid protein.

Alzheimer's beta-amyloid protein (A beta) is the main component of senile plaques, which are characteristic hallmarks of the Alzheimer's disease brain. Recently, there have been several reports that A beta has toxic effects on both cultured neurons and in the brain. We confirmed the neurotoxicity of A beta in vitro and found a new compound, called AZ36041 (4-chloro-N-(5-nitro-2-tiazoyl)benzenesulfone amide), which dramatically reduced A beta neurotoxicity. This compound was also found to have a neuroprotective effect against toxicity of glutamate and enhanced neuronal survival in the absence of neurotoxic compounds. AZ36041 may be a useful tool for investigating the mechanism of A beta neurotoxicity in vitro and in vivo.

Kunitz domain inhibitors of tissue factor-factor VIIa. II. Potent and specific inhibitors by competitive phage selection.

Active-site inhibitors of the human tissue factor-Factor VIIa complex (TF.FVIIa) that are both potent and specific have been selected from Alzheimer's amyloid beta-protein precursor inhibitor (APPI) Kunitz domain libraries using a strategy termed competitive phage selection. Phage display Libraries I-III, previously sorted by direct selection, were sorted by competitive selection against immobilized TF.FVIIa in which increasing amounts of Factor XIa (FXIa) were added to the selection buffer to remove phage with high affinity for FXIa. The most striking difference in the selected Kunitz domain sequences was at the P4' position where Lys was highly preferred instead of Leu which was found here by direct selection (Dennis, M. S., and Lazarus, R. A. (1994) J. Biol. Chem. 269, 22129-22136). In addition, both Lys and Arg were selected in the P1 position as opposed to Arg alone. A new library (Library IV) was constructed which contained all previously observed amino acids at 9 positions in the primary and secondary binding loops of APPI. Comparable results were obtained by sorting Library IV against immobilized TF.FVIIa in the presence of either FXIa alone or FXIa, plasma kallikrein, and plasmin. The Kunitz domains obtained by either competitive selection strategy potently inhibited TF.FVIIa, with Ki* values ranging from about 2 to 20 nM; the Ki* values were generally > 1 microM for FXIa and plasma kallikrein and ranged from 4 to 200 nM for plasmin. Variants selective for TF.FVIIa were assayed for free FVIIa and FXa inhibitory activity and characterized in coagulation assays. A rationale for the selection of Lys at both the P4' and P1 positions based upon comparison of sequences and structures of relevant serine proteases and inhibitors is presented.