Evelyn R. Stimson

Aluminum, Iron, and Zinc Ions Promote Aggregation of Physiological Concentrations of β‐Amyloid Peptide

Abstract: A major pathological feature of Alzheimers disease (AD) is the presence of a high density of amyloid plaques in the brain tissue of patients. The plaques are predominantly composed of human β‐amyloid peptide βA4, a 40‐mer whose neurotoxicity is related to its aggregation. Certain metals have been proposed as risk factors for AD, but the mechanism by which the metals may exert their effects is unclear. Radioiodinated human βA4 has been used to assess the effects of various metals on the aggregation of the peptide in dilute solution (10‐10M). In physiological buffers, 10‐3M calcium, cobalt, copper, manganese, magnesium, sodium, or potassium had no effect on the rate of βA4 aggregation. In sharp contrast, aluminum, iron, and zinc under the same conditions strongly promoted aggregation (rate enhancement of 100–1,000‐fold). The aggregation of βA4 induced by aluminum and iron is distinguishable from that induced by zinc in terms of rate, extent, pH and temperature dependence. These results suggest that high concentrations of certain metals may play a role in the pathogenesis of AD by promoting aggregation of βA4.

Amyloid beta-peptide is transported on lipoproteins and albumin in human plasma.

The amyloid β-peptide (Aβ) is the major constituent of neuritic plaques in Alzheimers disease and occurs as a soluble 40-42-residue peptide in cerebrospinal fluid and blood of both normal and AD subjects. It is unclear whether Aβ, once it is secreted by cells, remains free in biological fluids or is associated with other proteins and thus transported and metabolized with them. Such knowledge of the normal fate of Aβ is a prerequisite for understanding the changes that may lead to the pathological aggregation of soluble Aβ in vivo, the possible influence of certain extracellular proteins, particularly apolipoprotein E, on plaque formation, and the pharmacology of putative Aβ-lowering drugs. To address the question of Aβ distribution in human biological fluids, we incubated fresh human plasma from 38 subjects with physiological concentrations (0.5-0.7 nM) of radioiodinated Aβ1-40 and seven plasma samples with Aβ1-42. Lipoproteins and lipid-free proteins were separated and analyzed for bound iodinated Aβ1-40. We found that up to 5% of Aβ added to plasma is bound to selected lipoproteins: very low density, low density, and high density, but not lipoprotein(a). The large majority (≈89%), however, is bound to albumin, and very little Aβ is free. Aβ distribution in plasma was not significantly influenced by apolipoprotein E genotype. We conclude that Aβ is normally bound to and transported by albumin and specific lipoproteins in human plasma under physiological conditions.

Point Substitution in the Central Hydrophobic Cluster of a Human β-Amyloid Congener Disrupts Peptide Folding and Abolishes Plaque Competence†

Alzheimers disease (AD) is pathologically characterized by the presence of numerous insoluble amyloid plaques in the brain composed primarily of a 40-43 amino acid peptide, the human beta-amyloid peptide (A beta). The process of A beta deposition can be modeled in vitro by deposition of physiological concentrations of radiolabeled A beta onto preexisting amyloid in preparations of unfixed AD cerebral cortex. Using this model system, it has been shown that A beta deposition is biochemically distinct from A beta aggregation and occurs readily at physiological A beta concentrations, but which regions and conformations of A beta are essential to A beta deposition is poorly understood. We report here that an active congener, A beta (10-35)-NH2, displays time dependence, pH-activity profile, and kinetic order of deposition similar to A beta (1-40), and is sufficiently soluble for NMR spectroscopy in water under conditions where it actively deposits. To examine the importance of the central hydrophobic cluster of A beta (LVFFA, residues 17-21) for in vitro A beta deposition, an A beta (10-35)-NH2 analog with a single point substitution (F19T) in this region was synthesized and examined. Unlike A beta (10-35)-NH2, the F19T analog was plaque growth incompetent, and NMR analysis indicated that the mutant peptide was significantly less folded than wild-type A beta. These results support previous studies suggesting that the plaque competence of A beta correlates with peptide folding. Since compounds that alter A beta folding may reduce amyloid deposition, the central hydrophobic cluster of A beta will be a tempting target for structure-based drug design when high-resolution structural information becomes available.

Zinc-Induced Aggregation of Human and Rat β-Amyloid Peptides In Vitro

Abstract: The major pathological feature of Alzheimers disease is the presence of a high density of amyloid plaques in the brain tissue of patients. The plaques are predominantly composed of human β‐amyloid peptide (Aβ), a 39–43‐mer peptide the neurotoxicity of which is related to its aggregation state. Previous work has demonstrated that certain metals that have been implicated as risk factors for Alzheimers disease (Al, Fe, and Zn) also cause substantial aggregation of Aβ. In particular, we reported that zinc cations at concentrations of >10−4M dramatically accelerate the rate of Aβ aggregation at physiological peptide concentrations at 37°C in vitro. In the present study, we investigate the effect of Zn2+ on aggregation of radiolabeled and unlabeled human and rat Aβ over a wide range of peptide concentrations in the presence and absence of salt and blocking protein. Aggregation was assayed by centrifugation and filtration using amino acid analysis, immunoassay, and γ‐counting for quantification over a wide range of concentrations of Zn2+ and Aβ above and below physiological values. The results of this study demonstrate the following: (a) Radio‐iodinated Aβ accurately tracked unlabeled Aβ, (b) zinc concentrations of at least 10−4M were required to induce significant aggregation of Aβ, and (c) rat and human Aβ species were cleared from aqueous solutions by similar concentrations of zinc. These results stand in significant quantitative disagreement (∼100‐fold in zinc concentration) with one previous study that reported significant aggregation of Aβ by <1 µM Zn2+. Differences between the present study and the latter study from another laboratory appear to result from inappropriate reliance on optical density to measure Aβ concentrations and nonspecific loss of Aβ to plastic in the absence of blocking protein.

Aβ deposition inhibitor screen using synthetic amyloid

The formation, growth, and maturation of brain amyloid “senile” plaques are essential pathological processes in Alzheimers disease (AD) and key targets for therapeutic intervention. The process of in vitro deposition of Aβ at physiological concentrations onto plaques in AD brain preparations has been well characterized, but is cumbersome for drug discovery. We describe here a high-throughput screen for inhibitors of Aβ deposition onto a synthetic template (synthaloid) of fibrillar Aβ immobilized in a polymer matrix. Synthaloid is indistinguishable from plaques in AD brain (the natural template) in deposition kinetics, pH profile, and structure-activity relationships for both Aβ analogs and inhibitors. Synthaloid, in contrast to current Aβ aggregation screens, accurately predicted inhibitor potency for Aβ deposition onto AD cortex preparations, validating its use in searching for agents that can slow the progression of AD and exposing a previously inaccessible target for drug discovery.

Local Anesthetics Inhibit Substance P Binding and Evoked Increases in Intracellular Calcium sup 2

Background During spinal and epidural anesthesia, local anesthetics reach concentrations in cerebrospinal fluid and spinal cord tissues at which their actions may extend beyond the classic blockade of sodium channels. This study examines the effects of several clinical and experimental local anesthetics on the binding and actions of a peptide neurotransmitter, substance P, known to be important in nociceptive transmission in the dorsal horn. Methods The binding of radiolabeled (Bolton‐Hunter modified) substance P was studied in chick brain membranes in the presence of local anesthetics. The increase in intracellular calcium [Ca2+]in evoked by substance P was measured by the fluorescent indicator fura‐2 loaded in a murine cell line expressing substance P (NK1) receptors. Cells were preincubated with bupivacaine before and during the transient addition of substance P. Results Both substance P binding and Calcium2+ increase were inhibited half‐maximally by approximately 1 mM bupivacaine at pH 7.5, whereas tetracaine, lidocaine, and benzocaine were slightly less potent at inhibiting binding. Concentration‐dependent substance P‐binding studies showed that bupivacaines inhibition was not competitive. Inhibition of substance P binding by bupivacaine increased with increasing pH, but the protonated species appears to have some inhibitory activity, and quaternary lidocaine also inhibited binding. There was no stereoselectivity to the binding inhibition. Conclusions Because millimolar concentrations of local anesthetics are within the range measured in spinal cord during intrathecal and epidural procedures, these results are consistent with a direct action of local anesthetics on tachykinin‐mediated neurotransmission during regional anesthesia.

Intra-arterial infusion of [125I]A beta 1-40 labels amyloid deposits in the aged primate brain in vivo.

Alzheimers disease is characterized by extracellular amyloid deposits in the brain at both vascular sites (cerebrovascular amyloid, CVA) and within the neuropil (plaques). In the present study we demonstrated that brain amyloid of aged non-human primates is efficiently detected by [125I]A beta in vitro, and assessed the detection of that amyloid in vivo by intravascular infusion of [125I]A beta. Aged squirrel monkeys (Saimiri sciureus) were anesthetized and infused intra-arterially with [125I]A beta, and sacrificed 2 h later. Analysis of the anterior frontal and temporal cortices by autoradiography demonstrated that [125I]A beta was deposited on CVA and that essentially every amyloid deposit which could be detected with thioflavin S or anti-A beta antibodies was also labeled by [125I]A beta. These experiments suggest that intravascular infusion of radiolabeled A beta can be used to detect and image amyloid deposits in the human AD brain.

Stereochemical specificity of Alzheimer's disease β-peptide assembly

The formation and growth of insoluble amyloid deposits composed primarily of the human β-amyloid peptide (Aβ) in brain is an essentially invariant feature of Alzheimers disease (AD) and is widely believed to contribute to the progressive neurodegeneration of the disorder. To probe the specificity of amyloid formation and growth, we synthesized and examined the self-assembly of D- and L-stereoisomers of Aβ in vitro. While both enantiomers formed insoluble aggregates at similar rates with amyloid-like fibrillar morphology, deposition of soluble Aβ peptide onto preexisting Aβ aggregates was stereospecific. Although the L-peptide deposited readily onto immobilized L-Aβ aggregates with first-order kinetic dependence on soluble peptide concentration, essentially no association between the D-peptide and L-template was observed. Similarly, the D-peptide deposited with first-order kinetics onto a D-Aβ aggregate template but did not deposit onto a similar template composed of aggregates of the L-enantiomer. Furthermore, although the L-Aβ isomer deposited onto authentic AD amyloid in preparations of unfixed AD brain, no focal association between the D-peptide and brain amyloid was detected. These results establish that deposition of soluble Aβ onto preexisting amyloid template is stereospecific, likely involving direct docking interactions between peptide backbone and/or side chains rather than simple hydrophobic association.

Local Anesthetics Inhibit Substance P Binding and Evoked Increases in Intracellular Ca2

BACKGROUND During spinal and epidural anesthesia, local anesthetics reach concentrations in cerebrospinal fluid and spinal cord tissues at which their actions may extend beyond the classic blockade of sodium channels. This study examines the effects of several clinical and experimental local anesthetics on the binding and actions of a peptide neurotransmitter, substance P, known to be important in nociceptive transmission in the dorsal horn. METHODS The binding of radiolabeled (Bolton-Hunter modified) substance P was studied in chick brain membranes in the presence of local anesthetics. The increase in intracellular calcium [Ca2+]in evoked by substance P was measured by the fluorescent indicator fura-2 loaded in a murine cell line expressing substance P (NK1) receptors. Cells were preincubated with bupivacaine before and during the transient addition of substance P. RESULTS Both substance P binding and Ca2+ increase were inhibited half-maximally by approximately 1 mM bupivacaine at pH 7.5, whereas tetracaine, lidocaine, and benzocaine were slightly less potent at inhibiting binding. Concentration-dependent substance P-binding studies showed that bupivacaines inhibition was not competitive. Inhibition of substance P binding by bupivacaine increased with increasing pH, but the protonated species appears to have some inhibitory activity, and quaternary lidocaine also inhibited binding. There was no stereoselectively to the binding inhibition. CONCLUSIONS Because millimolar concentrations of local anesthetics are within the range measured in spinal cord during intrathecal and epidural procedures, these results are consistent with a direct action of local anesthetics on tachykinin-mediated neurotransmission during regional anesthesia.

Deposition of soluble amyloid-beta onto amyloid templates: with application for the identification of amyloid fibril extension inhibitors.

Publisher Summary Several human diseases are now recognized to be caused by the formation of insoluble amyloid from naturally occurring peptides and proteins. Amyloid-forming monomers are not intrinsically pathological; rather it is their conformation and assembly state that are most important to their cytotoxicity. Because the chemical information for forming these amyloids apparently lies in the primary structure of the monomers, there has been significant interest in using in vitro systems to study amyloid formation and growth. A β deposition, the process of amyloid growth by the association of individual soluble A β molecules with a preexisting amyloid template, can be modeled readily in vitro under physiologically relevant conditions and importantly at physiological (nM) A β concentrations. Such assays may elucidate key molecular interactions in amyloidosis and may be exploitable in the design of antiamyloid therapeutics. High-throughput A β deposition assays may provide a means for the identification and optimization of such compounds. The methods described in this chapter have been developed for the examination of A β deposition, but are adaptable for the study of other amyloids.