Paul A. Hyslop

Functional gamma‐secretase inhibitors reduce beta‐amyloid peptide levels in brain

Converging lines of evidence implicate the beta‐amyloid peptide (Aβ) as causative in Alzheimers disease. We describe a novel class of compounds that reduce Aβ production by functionally inhibiting γ‐secretase, the activity responsible for the carboxy‐terminal cleavage required for Aβ production. These molecules are active in both 293 HEK cells and neuronal cultures, and exert their effect upon Aβ production without affecting protein secretion, most notably in the secreted forms of the amyloid precursor protein (APP). Oral administration of one of these compounds, N‐[N‐(3,5‐difluorophenacetyl)‐l‐alanyl]‐S‐phenylglycine t‐butyl ester, to mice transgenic for human APPV717F reduces brain levels of Aβ in a dose‐dependent manner within 3 h. These studies represent the first demonstration of a reduction of brain Aβin vivo. Development of such novel functional γ‐secretase inhibitors will enable a clinical examination of the Aβ hypothesis that Aβ peptide drives the neuropathology observed in Alzheimers disease.

Measurement of striatal H2O2 by microdialysis following global forebrain ischemia and reperfusion in the rat : correlation with the cytotoxic potential of H2O2 in vitro

Toxic reactive oxygen species have been implicated as important mediators of tissue injury after reperfusion of ischemic organs. When rats are subject to 30 min global forebrain ischemia, 24 h following this insult, there is substantial loss of medium-sized neurones as revealed by histological sectioning of the striatal region of the forebrain. The goal of this study was to utilize microdialysis to directly measure one of the more stable intermediates of reduced molecular oxygen, H2O2 in the rat striatum following 4-vessel occlusion and reperfusion, and to correlate these levels with H2O2 toxicity to neurones grown in culture. A significant rise in striatal H2O2 levels was observed for about 1 h during reperfusion, amounting to an increase of approximately 100 microM at the peak. In control experiments where the dialysis probe was embedded in cortical regions surrounding the striatum (where there is no neuronal loss due to the ischemic episode), there was no measurable increase in tissue H2O2 levels. H2O2 has been previously shown to be neurotoxic to PC12 cells as well as rat primary hippocampal neurones at comparable concentrations striatal neurones experience during reperfusion. We demonstrate that H2O2 is also neurotoxic to the human cortical neuronal cell line, HCN-1A. These experiments establish an important link between oxidant generation and neuronal loss in this tissue following global forebrain ischemia.

RAGE potentiates Aβ‐induced perturbation of neuronal function in transgenic mice

Receptor for Advanced Glycation Endproducts (RAGE), a multiligand receptor in the immunoglobulin superfamily, functions as a signal‐transducing cell surface acceptor for amyloid‐beta peptide (Aβ). In view of increased neuronal expression of RAGE in Alzheimers disease, a murine model was developed to assess the impact of RAGE in an Aβ‐rich environment, employing transgenics (Tgs) with targeted neuronal overexpression of RAGE and mutant amyloid precursor protein (APP). Double Tgs (mutant APP (mAPP)/RAGE) displayed early abnormalities in spatial learning/memory, accompanied by altered activation of markers of synaptic plasticity and exaggerated neuropathologic findings, before such changes were found in mAPP mice. In contrast, Tg mice bearing a dominant‐negative RAGE construct targeted to neurons crossed with mAPP animals displayed preservation of spatial learning/memory and diminished neuropathologic changes. These data indicate that RAGE is a cofactor for Aβ‐induced neuronal perturbation in a model of Alzheimers‐type pathology, and suggest its potential as a therapeutic target to ameliorate cellular dysfunction.

α2-Macroglobulin as a β-amyloid peptide-binding plasma protein

Abstract: The β‐amyloid peptide (Aβ) is a normal proteolytic processing product of the amyloid precursor protein, which is constitutively expressed by many, if not most, cells. For reasons that are still unclear, Aβ is deposited in an aggregated fibrillar form in both diffuse and senile plaques in the brains of patients with Alzheimers disease (AD). The factor(s) responsible for the clearance of soluble Aβ from biological fluids or tissues are poorly understood. We now report that human α2‐macroglobulin (α2M), a major circulating endoproteinase inhibitor, which has recently been shown to be present in senile plaques in AD, binds 125I‐Aβ(1–42) with high affinity (apparent dissociation constant of 3.8 × 10−10M). Approximately 1 mol of Aβ is bound per mole of α2M. Both native and methylamine‐activated α2M bind 125I‐Aβ(1–42). The binding of 125I‐Aβ(1–42) to α2M is enhanced by micromolar concentrations of Zn2+ (but not Ca2+) and is inhibited by noniodinated Aβ(1–42) and Aβ(1–40) but not by the reverse peptide Aβ(40‐1) or the cytokines interleukin 1β or interleukin 2. α1‐Antichymotrypsin, another plaque‐associated protein, inhibits both the binding of 125I‐Aβ(1–42) to α2M as well as the degradation of 125I‐Aβ(1–42) by proteinase‐activated α2M. Moreover, the binding of 125I‐Aβ(1–42) to α2M protects the peptide from proteolysis by exogenous trypsin. These data suggest that α2M may function as a carrier protein for Aβ and could serve to either facilitate or impede clearance of Aβ from tissues such as the brain.

Hydrogen peroxide as a potent bacteriostatic antibiotic: Implications for host defense

Host defense against bacterial pathogens in higher organisms is mediated in part by the generation of reactive oxygen species (ROS) by PMN. In this study, we determined the following effects of exposure of constant concentrations of H2O2 on E. coli in a culture continuously monitored for H2O2 concentration, numbers, and viabilities of cells: (1) E. coli growth rates monitored for 1 h were profoundly affected by concentrations of H2O2, between 25-50 microM. (2) Complete bacteriostasis was observed at 100 microM. (3) Significant cell killing was not observed until the concentration of H2O2 was greater than 500 microM. (4) Bacteriostatic (25-50 microM) concentrations of H2O2 appeared not to be toxic to human skin fibroblasts for a 2-h exposure. (4) Bacteriostasis by H2O2 could not be explained by metabolic inhibition, because intracellular ATP levels were not compromised at bacteriostatic doses of H2O2. (5) Measurements of H2O2 concentrations in subcutaneous abscess fluid infected with both E. coli and S. aureus indicated prevailing concentrations of the oxidant consistent with a proposed role of H2O2 in host defense.

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.

Human cortical neuronal (HCN) cell lines: a model for amyloid β neurotoxicity

Abstract Human cortical neuronal cell lines HCN-1A and HCN-2 are killed following exposure of the differentiated cells to amyloid β-peptide(1–40), a component of senile plaques and other amyloid deposits in brains from Alzheimers patients. We present a model of Aβ toxicity uncomplicated by the presence of other cell types that can be used to address the mechanism of Aβ neurotoxicity. This model will be useful in the evaluation of neuroprotective compounds which may attenuate cortical neuronal loss in Alzheimers disease.

Impairment of integrin-mediated cell-matrix adhesion in oxidant-stressed PC12 cells

Oxidants are believed to play an important and complex role in neuronal injury and death in the aging process and various neurode generative diseases. We studied the effect of oxidative stress on integrin-mediated cell-extracellular matrix (ECM) interactions using the PC12 neuronal cell line. In assays in which attachment was measured between 30 and 90 min, addition of hydrogen peroxide (H2O2) to the attachment medium resulted in a dose-dependent inhibition of initial cell attachment to collagen. Addition of H2O2 also caused previously attached cells to detach from collagen. The inhibition by H2O2 was specific for integrin-mediated adhesion, since attachment to substrata coated with non-ECM molecules was much less affected. Exposure of cells to H2O2 resulted in a rapid and profound reduction of intracellular ATP, accompanied by only a slight increase in intracellular free Ca2+ concentration ([Ca2+]i). Treatment of cells with the microfilament-disrupting agent, cytochalasin B, like that with H2O2, inhibited cell adhesion to collagen. We propose that integrin-mediated cell adhesion, which requires interactions between cytoplasmic portions of integrin subunits and cytoskeletal microfilaments, is impaired by oxidative stress as a result of the depletion of intracellular ATP and that such depletion is an early event in the process of oxidant-induced neuronal injury.

Antiphospholipid autoantibodies as blood biomarkers for detection of early stage Alzheimer’s disease

Abstract A robust blood biomarker is urgently needed to facilitate early prognosis for those at risk for Alzheimer’s disease (AD). Redox reactive autoantibodies (R-RAAs) represent a novel family of antibodies detectable only after exposure of cerebrospinal fluid (CSF), serum, plasma or immunoglobulin fractions to oxidizing agents. We have previously reported that R-RAA antiphospholipid antibodies (aPLs) are significantly decreased in the CSF and serum of AD patients compared to healthy controls (HCs). These studies were extended to measure R-RAA aPL in serum samples obtained from Alzheimer’s Disease Neuroimaging Initiative (ADNI). Serum samples from the ADNI-1 diagnostic groups from participants with mild cognitive impairment (MCI), AD and HCs were blinded for diagnosis and analyzed for R-RAA aPL by ELISA. Demographics, cognitive data at baseline and yearly follow-up were subsequently provided by ADNI after posting assay data. As observed in CSF, R-RAA aPL in sera from the AD diagnostic group were significantly reduced compared to HC. However, the sera from the MCI population contained significantly elevated R-RAA aPL activity relative to AD patient and/or HC sera. The data presented in this study indicate that R-RAA aPL show promise as a blood biomarker for detection of early AD, and warrant replication in a larger sample. Longitudinal testing of an individual for increases in R-RAA aPL over a previously established baseline may serve as a useful early sero-epidemiologic blood biomarker for individuals at risk for developing dementia of the Alzheimer’s type.

Methods for Sample Preparation for Direct Immunoassay Measurement of Analytes in Tissue Homogenates: ELISA Assay of Amyloid β‐Peptides

Use of low abundance analytes in whole tissue homogenates has been realized with the development of assays in which a specific analyte is captured and detected using immunological reagents. One of the many advantages of analyte immunoassay in crude homogenates is its relative simplicity, allowing high throughput analysis of samples. In this unit, some major key determinants in sample and standard preparation and handling are described that have been shown to improve the performance and reliability of these assay systems. The ELISA assay of amyloid peptides from brain tissue is described as an example, since the protocols for this analysis exemplify many of the techniques and problems that are encountered in the development of new assays.