Search PubMed⌕ Search

Biomedical subjects

A I Bush

Publications and source records attributed to A I Bush.

At least 37 records · Page 2Linked to original sources
PubMed1999

The Alzheimer's disease amyloid precursor protein modulates copper-induced toxicity and oxidative stress in primary neuronal cultures.

The amyloid precursor protein (APP) of Alzheimer's disease can reduce copper (II) to copper (I) in a cell-free system potentially leading to increased oxidative stress in neurons. We used neuronal cultures derived from APP knock-out (APP(-/-)) and wild-type (WT) mice to examine the role of APP in copper neurotoxicity. WT cortical, cerebellar, and hippocampal neurons were significantly more susceptible than their respective APP(-/-) neurons to toxicity induced by physiological concentrations of copper but not by zinc or iron. There was no difference in copper toxicity between APLP2(-/-) and WT neurons, demonstrating specificity for APP-associated copper toxicity. Copper uptake was the same in WT and APP(-/-) neurons, suggesting APP may interact with copper to induce a localized increase in oxidative stress through copper (I) production. This was supported by significantly higher levels of copper-induced lipid peroxidation in WT neurons. Treatment of neuronal cultures with a peptide corresponding to the human APP copper-binding domain (APP142-166) potentiated copper but not iron or zinc toxicity. Incubation of APP142-166 with low-density lipoprotein (LDL) and copper resulted in significantly increased lipid peroxidation compared to copper and LDL alone. Substitution of the copper coordinating histidine residues with asparagines (APP142-166(H147N, H149N, H151N)) abrogated the toxic effects. A peptide corresponding to the zinc-binding domain (APP181-208) failed to induce copper or zinc toxicity in neuronal cultures. These data support a role for the APP copper-binding domain in APP-mediated copper (I) generation and toxicity in primary neurons, a process that has important implications for Alzheimer's disease and other neurodegenerative disorders.

Amino Acid Sequence↗
PubMed1999

Copper levels are increased in the cerebral cortex and liver of APP and APLP2 knockout mice.

The pathological process in Alzheimer's disease (AD) involves amyloid beta (Abeta) deposition and neuronal cell degeneration. The neurotoxic Abeta peptide is derived from the amyloid precursor protein (APP), a member of a larger gene family including the amyloid precursor-like proteins, APLP1 and APLP2. The APP and APLP2 molecules contain metal binding sites for copper and zinc. The zinc binding domain (ZnBD) is believed to have a structural rather than a catalytic role. The activity of the copper binding domain (CuBD) is unknown, however, APP reduces copper (II) to copper (I) and this activity could promote copper-mediated neurotoxicity. The expression of APP and APLP2 in the brain suggests they could have an important direct or indirect role in neuronal metal homeostasis. To examine this, we measured copper, zinc and iron levels in the cerebral cortex, cerebellum and selected non-neuronal tissues from APP (APP(-/-)) and APLP2 (APLP2(-/-)) knockout mice using atomic absorption spectrophotometry. Compared with matched wild-type (WT) mice, copper levels were significantly elevated in both APP(-/-) and APLP2(-/-) cerebral cortex (40% and 16%, respectively) and liver (80% and 36%, respectively). Copper levels were not significantly different between knockout and WT cerebellum, spleen or serum samples. There were no significant differences observed between APP(-/-), APLP2(-/-) and WT mice zinc or iron levels in any tissue examined. These findings indicate APP and APLP2 expression specifically modulates copper homeostasis in the liver and cerebral cortex, the latter being a region of the brain particularly involved in AD. Perturbations to APP metabolism and in particular, its secretion or release from neurons may alter copper homeostasis resulting in increased Abeta accumulation and free radical generation. These data support a novel mechanism in the APP/Abeta pathway which leads to AD.

Alzheimer Disease↗
PubMed1999

Aqueous dissolution of Alzheimer's disease Abeta amyloid deposits by biometal depletion.

Zn(II) and Cu(II) precipitate Abeta in vitro into insoluble aggregates that are dissolved by metal chelators. We now report evidence that these biometals also mediate the deposition of Abeta amyloid in Alzheimer's disease, since the solubilization of Abeta from post-mortem brain tissue was significantly increased by the presence of chelators, EGTA, N,N,N',N'-tetrakis(2-pyridyl-methyl) ethylene diamine, and bathocuproine. Efficient extraction of Abeta also required Mg(II) and Ca(II). The chelators were more effective in extracting Abeta from Alzheimer's disease brain tissue than age-matched controls, suggesting that metal ions differentiate the chemical architecture of amyloid in Alzheimer's disease. Agents that specifically chelate copper and zinc ions but preserve Mg(II) and Ca(II) may be of therapeutic value in Alzheimer's disease.

Alzheimer Disease↗
PubMed1999

The A beta peptide of Alzheimer's disease directly produces hydrogen peroxide through metal ion reduction.

Oxidative stress markers characterize the neuropathology both of Alzheimer's disease and of amyloid-bearing transgenic mice. The neurotoxicity of amyloid A beta peptides has been linked to peroxide generation in cell cultures by an unknown mechanism. We now show that human A beta directly produces hydrogen peroxide (H2O2) by a mechanism that involves the reduction of metal ions, Fe(III) or Cu(II), setting up conditions for Fenton-type chemistry. Spectrophotometric experiments establish that the A beta peptide reduces Fe(III) and Cu(II) to Fe(II) and Cu(I), respectively. Spectrochemical techniques are used to show that molecular oxygen is then trapped by A beta and reduced to H2O2 in a reaction that is driven by substoichiometric amounts of Fe(II) or Cu(I). In the presence of Cu(II) or Fe(III), A beta produces a positive thiobarbituric-reactive substance (TBARS) assay, compatible with the generation of the hydroxyl radical (OH.). The amounts of both reduced metal and TBARS reactivity are greatest when generated by A beta 1-42 >> A beta 1-40 > rat A beta 1-40, a chemical relationship that correlates with the participation of the native peptides in amyloid pathology. These findings indicate that the accumulation of A beta could be a direct source of oxidative stress in Alzheimer's disease.

Alzheimer Disease↗
PubMed1999

Differential effects of apolipoprotein E isoforms on metal-induced aggregation of A beta using physiological concentrations.

The epsilon 4 allele of apolipoprotein E (APOE) has been found to be a risk factor for late-onset Alzheimer's disease (AD). While the pathogenic mechanism of APOE in AD is not yet clear, APOE isoforms appear to differentially influence the aggregation of A beta, the principal component of Alzheimer-associated beta-amyloid deposits. To date, no data are available for the propensity of A beta to aggregate in the presence of APOE under conditions where these components are at physiological concentrations (in cerebrospinal fluid, APOE and A beta are approximately 100 nM and approximately 5 nM, respectively). We employed a novel in vitro filtration assay for detecting zinc(II)- and copper(II)-induced aggregation of A beta in solutions containing concentrations of the peptide that are similar to those reported for human cerebrospinal fluid. The potential for resolubilization with EDTA and the relative densities of zinc- and copper-induced A beta aggregates were also compared. Zinc-induced A beta aggregates were found to be denser and less easily resolubilized than copper-induced precipitates. Metal-induced aggregation of A beta was studied in the presence of purified apolipoprotein E2, apolipoprotein E3, and apolipoprotein E4 under conditions that approximate the physiological concentrations and ratios of these proteins. In the presence of all three APOE isoforms, zinc-induced aggregation of A beta was attenuated, while precipitation with copper was enhanced. Consistent with the increased risk for AD associated with the epsilon 4 allele of APOE, metal-induced aggregation of A beta was highest for both zinc and copper in the presence of apolipoprotein E4. Our data are consistent with a role for APOE as an in vivo molecular chaperone for A beta.

Amyloid beta-Peptides↗
PubMed1999

Soluble pool of Abeta amyloid as a determinant of severity of neurodegeneration in Alzheimer's disease.

Genetic evidence strongly supports the view that Abeta amyloid production is central to the cause of Alzheimer's disease. The kinetics, compartmentation, and form of Abeta and its temporal relation to the neurodegenerative process remain uncertain. The levels of soluble and insoluble Abeta were determined by using western blot techniques, and the findings were assessed in relation to indices of severity of disease. The mean level of soluble Abeta is increased threefold in Alzheimer's disease and correlates highly with markers of disease severity. In contrast, the level of insoluble Abeta (also a measure of total amyloid load) is found only to discriminate Alzheimer's disease from controls, and does not correlate with disease severity or numbers of amyloid plaques. These findings support the concept of several interacting pools of Abeta, that is, a large relatively static insoluble pool that is derived from a constantly turning over smaller soluble pool. The latter may exist in both intracellular and extracellular compartments, and contain the basic forms of Abeta that cause neurodegeneration. Reducing the levels of these soluble Abeta species by threefold to levels found in normal controls might prove to be a goal of future therapeutic intervention.

Aged↗
PubMed1999

Exacerbation of copper toxicity in primary neuronal cultures depleted of cellular glutathione.

Perturbations to glutathione (GSH) metabolism may play an important role in neurodegenerative disorders such as Alzheimer's, Parkinson's, and prion diseases. A primary function of GSH is to prevent the toxic interaction between free radicals and reactive transition metals such as copper (Cu). Due to the potential role of Cu in neurodegeneration, we examined the effect of GSH depletion on Cu toxicity in murine primary neuronal cultures. Depletion of cellular GSH with L-buthionine-[S,R]-sulfoximine resulted in a dramatic potentiation of Cu toxicity in neurons without effect on iron (Fe) toxicity. Similarly, inhibition of glutathione reductase (GR) activity with 1,3-bis(2-chloroethyl)-1-nitrosurea also increased Cu toxicity in neurons. To determine if the Alzheimer's amyloid-beta (Abeta) peptide can affect neuronal resistance to transition metal toxicity, we exposed cultures to nontoxic concentrations of Abeta25-35 in the presence or absence of Cu or Fe. Abeta25-35 pretreatment was found to deplete neuronal GSH and increase GR activity, confirming the ability of Abeta to perturb neuronal GSH homeostasis. Abeta25-35 pretreatment potently increased Cu toxicity but had no effect on Fe toxicity. These studies demonstrate an important role for neuronal GSH homeostasis in selective protection against Cu toxicity, a finding with widespread implications for neurodegenerative disorders.

Amyloid beta-Peptides↗
PubMed1999

Intracellular accumulation of detergent-soluble amyloidogenic A beta fragment of Alzheimer's disease precursor protein in the hippocampus of aged transgenic mice.

To study amyloid beta-protein (A beta) production and aggregation in vivo, we created two transgenic (Tg) mouse lines expressing the C-terminal 100 amino acids of human amyloid precursor protein (APP): Tg C100.V717F and Tg C100.WT. Western blot analysis showed that human APP-C100 and A beta were produced in brain and some peripheral tissues and A beta was produced in serum. Using antibodies specific for the A beta C terminus we found that Tg C100.V717F produced a 1.6-fold increase in A beta42/A beta40 compared with Tg C100.WT. Approximately 30% of total brain A beta (approximately 122 ng/g of wet tissue) was water-soluble. The remaining 70% of A beta partitioned into the particulate fraction and was completely sodium dodecyl sulfate-soluble. In contrast, human Alzheimer's disease brain has predominantly sodium dodecyl sulfate-insoluble A beta. Immunohistochemistry with an A beta(5-8) antibody showed that A beta or A beta-containing fragments accumulated intracellularly in the hippocampus of aged Tg C100.V717F mice. The soluble A beta levels in Tg brain are similar to those in normal human brain, and this may explain the lack of microscopic amyloid deposits in the Tg mice. However, this mouse model provides a system to study the intracellular processing and accumulation of A beta or A beta-containing fragments and to screen for compounds directed at the gamma-secretase activity.

Aging↗
PubMed1998

Dramatic aggregation of Alzheimer abeta by Cu(II) is induced by conditions representing physiological acidosis.

The cortical deposition of Abeta is an event that occurs in Alzheimer's disease, Down's syndrome, head injury, and normal aging. Previously, in appraising the effects of different neurochemical factors that impact upon the solubility of Abeta, we observed that Zn2+ was the predominant bioessential metal to induce the aggregation of soluble Abeta at pH 7.4 in vitro and that this reaction is totally reversible with chelation. We now report that unlike other biometals tested at maximal biological concentrations, marked Cu2+-induced aggregation of Abeta1-40 emerged as the solution pH was lowered from 7.4 to 6.8 and that the reaction was completely reversible with either chelation or alkalinization. This interaction was comparable to the pH-dependent effect of Cu2+ on insulin aggregation but was not seen for aprotinin or albumin. Abeta1-40 bound three to four Cu2+ ions when precipitated at pH 7.0. Rapid, pH-sensitive aggregation occurred at low nanomolar concentrations of both Abeta1-40 and Abeta1-42 with submicromolar concentrations of Cu2+. Unlike Abeta1-40, Abeta1-42 was precipitated by submicromolar Cu2+ concentrations at pH 7.4. Rat Abeta1-40 and histidine-modified human Abeta1-40 were not aggregated by Zn2+, Cu2+, or Fe3+, indicating that histidine residues are essential for metal-mediated Abeta assembly. These results indicate that H+-induced conformational changes unmask a metal-binding site on Abeta that mediates reversible assembly of the peptide. Since a mildly acidic environment together with increased Zn2+ and Cu2+ are common features of inflammation, we propose that Abeta aggregation by these factors may be a response to local injury. Cu2+, Zn2+, and Fe3+ association with Abeta explains the recently reported enrichment of these metal ions in amyloid plaques in Alzheimer's disease.

Acidosis↗
PubMed1998

Relative increase in Alzheimer's disease of soluble forms of cerebral Abeta amyloid protein precursor containing the Kunitz protease inhibitory domain.

Although a number of studies have examined amyloid precursor protein (APP) mRNA levels in Alzheimer's disease (AD), no clear consensus has emerged as to whether the levels of transcripts for isoforms containing a Kunitz protease inhibitory (KPI)-encoded region are increased or decreased in AD. Here we compare AD and control brain for the relative amounts of APP protein containing KPI to APP protein lacking this domain. APP protein was purified from the soluble subcellular fraction and Triton X-100 membrane pellet extract of one hemisphere of AD (n = 10), normal (n = 7), and neurological control (n = 5) brains. The amount of KPI-containing APP in the purified protein samples was determined using two independent assay methods. The first assay exploited the inhibitory action of KPI-containing APP on trypsin. The second assay employed reflectance analysis of Western blots. The proportion of KPI-containing forms of APP in the soluble subcellular fraction of AD brains is significantly elevated (p < 0.01) compared with controls. Species containing a KPI domain comprise 32-41 and 76-77% of purified soluble APP from control and AD brains, respectively. For purified membrane-associated APP, 72-77 and 65-82% of control and AD samples, respectively, contain a KPI domain. Since KPI-containing species of APP may be more amyloidogenic (Ho, L., Fukuchi, K., and Yonkin, S. G. (1996) J. Biol. Chem. 271, 30929-30934), our findings support an imbalance of isoforms as one possible mechanism for amyloid deposition in sporadic AD.

Alternative Splicing↗
PubMed1997

Zinc-induced Alzheimer's Abeta1-40 aggregation is mediated by conformational factors.

The heterogeneous precipitates of Abeta that accumulate in the brain cortex in Alzheimer's disease possess varying degrees of resistance to resolubilization. We previously found that Abeta1-40 is rapidly precipitated in vitro by physiological concentrations of zinc, a neurochemical that is highly abundant in brain compartments where Abeta is most likely to precipitate. We now present evidence that the zinc-induced precipitation of Abeta is mediated by a peptide dimer and favored by conditions that promote alpha-helical and diminish beta-sheet conformations. The manner in which the synthetic peptide is solubilized was critical to its behavior in vitro. Zinc-induced Abeta aggregation was dependent upon the presence of NaCl, was enhanced by alpha-helical-promoting solvents, but was abolished when the peptide stock solution was stored frozen. The Abeta aggregates induced by zinc were reversible by chelation, but could then be reprecipitated by zinc for several cycles, indicating that the peptide's conformation is probably preserved in the zinc-mediated assembly. In contrast, Abeta aggregates induced by low pH (5.5) were not resolubilized by returning the pH milieu to 7.4. The zinc-Abeta interaction exhibits features resembling the gelation process of zinc-mediated fibrin assembly, suggesting that, in events such as clot formation or injury, reversible Abeta assembly could be physiologically purposive. Such a mechanism is contemplated in the early evolution of diffuse plaques in Alzheimer's disease and suggests a possible therapeutic strategy for the resolubilization of some forms of Abeta deposit in the disease.

Alzheimer Disease↗
PubMed1997

Metallothioneins in brain--the role in physiology and pathology.

A symposium on the role of brain metallothioneins (MTs) in physiology and pathology was held at the 1996 Annual Society of Toxicology Meeting in Anaheim, California. The objectives of this symposium were to: (1) review the physiologic function of MTs, (2) examine the distribution of brain MTs with particular emphasis on cell-specific localization (neurons vs neuroglia), (3) discuss MT gene responsiveness upon toxic insult with metals, and (4) discuss the potential role of MTs in the etiology of neurodegenerative disorders. Dr. Cherian discussed the biochemical properties of the MTs, emphasizing structural similarities and differences between the MTs. Dr. Klaassen addressed the expression and distribution of the MTs in brains with special reference to the cell-specific localization of MTs. Dr. Aschner provided data illustrating a potential role for MTs in attenuating the cytotoxicity caused by methylmercury (MeHg) in cultured neonatal astrocytes. Dr. Palmiter discussed the properties of MT-III and the increased sensitivity of MT-III knockout mice to kainate-induced seizures. Cerebral zinc metabolism, its relationship to MT homeostasis, and its pathogenic potential in Alzheimer's disease was addressed by Dr. Bush.

Animals↗
PubMed1995

LDL receptor-related protein, a multifunctional ApoE receptor, binds secreted beta-amyloid precursor protein and mediates its degradation.

The secreted form of beta-amyloid precursor protein (APP) containing the Kunitz proteinase inhibitor (KPI) domain, also called protease nexin II, is internalized and degraded by cells. We show that the low density lipoprotein (LDL) receptor-related protein (LRP) is responsible for the endocytosis of secreted APP. APPs770 degradation is inhibited by an LRP antagonist called the receptor-associated protein (RAP) and by LRP antibodies and is greatly diminished in fibroblasts genetically deficient in LRP. APPs695, which lacks the KPI domain, is a poor LRP ligand. Since LRP also binds apolipoprotein E (apoE)-enriched lipoproteins and inheritance of the epsilon 4 allele of the apoE gene is a risk factor for Alzheimer's disease (AD), these data link in a single metabolic pathway two molecules strongly implicated in the pathophysiology of AD.

Amyloid beta-Protein Precursor↗
PubMed1994

Interaction between the zinc (II) and the heparin binding site of the Alzheimer's disease beta A4 amyloid precursor protein (APP).

The Alzheimer's disease beta A4 amyloid precursor protein (APP) has been suggested to be involved in regulation of cell growth, neurite outgrowth and adhesiveness through binding to heparin sulfate proteoglycans. In order to unravel the molecular mechanisms underlying those functions in vitro we show that APP binds in a time dependent and saturable manner to the glycosaminoglycan side-chains of proteoglycans but not to chondroitinsulfate. We also demonstrate an interaction between the high affinity heparin binding site within the carbohydrate domain of APP and the zinc(II) binding site of APP. We show that the affinity for heparin is increased two- to four-fold in the presence of micromolar zinc(II). Thus micromolar concentrations of zinc(II) appear to be able to modulate the binding of APP to heparin side-chains of proteoglycans and as shown previously [Science 265 (1994) 1464-1467] to induce the aggregation of soluble amyloid beta A4 protein.

Allosteric Site↗
PubMed1994

The amyloid beta-protein precursor and its mammalian homologues. Evidence for a zinc-modulated heparin-binding superfamily.

The Alzheimer beta-amyloid precursor protein (APP) contains an ectodomain zinc binding site that has been reported to modulate the heparin affinity and protease-inhibitory properties of the molecule. This motif, GVEFVCCP, is highly conserved in amyloid precursor-like proteins 1 and 2 (APLP1 and APLP2), as well as in the Drosophila and Caenorhabditis elegans APP-like proteins (APPL and APL-1). To determine whether the function of this domain is preserved in the human APP-like proteins, the effect of zinc in modulating the elution profile of these proteins upon heparin-Sepharose chromatography was studied. Both APLP1 and APLP2 bound heparin-Sepharose and had NaCl elution profiles similar to that of APP. As previously reported for APP, zinc increased the recovery of APLP1 and APLP2 upon heparin-Sepharose chromatography. APP, APLP1, and APLP2 all bind zinc-chelating Sepharose, indicating that the zinc binding motif may be functionally conserved in these proteins. Additionally, APP, APLP1, and APLP2 migrate at higher molecular sizes (approximately 40 kDa) on SDS-polyacrylamide gel electrophoresis than their predicted molecular sizes. We report data that compare the physicochemical properties of APP to its novel APLP homologues and indicate that these molecules behave as a family of zinc-modulated, heparin-binding proteins.

Amino Acid Sequence↗