Activation of epidermal growth factor receptor by metal-ligand complexes decreases levels of extracellular amyloid beta peptide

The epidermal growth factor receptor is a receptor tyrosine kinase expressed in a range of tissues and cell-types. Activation of the epidermal growth factor receptor by a number of ligands induces downstream signalling that modulates critical cell functions including growth, survival and differentiation. Abnormal epidermal growth factor receptor expression and activation is also involved in a number of cancers. In addition to its cognate ligands, the epidermal growth factor receptor can be activated by metals such as zinc (Zn) and copper (Cu). Due to the important role of these metals in a number of diseases including neurodegenerative disorders, therapeutic approaches are being developed based on the use of lipid permeable metal-complexing molecules. While these agents are showing promising results in animal models and clinical trials, little is known about the effects of metal-ligand complexes on cell signalling pathways. In this study, we investigated the effects of clioquinol (CQ)-metal complexes on activation of epidermal growth factor receptor. We show here that CQ-Cu complexes induced potent epidermal growth factor receptor phosphorylation resulting in downstream activation of extracellular signal-regulated kinase. Similar levels of epidermal growth factor receptor activation were observed with alternative lipid permeable metal-ligands including neocuproine and pyrrolidine dithiocarbamate. We found that CQ-Cu complexes induced a significant reduction in the level of extracellular Abeta1-40 in cell culture. Inhibition of epidermal growth factor receptor activation by PD153035 blocked extracellular signal-regulated kinase phosphorylation and restored Abeta1-40 levels. Activation of the epidermal growth factor receptor by CQ-Cu was mediated through up-regulation of src kinase activity by a cognate ligand-independent process involving membrane integrins. These findings provide the first evidence that metal-ligand complexes can activate the epidermal growth factor receptor with potentially neuroprotective effects.     

Autoantibody-catalyzed hydrolysis of amyloid beta peptide

We describe IgM class human autoantibodies that hydrolyze amyloid beta peptide 1-40 (Abeta40). A monoclonal IgM from a patient with Waldenstr?m's macroglobulinemia hydrolyzed Abeta40 at the Lys-28-Gly-29 bond and Lys-16-Ala-17 bonds. The catalytic activity was inhibited stoichiometrically by an electrophilic serine protease inhibitor. Treatment with the catalytic IgM blocked the aggregation and toxicity of Abeta40 in neuronal cell cultures. IgMs purified from the sera of patients with Alzheimer disease (AD) hydrolyzed Abeta40 at rates superior to IgMs from age-matched humans without dementia. IgMs from non-elderly humans expressed the least catalytic activity. The reaction rate was sufficient to afford appreciable degradation at physiological Abeta and IgM concentrations found in peripheral circulation. Increased Abeta concentrations in the AD brain are thought to induce neurodegenerative effects. Peripheral administration of Abeta binding antibodies has been suggested as a potential treatment of AD. Our results suggest that catalytic IgM autoantibodies can help clear Abeta, and they open the possibility of using catalytic Abs for AD immunotherapy.     

Copper complexing decreases the ability of amyloid beta peptide to cross the BBB and enter brain parenchyma

The amyloid hypothesis states that amyloid beta protein (Abeta) plays a major causal role in the onset of Alzheimer's disease. Toxicity of Abeta can be modified by metal ions. Two mechanisms by which such Abeta and metal ions could interact are by enhanced oxidative stress or by altered fibrillation. Specifically, Abeta fibrillation is increased by aluminum (Al) and copper (Cu) and Al also increases Abeta uptake into brain. Here, we determined whether chelation with Cu would alter uptake of the human or rat 1-42 form of Abeta (Abeta42) by brain or alter Abeta-induced oxidative stress in an immortalized line of rat brain endothelial cells (RBE4). We found that Cu enhanced cytotoxicity of rat, but not of human Abeta, had no effect on glutathione (GSH) or cysteine (CYS) levels. Cu significantly decreased homocysteine (HCYS) levels when complexed with Abeta. Cu chelation did not alter Abeta uptake into brain or other tissues (except for kidney) or alter clearance from blood or brain in vivo, but did increase efflux in an in vitro model of the blood-brain barrier (BBB). Chelation to Cu also impaired the capillary to brain transport of Abeta, an effect opposite to that previously found for chelation of Abeta to Al. These results show that metal ions have varied effects on Abeta uptake by brain and that Cu could be protective against the neurotoxic effects of circulating Abeta.     

Interaction between amyloid beta peptide and an aggregation blocker peptide mimicking islet amyloid polypeptide

Assembly of amyloid-beta peptide (Aβ) into cytotoxic oligomeric and fibrillar aggregates is believed to be a major pathologic event in Alzheimer's disease (AD) and interfering with Aβ aggregation is an important strategy in the development of novel therapeutic approaches. Prior studies have shown that the double N-methylated analogue of islet amyloid polypeptide (IAPP) IAPP-GI, which is a conformationally constrained IAPP analogue mimicking a non-amyloidogenic IAPP conformation, is capable of blocking cytotoxic self-assembly of Aβ. Here we investigate the interaction of IAPP-GI with Aβ40 and Aβ42 using NMR spectroscopy. The most pronounced NMR chemical shift changes were observed for residues 13-20, while residues 7-9, 15-16 as well as the C-terminal half of Aβ--that is both regions of the Aβ sequence that are converted into β-strands in amyloid fibrils--were less accessible to solvent in the presence of IAPP-GI. At the same time, interaction of IAPP-GI with Aβ resulted in a concentration-dependent co-aggregation of Aβ and IAPP-GI that was enhanced for the more aggregation prone Aβ42 peptide. On the basis of the reduced toxicity of the Aβ peptide in the presence of IAPP-GI, our data are consistent with the suggestion that IAPP-GI redirects Aβ into nontoxic "off-pathway" aggregates.     

Lysophosphatidylcholine modulates fibril formation of amyloid beta peptide

Phospholipids are known to influence fibril formation of amyloid beta (Aβ) peptide. Here, we show that lysophosphatidylcholine (LPC), a polar phospholipid, enhances Aβ(1-42) fibril formation, by decreasing the lag time and the critical peptide concentration required for fibril formation, and increasing the fibril elongation rate. Conversely, LPC did not have an enhancing effect on Aβ(1-40) fibril formation, and appeared to be inhibitory. Tyrosine fluorescence spectroscopy showed that LPC altered the fluorescence spectra of Aβ(1-40) and Aβ(1-42) in opposite ways. Further, 8-anilino-1-naphthalene sulfonic acid fluorescence spectroscopy showed that LPC significantly increased the hydrophobicity of Aβ(1-42), but not of Aβ(1-40). Tris-tricine gradient SDS/PAGE revealed that LPC increased the formation of higher-molecular-weight species of Aβ(1-42), including trimers and tetramers. LPC had no such effect on Aβ(1-40), and thus may specifically influence the oligomerization and nucleation processes of Aβ(1-42) in a manner dependent on its native structure. Dot-blot assays confirmed that LPC induced Aβ(1-42) oligomer formation at an early time point. Thus our results indicate that LPC specifically enhances the formation of Aβ(1-42) fibrils, the main component of senile plaques in Alzheimer's disease patients, and may be involved in Alzheimer's disease pathology.     

Expression of BRI-amyloid beta peptide fusion proteins: a novel method for specific high-level expression of amyloid beta peptides

In order to develop transgenic animal models that selectively overexpress various Abeta peptides, we have developed a novel expression system that selectively expresses Abeta40 or Abeta42 in the secretory pathway. This system utilizes fusion constructs in which the sequence encoding the 23-amino-acid ABri peptide at the carboxyl terminus of the 266-amino-acid type 2 transmembrane protein BRI is replaced with a sequence encoding either Abeta40 or Abeta42. Constitutive processing of the resultant BRI-Abeta fusion proteins in transfected cells results in high-level expression and secretion of the encoded Abeta peptide. Significantly, expression of Abeta42 from the BRI-Abeta42 construct resulted in no increase in secreted Abeta40, suggesting that the majority of Abeta42 is not trimmed by carboxypeptidase to Abeta40 in the secretory pathway.     

Enhanced toxicity and cellular binding of a modified amyloid beta peptide with a methionine to valine substitution

The amyloid beta peptide (Abeta) is toxic to neuronal cells, and it is probable that this toxicity is responsible for the progressive cognitive decline associated with Alzheimer's disease. However, the nature of the toxic Abeta species and its precise mechanism of action remain to be determined. It has been reported that the methionine residue at position 35 has a pivotal role to play in the toxicity of Abeta. We examined the effect of mutating the methionine to valine in Abeta42 (AbetaM35V). The neurotoxic activity of AbetaM35V on primary mouse neuronal cortical cells was enhanced, and this diminished cell viability occurred at an accelerated rate compared with Abeta42. AbetaM35V binds Cu2+ and produces similar amounts of H2O2 as Abeta42 in vitro, and the neurotoxic activity was attenuated by the H2O2 scavenger catalase. The increased toxicity of AbetaM35V was associated with increased binding of this mutated peptide to cortical cells. The M35V mutation altered the interaction between Abeta and copper in a lipid environment as shown by EPR analysis, which indicated that the valine substitution made the peptide less rigid in the bilayer region with a resulting higher affinity for the bilayer. Circular dichroism spectroscopy showed that both Abeta42 and AbetaM35V displayed a mixture of alpha-helical and beta-sheet conformations. These findings provide further evidence that the toxicity of Abeta is regulated by binding to neuronal cells.     

Lipocalin 2 modulates the cellular response to amyloid beta

The production, accumulation and aggregation of amyloid beta (Aβ) peptides in Alzheimer''s disease (AD) are influenced by different modulators. Among these are iron and iron-related proteins, given their ability to modulate the expression of the amyloid precursor protein and to drive Aβ aggregation. Herein, we describe that lipocalin 2 (LCN2), a mammalian acute-phase protein involved in iron homeostasis, is highly produced in response to Aβ_1-42 by choroid plexus epithelial cells and astrocytes, but not by microglia or neurons. Although Aβ_1-42 stimulation decreases the dehydrogenase activity and survival of wild-type astrocytes, astrocytes lacking the expression of Lcn2 are not affected. This protection results from a lower expression of the proapoptotic gene Bim and a decreased inflammatory response. Altogether, these findings show that Aβ toxicity to astrocytes requires LCN2, which represents a novel mechanism to target when addressing AD.One of the pathological hallmarks of Alzheimer''s disease (AD) is the increased production and accumulation of amyloid beta (Aβ) peptides in the brain, which result from the misprocessing of the membrane amyloid precursor protein. Through an unidentified combination of events, Aβ peptides, initially soluble, aggregate into oligomers, which are highly toxic to brain cells. Oligomers of Aβ ultimately deposit in different brain regions and form amyloid plaques.¹ The steps that drive the amyloidogenic pathway are still unclear, but the aggregation of Aβ into dimers, trimers and other toxic oligomeric forms seems to be decisive. This process was shown to be influenced by many factors, among which is iron, described to favor the formation and stabilization of toxic Aβ oligomers.^{2, 3} Notably, iron accumulates with age in brain areas that are preferentially affected in AD patients, such as the hippocampus and the cortex.⁴ In these areas, iron and iron-binding proteins were shown to accumulate in the amyloid plaques.⁵ Interestingly, recent evidence points to alterations in the level of iron metabolism-related proteins, such as ferritin, and their impact on iron homeostasis as probable causes of increased amyloid precursor protein expression and misprocessing, as well as increased aggregation of Aβ into toxic oligomers.^{6, 7}Recently, the iron-associated protein lipocalin 2 (LCN2) was implicated in AD.⁸ LCN2, a member of the lipocalin family of soluble proteins, was originally identified as a constituent of granules in human neutrophils.⁹ It was first described as an acute-phase protein¹⁰ able to bind and sequester bacterial iron-loaded siderophores, thus preventing the growth and dissemination of the infectious agents.¹¹ In addition, LCN2 has been described also to mediate transferrin-independent iron delivery^{12, 13} and removal from cells,¹⁴ which is associated with cell proliferation and apoptosis, respectively. Although the pathway through which LCN2 influences cell proliferation remains uncertain, LCN2-mediated apoptosis involves the proapoptotic protein BCL2-like 11 (BCL2L11 or BIM).^{14, 15} Of notice, different cells from the central nervous system (CNS), namely choroid plexus (CP) epithelial cells and astrocytes, have been shown to produce LCN2 in response to various stimuli.^{15, 16, 17, 18, 19} Importantly, a recent study demonstrated that LCN2, produced in response to tumor necrosis factor (TNF), is able to interfere with TNF receptor protective signaling and to enhance the toxicity of glutamate and Aβ.⁸ The present study investigated the mechanism through which LCN2 contributes to the modulation of brain cell metabolism and survival in response to Aβ.     

Activation of Toll-like receptor 2 on microglia promotes cell uptake of Alzheimer disease-associated amyloid beta peptide

The human G-protein-coupled formyl peptide receptor-like 1 (FPRL1) and its mouse homologue mFPR2 mediate the chemotactic activity of a variety of polypeptides associated with inflammation and bacterial infection, including the 42-amino acid form of amyloid beta peptide (Abeta42), a pathogenic factor in Alzheimer disease. Because mFPR2 was inducible in mouse microglial cells by proinflammatory stimulants, such as bacterial lipopolysaccharide, a ligand for the Toll-like receptor 4 (TLR4), we investigated the role of TLR2 in the regulation of mFPR2. We found that a TLR2 agonist, peptidoglycan (PGN) derived from Gram-positive bacterium Staphylococcus aureus, induced considerable mFpr2 mRNA expression in a mouse microglial cell line and primary microglial cells. This was associated with a markedly increased chemotaxis of the cells in response to mFPR2 agonist peptides. In addition, activation of TLR2 markedly enhanced mFPR2-mediated uptake of Abeta42 by microglia. Studies of the mechanistic basis showed that PGN activates MAPK and IkappaBalpha, and the effect of PGN on induction of mFPR2 was dependent on signaling pathways via ERK1/2 and p38 MAPKs. The use of TLR2 on microglial cells by PGN was supported by the fact that N9 cells transfected with short interfering RNA targeting mouse TLR2 failed to show increased expression of functional mFPR2 after stimulation with PGN. Our results demonstrated a potentially important role for TLR2 in microglial cells of promoting cell responses to chemoattractants produced in lesions of inflammatory and neurodegenerative diseases in the brain.     

Phosphorylation of liver X receptor alpha selectively regulates target gene expression in macrophages

Dysregulation of liver X receptor alpha (LXRalpha) activity has been linked to cardiovascular and metabolic diseases. Here, we show that LXRalpha target gene selectivity is achieved by modulation of LXRalpha phosphorylation. Under basal conditions, LXRalpha is phosphorylated at S198; phosphorylation is enhanced by LXR ligands and reduced both by casein kinase 2 (CK2) inhibitors and by activation of its heterodimeric partner RXR with 9-cis-retinoic acid (9cRA). Expression of some (AIM and LPL), but not other (ABCA1 or SREBPc1) established LXR target genes is increased in RAW 264.7 cells expressing the LXRalpha S198A phosphorylation-deficient mutant compared to those with WT receptors. Surprisingly, a gene normally not expressed in macrophages, the chemokine CCL24, is activated specifically in cells expressing LXRalpha S198A. Furthermore, inhibition of S198 phosphorylation by 9cRA or by a CK2 inhibitor similarly promotes CCL24 expression, thereby phenocopying the S198A mutation. Thus, our findings reveal a previously unrecognized role for phosphorylation in restricting the repertoire of LXRalpha-responsive genes.     

The redox chemistry of the Alzheimer's disease amyloid beta peptide

There is a growing body of evidence to support a role for oxidative stress in Alzheimer's disease (AD), with increased levels of lipid peroxidation, DNA and protein oxidation products (HNE, 8-HO-guanidine and protein carbonyls respectively) in AD brains. The brain is a highly oxidative organ consuming 20% of the body's oxygen despite accounting for only 2% of the total body weight. With normal ageing the brain accumulates metals ions such iron (Fe), zinc (Zn) and copper (Cu). Consequently the brain is abundant in antioxidants to control and prevent the detrimental formation of reactive oxygen species (ROS) generated via Fenton chemistry involving redox active metal ion reduction and activation of molecular oxygen. In AD there is an over accumulation of the Amyloid beta peptide (Abeta), this is the result of either an elevated generation from amyloid precursor protein (APP) or inefficient clearance of Abeta from the brain. Abeta can efficiently generate reactive oxygen species in the presence of the transition metals copper and iron in vitro. Under oxidative conditions Abeta will form stable dityrosine cross-linked dimers which are generated from free radical attack on the tyrosine residue at position 10. There are elevated levels of urea and SDS resistant stable linked Abeta oligomers as well as dityrosine cross-linked peptides and proteins in AD brain. Since soluble Abeta levels correlate best with the degree of degeneration [C.A. McLean, R.A. Cherny, F.W. Fraser, S.J. Fuller, M.J. Smith, K. Beyreuther, A.I. Bush, C.L. Masters, Soluble pool of Abeta amyloid as a determinant of severity of neurodegeneration in Alzheimer's disease, Ann. Neurol. 46 (1999) 860-866] we suggest that the toxic Abeta species corresponds to a soluble dityrosine cross-linked oligomer. Current therapeutic strategies using metal chelators such as clioquinol and desferrioxamine have had some success in altering the progression of AD symptoms. Similarly, natural antioxidants curcumin and ginkgo extract have modest but positive effects in slowing AD development. Therefore, drugs that target the oxidative pathways in AD could have genuine therapeutic efficacy.     

Interaction of calreticulin with amyloid beta peptide 1-42

The interaction of calreticulin with amyloid beta (Abeta) was investigated using solid phase and solution binding assays. Calreticulin bound Abeta 1-42 in a time and concentration dependent fashion. The binding was optimal at pH 5 and was stimulated by Ca2+ and inhibited by Zn2+ at pH 7. Interaction took place through the hydrophobic C-terminus of Abeta 1-42 and the polypeptide binding site of calreticulin. The results are discussed in the light of a reported role of calreticulin as a cell surface scavenger receptor.     

Degradation of the Alzheimer's amyloid beta peptide by endothelin-converting enzyme

Deposition of beta-amyloid (Abeta) peptides in the brain is an early and invariant feature of all forms of Alzheimer's disease. As with any secreted protein, the extracellular concentration of Abeta is determined not only by its production but also by its catabolism. A major focus of Alzheimer's research has been the elucidation of the mechanisms responsible for the generation of Abeta. Much less, however, is known about the mechanisms responsible for Abeta removal in the brain. In this report, we describe the identification of endothelin-converting enzyme-1 (ECE-1) as a novel Abeta-degrading enzyme. We show that treatment of endogenous ECE-expressing cell lines with the metalloprotease inhibitor phosphoramidon causes a 2-3-fold elevation in extracellular Abeta concentration that appears to be due to inhibition of intracellular Abeta degradation. Furthermore, we show that overexpression of ECE-1 in Chinese hamster ovary cells, which lack endogenous ECE activity, reduces extracellular Abeta concentration by up to 90% and that this effect is completely reversed by treatment of the cells with phosphoramidon. Finally, we show that recombinant soluble ECE-1 is capable of hydrolyzing synthetic Abeta40 and Abeta42 in vitro at multiple sites.     

A liver X receptor and retinoid X receptor heterodimer mediates apolipoprotein E expression, secretion and cholesterol homeostasis in astrocytes

Apolipoprotein E (apoE) is an important protein involved in lipoprotein clearance and cholesterol redistribution. ApoE is abundantly expressed in astrocytes in the brain and is closely linked to the pathogenesis of Alzheimer's disease (AD). We report here that small molecule ligands that activate either liver X receptors (LXR) or retinoid X receptor (RXR) lead to a dramatic increase in apoE mRNA and protein expression as well as secretion of apoE in a human astrocytoma cell line (CCF-STTG1 cells). Examination of primary mouse astrocytes also revealed significant induction of apoE mRNA, and protein expression and secretion following incubation with LXR/RXR agonists. Moreover, treatment of mice with a specific synthetic LXR agonist T0901317 resulted in up-regulation of apoE mRNA and protein in both hippocampus and cerebral cortex, indicating that apoE expression in brain can be up-regulated by LXR agonists in vivo. Along with a dramatic induction of ABCA1 cholesterol transporter expression, these ligands effectively mediate cholesterol efflux in both CCF-STTG1 cells and mouse astrocytes in the presence or absence of apolipoprotein AI (apoAI). Our studies provide strong evidence that small molecule LXR/RXR agonists can effectively mediate apoE synthesis and secretion as well as cholesterol homeostasis in astrocytes. LXR/RXR agonists may have significant impact on the pathogenesis of multiple neurological diseases, including AD.