Deborah J. Tew

Tyrosine gated electron transfer is key to the toxic mechanism of Alzheimer's disease β-amyloid

Alzheimers disease (AD) is characterized by the presence of neurofibrillary tangles and amyloid plaques, which are abnormal protein deposits. The major constituent of the plaques is the neurotoxic β‐amyloid peptide (Aβ); the genetics of familial AD support a direct role for this peptide in AD. Aβ neurotoxicity is linked to hydrogen peroxide formation. Aβ coordinates the redox active transition metals, copper and iron, to catalytically generate reactive oxygen species. The chemical mechanism underlying this process is not well defined. With the use of density functional theory calculations to delineate the chemical mechanisms that drive the catalytic production of H2O2 by Aβ/Cu, tyrosine10 (Y10) was identified as a pivotal residue for this reaction to proceed. The relative stability of tyrosyl radicals facilitates the electron transfers that are required to drive the reaction. Confirming the theoretical results, mutation of the tyrosine residue to alanine inhibited H2O2 production, Cu‐induced radicalization, dityrosine cross‐linking, and neurotoxicity.

Dopamine promotes α-synuclein aggregation into SDS-resistant soluble oligomers via a distinct folding pathway

Dopamine (DA) and α‐synuclein (α‐SN) are two key molecules associated with Parkinsons disease (PD). We have identified a novel action of DA in the initial phase of α‐SN aggregation and demonstrate that DA induces α‐SN to form soluble, SDS‐resistant oligomers. The DA:α‐SN oligomeric species are not amyloidogenic as they do not react with thioflavin T and lack the typical amyloid fibril structures as visualized with electron microscopy. Circular dichroism studies indicate that in the presence of lipid membranes DA interacts with α‐SN, causing an alteration to the structure of the protein. Furthermore, DA inhibited the formation of iron‐induced α‐SN amyloidogenic aggregates, suggesting that DA acts as a dominant modulator of α‐SN aggregation. These observations support the paradigm emerging for other neurodegenerative diseases that the toxic species is represented by a soluble oligomer and not the insoluble fibril.

Neurotoxic, Redox-competent Alzheimer's β-Amyloid Is Released from Lipid Membrane by Methionine Oxidation

The amyloid β peptide is toxic to neurons, and it is believed that this toxicity plays a central role in the progression of Alzheimers disease. The mechanism of this toxicity is contentious. Here we report that an Aβ peptide with the sulfur atom of Met-35 oxidized to a sulfoxide (Met(O)Aβ) is toxic to neuronal cells, and this toxicity is attenuated by the metal chelator clioquinol and completely rescued by catalase implicating the same toxicity mechanism as reduced Aβ. However, unlike the unoxidized peptide, Met(O)Aβ is unable to penetrate lipid membranes to form ion channel-like structures, and β-sheet formation is inhibited, phenomena that are central to some theories for Aβ toxicity. Our results show that, like the unoxidized peptide, Met(O)Aβ will coordinate Cu2+ and reduce the oxidation state of the metal and still produce H2O2. We hypothesize that Met(O)Aβ production contributes to the elevation of soluble Aβ seen in the brain in Alzheimers disease.

Amyloid-β Peptide (Aβ) Neurotoxicity Is Modulated by the Rate of Peptide Aggregation: Aβ Dimers and Trimers Correlate with Neurotoxicity

Alzheimers disease is an age-related neurodegenerative disorder with its toxicity linked to the generation of amyloid-β peptide (Aβ). Within the Aβ sequence, there is a systemic repeat of a GxxxG motif, which theoretical studies have suggested may be involved in both peptide aggregation and membrane perturbation, processes that have been implicated in Aβ toxicity. We synthesized modified Aβ peptides, substituting glycine for leucine residues within the GxxxG repeat motif (GSL peptides). These GSL peptides undergo β-sheet and fibril formation at an increased rate compared with wild-type Aβ. The accelerated rate of amyloid fibril formation resulted in a decrease in the presence of small soluble oligomers such as dimeric and trimeric forms of Aβ in solution, as detected by mass spectrometry. This reduction in the presence of small soluble oligomers resulted in reduced binding to lipid membranes and attenuated toxicity for the GSL peptides. The potential role that dimer and trimer species binding to lipid plays in Aβ toxicity was further highlighted when it was observed that annexin V, a protein that inhibits Aβ toxicity, specifically inhibited Aβ dimers from binding to lipid membranes.

Copper-mediated Amyloid-β Toxicity Is Associated with an Intermolecular Histidine Bridge

Amyloid-β peptide (Aβ) is pivotal to the pathogenesis of Alzheimer disease. Here we report the formation of a toxic Aβ-Cu2+ complex formed via a histidine-bridged dimer, as observed at Cu2+/peptide ratios of >0.6:1 by EPR spectroscopy. The toxicity of the Aβ-Cu2+ complex to cultured primary cortical neurons was attenuated when either the π -or τ-nitrogen of the imidazole side chains of His were methylated, thereby inhibiting formation of the His bridge. Toxicity did not correlate with the ability to form amyloid or perturb the acyl-chain region of a lipid membrane as measured by diphenyl-1,3,5-hexatriene anisotropy, but did correlate with lipid peroxidation and dityrosine formation. 31P magic angle spinning solid-state NMR showed that Aβ and Aβ-Cu2+ complexes interacted at the surface of a lipid membrane. These findings indicate that the generation of the Aβ toxic species is modulated by the Cu2+ concentration and the ability to form an intermolecular His bridge.

Platinum-based inhibitors of amyloid-β as therapeutic agents for Alzheimer's disease

Amelyoid-β peptide (Aβ) is a major causative agent responsible for Alzheimers disease (AD). Aβ contains a high affinity metal binding site that modulates peptide aggregation and toxicity. Therefore, identifying molecules targeting this site represents a valid therapeutic strategy. To test this hypothesis, a range of L-PtCl2 (L = 1,10-phenanthroline derivatives) complexes were examined and shown to bind to Aβ, inhibit neurotoxicity and rescue Aβ-induced synaptotoxicity in mouse hippocampal slices. Coordination of the complexes to Aβ altered the chemical properties of the peptide inhibiting amyloid formation and the generation of reactive oxygen species. In comparison, the classic anticancer drug cisplatin did not affect any of the biochemical and cellular effects of Aβ. This implies that the planar aromatic 1,10-phenanthroline ligands L confer some specificity for Aβ onto the platinum complexes. The potent effect of the L-PtCl2 complexes identifies this class of compounds as therapeutic agents for AD.

Methylation of the Imidazole Side Chains of the Alzheimer Disease Amyloid-β Peptide Results in Abolition of Superoxide Dismutase-like Structures and Inhibition of Neurotoxicity

The toxicity of the amyloid-β peptide (Aβ) is thought to be responsible for the neurodegeneration associated with Alzheimer disease. Generation of hydrogen peroxide has been implicated as a key step in the toxic pathway. Aβ coordinates the redox active metal ion Cu2+ to catalytically generate H2O2. Structural studies on the interaction of Aβ with Cu have suggested that the coordination sphere about the Cu2+ resembles the active site of superoxide dismutase 1. To investigate the potential role for such structures in the toxicity of Aβ, two novel Aβ40 peptides, Aβ40(HisτMe) and Aβ40(HisπMe), have been prepared, in which the histidine residues 6, 13, and 14 have been substituted with modified histidines where either the π- or τ-nitrogen of the imidazole side chain is methylated to prevent the formation of bridging histidine moieties. These modifications did not inhibit the ability of these peptides to form fibrils. However, the modified peptides were four times more effective at generating H2O2 than the native sequence. Despite the ability to generate more H2O2, these peptides were not neurotoxic. Whereas the modifications to the peptide altered the metal binding properties, they also inhibited the interaction between the peptides and cell surface membranes. This is consistent with the notion that Aβ-membrane interactions are important for neurotoxicity and that inhibiting these interactions has therapeutic potential.

Enhanced Toxicity and Cellular Binding of a Modified Amyloid β Peptide with a Methionine to Valine Substitution

The amyloid β peptide (Aβ) is toxic to neuronal cells, and it is probable that this toxicity is responsible for the progressive cognitive decline associated with Alzheimers disease. However, the nature of the toxic Aβ 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 Aβ. We examined the effect of mutating the methionine to valine in Aβ42 (AβM35V). The neurotoxic activity of AβM35V on primary mouse neuronal cortical cells was enhanced, and this diminished cell viability occurred at an accelerated rate compared with Aβ42. AβM35V binds Cu2+ and produces similar amounts of H2O2 as Aβ42 in vitro, and the neurotoxic activity was attenuated by the H2O2 scavenger catalase. The increased toxicity of AβM35V was associated with increased binding of this mutated peptide to cortical cells. The M35V mutation altered the interaction between Aβ 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 Aβ42 and AβM35V displayed a mixture of α-helical and β-sheet conformations. These findings provide further evidence that the toxicity of Aβ is regulated by binding to neuronal cells.

Cu2+ Binding Modes of Recombinant α-Synuclein − Insights from EPR Spectroscopy

The interaction of the small (140 amino acid) protein, alpha-synuclein (alphaS), with Cu(2+) has been proposed to play a role in Parkinsons disease (PD). While some insight from truncated model complexes has been gained, the nature of the corresponding Cu(2+) binding modes in the full length protein remains comparatively less well characterized. This work examined the Cu(2+) binding of recombinant human alphaS using Electron Paramagnetic Resonance (EPR) spectroscopy. Wild type (wt) alphaS was shown to bind stoichiometric Cu(2+) via two N-terminal binding modes at physiological pH. An H50N mutation isolated one binding mode, whose g parallel, A parallel, and metal-ligand hyperfine parameters correlated well with a {NH2, N(-), beta-COO(-), H2O} mode previously identified in truncated model fragments. Electron spin-echo envelope modulation (ESEEM) studies of wt alphaS confirmed the second binding mode at pH 7.4 involved coordination of His50 and its g parallel and A parallel parameters correlated with either {NH2, N(-), beta-COO(-), N(Im)} or {N(Im), 2 N(-)} coordination observed in alphaS fragments. At pH 5.0, His50-anchored Cu(2+) binding was greatly diminished, while {NH2, N(-), beta-COO(-), H2O} binding persisted in conjunction with another two binding modes. Metal-ligand hyperfine interactions from one of these indicated a 1N3O coordination sphere, which was ascribed to a {NH2, CO} binding mode. The other was characterized by a spectrum similar to that previously observed for diethylpyrocarbonate-treated alphaS and was attributed to C-terminal binding centered on Asp121. In total, four Cu(2+) binding modes were identified within pH 5.0-7.4, providing a more comprehensive picture of the Cu(2+) binding properties of recombinant alphaS.

The Caenorhabditis elegans Aβ1–42 Model of Alzheimer Disease Predominantly Expresses Aβ3–42

Transgenic expression of human amyloid β (Aβ) peptide in body wall muscle cells of Caenorhabditis elegans has been used to better understand aspects of Alzheimer disease (AD). In human aging and AD, Aβ undergoes post-translational changes including covalent modifications, truncations, and oligomerization. Amino truncated Aβ is increasingly recognized as potentially contributing to AD pathogenesis. Here we describe surface-enhanced laser desorption ionization-time of flight mass spectrometry mass spectrometry of Aβ peptide in established transgenic C. elegans lines. Surprisingly, the Aβ being expressed is not full-length 1–42 (amino acids) as expected but rather a 3–42 truncation product. In vitro analysis demonstrates that Aβ3–42 self-aggregates like Aβ1–42, but more rapidly, and forms fibrillar structures. Similarly, Aβ3–42 is also the more potent initiator of Aβ1–40 aggregation. Seeded aggregation via Aβ3–42 is further enhanced via co-incubation with the transition metal Cu(II). Although unexpected, the C. elegans model of Aβ expression can now be co-opted to study the proteotoxic effects and processing of Aβ3–42.