Biochemistry and Pharmacology - Research Publications
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ItemThe hypoxia imaging agent CuII(atsm) is neuroprotective and improves motor and cognitive functions in multiple animal models of Parkinson's disease(ROCKEFELLER UNIV PRESS, 2012-04-09)Parkinson's disease (PD) is a progressive, chronic disease characterized by dyskinesia, rigidity, instability, and tremors. The disease is defined by the presence of Lewy bodies, which primarily consist of aggregated α-synuclein protein, and is accompanied by the loss of monoaminergic neurons. Current therapeutic strategies only give symptomatic relief of motor impairment and do not address the underlying neurodegeneration. Hence, we have identified Cu(II)(atsm) as a potential therapeutic for PD. Drug administration to four different animal models of PD resulted in improved motor and cognition function, rescued nigral cell loss, and improved dopamine metabolism. In vitro, this compound is able to inhibit the effects of peroxynitrite-driven toxicity, including the formation of nitrated α-synuclein oligomers. Our results show that Cu(II)(atsm) is effective in reversing parkinsonian defects in animal models and has the potential to be a successful treatment of PD.
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ItemSmall angle X-ray scattering analysis of Cu2+-induced oligomers of the Alzheimer's amyloid β peptide(ROYAL SOC CHEMISTRY, 2015)Research into causes of Alzheimer's disease and its treatment has produced a tantalising array of hypotheses about the role of transition metal dyshomeostasis, many of them on the interaction of these metals with the neurotoxic amyloid-β peptide (Aβ). Here, we have used small angle X-ray scattering (SAXS) to study the effect of the molar ratio, Cu(2+)/Aβ, on the early three-dimensional structures of the Aβ1-40 and Cu(2+)/Aβ1-42 peptides in solution. We found that at molar ratios of 0.5 copper to peptide Aβ1-40 aggregated, while Aβ1-42 adopted a relatively monodisperse cylindrical shape, and at a ratio of 1.5 copper to peptide Aβ1-40 adopted a monodisperse cylindrical shape, while Aβ1-42 adopted the shape of an ellipsoid of rotation. We also found, via in-line rapid mixing SAXS analysis, that both peptides in the absence of copper were monodisperse at very short timeframes (<2 s). Kratky plots of these scattering profiles indicated that immediately after mixing both were intrinsically disordered. Ensemble optimisation modelling reflected this, indicating a wide range of structural conformers. These data reflect the ensembles from which the Cu(2+)-promoted oligomers were derived. Our results are discussed in the light of other studies that have shown that the Cu(2+)/Aβ has a marked effect on fibril and oligomer formation by this peptide, with a higher ratio favouring the formation of cytotoxic non-amyloid oligomers. Our results are relatively consistent with previous two-dimensional studies of the conformations of these Cu(2+)-induced entities, made on a much longer time-scale than SAXS, by transmission electron microscopy and atomic force microscopy, which showed that a range of oligomeric species are formed. We propose that SAXS carried out on a modern synchrotron beamline enables studies on initial events in disordered protein folding on physiologically-relevant time-scales, and will likely provide great insight into the initiating processes of the Aβ misfolding, oligomerisation and amyloid formation.
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ItemStabilization of Nontoxic Aβ-Oligomers: Insights into the Mechanism of Action of Hydroxyquinolines in Alzheimer's Disease(SOC NEUROSCIENCE, 2015-02-18)The extracellular accumulation of amyloid β (Aβ) peptides is characteristic of Alzheimer's disease (AD). However, formation of diffusible, oligomeric forms of Aβ, both on and off pathways to amyloid fibrils, is thought to include neurotoxic species responsible for synaptic loss and neurodegeneration, rather than polymeric amyloid aggregates. The 8-hydroxyquinolines (8-HQ) clioquinol (CQ) and PBT2 were developed for their ability to inhibit metal-mediated generation of reactive oxygen species from Aβ:Cu complexes and have both undergone preclinical and Phase II clinical development for the treatment of AD. Their respective modes of action are not fully understood and may include both inhibition of Aβ fibrillar polymerization and direct depolymerization of existing Aβ fibrils. In the present study, we find that CQ and PBT2 can interact directly with Aβ and affect its propensity to aggregate. Using a combination of biophysical techniques, we demonstrate that, in the presence of these 8-HQs and in the absence of metal ions, Aβ associates with two 8-HQ molecules and forms a dimer. Furthermore, 8-HQ bind Aβ with an affinity of 1-10 μm and suppress the formation of large (>30 kDa) oligomers. The stabilized low molecular weight species are nontoxic. Treatment with 8-HQs also reduces the levels of in vivo soluble oligomers in a Caenorhabditis elegans model of Aβ toxicity. We propose that 8-HQs possess an additional mechanism of action that neutralizes neurotoxic Aβ oligomer formation through stabilization of small (dimeric) nontoxic Aβ conformers.
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ItemUtility of an improved model of amyloid-beta (Aβ1-42) toxicity in Caenorhabditis elegans for drug screening for Alzheimer's disease(BMC, 2012-11-21)BACKGROUND: The definitive indicator of Alzheimer's disease (AD) pathology is the profuse accumulation of amyloid-ß (Aß) within the brain. Various in vitro and cell-based models have been proposed for high throughput drug screening for potential therapeutic benefit in diseases of protein misfolding. Caenorhabditis elegans offers a convenient in vivo system for examination of Aß accumulation and toxicity in a complex multicellular organism. Ease of culturing and a short life cycle make this animal model well suited to rapid screening of candidate compounds. RESULTS: We have generated a new transgenic strain of C. elegans that expresses full length Aß₁₋₄₂. This strain differs from existing Aß models that predominantly express amino-truncated Aß₃₋₄₂. The Aß₁₋₄₂ is expressed in body wall muscle cells, where it oligomerizes, aggregates and results in severe, and fully penetrant, age progressive-paralysis. The in vivo accumulation of Aß₁₋₄₂ also stains positive for amyloid dyes, consistent with in vivo fibril formation. The utility of this model for identification of potential protective compounds was examined using the investigational Alzheimer's therapeutic PBT2, shown to be neuroprotective in mouse models of AD and significantly improve cognition in AD patients. We observed that treatment with PBT2 provided rapid and significant protection against the Aß-induced toxicity in C. elegans. CONCLUSION: This C. elegans model of full length Aß₁₋₄₂ expression can now be adopted for use in screens to rapidly identify and assist in development of potential therapeutics and to study underlying toxic mechanism(s) of Aß.
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ItemSynthetic dityrosine-linked β-amyloid dimers form stable, soluble, neurotoxic oligomers(ROYAL SOC CHEMISTRY, 2013)
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ItemGuanidine hydrochloride denaturation of dopamine-induced α-synuclein oligomers: A small-angle X-ray scattering study(WILEY, 2014-01)Alpha-synuclein (α-syn) forms the amyloid-containing Lewy bodies found in the brain in Parkinson's disease. The neurotransmitter dopamine (DA) reacts with α-syn to form SDS-resistant soluble, non-amyloid, and melanin-containing oligomers. Their toxicity is debated, as is the nature of their structure and their relation to amyloid-forming conformers of α-syn. The small-angle X-ray scattering technique in combination with modeling by the ensemble optimization method showed that the un-reacted native protein populated three broad classes of conformer, while reaction with DA gave a restricted ensemble range suggesting that the rigid melanin molecule played an important part in their structure. We found that 6 M guanidine hydrochloride did not dissociate α-syn DA-reacted dimers and trimers, suggesting covalent linkages. The pathological significance of covalent association is that if they are non-toxic, the oligomers would act as a sink for toxic excess DA and α-syn; if toxic, their stability could enhance their toxicity. We argue it is essential, therefore, to resolve the question of whether they are toxic or not.