Pharmacology and Therapeutics - Research Publications
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ItemDiacetylbis(N(4)-methylthiosemicarbazonato) Copper(II) (CuII(atsm)) Protects against Peroxynitrite-induced Nitrosative Damage and Prolongs Survival in Amyotrophic Lateral Sclerosis Mouse Model(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2011-12-23)Amyotrophic lateral sclerosis (ALS) is a progressive paralyzing disease characterized by tissue oxidative damage and motor neuron degeneration. This study investigated the in vivo effect of diacetylbis(N(4)-methylthiosemicarbazonato) copper(II) (CuII(atsm)), which is an orally bioavailable, blood-brain barrier-permeable complex. In vitro the compound inhibits the action of peroxynitrite on Cu,Zn-superoxide dismutase (SOD1) and subsequent nitration of cellular proteins. Oral treatment of transgenic SOD1G93A mice with CuII(atsm) at presymptomatic and symptomatic ages was performed. The mice were examined for improvement in lifespan and motor function, as well as histological and biochemical changes to key disease markers. Systemic treatment of SOD1G93A mice significantly delayed onset of paralysis and prolonged lifespan, even when administered to symptomatic animals. Consistent with the properties of this compound, treated mice had reduced protein nitration and carbonylation, as well as increased antioxidant activity in spinal cord. Treatment also significantly preserved motor neurons and attenuated astrocyte and microglial activation in mice. Furthermore, CuII(atsm) prevented the accumulation of abnormally phosphorylated and fragmented TAR DNA-binding protein-43 (TDP-43) in spinal cord, a protein pivotal to the development of ALS. CuII(atsm) therefore represents a potential new class of neuroprotective agents targeting multiple major disease pathways of motor neurons with therapeutic potential for ALS.
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ItemNo Preview AvailableEffect of Metal Chelators on γ-Secretase Indicates That Calcium and Magnesium Ions Facilitate Cleavage of Alzheimer Amyloid Precursor Substrate.(Hindawi Limited, 2010-12-28)Gamma-secretase is involved in the production of Aβ amyloid peptides. It cleaves the transmembrane domain of the amyloid precursor protein (APP) at alternative sites to produce Aβ and the APP intracellular domain (AICD). Metal ions play an important role in Aβ aggregation and metabolism, thus metal chelators and ligands represent potential therapeutic agents for AD treatment. A direct effect of metal chelators on γ-secretase has not yet been investigated. The authors used an in vitro γ-secretase assay consisting of cleavage of APP C100-3XFLAG by endogenous γ-secretase from rodent brains and human neuroblastoma SH-SY5Y, and detected AICD production by western blotting. Adding metalloprotease inhibitors to the reaction showed that clioquinol, phosphoramidon, and zinc metalloprotease inhibitors had no significant effect on γ-secretase activity. In contrast, phenanthroline, EDTA, and EGTA markedly decreased γ-secretase activity that could be restored by adding back calcium and magnesium ions. Mg(2+) stabilized a 1,000 kDa presenilin 1 complex through blue native gel electrophoresis and size-exclusion chromatography. Data suggest that Ca(2+) and Mg(2+) stabilize γ-secretase and enhance its activity.
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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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ItemNo Preview AvailableCopper Promotes the Trafficking of the Amyloid Precursor Protein(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2011-03-11)Accumulation of the amyloid β peptide in the cortical and hippocampal regions of the brain is a major pathological feature of Alzheimer disease. Amyloid β peptide is generated from the sequential protease cleavage of the amyloid precursor protein (APP). We reported previously that copper increases the level of APP at the cell surface. Here we report that copper, but not iron or zinc, promotes APP trafficking in cultured polarized epithelial cells and neuronal cells. In SH-SY5Y neuronal cells and primary cortical neurons, copper promoted a redistribution of APP from a perinuclear localization to a wider distribution, including neurites. Importantly, a change in APP localization was not attributed to an up-regulation of APP protein synthesis. Using live cell imaging and endocytosis assays, we found that copper promotes an increase in cell surface APP by increasing its exocytosis and reducing its endocytosis, respectively. This study identifies a novel mechanism by which copper regulates the localization and presumably the function of APP, which is of major significance for understanding the role of APP in copper homeostasis and the role of copper in Alzheimer disease.
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ItemVariability in Blood-Based Amyloid-β Assays: The Need for Consensus on Pre-Analytical Processing(IOS PRESS, 2012)Effective therapeutic interventions for Alzheimer's disease (AD) will require treatment regimes to move toward the earliest stages of the disease. For this to occur the field has to identify biomarkers that are able to accurately identify individuals at risk for progression toward AD in the presymptomatic stage. One very significant implication is that some form of population-based screening will need to be undertaken in order to identify those at risk. To date, efforts in neuroimaging brain amyloid-β (Aβ) and changes in cerebrospinal fluid Aβ and tau levels shows promise, however, it is questionable as to whether these methods are applicable for screening the general population. The Aβ peptide is also found in blood which is the most economical and efficient biological fluid to analyze. Unfortunately, investigations into blood-based diagnostic markers have produced mixed results. This variability is likely to be the result of differences in the preanalytical processing of samples and as such is delaying progress in the field. Reported preanalytical processing techniques from 87 recent articles focusing on the measurement of Aβ in blood were compared, to investigate whether basic sample-handling techniques were comparable between studies. This comparison revealed that not only is it likely that some of the variability in blood-based results is attributable to discrepancies in preanalytical methodologies but also that the field is failing to adequately report sample processing techniques. This review highlights the current shortcomings in methodological reporting and recommends a standardized blood collection methodology based on the limited consensus of the reviewed articles.
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ItemPeripheral α-Defensins 1 and 2 are Elevated in Alzheimer's Disease(IOS PRESS, 2015)Biomarkers enabling the preclinical identification of Alzheimer's disease (AD) remain one of the major unmet challenges in the field. The blood cellular fractions offer a viable alternative to current cerebrospinal fluid and neuroimaging modalities. The current study aimed to replicate our earlier reports of altered binding within the AD-affected blood cellular fraction to copper-loaded immobilized metal affinity capture (IMAC) arrays. IMAC and anti-amyloid-β (Aβ) antibody arrays coupled with mass spectrometry were used to analyze blood samples collected from 218 participants from within the AIBL Study of Aging. Peripheral Aβ was fragile and prone to degradation in the AIBL samples, even when stored at -80°C. IMAC analysis of the AIBL samples lead to the isolation and identification of alpha-defensins 1 and 2 at elevated levels in the AD periphery, validating earlier findings. Alpha-defensins 1 and 2 were elevated in AD patients indicating that an inflammatory phenotype is present in the AD periphery; however, peripheral Aβ levels are required to supplement their prognostic power.
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ItemC-Jun N-terminal kinase controls TDP-43 accumulation in stress granules induced by oxidative stress(BIOMED CENTRAL LTD, 2011-08-08)BACKGROUND: TDP-43 proteinopathies are characterized by loss of nuclear TDP-43 expression and formation of C-terminal TDP-43 fragmentation and accumulation in the cytoplasm. Recent studies have shown that TDP-43 can accumulate in RNA stress granules (SGs) in response to cell stresses and this could be associated with subsequent formation of TDP-43 ubiquinated protein aggregates. However, the initial mechanisms controlling endogenous TDP-43 accumulation in SGs during chronic disease are not understood. In this study we investigated the mechanism of TDP-43 processing and accumulation in SGs in SH-SY5Y neuronal-like cells exposed to chronic oxidative stress. Cell cultures were treated overnight with the mitochondrial inhibitor paraquat and examined for TDP-43 and SG processing. RESULTS: We found that mild stress induced by paraquat led to formation of TDP-43 and HuR-positive SGs, a proportion of which were ubiquitinated. The co-localization of TDP-43 with SGs could be fully prevented by inhibition of c-Jun N-terminal kinase (JNK). JNK inhibition did not prevent formation of HuR-positive SGs and did not prevent diffuse TDP-43 accumulation in the cytosol. In contrast, ERK or p38 inhibition prevented formation of both TDP-43 and HuR-positive SGs. JNK inhibition also inhibited TDP-43 SG localization in cells acutely treated with sodium arsenite and reduced the number of aggregates per cell in cultures transfected with C-terminal TDP-43 162-414 and 219-414 constructs. CONCLUSIONS: Our studies are the first to demonstrate a critical role for kinase control of TDP-43 accumulation in SGs and may have important implications for development of treatments for FTD and ALS, targeting cell signal pathway control of TDP-43 aggregation.
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ItemHigh Order W02-Reactive Stable Oligomers of Amyloid-β are Produced in vivo and in vitro via Dialysis and Filtration of Synthetic Amyloid-β Monomer(IOS PRESS, 2015)Oligomeric forms of amyloid-β (Aβ) are thought to be responsible for the pathogenesis of Alzheimer's disease. While many oligomers of Aβ are thought to be naturally occurring in the brain of humans and/or transgenic animals, it is well known that Aβ oligomers are also readily produced in vitro in the laboratory. In recent studies, we discovered that synthetic monomeric Aβ (4.7 kDa) could be transformed by microdialysis to higher molecular weight species (approximately 56 kDa, by western blot). Surface-enhanced laser desorption/ionization mass spectrometry and electron microscopy further identified these species' as potential Aβ oligomers. The production of similar species could also be produced by centrifugal filtration and this formation was concentration and pore-size dependent. These higher order species of Aβ were resistant to dissolution in NaOH, HFIP, formic acid, urea, and guanidine. We postulate that we have identified a novel way of producing a high order species of oligomeric Aβ and we provide evidence to suggest that Aβ oligomers can quite easily be a product of normal laboratory practices. These data suggest that the experimental detection of higher order oligomers in tissues derived from Alzheimer's disease brains or from animal models of disease could, in some cases, be a product the method of analysis.
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ItemAmyloid-Beta Peptide Aβ3pE-42 Induces Lipid Peroxidation, Membrane Permeabilization and Calcium-Influx in Neurons(American Society for Biochemistry and Molecular Biology, 2016)Pyroglutamate-modified amyloid-β (pE-Aβ) is a highly neurotoxic amyloid-β (Aβ) isoform and is enriched in the brains of individuals with Alzheimer disease compared with healthy aged controls. Pyroglutamate formation increases the rate of Aβ oligomerization and alters the interactions of Aβ with Cu(2+) and lipids; however, a link between these properties and the toxicity of pE-Aβ peptides has not been established. We report here that Aβ3pE-42 has an enhanced capacity to cause lipid peroxidation in primary cortical mouse neurons compared with the full-length isoform (Aβ(1-42)). In contrast, Aβ(1-42) caused a significant elevation in cytosolic reactive oxygen species, whereas Aβ3pE-42 did not. We also report that Aβ3pE-42 preferentially associates with neuronal membranes and triggers Ca(2+) influx that can be partially blocked by the N-methyl-d-aspartate receptor antagonist MK-801. Aβ3pE-42 further caused a loss of plasma membrane integrity and remained bound to neurons at significantly higher levels than Aβ(1-42) over extended incubations. Pyroglutamate formation was additionally found to increase the relative efficiency of Aβ-dityrosine oligomer formation mediated by copper-redox cycling.
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ItemAlpha-synuclein oligomers and fibrils originate in two distinct conformer pools: a small angle X-ray scattering and ensemble optimisation modelling study(ROYAL SOC CHEMISTRY, 2015-01)The 140 residue intrinsically disordered protein α-synuclein (α-syn) self-associates to form fibrils that are the major constituent of the Lewy body intracellular protein inclusions, and neurotoxic oligomers. Both of these macromolecular structures are associated with a number of neurodegenerative diseases, including Parkinson's disease and dementia with Lewy bodies. Using ensemble optimisation modelling (EOM) and small angle X-ray scattering (SAXS) on a size-exclusion column equipped beamline, we studied how the distribution of structural conformers in α-syn may be influenced by the presence of the familial early-onset mutations A30P, E45K and A53T, by substituting the four methionine residues with alanines and by reaction with copper (Cu2+) or an anti-fibril organic platinum (Pt) complex. We found that the WT had two major conformer groups, representing ensembles of compact and extended structures. The population of the extended group was increased in the more rapidly fibril-forming E45K and A53T mutants, while the compact group was enlarged in the oligomer-forming A30P mutant. Addition of Cu2+ resulted in the formation of an ensemble of compact conformers, while the anti-fibril agent and alanine substitution substantially reduced the population of extended conformers. Since our observations with the mutants suggest that fibrils may be drawn from the extended conformer ensemble, we propose that the compact and extended ensembles represent the beginning of oligomer and fibril formation pathways respectively, both of which have been reported to lead to a toxic gain of function. Manipulating these pathways and monitoring the results by EOM and SAXS may be useful in the development of anti-Parkinson's disease therapies.