Florey Department of Neuroscience and Mental Health - Research Publications
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ItemA randomized, exploratory molecular imaging study targeting amyloid β with a novel 8-OH quinoline in Alzheimer's disease: The PBT2-204 IMAGINE study.(Wiley, 2017-11)INTRODUCTION: We are developing a second generation 8-OH quinoline (2-(dimethylamino) methyl-5, 7-dichloro-8-hydroxyquinoline [PBT2, Prana Biotechnology]) for targeting amyloid β (Aβ) in Alzheimer's disease (AD). In an earlier phase IIa, 3 month trial, PBT2 lowered cerebrospinal fluid Aβ by 13% and improved cognition (executive function) in a dose-related fashion in early AD. We, therefore, sought to learn whether PBT2 could alter the Aβ-PET signal in subjects with prodromal or mild AD, in an exploratory randomized study over a 12-month phase in a double-blind and a 12-month open label extension phase trial design. METHODS: For inclusion, the usual clinical criteria for prodromal or probable AD, Mini-Mental State Examination ≥20, and global Pittsburgh compound B (PiB)-PET standardized uptake volume ratio (SUVR) >1.7 were used. As this was an exploratory study, we included contemporaneous matched control data from the Australian Imaging Biomarker and Lifestyle Study (AIBL). Other measures included fluorodeoxyglucose-positron emission tomography, magnetic resonance imaging volumetrics, blood Aβ biomarkers, and cognition and function. RESULTS: Forty subjects completed the first 12-month double-blind phase (placebo = 15, PBT2 = 25), and 27 subjects completed the 12-month open label extension phase (placebo = 11, PBT2 = 16). Overall, PTB2 250 mg/day was safe and well tolerated. The mean PiB-PET SUVR at baseline was 2.51 ± 0.59. After adjusting for baseline SUVR, in the double-blind phase, the placebo group showed a nonsignificant decline in PiB-PET SUVR, whereas the PBT2 group declined significantly (P = .048). Subjects who did not enter or complete the extension study had a significantly higher 12-month Aβ-PET SUVR (2.68 ± 0.55) compared with those who completed (2.29 ± 0.48). Both groups differed significantly from the rate of change over 12 months in the AIBL control group. In the open label 12-month extension study, the PiB-SUVR stabilized. There were no significant differences between PBT2 and controls in fluorodeoxyglucose-positron emission tomography, magnetic resonance imaging volumetrics, blood Aβ biomarkers, or cognition/function over the course of the double-blind phase. DISCUSSION: There was no significant difference between PBT2 and controls at 12 months, likely due to the large individual variances over a relatively small number of subjects. PBT2 was associated with a significant 3% PiB-PET SUVR decline in the double-blind phase and a stabilization of SUVR in the open-label phase. From this exploratory study, we have learned that the entry criterion of SUVR should have been set at ≥ 1.5 and <2.0, where we know from the AIBL study that subjects in this band are accumulating Aβ in a linear fashion and that subjects who withdrew from this type of study have much higher SUVRs, which if not taken into account, could distort the final results. Because of large individual variations in SUVR, future studies of PBT2 will require larger numbers of subjects (n > 90 per arm) over a longer period (18 months or more). Further evaluation of higher doses of PBT2 in earlier stages of AD is warranted. TRIAL REGISTRATION: ACTRN 12611001008910 and ACTRN 12613000777796.
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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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ItemThe novel compound PBT434 prevents iron mediated neurodegeneration and alpha-synuclein toxicity in multiple models of Parkinson's disease(BMC, 2017-06-28)Elevated iron in the SNpc may play a key role in Parkinson's disease (PD) neurodegeneration since drug candidates with high iron affinity rescue PD animal models, and one candidate, deferirpone, has shown efficacy recently in a phase two clinical trial. However, strong iron chelators may perturb essential iron metabolism, and it is not yet known whether the damage associated with iron is mediated by a tightly bound (eg ferritin) or lower-affinity, labile, iron pool. Here we report the preclinical characterization of PBT434, a novel quinazolinone compound bearing a moderate affinity metal-binding motif, which is in development for Parkinsonian conditions. In vitro, PBT434 was far less potent than deferiprone or deferoxamine at lowering cellular iron levels, yet was found to inhibit iron-mediated redox activity and iron-mediated aggregation of α-synuclein, a protein that aggregates in the neuropathology. In vivo, PBT434 did not deplete tissue iron stores in normal rodents, yet prevented loss of substantia nigra pars compacta neurons (SNpc), lowered nigral α-synuclein accumulation, and rescued motor performance in mice exposed to the Parkinsonian toxins 6-OHDA and MPTP, and in a transgenic animal model (hA53T α-synuclein) of PD. These improvements were associated with reduced markers of oxidative damage, and increased levels of ferroportin (an iron exporter) and DJ-1. We conclude that compounds designed to target a pool of pathological iron that is not held in high-affinity complexes in the tissue can maintain the survival of SNpc neurons and could be disease-modifying in PD.
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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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ItemThe effect of paraformaldehyde fixation and sucrose cryoprotection on metal concentration in murine neurological tissue(ROYAL SOC CHEMISTRY, 2014-03)
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ItemProfiling the iron, copper and zinc content in primary neuron and astrocyte cultures by rapid online quantitative size exclusion chromatography-inductively coupled plasma-mass spectrometry(ROYAL SOC CHEMISTRY, 2013)Metals often determine the chemical reactivity of the proteins to which they are bound. Each cell in the body tightly maintains a unique metalloproteomic profile, mostly dependent on function. This paper describes an analytical online flow injection quantitative size exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS) method, which was applied to profiling the metal-binding proteins found in primary cultures of neurons and astrocytes. This method can be conducted using similar amounts of sample to those used for Western blotting (20-150 μg protein), and has a turnaround time of <15 minutes. Metalloprotein standards for Fe (as ferritin), Cu and Zn (as superoxide dismutase-1) were used to construct multi-point calibration curves for online quantification of metalloproteins by SEC-ICP-MS. Homogenates of primary neuron and astrocyte cultures were analysed by SEC-ICP-MS. Online quantification by external calibration with metalloprotein standards determined the mass of metal eluting from the column relative to time (as pg s(-1)). Total on-column Fe, Cu and Zn detection limits ranged from 0.825 ± 0.005 ng to 13.6 ± 0.7 pg. Neurons and astrocytes exhibited distinct metalloprotein profiles, featuring both ubiquitous and unique metalloprotein species. Separation and detection by SEC-ICP-MS allows appraisal of these metalloproteins in their native state, and online quantification was achieved using this relatively simple external calibration process.
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ItemDecreased serum zinc is an effect of ageing and not Alzheimer's disease(ROYAL SOC CHEMISTRY, 2014)We examined the distribution of zinc in the periphery (erythrocytes and serum) in a large, well-characterised cohort, the Australian Imaging, Biomarkers and Lifestyle (AIBL) study, in order to determine if there is systemic perturbation in zinc homeostasis in Alzheimer's disease (AD). We observed an age dependent decrease in serum zinc of approximately 0.4% per year. When correcting for the age dependent decline in serum zinc no significant difference between healthy controls (HC), mildly cognitively impaired (MCI) or AD subjects was observed.
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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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ItemIntracellular amyloid formation in muscle cells of Aβ-transgenic Caenorhabditis elegans: determinants and physiological role in copper detoxification(BMC, 2009-01-06)BACKGROUND: The amyloid beta-peptide is a ubiquitous peptide, which is prone to aggregate forming soluble toxic oligomers and insoluble less-toxic aggregates. The intrinsic and external/environmental factors that determine Abeta aggregation in vivo are poorly understood, as well as the cellular meaning of this process itself. Genetic data as well as cell biological and biochemical evidence strongly support the hypothesis that Abeta is a major player in the onset and development of Alzheimer's disease. In addition, it is also known that Abeta is involved in Inclusion Body Myositis, a common myopathy of the elderly in which the peptide accumulates intracellularly. RESULTS: In the present work, we found that intracellular Abeta aggregation in muscle cells of Caenorhabditis elegans overexpressing Abeta peptide is affected by two single amino acid substitutions, E22G (Arctic) and V18A (NIC). Both variations show decrease intracellular amyloidogenesis compared to wild type Abeta. We show that intracellular amyloid aggregation of wild type Abeta is accelerated by Cu2+ and diminished by copper chelators. Moreover, we demonstrate through toxicity and behavioral assays that Abeta-transgenic worms display a higher tolerance to Cu2+ toxic effects and that this resistance may be linked to the formation of amyloid aggregates. CONCLUSION: Our data show that intracellular Abeta amyloid aggregates may trap excess of free Cu2+ buffering its cytotoxic effects and that accelerated intracellular Abeta aggregation may be part of a cell protective mechanism.