Publications by authors named "Blaine R Roberts"

66 Publications

Utilizing Ion Mobility-Mass Spectrometry to Investigate the Unfolding Pathway of Cu/Zn Superoxide Dismutase.

Front Chem 2021 9;9:614595. Epub 2021 Feb 9.

Department of Biochemistry, Emory University School of Medicine, Atlanta, GA, United States.

Native mass spectrometry has emerged as a powerful tool for structural biology as it enables the evaluation of molecules as they occur in their physiological conditions. Ion mobility spectrometry-mass spectrometry (IMS-MS) has shown essential in these analyses as it allows the measurement of the shape of a molecule, denoted as its collision cross section (CCS), and mass. The structural information garnered from native IMS-MS provides insight into the tertiary and quaternary structure of proteins and can be used to validate NMR or crystallographic X-ray structures. Additionally, due to the rapid nature (millisecond measurements) and ability of IMS-MS to analyze heterogeneous solutions, it can be used to address structural questions not possible with traditional structural approaches. Herein, we applied multiple solution conditions to systematically denature bovine Cu/Zn-superoxide dismutase (SOD1) and assess its unfolding pathway from the holo-dimer to the holo-monomer, single-metal monomer, and apo-monomer. Additionally, we compared and noted 1-2% agreement between CCS values from both drift tube IMS and trapped IMS for the SOD1 holo-monomer and holo-dimer. The observed CCS values were in excellent agreement with computational CCS values predicted from the homo-dimer crystal structure, showcasing the ability to use both IMS-MS platforms to provide valuable structural information for molecular modeling of protein interactions and structural assessments.
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http://dx.doi.org/10.3389/fchem.2021.614595DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7900566PMC
February 2021

Cadmium stress dictates central carbon flux and alters membrane composition in Streptococcus pneumoniae.

Commun Biol 2020 Nov 19;3(1):694. Epub 2020 Nov 19.

Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, The University of Melbourne, Melbourne, VIC, Australia.

Metal ion homeostasis is essential for all forms of life. However, the breadth of intracellular impacts that arise upon dysregulation of metal ion homeostasis remain to be elucidated. Here, we used cadmium, a non-physiological metal ion, to investigate how the bacterial pathogen, Streptococcus pneumoniae, resists metal ion stress and dyshomeostasis. By combining transcriptomics, metabolomics and metalloproteomics, we reveal that cadmium stress dysregulates numerous essential cellular pathways including central carbon metabolism, lipid membrane biogenesis and homeostasis, and capsule production at the transcriptional and/or functional level. Despite the breadth of cellular pathways susceptible to metal intoxication, we show that S. pneumoniae is able to maintain viability by utilizing cellular pathways that are predominately metal-independent, such as the pentose phosphate pathway to maintain energy production. Collectively, this work provides insight into the cellular processes impacted by cadmium and how resistance to metal ion toxicity is achieved in S. pneumoniae.
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http://dx.doi.org/10.1038/s42003-020-01417-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7678824PMC
November 2020

Increased glutaminyl cyclase activity in brains of Alzheimer's disease individuals.

J Neurochem 2021 Mar 20;156(6):979-987. Epub 2020 Jul 20.

Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, Vic, Australia.

Glutaminyl cyclases (QC) catalyze the formation of neurotoxic pGlu-modified amyloid-β peptides found in the brains of people with Alzheimer's disease (AD). Reports of several-fold increases in soluble QC (sQC) expression in the brain and peripheral circulation of AD individuals has prompted the development of QC inhibitors as potential AD therapeutics. There is, however, a lack of standardized quantitative data on QC expression in human tissues, precluding inter-laboratory comparison and validation. We tested the hypothesis that QC is elevated in AD tissues by quantifying levels of sQC protein and activity in post-mortem brain tissues from AD and age-matched control individuals. We found a modest but statistically significant increase in sQC protein, which paralleled a similar increase in enzyme activity. In plasma samples sourced from the Australian Imaging, Biomarker and Lifestyle study we determined that QC activity was not different between the AD and control group, though a modest increase was observed in female AD individuals compared to controls. Plasma QC activity was further correlated with levels of circulating monocytes in AD individuals. These data provide quantitative evidence that alterations in QC expression are associated with AD pathology.
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http://dx.doi.org/10.1111/jnc.15114DOI Listing
March 2021

PrPSc Oligomerization Appears Dynamic, Quickly Engendering Inherent M1000 Acute Synaptotoxicity.

Biophys J 2020 07 10;119(1):128-141. Epub 2020 Jun 10.

Department of Medicine (RMH), The University of Melbourne, Parkville, Australia; Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, Australia. Electronic address:

Prion diseases are neurodegenerative disorders pathogenically linked to cellular prion protein (PrPC) misfolding into abnormal conformers (PrPSc), with PrPSc underpinning both transmission and synaptotoxicity. Although the biophysical features of PrPSc required to induce acute synaptic dysfunction remain incompletely defined, we recently reported that acutely synaptotoxic PrPSc appeared to be oligomeric. We herein provide further insights into the kinetic and requisite biophysical characteristics of acutely synaptotoxic ex vivo PrPSc derived from the brains of mice dying from M1000 prion disease. Pooled fractions of M1000 PrPSc located within the molecular weight range approximating monomeric PrP (mM1000) generated through size exclusion chromatography were found to harbor acute synaptotoxicity equivalent to preformed oligomeric fractions (oM1000). Subsequent investigation showed mM1000 corresponded to PrPSc rapidly concatenating in physiological buffer to exist as predominantly, closely associated, small oligomers. The oligomerization of PrP in mM1000 could be substantially mitigated by treatment with the antiaggregation compound epigallocatechin gallate, thereby maintaining the PrPSc as primarily nonoligomeric with completely abrogated acute synaptotoxicity; moreover, despite epigallocatechin gallate treatment, pooled oM1000 remained oligomeric and acutely synaptotoxic. A similar tendency to rapid formation of oligomers was observed for PrPC when monomeric fractions derived from size exclusion chromatography of normal brain homogenates (mNBH) were pooled, but neither mNBH nor preformed higher-order NBH complexes (oNBH) were acutely synaptotoxic. Oligomers formed from mNBH could be reduced to mainly monomers (<100 kDa) after enzymatic digestion of nucleic acids, whereas higher-order PrP assemblies derived from pooled mM1000, oM1000, and oNBH resisted such treatment. Collectively, these findings support that oligomerization of PrPSc into small multimeric assemblies appears to be a critical biophysical feature for engendering inherent acute synaptotoxicity, with preformed oligomers found in oM1000 appearing to be stable, tightly self-associated ensembles that coexist in dynamic equilibrium with mM1000, with the latter appearing capable of rapid aggregation, albeit initially forming smaller, weakly self-associated, acutely synaptotoxic oligomers.
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http://dx.doi.org/10.1016/j.bpj.2020.04.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7335956PMC
July 2020

Human glutaredoxin-1 can transfer copper to isolated metal binding domains of the P-type ATPase, ATP7B.

Sci Rep 2020 03 5;10(1):4157. Epub 2020 Mar 5.

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, Australia.

Intracellular copper (Cu) in eukaryotic organisms is regulated by homeostatic systems, which rely on the activities of soluble metallochaperones that participate in Cu exchange through highly tuned protein-protein interactions. Recently, the human enzyme glutaredoxin-1 (hGrx1) has been shown to possess Cu metallochaperone activity. The aim of this study was to ascertain whether hGrx1 can act in Cu delivery to the metal binding domains (MBDs) of the P-type ATPase ATP7B and to determine the thermodynamic factors that underpin this activity. hGrx1 can transfer Cu to the metallochaperone Atox1 and to the MBDs 5-6 of ATP7B (WLN5-6). This exchange is irreversible. In a mixture of the three proteins, Cu is delivered to the WLN5-6 preferentially, despite the presence of Atox1. This preferential Cu exchange appears to be driven by both the thermodynamics of the interactions between the proteins pairs and of the proteins with Cu(I). Crucially, protein-protein interactions between hGrx1, Atox1 and WLN5-6 were detected by NMR spectroscopy both in the presence and absence of Cu at a common interface. This study augments the possible activities of hGrx1 in intracellular Cu homeostasis and suggests a potential redundancy in this system, where hGrx1 has the potential to act under cellular conditions where the activity of Atox1 in Cu regulation is attenuated.
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http://dx.doi.org/10.1038/s41598-020-60953-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7057996PMC
March 2020

Optimizing red blood cell protein extraction for biomarker quantitation with mass spectrometry.

Anal Bioanal Chem 2020 Mar 6;412(8):1879-1892. Epub 2020 Feb 6.

The Florey Institute of Neuroscience and Mental Health, Melbourne Dementia Research Centre, The University of Melbourne, 30 Royal Parade, Parkville, Victoria, 3052, Australia.

Red blood cells (RBC) are the most common cell type found in blood. They might serve as reservoir for biomarker research as they are anuclear and lack the ability to synthesize proteins. Not many biomarker assays, however, have been conducted on RBC because of their large dynamic range of proteins, high abundance of lipids, and hemoglobin interferences. Here, we developed a semiquantitative mass spectrometry-based assay that targeted 144 proteins and compared the efficiency of urea, sodium deoxycholate, acetonitrile, and HemoVoid™ in their extraction of the RBC proteome. Our results indicate that protein extraction with HemoVoid™ led to hemoglobin reduction and increased detection of low abundance proteins. Although hemoglobin interference after deoxycholate and urea extraction was high, there were adequate amounts of low abundance proteins for quantitation. Extraction with acetonitrile led to an overall decrease in protein abundances probably as a result of precipitation. Overall, the best compromise in sensitivity and sample processing time was achieved with the urea-trypsin digestion protocol. This provided the basis for large-scale evaluations of protein targets as potential blood-based biomarkers. As a proof of concept, we applied this assay to determine that alpha-synuclein, a prominent marker in Parkinson's disease, has an average concentration of approximately 40 μg mL in RBC. This is important to know as the concentration of alpha-synuclein in plasma, typically in the picogram per milliliter range, might be partially derived from lysed RBC. Utilization of this assay will prove useful for future biomarker studies and provide a more complete analytical toolbox for the measurement of blood-derived proteins. Graphical abstract.
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http://dx.doi.org/10.1007/s00216-020-02439-5DOI Listing
March 2020

Zinc Transporter-3 Knockout Mice Demonstrate Age-Dependent Alterations in the Metalloproteome.

Int J Mol Sci 2020 Jan 28;21(3). Epub 2020 Jan 28.

The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Melbourne, VIC 3010, Australia.

Metals are critical cellular elements that are involved in a variety of cellular processes, with recent literature demonstrating that zinc, and the synaptic zinc transporter (ZnT3), are specifically involved in learning and memory and may also be key players in age-related neurodegenerative disorders such as Alzheimer's disease. Whilst the cellular content and location of metals is critical, recent data has demonstrated that the metalation state of proteins is a determinant of protein function and potential toxicity. As we have previously reported that ZnT3 knockout (KO) mice have deficits in total zinc levels at both 3 and 6 months of age, we were interested in whether there might be changes in the metalloproteomic profile in these animals. To do this, we utilised size exclusion chromatography-inductively coupled plasma mass spectrometry (SEC-ICP-MS) and examined hippocampal homogenates from ZnT3 KO and age-matched wild-type mice at 3, 6 and 18 months of age. Our data suggest that there are alterations in specific metal binding proteins, for zinc, copper and iron all being modulated in the ZnT3 KO mice compared to wild-type (WT). These data suggest that ZnT3 KO mice may have impairments in the levels or localisation of multiple transition metals, and that copper- and iron-dependent cellular pathways may also be impacted in these mice.
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http://dx.doi.org/10.3390/ijms21030839DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7037208PMC
January 2020

In situ 3D visualization of biomineralization matrix proteins.

J Struct Biol 2020 03 9;209(3):107448. Epub 2020 Jan 9.

School of BioSciences, The University of Melbourne, Parkville Campus, Victoria 3010, Australia.

Calcium biominerals occur in all major animal phyla, and through biomolecular control, exhibit such diverse structures as exoskeletons, shells, bones, teeth and earstones (otoliths). Determining the three-dimensional expression of key biomineral proteins, however, has proven challenging as typical protein identification methods either lose spatial resolution during dissolution of the mineral phase or are costly and limited to two-dimensional expression of high abundance proteins. Here we present a modification of the CLARITY and ACT-PRESTO protocols to visualize and confirm, for the first time, the timing of expression and function of two key regulators of biomineralization.
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http://dx.doi.org/10.1016/j.jsb.2020.107448DOI Listing
March 2020

Choice of mobile phase: Implications for size exclusion chromatography-inductively coupled plasma-mass spectrometry analyses of copper, zinc and iron metalloproteins.

J Chromatogr A 2020 Apr 18;1616:460806. Epub 2019 Dec 18.

Department of Biochemistry, Emory University School of Medicine, Atlanta, GA, 30322 USA. Electronic address:

The correct identification of the metalloproteins present in human tissues and fluids is essential to our understanding of the cellular mechanisms underpinning a host of health disorders. Separation and analysis of biological samples are typically done via size exclusion chromatography hyphenated with inductively coupled plasma-mass spectrometry (SEC-ICP-MS). Although this technique can be extremely effective in identification of potential metalloproteins, the choice of mobile phase may have a marked effect on results, results by adversely affecting metal-protein bonds of the metalloproteins of interest. To assess the choice of mobile phase on SEC-ICP-MS resolution and the resulting metalloproteome pattern, we analysed several different sample types (brain homogenate; Cu/Zn-superoxide dismutase (SOD1); a molecular weight standard mix containing ferritin (Ft), ceruloplasmin (Cp), cytochrome c (CytC), vitamin B12 (B12) and thyroglobulin (Tg) using six different mobile phase conditions (200 mM, pH 7.5 solutions of ammonium salts nitrate, acetate, and sulfate; HEPES, MOPS and Tris-HCl). Our findings suggest that ammonium nitrate, ammonium acetate and Tris-HCl are optimal choices for the mobile phase, with the specific choice being dependent on both the number of samples and method of detection that is hyphenated with separation. Furthermore, we found that MOPS, HEPES and ammonium sulfate mobile phases all caused significant changes to peak resolution, retention time and overall profile shape. MOPS and HEPES, in particular, produced additional Fe peaks that were not detected with any of the other mobile phases that were investigated. As well as this, MOPS and HEPES both caused significant concentration dependent matrix suppression of the internal standard.
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http://dx.doi.org/10.1016/j.chroma.2019.460806DOI Listing
April 2020

Regional iron distribution and soluble ferroprotein profiles in the healthy human brain.

Prog Neurobiol 2020 03 20;186:101744. Epub 2019 Dec 20.

The Florey Institute of Neuroscience and Mental Health, Parkville, Victoria, Australia. Electronic address:

Iron is essential for brain development and health where its redox properties are used for a number of neurological processes. However, iron is also a major driver of oxidative stress if not properly controlled. Brain iron distribution is highly compartmentalised and regulated by a number of proteins and small biomolecules. Here, we examine heterogeneity in regional iron levels in 10 anatomical structures from seven post-mortem human brains with no apparent neuropathology. Putamen contained the highest levels, and most case-to-case variability, of iron compared with the other regions examined. Partitioning of iron between cytosolic and membrane-bound iron was generally consistent in each region, with a slightly higher proportion (55 %) in the 'insoluble' phase. We expand on this using the Allen Human Brain Atlas to examine patterns between iron levels and transcriptomic expression of iron regulatory proteins and using quantitative size exclusion chromatography-inductively coupled plasma-mass spectrometry to assess regional differences in the molecular masses to which cytosolic iron predominantly binds. Approximately 60 % was associated with ferritin, equating to approximately 25 % of total tissue iron essentially in storage. This study is the first of its kind in human brain tissue, providing a valuable resource and new insight for iron biologists and neuroscientists, alike.
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http://dx.doi.org/10.1016/j.pneurobio.2019.101744DOI Listing
March 2020

Expanding beyond ICP-MS to better understand selenium biochemistry.

Metallomics 2019 12;11(12):1974-1983

Deakin University, Institute for Physical Activity and Nutrition Research (IPAN), School of Exercise and Nutrition Sciences, Geelong, Australia.

Selenium is an essential trace element in human health and therefore its concentration in biological samples (biofluids and tissues) is used as an indicator of health and nutritional status. In humans, selenium's biological activity occurs through the 25 identified selenoproteins. As total selenium concentration encompasses both functional selenoproteins, small selenocompounds and other selenium-binding proteins, selenium speciation, rather than total concentration, is critical in order to assess functional selenium. Previously, quantitative analysis of selenoproteins required laborious techniques that were often slow and costly. However, more recent advancements in tandem mass spectrometry have facilitated the qualitative and quantitative identification of these proteins. In light of the current alternatives for understanding selenium biochemistry, we aim to provide a review of the modern applications of electrospray ionisation mass spectrometry (ESI-MS) as an alternative to inductively coupled plasma mass spectrometry (ICP-MS) for qualitative and quantitative selenium speciation.
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http://dx.doi.org/10.1039/c9mt00201dDOI Listing
December 2019

Greater Circulating Copper Concentrations and Copper/Zinc Ratios are Associated with Lower Psychological Distress, But Not Cognitive Performance, in a Sample of Australian Older Adults.

Nutrients 2019 10 17;11(10). Epub 2019 Oct 17.

Institute for Physical Activity and Nutrition (IPAN), School of Exercise and Nutrition Sciences, Deakin University, Geelong, VIC 3220, Australia.

Dyshomeostasis of copper and zinc is linked to neurodegeneration. This study investigated the relationship between circulating copper and zinc and copper/zinc ratios and cognitive function, symptoms of depression and anxiety, and neurotrophic factors in older Australian adults. In this cross-sectional study ( = 139), plasma copper, serum zinc, and neurotrophic factors (brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor, and insulin-like growth factor-1) were assessed. Cognition was assessed using the Cogstate battery and the Behavior Rating Inventory (BRI) of Executive Function (Adult version). Symptoms of anxiety and depression were assessed with the Hospital Anxiety and Depression Scale. Copper (β = -0.024; 95% CI = -0.044, -0.004; = 0.019) and copper/zinc ratio (β = -1.99; 95% CI = -3.41, -0.57; = 0.006) were associated with lower depressive symptoms, but not cognition. Plasma copper had a modest positive association with BDNF (β = -0.004; 95% CI = 0.000, 0.007; = 0.021). Zinc was not associated with any of the outcomes. In conclusion, greater circulating copper concentrations and higher copper/zinc ratios were associated with lower depressive symptoms (but not cognition), with copper also positively associated with BDNF concentration, in a sample of community-dwelling older adults.
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http://dx.doi.org/10.3390/nu11102503DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6836146PMC
October 2019

Establishing Signature Fragments for Identification and Sequencing of Dityrosine Cross-Linked Peptides Using Ultraviolet Photodissociation Mass Spectrometry.

Anal Chem 2019 10 11;91(19):12129-12133. Epub 2019 Sep 11.

Florey Institute of Neuroscience and Mental Health , University of Melbourne , Melbourne , Victoria 3010 , Australia.

Dityrosine cross-linking of Aβ peptides and α-synuclein is increasingly becoming recognized as a biomarker of neuropathological diseases. However, there remains a need for the development of analytical methods that enable the specific and selective identification of dityrosine cross-linked proteins and peptides in complex biological samples. Here, we report that the gas-phase fragmentation of protonated dityrosine cross-linked peptides under ultraviolet photodissociation (UVPD) tandem mass spectrometry (MS/MS) conditions results in the cleavage across C and C atoms of the dityrosine residue. This C-C cleavage in UVPD-MS/MS results in the formation of diagnostic pairs of product ions, providing information on the two individual peptides involved in the cross-linking, resolving the intrinsic " problem" plaguing the identification of this post-translational modification (PTM) by tandem mass spectrometry. Sequencing of a heterodimeric dityrosine cross-linked peptide was demonstrated using hybrid UVPD-MS/MS and CID-MS on a diagnostic pair of product ions. In combination with dedicated MS-cleavable MS software, UVPD-MS therefore provides an avenue to selectively discover and describe dityrosine cross-linked peptides. Additionally, observation of dityrosine-specific "reporter ions" at / 240.1019 and / 223.0752 in UVPD-MS/MS will be useful for the validation of the dityrosine cross-linked peptides.
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http://dx.doi.org/10.1021/acs.analchem.9b02986DOI Listing
October 2019

The inner ear proteome of fish.

FEBS J 2019 01 21;286(1):66-81. Epub 2018 Dec 21.

The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, Vic., Australia.

The mechanisms that underpin the formation, growth and composition of otoliths, the biomineralized stones in the inner ear of fish, are largely unknown, as only a few fish inner ear proteins have been reported. Using a partial transcriptome for the inner ear of black bream (Acanthopagrus butcheri), in conjunction with proteomic data, we discovered hundreds of previously unknown proteins in the otolith. This allowed us to develop hypotheses to explain the mechanisms of inorganic material supply and daily formation of growth bands. We further identified a likely protein mediator of crystal nucleation and an explanation for the apparent metabolic inertness of the otolith. Due to the formation of both daily and annual increments, otoliths are routinely employed as natural chronometers, being used for age and growth estimation, fisheries stock assessments, and the reconstruction of habitat use, movement, diet and the impacts of climate change. Our findings provide an unprecedented view of otolith molecular machinery, aiding in the interpretation of these essential archived data.
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http://dx.doi.org/10.1111/febs.14715DOI Listing
January 2019

Selenium Status Is Not Associated with Cognitive Performance: A Cross-Sectional Study in 154 Older Australian Adults.

Nutrients 2018 Dec 1;10(12). Epub 2018 Dec 1.

Institute for Physical Activity and Nutrition (IPAN), School of Exercise and Nutrition Sciences, Deakin University, 3220 Geelong, Australia.

Selenium was suggested to play a role in modulating cognitive performance and dementia risk. Thus, this study aimed to investigate the association between selenium status and cognitive performance, as well as inflammatory and neurotrophic markers in healthy older adults. This cross-sectional study included 154 older adults (≥60 years) from Victoria, Australia. Participants were assessed for cognitive performance (Cogstate battery), dietary selenium intake (two 24-h food recalls), plasma selenium concentration, inflammatory markers (interleukin (IL)-6, -8, -10, tumor necrosis factor-alpha and adiponectin) and neurotrophic factors (brain-derived neurotrophic factor, vascular endothelial growth factor and insulin-like growth factor 1). Dietary selenium intake was adequate for 85% of all participants. The prevalence of selenium deficiency was low; only 8.4% did not have the minimum concentration in plasma required for optimization of iodothyronine 5' deiodinases activity. Multiple linear regression analysis revealed that plasma selenium was not associated with cognitive performance, inflammatory markers nor neurotrophic factors, independent of age, sex, body mass index (BMI), habitual physical activity, status, education, and history of cardiovascular disease. The lack of association might be due to the optimization of selenoproteins synthesis as a result of adequate selenium intake. Future prospective studies are recommended to explore potential associations of selenium status with age-associated cognitive decline.
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http://dx.doi.org/10.3390/nu10121847DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6315874PMC
December 2018

Characterization of the metal status of natively purified alpha-synuclein from human blood, brain tissue, or recombinant sources using size exclusion ICP-MS reveals no significant binding of Cu, Fe or Zn.

Metallomics 2019 01;11(1):128-140

The Florey Institute of Neuroscience and Mental Health, University of Melbourne, 30 Royal Parade, Genetics Lane, Parkville 3052, Australia.

Abnormal protein structure and function have been implicated as the toxic species in many diseases including neurodegenerative diseases, such as Parkinson's. One key pathological hallmark in Parkinson's disease is the formation of Lewy bodies, of which alpha-synuclein is the major component. These Lewy bodies are formed by the aggregation and oligomerization of alpha-synuclein. The oligomeric form of the protein is suspected to be the main contributor to the neurotoxicity seen in the disease. The formation of toxic oligomers has been shown to occur through reactions with lipids, dopamine, hydrogen peroxide as well as metals. The interplay between metals and alpha-synuclein has also been proposed to cause oxidative stress, which promotes the formation of protein aggregates. Most studies investigating the relationship of Cu, Fe and Zn with alpha-synuclein have relied on the use of recombinant protein and there is little evidence that the interaction between metals and alpha-synuclein are physiologically relevant. To address this gap in our knowledge we have characterized the metal content and metal binding capacity of alpha-synuclein purified from human erythrocytes and brain tissue. In addition, we examined the ability of dityrosine cross-linked alpha-synuclein oligomers to bind Cu, Fe and Zn. Using size exclusion chromatography-inductively coupled plasma-mass spectrometry we demonstrated that native human alpha-synuclein, recombinant familial mutants and oligomers do not bind to significant amounts of metal even when they are added to the protein in excess.
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http://dx.doi.org/10.1039/c8mt00223aDOI Listing
January 2019

Supranutritional Sodium Selenate Supplementation Delivers Selenium to the Central Nervous System: Results from a Randomized Controlled Pilot Trial in Alzheimer's Disease.

Neurotherapeutics 2019 01;16(1):192-202

Melbourne Dementia Research Centre, The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, 30 Royal Parade, Parkville, Victoria, 3052, Australia.

Insufficient supply of selenium to antioxidant enzymes in the brain may contribute to Alzheimer's disease (AD) pathophysiology; therefore, oral supplementation may potentially slow neurodegeneration. We examined selenium and selenoproteins in serum and cerebrospinal fluid (CSF) from a dual-dose 24-week randomized controlled trial of sodium selenate in AD patients, to assess tolerability, and efficacy of selenate in modulating selenium concentration in the central nervous system (CNS). A pilot study of 40 AD cases was randomized to placebo, nutritional (0.32 mg sodium selenate, 3 times daily), or supranutritional (10 mg, 3 times daily) groups. We measured total selenium, selenoproteins, and inorganic selenium levels, in serum and CSF, and compared against cognitive outcomes. Supranutritional selenium supplementation was well tolerated and yielded a significant (p < 0.001) but variable (95% CI = 13.4-24.8 μg/L) increase in CSF selenium, distributed across selenoproteins and inorganic species. Reclassifying subjects as either responsive or non-responsive based on elevation in CSF selenium concentrations revealed that responsive group did not deteriorate in Mini-Mental Status Examination (MMSE) as non-responsive group (p = 0.03). Pooled analysis of all samples revealed that CSF selenium could predict change in MMSE performance (Spearman's rho = 0.403; p = 0.023). High-dose sodium selenate supplementation is well tolerated and can modulate CNS selenium concentration, although individual variation in selenium metabolism must be considered to optimize potential benefits in AD. The Vel002 study is listed on the Australian and New Zealand Clinical Trials Registry ( http://www.anzctr.org.au /), ID: ACTRN12611001200976.
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http://dx.doi.org/10.1007/s13311-018-0662-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6361071PMC
January 2019

Marked Age-Related Changes in Brain Iron Homeostasis in Amyloid Protein Precursor Knockout Mice.

Neurotherapeutics 2018 10;15(4):1055-1062

Melbourne Dementia Research Centre, The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, 30 Royal Parade, Parkville, 3052, Victoria, Australia.

Proteolytic cleavage of the amyloid precursor protein (APP) into the Aβ peptide has been an extensively researched mechanism for Alzheimer's disease, but the normal function of the protein is less understood. APP functions to regulate neuronal iron content by stabilizing the surface presentation of ferroportin-the only iron exporter channel of cells. The present study aims to quantify the contribution of APP to brain and peripheral iron by examining the lifetime impact on brain and liver iron levels in APP knockout mice. Consistent with previous reports, we found that wild-type mice exhibited an age-dependent increase in iron and ferritin in the brain, while no age-dependent changes were observed in the liver. APP ablation resulted in an exaggeration of age-dependent iron accumulation in the brain and liver in mice that was assessed at 8, 12, 18, and 22 months of age. Brain ferroportin levels were decreased in APP knockout mice, consistent with a mechanistic role for APP in stabilizing this iron export protein in the brain. Iron elevation in the brain and liver of APP knockout mice correlated with decreased transferrin receptor 1 and increased ferritin protein levels. However, no age-dependent increase in brain ferritin iron saturation was observed in APP-KO mice despite similar protein expression levels potentially explaining the vulnerability of APP-KO mice to parkinsonism and traumatic brain sequelae. Our results support a crucial role of APP in regulating brain and peripheral iron, and show that APP may act to oppose brain iron elevation during aging.
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http://dx.doi.org/10.1007/s13311-018-0656-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6277293PMC
October 2018

Subcellular compartmentalisation of copper, iron, manganese, and zinc in the Parkinson's disease brain.

Metallomics 2017 10;9(10):1447-1455

Discipline of Biomedical Science and Brain and Mind Centre, Sydney Medical School, The University of Sydney, Camperdown, NSW 2006, Australia.

Elevated iron and decreased copper levels are cardinal features of the degenerating substantia nigra pars compacta in the Parkinson's disease brain. Both of these redox-active metals, and fellow transition metals manganese and zinc, are found at high concentrations within the midbrain and participate in a range of unique biological reactions. We examined the total metal content and cellular compartmentalisation of manganese, iron, copper and zinc in the degenerating substantia nigra, disease-affected but non-degenerating fusiform gyrus, and unaffected occipital cortex in the post mortem Parkinson's disease brain compared with age-matched controls. An expected increase in iron and a decrease in copper concentration was isolated to the soluble cellular fraction, encompassing both interstitial and cytosolic metals and metal-binding proteins, rather than the membrane-associated or insoluble fractions. Manganese and zinc levels did not differ between experimental groups. Altered Fe and Cu levels were unrelated to Braak pathological staging in our cases of late-stage (Braak stage V and VI) disease. The data supports our hypothesis that regional alterations in Fe and Cu, and in proteins that utilise these metals, contribute to the regional selectively of neuronal vulnerability in this disorder.
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http://dx.doi.org/10.1039/c7mt00244kDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5647261PMC
October 2017

The influence of the ethane-1,2-diamine ligand on the activity of a monofunctional platinum complex.

J Inorg Biochem 2017 12 29;177:328-334. Epub 2017 Jul 29.

School of Chemistry, The University of Sydney, NSW 2006, Australia. Electronic address:

The continued use of platinum-based chemotherapeutic drugs in the clinic mandates the need for further investigation of the biological activity of structural analogues of the clinically approved complexes. Of interest are monofunctional platinum(II) complexes, which bear only one labile ligand, for which it is believed that each complex binds to DNA only once. Pyriplatin ([PtCl(NH)(py)]) and enpyriplatin ([PtCl(en)(py)]) are both monofunctional platinum(II) complexes that bear a pyridine ligand and a labile chlorido ligand, differing in their cis‑ammine and ethane-1,2-diamine (en) ligands respectively. Despite their similar structure, the complexes exhibit dramatically different cytotoxicities. In this study, we synthesized and characterized both complexes in terms of their cytotoxicity, lipophilicity, DNA binding and cellular accumulation. There was no significant difference between the lipophilicities of the complexes and both complexes exhibited monofunctional type binding, but it was the temporal accumulation profiles of the two complexes which differed greatly. The complexes were further analyzed with size exclusion chromatography coupled with inductively coupled plasma mass spectrometry (SEC-ICP-MS) to determine the platination state of the proteins. Consistent with the accumulation studies, pyriplatin bound to proteins in far greater amounts than enpyriplatin, and this study also revealed some different protein targets between the bifunctional cisplatin and monofunctional pyriplatin. This study highlights the need for more sophisticated techniques, such as SEC-ICP-MS, to determine not only how much of a platinum complex accumulates in cells, but also the speciation and metabolites of platinum anticancer drugs.
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http://dx.doi.org/10.1016/j.jinorgbio.2017.07.029DOI Listing
December 2017

The APOE ε4 Allele Is Associated with Lower Selenium Levels in the Brain: Implications for Alzheimer's Disease.

ACS Chem Neurosci 2017 07 3;8(7):1459-1464. Epub 2017 May 3.

The Florey Institute of Neuroscience and Mental Health, The University of Melbourne , Parkville, Victoria 3052, Australia.

The antioxidant activity of selenium, which is mainly conferred by its incorporation into dedicated selenoproteins, has been suggested as a possible neuroprotective approach for mitigating neuronal loss in Alzheimer's disease. However, there is inconsistent information with respect to selenium levels in the Alzheimer's disease brain. We examined the concentration and cellular compartmentalization of selenium in the temporal cortex of Alzheimer's disease and control brain tissue. We found that Alzheimer's disease was associated with decreased selenium concentration in both soluble (i.e., cytosolic) and insoluble (i.e., plaques and tangles) fractions of brain homogenates. The presence of the APOE ε4 allele correlated with lower total selenium levels in the temporal cortex and a higher concentration of soluble selenium. Additionally, we found that age significantly contributed to lower selenium concentrations in the peripheral membrane-bound and vesicular fractions. Our findings suggest a relevant interaction between APOE ε4 and selenium delivery into brain, and show changes in cellular selenium distribution in the Alzheimer's disease brain.
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http://dx.doi.org/10.1021/acschemneuro.7b00014DOI Listing
July 2017

Characterization and Identification of Dityrosine Cross-Linked Peptides Using Tandem Mass Spectrometry.

Anal Chem 2017 06 18;89(11):6136-6145. Epub 2017 May 18.

The Florey Institute of Neuroscience and Mental Health, The University of Melbourne , 30 Royal Parade, Parkville, Victoria 3052, Australia.

The use of mass spectrometry coupled with chemical cross-linking of proteins has become a powerful tool for proteins structure and interactions studies. Unlike structural analysis of proteins using chemical reagents specific for lysine or cysteine residues, identification of gas-phase fragmentation patterns of endogenous dityrosine cross-linked peptides have not been investigated. Dityrosine cross-linking in proteins and peptides are clinical markers of oxidative stress, aging, and neurodegenerative diseases including Alzheimer's disease and Parkinson's disease. In this study, we investigated and characterized the fragmentation pattern of a synthetically prepared dityrosine cross-linked dimer of Aβ(1-16) using ESI tandem mass spectrometry. We then detailed the fragmentation pattern of dityrosine cross-linked Aβ(1-16), using collision induced dissociation (CID), higher-energy collision induced dissociation (HCD), electron transfer dissociation (ETD), and electron capture dissociation (ECD). Application of these generic fragmentation rules of dityrosine cross-linked peptides allowed for the identification of dityrosine cross-links in peptides of Aβ and α-synuclein generated in vitro by enzymatic peroxidation. We report, for the first time, the dityrosine cross-linked residues in human hemoglobin and α-synuclein under oxidative conditions. Together these tools open up the potential for automated analysis of this naturally occurring post-translation modification in neurodegenerative diseases as well as other pathological conditions.
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http://dx.doi.org/10.1021/acs.analchem.7b00941DOI Listing
June 2017

Molecular Aspects of a Robust Assay for Ferroxidase Function of Ceruloplasmin.

Inorg Chem 2017 May 17;56(9):5275-5284. Epub 2017 Apr 17.

School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne , Parkville, Victoria 3010, Australia.

Ceruloplasmin (Cp) is one of the most complex multicopper oxidase enzymes and plays an essential role in the metabolism of iron in mammals. Ferrous ion supplied by the ferroportin exporter is converted by Cp to ferric ion that is accepted by plasma metallo-chaperone transferrin. Study of the enzyme at the atomic and molecular level has been hampered by the lack of a suitable ferrous substrate. We have developed the classic chromophoric complex FeH(Tar) (HTar, 4-(2-thiazolylazo)resorcinol; x = 0-2; overall charge omitted) as a robust substrate for evaluation of the ferroxidase function of Cp and related enzymes. The catalysis can be followed conveniently in real time by monitoring the solution absorbance at 720 nm, a fingerprint of FeH(Tar). The complex is oxidized to its ferric form FeH(Tar) via the overall reaction sequence FeH(Tar) → Fe-Cp → Fe-Cp → FeH(Tar): i.e., Fe(II) is transferred formally from FeH(Tar) to the substrate docking/oxidation (SDO) site(s) in Cp, followed by oxidation to product Fe(III) that is trapped again by the ligand. Each Tar ligand in the above bis-complex coordinates the metal center in a meridional tridentate mode involving a pH-sensitive -OH group (pK > 12), and this imposes rapid Fe(II) and Fe(III) transfer kinetics to facilitate the catalytic process. The formation constants of both the ferrous and ferric complexes at pH 7.0 were determined (log β' = 13.6 and 21.6, respectively), as well as an average dissociation constant of the SDO site(s) in Cp (log K' = -7.2).
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http://dx.doi.org/10.1021/acs.inorgchem.7b00372DOI Listing
May 2017

Biochemically-defined pools of amyloid-β in sporadic Alzheimer's disease: correlation with amyloid PET.

Brain 2017 May;140(5):1486-1498

The Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia.

We fractionated frontal cortical grey matter from human Alzheimer's disease and control subjects into four biochemically defined pools that represent four distinct compartments: soluble/cytosolic, peripheral membrane/vesicular cargo, integral lipid/membranous pools and aggregated/insoluble debris. Most of the readily extractable amyloid-β remains associated with a lipid/membranous compartment. There is an exchange of amyloid-β between the biochemical pools that was lost for the amyloid-β42 species in Alzheimer's disease, consistent with the peptide being irreversibly trapped in extracellular deposits. The quantitative amyloid-β data, combined with magnetic resonance imaging volumetric analysis of the amount of cortical grey matter in brain, allowed us to estimate the total mass of amyloid-β in Alzheimer's disease (6.5 mg) and control (1.7 mg) brains. The threshold positron emission tomography standard uptake value ratio of 1.4 equates to 5.0 μg amyloid-β/g of grey matter and the mean Alzheimer's disease dementia standard uptake value ratio level of 2.3 equates to 11.20 μg amyloid-β/g of grey matter. It takes 19 years to accumulate amyloid from the threshold positron emission tomography standard uptake value ratio to the mean value observed for Alzheimer's disease dementia. This accumulation time window combined with the difference of 4.8 mg of amyloid-β between Alzheimer's disease and control brain allows for a first approximation of amyloid-β accumulation of 28 ng/h. This equates to an estimated 2-5% of the total amyloid-β production being deposited as insoluble plaques. Understanding these rates of amyloid-β accumulation allows for a more quantitative approach in targeting the failure of amyloid-β clearance in sporadic Alzheimer's disease.
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http://dx.doi.org/10.1093/brain/awx057DOI Listing
May 2017

Selenium Levels in Serum, Red Blood Cells, and Cerebrospinal Fluid of Alzheimer's Disease Patients: A Report from the Australian Imaging, Biomarker & Lifestyle Flagship Study of Ageing (AIBL).

J Alzheimers Dis 2017 ;57(1):183-193

The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, VIC, Australia.

Selenium (Se) protects cells against oxidative stress damage through a range of bioactive selenoproteins. Increased oxidative stress is a prominent feature of Alzheimer's disease (AD), and previous studies have shown that Se deficiency is associated with age-related cognitive decline. In this study, we assessed Se status in different biofluids from a subgroup of participants in the Australian Imaging, Biomarkers and Lifestyle Flagship Study of Ageing. As Se in humans can either be an active component of selenoproteins or inactive via non-specific incorporation into other proteins, we used both size exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS) and tandem mass spectrometry to characterize selenoproteins in serum. We observed no differences in total Se concentration in serum or cerebrospinal fluid of AD subjects compared to mildly cognitively impairment patients and healthy controls. However, Se levels in erythrocytes were decreased in AD compared to controls. SEC-ICP-MS analysis revealed a dominant Se-containing fraction. This fraction was subjected to standard protein purification and a bottom-up proteomics approach to confirm that the abundant Se in the fraction was due, in part, to selenoprotein P. The lack of change in the Se level is at odds with our previous observations in a Brazilian population deficient in Se, and we attribute this to the Australian cohort being Se-replete.
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http://dx.doi.org/10.3233/JAD-160622DOI Listing
February 2018

Cu(atsm) improves the neurological phenotype and survival of SOD1 mice and selectively increases enzymatically active SOD1 in the spinal cord.

Sci Rep 2017 02 13;7:42292. Epub 2017 Feb 13.

Department of Pathology, the University of Melbourne, Parkville, Victoria, Australia.

Ubiquitous expression of mutant Cu/Zn-superoxide dismutase (SOD1) selectively affects motor neurons in the central nervous system (CNS), causing the adult-onset degenerative disease amyotrophic lateral sclerosis (ALS). The CNS-specific impact of ubiquitous mutant SOD1 expression is recapitulated in transgenic mouse models of the disease. Here we present outcomes for the metallo-complex Cu(atsm) tested for therapeutic efficacy in mice expressing SOD1 on a mixed genetic background. Oral administration of Cu(atsm) delayed the onset of neurological symptoms, improved locomotive capacity and extended overall survival. Although the ALS-like phenotype of SOD1 mice is instigated by expression of the mutant SOD1, we show the improved phenotype of the Cu(atsm)-treated animals involves an increase in mature mutant SOD1 protein in the disease-affected spinal cord, where concomitant increases in copper and SOD1 activity are also evident. In contrast to these effects in the spinal cord, treating with Cu(atsm) had no effect in liver on either mutant SOD1 protein levels or its activity, indicating a CNS-selective SOD1 response to the drug. These data provide support for Cu(atsm) as a treatment option for ALS as well as insight to the CNS-selective effects of mutant SOD1.
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http://dx.doi.org/10.1038/srep42292DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5304223PMC
February 2017

Trace element-protein interactions in endolymph from the inner ear of fish: implications for environmental reconstructions using fish otolith chemistry.

Metallomics 2017 03;9(3):239-249

School of BioSciences, The University of Melbourne, BioSciences 4, Royal Parade, Parkville, Victoria 3052, Australia.

Otoliths, the biomineralised hearing "ear stones" from the inner ear of fish, grow throughout the lifespan of an individual, with deposition of alternating calciferous and proteinaceous bands occurring daily. Trace element : calcium ratios within daily increments measured by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) are often used in fisheries science to reconstruct environmental histories. There is, however, considerable uncertainty as to which elements are interacting with either the proteinaceous or calciferous zones of the otolith, and thus their utility as indicators of environmental change. To answer this, we used size exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS) of endolymph, the otolith growth medium, to determine the binding interactions for a range of elements. In addition, we used solution ICP-MS to quantify element concentrations in paired otolith and endolymph samples and determined relative enrichment factors for each. We found 12 elements that are present only in the proteinaceous fraction, 6 that are present only in the salt fraction, and 4 that are present in both. These findings have important implications for the reconstruction of environmental histories based on changes in otolith elemental composition: (1) elements occurring only in the salt fraction are most likely to reflect changes in the physico-chemical environment experienced during life; (2) elements occurring only in the proteinaceous fraction are more likely to reflect physiological rather than environmental events; and (3) elements occurring in both the salt and proteinaceous fractions are likely to be informative about both endogenous and exogenous processes, potentially reducing their utility in environmental reconstructions.
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http://dx.doi.org/10.1039/c6mt00189kDOI Listing
March 2017

Rubidium and potassium levels are altered in Alzheimer's disease brain and blood but not in cerebrospinal fluid.

Acta Neuropathol Commun 2016 11 14;4(1):119. Epub 2016 Nov 14.

The Florey Institute of Neuroscience, The University of Melbourne, Parkville, VIC, Australia.

Loss of intracellular compartmentalization of potassium is a biochemical feature of Alzheimer's disease indicating a loss of membrane integrity and mitochondrial dysfunction. We examined potassium and rubidium (a biological proxy for potassium) in brain tissue, blood fractions and cerebrospinal fluid from Alzheimer's disease and healthy control subjects to investigate the diagnostic potential of these two metal ions. We found that both potassium and rubidium levels were significantly decreased across all intracellular compartments in the Alzheimer's disease brain. Serum from over 1000 participants in the Australian Imaging, Biomarkers and Lifestyle Flagship Study of Ageing (AIBL), showed minor changes according to disease state. Potassium and rubidium levels in erythrocytes and cerebrospinal fluid were not significantly different according to disease state, and rubidium was slightly decreased in Alzheimer's disease patients compared to healthy controls. Our data provides evidence that contrasts the hypothesized disruption of the blood-brain barrier in Alzheimer's disease, with the systemic decrease in cortical potassium and rubidium levels suggesting influx of ions from the blood is minimal and that the observed changes are more likely indicative of an internal energy crisis within the brain. These findings may be the basis for potential diagnostic imaging studies using radioactive potassium and rubidium tracers.
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http://dx.doi.org/10.1186/s40478-016-0390-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5109650PMC
November 2016

The Neurobiology and Age-Related Prevalence of the ε4 Allele of Apolipoprotein E in Alzheimer's Disease Cohorts.

J Mol Neurosci 2016 Nov 6;60(3):316-324. Epub 2016 Aug 6.

University of Melbourne, The Florey Institute of Neuroscience and Mental Health, 30 Royal Parade, Parkville, VIC, 3052, Australia.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterised by amyloid beta (Aβ) plaques and tau neurofibrillary tangles in the brain. Human apolipoprotein E (ApoE) is a lipid transport protein coded by the polymorphic APOE gene, with three major alleles: ε2, ε3 and ε4. After age, the ε4 allele is the greatest risk factor for developing sporadic AD, conferring an increased risk of 3-4 and 8-12 times for one or two copies of the allele, respectively. This risk is reported to vary by demographic factors including sex, ethnicity and geography. In order to understand the risk of ApoE ε4 in relation to age, the primary risk factor for developing AD, we need to understand how the prevalence of APOE genotypes changes with age. Here, we present the first data on age-related prevalence of APOE ε4 in AD in three AD cohorts in Australia and the USA. There is a significant association between age and ε4 prevalence, particularly for ε4 homozygotes, such that as age increases the prevalence of ε4 decreases. Further studies on a random, population-based sample of the population are needed to provide more generalizable data, particularly in the >90-year-old age group.
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http://dx.doi.org/10.1007/s12031-016-0804-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5531868PMC
November 2016