The APOE Îµ4 Allele Is Associated with Lower Selenium Levels in the

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Letter

The APOE #4 allele is associated with lower selenium levels in the brain: implications in Alzheimer’s disease Barbara Rita Cardoso, Dominic J Hare, Monica Lind, Catriona A. McLean, Irene Volitakis, Simon M. Laws, Colin L. Masters, Ashley I. Bush, and Blaine R Roberts ACS Chem. Neurosci., Just Accepted Manuscript • Publication Date (Web): 28 Apr 2017 Downloaded from http://pubs.acs.org on May 3, 2017

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ACS Chemical Neuroscience

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The APOE ε4 allele is associated with lower selenium levels in the brain: implications in Alzheimer’s disease

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Bárbara R. Cardoso1,2*, Dominic J. Hare1,3, Monica Lind1, Catriona A. McLean1,4, Irene Volitakis1, Simon M. Laws5,6, Colin L. Masters1,6, Ashley I. Bush1,6, Blaine R. Roberts1,6*

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Faculty of Pharmaceutical Sciences, Department of Food and Experimental Nutrition, University of São Paulo, São Paulo, Brazil

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Elemental Bio-imaging Facility, University of Technology Sydney, Broadway, New South Wales, Australia

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Department of Anatomical Pathology, Alfred Hospital, Prahran, Victoria, Australia

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

Centre of Excellence for Alzheimer’s Disease Research and Care, School of Medical and Health Sciences, Edith Cowan University, Western Australia, Australia Co-operative Research Centre for Mental Health, http://www.mentalhealthcrc.com

16 17 18 Running title: Selenium distribution in brain 19 Key-words: selenium, Alzheimer’s disease, APOE ε4 20 21

* Corresponding authors:

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Bárbara R. Cardoso, The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, 30 Royal Parade, Parkville, Victoria, 3052, Australia. Ph +61 450 633 537; Email: [email protected]

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Blaine Roberts, The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, 30 Royal Parade, Parkville, Victoria, 3052, Australia. Ph +61 450 633 537; Email: [email protected]

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ACS Paragon Plus Environment

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Abstract

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The antioxidant activity of selenium, which is mainly conferred by its incorporation into

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dedicated selenoproteins, has been suggested as a possible neuroprotective approach for

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mitigating neuronal loss in Alzheimer’s disease. However, there is inconsistent information

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with respect to selenium levels in the Alzheimer’s disease brain. We examined the

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concentration and cellular compartmentalization of selenium in the temporal cortex of

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Alzheimer’s disease and control brain tissue. We found that Alzheimer’s disease was

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associated with decreased selenium concentration in both soluble (i.e. cytosolic) and

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insoluble (i.e. plaques and tangles) fractions of brain homogenates. The presence of the

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APOE ε4 allele correlated with lower total selenium levels in the temporal cortex and a

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higher concentration of soluble selenium. Additionally, we found that age significantly

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contributed to lower selenium concentrations in the peripheral membrane-bound and

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vesicular fractions. Our findings suggest a relevant interaction between APOE ε4 and

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selenium delivery into brain, and show changes in cellular selenium distribution in the

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Alzheimer’s disease brain.

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Selenium is an essential micronutrient known for its antioxidant role via the incorporation of

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selenocysteine (SeCys) in 25 thus-far identified human selenoproteins. In the brain, the

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importance of selenium in regulating oxidative stress in the central nervous system is

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reinforced by the position of the brain atop a selenium hierarchy, as it is the typically last

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organ to be depleted of stores in cases of deficiency, and the first to revert to normal levels

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when selenium levels become replete.1, 2 Selenium has multiple roles in other systems which

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may influence accessibility of the brain to this essential micronutrient; for instance, the

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important role of selenium in male fertility can result in ‘competition’ between the brain and

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testes during situations of extreme deficiency.3

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Selenium status has been associated with cognitive performance, although there are

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conflicting opinions regarding the most reliable biomarker for assessing selenium levels and

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need.4-6 Several trials have been conducted that focused on selenium status as possible

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therapeutic target for Alzheimer’s disease. A systematic review performed in 2011

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highlighted that while there was no direct evidence supporting a role for selenium in treating

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the disease, noted that the base of knowledge regarding selenium in Alzheimer’s disease was

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small, and that future therapeutic benefits could not be ruled out.7 Subsequent clinical trials

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have reached a similar conclusion, failing to identify significant clinical outcomes, yet

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observing small trends that indicated some degree of neurological efficacy of selenium

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supplementation.8 A common conclusion reached by those conducting clinical trials

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examining selenium status in Alzheimer’s disease is that longer-term treatment may prove to

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have more significant effects. Taken together, these studies suggest that selenium has a more

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nuanced role in Alzheimer’s disease pathology, and that a better understanding of the role

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selenium plays in the molecular basis of neurodegeneration is required to design future, long-

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term targeted therapies.

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The selenoprotein glutathione peroxidase 4 (GPx4) is an important inhibitor of ferroptosis in

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neurodegeneration, a non-apoptotic form of cell death that features increased oxidative stress

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by increases in labile cellular iron levels.9 GPx4 activity has been implicated in ferroptotic

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cancer cell death10 and acute renal failure in mice,11 and the long-standing association

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between iron and neurodegeneration suggests that ferroptosis is a mechanism intrinsic to cell

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loss in Parkinson’s and Alzheimer’s disease.12 GPx4 has been identified as a component in an

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oxidative stress-induced neurodegenerative process that is now widely known as

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ferroptosis.13 As selenium is a key factor for GPx4 expression and activity, we recently

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suggested that availability of this element to the brain is essential for neuroprotection and


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may represent a specific target for treating Alzheimer’s disease via modulation of the brain

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selenoproteome.14

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Specific data directly assessing selenium levels in the Alzheimer’s disease brain is scarce.

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Ramos et al.15 reported a specific decrease in hippocampal selenium levels and increase in the

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superior temporal gyrus, frontal cortex, midbrain and putamen, though (as the authors

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acknowledged), the very small sample size (n = 2) of Alzheimer’s disease tissue limits

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interpretation of these data. Morris and colleagues16 conducted a more extensive study,

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examining the relationship between seafood consumption, brain mercury and selenium levels

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and ApoE ε4 status in 286 elderly brains. Increased selenium was associated with higher

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neurofibrillary tangle severity according to Braak pathological staging.

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In light of the mounting evidence regarding the importance of selenium in normal brain

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function, and the recent literature reporting possible neuroprotective benefits in Alzheimer’s

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disease, we investigated the cellular distribution and concentration of selenium in the

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temporal cortex of post mortem Alzheimer’s disease subjects compared to control samples;

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and examined the relevance of the high-risk APOE ε4 genotype on selenium distribution.

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Results and Discussion

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We analysed 38 Alzheimer’s disease brain samples and 33 control samples. Subject

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demographics for both groups are presented in Table 1. This study describes the distribution

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of selenium in cellular fractions within the temporal cortex of Alzheimer’s disease subjects,

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compared to control samples (Figure 1). Selenium was most concentrated in the membrane

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fraction (64.67 ± 20.56 and 53.99 ± 17.71 ng g-1 of wet tissue in control and Alzheimer’s

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disease samples, respectively), where the selenoproteins iodothyronine deiodinase 217 and

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selenoprotein K18 are most abundant. GPx4 is a membrane-associated protein enriched in

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mitochondria,19 though its activity is also detectable to a lesser extent in the cytosol.20 The

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insoluble formic acid fraction, that most likely corresponds to proteinaceous aggregates,

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including Aβ plaques and tau-containing neurofibrillary tangles, contained the lowest

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selenium concentration (6.28 ± 2.56 and 4.42 ± 2.60 ng g-1 in control and Alzheimer’s

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disease samples, respectively). While it is possible that interactions between selenoproteins

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and solvents used (e.g. loss of hydrophilic selenoproteins from insoluble plaques into the

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‘cytosolic’ fraction) may contribute some degree of redistribution and overlap, all samples


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were prepared in an identical fashion, and the low concentrations of selenium in each fraction

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suggests potential confounding effects were negligible.

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Alzheimer’s brain samples had lower total selenium concentration, which was reflected in

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lower levels in the soluble, membrane and insoluble fractions (Fig. 1C). However, when we

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further investigated the differences in each selenium variable across the diagnostic groups

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using linear regression models that considered the presence of the APOE ε4 allele and age as

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potential confounders, Alzheimer’s disease was not associated with a change in total

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selenium levels, but was negatively associated with selenium content in soluble and insoluble

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fractions (Table 2). These findings do not corroborate previous studies that reported an

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association between selenium concentration and neurofibrillary tangle severity in two cortical

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areas16 and the presence of selenoprotein P in plaques21 (although it is difficult to distinguish

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if this colocalization represents a role of selenoprotein P in plaque formation or is merely the

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consequence of a protein-dense aggregate). However, our findings do support the hypothesis

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that the ratio Sec:Cys in selenoproteins is decreased, and even though selenoprotein P levels

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are increased in Alzheimer’s disease brains, it is not delivering selenium efficiently to

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promote synthesis of other selenoproteins in neurons. This process would be associated with

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the selenoprotein synthesis hierarchy disturbance, unsuccessfully neutralizing localized

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oxidative stress in neurons. Consistent with this hypothesis, we observed an effect of ageing

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on selenium distribution among different brain fractions, as age was negatively associated

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with selenium levels in peripheral membranes and vesicles, though other studies have

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reported higher expression of selenoprotein P associated with ageing 22, 23.

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The results presented here are consistent with our hypothesis that reduced availability of

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selenium to neurons limits the expression of selenoproteins, such as the antioxidant and

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ferroptotic regulator GPx4, which causes impaired neurological function with implications

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beyond just Alzheimer’s disease. Both in and ex vivo depletion of GPx4 causes neuron-

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specific degeneration.13 GPx4 is essential for the development of parvalbumin-positive

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interneuron development, and genetic ablation causes seizures, ataxia24 and rapid

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neurodegeneration.25 Dysfunction of the innate mechanism regulating selenoprotein synthesis

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causes marked neurodevelopmental deficits, primarily via the negative effect on

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paravalbumin-positive neuron proliferation.26

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We also report that APOE ߝ4 allele carriers had both decreased total selenium levels and

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reduced selenium concentrations in the membrane fraction when compared to non-ߝ4-

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carriers, irrespective of clinical classification (Fig. 1D). Nonetheless, univariate statistical


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modelling revealed that total selenium levels in the temporal cortex are independently

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influenced by APOE genotype, and not the disease. Although this analysis does not confirm

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an association between APOE ߝ4 allele and selenium content in membrane fraction, it does

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demonstrate a positive correlation between the APOE ߝ4 allele and selenium content in the

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soluble fraction (Table 2). This genotype is the most significant genetic risk factor for late-

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onset Alzheimer’s disease,27-29 and is relevant to selenium metabolism as delivery of

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selenium into the brain is modulated by ApoE receptor 2 (ApoER2). This receptor interacts

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with selenoprotein P either at the blood-brain barrier or inside the brain to supply selenium to

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neurons and glial cells.30 One previous study has shown that the ApoE4 isoform has impaired

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recycling capacity in embryonic cortical neurons, which contributes to increased

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sequestration of ApoER2 into intracellular compartments. As a consequence, the receptor

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presents reduced surface expression at the plasma membrane.31 Furthermore, ApoE4

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selectively antagonizes ApoE3-ApoER2 in endothelial cells, affecting eNOS and kinase

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activation, as well as inhibiting cell migration, likely due to a conformational change in

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ApoER2.32 From the data presented here, we hypothesize that the presence of the ApoE4

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isoform may modify or reduce expression of ApoER2, leading to reduced binding with

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selenoprotein P and decreased selenium delivery to the brain. As consequence, the APOE ߝ4

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genotype could also result in increased selenoprotein P concentration in CSF, however such

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association needs to be further investigated.

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We have previously reported that a selenium deficient intake may increase risk for

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Alzheimer’s disease,5 and incremented intake with selenium-rich food source contributes to

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better cognitive performance.33 This indicates an association between diet, selenium status

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and neurodegeneration. Hence, selenium nutritional status and diet aspects should be

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carefully considered when selenium levels in brain are assessed regarding to

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neurodegeneration. Our previous study has shown that elderly from two different areas in

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Australia did not present selenium deficiency,34 which is in agreement with other studies that

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show Australians have a selenium-replete diet.35,

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significantly influenced by selenium intake, but that the APOE ε4 allele may be associated

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Alzheimer’s disease pathogenesis due to its negative effects on selenium delivery into brain.

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Additionally, APOE ε4 allele status was associated with higher selenium concentrations in

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the soluble fraction, and this can lead to misinterpretation of selenium data if not taken into

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account. We therefore highlight the importance of considering this genotype in further studies

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Thus, we believe our findings are not


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that aim to investigate the association of selenium concentration in brain and Alzheimer’s

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disease.

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Methods

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Human post-mortem tissue collection. Temporal cortex samples were obtained

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from the Victorian Brain Bank Network. Control brains were defined as free from

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Alzheimer’s disease lesions or significant neuropathology by a trained neuropathologist (i.e.

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numbers of plaques and tangles were below the defined cut-off values for the NIA Regan

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criteria for diagnosis

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neuropathological examination. All procedures were approved by the University of

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Melbourne Health Sciences, Human Ethics subcommittee (ID1136882).

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). Alzheimer’s disease brain tissues were similarly confirmed by

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Tissue fractionation. Hemisected frozen brains were thawed from −80 ˚C to −20 ˚C

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and the meninges were removed from approximately 5 g of temporal cortex (Brodmann area

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9). Grey matter was dissected in to 0.2 – 0.5 g aliquots and stored at −80 ˚C. The tissue was

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weighed and thawed on ice, then homogenised using a BioMasher (Omni International) and

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centrifuged at 100,000 g with a benchtop centrifuge. Tris buffered saline (TBS, 50 mM Tris

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pH 8.0, 100 mM NaCl) containing EDTA free protease inhibitors (Roche, 05056489001) was

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then added at a ratio of 1:4 (tissue:buffer; w/v). The cytosolic, or ‘soluble’ fraction (all

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material soluble in the TBS buffer) was collected as the supernatant after centrifugation at

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100,000 g for 30 minutes at 4 ˚C. The remaining pellet was re-suspended in 100 mM NaCO3

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pH 11.0 (1:4 tissue:buffer; w/v). Samples were incubated on ice for 20 min before

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centrifugation at 100,000 g for 30 minutes at 4 ˚C. The supernatant containing peripheral

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membrane proteins and vesicular (the ‘peripheral membrane/vesicular’ fraction) material was

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removed. The resultant pellet was re-suspended with 7 M urea, 2 M thiourea, 4% CHAPS, 30

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mM bicine, pH 8.5 and centrifuged (100,000 g for 30 minutes at 4 ˚C) and the supernatant

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(the ‘membrane’ fraction) was retained. Finally, the ‘insoluble’ fraction was obtained by

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digesting the remaining material in 70% formic acid for 16 – 18 hours and centrifuged as

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above. The supernatant was collected, with negligible material remaining. All fractions were

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stored at -80 ˚C until analysis.

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APOE genotyping. DNA was extracted from fresh frozen brain tissue using the

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PureLink® Genomic DNA kit (ThermoFisher Scientific) following manufacturer’s protocol.

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To ascertain APOE genotype TaqMan® genotyping assays (rs7412, assay ID:


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C____904973_10; rs429358, assay ID: C___3084793_20; Life Technologies, Carlsbad, CA)

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were performed on a QuantStudio 12K Flex™ Real-Time-PCR systems (Applied

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Biosystems) using TaqMan® GTXpress™ Master Mix (Life Technologies) following

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manufacturer’s instructions. In this study APOE carrier status was defined by the presence (1

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or 2 copies; carrier) or absence (0 copies; non-carrier) of the APOE ε4 allele.

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Selenium assay. Aliquots of brain fractions were diluted with 1% HNO3 at the

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following ratios (all v/v): soluble, peripheral membrane/vesicular and membrane fractions

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1:15; insoluble fraction 1:30. Selenium-78 was chosen as the isotope to be monitored using

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the Agilent Technologies 7700x Series inductively coupled plasma-mass spectrometry (ICP-

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MS) system (Agilent Technologies, Mulgrave, Australia). Hydrogen was used as reaction gas

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for analysis in order to reduce polyatomic interferences on the chosen selenium isotope 38. A

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calibration curve was prepared using a multielement standard solution containing 0, 0.1, 0.3,

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0.5, 1, 5, 10 µg L-1 of selenium. An internal standard solution containing 200 µg L-1 of

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yttrium (89Y) in 1% HNO3 was used as a reference element and was introduced on-line via a

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T-piece. The method validity was confirmed by recovery analysis of a certified lyophilised

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human reference control (Seronorm Trace Elements Serum and Whole Blood, Sero AS). For

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each analysis, the accepted recovery of the reference material was > 90%.

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Statistical analysis. All results are expressed as the mean ± standard deviation (SD)

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or continuous variables, and as a percentage for categorical data. A Kolmogorov-Smirnov

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test was used to determine the normality of the data. The values from membrane fraction

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were not normally distributed and were thus log-transformed. Two-tailed unpaired Student’s t

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tests were performed to compare selenium concentration between diagnosis groups and

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APOE genotypes, according to ε4 allele carrier status. To further assess the differences in

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each selenium variable across the diagnostic groups, multiple regression models were

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developed to obtain the minimum model factoring for APOE genotype (presence/absence of

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the ε4 allele), age and sex as covariates. For all dependent variables, the resultant minimum

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model contained all covariates excluding sex. Postmortem interval was not considered for

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multiple regression models as preliminary analysis with correlation tests (Pearson or

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Spearman) did not identify any correlation between age and selenium. A p-value less than

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0.05 was considered statistically significant. All statistical analyses were carried out using the

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IBM Statistical Package for the Social Sciences (SPSS) software, v 23.0 (IBM Corporation)

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and Prism v6.0h (GraphPad) was used to construct figures.

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Funding sources:

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BRC was supported by a Science without Borders (Ciência sem Fronteiras) Fellowship. DJH

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is a National Health and Medical Research Council Career Development Fellow

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(APP1122981). SML, BRR, AIB and CLM received financial support from the Cooperative

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Research Centre (CRC) for Mental Health (Grant ID: 20100104), an Australian Government

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Initiative. Tissues were received from the Victorian Brain Bank Network, supported by The

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University of Melbourne, Alfred Hospital, the Victorian Forensic Institute of Medicine, the

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National Health and Medical Research Council. We acknowledge funding from the Victorian

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Government’s Operational Infrastructure Support Program and the Australian Research

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Council Linkage Projects Scheme (LP140100095, with Agilent Technologies).

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Author contributions:

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BRC designed and performed experiments, analysed data and wrote the paper. DJH: analysed

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data and wrote the paper. ML: prepared tissue samples for analysis. CAM: Conducted

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pathological examination and diagnosis of brain tissue. CLM: supervised and provided

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financial support for ML. AIB: supervised the project. SML: provided APOE genotyping. IV:

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prepared tissue samples for analysis. BRR: designed the experiments, wrote the paper and

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supervised the project. All authors contributed to, edited and approved the manuscript.

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Conflict of interest:

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B.R.R. and D.J.H. receive research support from Agilent Technologies. A.I.B. is a

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shareholder of Cogstate Ltd, Prana Biotechnology Ltd, Mesoblast Ltd, Collaborative

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Medicinal Development Pty Ltd, Grunbiotics Pty Ltd, Brighton Biotech LLC, and a paid

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consultant for Collaborative Medicinal Development Pty Ltd. CLM is a shareholder for Prana

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Biotechnology and consultant for Eli Lilly & Co; all other authors declare no competing

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financial interests.

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References: [1] Cardoso, B. R., Roberts, B. R., Bush, A. I., and Hare, D. J. (2015) Selenium, selenoproteins and neurodegenerative diseases., Metallomics 7, 1213-1228. [2] Nakayama, A., Hill, K. E., Austin, L. M., Motley, A. K., and Burk, R. F. (2007) All regions of mouse brain are dependent on selenoprotein P for maintenance of selenium, J. Nutr. 137, 690-693.


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401


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402 403

Fig. 1: Selenium content in human brain fractions expressed as percentage distribution: A)

404

Control and B) Alzheimer’s disease brains. Selenium content in human brain fractions

405

stratified by: C) diagnosis; and D) APOE ε4 genotypes. AD: Alzheimer’s disease; CO:

406

control; PM / vesicular. Student t test: * p < 0.05; ** p ≤ 0.001; ✧Student t test performed

407

with log transformed data.

408


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409

Table 1. Demographics for individuals from control (n = 33) and Alzheimer’s disease (n =

410

38) samples. Control

Alzheimer’s disease

p

Age (years)

70.81 ± 12.89

79.38 ± 9.95

0.003a

Sex (% female)

24.4

34.4

0.359b

APOE ε4 carriers (%)

45.5

60.5

0.204b

PMI (hours)

43.09 ±17.46

29.78 ±18.60

0.003a

411

Mean ± SD unless noted above as otherwise. a Student t-test; b Chi-square test. APOE ߝ4:

412

Apolipoprotein E epsilon 4, PMI: postmortem interval.

413 414

Table 2: Modelling the association of selenium concentration and distribution in brain by

415

subject demographics. Univariate analysis Alzheimer diseasea

APOE ε4 alleleb

Agec

β

p

β

p

β

p

Total

-11.228

0.210

-16.772

0.045

-0.415

0.249

Soluble

-10.061

0.000

5.259

0.045

-0.062

0.584

Peripheral

-1.058

3.472

-1.935

0.565

-0.351

0.019

Membrane

-0.048

0.214

-0.058

0.113

-0.002

0.156

FA insoluble

-1.830

0.022

0.371

0.616

-0.021

0.538

membrane / vesicular

416

a

417

and age as covariates.

418

b

419

and age as covariates

420

c

421

diagnostic groups as covariates.

Diagnostic group as dependent variable, APOE genotype (presence/absence of the ε4 allele)

APOE genotype (presence/absence of the ε4 allele) as dependent variable, diagnostic group

Age as dependent variable, APOE genotype (presence/absence of the ε4 allele) and

422 423


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The APOE ε4 allele is associated with lower selenium levels in the brain: implications in Alzheimer’s disease Bárbara R. Cardoso, Dominic J. Hare, Monica Lind, Catriona A. McLean, Colin L. Masters, Ashley I. Bush, Simon M. Laws, Irene Volitakis, Blaine R. Roberts


The APOE Îµ4 Allele Is Associated with Lower Selenium Levels in the

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