Metal regulation of extra-neuronal tau

© 2018 Dr Luz Fernanda Yévenes UgarteMetal dyshomeostasis is an important neurodegenerative event that can affect the structure and function of proteins, such as tau. Tau binds metals including copper, iron and zinc, and may undergo conformational changes that promote its aggregation, leading to neurodegeneration. Tau is mainly an axonal microtubule stabilising protein. However, tau is also located to dendrites where it mediates the transport of proteins to the postsynaptic terminal and may modulate the neuronal susceptibility to excitotoxicity. Furthermore, tau is present in bodily fluids, which can be used for detection of pathological biomarkers. The finding of tau in extracellular fluids and dendrites has directed its current research focus to the study of tau mechanisms of secretion and spreading. The aim of this project is to investigate the role of metals in the modulation of tau secretion, and to analyse if platelet tau and metal levels could serve as neurodegenerative blood biomarkers. To determine if metals could modulate tau secretion, primary cortical cell cultures from tau-knockout and wild-type mice were treated with copper, iron, zinc, glutamate and clioquinol, an 8-hydroxyquinoline derivative with ionophore activity. The results showed that copper, iron and clioquinol decreased tau secretion from cells whilst zinc and glutamate increased it. Copper and iron decreased APP secretion from cells whilst glutamate and clioquinol increased it. Additionally, tau-knockout cells showed decreased APP secretion compared to wild-type controls and metals did not alter their APP secretion. To further investigate if this occurs also in vivo, interstitial fluid (ISF) was collected using microdialysis on wild-type mice fed either with copper, iron or zinc. Zinc and iron increased tau ISF levels whilst copper decreased it. The analysis of baseline ISF tau levels collected over time from untreated animals suggested that tau could be involved in circadian rhythm. To evaluate if Alzheimer’s disease (AD) platelets present tau abnormalities and metal dyshomeostasis, platelet samples from 55 healthy and 45 AD subjects from the Australian Imaging, Biomarkers and Lifestyle flagship study of ageing (AIBL) were analysed by inductively coupled plasma-mass spectrometry (ICP-MS) and Western blotting. The results showed that copper is decreased in AD platelets. Additionally, healthy controls who are ApoEε4 carriers had the highest platelet copper content. Iron levels were decreased in AD platelets. Platelet zinc levels positively correlated with verbal fluency. ROC curves showed that copper and iron were poor biomarker candidates. There were no changes in total and GSK3β pS9 levels in AD platelets. To characterise platelet tau species, mouse brain and platelets from human, wild-type and tau-knockout mice were analysed by Western blotting and immunofluorescence. The results showed that tau immunoreactivity might be an antibody artefact. To further investigate this, tandem-MS was used to identify tau in a human platelet sample, using brain samples from wild-type and transgenic tau overexpressing mouse as positive controls. Tau was identified in mouse brain samples but not in human platelets. Additionally, a qRT-PCR was performed to platelets from humans, wild-type and tau-knockout mice to determine tau mRNA levels. The results showed that MAPT was expressed in wild-type and tau-knockout mice tissues but was not detected in either human or mouse platelets. The conclusions drawn from this thesis are that: (1) extracellular zinc may modulate tau secretion; (2) APP secretion may be tau dependent; (3) in AD platelets, there was no detectable level of tau and there was no change in GSK3β levels and activity; (4) platelets from AD patients exhibit metal dyshomeostasis, resulting in decreased copper and iron content. This change in platelet copper and iron concentrations alone do not provide sufficient sensitivity and specificity as AD biomarkers. Further studies are required to identify a panel of platelet biomarkers, together with changes in copper and iron that will provide high sensitivity and specificity as biomarkers for AD