Immunohistochemical analysis of Ank1 in Tg4510 and J20 mouse models

Abstract

Alzheimer’s disease is a neurodegenerative disease affecting over 55 million people worldwide. The disease is characterised by the presence of amyloid beta plaques and neurofibrillary tangles of hyperphosphorylated tau protein in the brain. There are currently no disease-modifying treatments, largely because the mechanisms underlying disease are not fully understood. Increasing evidence suggests a role for epigenetic alterations in Alzheimer’s disease. Recent findings have discovered neuropathology-associated DNA hypermethylation in ANK1, a gene coding for a scaffolding protein, Ankyrin 1 that has not been investigated in the diseased brain. We used immunohistochemistry in Tg4510 and J20 transgenic mouse models and their controls to determine the location of Ankyrin 1 within the brain and investigate any association with specific cell types and/or disease. Staining was completed to determine affiliations between two Ankyrin 1 antibodies and three cellular markers marking astrocytes, neuronal nuclei and microglia. To determine if Ankyrin 1 expression was associated with pathological markers of AD, tissue sections were stained with antisera directed against tau and amyloid beta. The localisation of Ankyrin 1 was analysed using the Vectra Polaris (Akoya) imaging platform and Indica HALO image analysis platform. Whole-slide section scanning allowed visualisation of immunostaining across brain regions and the HALO Highplex FL analysis module allowed localisation of Ankyrin1 to specific cell types and quantification of protein expression. While identifying differences between mouse models, we determine that Ankyrin 1 is seen in all regions of the brain. We show that two Ankyrin 1 antibodies identify distinct cell populations suggesting two different isoforms are detected. We determined disease associated differences in the number of cells expressing Ankyrin 1 and expression intensity, with a trend of increased expression in disease. Through HALO analysis, we also determined that Ankyrin 1 is expressed across all cell types tested, with expression level being dependant on pathology. A positive correlation between tau and Ankyrin 1 expression was determined, further supporting the association between Ankyrin 1 and Alzheimer’s disease pathology. The main outcomes of this study were developing methods to evaluate a range of phenotypes for Ankyrin 1. This study is a report of an exploratory study following up on an AD EWAS finding and providing novel preliminary data to expand on in the future and as such is of considerable interest to the field.