Age-related neurodegenerative changes in spontaneous mammalian models provide insights into diseases like Alzheimer’s disease (AD), which is characterised by amyloid-beta plaques, neurofibrillary tangles (tau pathology), and neuroinflammation. We explored age-related morphological changes in astrocytes and microglia in cat and sheep brains, which can develop AD-like neuropathology. We also investigated comparative sequences of APOE (a major risk factor for human AD) homologues in these species, to identify variants associated with increased neuropathology and/or cognitive dysfunction.

Transverse sections of brain regions were cut from formalin fixed, paraffin embedded blocks from cats and sheep of various ages, including cats diagnosed with cognitive dysfunction syndrome (CDS). Age groups for sheep were young (2-4 years, n=8), middle-aged (6-7 years, n=8) and old (11-12 years, n=7). Cats were divided into young (2-6 years, n=5), aged (14-20 years, n=7) and an older group of cats diagnosed with CDS (16-19 years n=5). Sections were labelled using antibodies for glial cell markers (Iba1 for microglia; GFAP for astrocytes) using standard immunohistochemical techniques. For cats, images of grey and white matter were captured from the parietal and rostral cortex, and the dentate gyrus. In sheep, analysis was performed in the frontal cortex, parietal cortex, temporal cortex and dentate gyrus. ImageJ macros were used to determine cell size and density. Analysis of microglia process length and number was also performed. Statistical analysis was performed in R studio using the lme4 and lmerTest packages to allow for Linear Mixed-effect Models and Generalised Linear Mixed-effect Models to be fitted. A region of the APOE gene, including exons 4 and 5, was amplified by PCR from genomic DNA of 24 Cheviot sheep included in histopathology analyses, and sequenced by Sanger sequencing. In addition, genomic and cDNA sequences of APOE from 18 different sheep breeds were retrieved from annotated genomes available in Ensembl rapid, and from whole genome sequences of 46 Scottish Blackface sheep. Multiple sequence alignments were performed on Geneious Prime to analyse APOE.

In sheep, astrocyte density increased with age in grey matter cortical areas and the dentate gyrus. Tau tangles were associated with higher astrocyte density in specific cortical areas. In older cats with CDS (16-19 years) decreased microglial branch length (indicating activation) was observed in the parietal and rostral cortex compared to younger cats (2-6 years). Older cats with both amyloid-beta and tau pathology had higher microglial density than those with only amyloid-beta plaques or no identified pathologies in parietal cortex white matter. These glial changes may indicate cell activation and/or neuroinflammation, contributing to neuropathology similar to human AD. Analysis of APOE in the sheep breeds revealed multiple variants, and demonstrated all sheep encode identical amino acids at positions 112 and 158 to those that define the human APOE ε4 allele.

This research offers approaches for comparative investigations to understand mechanisms underlying neurodegenerative disease. Future investigations should explore these age-related and pathology-related changes to astrocytes and microglial cells using techniques that could target pathways of glial reactivity to reveal mechanisms of senescence, and provide insight into the glial cell phenotypes.