Introduction:

Alzheimer’s Disease (AD) is a progressive neurodegenerative disorder involving chronic neuroinflammation and glial dysfunction, particularly of microglia and astrocytes. A striking feature of AD is its sex disparity, meaning women represent nearly two-thirds of cases and experience more severe cognitive decline. Emerging evidence suggests that astrocytes may contribute to this difference through sex-biased transcriptional and functional changes (1). To investigate these changes, zebrafish are an emerging in vivo model for astrocyte research, offering rapid development, optical transparency, and conserved glial biology, making them ideal for studying sex- and age-dependent glial responses.

Aims:

This study aimed to 1) identify sex-biased gene expression differences in astrocytes using publicly available single-cell RNA-seq datasets from human AD patients and 2) explore astrocytic changes between adult male and female zebrafish, in order to validate the model for future studies.

Methodology:

The publicly available RNAseq datasets: GSE157827 (2), syn21670836 (3), and GSE167494 (4) were utilised. Dataset metadata was used to stratify each dataset’s samples by sex and disease status using BRAAK staging, with age- and stage-matched cohorts (minimum n = 3 per sex and condition). UMAP clustering was used for visualisation in RStudio, and astrocytes were identified based on GFAP, AQP4, and SLC1A3 expression. Differential gene expression between astrocytes from female and male patient samples was assessed using the Wilcoxon signed-rank test separately for control and AD samples. Genes similarly regulated in both conditions were excluded, and differentially expressed genes were compared across datasets.

Astrocytes abundance and morphology  were analysed in male versus female zebrafish (4 months, 1 year, and 2 years; n = 5 per group) using immunofluorescence and RT-qPCR targeting GFAP. All animal experiments were conducted in accordance with EU and HPRA regulations for the use of animals for scientific purposes.

Results:

Bioinformatic analyses identified a set of genes upregulated in astrocytes from female relative to male AD patients . Analyses using Ingenuity Pathway Analysis and Gene Ontology revealed dysregulation of pathways associated with reactive astrocyte phenotypes and cytoskeletal remodelling. Notably, HSPB8 was consistently upregulated in astrocytes from female compared to male AD patients. In zebrafish, astrocyte numbers were comparable between sexes across ages; however, a significant increase in GFAP-positive area was observed in specific brain regions of 2-year-old female zebrafish (p < 0.05; two-way ANOVA with Tukey’s HSD correction).

Conclusions:

My work identifies sex-specific astrocytic transcriptional and morphological changes that may contribute to the increased vulnerability of females to Alzheimer’s disease. Integrated analysis of human single-cell RNA-seq datasets revealed a consistently dysregulated set of genes in female AD astrocytes. Notably, the consistent upregulation across all datasets of HSPB8 highlights autophagic regulation as a potential mechanism underlying sex-biased disease susceptibility, as together with its chaperone partner BAG3, HSPB8 has recently been implicated in the regulation of autophagic flux in AD (5). Additionally, analysis in zebrafish demonstrates age- and sex-dependent differences in astrocyte morphology without changes in overall astrocyte abundance, supporting the translational relevance of this model for future investigation of the impact of HSPB8 dysregulation on astrocyte phenotype and function.