Differential Expression Analysis of the SLIT Family in Different Brain Regions
The ageing human brain undergoes extensive remodelling of its extracellular matrix (ECM), yet the regional specificity and molecular drivers remain poorly defined. Here we combine bulk RNA-sequencing of post-mortem tissue (n = 63 across eight regions) with independent validation in the Genotype-Tissue Expression (GTEx) cohort (n = 962) to chart the expression landscape of the 239-member core matrisome. Hippocampus emerged as a major ECM outlier, with 114 matrisome genes—dominated by glycoproteins—up-regulated compared to the other seven regions (Fisher's combined test, FDR < 0.05). Genes coding the axon-guidance ligands SLIT1, SLIT2 and SLIT3 ranked among the top hippocampal signatures and displayed strong co-expression with ROBO2 (r > 0.70). Age-stratified analysis in GTEx revealed a concerted decline of the SLIT-ROBO2 axis after 60y, aligning with hallmarks of cognitive ageing. Immunohistochemistry and western blotting (n = 3 per region) supported regional and subcellular patterns of SLITs ROBO2. These findings identify the SLIT-ROBO2 module as a hippocampus-centric, age-labile component of the human matrisome. We propose that sustained SLIT-ROBO signalling supports hippocampal synaptic plasticity, whereas its late-life attenuation may diminish structural resilience and heighten vulnerability to neurodegeneration. Our resource provides a framework for targeting ECM pathways to preserve brain connectivity during ageing. Overall design: This study employed a comparative bulk RNA-sequencing analysis of human post-mortem brain tissue focused on the core matrisome (comprising ECM glycoproteins, collagens, and proteoglycans). Samples: The submission includes sixty-three autopsy specimens (n=63) derived from a local research group's biorepository. Inclusion criteria required death from natural causes, while cases of neurodegenerative disease, stroke, or infectious conditions were excluded. Experimental Conditions and Variables: The primary variable under investigation was the anatomical brain region. Samples were collected from eight distinct regions: Cerebellum (CER, n=8); Hippocampus (HCP, n=8); Hypothalamus (HTL, n=8); Substantia Nigra (SN, n=5); Temporal Cortex (TEMP, n=5); Occipital Cortex (OCC, n=5); Frontal Cortex (FRO, n=8); Parietal Cortex (PAR, n=16). Replicates and Controls: The design utilized paired samples for differential expression analysis across regions, meaning multiple samples (regions) were obtained from the same subject. This pairing was controlled for statistically by including the Subject Identifier as a covariate in the model, controlling for inter-individual variability. Reference/Comparison Samples: The Hippocampus (HCP) served as the critical comparison site, as the study aimed to evaluate differential expression in the matrisome genes in HCP relative to each of the other seven brain regions. HCP was determined to be a major ECM outlier site. Statistical comparisons were performed gene-wise, with the alternative hypothesis that the median expression in HCP exceeds that of the comparison site.