Age-related alterations on ultrastructure and gene expression profile of the female Blood-Brain Barrier

Abstract

Blood-Brain Barrier (BBB) breakdown occurs in ageing and neurodegenerative disorders and affects several brain regions including cortex and hippocampus. During ageing, structural and functional changes affecting the main BBB components (brain endothelial cells (BECs), pericytes and astrocytes), appear to be associated with altered expression of genes and microRNAs (miRNAs) potentially related to development, protein synthesis or longevity pathways. However, little is known about the age-related BBB dysfunction in females. In this study, we aimed to assess the relation between ultrastructural and transcriptional changes in the ageing female BBB. A combination of transmission electron microscopy (TEM) and 3D reconstruction was used to study microvessel ultrastructure in 6- and 24-month-old female C57BL/6J mice. According to our results, the ageing female BBB shows a significant increase in basement membrane (BM) thickness, volume and number of BEC pseudopods, pericyte mitochondrial volume, pericyte – BEC contact and tight junction (TJ) tortuosity. Also, cortical capillaries appeared to be prominently more affected during ageing than hippocampal capillaries. These results suggest a higher impact of ageing on the cortical BBB in females, promoting changes that lead to a pro-inflammatory state, among other processes. In addition, sequencing results showed that the majority of upregulated genes in the ageing female BBB were involved in inflammation and immune response pathways, whereas the downregulated genes were mostly related to metabolism and signalling pathways. Amongst the age-deregulated mRNAs and miRNAs, miR-144-3p (upregulated) and Dnmt3a (downregulated) were selected for functional analysis in a human BEC line (hCMEC/D3), where their inverse correlation was confirmed. However, DNMT3A, not miR-144-3p, was shown to influence BEC function when deregulated, thereby promoting higher leukocyte adhesion and mRNA levels of adhesion molecules (ICAM-1, VCAM-1) and the chemokine CCL5. Age-induced increase of miR-144-3p appears to modulate DNMT3A expression, but DNMT3A might be independently contributing more to switching BECs into a pro-inflammatory state.