Study of transporter and tight junction modification during ageing, cerebral amyloid angiopathy and ischaemia at the blood-brain barrier

Abstract

Cerebral amyloid angiopathy (CAA), Alzheimer’s disease (AD) and ischaemic stroke are diseases that often co-occur in the ageing population. Emerging evidence suggests that ischaemia and amyloid β (Aβ) interact in a mutually detrimental way, though the exact mechanisms remain unclear. With the blood-brain barrier (BBB) as a major constituent of cerebrovascular function and Aβ clearance, this work focuses on changes of BBB transporters and tight junctions (TJs) during ageing, AD/CAA and ischaemia in order to broaden the understanding of these diseases’ development and progression. Two ATP-binding cassette (ABC) transporters that have been implicated in Aβ clearance are p-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp). Using intact isolated capillaries as an ex-vivo model of the BBB, age- and sex-dependent changes in P-gp and Bcrp activity were observed in APP23 mice, a transgenic model of AD/CAA. In wild-type (WT) mice, P-gp activity declined with age in both sexes (3 versus 20−22 months), in agreement with current literature. However, while Bcrp activity was also lower in old male mice, an up-regulation was noted for females. Protein expression reflected the functional results found in females. This sex-specific difference at the BBB might explain some of the disparities observed between men and women in age-associated diseases such as AD and stroke. Since P-gp and Bcrp are both linked to Aβ transport, age-associated decline in their activity may contribute to the onset of AD/CAA. Indeed, it was found that transport of Aβ also decreased with age. In APP23 mice, P-gp and Bcrp activity was elevated in young and middle-aged males, with the old mice showing non-significant differences (age range: 2−5, 10−16 and 23−24 months). In contrast, P-gp and Bcrp activity was down-regulated in female APP23 mice at all ages. The tendency for elevated P-gp and Bcrp activity in male mice could point to a stronger compensatory action in response to elevated Aβ levels and emphasize the importance of separating by sex when studying AD/CAA. No difference was found in Aβ transport between old WT and APP23 mice in either males or females, although both P-gp and Bcrp were down-regulated in female APP23 mice. This may be linked to the presence of compensatory actions by other Aβ transport mechanisms. Although there is some evidence of disruption of BBB integrity during ageing and AD/CAA, western blot analysis of capillary TJs claudin 5 (Cldn-5), occludin (Ocln) and zona occludens 1 (ZO-1) generally showed increased expression in brain capillary for both conditions in middle-aged mice (age range: 10−14.5 months). Potentially, TJ disruption either sets in during later stages of ageing and AD/CAA, or is mitigated by factors other than protein expression, such as localisation. To probe the interaction of AD/CAA and ischaemia at the BBB directly, primary mouse brain microvascular endothelial cell (BMEC) cultures were treated with Aβ and oxygen-glucose deprivation (OGD), followed by a reoxygenation period to simulate reperfusion. Aβ1-40 was used at 1 and 12 nM, while OGD intervals were 12 and 24 h, with 0, 2, 4, 6 and 18 h of reoxygenation. qPCR and western blot analysis of P-gp, Bcrp, glucose transporter 1 (Glut-1), TJs and hypoxia-inducible factor 1α (HIF-1α) showed that their expression is highly dependent on concentration, order of treatment, and the duration of treatment and reoxygenation periods. The combination of OGD and Aβ often resulted in a non-additive effect with the response differing between WT and APP23 BMECs, revealing an intricate relationship between ischaemia and Aβ. Interestingly, the addition of Aβ during OGD in WT BMECs tended to counteract the down-regulation brought on by OGD when followed by either 0 or 2 h of reoxygenation, supporting previous studies suggesting possible beneficial roles for Aβ. Overall, the findings of this work help to identify age-dependent changes at the BBB that may contribute to AD/CAA, as well as denote the presence of important sex-dependent differences. Additionally, the complex interaction between OGD and Aβ shown at the BBB suggests the need for more systematic studies in order to fully understand the relationship between ischaemic stroke and AD/CAA.