Endothelium5-HT3A receptor as a function of Age, ApoE, and ADRD pathology

Abstract

Alzheimer’s disease (AD) is the most common type of dementia, which is clinically characterized by cognitive and memory impairment. One of the major hallmarks of AD is the blood-brain barrier (BBB) dysfunction. The BBB is a collection of cells that protects the brain by providing a barrier with highly selective transport into and out of the brain. Our previous research demonstrated that 5-hydroxytryptamine receptor 3A (5-HT3AR) antagonism improved BBB function in vitro and in vivo in an AD mouse model, suggesting that 5-HT3A R is associated in the BBB-endothelium (ECs). Therefore, in this project, we aimed to investigate ECs-5-HT3AR expression as a function of age, AD pathology, and apolipoprotein E (ApoE) isoform. To achieve the objectives of this work, we optimized immunostaining protocols to evaluate ECs-5-HT3A R expression in mice and in postmortem human brain tissues across different brain regions. We optimized the staining protocol to achieve “rigorous and reproducible staining,” defined as obtaining quantifiable microscope images with minimal to no background, minimal autofluorescence, and consistent antigen retrieval. We also tested and validated several antibodies and optimized the quantification method to reduce subjectivity and increase robustness. We then focused on quantifying ECs-5-HT3AR expression in mice and human brain tissues. Our data from postmortem human brain tissues demonstrated regional differences in ECs-5-HT3AR expression as a function of age. AD pathology was associated with reduced ECs-5-HT3A R expression in the hippocampus and increased expression in the occipital cortex, with no alterations in the frontal cortex. However, the reduction observed in the hippocampus as a function of AD/CAA pathology was similar to that of the aging group, suggesting observed effect is that of age. Collectively, these findings suggest that ECs-5-HT3A R expression declines with aging; however, the effect of pathology differed, with receptor expression significantly increased in the occipital cortex, a region primarily affected by CAA. Furthermore, our findings showed that ECs-5-HT3AR increases in humans carrying the ApoE4 isoform, independently of AD pathology and through an interaction with pathology, as observed when data were analyzed using weighted linear regression, with an effect size for the ApoE isoform of 0.095, compared to the pathology effect of 0.019. The addition of sex as a variable to the regression model increased the proportion of variance explained (R2), from 0.15 to 0.25, with sex showing the strongest association with ECs-5-HT3AR expression, compared with ApoE, AD/CAA pathology, and their interactions. Similarly, we assessed ECs-5-HT3AR expression in mouse brains based on age and pathology. Consistent with our findings in the human hippocampus, ECs-5-HT3AR levels in mice decrease with age. However, there was no impact of amyloid pathology on ECs-5-HT3AR expression in 5xFAD and TgSwDI mice. Additionally, ECs-5-HT3AR increased in mice carrying the human ApoE4 isoform regardless of AD pathology, but no interaction with amyloid pathology was observed. In summary, these results show that ECs-5-HT3AR expression is influenced by aging, AD pathology, ApoE isoform, and sex. These findings suggest that ECs-5-HT3AR could be a promising therapeutic target, and that sex and ApoE isoform should be considered when developing treatments targeting ECs-5-HT3AR.