(M1230-04-23) Molecular Mechanisms Driving Sex-Dependent Cerebrovascular Inflammation in Alzheimer's disease

Purpose: Epidemiological studies demonstrate that Alzheimer's disease (AD) is more prevalent in women compared to men. In the US, women make up almost 4 million out of more than 6 million Alzheimer's patients, which accounts for nearly two-thirds of the total cases. Furthermore, AD female patients were found to show an upregulation of inflammation pathway related genes than males. A majority of AD patients have vascular dysfunction and manifest cerebrovascular inflammation, which is believed to be triggered by toxic amyloid beta (Aβ) exposure. It is thus crucial to understand the molecular mechanisms underlying sex related differences driving cerebrovascular inflammation in Alzheimer’s disease, which may help elucidate molecular basis for sex-dependent differences in AD pathogenesis.

Methods: We investigated the effect of amyloid burden on the expression of vascular inflammatory marker, VCAM-1, in the brains of male and female APP/PS1 delta E9 transgenic mice (app+) and non-transgenic littermates (app-) at 12-months-of-age. The femoral vein was catheterized under general anesthesia. The mice were bolus injected with ¹²⁵I-VCAM-1 IgG (150 μCi) via the femoral vein and allowed to circulate for 40 minutes. The mice were transcardially perfused with PBS and euthanized. The brain was harvested and dissected into different brain regions. ¹²⁵I activity in each brain region was determined using a gamma counter. Additionally, APP/PS1 delta E9 male and female mice were transcardially perfused with PBS followed by 4 % PFA. The brains were harvested from the cranial cavity, embedded in paraffin blocks, and sliced into 5 μm sections. The brain slices were used to stain for VCAM-1 using immunohistochemistry to evaluate gender differences in VCAM-1 expression. In addition, we employed NanoString digital spatiolomics to obtain a spatially resolved, high-plex (10s -10,000s) digital quantitation of mRNA in tissue. The DSP assay utilized RNA probes coupled to UV photocleavable oligonucleotide barcodes. The formalin-fixed paraffin-embedded (FFPE) brain slices were incubated with GeoMx reagents along with visualization reagents. We then scanned and imaged the brain slices for CD31 (endothelial cell marker), Aβ (4G8 antibody), and DAPI (nuclei) (Fig.1). Based on the scan, the region of interest (ROI) was selected, and the oligonucleotides from the ROI were quantified using standard NanoString nCounter®. The spatial resolution allowed for selection of ROIs on brain slices that are positive for CD31 cells (endothelial marker), thereby aiding the identification of genes driving cerebrovascular inflammation in areas that are ridden with vascular Aβ deposits and positive for CD31 cells (Fig.2). Ultimately, this approach helped evaluate the genes driving gender differences in cerebrovascular inflammation.

Results: There was an increase in the brain accumulation (% injected dose/brain weight) of ¹²⁵I-VCAM-1 IgG was observed in female app+ mice compared to male app+ mice. Immunohistochemistry studies have demonstrated that male app- mice has a lower VCAM-1 expression than male or female app+ mice. The app- female mice demonstrated higher VCAM-1 expression compared to male app- mice (Fig 3). Moreover, the VCAM-1 staining is similar in female app- and app+ mice, which suggests that female male is more susceptible to age-related vascular changes than the male mice.

Conclusion: In summary, the current research demonstrates sex differences in cerebrovascular inflammation in AD transgenic and non-transgenic mouse models as evidenced by changes in VCAM-1 expression. Furthermore, NanoString DSP protein panel allows us to elucidate molecular mechanisms underlying sex differences in blood-brain barrier integrity and function.

References: Beam CR, Kaneshiro C, Jang JY, Reynolds CA, Pedersen NL, Gatz M. Differences Between Women and Men in Incidence Rates of Dementia and Alzheimer's Disease. J Alzheimers Dis. 2018;64(4):1077-1083. doi: 10.3233/JAD-180141. PMID: 30010124; PMCID: PMC6226313.
Attems, J., Jellinger, K.A. The overlap between vascular disease and Alzheimer’s disease - lessons from pathology. BMC Med 12, 206 (2014). https://doi.org/10.1186/s12916-014-0206-2

Staining for CD31 (BBB endothelial marker) and 4G8 (amyloid marker) in APP + female mice. 001 and 002 are the ROI selected areas that are positive for CD31 and 4G8.

Segmentation of ROI to isolate CD31 + positive cells.

VCAM-1(dark brown) expression in APP/PS1 delta E9 mice and non-transgenic littermates.