Effects of Steroidal Na/K‐ATPase Inhibitor Marinobufagenin on Neurodegeneration, Neuroinflammation and Cognition in a Mouse Model of Alzheimer's Disease and Cardiovascular Amyloidosis

Abstract

Background Na/K-ATPase is a keystone enzyme in all living cells. Steroidal inhibitor of Na/K-ATPase, marinobufagenin (MBG), participates in regulating cardiac function, vessel architecture, intracellular signaling transduction, and anti-neuroinflammatory response. The aged double-transgene APPswe/PS1dE9 mice, a model of Alzheimer's disease (AD), exhibit lower levels of urine MBG compared to wild-type (WT) mice, impairments in cognitive function and amyloid β (Aβ) pathologies. Compromised blood supply to the brain may contribute to AD development and Aβ pathologies. Here we examined Aβ pathology in the cerebral and central vasculature of aged AD mice and whether MBG treatment affects neuroinflammation, neurodegeneration, and cognitive function. Methods To aged male AD (n=8; 15-mo old) and age-matched WT mice (n=4) MBG (100 µg/day/kg body weight) was administered via subcutaneous ALZET minipumps for 3-mo. Age-matched control mice received saline (AD-C, n=8; WT-C, n=4). Systolic blood pressure (SBP; by tail cuff plethysmography), cognitive flexibility (reversal learning in a water T-maze), plasma (for MBG measurement), brain and tissue samples (for qPCR, 3D green light laser microscopy and immunohistochemistry analysis) were collected at 18-mo. Data was analyzed by 2-way ANOVA and presented as mean±SE. Results In aged AD mice, we observed granulated Aβ plaques and Aβ localization around small cerebral blood vessels (Fig 1B; white arrows). Aβ protein was also found in significantly larger abundance in aortae vessel wall of old AD mice vs. WT mice (Fig 2; blue stain). SBP was not affected by treatment and did not differ between AD and WT mice. Endogenous plasma MBG was lower in AD-C vs. WT-C (125±21 vs. 223±41 pmol/L; p<0.05). Plasma MBG was elevated in both WT-MBG and AD-MBG (483±116 and 657±176 pmol/L; p<0.05 vs. WT-C and AD-C, correspondently). Hippocampal mRNA expression of inflammatory marker IL6 was upregulated 1.5-fold in AD-C vs. WT-C (p<0.05); MBG reduced this expression in AD-MBG vs. AD-C (p<0.01). Hippocampal mRNA expression of amyloid precursor protein (APP) was upregulated 2.6-fold in AD-C vs. WT-C (p<0.01); MBG reduced this expression in AD-MBG vs. AD-C (p<0.01). AD mice showed normal learning for a turn-based discrimination rule but were impaired during the reversal phase vs. WT mice. MBG treatment did not affect the amount of Aβ plaque load in the brains of AD-MBG vs. AD-C. Conclusion Old AD mice exhibited lower endogenous plasma MBG, impaired cognitive flexibility, higher hippocampal IL6 and APP mRNAs, and Aβ accumulation in cerebral vessels and aortae relative to WT counterparts. MBG treatment significantly reduced hippocampal APP and inflammatory mRNA markers; however, it had no effect on existing Aβ load. In future experiments, we will examine how MBG treatment affects the formation of brain Aβ plaques in younger AD mice.