Steroidal Inhibitors of Na/K‐ATPase in a Mouse Model of Alzheimer’s Disease
Authors: Zahariadis Eleni Nicole , McDevitt Ross , Grigorova Yulia , Zernetkina Valentina , Zheng Lucy , Petrashevskaya Natalia , Wei Wen , Camandola Simonetta , Mattson Mark , Lakatta Edward , Fedorova Olga V. 
Published: April 2020

BackgroundNeurons rely on Na/K‐ATPase (NKA) to create the electrochemical gradient that allows them to fire action potentials. The NKA also acts as a receptor for cardiotonic steroids (CTS), which have been shown to participate in intracellular signaling transduction, cardiovascular function, and have potential neuroprotective effects. Despite the importance of the NKA and CTS in brain physiology and pathology, their potential roles in Alzheimer’s disease (AD) pathology are not well studied. Marinobufagenin (MBG) is a bufadienolide CTS, and its potential neuroprotective effect has never been studied. MBG levels were significantly lower in 15‐month old double‐mutant Alzheimer’s mice (APPswe/PS1dE9; 2xTg‐AD; AD) compared to age‐matched, non‐transgenic controls. The present study examined whether the treatment of AD mice by MBG affects cardiovascular and behavioral parameters.MethodsMale AD (n=16) and wild type (WT; n=8) mice (15‐mo old) were included in the experimental design. Mice were administered MBG (100 μg/day/kg body weight; AD‐MBG, n=8; WT‐MBG, n=4) or vehicle (control: AD‐C, n=8; WT‐C, n=4) treatment using subcutaneous ALZET osmotic minipumps over the course of 12 weeks before data (Systolic blood pressure (SBP; by tail cuff plethysmography), open field test behavior (OFT), and plasma, tissues samples) was collected. Data was analyzed by 2‐way ANOVA and t‐test and presented as mean±SEM.ResultsSBP did not differ between WT‐C and AD‐C mice. AD mice weighed more than WT (P<0.05). MBG did not affect either parameters (Table
1). WT‐MBG mice had higher heart rates than WT‐C mice (Table
1, P<0.05). Plasma MBG was lower in AD‐C mice vs. WT‐C (125±21 pmol/L vs. 223±41 pmol/L, P<0.05, t‐test). MBG increased plasma levels of this steroid in both WT‐MBG and AD‐MBG mice, which indicated the efficiency of the drug delivery (Table
1). AD‐C mice had 1.6‐fold higher brain cortical expression of inflammatory marker interleukin‐6 (IL6) mRNA vs. WT‐C; MBG significantly reduced cortical mRNA IL6 expression in AD‐MBG mice (Fig
1). No differences were found for total distance traveled, nor for time spent in the center during first 90 seconds of OFT between AD‐C and WT‐C, and between AD‐MBG and AD‐C. Distance traveled in the center showed an effect of strain (P<0.05), an effect of treatment (P<0.05), and a trend‐level treatment:strain interaction (P<0.1). WT traveled farther in the center of the field vs. AD mice. MBG treated mice traveled less in the center vs. non‐treated mice. MBG caused a greater reduction in distance traveled in the center for WT mice than it did for AD mice (Fig
1).ConclusionAD mice had lower endogenous plasma MBG and higher brain pro‐inflammatory mRNA marker IL6 vs. WT mice. Treatment with MBG significantly reduced levels of this inflammatory marker. WT mice exhibited more exploratory behavior than AD mice in the OFT; whereas, MBG decreased exploratory behavior. Exploratory behavior in WT‐MBG mice was more affected than AD‐MBG mice. The association of the brain inflammatory response, cognitive functions and MBG will be further explored in this AD mouse model.Support or Funding InformationSupported by the NIH/NIA Intramural Research ProgramEffect of MBG treatment on expression of IL6 mRNA and OFT performance during first 90 seconds of task. IL6 mRNA (a), time spent in center (b), distance traveled in center (c), and total distance traveled (d) in wild type (WT) and 2xTg‐AD (AD) mice. By unpaired T‐test (a) †P<0.05, WT vs. AD, #P<0.05 AD‐C vs AD‐MBG; by 2‐way ANOVA Linear Mixed Effects followed by Tukey post‐test (b–d): *P<0.05 WT vs. AD; ##P<0.05 Vehicle vs. MBG: drug x strain interaction: P=0.06.Figure 1
Clinical, physiological and behavioral parameters in 15 months old AD mice and the wild type control mice with and without 12‐week treatment with MBG

WT

AD

Control (n=4)

MBG (n=4)

Control (n=8)

MBG (n=8)

Body weight, g

35.0 ± 1.3

34.5 ± 1.8

38.1 ± 1.6
*

38.1 ± 0.5

SBP, mm Hg

98.2 ± 4.3

100.0 ± 4.3

91.6 ± 1.0

103.6 ± 5.9

HR, beats/min

457 ± 11

594 ± 4

513 ± 29

557 ± 19
#

Plasma MBG, pmol/L

223 ± 41

483 ± 116

125 ± 21
†

657 ± 176
#

OFT distance center, m

0.44 ± 0.14

0.11 ± 0.04

0.17 ± 0.04
*

0.15 ± 0.03
#

OFT distance total, m

3.66 ± 0.48

2.56 ± 0.47

2.37 ± 0.48

2.79 ± 1.09

OFT time center, s

4.48 ± 1.84

2.48 ± 1.44

3.01 ± 1.38

0.92 ± 0.38

Data presented as Mean ± SEM.
By 2‐way ANOVA followed by Tukey post‐test:
*P<0.05, AD vs. WT;
#P<0.05. MBG vs. control.
By unpaired t‐test:
†P<0.05. AD vs. WT.
For OFT distance center, effect of strain: P=0.03: effect of drug, P=0.02; drug x strain interaction: P=0.06. Post‐hoc: WT‐MBG HR higher than WT‐C HR (P<0.05). WT, wild type mice: AD, 2xTg‐AD mice; MBG, marinobufagenin; SBP, systolic blood pressure; HR, heart rate: OFT, openfield test.