Blockade of A2AR improved brain perfusion and cognitive function in a mouse model of Alzheimer's disease

Abstract

Alzheimer's disease (AD) is a neurodegenerative disorder that affects more than 6.2 million Americans aged 65 and older, particularly women. Along with AD's main hallmarks (formation of β-amyloid plaques and tau neurofibrillary tangles), there are vascular alterations that occurs in AD pathology. Adenosine A2 receptor (A_2AR) is one of the key factors of brain vascular autoregulation and is overexpressed in AD patients. Our previous findings suggest that protein arginine methyltransferase 4 (PRMT4) is overexpressed in AD, which leads to decrease in cerebral blood flow in aged female 3xTg mice. We aimed to investigate the mechanism behind A_2AR signaling in the regulation of brain perfusion and blood-brain barrier integrity in age and sex-dependent 3xTg mice, and if it is related to PRMT4. Istradefylline, a highly selective A_2AR antagonist, was used to modulate A_2AR signaling. Aged female 3xTg and C57BL/6 J mice were evaluated for brain perfusion (via laser speckle) and cognitive function (via open field, T-maze and novel object recognition). Our results suggest that modulation of A_2AR signaling in aged female 3xTg increased cerebral perfusion by decreasing PRMT4 expression, restored the levels of APP and tau, maintained blood-brain barrier integrity by maintaining the expression of tight junction proteins, and preserved functional learning/memory.

Keywords: Adenosine A2 Receptor; Alzheimer's Disease; Brain Perfusion; Protein Arginine Methyltransferase 4.

Fig. 1

Cerebral hypoperfusion of aged 3xTg female mice was restored by A_2AR antagonist. A Brain perfusion was measured via laser speckle imaging in the brain of male/female of young (3-month-old)/aged (12 to 15-month-old) 3xTg mice (no treatment). *p < 0.05, **p < 0.01 comparing young vs aged mice; ^#p < 0.05 comparing male vs female mice, via two-way ANOVA followed by Tukey’s post-hoc test. B Inhibition of A_2AR with istradefylline (a specific A_2AR antagonist—2 mg/Kg/day, 7 days, IP) restored cerebral perfusion in aged 3xTg female mice. ^@p < 0.01 v. aged C57BL/6 J (C57—gray bar), young (white bar) and aged 3xTg female mice treated with istradefylline (green bar) via one-way ANOVA with Tukey’s post-hoc test. C-D Representative mice cortical images of brain perfusion. (n = 7–10/group)

Fig. 2

A_2AR protein levels are elevated in aged 3xTg female mice. Proteins were quantified by capillary electrophoresis (ProteinSimple) in the cortex (A) and hippocampus (B) of aged (12 to 15-month-old) C57 female mice with saline treatment (gray bars), and aged 3xTg female mice treated with saline (red bars) or istradefylline (2 mg/Kg, 7 days, IP—green bars). Representative pseudoblots are presented below each bar graph. Protein levels were quantified via capillary electrophoresis and normalized to total protein. *p < 0.01 via one-way ANOVA with Tukey’s post-hoc test (n = 6–8/group)

Fig. 3

Blockade of A_2AR restored cortical iNOS levels in aged 3xTg female mice. Protein levels were quantified via capillary electrophoresis and normalized to total protein (ProteinSimple) in the cortex (A-B) and hippocampus (C-D) of aged (12 to 15-month-old) C57 female mice (gray bars), young (3-month-old) 3xTg female mice (white bars), and aged (12 to 15-month-old) 3xTg female mice treated with saline (red bars) or istradefylline (2 mg/Kg/day, 7 days, IP—green bars). Representative pseudoblots are presented (E). ^@p < 0.05 vs aged C57BL/6 J mice; *p < 0.05 between 3xTg mice, via one-way ANOVA, with Tukey’s post-hoc test. (n = 7–8/group)

Fig. 4

Blockade of A_2AR reduced APP and Tau levels in aged 3xTg female mice brain. Protein levels were quantified via capillary electrophoresis and normalized to total protein (ProteinSimple) in the cortex (A-C) and hippocampus (D-F) of young (3-month-old) 3xTg female mice (white bars), and aged (12 to 15-month-old) 3xTg female mice treated with saline (red bars) or istradefylline (2 mg/Kg/day, 7 days, IP – green bars). G Representative pseudoblots are presented. *p < 0.05, **p < 0.01, ***p < 0.001 via one-way ANOVA with Tukey’s post-hoc test. (n = 6/group)

Fig. 5

Blockade of A_2AR reduced PRMT4 and ADMA in aged 3xTg female mice. Protein levels were quantified via capillary electrophoresis and normalized to total protein (ProteinSimple) in the cortex (A-B) and hippocampus (C-D) of aged (12 to 15-month-old) C57 female mice (gray bars), young (3-month-old) 3xTg female mice (white bars), and aged (12 to 15-month-old) 3xTg female mice treated with saline (red bars) or istradefylline (2 mg/Kg/day, 7 days, IP – green bars). Representative pseudoblots are presented in E. ^@p < 0.05 vs aged C57BL/6 J female mice; *p < 0.05, **p < 0.01, ***p < 0.001 comparison between 3xTg female mice via one-way ANOVA, with Tukey’s post-hoc test. (n = 6–8/group)

Fig. 6

Blockade of A_2AR restored cortical tight junction proteins. Occludin and ZO-1 protein levels were quantified via capillary electrophoresis and normalized to total protein (ProteinSimple) in the cortex A-B and hippocampus C-D of aged (12 to 15-month-old) C57 female mice (gray bars), young (3-month-old) 3xTg female mice (white bars), and aged (12 to 15-month-old) 3xTg female mice treated with saline (red bars) or istradefylline (2 mg/Kg/day, 7 days, IP – green bars). Representative pseudoblots are presented in E. ^@p < 0.0001 vs aged C57BL/6 J female mice; *p < 0.05, **p < 0.01, ***p < 0.001 between 3xTg female mice, via one-way ANOVA with Tukey’s post hoc test. (n = 8/group)

Fig. 7

Blockade of A_2AR improved exploratory behavior in aged 3xTg-AD female mice. Exploratory behavior was measured via open field before (basal) and after 7 days of treatment, in young (3-month-old) 3xTg female mice (A), and aged (12 to 15-month-old) 3xTg female mice treated with saline (B) or istradefylline (C); *p < 0.05, **p < 0.01 via paired t-student test (n = 6/group). D Representative tracking of distance traveled in open-field arena, tracked by ANY-maze software. (n = 6–7/group)

Fig. 8

Blockade of A_2AR prevented age-related memory decline in 3xTg-AD female mice. Working memory was evaluated via T-maze test before (basal) and after 7 days of treatment (A) on young (3-month-old) 3xTg female mice (black line), and aged (12 to 15-month-old) 3xTg female mice treated with saline (red line) or istradefylline (2 mg/Kg/day, 7 days, IP—green line); **p < 0.01 via two-way ANOVA, followed by Tukey’s post-hoc test. B Percentage of spontaneous alternation of young (white bar) and aged (12 to 15-month-old) 3xTg female mice after 7 days of treatment with saline (red) or istradefylline (green); *p < 0.05, **p < 0.01 via one-way ANOVA followed by Tukey’s post hoc test. C Long-term memory was evaluated via novel object recognition on young (white bar), on aged (12 to 15-month-old) C57 female (gray bars), and on aged 3xTg female mice after 7 days of treatment with saline (red) or istradefylline (green). ^@p < 0.01 compared to aged C57BL/6 J female mice, **p < 0.01 compared to aged 3xTg female mice treated with istradefylline via one-way ANOVA with Tukey’s post hoc test. (n = 5–8/group)