Figure 1. Astrocytic A_2A receptor expression is increased in humans with AD.

(a) Levels of A_2A receptor protein in hippocampi (HP) and frontal cortices (FC) of human control (Con) and AD cases as determined by western blotting. Ratios of A_2A/actin were normalized to the average ratio in Con samples. Student’s t-test with Welch’s correction (Con vs. AD): P = 0.001 (HP); P = 0.70 (FC). n = 5 (Con HP), 17 (AD HP), 5 (Con FC), and 16 (AD FC) cases per group. (b) Levels of ADORA2A and GFAP mRNA in the dentate gyri from Con and AD cases as determined by qRT-PCR. GAPDH mRNA served as a loading control. Student’s t-test with Welch’s correction (Con vs. AD): P = 0.0008 (ADORA2A); P < 0.0001 (GFAP). n = 3 (Con ADORA2A), 15 (AD ADORA2A), 3 (Con GFAP), and 16 (AD GFAP) cases per group. (c) Levels of ADORA2A mRNA in the dentate gyri of humans with AD ranging from mild to severe based on pathological analysis and Braak scoring.¹² ADORA2A/GAPDH ratios were plotted as a function of Braak score. Spearman rank correlation, R = 0.76, P = 0.0063, n = 11 cases. Red line shows the linear regression curve and dotted lines show the 95% confidence intervals. (d) Levels of ADORA2A mRNA in the dentate gyri of humans with AD plotted as a function of GFAP mRNA levels. Spearman rank correlation, R = 0.57, P = 0.027, n = 15 cases. GAPDH mRNA served as a loading control. (e–f) Representative photomicrographs of A_2A receptor immunoreactivity in hippocampal sections from two independent cohorts of human cases with the indicated Braak stages. Severity of cognitive impairment is indicated by Mini Mental State Examination (MMSE) or Blessed Dementia Scale (BDS) scores. Insets (i–iv) show magnified views of the boxed regions. (g) Representative photomicrographs of a hippocampal section from an AD case co-immunostained for the A_2A receptor (green) and the astrocyte marker Aldh1L1 (red). Cell nuclei were labeled with DAPI (blue). The overlay shows co-localization of A_2A and Aldh1L1 immunoreactivities. Scale bars: 100 μm (e–g), 25 μm (bottom insets in g). n = 10 Con and 19 AD cases. Insets (i–vi) show magnified views of the boxed regions. ***P < 0.001 (Student’s t-test with Welch’s correction). Values are means ± s.e.m.