CD73 is a major regulator of adenosinergic signalling in mouse brain

Abstract

CD73 (ecto-5'-nucleotidase) is a cell surface enzyme that regulates purinergic signalling by desphosphorylating extracellular AMP to adenosine. 5'-nucleotidases are known to be expressed in brain, but the expression of CD73 and its putative physiological functions at this location remain elusive. Here we found, using immunohistochemistry of wild-type and CD73 deficient mice, that CD73 is prominently expressed in the basal ganglia core comprised of striatum (caudate nucleus and putamen) and globus pallidus. Furthermore, meninges and the olfactory tubercle were found to specifically express CD73. Analysis of wild type (wt) and CD73 deficient mice revealed that CD73 confers the majority of 5'-nucleotidase activity in several areas of the brain. In a battery of behavioural tests and in IntelliCage studies, the CD73 deficient mice demonstrated significantly enhanced exploratory locomotor activity, which probably reflects the prominent expression of CD73 in striatum and globus pallidus that are known to control locomotion. Furthermore, the CD73 deficient mice displayed altered social behaviour. Overall, our data provide a novel mechanistic insight into adenosinergic signalling in brain, which is implicated in the regulation of normal and pathological behaviour.

Figure 1. Immunohistochemical localization of CD73 in mouse brain.

Coronal sections of WT (panels A, C and E) and CD73 deficient (panels B, D and F) mouse cerebrums stained with CD73 antibody. The specific CD73 staining is detected in caudoputamen (CP), olfactory tubercle (OT), globus pallidus (GP) and meninx (arrow). Scale bar 500 μm.

Figure 2. CD73 is the dominant 5’nucleotidase in the brain.

(A) A scheme of the major extracellular nucleotide-converting pathways. The inactivating cascade is composed of NTPDase (1,2), and CD73/ecto-5’-nucleotidase (3), whereas the backward ATP-generating pathway is regulated by adenylate kinase (4) and NDP kinase (5). Brain lysates were isolated from forebrain, middle brain and cerebellum of wt and CD73 deficient mice and assayed for 5’-nucleotidase (B) ATPase (C), ADPase (D) and adenylate kinase (E) activities, as specified in Materials and Methods (mean±SEM; n = 4–5). *P<0.05 as compared with corresponding wt controls.

Figure 3. CD73 deficient and wt mice display similar pre-pulse inhibition but startle response is enhanced in CD73 deficient mice compared to wt mice.

Percentage of pre-pulse inhibition averaged across all pre-pulse intensities. Insert bar graphs represent percentage of alteration of startle response without pre-pulse stimulus from the wild type animals’ startle response level (wt mice, n = 38; CD73 deficient mice, n = 29). The results from both sexes are combined, because there was no sex effect or sex X genotype interaction. Mean values are plotted with SEM, **p<0.01 t-test.

Figure 4. CD73 deficient mice do not differ from wt mice in olfactory discrimination test.

(A) Latency to sniffing and (B) time spent in olfactory investigation of capsules with odors during four repeated trials (with cinnamon) and one trial of dishabituation (cocoa) (wt female mice, n = 6; CD73 deficient female mice, n = 7).

Figure 5. Learning in water maze and in IntelliCage is comparable in wt and CD73 deficient mice.

(A) Escape latency to hidden platform during training and reversed training with moved platform in Morris water maze (wt mice, n = 38; CD73 deficient mice, n = 29). The results from both sexes are combined, because there was no sex effect or sex X genotype interaction. (B) Percentage of visits to the “correct” corner during corner preference learning and reversal learning in IntelliCage (CD73 deficient female mice, n = 9; wt female mice, n = 8).

Figure 6. CD73 deficient mice demonstrate increased exploratory activity.

The distances travelled by wt (n = 38 mice) and CD73 deficient (n = 29) mice in (A) the elevated plus maze and (B) open field tests. The results from both sexes are combined, because there was no sex effect or sex X genotype interaction. In Intellicage analyses the numbers of corner visits (mean ± SEM) are plotted between 1 and 6 hr (C). The IntelliCage data are from females only (CD73 deficient mice, n = 9; wt mice, n = 8). *p<0.05 t-test, **p<0.01 t-test, # p<0.05 ANOVA, ## p<0.01 ANOVA.

Figure 7. CD73 controls circadian changes in locomotor activity in isolated mice.

(A) Locomotor activity presented as activity counts during 72 hr observation period, when the mice were separated in individual cages. These data are presented for both sexes together, because there was no sex effect or sex X genotype interaction (CD73 deficient mice, n = 25; wt mice, n = 30). (B) Locomotor activity measured as corner visits in IntelliCage. In IntelliCage analyses only females were used (CD73 deficient, n = 9; wt, n = 8). Mean values are plotted with SEM, # p<0.05 and ## p<0.01, repeated ANOVA.

Figure 8. Mouse with hair loss (barbering).

An example of a mouse with bald patches due to barbering.

Figure 9. CD73 deficient mice display altered social behaviour.

(A) Percentages of the times pushed out in the tube test (32 mice/group in both genotypes). (B) Percentage of the time spent in social activity towards the intruder in the resident-intruder test (wt mice, n = 15; CD73 deficient mice, n = 13). (C) Time spent in sniffing the stranger in the social novelty preference test, and (D) time spent in sniffing a new and an old stranger in the sociability test (wt, n = 16; CD73 deficient, n = 16). Mean values are plotted with SEM, *p<0.05 t-test, **p<0.01 t-test.