The adenosine generating enzymes CD39/CD73 control microglial processes ramification in the mouse brain.

Marina Matyash,Vitali Matyash,Oleksandr Zabiegalov,Helmut Kettenmann,Stefan Wendt,Michelle L Block

doi:10.1371/journal.pone.0175012

Marina Matyash, Vitali Matyash + Show 4 more

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https://doi.org/10.1371/journal.pone.0175012

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Abstract

Microglial cells invade the brain as amoeboid precursors and acquire a highly ramified morphology in the postnatal brain. Microglia express all essential purinergic elements such as receptors, nucleoside transporters and ecto-enzymes, including CD39 (NTPDase1) and CD73 (5'-nucleotidase), which sequentially degrade extracellular ATP to adenosine. Here, we show that constitutive deletion of CD39 and CD73 or both caused an inhibition of the microglia ramified phenotype in the brain with a reduction in the length of processes, branching frequency and number of intersections with Sholl spheres. In vitro, unlike wild-type microglia, cd39-/- and cd73-/- microglial cells were less complex and did not respond to ATP with the transformation into a more ramified phenotype. In acute brain slices, wild-type microglia retracted approximately 50% of their processes within 15 min after slicing of the brain, and this phenomenon was augmented in cd39-/- mice; moreover, the elongation of microglial processes towards the source of ATP or towards a laser lesion was observed only in wild-type but not in cd39-/- microglia. An elevation of extracellular adenosine 1) by the inhibition of adenosine transport with dipyridamole, 2) by application of exogenous adenosine or 3) by degradation of endogenous ATP/ADP with apyrase enhanced spontaneous and ATP-induced ramification of cd39-/- microglia in acute brain slices and facilitated the transformation of cd39-/- and cd73-/- microglia into a ramified process-bearing phenotype in vitro. These data indicate that under normal physiological conditions, CD39 and CD73 nucleotidases together with equilibrative nucleoside transporter 1 (ENT1) control the fate of extracellular adenosine and thereby the ramification of microglial processes.

Highlights

Microglia mediate innate immune responses in the brain and, as recently became evident, are involved in tissue remodeling during development as well as in synaptic plasticity in the adult brain [1, 2]
The cumulative length of microglial processes in wild-type mice was 1500 ± 200 μm, but it was significantly reduced to 713 ± 147 μm and 904 ± 154 μm in cd39-/- and cd73-/- mice, respectively (Fig 2A)
Regardless of the genotype, the presence of ticagrelor (5 μM) in the cell culture medium prevented a transformation of microglia into the more ramified phenotype but caused edges and protrusions to retract (Fig 9A). These results indicate that the ramified phenotype of cultured microglia is promoted by extracellular adenosine, and its availability is regulated by CD39, CD73 and equilibrative nucleoside transporter 1 (ENT1)

Summary

Introduction

Microglia mediate innate immune responses in the brain and, as recently became evident, are involved in tissue remodeling during development as well as in synaptic plasticity in the adult brain [1, 2]. In response to a focal injury, microglia change its motility mode by moving the processes toward the injury site [5, 7]. This microglial response plays a protective role as its inhibition leads to increased areas of tissue damage [8]. Microglia contact synapses and can thereby control their functional state [9, 10] Both motility and phagocytosis of microglia are tightly regulated by purinergic signaling. The targeted extension of the microglia process is controlled by ATP and is mediated by activation of the P2Y12 subtype of purinergic receptors [7, 11]. Activation of A2A adenosine receptors mediates microglial process retraction during inflammation [15]

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