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The P2Y12 receptor regulates microglial activation by extracellular nucleotides


Microglia are primary immune sentinels of the CNS. Following injury, these cells migrate or extend processes toward sites of tissue damage. CNS injury is accompanied by release of nucleotides, serving as signals for microglial activation or chemotaxis. Microglia express several purinoceptors, including a Gi-coupled subtype that has been implicated in ATP- and ADP-mediated migration in vitro. Here we show that microglia from mice lacking Gi-coupled P2Y12 receptors exhibit normal baseline motility but are unable to polarize, migrate or extend processes toward nucleotides in vitro or in vivo. Microglia in P2ry12−/− mice show significantly diminished directional branch extension toward sites of cortical damage in the living mouse. Moreover, P2Y12 expression is robust in the 'resting' state, but dramatically reduced after microglial activation. These results imply that P2Y12 is a primary site at which nucleotides act to induce microglial chemotaxis at early stages of the response to local CNS injury.

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Figure 1: P2Y12 immunoreactivity localized to the surface of CNS microglia.
Figure 2: Loss of P2Y12 expression accompanies microglial transformation from highly ramified to amoeboid state.
Figure 3: Activated microglia lack P2Y12 expression in vivo.
Figure 4: Nucleotide-evoked membrane ruffling and chemotaxis are abolished in cultured microglia lacking P2Y12 receptors.
Figure 5: P2Y12-deficient microglia do not respond to exogenous nucleotides in brain slices.
Figure 6: Loss of P2Y12 receptors abrogates response of microglia to ATP injection or focal laser ablation in the living brain.


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We are grateful to L. Fuller, H. Morisaki, J. Braz and A. So for experimental advice and assistance, and to D. Littman (Skirball Institute, New York University) for providing Cx3cr1+/GFP mice. This work was supported by predoctoral fellowships from the US National Institute of General Medical Sciences (GM56847 for S.H.) and the American Heart Association (G.H.) and by research grants from the US National Institutes of Health (NS43468 to M.D.), the National Institute of Mental Health (Silvio Conte Center for Neuroscience Research at UCSF to D.J.) and the Christopher Reeve Paralysis Foundation (to D.J.).

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Correspondence to David Julius.

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Supplementary information

Supplementary Fig. 1

Microglia from P2Y12-deficient mice have normal morphology and prevalence within the CNS. (PDF 15 kb)

Supplementary Fig. 2

Pretreatment with LPS diminishes ATP-induced microglial ruffling. (PDF 44 kb)

Supplementary Fig. 3

Microglia express P2Y13 transcripts, but not detectable receptor protein. (PDF 93 kb)

Supplementary Video 1

Wild-type (left) microglia in the Dunn chemotaxis chamber undergo membrane ruffling, polarization, and directed motility in the presence of a 50 to 0 μM ATP gradient (top to bottom) whereas P2Y12-deficient (right) microglia show dramatically reduced stimulation. The chemotaxis chamber was placed in a heated, C02 buffered humidified microscope incubator and phase contrast images were acquired every 150 s for 30 min. Scale bar represents 20 μm. (MOV 540 kb)

Supplementary Video 2

Acutely prepared hippocampal slices from P7 neonatal wild-type (left) and P2Y12-deficient (right) mice were bathed in imaging media with 1 mM ADP. Wild-type microglia respond within minutes by sending out long, cellular projections towards the periphery of the slice (top, right, and bottom edges of field of view). Some cells actually undergo whole-cell body displacement as they travel towards the nucleotide source while other cells extend elaborately branched ramifications. Some cells first respond by sending out branches before exhibiting whole cell locomotion. In striking contrast, microglia from P2Y12-deficient animals show a dramatically diminished behavioral response towards the nucleotide source. Images show the CA3 layer of the hippocampal slice. Cells were visualized by GFP expression, images were taken at 5 min intervals for 6.75 h, and a maximum projection of 15 z-steps spaced 2 μm apart (30 μm total thickness) was constructed for each frame. Scale bar represents 100 μm. (MOV 9980 kb)

Supplementary Video 3

In vivo imaging of GFP labelled microglia in wild-type (left) and P2Y12-deficient (right) mice after injection of 20 mM ATP (red needle). Wild-type microglia displayed robust branch extension towards the site of injection whereas P2Y12-deficient microglia did not. Experiment is 40 min, 4 min intervals. Scale bar represents 20 μm. (MOV 2432 kb)

Supplementary Video 4

In vivo imaging of GFP labelled microglia in wild-type (left) and P2Y12-deficient (right) mice after focal laser ablation induced by the two-photon laser. Wild-type microglia displayed robust process extension towards the site of injury whereas P2Y12-deficient microglia showed a dramatically reduced and time-delayed response. Experiment is 40 min, 4 min intervals. Scale bar represents 20 μm. (MOV 5589 kb)

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Haynes, S., Hollopeter, G., Yang, G. et al. The P2Y12 receptor regulates microglial activation by extracellular nucleotides. Nat Neurosci 9, 1512–1519 (2006).

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