Although microglia have been implicated in nerve injury–induced neuropathic pain, the manner by which injured sensory neurons engage microglia remains unclear. We found that peripheral nerve injury induced de novo expression of colony-stimulating factor 1 (CSF1) in injured sensory neurons. CSF1 was transported to the spinal cord, where it targeted the microglial CSF1 receptor (CSF1R). Cre-mediated sensory neuron deletion of Csf1 completely prevented nerve injury–induced mechanical hypersensitivity and reduced microglial activation and proliferation. In contrast, intrathecal injection of CSF1 induced mechanical hypersensitivity and microglial proliferation. Nerve injury also upregulated CSF1 in motoneurons, where it was required for ventral horn microglial activation and proliferation. Downstream of CSF1R, we found that the microglial membrane adaptor protein DAP12 was required for both nerve injury– and intrathecal CSF1–induced upregulation of pain-related microglial genes and the ensuing pain, but not for microglial proliferation. Thus, both CSF1 and DAP12 are potential targets for the pharmacotherapy of neuropathic pain.
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We thank N. Shah (University of California, San Francisco) for providing Nestin-Cre mice, and D.L. Davies (University of Southern California) and F. Rassendren (Inst. Genomique Functionnelle) for providing the P2X4−/− mice. We also thank E.K. Unger for illustrating Supplementary Figure 13 and J. Leff for technical support. This work was supported by grants from the Foundation for Anesthesia Education and Research (FAER) MRTG-BS-02/15/2010-G and the US National Institutes of Health (K08NS078050) to Z.G., the DFG (KU 3039/1-1) to J.A.K., NIH-DE022001 to A.I.B. and S.L., NIH-NS14627 and the Wellcome Trust to A.I.B., NIH-AG045040 to S.L.A.-W. and NIH-AI068129 to L.L.L.
The authors have submitted a patent, “Targeted disruption of a CSF1-DAP12 pathway member gene for the treatment of neuropathic pain” (PCT/US2015/054704), based on the work described in this article.
Integrated supplementary information
(a) Double immunolabeling for ATF3 and CSF1 in sensory neurons at different time points after nerve injury; (b) Percentage of ATF3-expressing neurons that co-express CSF1. CSF1 is detected in a subset of ATF3- expressing neurons within 18 h of nerve injury and persists for at least 3 weeks. At least 150 ATF3-expressing neurons were counted in each mouse. N = 3 mice per time point. Scale bar represents 100 µm. Data are presented as mean ± s.e.m.
Supplementary Figure 2 CSF1 is induced in injured sensory neurons and co-transported with NPY to the spinal cord, where CSF1R is expressed in microglia.
(a) CSF1 induction in DRG neurons ipsilateral to the nerve injury co-localizes with NPY (Inset), a neuropeptide that is only expressed in sensory neurons after injury (1 d). Scale bars represent 200 μm and 10 μm (Inset); (b) Accumulation of CSF1 and NPY at the dorsal root ligature (4d post injury). Co-localization of CSF1 with NPY establishes that the CSF1 transport is intra-axonal. Dashed line denotes ligature. Scale bar represents 200 μm; (c) CSF1R (immunostaining) co-localizes with the microglial marker CD11b and both markers are induced in the dorsal horn after nerve injury (3 d post injury). Scale bar represents 100 μm; (d) There is complete CSF1R-GFP co-localization with the microglial marker Iba1, and none with the neuronal marker, NeuN. Both CSF1R-GFP and Iba1 are induced in the dorsal horn after nerve injury (3 d post injury). White square shows enlarged region. Scale bar represents 100 μm; (e) Quantification of CSF1R immunostaining in CD11b positive cells in the superficial dorsal horn 3 days after nerve injury; (f) Quantification of GFP intensity in Iba1 positive cells in the superficial dorsal horn 3 days after nerve injury. N = 3–4 mice/group. Data are presented as mean ± s.e.m. *p≤0.05, ***p≤0.001.
Supplementary Figure 3 CSF1 is both necessary and sufficient for microglia activation in the dorsal horn.
(a) Quantification of dorsal horn microglial Iba1 immunoreactivity in control and Adv-CSF1 KO mice 3 days after nerve injury; (b) Iba1 quantification in dorsal horn microglia 3 days after intrathecal injection of CSF1 or PBS. N = 3 mice/group. Data are presented as mean ± s.e.m. *p≤0.05, **p≤0.01.
Supplementary Figure 4 Csf1 deletion from sensory neurons prevents nerve injury-induced mechanical hypersensitivity for at least 4 weeks.
(a) Mice with Advillin-Cre-mediated deletion of Csf1 from sensory neurons do not develop mechanical hypersensitivity for at least 4 weeks after nerve injury (n = 4–5 mice/group). These mutant mice have normal body weight (n = 7 mice/group) (b), normal Rotarod motor function (n = 7 mice/group) (c), normal behavioral responses to noxious heat (n = 7 and 6 mice/group) (d,e), normal nocifensive behavior in an inflammatory pain (hindpaw formalin) model (n = 6 mice/group) (f), and normal numbers of CGRP- and NF200-expressing DRG neurons (n = 3 mice/group) (g,h). In the box plots, the box limits show the first and third quartile, the center line is the median and the whiskers represent the minimum and maximum values. Other data are presented as mean ± s.e.m. *** p≤0.001, **** p≤0.0001.
(a) At 2 hours after CSF1 intrathecal injection, we observed an amoeboid morphology of activated dorsal horn microglia (inset reveals shorter, thickened processes, with larger cell body), and (b) a slight, but significant increase of Iba1 expression (n = 6 for PBS, n = 4 for CSF1). (c) The microglia inhibitor, minocycline, prevents the mechanical hypersensitivity produced by intrathecal CSF1 (n = 6). Scale bar represents 100 µm. In the box plots, the box limits show the first and third quartile, the center line is the median and the whiskers represent the minimum and maximum values. Other data are presented as mean ± s.e.m. * p≤0.05, ** p≤0.01.
(a) Upregulation of Tyrobp mRNA in the dorsal horn persists 7 d post-nerve injury (n = 3 mice/group). (b) CSF1 induction is preserved in injured (ATF3+) sensory neurons of Tyrobp–/– mice (1 d post injury). Scale bar represents 50 μm. Tyrobp–/– mice have normal Rotarod motor activity (c), normal responses to noxious heat in the Hargreaves (d) and hot plate tests (e). N = 6 mice/group (c-e). Data are presented as mean ± s.e.m. ***p≤0.001.
Supplementary Figure 7 Increased expression of DAP12 mRNA correlates with a higher incidence of autotomy in the rat.
Tyrobp mRNA levels in the spinal cord are elevated in a rat strain with high autotomy scores (self-mutilation of a denervated limb) compared to a rat strain with low autotomy scores. These differences are present both before and after denervation (n = 4 rats/group). The box limits show the first and third quartile, the center line is the median and the whiskers represent the minimum and maximum values. * p≤0.05.
Supplementary Figure 8 Microglia–enriched genes are induced in the dorsal cord after nerve injury; monocyte–specific genes are not.
qRT-PCR illustrates that microglia-enriched genes are induced in the ipsilateral dorsal cord 3 d after nerve injury; the levels of monocyte specific genes remain undetectable. (n = 3 mice/group). Data are presented as mean ± s.e.m. **** p≤0.0001.
Supplementary Figure 9 Nerve injury and CSF1-induced microglia proliferation in the dorsal horn is DAP12-independent.
(a) Advillin-Cre-mediated deletion of Csf1 from sensory neurons decreases nerve injury-induced dorsal horn microglia proliferation (3 d post injury; 3 mice per group); (b) Microglial proliferation 3 d post injury persists in Tyrobp–/– mice (3 d post injury, n = 4 mice); (c) Dorsal horn microglia proliferation after intrathecal CSF1 (3 d); (d) Intrathecal CSF1-induced microglia proliferation persists in Tyrobp–/– mice. Scale bar represents 100 μm.
Nerve injury induces CSF1 expression in the injured (ATF3-expressing) motoneurons within 18 h of the injury (left) and the induction persists for at least 3 weeks (right). Scale bar represents 100 μm.
Supplementary Figure 11 Nerve injury-induced CSF1 in injured sensory neurons is preserved in Nestin-Cre; Csf1fl/fl mice.
CSF1 induction in DRG neurons ipsilateral to the nerve injury is preserved in Nestin-Cre;Csf1fl/fl mice (8 d post injury), indicating that Nestin-Cre is not expressed in DRG neurons. Scale bar represents 50 μm.
Supplementary Figure 12 DAP12 is required for CSF1-induced upregulation of neuropathic pain-related microglial genes.
(a) Intrathecal CSF1 upregulates Irf8, Irf5, and P2X4 in the spinal cord and (b) this induction is completely prevented in Tyrobp–/– mice (n = 3–4 mice/group). Data are presented as mean ± s.e.m. * p≤0.05, ** p≤0.01.
Supplementary Figure 13 De novo CSF1 expression in injured sensory neurons triggers a DAP12-independent self-renewal of microglia and a DAP12-dependent upregulation of microglial genes that contribute to the neuropathic pain phenotype.
CSF1 is induced in injured (ATF3-positive) sensory neurons within 1 d of injury and is transported to the spinal cord, where it interacts with microglial CSF1R. Stimulated microglia, in turn, undergo a DAP12-independent proliferation/self-renewal and a DAP12-dependent neuropathic pain–associated gene induction, including BDNF and cathepsin S (CatS). The microglial–derived BDNF contributes to reduced GABAergic inhibitory control and a consequent hyperexcitability of dorsal horn pain transmission neurons. By cleaving CX3CL1 (fractalkine) from neuronal cell membranes, cathepsin S amplifies the activation of microglia. Whether the neuropathic pain phenotype is exacerbated by the concurrent CSF1-induced microglia self-renewal/proliferation and whether DAP12 contributes to that process remains to be determined.
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Guan, Z., Kuhn, J., Wang, X. et al. Injured sensory neuron–derived CSF1 induces microglial proliferation and DAP12-dependent pain. Nat Neurosci 19, 94–101 (2016). https://doi.org/10.1038/nn.4189
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