Extracellular caspase-6 drives murine inflammatory pain via microglial TNF-α secretion
Temugin Berta, … , Yen-Chin Liu, Ru-Rong Ji
Temugin Berta, … , Yen-Chin Liu, Ru-Rong Ji
Published February 17, 2014
Citation Information: J Clin Invest. 2014;124(3):1173-1186. https://doi.org/10.1172/JCI72230.
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Research Article Neuroscience

Extracellular caspase-6 drives murine inflammatory pain via microglial TNF-α secretion

Abstract

Increasing evidence indicates that the pathogenesis of neuropathic pain is mediated through spinal cord microglia activation. The intracellular protease caspase-6 (CASP6) is known to regulate neuronal apoptosis and axonal degeneration; however, the contribution of microglia and CASP6 in modulating synaptic transmission and pain is unclear. Here, we found that CASP6 is expressed specifically in C-fiber axonal terminals in the superficial spinal cord dorsal horn. Animals exposed to intraplantar formalin or bradykinin injection exhibited CASP6 activation in the dorsal horn. Casp6-null mice had normal baseline pain, but impaired inflammatory pain responses. Furthermore, formalin-induced second-phase pain was suppressed by spinal injection of CASP6 inhibitor or CASP6-neutralizing antibody, as well as perisciatic nerve injection of CASP6 siRNA. Recombinant CASP6 (rCASP6) induced marked TNF-α release in microglial cultures, and most microglia within the spinal cord expressed Tnfa. Spinal injection of rCASP6 elicited TNF-α production and microglia-dependent pain hypersensitivity. Evaluation of excitatory postsynaptic currents (EPSCs) revealed that rCASP6 rapidly increased synaptic transmission in spinal cord slices via TNF-α release. Interestingly, the microglial inhibitor minocycline suppressed rCASP6 but not TNF-α–induced synaptic potentiation. Finally, rCASP6-activated microglial culture medium increased EPSCs in spinal cord slices via TNF-α. Together, these data suggest that CASP6 released from axonal terminals regulates microglial TNF-α secretion, synaptic plasticity, and inflammatory pain.

Authors

Temugin Berta, Chul-Kyu Park, Zhen-Zhong Xu, Ruo-Gang Xie, Tong Liu, Ning Lü, Yen-Chin Liu, Ru-Rong Ji

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Figure 3

Formalin-induced second-phase inflammatory pain is reduced after deletion of Casp6, spinal inhibition of CASP6 with inhibitor or neutralizing antibody, or DRG knockdown of Casp6 with specific siRNA.

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Formalin-induced second-phase inflammatory pain is reduced after deletio...
(A–C) Casp6–/– mice exhibit normal thermal pain (A, tail immersion test), mechanical pain (B, Randall-Selitto test), and motor function (C, rotarod test). n = 6 mice. (D) Time course (0–45 minutes) of formalin-induced spontaneous pain in WT and Casp6–/– mice. *P < 0.05, compared with WT mice, n = 6–8 mice. (E) Formalin-induced first-phase (1–10 minutes) and second-phase nociceptive responses (10–45 minutes) in WT and Casp6–/– mice. *P < 0.05, n = 6–8 mice. (F) Spinal injection of the CASP6 inhibitor ZVEID (i.t., 1–10 μg) reduces the second-phase pain. *P < 0.05, compared with vehicle (10% DMSO), n = 5–6 mice. (G) Spinal injection of CASP6-neutralizing antibody (i.t., 1 μg) reduces the second-phase pain. *P < 0.05, n = 5–7 mice. (H and I) Perisciatic delivery of Casp6 siRNA (2 μg, 6 μl, mixed with the RVG peptide) suppresses the formalin-induced second-phase pain (H) and reduces the expression of Casp6 but not Casp1 and Casp3 in DRGs (I). Scramble siRNA served as control siRNA. *P < 0.05, n = 5–8 mice.