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 8

rCASP6 increases EPSCs in lamina IIo neurons of spinal cord slices via TNF-α signaling.

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rCASP6 increases EPSCs in lamina IIo neurons of spinal cord slices via T...
(A) Traces of sEPSCs in lamina II neurons of spinal cord slices with and without rCASP6 (5 U/ml) and TNF-α (10 ng/ml) incubation. Bottom panels: Enlargement of traces 1–4. Note the same neuron responds to both rCASP6 and TNF-α. (B) Frequency and amplitude of sEPSCs as shown in A. Note that rCASP6 and TNF-α only increase the sEPSC frequency. *P < 0.05, n = 6–8 neurons. (C) Traces of sEPSCs before and after rCASP3 (5 U/ml) and TNF-α (10 ng/ml) incubation. Note that rCASP3 does not change sEPSCs. Bottom panels: Frequency and amplitude of sEPSCs. n = 5 neurons. (D) Traces of sEPSCs in spinal cord slices of Tnfr1/2 DKO mice before and after rCASP6 (5 U/ml) incubation. (E) Frequency and amplitude of sEPSCs in Tnfr1/2 DKO mice before and after rCASP6 treatment. Note that rCASP6 does not alter sEPSCs in the DKO mice. n = 6 neurons. (F) Traces of eEPSCs in lamina IIo neurons of spinal cord slices following dorsal root stimulation before and after TNF-α (10 ng/ml) and rCASP6 (5 U/ml). Amplitude of eEPSCs as fold difference relative to control. *P < 0.05, n = 7–15 neurons.