Lyn tyrosine kinase is required for P2X₄ receptor upregulation and neuropathic pain after peripheral nerve injury

Abstract

Neuropathic pain, a debilitating chronic pain following nerve damage, is a reflection of the aberrant functioning of a pathologically altered nervous system. One hallmark is abnormal pain hypersensitivity to innocuous stimuli (tactile allodynia), for which effective therapy is lacking, and the underlying mechanisms of which remain to be determined. Here we show that Lyn, a member of the Src family kinases (SFKs), plays an important role in the pathogenesis of neuropathic pain. Nerve injury, but not peripheral inflammation, increased immunoreactivity for active SFKs that were autophosphorylated in the kinase domain (phospho-SFK-IR) in spinal microglia. In spinally derived microglial cells, we identified Lyn as the predominant SFK among the five members (Src, Fyn, Yes, Lck, and Lyn) known to be expressed in the CNS. Lyn expression in the spinal cord was highly restricted to microglia, and its level was increased after nerve injury. We found that mice lacking lyn (lyn(-/-)) exhibit a striking reduction in the levels of phospho-SFK-IR and tactile allodynia after nerve injury, without any change in basal mechanical sensitivity or inflammatory pain. Importantly, lyn(-/-) mice displayed impaired upregulation of the ionotropic ATP receptor subtype P2X(4) receptors (P2X(4)R) in the spinal cord after nerve injury, which is crucial for tactile allodynia. Microglial cells from lyn(-/-) mice showed a deficit in their ability to increase P2X(4)R expression in response to fibronectin, a factor implicated as a microglial P2X(4)R upregulator in allodynia. Together, our findings suggest that Lyn may be a critical kinase mediating nerve injury-induced P2X(4)R upregulation and neuropathic pain.

ATP receptors gate microglia signaling in neuropathic pain

“…Also critical for upregulating expression of P2X4 receptors is the extracellular matrix molecule fibronectin, which through the Lyn kinase signaling pathway modulates the transcriptional and post-transcriptional levels of P2X4 receptor expression in microglia (Nasu-Tada et al, 2006;Tsuda et al, 2008a;Tsuda et al, 2008b;Tsuda et al, 2009c). Similarly, activation of mu-opioid receptors by morphine can drive P2X4 receptor expression in states of opioid tolerance (Horvath and DeLeo, 2009;Horvath et al, 2010) and opioid-induced hyperalgesia (Ferrini et al, 2010).…”

“…Thus, the upregulation of these microglial markers after peripheral nerve injury is not dependent upon P2X4 receptors. Definitive evidence that stimulation of P2X4 receptors expressed on microglia is sufficient to elicit pain hypersensitivity comes from the finding that intrathecal injection of P2X4 receptor-stimulated cultured microglia elicits robust mechanical allodynia in non-nerve injured animals (Coull et al, 2005;Tsuda et al, 2003;Tsuda et al, 2008b). Taken together, the pharmacological, genetic, and behavioral findings indicate that activity of P2X4 receptors expressed on spinal microglia is both necessary and sufficient, and therefore logically causative, to induce tactile allodynia after peripheral nerve injury.…”

Microglia–neuronal signalling in neuropathic pain hypersensitivity 2.0

“…The subsequent signalling to spinal microglia has been suggested to involve a number of ligand/receptor systems, including fibronectin/integrin 18,19 , MCP1/ CCR2 20 , interferon/IFG receptor signalling 21 . Also critical for increasing expression of P2X4Rs is the tyrosine kinase Lyn 22 . Thus several key signalling elements necessary for the upregulation of P2X4Rs have been identified, and thus the next major challenges will be to determine how these elements are causally connected, and whether these encompass the entirety of the necessary pathways, or where other pathways are also required.…”

P2X4 purinoceptor signaling in chronic pain

“…Likewise, microglia grown in culture in the presence of the extracellular matrix molecule fibronectin show a marked increase in P2X4R levels and a consequent enhanced Ca 2+ response to ATP stimulation [52]. Fibronectin expression was elevated in the spinal cord of nerve injured animals and blockade of fibronectin receptors suppressed both the increase in P2X4Rs and tactile allodynia [52,[76][77][78]. Fibronectin induced increase in microglial P2X4R requires activity of the tyrosine kinase Lyn and downstream activation of intracellular signaling pathways involving phosphatidylinositol 3-kinase (PI3K)-Akt and mitogen-activated protein kinase kinase (MAPK kinase, MEK)-extracellular signal-regulated kinase (ERK), which have distinct roles in the up-regulation of P2X4R expression in microglia at the transcriptional and post-transcriptional levels, respectively [77].…”