Dysregulation of voltage-gated sodium channels by ubiquitin ligase NEDD4-2 in neuropathic pain
Cédric J. Laedermann, … , Hugues Abriel, Isabelle Decosterd
Cédric J. Laedermann, … , Hugues Abriel, Isabelle Decosterd
Published June 17, 2013
Citation Information: J Clin Invest. 2013;123(7):3002-3013. https://doi.org/10.1172/JCI68996.
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Research Article Neuroscience

Dysregulation of voltage-gated sodium channels by ubiquitin ligase NEDD4-2 in neuropathic pain

Abstract

Peripheral neuropathic pain is a disabling condition resulting from nerve injury. It is characterized by the dysregulation of voltage-gated sodium channels (Navs) expressed in dorsal root ganglion (DRG) sensory neurons. The mechanisms underlying the altered expression of Navs remain unknown. This study investigated the role of the E3 ubiquitin ligase NEDD4-2, which is known to ubiquitylate Navs, in the pathogenesis of neuropathic pain in mice. The spared nerve injury (SNI) model of traumatic nerve injury–induced neuropathic pain was used, and an Nav1.7-specific inhibitor, ProTxII, allowed the isolation of Nav1.7-mediated currents. SNI decreased NEDD4-2 expression in DRG cells and increased the amplitude of Nav1.7 and Nav1.8 currents. The redistribution of Nav1.7 channels toward peripheral axons was also observed. Similar changes were observed in the nociceptive DRG neurons of Nedd4L knockout mice (SNS-Nedd4L–/–). SNS-Nedd4L–/– mice exhibited thermal hypersensitivity and an enhanced second pain phase after formalin injection. Restoration of NEDD4-2 expression in DRG neurons using recombinant adenoassociated virus (rAAV2/6) not only reduced Nav1.7 and Nav1.8 current amplitudes, but also alleviated SNI-induced mechanical allodynia. These findings demonstrate that NEDD4-2 is a potent posttranslational regulator of Navs and that downregulation of NEDD4-2 leads to the hyperexcitability of DRG neurons and contributes to the genesis of pathological pain.

Authors

Cédric J. Laedermann, Matthieu Cachemaille, Guylène Kirschmann, Marie Pertin, Romain-Daniel Gosselin, Isabelle Chang, Maxime Albesa, Chris Towne, Bernard L. Schneider, Stephan Kellenberger, Hugues Abriel, Isabelle Decosterd

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Figure 2

NEDD4-2 downregulates membrane Nav1.7.

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NEDD4-2 downregulates membrane Nav1.7. (A) Representative current trac...
(A) Representative current traces obtained with a V-I protocol (see Methods) on HEK293 cells after Nav1.7 transfection or cotransfection with NEDD4-2. (B) Quantification of current densities from A. NEDD4-2 reduced Nav1.7 current density (***P < 0.001). See Supplemental Figure 1A and Supplemental Table 1 for values and biophysical properties. (C) Surface biotinylation of HEK293 cells and their associated quantification. In membrane fractions, NEDD4-2 reduced the fully glycosylated form of Nav1.7 (***P < 0.001), whereas the core glycosylated form remained unchanged (P = 0.416). Nav1.7 total expression was unchanged (P = 0.337; Input). The β1 subunit of the NaK/ATPase and actin were used as loading controls in input and biotinylation fractions, respectively. Deglycosylation experiments are presented in Supplemental Figure 1B. NEDD4-2 antibody recognizes both endogenous (120 kDa) and transfected (100 kDa) proteins. (D) GST pull-down experiment showing Nav1.7 PY motif interaction with NEDD4-2. HEK293 cells were transfected with NEDD4-2, and soluble fractions were mixed GST proteins or GST-Cter-Nav1.7 fusion proteins. Bound NEDD4-2 was analyzed by Western blot. The entire Western blot with PY motif mutants can be seen in Supplemental Figure 2E. (E) NEDD4-2–mediated ubiquitylation. HEK293 cells were transfected with Nav1.7 or cotransfected with NEDD4-2, and soluble fractions were mixed with GST-S5A proteins to pull down ubiquitylated proteins. Bound Nav1.7 was analyzed by Western blotting. The entire Western blot with PY motif mutants can be seen in Supplemental Figure 2F.