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Abstracts and Programme: EUROANAESTHESIA 2011: The European Anaesthesiology Congress: ESA Best Abstract Prize Competition (BAPC)

The ubiquitin ligase Nedd4-2 is a potent regulator of the voltage-gated sodium channel Nav1.7 and is implicated in neuropathic pain


Laedermann, C.; Cachemaille, M.; Abriel, H.; Decosterd, I.

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European Journal of Anaesthesiology: June 2011 - Volume 28 - Issue - p 2
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Background and Goal of Study: Neuropathic pain (NP) occurs after a lesion of the nervous system and is associated with nervous system hyperexcitability. Peripheral nerve activity is mainly carried by voltage-gated sodium channels (VGSC), with Nav1.7 isoform being important for nociception. Ubiquitin ligases from the Nedd4 family are proteins that downregulate the expression of many membrane proteins such as VGSC. We hypothesized that Nedd4-2 is decreased in NP, leading to an increase of functional Nav1.7 at the membrane of nociceptors, in turn responsible for the hypersensitivity observed in NP.

Materials and Methods: We used the spared nerve injury (SNI) as an animal model of NP. Allodynia after SNI was assessed with Von Frey filament. Subcellular fractionation of dorsal root ganglia (DRG) allowing the separation of membrane and cytosol enriched fraction was performed to study the targeting of Nav1.7 after SNI. Nedd4-2 expression after SNI was investigated using both immunohistochemistry and westerblotting. In vitro whole cell patch clamp on HEK293 transiently transfected with Nav1.7 alone, or Nav1.7 and Nedd4-2 was used to record sodium currents (INa), where the peak current of INa reflects the quantity of functional Nav1.7 expressed at the membrane. Nav1.7 currents were also recorded in DRG cells in culture.

We used an adeno-associated virus intrathecally injected to exogenously overexpress Nedd4-2 in the DRG. T-test was used to compare SNI to Sham groups with GraphPadPrism software.

Results and Discussion: The subcellular fractionation of DRG showed that Nav1.7 was only increased in the membrane enriched fractions. Nedd4-2 was decreased in DRG after SNI in immunohistochemistry (40%, p=0.0045) and westernbloting (50%, p=0.0078). Co-transfection of Nav1.7 with Nedd4-2 reduced Nav1.7 current amplitude by ˜80% (p=0.0023) confirming that Nedd4-2 downregulates Nav1.7 at the membrane.

Viral overexpression of Nedd4-2 in nociceptors decreased both the mechanical allodynia and Nav1.7 current (43%, p=0.027) in dissociated nociceptors observed after SNI, indicating that Nedd4-2 is sufficient for counteracting full development of allodynia.

Conclusion(s): Our observations pave the way for new avenues in therapeutics against neuropathic pain. We could decrease NP behaviour and restore normal level of excitability by acting on regulatory mechanisms such as the ubiquitin ligase pathway instead of directly targeting VGSC.

Acknowledgements: Supported by ESA and Synapsis Foundation

© 2011 European Society of Anaesthesiology