The role of RhoA GTPase activating protein DLC2 in painful diabetic neuropathy

Abstract

Neuropathy is a major complication that affects nearly half of all patients with diabetes, greatly decreasing their quality of life. Patients experience a wide range of symptoms including pain, numbness, weakness and other morbidities. While its pathogenesis has been the focus of extensive research, there are still few effective treatment options available for this disease. The discovery of novel molecular targets underlying this diabetic neuropathy may lead to the development of new, more effective therapeutics.

DLC2, a Rho GTPase-activating protein with specific activity for RhoA, was shown to be involved in pain signaling. Mice deficient for this protein (DLC2-/-) have increased RhoA activity in their peripheral nerves, and have heightened pain responses compared to wild type (DLC2+/+) in acute pain tests, displaying increased sensitivity to noxious thermal and inflammatory stimuli. DLC2-/- mice also show elevated blood glucose levels, lower body weight and increased sensitivity to blood glucose compared to wild type. Because of the hyperalgesia to acute pain displayed by DLC2-/- mice compared to wild type, and since the RhoA pathway is known to be involved in the pathogeneses and maintenance of diabetes and its complications, these mice were used to investigate more clinically relevant, chronic pain in a model of diabetic neuropathy. Streptozotocin (STZ), given in multiple low doses over five days (MLDS treatment), was used to induce diabetes in DLC2+/+ and DLC2-/- mice, and their pain responses were tested 8 weeks later. Diabetic DLC2-/- mice (DLC2-/--STZ) were hyperalgesic to thermal stimuli from the hot plate test compared to diabetic DLC2 wild type mice (DLC2+/+-STZ) and vehicle-treated controls of both genotypes (DLC2-/--Veh and DLC2+/+-Veh. Similar responses were seen from the von Frey filament test, where the DLC2-/--STZ group exhibited mechanical allodynia compared to the DLC2+/+-STZ group and both control groups. Dorsal root ganglia (DRG) were dissected from these four groups of mice for qPCR screening and protein analysis. DLC2-/--STZ mice showed significantly higher gene expression of the voltage-gated sodium channel Nav 1.9 compared to DLC2+/+-STZ mice, while there was a strong trend of increased levels in the DLC2-/--STZ group compared to both non-diabetic groups. Western blot analysis of the DRG from these mice shows increased levels of COX-2 expression of DLC2-/--STZ mice compared to DLC2+/+-Veh, and elevated levels of phosphorylated ERK (pERK) in DLC2-/--Veh and both diabetic groups compared to DLC2+/+-Veh. Overall, diabetic DLC2-/- mice have more severe painful diabetic neuropathy, with thermal hyperalgesia and mechanical allodynia. Increased RhoA activity and pERK, which are known to be involved in regulation, transcription and trafficking of sodium channels, may lead to increased Nav1.9 mRNA levels and activation. Localized mainly to nociceptors of the DRG, Nav1.9 is known to play a role in sensitizing neurons through lowering the threshold for action potentials, possibly leading to the observed heightened pain response. Additionally, elevated COX-2 levels in DLC2-/--STZ mice may lead to further deficits through activation of inflammatory responses. Future studies will further investigate how these mechanisms are involved in the altered pain response from diabetes.