Vector-mediated release of GABA attenuates pain-related behaviors and reduces Na(V)1.7 in DRG neurons

Abstract

Pain is a common and debilitating accompaniment of neuropathy that occurs as a complication of diabetes. In the current study, we examined the effect of continuous release of gamma amino butyric acid (GABA), achieved by gene transfer of glutamic acid decarboxylase (GAD67) to dorsal root ganglia (DRG) in vivo using a non-replicating herpes simplex virus (HSV)-based vector (vG) in a rat model of painful diabetic neuropathy (PDN). Subcutaneous inoculation of vG reduced mechanical hyperalgesia, thermal hyperalgesia and cold allodynia in rats with PDN. Continuous release of GABA from vector transduced cells in vivo prevented the increase in the voltage-gated sodium channel isoform 1.7 (Na(V)1.7) protein that is characteristic of PDN. In vitro, infection of primary DRG neurons with vG prevented the increase in Na(V)1.7 resulting from exposure to hyperglycemia. The effect of vector-mediated GABA on Na(V)1.7 levels in vitro was blocked by phaclofen but not by bicuculline, a GABA(B) receptor effect that was blocked by pertussis toxin-(PTX) interference with Gα((i/o)) function. Taken in conjunction with our previous observation that continuous activation of delta opioid receptors by vector-mediated release of enkephalin also prevents the increase in Na(V)1.7 in DRG exposed to hyperglycemia in vitro or in vivo, the observations in this report suggest a novel common mechanism through which activation of G protein coupled receptors (GPCR) in DRG neurons regulate the phenotype of the primary afferent.

Figure 1. Vector mediated expression of GABA reverses pain-related behaviors in animals with PDN

(a) Thermal withdrawal latency (Hargreave’s test) in seconds. (b) Mechanical hyperalgesia (Randall Selitto test) threshold in grams. (c) Cold allodynia in seconds. C – naïve control; D - diabetic; DvG - diabetic inoculated with vG; DvZ - diabetic inoculated with vZ; all data presented as mean ± SEM, n = 6–8 per group. #P < 0.0001 or ***P < 0.001 vs. untreated diabetic animals or DvZ.

Figure 2. vG mediated GABA expression attenuates the increase in Na_V1.7 in DRG in vivo characteristic of PDN

Amount of Na_V1.7 determined by Western blot, normalized to D (a) or DvZ (b). C - control; D - diabetic; DvG - diabetic inoculated with vG; DvZ - diabetic inoculated with vZ; ***P < 0.001 or *P < 0.01; n = 5 animals per group. Representative Western blots showing one sample from each group are presented above each graph.

Figure 3. Inoculation of vG prevents phosphorylation of PKC in diabetic DRG in vivo

Amount of pPKCα/β determined by Western blot, normalized to D (a) or DvZ (b). **P <0.005; n = 5 animals per group. C - Control; D - diabetic; DvG - diabetic inoculated with vG; DvZ - diabetic inoculated with vZ. Representative Western blots showing one sample from each group are presented above each graph.

Figure 4. Na_V1.7 levels are increased in adult DRG neurons in culture exposed to hyperglycemic conditions

Adult DRG neurons in culture were exposed to medium containing 45 mM glucose for 18 hrs, and the amount of NaV1.7 determined by Western blot.

Figure 5. Transgene-mediated expression of GABA prevents the increase in Na_V1.7 in primary DRG neurons exposed to hyperglycemic conditions in vitro

(a) Significant reduction in the amount of Na_V1.7 in hyperglycemic neurons treated with vG as determined by Western blot, normalized to G. (b) Amount of pPKCα/β determined by Western blot, normalized to C. **P < 0.005 or *P < 0.01; C - control; G - hyperglycemia; GvG – hyperglycemia, transfected with vG. Representative Western blots showing one sample from each group are presented above each graph.

Figure 6. Phaclofen but not bicuculline blocks the effect of vG on the amount Na_V1.7 in DRG neurons exposed to hyperglycemic conditions in vitro

(a) No substantial changes in the amount of Na_V1.7 in primary DRG neurons exposed to hyperglycemia, transfected with vG in the presence of 50 μM of bicuculline (vG + bic). (b) Significant changes in the amount of Na_V1.7 in primary DRG neurons exposed to hyperglycemia and transfected with vG in the presence of 500 μM of phaclofen (vG + phac), normalized to vG; (c) Substantial changes in the amount of Na_V1.7 in primary DRG neurons exposed to hyperglycemia in the presence of 10 μM of baclofen (G + bac). G - hyperglycemia; vG – cells transfected with vector; vG + phac- cells transfected with vG in presence of phaclofen; vG + bic- cells transfected with vG in presence of bicuculline, G + bac- hyperglycemia, in presence of baclofen. *P < 0.01, **P < 0.005. Representative Western blots showing one sample from each group are presented above each graph.

Figure 7. Phosphorylation of PKC in hyperglycemic DRG neurons is blocked by a GABA_BR antagonist

(a) No changes in pPKCα/β in primary DRG neurons exposed to 45 mM glucose and transfected with vG in the presence of 50 μM bicuculline (vG + bic) compared to vG treated neurons;. (b) There were significant changes in the amount of pPKCα/β in primary DRG neurons exposed to 45 mM glucose and transfected with vG in the presence of 500 μM phaclofen (vG + phac) compared to vG treated neurons; *P < 0.01, **P < 0.005; n = 3 wells per group. Representative Western blots showing one sample from each group are presented above each graph.

Figure 8. Effect of continuous activation of GABA_BR by vG on levels of Na_V1.7 is mediated through G_i/o proteins

(a) Treatment with of vG-infected neurons exposed to hyperglycemic conditions with pertussis toxin (PTX; 250 ng/ml) reversed the effect of GABA_BR activation on the amount of Na_V1.7 compared to vG infected neurons exposed to hyperglycemic conditions ** P < 0.005. (b) Treatment with cholera toxin (CTX) in similar conditions did not result in any change in amount of Na_V1.7 in vG infected DRG neurons * P < 0.01.