Munmun Chattopadhyay

Continuous δ-Opioid Receptor Activation Reduces Neuronal Voltage-Gated Sodium Channel (NaV1.7) Levels through Activation of Protein Kinase C in Painful Diabetic Neuropathy

The NaV1.7 tetrodotoxin-sensitive voltage-gated sodium channel isoform plays a critical role in nociception. In rodent models of diabetic neuropathy, increased NaV1.7 in dorsal root ganglia (DRG) neurons correlates with the emergence of pain-related behaviors characteristic of painful diabetic neuropathy (PDN). We examined the effect of transgene-mediated expression of enkephalin on pain-related behaviors and their biochemical correlates in DRG neurons. Transfection of DRG neurons by subcutaneous inoculation of a herpes simplex virus-based vector expressing proenkephalin reversed nocisponsive behavioral responses to heat, cold, and mechanical pressure characteristic of PDN. Vector-mediated enkephalin production in vivo prevented the increase in DRG NaV1.7 observed in PDN, an effect that correlated with inhibition of phosphorylation of p38 MAPK (mitogen-activated protein kinase) and protein kinase C (PKC). Primary DRG neurons in vitro exposed to 45 mm glucose for 18 h also demonstrated an increase in NaV1.7 and increased phosphorylation of p38 and PKC; these changes were prevented by transfection in vitro with the enkephalin-expressing vector. The effect of hyperglycemia on NaV1.7 production in vitro was mimicked by exposure to PMA and blocked by the myristolated PKC inhibitor 20-28 or the p38 inhibitor SB202190 [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole]; the effect of vector-mediated enkephalin on NaV1.7 levels was prevented by naltrindole. The results of these studies suggest that activation of the presynaptic δ-opioid receptor by enkephalin prevents the increase in neuronal NaV1.7 in DRG through inhibition of PKC and p38. These results establish a novel interaction between the δ-opioid receptor and voltage-gated sodium channels.

In vivo gene therapy for pyridoxine-induced neuropathy by herpes simplex virus-mediated gene transfer of neurotrophin-3

Neurotrophic factors have been demonstrated to prevent the development of peripheral neuropathy in animal models, but the therapeutic use of these factors in human disease has been limited by the short serum half‐life and dose‐limiting side effects of these potent peptides. We used peripheral subcutaneous inoculation with a replication‐incompetent, genomic herpes simplex virus‐based vector containing the coding sequence for neurotrophin‐3 to transduce sensory neurons of the rat dorsal root ganglion in vivo, and found that expression of neurotrophin‐3 from the vector protected peripheral sensory axons from neuropathy induced by intoxication with pyridoxine assessed by electrophysiological (foot sensory response amplitude, and conduction velocity, and H‐wave), histological (nerve morphology and morphometry), and behavioral measures of proprioceptive function. In vivo gene transfer using herpes simplex virus vectors provides a unique option for treatment of diseases of the sensory peripheral nervous system.

HSV-mediated gene transfer of vascular endothelial growth factor to dorsal root ganglia prevents diabetic neuropathy

We examined the utility of herpes simplex virus (HSV) vector-mediated gene transfer of vascular endothelial growth factor (VEGF) in a mouse model of diabetic neuropathy. A replication-incompetent HSV vector with VEGF under the control of the HSV ICP0 promoter (vector T0VEGF) was constructed. T0VEGF expressed and released VEGF from primary dorsal root ganglion (DRG) neurons in vitro, and following subcutaneous inoculation in the foot, expressed VEGF in DRG and nerve in vivo. At 2 weeks after induction of diabetes, subcutaneous inoculation of T0VEGF prevented the reduction in sensory nerve amplitude characteristic of diabetic neuropathy measured 4 weeks later, preserved autonomic function measured by pilocarpine-induced sweating, and prevented the loss of nerve fibers in the skin and reduction of neuropeptide calcitonin gene-related peptide and substance P in DRG neurons of the diabetic mice. HSV-mediated transfer of VEGF to DRG may prove useful in treatment of diabetic neuropathy.

Reduction of voltage gated sodium channel protein in DRG by vector mediated miRNA reduces pain in rats with painful diabetic neuropathy

BackgroundPainful neuropathy is a common complication of diabetes. Previous studies have identified significant increases in the amount of voltage gated sodium channel isoforms NaV1.7 and NaV1.3 protein in the dorsal root ganglia (DRG) of rats with streptozotocin (STZ)-induced diabetes. We found that gene transfer-mediated release of the inhibitory neurotransmitters enkephalin or gamma amino butyric acid (GABA) from DRG neurons in diabetic animals reduced pain-related behaviors coincident with a reduction in NaV1.7 protein levels in DRG in vivo. To further evaluate the role of NaVα subunit levels in DRG in the pathogenesis of pain in diabetic neuropathy, we constructed a non-replicating herpes simplex virus (HSV)-based vector expressing a microRNA (miRNA) against NaVα subunits.ResultsSubcutaneous inoculation of the miRNA-expressing HSV vector into the feet of diabetic rats to transduce DRG resulted in a reduction in NaVα subunit levels in DRG neurons, coincident with a reduction in cold allodynia, thermal hyperalgesia and mechanical hyperalgesia.ConclusionsThese data support the role of increased NaVα protein in DRG in the pathogenesis of pain in diabetic neuropathy, and provide a proof-of-principle demonstration for the development of a novel therapy that could be used to treat intractable pain in patients with diabetic neuropathy.

Neuroprotective effect of herpes simplex virus-mediated gene transfer of erythropoietin in hyperglycemic dorsal root ganglion neurons

We examined the efficacy of herpes simplex virus vector-mediated gene transfer of erythropoietin in preventing neuropathy in mouse model of streptozotocin-diabetes. A replication-incompetent herpes simplex virus vector with erythropoietin under the control of the human cytomegalovirus promoter (vector DHEPO) was constructed. DHEPO expressed and released erythropoietin from primary dorsal root ganglion neurons in vitro, and following subcutaneous inoculation in the foot, expressed erythropoietin in dorsal root ganglion neurons in vivo. At 2 weeks after induction of diabetes, subcutaneous inoculation of erythropoietin prevented the reduction in sensory nerve amplitude characteristic of diabetic neuropathy measured 4 weeks later, preserved autonomic function measured by pilocarpine-induced sweating, and prevented the loss of nerve fibres in the skin and reduction of neuropeptide calcitonin gene-related peptide in the dorsal horn of spinal cord of the diabetic mice. We further investigated whether vector-mediated local expression of erythropoietin in dorsal root ganglion neurons can protect in vivo as well as in vitro hyperglycemia-induced axonal degeneration. Our findings show that the AKT/GSK-3beta dependent pathway plays an important role in mediating the protection of erythropoietin against diabetic neuropathy. Herpes simplex virus-mediated transfer of erythropoietin to dorsal root ganglia may prove useful in treatment of diabetic neuropathy.

Increases in inflammatory mediators in DRG implicate in the pathogenesis of painful neuropathy in Type 2 diabetes

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editor-in-Chief. Image duplication has been observed within Figure 1g-h. The corresponding author has been asked to provide an acceptable explanation for this duplication but has not been able to do so neither have the original source files been supplied.

Vector-mediated release of GABA attenuates pain-related behaviors and reduces NaV1.7 in DRG neurons

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 (NaV1.7) protein that is characteristic of PDN. In vitro, infection of primary DRG neurons with vG prevented the increase in NaV1.7 resulting from exposure to hyperglycemia. The effect of vector‐mediated GABA on NaV1.7 levels in vitro was blocked by phaclofen but not by bicuculline, a GABAB 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 NaV1.7 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.

Gene therapy for the treatment of sensory neuropathy

Sensory polyneuropathy can be a serious problem, but for the majority of clinically important neuropathies there are no available therapies. Neurotrophic and neuroprotective peptide factors have been identified that prevent or reverse neuropathy in rodent models of disease, but delivery of these highly pleiotropic peptides has posed an obstacle for translation into effective human therapies. Gene transfer into muscle using viral or non-viral vectors, or into neurons of the dorsal root ganglion using herpes simplex virus-based vectors, provides an alternative means to achieve this end. Studies in animal models have been promising, and the first human trial, using a plasmid to transfer the gene coding for vascular endothelial growth factor into muscle for the treatment of diabetic neuropathy, is now underway. Evidence supporting the trial and the challenges facing this therapy are reviewed.

Decrease in neuroimmune activation by HSV-mediated gene transfer of TNFα soluble receptor alleviates pain in rats with diabetic neuropathy.

The mechanisms of diabetic painful neuropathy are complicated and comprise of peripheral and central pathophysiological phenomena. A number of proinflammatory cytokines are involved in this process. Tumor necrosis factor α (TNF-α) is considered to be one of the major contributors of neuropathic pain. In order to explore the potential role of inflammation in the peripheral nervous system of Type 1 diabetic animals with painful neuropathy, we investigated whether TNF-α is a key inflammatory mediator to the diabetic neuropathic pain and whether continuous delivery of TNFα soluble receptor from damaged axons achieved by HSV vector mediated transduction of DRG would block or alter the pain perception in animals with diabetic neuropathy. Diabetic animals exhibited changes in threshold of mechanical and thermal pain perception compared to control rats and also demonstrated increases in TNFα in the DRG, spinal cord dorsal horn, sciatic nerve and in the foot skin, 6 weeks after the onset of diabetes. Therapeutic approaches by HSV mediated expression of p55 TNF soluble receptor significantly attenuated the diabetes-induced hyperalgesia and decreased the expression of TNFα with reduction in the phosphorylation of p38MAPK in the spinal cord dorsal horn and DRG. The overall outcome of this study suggests that neuroinflammatory activation in the peripheral nervous system may be involved in the pathogenesis of painful neuropathy in Type 1 diabetes which can be alleviated by local expression of HSV vector expressing p55 TNF soluble receptor.