Understanding hyperexcitability of dorsal root ganglion neurons
Picchione, Kelly Elizabeth
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Neuropathic pain is a disease characterized as a burning, stabbing, chronic pain that is resistant to typical analgesics. Although the mechanism for maintenance of neuropathic pain is unknown, changes in ion-channel expression in the sensory neurons of the dorsal root ganglia (DRG) have been implicated. It is important to understand the intrinsic plasticity in the DRG to find effective analgesic targets to treat neuropathic pain. Numerous ion-channels have been studied in hopes of identifying a target to ameliorate the effects of neuropathic pain. Sodium-activated potassium channels (K Na ) are highly expressed in DRG neurons and have proposed functions including, setting and regulating the resting membrane potential (RMP), and controlling firing accommodation. Unfortunately, because there are no specific blockers it is difficult to determine the functions of these channels and examine their possible role in regulating DRG intrinsic plasticity. In addition, complications due to transfection and adenoviral delivery made studying the excitability of these neurons using RNA interference (RNAi) difficult. In this study, first I characterized the expression of K Na channels in adult and cultured DRG neurons. After overcoming the difficulties associated with transgene delivery, I used cultured DRG neurons as a model system to determine the contribution of Slack channels to neuronal excitability. Concurrently, while investigating the problems associated with adenoviral transgene delivery I studied a histone deacetylase, Sirt1, and its ability to regulate viral transgene expression. Although transfection and adenoviral transgene delivery has been described as a mostly innocuous delivery method in-vitro , I provide evidence to the contrary. While studying the physiology of the Slack channel, using current-clamp recordings, I found that transfection and transduction of exogenous DNA induced DRG depolarization and hyperexcitability. This hyperexcitability is associated with an increase in Ser/Thr phosphorylation and Slack channel trafficking. By an unclear mechanism, supplementing media with 1 mM nicotinamide overcame the effects of transgene introduction. Therefore, Slack channel knockdown experiments were performed in the presence of nicotinamide. These experiments showed that Slack is significantly involved in regulating the RMP and firing properties of DRG neurons. In addition to attenuating the DNA induced hyperexcitability of DRG neurons, I found that nicotinamide modulated transgene expression from adenovirus. This change in expression of the transduction marker Zoanthus sp. Green fluorescent protein (ZsGreen) was due to inhibition of Sirt1. Sirt1 is a NAD + -dependent histone deacetylase important for transcription regulation. In addition, other modulators of Sirt1, including activators resveratrol and SRT1720, had the opposite effect of nicotinamide. Understanding Sirt1’s role in regulation of viral gene expression is key because it has implications in neurotropic viral reactivation and regulation.