Investigating the Function of Adult DRG Neuron Axons Using an In Vitro Microfluidic Culture System

A critical role of the peripheral axons of nociceptors of the dorsal root ganglion (DRG) is the conduction of all-or-nothing action potentials from peripheral nerve endings to the central nervous system for the perception of noxious stimuli. Plasticity along multiple sites along the pain axis has now been widely implicated in the maladaptive changes that occur in pathological pain states such as neuropathic and inflammatory pain. Notably, increasing evidence suggests that nociceptive axons actively participate through the local expression of ion channels, receptors, and signal transduction molecules through axonal mRNA translation machinery that is independent of the soma component. In this report, we explore the sensitization of sensory neurons through the treatment of compartmentalized axon-like structures spanning microchannels that have been treated with the cytokine IL-6 and, subsequently, capsaicin. These data demonstrate the utility of isolating DRG axon-like structures using microfluidic systems, laying the groundwork for constructing the complex in vitro models of cellular networks that are involved in pain signaling for targeted pharmacological and genetic perturbations.

Exendin-4 Promotes Schwann Cell Survival/Migration and Myelination In Vitro

Besides its insulinotropic actions on pancreatic β cells, neuroprotective activities of glucagon-like peptide-1 (GLP-1) have attracted attention. The efficacy of a GLP-1 receptor (GLP-1R) agonist exendin-4 (Ex-4) for functional repair after sciatic nerve injury and amelioration of diabetic peripheral neuropathy (DPN) has been reported; however, the underlying mechanisms remain unclear. In this study, the bioactivities of Ex-4 on immortalized adult rat Schwann cells IFRS1 and adult rat dorsal root ganglion (DRG) neuron–IFRS1 co-culture system were investigated. Localization of GLP-1R in both DRG neurons and IFRS1 cells were confirmed using knockout-validated monoclonal Mab7F38 antibody. Treatment with 100 nM Ex-4 significantly enhanced survival/proliferation and migration of IFRS1 cells, as well as stimulated the movement of IFRS1 cells toward neurites emerging from DRG neuron cell bodies in the co-culture with the upregulation of myelin protein 22 and myelin protein zero. Because Ex-4 induced phosphorylation of serine/threonine-specific protein kinase AKT in these cells and its effects on DRG neurons and IFRS1 cells were attenuated by phosphatidyl inositol-3′-phosphate-kinase (PI3K) inhibitor LY294002, Ex-4 might act on both cells to activate PI3K/AKT signaling pathway, thereby promoting myelination in the co-culture. These findings imply the potential efficacy of Ex-4 toward DPN and other peripheral nerve lesions.

A10 FOOD ANTIGEN-STRESS INTERACTION LEADS TO INCREASE PAIN SIGNALING IN ILEUM AND COLON VIA STAT6 IN AN IBS MODEL

Background Abdominal pain can be triggered by food ingestion in IBS patients. Previously we have shown that a food antigen induces local release of immune mediators in the colon that increase dorsal root ganglion (DRG) neuron excitability when there is previous antigen exposure in the presence of psychological stress. However, it is unknown if this effect is limited to the colon. Furthermore, the involvement of histamine in the neuronal hyperexcitability suggests that the stress-food antigen interaction evokes a Th2 immune response. Thus, we sought to investigate the role of STAT6, a transcription factor downstream of Th2 cytokines and important for IgE production. Aims 1) Determine if stress-food antigen interaction leads to release of mediators within the small intestine that increase DRG neuron excitability. 2) Determine the involvement of STAT6 on neuronal hyperexcitability induced by the stress-food antigen interaction. Methods BALB/c mice were exposed to water avoidance stress (WAS) or sham stress (SHAM) for 1 hr daily for 10 days. On day 2–10, mice were exposed to ovalbumin (OVA) or saline (SAL). Seven days later, mice were re-exposed to either OVA or SAL every 2 days for 2 weeks yielding 3 groups: WAS/OVA+OVA, WAS/SAL+OVA, and SHAM/OVA+OVA. STAT6 deficient mice were also exposed to WAS/OVA+OVA protocol. Ileum or colonic supernatants were obtained 4 hours after tissue collection. DRG neurons were incubated overnight with supernatants prior to perforated patch clamp recordings. Neuronal excitability was evaluated by measuring the rheobase (minimum current to elicit an action potential, decreased rheobase indicates increased excitability). Mechanosensitivity of extrinsic afferent nerves innervating distal ileum was examined using ex vivo extracellular afferent nerve recordings. Data was analyzed by one or two-way ANOVA with Bonferroni post-hoc test. Results Ileum supernatants from WAS/OVA+OVA mice increased DRG neuron excitability compared to WAS/SAL+OVA and SHAM/OVA+OVA mice (63.3 ± 6.2 pA vs 83.2 ± 5.4 pA, 86.7 ± 4.5 pA, p<0.05). Ileum afferent nerve response to distention was significantly augmented in WAS/OVA+OVA mice compared to WAS/SAL+OVA and SHAM/OVA+OVA (P<0.05, n=4–7). DRG neurons incubated with WAS/OVA+OVA supernatant from STAT6 deficient mice were less excitable compared to neurons incubated with colonic supernatants from wild type mice (86.5 ± 4.1 pA vs 67.6 ± 4.8 pA, p<0.05). Conclusions Stress-food antigen interaction releases mediators in both the small intestine and colon to increase nociceptive signaling, an important finding as IBS can involve both areas. The release of excitatory mediators within the gut appears to involve STAT6. Thus, a stress-food antigen interaction evoking a Th2 immune response in the gut may be a mechanism underlying food induced symptoms in IBS. Funding Agencies Queen’s University, Department of Medicine

JAK1 palmitoylation by ZDHHC3/7 is Essential for Neuropoietic Cytokine Signaling and DRG Neuron Survival

Janus Kinase-1 (JAK1) plays key roles in pro-survival signaling during neurodevelopment and in responses to neuronal injury. JAK1 was identified as a potential palmitoyl-protein in high-throughput studies, but the importance of palmitoylation for JAK1’s roles in neurons has not been addressed. Here, we report that JAK1 is endogenously palmitoylated in cultured Dorsal Root Ganglion (DRG) neurons and, using an shRNA knockdown/rescue approach, reveal that JAK1 palmitoylation is important for neuropoietic cytokine-dependent signaling and neuronal survival. We further identify the related palmitoyl acyltransferases (PATs) ZDHHC3 and ZDHHC7 as dominant regulators of JAK1 palmitoylation in transfected non-neuronal cells and endogenously in neurons. At the molecular level, palmitoylation is critical for JAK1’s kinase activity in transfected cells and even in vitro, likely because palmitoylation facilitates transphosphorylation of key sites in the JAK1 activation loop. These findings provide new insights into palmitoylation-dependent control of neuronal development and survival.

A New Model of Sensorial Neuron-Like Cells for HTS of Novel Analgesics for Neuropathic Pain

In this study we developed a new translational phenotypic in vitro model for high-throughput screening (HTS) of novel analgesics for treating neuropathic pain, in order to address the poor translation of traditional recombinant models. The immortalized dorsal root ganglia (DRG) neuron-like F11 cell line was selected based on its phenotype after differentiation. The acquisition of neuronal characteristics was evaluated by measuring the expression of TrkA as a DRG neuron marker ( p < 0.01) as well as by measuring the global neurite length ( p < 0.001). The response of F11 cells to ATP and KCl was obtained by measuring intracellular calcium concentration, dynamic mass redistribution, and membrane potential. A KCl-induced increase of intracellular calcium levels was chosen as the readout because of the better signal quality, higher reproducibility, and greater compatibility with HTS assay requirements compared with other methods. The response to KCl differed significantly between differentiated and undifferentiated cells ( p < 0.05), with an EC50 value of 5 mM in differentiated cells. The model was validated by screening the Prestwick Chemical Library. Five hits already proposed for neuropathic-related pain were identified, with IC50 values between 1 and 7 µM. This cell model provides a new tool for screening novel analgesics for the relief of neuropathic pain.