Differentiation of the 50B11 dorsal ganglion cells into NGF and GDNF responsive nociceptor subtypes.

The embryonic rat dorsal root ganglion (DRG) neuron-derived 50B11 cell line is a promising sensory neuron model expressing markers characteristic of NGF and GDNF-dependent C-fibre nociceptors. Whether these cells have the capacity to develop into distinct nociceptive subtypes based on NGF- or GDNF-dependence has not been investigated. Here we show that by augmenting forskolin (FSK) and growth factor supplementation with NGF or GDNF, 50B11 cultures can be driven to acquire differential functional responses to common nociceptive agonists capsaicin and ATP respectively. In addition, to previous studies, we also demonstrate that a differentiated neuronal phenotype can be maintained for up to 7 days. Western blot analysis of nociceptive marker proteins further demonstrates that the 50B11 cells partially recapitulate the functional phenotypes of classical NGF-dependent (peptidergic) and GDNF-dependent (non-peptidergic) neuronal subtypes described in DRGs. Further, 50B11 cells differentiated with NGF/FSK, but not GDNF/FSK, show sensitization to acute prostaglandin E2 treatment. Finally, RNA-Seq analysis confirms that differentiation with NGF/FSK or GDNF/FSK produces two 50B11 cell subtypes with distinct transcriptome expression profiles. Gene ontology comparison of the two subtypes of differentiated 50B11 cells to rodent DRG neurons studies shows significant overlap in matching or partially matching categories. This transcriptomic analysis will aid future suitability assessment of the 50B11 cells as a high-throughput nociceptor model for a broad range of experimental applications. In conclusion, this study shows that the 50B11 cell line is capable of partially recapitulating features of two distinct types of embryonic NGF and GDNF-dependent nociceptor-like cells.

TMI-1, TNF-α-Converting Enzyme Inhibitor, Protects Against Paclitaxel-Induced Neurotoxicity in the DRG Neuronal Cells In Vitro.

Background: Chemotherapy-induced peripheral neuropathy (CIPN) negatively impacts cancer survivors' quality of life and is challenging to treat with existing drugs for neuropathic pain. TNF-α is known to potentiate TRPV1 activity, which contributes to CIPN. Here, we assessed the role of TMI-1, a TNF-α-converting enzyme inhibitor, in paclitaxel (PAC)-induced neurotoxicity in dorsal root ganglion (DRG) cells. Materials and Methods: Immortalized DRG neuronal 50B11 cells were cultured and treated with PAC or PAC with TMI-1 following neuronal differentiation. Cell viability, analysis of neurite growth, immunofluorescence, calcium flow cytometry, western blotting, quantitative RT-PCR, and cytokine quantitation by ELISA were performed to determine the role of TMI-1 in neurotoxicity in neuronal cells. Results: PAC administration decreased the length of neurites and upregulated the expression of TRPV1 in 50B11 cells. TMI-1 administration showed a protective effect by suppressing inflammatory signaling, and secretion of TNF-α. Conclusion: TMI-1 partially protects against paclitaxel-induced neurotoxicity by reversing the upregulation of TRPV1 and decreasing levels of inflammatory cytokines, including TNF-α, IL-1ß, and IL-6 in neuronal cells.

Generation of an in vitro model for peripheral neuropathy in Fabry disease using CRISPR-Cas9 in the nociceptive dorsal root ganglion cell line 50B11.

Fabry disease is a glycosphingolipid storage disorder that is caused by a genetic deficiency of the lysosomal enzyme alpha-galactosidase A (AGA, EC 3.2.1.22). As a result, the glycolipid substrate, globotriaosylceramide (Gb3) accumulates in various cell types throughout the body producing a multisystem disease that affects the vascular, cardiac, renal, and nervous systems. A hallmark of this disorder is neuropathic pain that occurs in up to 80% of Fabry patients and has been characterized as a small fiber neuropathy. The molecular mechanism by which changes in AGA activity produce neuropathic pain is not clear, in part due to a lack of relevant model systems. Using 50B11 cells, an immortalized dorsal root ganglion neuron with nociceptive characteristics derived from rat, we used CRISPR-Cas9 gene editing of the galactosidase alpha (GLA) gene for AGA to create two stable knock-out clones that have the phenotypic characteristics of Fabry cells. The cell lines show severely reduced lysosomal AGA activity in homogenates as well as impaired degradation of Gb3 in cultured cells. This phenotype is stable over long-term culture. Similar to the unedited 50B11 cell line, the clones differentiate in response to forskolin and extend neurites. Flow cytometry experiments demonstrate that the gene-edited cells express TRPV1 pain receptor at increased levels compared to control, suggesting a possible mechanism for increased pain sensitization in Fabry patients. Our 50B11 cell lines show phenotypic characteristics of Fabry disease and grow well under standard cell culture conditions. These cell lines can provide a convenient model system to help elucidate the molecular mechanism of pain in Fabry patients.

Glucagon Prevents Cytotoxicity Induced by Methylglyoxal in a Rat Neuronal Cell Line Model.

Although diabetic polyneuropathy (DPN) is a frequent diabetic complication, no effective therapeutic approach has been established. Glucagon is a crucial hormone for glucose homeostasis but has pleiotropic effects, including neuroprotective effects in the central nervous system. However, the importance of glucagon in the peripheral nervous system (PNS) has not been clarified. Here, we hypothesized that glucagon might have a neuroprotective function in the PNS. The immortalized rat dorsal root ganglion (DRG) neuronal cell line 50B11 was treated with methylglyoxal (MG) to mimic an in vitro DPN model. The cells were cultured with or without glucagon or MG. Neurotoxicity, survival, apoptosis, neurite projection, cyclic adenosine monophosphate (cAMP), and protein kinase A (PKA) were examined. Glucagon had no cytotoxicity and rescued the cells from neurotoxicity. Cell survival was increased by glucagon. The ratio of apoptotic cells, which was increased by MG, was reduced by glucagon. Neurite outgrowth was accelerated in glucagon-treated cells. Cyclic AMP and PKA accumulated in the cells after glucagon stimulation. In conclusion, glucagon protected the DRG neuronal cells from MG-induced cellular stress. The cAMP/PKA pathway may have significant roles in those protective effects of glucagon. Glucagon may be a potential target for the treatment of DPN.

Immortalized Dorsal Root Ganglion Neuron Cell Lines.

Pain is one of the most significant causes of suffering and disability world-wide, and arguably the most burdensome global health challenge. The growing number of patients suffering from chronic pain conditions such as fibromyalgia, complex regional pain syndrome, migraine and irritable bowel syndrome, not only reflect the complexity and heterogeneity of pain types, but also our lack of understanding of the underlying mechanisms. Sensory neurons within the dorsal root ganglia (DRG) have emerged as viable targets for effective chronic pain therapy. However, DRG's contain different classes of primary sensory neurons including pain-associated nociceptive neurons, non-nociceptive temperature sensing, mechanosensory and chemoreceptive neurons, as well as multiple types of immune and endothelial cells. This cell-population heterogeneity makes investigations of individual subgroups of DRG neurons, such as nociceptors, difficult. In attempts to overcome some of these difficulties, a limited number of immortalized DRG-derived cell lines have been generated over the past few decades. In vitro experiments using DRG-derived cell lines have been useful in understanding sensory neuron function. In addition to retaining phenotypic similarities to primary cultured DRG neurons, these cells offer greater suitability for high throughput assays due to ease of culture, maintenance, growth efficiency and cost-effectiveness. For accurate interpretation and translation of results it is critical, however, that phenotypic similarities and differences of DRG-derived cells lines are methodically compared to native neurons. Published reports to date show notable variability in how these DRG-derived cells are maintained and differentiated. Understanding the cellular and molecular differences stemming from different culture methods, is essential to validate past and future experiments, and enable these cells to be used to their full potential. This review describes currently available DRG-derived cell lines, their known sensory and nociceptor specific molecular profiles, and summarize their morphological features related to differentiation and neurite outgrowth.