Cyclosporin A as an Alternative Neuroimmune Strategy to Control Neurites and Recover Neuronal Tissues in Leprosy

Leprosy, also known as Hansen’s disease, continues to have a substantial impact on infectious diseases throughout the world. Leprosy is a chronic granulomatous infection caused by <i>Mycobacterium leprae</i> and shows a wide clinical and immunopathological spectrum related to the immune response of the host. This disease affects the skin and other internal organs with a predilection to infect Schwann cells, which play an active role during axonal degeneration, affecting peripheral nerves and promoting neurological damage. This chronic inflammation influences immune function, leading to neuroimmune disorders. Leprosy is also associated with neuroimmune reactions, including type 1 (reverse) and type 2 (erythema nodosum leprosum) reactions, which are immune-mediated inflammatory complications that can occur during the disease and appear to worsen dramatically; these complications are the main concerns of patients. The reactions may induce neuritis and neuropathic pain that progressively worsen with irreversible deformity and disabilities responsible for the immunopathological damage and glial/neuronal death. However, the neuronal damage is not always associated with the reactional episode. Also, the efficacy in the treatment of reactions remains low because of the nonexistence of a specific treatment and missing informations about the immunopathogenesis of the reactional episode. There is increasing evidence that peripheral neuron dysfunction strongly depends on the activity of neurotrophins. The most important neurotrophin in leprosy is nerve growth factor (NGF), which is decreased in the course of leprosy, as well as the presence of autoantibodies against NGF in all clinical forms of leprosy and neuroimmune reactions. The levels of autoantibodies against NGF are decreased by the immunomodulatory activity of cyclosporin A, which mainly controls pain and improves motor function and sensitivity. Therefore, the suppression of anti-NGF and the regulation of NGF levels can be attractive targets for immunomodulatory treatment and for controlling the neuroimmune reactions of leprosy, although further studies are needed to clarify this point.

TRPV2 interacts with actin and reorganizes submembranous actin cytoskeleton

The understanding of molecules and their role in neurite initiation and/or extension is not only helpful to prevent different neurodegenerative diseases but also can be important in neuronal damage repair. In this work, we explored the role of transient receptor potential vanilloid 2 (TRPV2), a non-selective cation channel in the context of neurite functions. We confirm that functional TRPV2 is endogenously present in F11 cell line, a model system mimicking peripheral neuron. In F11 cells, TRPV2 localizes in specific subcellular regions enriched with filamentous actin, such as in growth cone, filopodia, lamellipodia and in neurites. TRPV2 regulates actin cytoskeleton and also interacts with soluble actin. Ectopic expression of TRPV2-GFP in F11 cell induces more primary and secondary neurites, confirming its role in neurite initiation, extension and branching events. TRPV2-mediated neuritogenesis is dependent on wildtype TRPV2 as cells expressing TRPV2 mutants reveal no neuritogenesis. These findings are relevant to understand the sprouting of new neurites, neuroregeneration and neuronal plasticity at the cellular, subcellular and molecular levels. Such understanding may have further implications in neurodegeneration and peripheral neuropathy.

In Vitro Differentiation of Human Skin-Derived Cells into Functional Sensory Neurons-Like

Skin-derived precursor cells (SKPs) are neural crest stem cells that persist in certain adult tissues, particularly in the skin. They can generate a large type of cell in vitro, including neurons. SKPs were induced to differentiate into sensory neurons (SNs) by molecules that were previously shown to be important for the generation of SNs: purmorphamine, CHIR99021, BMP4, GDNF, BDNF, and NGF. We showed that the differentiation of SKPs induced the upregulation of neurogenins. At the end of the differentiation protocol, transcriptional analysis was performed on BRN3A and a marker of pain-sensing nerve cell PRDM12 genes: 1000 times higher for PRDM12 and 2500 times higher for BRN3A in differentiated cells than they were in undifferentiated SKPs. Using immunostaining, we showed that 65% and 80% of cells expressed peripheral neuron markers BRN3A and PERIPHERIN, respectively. Furthermore, differentiated cells expressed TRPV1, PAR2, TRPA1, substance P, CGRP, HR1. Using calcium imaging, we observed that a proportion of cells responded to histamine, SLIGKV (a specific agonist of PAR2), polygodial (a specific agonist of TRPA1), and capsaicin (a specific agonist of TRPV1). In conclusion, SKPs are able to differentiate directly into functional SNs. These differentiated cells will be very useful for further in vitro studies.

SUN-245 Sensory Neuron Metabolism Mediates Changes in Ovarian Function via LKB1

Bi-directional communication between sensory neurons to peripheral tissue to mediate physiology has become an area of interest. Previous research on peripheral neuron control of ovarian function shows that lesioning the superior ovarian nerve resulted in decreased estradiol release from the ovary; but there has been little research on how sensory neurons affect ovarian physiology. Interestingly, the metabolic activity of sensory neurons that control ovarian and follicular function may have profound effects on tissues they innervate. LKB1 (Liver Kinase B1) is a metabolic kinase that regulates cell growth and polarity. When downregulated in cortical neurons in vitro, leads to shorter axons with less branching. Female mice with LKB1 removed from their peripheral sensory neurons have litters more often and increased litter sizes compared to wild-type mice, which led to the investigation into the mechanisms into the role of LKB1 in sensory neurons in ovarian function. Nav1.8cre-LKB1fl/fl mice were used to assess the removal of LKB1 from Nav1.8-expressing neurons on the mechanisms behind reproductive viability. The estrus cycle was tracked by using vaginal lavage to collect cells from the vagina twice per day for ten days, and cytology was assessed to determine phase. Ovaries were collected from mice in all phases of the estrus cycle, sectioned at 8 micron thickness, stained with H&E. Follicle sizes and numbers were measured on 8 sections per ovary. Sensory innervation was measured by clearing whole ovary using ScaleS1 and using confocal microscopy to image through the whole tissue for sensory neurons tagged with tdTomato and DAPI. Our data indicate that Nav1.8-expressing neurons innervate the ovary from celiac ganglia, upper lumbar, and lower thoracic dorsal root ganglia. Nav1.8cre-LKB1fl/fl mice have larger litters and breed more frequently compared to cre-negative litter mates (WT). Nav1.8cre-LKB1fl/fl mice have larger ovaries, spend less time in proestrus, and have greater follicular turnover compared to WT mice. Phase-matched ovaries from Nav1.8cre-LKB1fl/fl mice in proestrus show greater numbers of antral and degenerating follicles than WT mice, but similar numbers of immature follicles; however, the size of all follicles from Nav1.8cre-LKB1fl/fl mice are smaller than follicles from WT mice. Nav1.8cre-LKB1fl/fl mice have improved reproductive viability by increased follicular turnover rate, more mature follicles, and shortened estrus cycle length.

TRPV2 activation reorganizes actin cytoskeleton, induces neurite initiation and branching by altering cAMP levels

Understanding of molecules and their role in neurite initiation and/or extension is not only helpful to prevent different neurodegenerative diseases but also can be important by which neuronal damages can be repaired. In this work we explored the role of TRPV2, a non-selective cation channel in the context of neurite functions. Using western blot, immunofluorescence, and live cell Ca2+-imaging; we confirm that functional TRPV2 is endogenously present in the F11 cell, a model system mimicking peripheral neuron. In F11 cells TRPV2 localizes in specific sub-cellular regions enriched with filamentous actin, such as in growth cone, filopodia, lamellipodia and in neurites. TRPV2 regulates actin cytoskeleton and also interacts with soluble actin. Ectopic expression of TRPV2-GFP but not GFP-only in F11 cell induces more primary and secondary neurites, confirming its role in neurite initiation, extension and branching events. TRPV2-mediated neuritogenesis is dependent on wild-type TRPV2 as cells expressing TRPV2 mutants reveal no neuritogenesis. However, TRPV2-mediated neuritogenesis is unperturbed by the chelation of intracellular Ca2+ by BAPTA-AM, and thus involves Ca2+-independent signaling events also. We demonstrate that pharmacological modulation of TRPV2 alters cellular cAMP levels. These findings are relevant to understand the sprouting of new neurites, neuroregeneration and neuronal plasticity at the cellular, subcellular and molecular level. Such understanding may have border implication in neurodegeneration and peripheral neuropathy.

Interleukin-17 regulates neuron-glial communications, inhibitory synaptic transmission and neuropathic pain after chemotherapy

The proinflammatory cytokine Interleukin-17 (IL-17) is produced mainly by Th17 cells and has been implicated in pain regulation. However, synaptic mechanisms by which IL-17 regulates pain transmission are unknown. Here we report that glia-produced IL-17 suppresses inhibitory synaptic transmission in spinal cord pain circuit and drives chemotherapy-induced neuropathic pain. We observed respective expression of IL-17 and its receptor IL-17R in spinal cord astrocytes and neurons. Patch clamp recording in spinal cord slices revealed that IL-17 not only enhanced EPSCs but also suppressed IPSCs and GABA-induced currents in lamina IIo somatostatin-expressing neurons. Spinal IL-17 was upregulated after paclitaxel treatment, and intrathecal IL-17R blockade reduced paclitaxel-induced neuropathic pain. In dorsal root ganglia, respective IL-17 and IL-17R expression in satellite glial cells and neurons was sufficient and required for inducing neuronal hyperexcitability after paclitaxel. Together, our data show that IL-17/IL-17R mediate both central and peripheral neuron-glial interactions in chemotherapy-induced peripheral neuropathy.