Functional subgroups of rat and human sensory neurons: a systematic review of electrophysiological properties

Sensory neurons are responsible for the generation and transmission of nociceptive signals from the periphery to the central nervous system. They encompass a broadly heterogeneous population of highly specialized neurons. The understanding of the molecular choreography of individual subpopulations is essential to understand physiological and pathological pain states. Recently, it became evident that species differences limit transferability of research findings between human and rodents in pain research. Thus, it is necessary to systematically compare and categorize the electrophysiological data gained from human and rodent dorsal root ganglia neurons (DRGs). In this systematic review, we condense the available electrophysiological data defining subidentities in human and rat DRGs. A systematic search on PUBMED yielded 30 studies on rat and 3 studies on human sensory neurons. Defined outcome parameters included current clamp, voltage clamp, cell morphology, pharmacological readouts, and immune reactivity parameters. We compare evidence gathered for outcome markers to define subgroups, offer electrophysiological parameters for the definition of neuronal subtypes, and give a framework for the transferability of electrophysiological findings between species. A semiquantitative analysis revealed that for rat DRGs, there is an overarching consensus between studies that C-fiber linked sensory neurons display a lower action potential threshold, higher input resistance, a larger action potential overshoot, and a longer afterhyperpolarization duration compared to other sensory neurons. They are also more likely to display an infliction point in the falling phase of the action potential. This systematic review points out the need of more electrophysiological studies on human sensory neurons.

Expression of Cholinergic Markers and Characterization of Splice Variants during Ontogenesis of Rat Dorsal Root Ganglia Neurons

Dorsal root ganglia (DRG) neurons synthesize acetylcholine (ACh), in addition to their peptidergic nature. They also release ACh and are cholinoceptive, as they express cholinergic receptors. During gangliogenesis, ACh plays an important role in neuronal differentiation, modulating neuritic outgrowth and neurospecific gene expression. Starting from these data, we studied the expression of choline acetyltransferase (ChAT) and vesicular ACh transporter (VAChT) expression in rat DRG neurons. ChAT and VAChT genes are arranged in a “cholinergic locus”, and several splice variants have been described. Using selective primers, we characterized splice variants of these cholinergic markers, demonstrating that rat DRGs express R1, R2, M, and N variants for ChAT and V1, V2, R1, and R2 splice variants for VAChT. Moreover, by RT-PCR analysis, we observed a progressive decrease in ChAT and VAChT transcripts from the late embryonic developmental stage (E18) to postnatal P2 and P15 and in the adult DRG. Interestingly, Western blot analyses and activity assays demonstrated that ChAT levels significantly increased during DRG ontogenesis. The modulated expression of different ChAT and VAChT splice variants during development suggests a possible differential regulation of cholinergic marker expression in sensory neurons and confirms multiple roles for ACh in DRG neurons, both in the embryo stage and postnatally.

Neuroimmune Consequences of eIF4E Phosphorylation on Chemotherapy-Induced Peripheral Neuropathy

Chemotherapy-induced peripheral neuropathy (CIPN) is a major dose-limiting side effect that occurs in up to 63% of patients and has no known effective treatment. A majority of studies do not effectively assess sex differences in the onset and persistence of CIPN. Here we investigated the onset of CIPN, a point of therapeutic intervention where we may limit, or even prevent the development of CIPN. We hypothesized that cap-dependent translation mechanisms are important in early CIPN development and the bi-directional crosstalk between immune cells and nociceptors plays a complementary role to CIPN establishment and sex differences observed. In this study, we used wild type and eIF4E-mutant mice of both sexes to investigate the role of cap-dependent translation and the contribution of immune cells and nociceptors in the periphery and glia in the spinal cord during paclitaxel-induced peripheral neuropathy. We found that systemically administered paclitaxel induces pain-like behaviors in both sexes, increases helper T-lymphocytes, downregulates cytotoxic T-lymphocytes, and increases mitochondrial dysfunction in dorsal root ganglia neurons; all of which is eIF4E-dependent in both sexes. We identified a robust paclitaxel-induced, eIF4E-dependent increase in spinal astrocyte immunoreactivity in males, but not females. Taken together, our data reveals that cap-dependent translation may be a key pathway that presents relevant therapeutic targets during the early phase of CIPN. By targeting the eIF4E complex, we may reduce or reverse the negative effects associated with chemotherapeutic treatments.

Microvesicles from Schwann-Like Cells as a New Biomaterial Promote Axonal Growth

Schwann cells promote axonal regeneration following peripheral nerve injury. However, in terms of clinical treatment, the therapeutic effects of Schwann cells are limited by their source. The transmission of microvesicles from neuroglia cells to axons is a novel communication mechanism in axon regeneration.To evaluate the effect of microvesicles released from Schwann-like cells on axonal regeneration, neural stem cells derived from human embryonic stem cells differentiated into Schwann-like cells, which presented a typical morphology and characteristics similar to those of schwann cells. The glial markers like MBP, P0, P75NTR, PMP-22, GFAP, HNK-1 and S100 were upregulated, whereas the neural stem markers like NESTIN, SOX1 and SOX2 were significantly downregulated in schwann-like cells. Microvesicles enhanced axonal growth in dorsal root ganglia neurons and regulated GAP43 expression in neuron-like cells (N2A and PC12) through the PTEN/PI3 K/Akt signaling pathway. A 5 mm section of sciatic nerve was transected in Sprague-Dawley rats. With microvesicles transplantation, regenerative nerves were evaluated after 6 weeks. Microvesicles increased sciatic function index scores, delayed gastrocnemius muscle atrophy and elevated βIII-tubulin-labeled axons in vivo. Schwann-like cells serve as a convenient source and promote axonal growth by secreting microvesicles, which may potentially be used as bioengineering materials for nerve tissue repair.

Calcitriol increases frataxin levels and restores mitochondrial function in cell models of Friedreich Ataxia

Friedreich Ataxia (FA) is a neurodegenerative disease caused by the deficiency of frataxin, a mitochondrial protein. In primary cultures of dorsal root ganglia neurons, we showed that frataxin depletion resulted in decreased levels of the mitochondrial calcium exchanger NCLX, neurite degeneration and apoptotic cell death. Here we describe that frataxin-deficient dorsal root ganglia neurons display low levels of ferredoxin 1, a mitochondrial Fe/S cluster-containing protein that interacts with frataxin and, interestingly, is essential for the synthesis of calcitriol, the active form of vitamin D. We provide data that calcitriol supplementation, used at nanomolar concentrations, is able to reverse the molecular and cellular markers altered in DRG neurons. Calcitriol is able to recover both ferredoxin 1 and NCLX levels and restores mitochondrial membrane potential indicating an overall mitochondrial function improvement. Accordingly, reduction of apoptotic markers and neurite degeneration was observed and, as a result, cell survival was also recovered. All these beneficial effects would be explained by the finding that calcitriol is able to increase the mature frataxin levels in both, frataxin-deficient DRG neurons and cardiomyocytes; remarkably, this increase also occurs in lymphoblastoid cell lines derived from FA patients. In conclusion, these results provide molecular bases to consider calcitriol for an easy and affordable therapeutic approach for FA patients.

Interleukin-31: The ‘itchy’ cytokine in inflammation and therapy

Interleukin-31 has been implicated in the pathophysiology of multiple atopic disorders such as atopic dermatitis (AD), rhinitis and airway hyperreactivity. In AD, IL-31 has been identified as one of the main ‘drivers’ of its cardinal symptom pruritus. Here, we aim to summarize the mechanisms by which IL-31 modulates inflammatory and allergic diseases. TH2 cells play a central role in AD and release high levels of TH2-produced cytokines including IL-31, thereby mediating inflammatory responses, initiating immunoregulatory circuits, and stimulating itch and neuronal outgrowth through activation of the heterodimer receptor IL-31 receptor alpha (IL31RA)/Oncostatin M receptor β. IL31RA expression is found on human and murine dorsal root ganglia neurons, epithelial cells including keratinocytes as well as various innate immune cells. IL-31 is a critical cytokine involved in neuro-immune communication, which opens new avenues for cytokine modulation in neuroinflammatory diseases including AD/pruritus, as validated by recent clinical trials using an anti-IL-31 antibody. Accordingly, inhibition of IL-31 downstream signaling may be a beneficial approach for various inflammatory diseases including prurigo nodularis. For example, whether downstream JAK inhibitors directly block IL-31-mediated-signaling needs to be clarified. Targeting the IL-31/IL31RA/OSMRβ axis appears to be a promising approach for inflammatory, neuroinflammatory and pruritic disorders in the future.

3D micro-organisation printing of mammalian cells to generate biological tissues

Significant strides have been made in the development of in vitro systems for disease modelling. However, the requirement of microenvironment control has placed limitations on the generation of relevant models. Herein, we present a biological tissue printing approach that employs open-volume microfluidics to position individual cells in complex 2D and 3D patterns, as well as in single cell arrays. The variety of bioprinted cell types employed, including skin epithelial (HaCaT), skin cancer (A431), liver cancer (Hep G2), and fibroblast (3T3-J2) cells, all of which exhibited excellent viability and survivability, allowing printed structures to rapidly develop into confluent tissues. To demonstrate a simple 2D oncology model, A431 and HaCaT cells were printed and grown into tissues. Furthermore, a basic skin model was established to probe drug response. 3D tissue formation was demonstrated by co-printing Hep G2 and 3T3-J2 cells onto an established fibroblast layer, the functionality of which was probed by measuring albumin production, and was found to be higher in comparison to both 2D and monoculture approaches. Bioprinting of primary cells was tested using acutely isolated primary rat dorsal root ganglia neurons, which survived and established processes. The presented technique offers a novel open-volume microfluidics approach to bioprint cells for the generation of biological tissues.