Exposure to ionizing radiation induces a cascade of molecular events that ultimately impact endogenous metabolism. Qualitative and quantitative characterization of metabolomic profiles is a pragmatic approach to studying the risks of radiation exposure since it provides a phenotypic readout. Studies were conducted in irradiated nonhuman primates (NHP) to investigate metabolic changes in plasma and plasma-derived exosomes. Specifically, rhesus macaques (Macaca mulatta) were exposed to cobalt-60 gamma-radiation and plasma samples were collected prior to and after exposure to 5.8 Gy or 6.5 Gy radiation. Exosomes were isolated using ultracentrifugation and analyzed by untargeted profiling via ultra-performance liquid chromatography mass spectrometry (UPLC-MS) based metabolomic and lipidomic analyses, with the goal of identifying a molecular signature of irradiation. The enrichment of an exosomal fraction was confirmed using quantitative ELISA. Plasma profiling showed markers of dyslipidemia, inflammation and oxidative stress post-irradiation. Exosomal profiling, on the other hand, enabled detection and identification of low abundance metabolites that comprise exosomal cargo which would otherwise get obscured with plasma profiling. We discovered enrichment of different classes of metabolites including N-acyl-amino acids, Fatty Acid ester of Hydroxyl Fatty Acids (FAHFA's), glycolipids and triglycerides as compared to the plasma metabolome composition with implications in mediation of systemic response to radiation induced stress signaling.

Exposure to ionizing radiation induces a complex cascade of systemic and tissue-specific responses that lead to functional impairment over time in the surviving population. However, due to the lack of predictive biomarkers of tissue injury, current methods for the management of survivors of radiation exposure episodes involve monitoring of individuals over time for the development of adverse clinical symptoms and death. Herein, we report on changes in metabolomic and lipidomic profiles in multiple tissues of nonhuman primates (NHPs) that were exposed to a single dose of 7.2 Gy whole-body 60Co γ-radiation that either survived or succumbed to radiation toxicities over a 60-day period. This study involved the delineation of the radiation effects in the liver, kidney, jejunum, heart, lung, and spleen. We found robust metabolic changes in the kidney and liver and modest changes in other tissue types at the 60-day time point in a cohort of NHPs. Remarkably, we found significant elevation of long-chain acylcarnitines in animals that were exposed to radiation across multiple tissue types underscoring the role of this class of metabolites as a generic indicator of radiation-induced normal tissue injury. These studies underscore the utility of a metabolomics approach for delineating anticipatory biomarkers of exposure to ionizing radiation.

(1) Background: Recent studies have investigated the expression of taste-related genes in the organs of various animals, including humans; however, data for additional taxa are needed to facilitate comparative analyses within and among species. (2) Methods: We investigated the expression of taste-related genes in the intestines of rhesus macaques, the non-human primates most commonly used in experimental models. (3) Results: Based on RNAseq and qRT-PCR, genes encoding bitter taste receptors and the G-protein gustducin were expressed in the gut of rhesus macaques. RNAscope analysis showed that one of the bitter receptors, TAS2R38, was expressed in some cells in the small intestine, and immunohistochemical analysis revealed the presence of T2R38-positive cells in the villi of the intestines. (4) Conclusions: These results suggest that bitter receptors are expressed in the gut of rhesus macaques, supporting the use of macaques as a model for studies of human taste, including gut analyses.

Current technologies that are used for genome-wide microRNA (miRNA) prediction are mainly based on BLAST tool. They often produce a large number of false positives. Here, we describe an effective approach for identifying orthologous pre-miRNAs in several primates based on syntenic information. Some of them have been validated by small RNA high throughput sequencing data. This approach uses the synteny information and experimentally validated miRNAs of human, and incorporates currently available algorithms and tools to identify the pre-miRNAs in five other primates. First, we identified 929 potential pre-miRNAs in the marmoset in which miRNAs have not yet been reported. Then, we predicted the miRNAs in other primates, and we successfully re-identified most of the published miRNAs and found 721, 979, 650 and 639 new potential pre-miRNAs in chimpanzee, gorilla, orangutan and rhesus macaque, respectively. Furthermore, the miRNA transcriptome in the four primates have been re-analyzed and some novel predicted miRNAs have been supported by the small RNA sequencing data. Finally, we analyzed the potential functions of those validated miRNAs and explored the regulatory elements and transcription factors of some validated miRNA genes of interest. The results show that our approach can effectively identify novel miRNAs and some miRNAs that supported by small RNA sequencing data maybe play roles in the nervous system.

The development of radiation countermeasures for acute radiation syndrome (ARS) has been underway for the past six decades, leading to the identification of multiple classes of radiation countermeasures. However, to date, only two growth factors (Neupogen and Neulasta) have been approved by the United States Food and Drug Administration (US FDA) for the mitigation of hematopoietic acute radiation syndrome (H-ARS). No radioprotector for ARS has been approved by the FDA yet. Gamma-tocotrienol (GT3) has been demonstrated to have radioprotective efficacy in murine as well as nonhuman primate (NHP) models. Currently, GT3 is under advanced development as a radioprotector that can be administered prior to radiation exposure. We are studying this agent for its safety profile and efficacy using the NHP model. In this study, we analyzed global metabolomic and lipidomic changes using ultra-performance liquid chromatography (UPLC) quadrupole time-of-flight mass spectrometry (QTOF-MS) in serum samples of NHPs administered GT3. Our study, using 12 NHPs, demonstrates that alterations in metabolites manifest only 24 h after GT3 administration. Furthermore, metabolic changes are associated with transient increase in the bioavailability of antioxidants, including lactic acid and cholic acid and anti-inflammatory metabolites 3 deoxyvitamin D3, and docosahexaenoic acid. Taken together, our results show that the administration of GT3 to NHPs causes metabolic shifts that would provide an overall advantage to combat radiation injury. This initial assessment also highlights the utility of metabolomics and lipidomics to determine the underlying physiological mechanisms involved in the radioprotective efficacy of GT3.

Cerebrospinal fluid contacting neurons (CSF-cNs) are a specific type of neurons located around the ventricles in the brain and the central canal in the spinal cord and have been demonstrated to be intrinsic sensory neurons in the central nervous system. One of the important channels responsible for the sensory function is the polycystic kidney disease 2-like 1 (PKD2L1) channel. Most of the studies concerning the distribution and function of the PKD2L1-expressing CSF-cNs in the spinal cord have previously been performed in non-mammalian vertebrates. In the present study immunohistochemistry was performed to determine the distribution of PKD2L1-immunoreactive (IR) CSF-cNs in the spinal cords of four mammalian species: mouse, rat, cat, and macaque monkey. Here, we found that PKD2L1-expressing CSF-cNs were present at all levels of the spinal cord in these animal species. Although the distribution pattern was similar across these species, differences existed. Mice and rats presented a clear PKD2L1-IR cell body labeling, whereas in cats and macaques the PKD2L1-IR cell bodies were more weakly labeled. Ectopic PKD2L1-IR neurons away from the ependymal layer were observed in all the animal species although the abundance and the detailed locations varied. The apical dendritic protrusions with ciliated fibers were clearly seen in the lumen of the central canal in all the animal species, but the sizes of protrusion bulbs were different among the species. PKD2L1-IR cell bodies/dendrites were co-expressed with doublecortin, MAP2 (microtubule-associated protein 2), and aromatic L-amino acid decarboxylase, but not with NeuN (neuronal nuclear protein), indicating their immature properties and ability to synthesize monoamine transmitters. In addition, in situ hybridization performed in rats revealed PKD2L1 mRNA expression in the cells around the central canal. Our results indicate that the intrinsic sensory neurons are conserved across non-mammalian and mammalian vertebrates. The similar morphology of the dendritic bulbs with ciliated fibers (probably representing stereocilia and kinocilia) protruding into the central canal across different animal species supports the notion that PKD2L1 is a chemo- and mechanical sensory channel that responds to mechanical stimulations and maintains homeostasis of the spinal cord. However, the differences of PKD2L1 distribution and expression between the species suggest that PKD2L1-expressing neurons may receive and process sensory signals differently in different animal species.

Translational research often requires the testing of experimental therapies in primates, but research in non-human primates is now stringently controlled by law around the world. Tissues fixed in formaldehyde without glutaraldehyde have been thought to be inappropriate for use in electron microscopic analysis, particularly those of the brain. Here we report the immunoelectron microscopic characterization of arginine vasopressin (AVP)-producing neurons in macaque hypothalamo-pituitary axis tissues fixed by perfusion with 4% formaldehyde and stored at -25 °C for several years (4-6 years). The size difference of dense-cored vesicles between magnocellular and parvocellular AVP neurons was detectable in their cell bodies and perivascular nerve endings located, respectively, in the posterior pituitary and median eminence. Furthermore, glutamate and the vesicular glutamate transporter 2 could be colocalized with AVP in perivascular nerve endings of both the posterior pituitary and the external layer of the median eminence, suggesting that both magnocellular and parvocellular AVP neurons are glutamatergic in primates. Both ultrastructure and immunoreactivity can therefore be sufficiently preserved in macaque brain tissues stored long-term, initially for light microscopy. Taken together, these results suggest that this methodology could be applied to the human post-mortem brain and be very useful in translational research.

Ribosome-inactivating proteins (RIPs) hydrolyze the N-glycosidic bond and depurinate a specific adenine residue (A-4324 in rat 28S ribosomal RNA, rRNA) in the conserved α-sarcin/ricin loop (α-SRL) of rRNA. In this study, we have purified and characterized lyophyllin, an unconventional RIP from Lyophyllum shimeji, an edible mushroom. The protein resembles peptidase M35 domain of peptidyl-Lys metalloendopeptidases. Nevertheless, protein either from the mushroom or in recombinant form possessed N-glycosidase and protein synthesis inhibitory activities. A homology model of lyophyllin was constructed. It was found that the zinc binding pocket of this protein resembles the catalytic cleft of a classical RIP, with key amino acids that interact with the adenine substrate in the appropriate positions. Mutational studies showed that E122 may play a role in stabilizing the positively charged oxocarbenium ion and H121 for protonating N-3 of adenine. The tyrosine residues Y137 and Y104 may be used for stacking the target adenine ring. This work first shows a protein in the peptidase M35 superfamily based on conserved domain search possessing N-glycosidase activity.

Mutations in the GBA1 gene coding for glucocerebrosidase (GCase) are the main genetic risk factor for Parkinson's disease (PD). Indeed, identifying reduced GCase activity as a common feature underlying the typical neuropathological signatures of PD-even when considering idiopathic forms of PD-has recently paved the way for designing novel strategies focused on enhancing GCase activity to reduce alpha-synuclein burden and preventing dopaminergic cell death. Here we have performed bilateral injections of a viral vector coding for the mutated form of alpha-synuclein (rAAV9-SynA53T) for disease modeling purposes, both in mice as well as in nonhuman primates (NHPs), further inducing a progressive neuronal death in the substantia nigra pars compacta (SNpc). Next, another vector coding for the GBA1 gene (rAAV9-GBA1) was unilaterally delivered in the SNpc of mice and NHPs one month after the initial insult, together with the contralateral delivery of an empty/null rAAV9 for control purposes. Obtained results showed that GCase enhancement reduced alpha-synuclein burden, leading to improved survival of dopaminergic neurons. Data reported here support using GCase gene therapy as a disease-modifying treatment for PD and related synucleinopathies, including idiopathic forms of these disorders.

Hypoxia-induced mitogenic factor (HIMF), which is also known as resistin-like molecule α (RELM-α), found in inflammatory zone 1 (FIZZ1), or resistin-like alpha (retlna), is a cysteine-rich secretory protein and cytokine. HIMF has been investigated in the lung as a mediator of pulmonary fibrosis, inflammation and as a marker for alternatively activated macrophages. Although these macrophages have been found to have a role in acute liver injury and acetaminophen toxicity, few studies have investigated the role of HIMF in acute or immune-mediated liver injury. The aim of this focused review is to analyze the literature and examine the effects of HIMF and its human homolog in organ-specific inflammation in the lung and liver. We followed the guidelines set by PRISMA in constructing this review. The relevant checklist items from PRISMA were included. Items related to meta-analysis were excluded because there were no randomized controlled clinical trials. We found that HIMF was increased in most models of acute liver injury and reduced damage from acetaminophen-induced liver injury. We also found strong evidence for HIMF as a marker for alternatively activated macrophages. Our overall risk of bias assessment of all studies included revealed that 80% of manuscripts demonstrated some concerns in the randomization process. We also demonstrated some concerns (54.1%) and high risk (45.9%) of bias in the selection of the reported results. The need for randomization and reduction of bias in the reported results was similarly detected in the studies that focused on HIMF and the liver. In conclusion, we propose that HIMF could be utilized as a marker for M2 macrophages in immune-mediated liver injury. However, we also detected the need for randomized clinical trials and additional experimental and human prospective studies in order to fully comprehend the role of HIMF in acute or immune-mediated liver injury.

The cellular, macromolecular and neutral lipid composition of the atherosclerotic plaque has been extensively characterized. However, a comprehensive lipidomic analysis of the major lipid classes within atherosclerotic lesions has not been reported. The objective of this study was to produce a detailed framework of the lipids that comprise the atherosclerotic lesion of a widely used pre-clinical model of plaque progression. Male New Zealand White rabbits were administered regular chow supplemented with 0.5% cholesterol (HC) for 12 weeks to induce hypercholesterolemia and atherosclerosis. Our lipidomic analyses of plaques isolated from rabbits fed the HC diet, using ultra-performance liquid chromatography (UPLC) and high-resolution mass spectrometry, detected most of the major lipid classes including: Cholesteryl esters, triacylglycerols, phosphatidylcholines, sphingomyelins, diacylglycerols, fatty acids, phosphatidylserines, lysophosphatidylcholines, ceramides, phosphatidylglycerols, phosphatidylinositols and phosphatidylethanolamines. Given that cholesteryl esters, triacylglycerols and phosphatidylcholines comprise greater than 75% of total plasma lipids, we directed particular attention towards the qualitative and quantitative assessment of the fatty acid composition of these lipids. We additionally found that sphingomyelins were relatively abundant lipid class within lesions, and compared the abundance of sphingomyelins to their precursor phosphatidylcholines. The studies presented here are the first approach to a comprehensive characterization of the atherosclerotic plaque lipidome.

In eukaryotes three of the four ribosomal RNA (rRNA) molecules are transcribed as a long precursor that is processed into mature rRNAs concurrently with the assembly of ribosomal subunits. However, the relative timing of association of ribosomal proteins with the ribosomal precursor particles and the cleavage of the precursor rRNA into the subunit-specific moieties is not known. To address this question, we searched for ribosomal precursors containing components from both subunits. Particles containing specific ribosomal proteins were targeted by inducing synthesis of epitope-tagged ribosomal proteins followed by pull-down with antibodies targeting the tagged protein. By identifying other ribosomal proteins and internal rRNA transcribed spacers (ITS1 and ITS2) in the immuno-purified ribosomal particles, we showed that eS7/S7 and uL4/L4 bind to nascent ribosomes prior to the separation of 40S and 60S specific segments, while uS4/S9, uL22, and eL13/L13 are bound after, or simultaneously with, the separation. Thus, the incorporation of ribosomal proteins from the two subunits begins as a co-assembly with a single rRNA molecule, but is finished as an assembly onto separate precursors for the two subunits.

Ppz enzymes are type-1 related Ser/Thr protein phosphatases that are restricted to fungi. In S. cerevisiae and other fungi, Ppz1 is involved in cation homeostasis and is regulated by two structurally-related inhibitory subunits, Hal3 and Vhs3, with Hal3 being the most physiologically relevant. Remarkably, Hal3 and Vhs3 have moonlighting properties, as they participate in an atypical heterotrimeric phosphopantothenoyl cysteine decarboxylase (PPCDC), a key enzyme for Coenzyme A biosynthesis. Here we identify and functionally characterize Ppz1 phosphatase (UmPpz1) and its presumed regulatory subunit (UmHal3) in the plant pathogen fungus Ustilago maydis. UmPpz1 is not an essential protein in U. maydis and, although possibly related to the cell wall integrity pathway, is not involved in monovalent cation homeostasis. The expression of UmPpz1 in S. cerevisiae Ppz1-deficient cells partially mimics the functions of the endogenous enzyme. In contrast to what was found in C. albicans and A. fumigatus, UmPpz1 is not a virulence determinant. UmHal3, an unusually large protein, is the only functional PPCDC in U. maydis and, therefore, an essential protein. However, when overexpressed in U. maydis or S. cerevisiae, UmHal3 does not reproduce Ppz1-inhibitory phenotypes. Indeed, UmHal3 does not inhibit UmPpz1 in vitro (although ScHal3 does). Therefore, UmHal3 might not be a moonlighting protein.

The VPS13 family of proteins have emerged as key players in intracellular lipid transport and human health. Humans have four different VPS13 orthologs, the dysfunction of which leads to different diseases. Yeast has a single VPS13 gene, which encodes a protein that localizes to multiple different membrane contact sites. The yeast vps13Δ mutant is pleiotropic, exhibiting defects in sporulation, protein trafficking, endoplasmic reticulum (ER)-phagy and mitochondrial function. Non-null alleles resulting from missense mutations can be useful reagents for understanding the multiple functions of a gene. The exceptionally large size of Vps13 makes the identification of key residues challenging. As a means to identify critical residues in yeast Vps13, amino acid substitution mutations from VPS13A, B, C and D, associated with human disease, were introduced at the cognate positions of yeast VPS13, some of which created separation-of-function alleles. Phenotypic analyses of these mutants have revealed that the promotion of ER-phagy is a fourth, genetically separable role of VPS13 and provide evidence that co-adaptors at the endosome mediate the activity of VPS13 in vacuolar sorting.

Cigarette smoke has been shown to trigger aberrant signaling pathways and pathophysiological processes; however, the regulatory mechanisms underlying smoke-induced gene expression remain to be established. Herein, we observed that two smoke-responsive genes, HO-1 and CYP1A1, are robustly induced upon smoke by different mechanisms in human bronchial epithelia. CYP1A1 is mediated by aryl hydrocarbon receptor signaling, while induction of HO-1 is regulated by oxidative stress, and suppressed by N-acetylcysteine treatment. In light of a pivotal role of NRF2 and BACH1 in response to oxidative stress and regulation of HO-1, we examined if smoke-induced HO-1 expression is modulated through the NRF2/BACH1 axis. We demonstrated that smoke causes significant nuclear translocation of NRF2, but only a slight decrease in nuclear BACH1. Knockdown of NRF2 attenuated smoke-induced HO-1 expression while down-regulation of BACH1 had stimulatory effects on both basal and smoke-induced HO-1 with trivial influence on NRF2 nuclear translocation. Chromatin immunoprecipitation assays showed that smoke augments promoter-specific DNA binding of NRF2 but suppresses BACH1 binding to the HO-1 promoter ARE sites, two of which at -1.0 kb and -2.6 kb are newly identified. These results suggest that the regulation of NRF2 activator and BACH1 repressor binding to the ARE sites are critical for smoke-mediated HO-1 induction.

14-3-3 proteins are regulatory proteins found in all eukaryotes and are known to selectively interact with phosphorylated proteins to regulate physiological processes. Through an affinity purification screening, many light-related proteins were recovered as 14-3-3 candidate binding partners. Yeast two-hybrid analysis revealed that the 14-3-3 kappa isoform (14-3-3κ) could bind to PHYTOCHROME INTERACTING FACTOR3 (PIF3) and CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1). Further analysis by in vitro pull-down assay confirmed the interaction between 14-3-3κ and PIF3. Interruption of putative phosphorylation sites on the 14-3-3 binding motifs of PIF3 was not sufficient to inhibit 14-3-3κ from binding or to disturb nuclear localization of PIF3. It was also indicated that 14-3-3κ could bind to other members of the PIF family, such as PIF1 and PIF6, but not to LONG HYPOCOTYL IN FAR-RED1 (HFR1). 14-3-3 mutants, as well as the PIF3 overexpressor, displayed longer hypocotyls, and a pif3 mutant displayed shorter hypocotyls than the wild-type in red light, suggesting that 14-3-3 proteins are positive regulators of photomorphogenesis and function antagonistically with PIF3. Consequently, our results indicate that 14-3-3 proteins bind to PIFs and initiate photomorphogenesis in response to a light signal.

Dysregulated cross-talk between immune cells and epithelial compartments is responsible for the onset and amplification of pathogenic auto-inflammatory circuits occurring in psoriasis. NAMPT-mediated NAD salvage pathway has been recently described as an immunometabolic route having inflammatory function in several disorders, including arthritis and inflammatory bowel diseases. To date, the role of NAD salvage pathway has not been explored in the skin of patients affected by psoriasis. Here, we show that NAD content is enhanced in lesional skin of psoriatic patients and is associated to high NAMPT transcriptional levels. The latter are drastically reduced in psoriatic skin following treatment with the anti-IL-17A biologics secukinumab. We provide evidence that NAMPT-mediated NAD+ metabolism fuels the immune responses executed by resident skin cells in psoriatic skin. In particular, intracellular NAMPT, strongly induced by Th1/Th17-cytokines, acts on keratinocytes by inducing hyper-proliferation and impairing their terminal differentiation. Furthermore, NAMPT-mediated NAD+ boosting synergizes with psoriasis-related cytokines in the upregulation of inflammatory chemokines important for neutrophil and Th1/Th17 cell recruitment. In addition, extracellular NAMPT, abundantly released by keratinocytes and dermal fibroblasts, acts in a paracrine manner on endothelial cells by inducing their proliferation and migration, as well as the expression of ICAM-1 membrane molecule and chemokines important for leukocyte recruitment into inflamed skin. In conclusion, our results showed that NAMPT-mediated NAD salvage pathway contributes to psoriasis pathogenic processes by amplifying epithelial auto-inflammatory responses in psoriasis.

The loss of bone following tooth extraction poses a significant clinical problem for maxillofacial esthetics, function, and future implant placement. In the present study, the efficacy of an erythropoietin-impregnated collagen scaffold as an alveolar ridge augmentation material versus a conventional collagen scaffold and a BioOss inorganic bovine bone xenograft was examined. The collagen/Erythropoietin (EPO) scaffold exhibited significantly more rapid and complete osseous regeneration of the alveolar defect when compared to bone xenograft and the collagen membrane alone. The new EPO induced extracellular matrix was rich in Collagen I, Collagen III, Fibronectin (Fn) and E-cadherin, and featured significantly increased levels of the osteogenic transcription factors Runt-related transcription factor 2 (Runx2) and Osterix (Osx). Histomorphometric evaluation revealed a significant two-fold increase in the number of capillaries between the EPO and the BioOss group. Moreover, there was a highly significant 3.5-fold higher level of vascular endothelial growth factor (VEGF) in the collagen/EPO-treated group compared to controls. The significant effect of EPO on VEGF, FN, and RUNX2 upregulation was confirmed in vitro, and VEGF pathway analysis using VEGF inhibitors confirmed that EPO modulated extracellular matrix protein expression through VEGF even in the absence of blood vessels. Together, these data demonstrate the effectiveness of an EPO-impregnated collagen scaffold for bone regeneration as it induces rapid matrix production and osseoinduction adjacent to new capillaries via VEGF.

Alzheimer's disease (AD) is the most common form of dementia, yet there are no therapeutic treatments that can either cure or delay its onset. Currently, the pathogenesis of AD is still uncertain, especially with respect to how the disease develops from a normal healthy brain. Amyloid β oligomers (AβO) are highly neurotoxic proteins and are considered potential initiators to the pathogenesis of AD. Rat brains were exposed to AβO via bilateral intracerebroventricular injections. Rats were then euthanized at either 1, 3, 7 or 21-days post surgery. Rat behavioural testing was performed using the Morris water maze and open field tests. Post-mortem brain tissue was immunolabelled for Aβ, microglia, and cholinergic neurons. Rats exposed to AβO showed deficits in spatial learning and anxiety-like behaviour. Acute positive staining for Aβ was only observed in the corpus callosum surrounding the lateral ventricles. AβO exposed rat brains also showed a delayed increase in activated microglia within the corpus callosum and a decreased number of cholinergic neurons within the basal forebrain. Acute exposure to AβO resulted in mild learning and memory impairments with co-concomitant white matter pathology within the corpus callosum and cholinergic cell loss within the basal forebrain. Results suggest that acute exposure to AβO in the rat may be a useful tool in assessing the early phases for the pathogenesis of AD.

Ion channels included in the family of Connexins (Cx) help to control cell proliferation and differentiation of neuronal progenitors. Here we explored the role of Connexin 50 (Cx50) in cell fate modulation of adult spinal cord derived neural precursors located in the ependymal canal (epSPC). epSPC from non-injured animals showed high expression levels of Cx50 compared to epSPC from animals with spinal cord injury (SCI) (epSPCi). When epSPC or epSPCi were induced to spontaneously differentiate in vitro we found that Cx50 favors glial cell fate, since higher expression levels, endogenous or by over-expression of Cx50, augmented the expression of the astrocyte marker GFAP and impaired the neuronal marker Tuj1. Cx50 was found in both the cytoplasm and nucleus of glial cells, astrocytes and oligodendrocyte-derived cells. Similar expression patterns were found in primary cultures of mature astrocytes. In addition, opposite expression profile for nuclear Cx50 was observed when epSPC and activated epSPCi were conducted to differentiate into mature oligodendrocytes, suggesting a different role for this ion channel in spinal cord beyond cell-to-cell communication. In vivo detection of Cx50 by immunohistochemistry showed a defined location in gray matter in non-injured tissues and at the epicenter of the injury after SCI. epSPCi transplantation, which accelerates locomotion regeneration by a neuroprotective effect after acute SCI is associated with a lower signal of Cx50 within the injured area, suggesting a minor or detrimental contribution of this ion channel in spinal cord regeneration by activated epSPCi.