Vesicular nucleotide transporter is a molecular target of eicosapentaenoic acid for neuropathic and inflammatory pain treatment.

Eicosapentaenoic acid (EPA), an omega-3 (ω-3) polyunsaturated fatty acid, is an essential nutrient that exhibits antiinflammatory, neuroprotective, and cardiovascular-protective activities. Although EPA is used as a nutrient-based pharmaceutical agent or dietary supplement, its molecular target(s) is debatable. Here, we showed that EPA and its metabolites strongly and reversibly inhibit vesicular nucleotide transporter (VNUT), a key molecule for vesicular storage and release of adenosine triphosphate (ATP) in purinergic chemical transmission. In vitro analysis showed that EPA inhibits human VNUT-mediated ATP uptake at a half-maximal inhibitory concentration (IC50) of 67 nM, acting as an allosteric modulator through competition with Cl-. EPA impaired vesicular ATP release from neurons without affecting the vesicular release of other neurotransmitters. In vivo, VNUT-/- mice showed a delay in the onset of neuropathic pain and resistance to both neuropathic and inflammatory pain. EPA potently attenuated neuropathic and inflammatory pain in wild-type mice but not in VNUT-/- mice without affecting the basal nociception. The analgesic effect of EPA was canceled by the intrathecal injection of purinoceptor agonists and was stronger than that of existing drugs used for neuropathic pain treatment, with few side effects. Neuropathic pain impaired insulin sensitivity in previous studies, which was improved by EPA in the wild-type mice but not in the VNUT-/- mice. Our results showed that VNUT is a molecular target of EPA that attenuates neuropathic and inflammatory pain and insulin resistance. EPA may represent a unique nutrient-based treatment and prevention strategy for neurological, immunological, and metabolic diseases by targeting purinergic chemical transmission.

Effect of short-time treatment with TNF-α on stem cell activity and barrier function in enteroids.

Although tumor necrosis factor-α (TNF-α) is a known major inflammatory mediator in inflammatory bowel disease (IBD) and has various effects on intestinal epithelial cell (IEC) homeostasis, the changes in IECs in the early inflammatory state induced during short-time treatment (24 h) with TNF-α remain unclear. In this study, we investigated TNF-α-induced alterations in IECs in the early inflammatory state using mouse jejunal organoids (enteroids). Of the inflammatory cytokines, i.e., TNF-α, IL-1ß, IL-6, and IL-17, only TNF-α markedly increased the mRNA level of macrophage inflammatory protein 2 (MIP-2; the mouse homologue of interleukin-8), which is induced in the early stages of inflammation. TNF-α stimulation (3 h and 6 h) decreased the mRNA level of the stem cell markers leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) and polycomb group ring finger 4 and the progenitor cell marker prominin-1, which is also known as CD133. In addition, TNF-α treatment (24 h) decreased the number of Lgr5-positive cells and enteroid proliferation. TNF-α stimulation at 3 h and 6 h also decreased the mRNA level of chromogranin A and mucin 2, which are respective markers of enteroendocrine and goblet cells. Moreover, enteroids treated with TNF-α (24 h) not only decreased the integrity of tight junctions and cytoskeletal components but also increased intercellular permeability in an influx test with fluorescent dextran, indicating disrupted intestinal barrier function. Taken together, our findings indicate that short-time treatment with TNF-α promotes the inflammatory response and decreases intestinal stem cell activity and barrier function. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10616-021-00487-y.

Ngn3-Positive Cells Arise from Pancreatic Duct Cells.

The production of pancreatic ß cells is the most challenging step for curing diabetes using next-generation treatments. Adult pancreatic endocrine cells are thought to be maintained by the self-duplication of differentiated cells, and pancreatic endocrine neogenesis can only be observed when the tissue is severely damaged. Experimentally, this can be performed using a method named partial duct ligation (PDL). As the success rate of PDL surgery is low because of difficulties in identifying the pancreatic duct, we previously proposed a method for fluorescently labeling the duct in live animals. Using this method, we performed PDL on neurogenin3 (Ngn3)-GFP transgenic mice to determine the origin of endocrine precursor cells and evaluate their potential to differentiate into multiple cell types. Ngn3-activated cells, which were marked with GFP, appeared after PDL operation. Because some GFP-positive cells were aligned proximally to the duct, we hypothesized that Ngn3-positive cells arise from the pancreatic duct. Therefore, we next developed an in vitro pancreatic duct culture system using Ngn3-GFP mice and examined whether Ngn3-positive cells emerge from this duct. We observed GFP expressions in ductal organoid cultures. GFP expressions were correlated with Ngn3 expressions and endocrine cell lineage markers. Interestingly, tuft cell markers were also correlated with GFP expressions. Our results demonstrate that in adult mice, Ngn3-positive endocrine precursor cells arise from the pancreatic ducts both in vivo and in vitro experiments indicating that the pancreatic duct could be a potential donor for therapeutic use.

Mycotoxin Deoxynivalenol Has Different Impacts on Intestinal Barrier and Stem Cells by Its Route of Exposure.

The different effects of deoxynivalenol (DON) on intestinal barrier and stem cells by its route of exposure remain less known. We explored the toxic effects of DON on intestinal barrier functions and stem cells after DON microinjection (luminal exposure) or addition to a culture medium (basolateral exposure) using three-dimensional mouse intestinal organoids (enteroids). The influx test using fluorescein-labeled dextran showed that basolateral DON exposure (1 micromolar (µM) disrupted intestinal barrier functions in enteroids compared with luminal DON exposure at the same concentration. Moreover, an immunofluorescence experiment of intestinal epithelial proteins, such as E-cadherin, claudin, zonula occludens-1 (ZO-1), and occludin, exhibited that only basolateral DON exposure broke down intestinal epithelial integrity. A time-lapse analysis using enteroids from leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5)-enhanced green fluorescence protein (EGFP) transgenic mice and 5-ethynyl-2-deoxyuridine (EdU) assay indicated that only the basolateral DON exposure, but not luminal DON exposure, suppressed Lgr5+ stem cell count and proliferative cell ratio, respectively. These results revealed that basolateral DON exposure has larger impacts on intestinal barrier function and stem cells than luminal DON exposure. This is the first report that DON had different impacts on intestinal stem cells depending on the administration route. In addition, RNA sequencing analysis showed different expression of genes among enteroids after basolateral and luminal DON exposure.

Comparative analysis of enteroendocrine cells and their hormones between mouse intestinal organoids and native tissues.

Endocrine cells in the gastrointestinal tract secrete multiple hormones to maintain homeostasis in the body. In the present study, we generated intestinal organoids from the duodenum, jejunum, and ileum of Neurogenin 3 (Ngn3)-EGFP mice and examined how enteroendocrine cells (EECs) within organoid cultures resemble native epithelial cells in the gut. Transcriptome analysis of EGFP-positive cells from Ngn3-EGFP organoids showed gene expression pattern comparable to EECs in vivo. We also compared mRNAs of five major hormones, namely, ghrelin (Ghrl), cholecystokinin (Cck), Gip, secretin (Sct), and glucagon (Gcg) in organoids and small intestine along the longitudinal axis and found that expression patterns of these hormones in organoids were similar to those in native tissues. These findings suggest that an intestinal organoid culture system can be utilized as a suitable model to study enteroendocrine cell functions in vitro.

Dorsal horn neurons release extracellular ATP in a VNUT-dependent manner that underlies neuropathic pain.

Activation of purinergic receptors in the spinal cord by extracellular ATP is essential for neuropathic hypersensitivity after peripheral nerve injury (PNI). However, the cell type responsible for releasing ATP within the spinal cord after PNI is unknown. Here we show that PNI increases expression of vesicular nucleotide transporter (VNUT) in the spinal cord. Extracellular ATP content ([ATP]e) within the spinal cord was increased after PNI, and this increase was suppressed by exocytotic inhibitors. Mice lacking VNUT did not show PNI-induced increase in [ATP]e and had attenuated hypersensitivity. These phenotypes were recapitulated in mice with specific deletion of VNUT in spinal dorsal horn (SDH) neurons, but not in mice lacking VNUT in primary sensory neurons, microglia or astrocytes. Conversely, ectopic VNUT expression in SDH neurons of VNUT-deficient mice restored PNI-induced increase in [ATP]e and pain. Thus, VNUT is necessary for exocytotic ATP release from SDH neurons which contributes to neuropathic pain.

Urothelial ATP exocytosis: regulation of bladder compliance in the urine storage phase.

The bladder urothelium is more than just a barrier. When the bladder is distended, the urothelium functions as a sensor to initiate the voiding reflex, during which it releases ATP via multiple mechanisms. However, the mechanisms underlying this ATP release in response to the various stretch stimuli caused by bladder filling remain largely unknown. Therefore, the aim of this study was to elucidate these mechanisms. By comparing vesicular nucleotide transporter (VNUT)-deficient and wild-type male mice, we showed that ATP has a crucial role in urine storage through exocytosis via a VNUT-dependent mechanism. VNUT was abundantly expressed in the bladder urothelium, and when the urothelium was weakly stimulated (i.e. in the early filling stages), it released ATP by exocytosis. VNUT-deficient mice showed reduced bladder compliance from the early storage phase and displayed frequent urination in inappropriate places without a change in voiding function. We conclude that urothelial, VNUT-dependent ATP exocytosis is involved in urine storage mechanisms that promote the relaxation of the bladder during the early stages of filling.

Correlation Between Activation of the Prelimbic Cortex, Basolateral Amygdala, and Agranular Insular Cortex During Taste Memory Formation.

Conditioned taste aversion (CTA) is a well-established learning paradigm, whereby animals associate tastes with subsequent visceral illness. The prelimbic cortex (PL) has been shown to be involved in the association of events separated by time. However, the nature of PL activity and its functional network in the whole brain during CTA learning remain unknown. Here, using awake functional magnetic resonance imaging and fiber tracking, we analyzed functional brain connectivity during the association of tastes and visceral illness. The blood oxygen level-dependent (BOLD) signal significantly increased in the PL after tastant and lithium chloride (LiCl) infusions. The BOLD signal in the PL significantly correlated with those in the amygdala and agranular insular cortex (IC), which we found were also structurally connected to the PL by fiber tracking. To precisely examine these data, we then performed double immunofluorescence with a neuronal activity marker (c-Fos) and an inhibitory neuron marker (GAD67) combined with a fluorescent retrograde tracer in the PL. During CTA learning, we found an increase in the activity of excitatory neurons in the basolateral amygdala (BLA) or agranular IC that project to the PL. Taken together, these findings clearly identify a role of synchronized PL, agranular IC, and BLA activity in CTA learning.

Impairment of vesicular ATP release affects glucose metabolism and increases insulin sensitivity.

Neuroendocrine cells store ATP in secretory granules and release it along with hormones that may trigger a variety of cellular responses in a process called purinergic chemical transmission. Although the vesicular nucleotide transporter (VNUT) has been shown to be involved in vesicular storage and release of ATP, its physiological relevance in vivo is far less well understood. In Vnut knockout (Vnut(-/-)) mice, we found that the loss of functional VNUT in adrenal chromaffin granules and insulin granules in the islets of Langerhans led to several significant effects. Vesicular ATP accumulation and depolarization-dependent ATP release were absent in the chromaffin granules of Vnut(-/-) mice. Glucose-responsive ATP release was also absent in pancreatic ß-cells in Vnut(-/-) mice, while glucose-responsive insulin secretion was enhanced to a greater extent than that in wild-type tissue. Vnut(-/-) mice exhibited improved glucose tolerance and low blood glucose upon fasting due to increased insulin sensitivity. These results demonstrated an essential role of VNUT in vesicular storage and release of ATP in neuroendocrine cells in vivo and suggest that vesicular ATP and/or its degradation products act as feedback regulators in catecholamine and insulin secretion, thereby regulating blood glucose homeostasis.

Microglia trigger astrocyte-mediated neuroprotection via purinergic gliotransmission.

Microglia are highly sensitive to even small changes in the brain environment, such as invasion of non-hazardous toxicants or the presymptomatic state of diseases. However, the physiological or pathophysiological consequences of their responses remain unknown. Here, we report that cultured microglia sense low concentrations of the neurotoxicant methylmercury (MeHg(low)) and provide neuroprotection against MeHg, for which astrocytes are also required. When exposed to MeHg(low), microglia exocytosed ATP via p38 MAPK- and vesicular nucleotide transporter (VNUT)-dependent mechanisms. Astrocytes responded to the microglia-derived ATP via P2Y1 receptors and released interleukin-6 (IL-6), thereby protecting neurons against MeHg(low). These neuroprotective actions were also observed in organotypic hippocampal slices from wild-type mice, but not in slices prepared from VNUT knockout or P2Y1 receptor knockout mice. These findings suggest that microglia sense and respond to even non-hazardous toxicants such as MeHg(low) and change their phenotype into a neuroprotective one, for which astrocytic support is required.

Identification of the vesicular nucleotide transporter (VNUT) in taste cells.

Taste cells are chemosensory epithelial cells that sense distinct taste qualities. It is the type II taste cell that express G-protein coupled receptors to sense either umami, sweet, or bitter compounds. Whereas several reports have suggested involvement of ATP in taste signal transduction, there is a paucity of molecular information about how ATP is stored and being released. The recent discovery of a novel vesicular nucleotide transporter (VNUT) led us to examine whether VNUT exist in the taste tissue where ATP is to be released for taste signal transmission. Here, we report that VNUT is selectively expressed in type II cell but not in type III taste cell. In addition, we show that during taste bud development VNUT expression is always accompanied by the expression of type II taste cell markers. Our results, together with previous studies, strongly suggest that VNUT plays a role in type II taste cell.

A novel connection between rods and ON cone bipolar cells revealed by ectopic metabotropic glutamate receptor 7 (mGluR7) in mGluR6-deficient mouse retinas.

Since the discovery of direct chemical synapses between rod photoreceptor and OFF cone bipolar cells in mouse retinas, whether the ON cone bipolar cell also receive direct chemical input from rod has been a pending question. In finding that metabotropic glutamate receptor 7 (mGluR7) was uniquely expressed in dendrites of ON cone bipolar cells in the mGluR6-deficient mouse retina, we used this ectopic mGluR7 immunoreactivity as a specific marker for the ON cone bipolar to search for its rod connection. Here, we show that a certain type of ON cone bipolar cell forms ribbon-associated synapses not only with cones, but also rods. This finding was verified in the wild-type mouse retina by three-dimensional reconstruction of bipolar cells from serial electron micrographs. These ON cone bipolars were further identified as corresponding to type 7 of mouse bipolar cell described by Ghosh et al. (2004) and also to the green fluorescent protein (GFP)-labeled type 7 bipolars in the alpha-gustducin-GFP transgenic mouse. Our findings suggest that, in mice, rod signals bifurcate into a third ON and OFF pathway in addition to the two known routes to cone bipolar cells: (1) via rod chemical synapse --> rod bipolar --> AII amacrine --> ON and OFF cone bipolar cells; (2) via rod-cone gap junction --> cone chemical synapse --> ON and OFF cone bipolar cells; and (3) via rod chemical synapse --> ON and OFF cone bipolar cells. This third novel pathway is thought to transmit fast and moderately light-sensitive rod signals, functioning to smooth out the intensity changes at the scotopic-mesopic interface.

Wnt signaling interacts with Shh to regulate taste papilla development.

Wnt and Shh signaling pathways are critical for the development and maturation of many epithelial tissues. Both pathways have roles in stem cell maintenance, tissue development, and tumorigenesis. However, linkage between these pathways in mammalian systems had not been well established. Here, we report that Shh expression in fungiform papillae and formation of normal mature fungiform papillae depend on signaling through Wnt and beta-catenin. We observed that during fungiform papilla formation in mice, Shh and components of the Wnt/beta-catenin signaling pathway are expressed together in the developing placode. The elimination of Wnt/beta-catenin signaling in either Lef1 or Wnt10b knockout mice resulted in down-regulation of Shh expression. In addition, the size and number of fungiform papillae were greatly reduced in Lef1 knockout mice. By examining embryonic mouse tongues in culture we determined that activation of Wnt/beta-catenin signaling up-regulates Shh expression. We observed that blocking Shh signaling in cultured tongue explants enhanced papillae formation and was accompanied by an up-regulation of Wnt/beta-catenin signaling, indicating that Shh inhibits the Wnt/beta-catenin pathway. Exogenously added Shh suppressed expression of endogenous Shh and inhibited Wnt/beta-catenin signaling (assessed in TOPGAL mice), further implicating Shh as an inhibitor of the Wnt/beta-catenin pathway. Our observations indicate that Wnt/beta-catenin signaling and interactions between the Wnt and Shh pathways play essential roles in the development of fungiform papillae.

Runx1 promotes angiogenesis by downregulation of insulin-like growth factor-binding protein-3.

Mouse embryos lacking the Runx1 transcription factor exhibit an angiogenic defect accompanied by the absence of hematopoietic stem cells (HSCs). To ask whether Runx1 plays a direct role in angiogenesis, we established a novel endothelial progenitor cell line, designated AEL-DeltaR1, from the aorta-gonad-mesonephros (AGM) region of Runx1-null mouse. We introduced Runx1 cDNA into AEL-DeltaR1 cells under the doxycycline-inducible promoter. The ability of AEL-DeltaR1 cells to form vascular networks on matrigel was highly enhanced by the restored expression of Runx1. By molecular comparison of mRNAs in AEL-DeltaR1 cells before and after the induction of Runx1, we found that mRNA expression of insulin-like growth factor-binding protein 3 (IGFBP-3) is downregulated by Runx1. Gel retardation and reporter assays revealed that Runx1 binds to the promoter region of mouse IGFBP-3 gene and represses its transcription. When IGFBP-3 was exogenously added in the matrigel assay, the angiogenesis-enhancing activity of Runx1 was suppressed in a dose-dependent manner. These results demonstrate that Runx1 is directly involved in angiogenesis by repression of IGFBP-3 mRNA expression.

Expression and the biological activities of insulin-like growth factor-binding protein related protein 1 in rat uterus during the periimplantation period.

IGF binding protein-related protein 1 (IGFBP-rP1) is highly expressed in the rat uterus around the time of implantation. In the present study, we determined the periimplantation localization of IGFBP-rP1 mRNA and assessed the effects of recombinant IGFBP-rP1 on the proliferative and prostacyclin (PGI(2))-producing abilities of cultured endometrial cells early in pregnancy. IGFBP-rP1 mRNA was detected at high levels in endometrial stromal cells close to the smooth muscle of interimplantation sites around the time of implantation but absent from decidual zones surrounding the embryo. Differential uterine IGFBP-rP1 expression was also recognized in the delayed implanting pregnant model, but the level of mRNA decreased as decidual tissues formed in the decidualization model. Recombinant IGFBP-rP1 inhibited the proliferation of endometrial stromal cells in vitro and arrested them in the G(1) phase of the cell cycle. Furthermore, IGFBP-rP1 significantly stimulated PGI(2) synthesis and cyclooxygenase II mRNA expression in myometrial cells, both of which are essential molecules for successful implantation. These data suggest that IGFBP-rP1 is an implantation-associated protein and that it modulates the proliferation of rat uterine cells and their production of PGI(2) during the periimplantation period.

Cloning of cDNA encoding a regeneration-associated muscle protease whose expression is attenuated in cell lines derived from Duchenne muscular dystrophy patients.

In the dystrophin-mutant mdx mouse, an animal model for Duchenne muscular dystrophy (DMD), damaged skeletal muscles are efficiently regenerated and thus the animals thrive. The phenotypic differences between DMD patients and the mdx mice suggest the existence of factors that modulate the muscle wasting in the mdx mice. To identify these factors, we searched for mRNAs affected by the mdx mutation by using cDNA microarrays with newly established skeletal muscle cell lines from mdx and normal mice. We found that in the mdx muscle cell line, 12 genes, including L-arginine:glycine amidinotransferase and thymosin beta4, are up-regulated, whereas 7 genes, including selenoprotein P and a novel regeneration-associated muscle protease (RAMP), are down-regulated. Northern blot analysis and in situ hybridization revealed that RAMP mRNA is predominantly expressed in normal skeletal muscle and brain, and its production is enhanced in the regenerating area of injured skeletal muscle in mice. RAMP expression was much lower in individual muscle cell lines derived from biopsies of six DMD patients compared to a normal muscle cell line. These results suggest that RAMP may play a role in the regeneration of skeletal muscle and that its down-regulation could be involved in the progression of DMD in humans.

Complementary DNA cloning and characterization of RANDAM-2, a type I membrane molecule specifically expressed on glutamatergic neuronal cells in the mouse cerebrum.

A membrane-surface glycoprotein, RANDAM-2, is one of the neuronal cell lineage-specific antigens involved in the neuronal differentiation of P19 embryonic carcinoma (EC) cells and the mouse central nervous system (CNS). Complementary DNA cloning of RANDAM-2 indicated that its nucleotide sequence completely matched that of PA2.26 antigen, a sialomucin-like transmembrane glycoprotein previously found on tumorigenic keratinocytes. RANDAM-2 transcripts were detectable from the embryonic stage of 6.5 days, and then the expression continued throughout the remaining embryonic stages and adulthood, with a localization restricted to the CNS. In growth factor-induced neurospheres and adult cerebrum, RANDAM-2-expressing cells coincided well not only with nestin-positive cells but also with glutamate-positive neurons, but not with gamma-aminobutyric acid-positive ones. These results indicate that RANDAM-2 is one of the type I membrane surface antigens constitutively expressed on undifferentiated neuronal cells and the glutamatergic neuronal cells during mouse neurogenesis.

In vivo differentiation of stem cells in the aorta-gonad-mesonephros region of mouse embryo and adult bone marrow.

OBJECTIVE: Hematopoietic stem cells (HSCs) are thought to be generated from hemangioblasts, the common precursor cells for blood and endothelial cells, in the aorta-gonad-mesonephros (AGM) region of the mouse embryo. The genetic program of HSCs was recently demonstrated to be plastic, but the potential for AGM-region hemangioblasts to be transplanted and to differentiate in vivo has not been well described. Here we examined the fate of donor cells in mice transplanted with CD45(-) AGM cells, which presumably include hemangioblasts. MATERIALS AND METHODS: CD45(-) cells in the AGM region of embryos at 11.5 days post coitum or CD45(+)CD34(-) side population (SP) of cells in adult bone marrow (BM) derived from enhanced green fluorescent protein transgenic mice were transplanted into the liver of busulfan-treated neonatal mice. Two to 6 months after injection of the cells, the contribution of donor-derived cells in the hematopoietic compartment and in various organs was analyzed by flow cytometry and confocal microscopy. RESULTS: CD45(-) cells from the AGM region not only generated peripheral blood cells but also differentiated into endothelial and other nonhematopoietic cells in liver, kidney, lung, small intestine, and uterus in transplanted mice. A similar engrafting pattern was observed in the small intestine of mice transplanted with BM SP/CD45(+) cells, secondary BM-transplanted mice, and lethally irradiated adult mice that received intravenous injections of BM cells. CONCLUSION: A CD45(-) fraction of the AGM region and CD45(+) BM stem cells share the same in vivo potential to differentiate into hematopoietic, endothelial, smooth muscle, and stroma-like cells when transplanted in mice.