Diphtheria Toxin Receptor Research Articles

Heparin-binding EGF-like growth factor: mechanisms of biological activity and potential therapeutic applications

The diphtheria toxin receptor on sensitive mammalian cells is known as the membrane anchored precursor of heparin-binding EGF-like growth factor (HB-EGF). When the precursor is cleaved by metalloproteinases, a soluble form (sHB-EGF) is formed that can bind to the EGF receptors, resulting in activation of signaling pathways that regulate cell proliferation, differentiation, migration, and inhibition of apoptosis. The ability of HB-EGF to cause both positive and negative consequences for organism underscores the complexity of its biological functions and the need for a nuanced understanding of its role in health and disease. In this review the data on the HB-EGF structure, biological activity, involvement in the mechanism of diphtheria toxin action, wound healing, tumor progression as well as the methods of HB-EGF delivery are summarized. Keywords: cell proliferation, diphtheria toxin, EGF receptor, heparin-binding EGF-like growth factor, signal transduction, wound healing

MrgprA3+ Primary Sensory Neurons Mediate Acute Allergic Itch Responses in Atopic Dermatitis Model Mice.

Itch is a prominent symptom of atopic dermatitis (AD). However, the underlying mechanism remains complex and has not yet been fully elucidated. Mas-related G protein-coupled receptor A3 (MrgprA3) has emerged attention as a marker of primary sensory neurons that specifically transmit itch signals; however, its involvement in AD-related itch has not been extensively explored. In this study, we developed an AD itch mouse model by repeatedly applying house dust mite (HDM) extract to barrier-impaired skin via a special diet. To clarify the role of MrgprA3+ neurons in itch behavior in our AD model, we adopted a toxin receptor-mediated cell knockout strategy using transgenic mice in which the diphtheria toxin receptor (DTR) gene was placed under the control of the Mrgpra3 promoter. When the HDM extract was repeatedly applied to the face and back skin of special diet-fed mice, the mice exhibited AD-like dry and eczematous skin lesions accompanied by three types of itch-related behaviors:1) spontaneous scratching, 2) acute scratching after antigen challenge, and 3) light touch-evoked scratching. Upon diphtheria toxin administration, substantial depletion of DTR+/MrgprA3+ neurons was observed in the dorsal root ganglion. Ablation of MrgprA3+ neurons suppressed acute itch responses after HDM application, whereas spontaneous and touch-evoked itch behaviors remained unaffected. Our findings unequivocally demonstrate that in our AD model, MrgprA3+ primary sensory neurons mediate acute allergic itch responses, whereas these neurons are not involved in spontaneous itch or alloknesis.

Elimination of physiological senescent cutaneous cells in a novel p16-dependent senolytic mouse model impacts lipid metabolism in skin aging.

The evidence of the correlation between cellular senescence and aging has increased in research with animal models. These models have been intentionally generated to target and regulate cellular senescent cells with the promoter activity of p16Ink4a or p19Arf, genes that are highly expressed in aging cells. However, the senolytic efficiency in various organs and cells from these models represents unexpected variation and diversity in some cases. We have generated a novel knock-in model, p16tdT-hDTR mice, which possess tdTomato and human diphtheria toxin receptor (hDTR) downstream of Cdkn2a, an endogenous p16Ink4a gene. We successfully demonstrated that p16-derived tdTomato and hDTR expressions are observed in these mouse embryo fibroblasts and following treatment with diphtheria toxin (DT) eliminates those cells. Furthermore, we demonstrated the efficacy of eliminating p16-positive cells in vivo, and also observed a tendency to decrease their cutaneous SA-β-gal activity after subcutaneous DT injection into p16tdT-hDTR mice. In particular, comprehensive gene expression analysis in skin revealed that upregulated genes related to lipid metabolisms with aging exhibited remarkable expressions under the senolysis. These results clearly unveiled p16-positive senescent cells contribute to age-related changes in skin.

Presence of fetal microchimerisms in the heart and effect on cardiac repair.

Multiple complex biological processes take place during pregnancy, including the migration of fetal cells to maternal circulation and their subsequent engraftment in maternal tissues, where they form microchimerisms. Fetal microchimerisms have been identified in several tissues; nevertheless, their functional role remains largely unknown. Different reports suggest these cells contribute to tissue repair and modulate the immune response, but they have also been associated with pre-eclampsia and tumor formation. In the maternal heart, cells of fetal origin can contribute to different cell lineages after myocardial infarction. However, the functional role of these cells and their effect on cardiac function and repair are unknown. In this work, we found that microchimerisms of fetal origin are present in the maternal circulation and graft in the heart. To determine their functional role, WT female mice were crossed with male mice expressing the diphtheria toxin (DT) receptor. Mothers were treated with DT to eliminate microchimerisms and the response to myocardial infarction was investigated. We found that removal of microchimerisms improved cardiac contraction in postpartum and post-infarction model females compared to untreated mice, where DT administration had no significant effects. These results suggest that microchimerisms play a detrimental role in the mother following myocardial infarction.

Establishment and characterization of salivary gland-specific injury in transgenic mice model.

Many mouse models for autoimmune diseases also have lesions in non-target organs, which may make it difficult to determine whether the target organ lesion is primary or secondary. Hyposalivation has conventionally been studied using genetically modified mouse models for Sjogren's syndrome as well as spontaneous autoimmune mice with systemic lesions, none of which has salivary gland-specific injury. In this study, we established a salivary gland-specific injury mouse model using the TRECK system by gene modification with the transgene composed of the 5' untranslated region of human salivary mucin gene MUC7 (highly expressed specifically in human salivary gland) inserted at the upstream of hHB-EGF (diphtheria toxin receptor) in the TRECK vector. In this transgenic mouse model, we confirmed salivary gland-specific expression of hHB-EGF gene, and hyposalivation after treatment with diphtheria toxin. Histological assessment of the salivary gland from these mice showed granular convoluted tubule epithelial cells destruction at the same position as a positivity in TUNEL assay. This transgenic mouse model may become a useful tool for elucidating the mechanisms involved in hyposalivation and for developing pharmaceuticals and tissue regenerative medical products for this condition.

Toll-Like Receptors Mediate Opposing Dendritic Cell Effects on Treg/Th17 Balance in Mice With Intracerebral Hemorrhage.

Dendritic cells (DCs) regulate the immune response associated with T lymphocytes, but their role in stroke remains unclear. In this study, we investigated the causal relationship between DCs and T-cell response in intracerebral hemorrhage (ICH) by focusing on TLRs (toll-like receptors) that may modulate the function of DCs. We studied the effects of TLR4, TLR2, and TLR9 on DC-mediated T-cell response and the outcomes of ICH using male C57BL/6 and CD11c-DTx (diphtheria toxin) receptor mice. We administered specific agents intraperitoneally or orally and evaluated the results using flow cytometry, real-time polymerase chain reaction, Western blotting, immunofluorescence staining, histopathology, and behavioral tests. TLR4 and TLR2 activation induces DC maturation and reduces the ratio of regulatory T to T-helper 17 cells in the brain and periphery after ICH. When either of these receptors is activated, it can worsen neuroinflammation and exacerbate ICH outcomes. TLR9 also promotes DC maturation, stabilizing the number of DCs, particularly conventional DCs. TLR9 has the opposite effects on regulatory T/T-helper 17 balance, neuroinflammation, and ICH outcomes compared with TLR4 and TLR2. Upon stimulation, TLR4 and TLR9 may achieve these effects through the p38-MAPK (p38-mitogen-activated protein kinase)/MyD88 (myeloid differentiation primary response gene 88) and indoleamine 2,3-dioxygenase 1 (IDO1)/GCN2 (general control nonderepressible 2) signaling pathways, respectively. DCs act as intermediaries for TLR-mediated T-cell response. TLR-mediated opposing effects of DCs on T-cell response may provide novel strategies to treat ICH.

Abstract PO4-25-02: Long-range Deployment of Tumor-antigen Specific Cytotoxic T Lymphocytes Inhibits Lung Metastasis of Breast Cancer

Abstract Effector Immune Cell Deployment (EICD) refers to systemic deployment of anti-tumor effector immune cells in cancer patients, including the priming, circulation, trafficking, activity and fate of the immunocytes, and is a panorama to reflect the generation, distribution and development of anti-tumor immunity. Tumor antigen-specific T cells are the main component of effector immune cells in anti-tumor immune responses, but their systemic deployment in breast cancer patients and the underlying mechanisms remain largely unknown. Here, we identified a cluster of CD8+ T cell exhibiting tissue-resident memory (TRM) phenotype in the tumor-draining lymph nodes (TDLNs) of 487 breast cancer patients, whose abundancy specifically predicts improved lung, but not other target organ metastasis-free survival. Also, high lung CD8+ TRM infiltration is associated with lower metastatic burden in breast cancer patients. Using single-cell RNA sequencing, we observed that in multiple mouse cancer models, CD8+TRM accumulate at early tumor stages when lung metastasis was not identified. Functionally, the CD8+TRM isolated from pre-metastatic lungs of the animals were tumor antigen specific and cytotoxic to the tumor cells. Nevertheless, the abundancy of CD8+TRM in the lungs were dramatically decreased in the lungs upon metastasis establishment. Moreover, in Cd8cre/+ Itgaeloxp/+-diphtheria toxin receptor conditional knockout mice, we unambiguously demonstrated that specific depletion of the CD8+ TRM subset facilitates lung metastasis in vivo, while adoptive transfer of CD8+ TRM successfully inhibited lung metastasis and prolonged survival of the mice. Mechanistically, using cell-tracing techniques in the photo-convertible Kaede-Tg mice, we found that tumor-specific CD8+ TRM were generated in the TDLNs of the mice and were recruited to the lungs via CCL25/CCR9 signaling axis. However, the circulating exosomes secreted by primary tumor cells were taken up by alveolar macrophages and polarized them to release IDO1 and impaired anti-tumor T cell immunity, facilitating lung metastasis. More importantly, inhibition of IDO1 effectively retrieves TRM-mediated protection against lung metastasis. Collectively, we have revealed a cross-talk between tumor cells and the inflammatory environment of distant organs, which could drive lung metastasis by impairing EICD. Our findings also highlight the therapeutic potential of orchestrating EICD in turning “cold” metastatic tumor foci to “hot” ones. Citation Format: Yue Xing, Shicheng Su, Erwei Song. Long-range Deployment of Tumor-antigen Specific Cytotoxic T Lymphocytes Inhibits Lung Metastasis of Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-25-02.

Co-regulatory Proteins Integrate Metabolic Signals to Regulate ESRRG Transcriptional Transactivation in Stomach Parietal Cell Differentiation

Acid-producing stomach parietal cells (PC) are metabolically active and energy demanding cells. Aberrant PC function and census leads to complications ranging from peptic ulcers to stomach adenocarcinoma. Effcient energy expenditure and catabolic metabolism are required for normal PC differentiation from progenitor cells. Here, our data show that the master metabolic transcriptional factor, estrogen-related receptor gamma ( ESRRG), governs murine and human PC differentiation. We are further investigating how known metabolically controlled co-regulatory proteins — Sirtuin 1 (SIRT1), Nuclear Receptor Subfamily 0 Group B Member 2 (NR0B2), and CREB/ATF BZIP Transcription Factor (CREBZF, or SMILE) — integrate stimuli to enable ESRRG transcriptional activation during PC emergence from precursors. Specifically, our lab has developed an acute and reversible PC ablation model to monitor PC recovery in which Simian diphtheria toxin receptor (DTR) expression under the Atp4b promoter targets PC-specific ablation by diphtheria toxin (DT). Additionally, we employ high-dose tamoxifen (HD TAM), which causes a more general gastric injury (independent of tamoxifen effects on estrogen) from PC death along with reactive spasmolytic polypeptide expressing metaplasia (SPEM) of remaining cells. Immunofluorescence, western blotting, and qRT-PCR revealed SIRT1 is upregulated before emergence of early PC clones post-DT and decreases later, suggesting positive regulation of early PC lineage-commitment from stem cells. During the same early time frame, factors NR0B2 and CREBZF decreased, suggesting contrasting roles. Administration of Resv to mice accelerated PC recovery after injury and expanded PCs even at homeostasis. ChIP and ongoing ChIP-seq and Cut&Run assays show ESRRG binds cis-regulatory elements of the metabolic genes and the co-regulatory proteins to promote PC lineage commitment in both mice and in a human gastric cell line in which stable ESRRG-GFP is expressed under a doxycycline-inducible promoter. In other ongoing work, we are generating mice with PC-specific Sirt1/ Nr0b2 ablation to further dissect PC regeneration dynamics and conducting gastric organoid experiments with NR0B2/SIRT1 pharmacological modulators to monitor PC differentiation effects. These organoid samples will be further subjected to epigenetic analysis (Cut&Run, ATACseq) to study chromatin landscape changes. We conclude that cellular metabolic integration by co-regulators enables ESRRG-mediated transactivation during PC differentiation, and SIRT1 promotes early PC lineage-commitment that might offer a promising pro-PC differentiation therapeutic strategy. This work is supported by funding from the NIH through the National Institute of Diabetes and Digestive and Kidney Diseases and the National Cancer Institute. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Targeted depletion of PD-1-expressing cells induces immune tolerance through peripheral clonal deletion.

Thymic negative selection of the T cell receptor (TCR) repertoire is essential for establishing self-tolerance and acquired allograft tolerance following organ transplantation. However, it is unclear whether and how peripheral clonal deletion of alloreactive T cells induces transplantation tolerance. Here, we establish that programmed cell death protein 1 (PD-1) is a hallmark of alloreactive T cells and is associated with clonal expansion after alloantigen encounter. Moreover, we found that diphtheria toxin receptor (DTR)-mediated ablation of PD-1+ cells reshaped the TCR repertoire through peripheral clonal deletion of alloreactive T cells and promoted tolerance in mouse transplantation models. In addition, by using PD-1-specific depleting antibodies, we found that antibody-mediated depletion of PD-1+ cells prevented heart transplant rejection and the development of experimental autoimmune encephalomyelitis (EAE) in humanized PD-1 mice. Thus, these data suggest that PD-1 is an attractive target for peripheral clonal deletion and induction of immune tolerance.

A p21-ATD mouse model for monitoring and eliminating senescent cells and its application in liver regeneration post injury

Cellular senescence associates with pathological aging and tissue dysfunctions. Studies utilizing mouse models for cell lineage tracings have emphasized the importance of senescence heterogeneity in different organs and cell types. Here, we constructed a p21- [Akaluc - tdTomato - Diphtheria Toxin Receptor (DTR)] (ATD) mouse model to specifically study the undefined mechanism for p21-expressing senescent cells in the aged and liver injury animals. The successful expressions of these genes enabled in vitro flow cytometric sorting, in vivo tracing and eliminating of p21-expressing senescent cells. During the natural aging process, p21-expressing cells were found in various tissues of p21-ATD mice. Eliminating p21-expressing cells in the aged p21-ATD mice recovered their multiple biological functions. p21-ATD/Fah-/- mice, bred from p21-ATD mice and fumarylacetoacetate hydrolase (Fah)-/- mice of liver injury, showed that the majority of their senescent hepatocytes were the phenotype of p21+ rather than p16+. Furthermore, eliminating the p21-expressing hepatocytes significantly promoted the engraftment of grafted hepatocytes and facilitated the liver repopulation, resulting in the significant recovery from liver injury. Our p21-ATD mouse model serves as an optimal model for studying the pattern and function of p21-expressing senescent cells under the physical and pathological conditions during aging.

Generation of a human embryonic stem cell line (SMUDHe010-A-1B) carrying inducible DTA expression cassette in the AAVS1 locus by CRISPR/Cas9-mediated homologous recombination

Diphtheria toxin A (DTA) is an exotoxin secreted by Corynebacterium diphtheriae. After entering the cell through receptor-mediated manner, DTA can trigger the programmed cell death mechanism and lead to cell death. In 2001, Michiko Saito established a Diphtheria toxin receptor–mediated cell knockout system, which can conditional deplete specific cell type in transgenic mice. This system is not only very useful in the pathogenesis study of human diseases, but also has a wide application prospect in the study of organ development and regeneration. In 2008, David Voehringer described a newly generated mouse strain that encodes DTA under control of a loxP-flanked stop cassette in the ubiquitously expressed ROSA26 locus. Thereby, it can be used in combination with tissue-specific and/or inducible Cre-expressing mouse strains to achieve toxin-mediated cell ablation in vivo. The application of DTA–mediated cell knockout system in mice has been widely reported, but it has rarely been used in human cells. Accordingly, we generated a human embryonic stem cell line (SMUDHe010-A-1B) carrying inducible DTA expression cassette (loxp-stop-loxp-DTA, LSL-DTA) using CRISPR/Cas9-mediated homologous recombination. The cell line preserves normal karyotype, pluripotency and the ability to differentiate into all three germ layers. Moreover, the cell line can be used to prepare human organoid, which may provide a model for achieving conditional cell ablation in human tissues and organs.

Characterization of a new mouse line triggering transient oligodendrocyte progenitor depletion

Oligodendrocyte progenitor cells (OPC) are the main proliferative cells in the healthy adult brain. They produce new myelinating oligodendrocytes to ensure physiological myelin remodeling and regeneration after various pathological insults. Growing evidence suggests that OPC have other functions. Here, we aimed to develop an experimental model that allows the specific ablation of OPC at the adult stage to unravel possible new functions. We generated a transgenic mouse expressing a floxed human diphtheria toxin receptor under the control of the PDGFRa promoter, crossed with an Olig2Cre mouse to limit the recombination to the oligodendrocyte lineage in the central nervous system. We determined a diphtheria toxin dose to substantially decrease OPC density in the cortex and the corpus callosum without triggering side toxicity after a few daily injections. OPC density was normalized 7 days post-treatment, showing high repopulation capacity from few surviving OPC. We took advantage of this strong but transient depletion to show that OPC loss was associated with behavioral impairment, which was restored by OPC recovery, as well as disruption of the excitation/inhibition balance in the sensorimotor cortex, reinforcing the hypothesis of a neuromodulatory role of OPC in the adult brain.

Invited Session II: Retinal remodeling and regeneration: Partial cone loss triggers differential modification of inhibition across retinal pathways.

Cone loss up to 40-60% can be undetected by visual acuity or sensitivity metrics. These findings suggest that conventional diagnostics can be insensitive to cone loss below a threshold and/or that visual circuits can be resilient to cone loss up to a threshold. To distinguish between these possibilities, our lab studied the effects of partial cone loss in the mature mouse retina. We induced controlled cone loss by selectively expressing the diphtheria toxin receptor under the cone opsin promoter (cone-DTR). We examined the effects of 50% cone loss on the three of the most sensitive cell types in mouse retina: alpha ON sustained (AON-S), OFF sustained (AOFF-S), and OFF transient (AOFF-T) ganglion cells. With multiple cell types, we can discern between effects common across pathways, i.e., changes to common circuit components, and effects unique to pathways, i.e., changes to circuit components after the pathways diverge to each cell type. In AON-S ganglion cells, partial cone loss triggers inhibition to increase spatiotemporal integration, recover contrast gain, and receive increased synaptic inputs. While OFF pathways also exhibit modified spatiotemporal processing with fewer cones, the extent of functional adjustments was unique between the AOFF-S and AOFF-T. Cone loss caused differential modifications to inhibition in each of these retinal pathways. These findings demonstrate that partial cone loss induced circuitry changes after the divergence of OFF retinal pathways.

Abstract 16783: Discovery and Characterization of Transient Endothelial Stem-Like Cells Responsible for Rapid Lung Microvascular Regeneration Post Endothelial Ablation

Background: Single-cell RNA sequencing (scRNA-seq) has revealed two main types of alveolar capillary endothelial cells (ECs): aCap ECs (or aerocytes) express typical aCap markers, including apelin, whereas general (gCap) ECs express apelin receptor, Aplnr /APJ. Aerocytes constitute the air-blood barrier and are incapable of proliferation, unlike smaller gCap ECs that can proliferate. Hypothesis: After lung microvascular injury, gCap ECs give rise to stem-like ECs that can regenerate depleted lung EC populations, including highly specialized aerocytes. Approach: scRNA-seq was used to study depletion and regeneration of lung EC populations after intratracheal instillation of diphtheria toxin (DT) in transgenic mice harboring an EC-targeted human DT receptor. Results: DT resulted in ~80% loss of lung microvascular EC populations including aerocytes, associated with severe acute lung injury (ALI), and there was full spontaneous recovery by day 7. Interestingly, a novel EC population appeared at day 3 post injury in a transitional gCap EC cluster characterized by paradoxical expression of ‘aCap’ marker, apelin, together with endothelial progenitor/stem cell markers, Cd34 and endothelial protein C receptor ( Procr /EPCR). These ‘transient endothelial stem-like cells’ (TESCs) transitioned to highly proliferative progenitor-like FoxM1 + cells ultimately repopulating all depleted EC populations by day 7. Using flow cytometry of lung cells harvested at 3 days post DT instillation, TESCs were identified by unique co-expression of CD34 and EPCR (Figure), thereby allowing isolation and culture in vitro for further characterization of their proliferation and differentiation potential. Conclusions: Lung microvascular repair is orchestrated by the emergence of stem-like, gCap ECs (TESCs), transiently expressing aCap/aerocyte marker, apelin, together with Procr /EPCR, which have the capacity to regenerate lung microvasculature rapidly and efficiently.

Macrophage regulation of the "second brain": CD163 intestinal macrophages interact with inhibitory interneurons to regulate colonic motility - evidence from the Cx3cr1-Dtr rat model.

Intestinal macrophages are well-studied for their conventional roles in the immune response against pathogens and protecting the gut from chronic inflammation. However, these macrophages may also have additional functional roles in gastrointestinal motility under typical conditions. This is likely to occur via both direct and indirect influences on gastrointestinal motility through interaction with myenteric neurons that contribute to the gut-brain axis, but this mechanism is yet to be properly characterised. The CX3CR1 chemokine receptor is expressed in the majority of intestinal macrophages, so we used a conditional knockout Cx3cr1-Dtr (diphtheria toxin receptor) rat model to transiently ablate these cells. We then utilized ex vivo video imaging to evaluate colonic motility. Our previous studies in brain suggested that Cx3cr1-expressing cells repopulate by 7 days after depletion in this model, so we performed our experiments at both the 48 hr (macrophage depletion) and 7-day (macrophage repopulation) time points. We also investigated whether inhibitory neuronal input driven by nitric oxide from the enteric nervous system is required for the regulation of colonic motility by intestinal macrophages. Our results demonstrated that CD163-positive resident intestinal macrophages are important in regulating colonic motility in the absence of this major inhibitory neuronal input. In addition, we show that intestinal macrophages are indispensable in maintaining a healthy intestinal structure. Our study provides a novel understanding of the interplay between the enteric nervous system and intestinal macrophages in colonic motility. We highlight intestinal macrophages as a potential therapeutic target for gastrointestinal motility disorders when inhibitory neuronal input is suppressed.

Depletion of Neural Crest-Derived Cells Leads to Plasma Noradrenaline Decrease and Alters T Cell Development.

The differentiation of neural crest (NC) cells into various cell lineages contributes to the formation of many organs, including the thymus. In this study, we explored the role of NC cells in thymic T cell development. In double-transgenic mice expressing NC-specific Cre and the Cre-driven diphtheria toxin receptor, plasma noradrenaline and adrenaline levels were significantly reduced, as were thymic T cell progenitors, when NC-derived cells were ablated with short-term administration of diphtheria toxin. Additionally, yellow fluorescent protein+ NC-derived mesenchymal cells, perivascular cells, and tyrosine hydroxylase+ sympathetic nerves in the thymus significantly decreased. Furthermore, i.p. administration of 6-hydroxydopamine, a known neurotoxin for noradrenergic neurons, resulted in a significant decrease in thymic tyrosine hydroxylase+ nerves, a phenotype similar to that of depleted NC-derived cells, whereas administration of a noradrenaline precursor for ablating NC-derived cells or sympathetic nerves rarely rescued this phenotype. To clarify the role of NC-derived cells in the adult thymus, we transplanted thymus into the renal capsules of wild-type mice and observed abnormal T cell development in lethally irradiated thymus with ablation of NC-derived cells or sympathetic nerves, suggesting that NC-derived cells inside and outside of the thymus contribute to T cell development. In particular, the ablation of NC-derived mesenchymal cells in the thymus decreases the number of thymocytes and T cell progenitors. Overall, ablation of NC-derived cells, including sympathetic nerves, in the thymus leads to abnormal T cell development in part by lowering plasma noradrenalin levels. This study reveals that NC-derived cells including mesenchymal cells and sympathetic nerves within thymus regulate T cell development.

Prefrontal cortex astroglia modulate anhedonia-like behavior

Reductions of astroglia expressing glial fibrillary acidic protein (GFAP) are consistently found in the prefrontal cortex (PFC) of patients with depression and in rodent chronic stress models. Here, we examine the consequences of PFC GFAP+ cell depletion and cell activity enhancement on depressive-like behaviors in rodents. Using viral expression of diphtheria toxin receptor in PFC GFAP+ cells, which allows experimental depletion of these cells following diphtheria toxin administration, we demonstrated that PFC GFAP+ cell depletion induced anhedonia-like behavior within 2 days and lasting up to 8 days, but no anxiety-like deficits. Conversely, activating PFC GFAP+ cell activity for 3 weeks using designer receptor exclusively activated by designer drugs (DREADDs) reversed chronic restraint stress-induced anhedonia-like deficits, but not anxiety-like deficits. Our results highlight a critical role of cortical astroglia in the development of anhedonia and further support the idea of targeting astroglia for the treatment of depression.

196-OR: The Long-Chain Fatty-Acid Receptor FFA4 Stimulates Insulin Secretion via Inhibition of Somatostatin Release from the Delta Cell

The long-chain fatty-acid receptor FFA4 exerts beneficial effects on glucose homeostasis and insulin secretion and is considered a potential therapeutic target for type 2 diabetes. FFA4 is expressed in islets, but its precise mechanism of action remains unknown. Previous studies from our group suggest that FFA4 is expressed in delta cells and regulates somatostatin secretion. The objective of this study was to test the hypothesis that FFA4 agonists indirectly stimulate insulin secretion via inhibition of somatostatin release. In 1-h static incubations of isolated, wild-type mouse islets, the FFA4 agonist CpdA dose-dependently potentiated glucose-induced insulin secretion from 2.2±0.2 to 3.4±0.2 % of insulin content at the concentration of 50 μM (n=8; p<0.001), and simultaneously decreased somatostatin secretion from 28.4±2.1 to 10.5±0.9 pM (n=8; p<0.0001). No effect of CpdA on insulin or somatostatin secretion was observed in islets from mice that do not express somatostatin (insulin: 3.2±0.1 vs 2.9±0.4, n=6, NS; somatostatin: 11.4±1.2 vs 11.9±1.2, n=6, NS). No sex differences were observed. Likewise, in delta-cell deficient islets isolated from diphtheria toxin-treated male mice expressing the diphtheria toxin receptor under the somatostatin promoter, no effect of CpdA was observed on insulin or somatostatin release (insulin: 4.9±0.9 vs 4.1±0.8, n=3, NS; somatostatin: 10.1±4.9 vs 9.2±5.3, n=6, NS). We conclude that FFA4 stimulation of insulin secretion is exclusively mediated by inhibition of somatostatin secretion from delta cells. Disclosure L.Reininger: None. M.Ethier: None. C.Tremblay: None. J.Ghislain: None. M.Huising: Consultant; AstraZeneca, Research Support; Crinetics Pharmaceuticals, Inc. V.Poitout: Consultant; AstraZeneca. M.Flisher: None. Funding National Institutes of Health (R01DK132597)

Ablation of Projection Glutamatergic Neurons in the Lateral Cerebellar Nuclei Alters Motor Coordination in Vglut2-Cre+ Mice.

Cerebellar nuclei (CN) constitute the sole cerebellar output to the rest of the central nervous system and play a central role in cerebellar circuits. Accumulating evidence from both human genetics and animal studies point to a crucial role for CN connectivity in neurological diseases, including several types of ataxia. However, because of the compact and restricted topography and close functional connection between the CN and the cerebellar cortex, identifying cerebellar deficits exclusively linked to CN is challenging. In this study, we have experimentally ablated large projection glutamatergic neurons of the lateral CN and evaluated the impact of this selective manipulation on motor coordination in mice. To this end, through stereotaxic surgery, we injected the lateral CN of Vglut2-Cre+ mice with an adeno-associated virus (AAV) encoding a Cre-dependent diphtheria toxin receptor (DTR), followed by an intraperitoneal injection of diphtheria toxin (DT) to ablate the glutamatergic neurons of the lateral nucleus. Double immunostaining of cerebellar sections with anti-SMI32 and -GFP antibodies revealed GFP expression and provided evidence of SMI32+ neuron degeneration at the site of AAV injection in the lateral nucleus of Vglut2-Cre+ mice. No changes were observed in Vglut2-Cre negative mice. Analysis of motor coordination by rotarod test indicated that the latency to fall was significantly different before and after AAV/DT injection in the Vglut2-Cre+ group. Elapsed time and number of steps in the beam walking test were significantly higher in AAV/DT injected Vglut2-Cre+ AAV/DT mice compared to controls. We demonstrate for the first time that partial degeneration of glutamatergic neurons in the lateral CN is sufficient to induce an ataxic phenotype.

Breast metastatic tumors in lung can be substituted by lung-derived malignant cells transformed by alternative splicing H19 lncRNA

Metastasis accounts for most cancer-associated deaths; yet, this complex process remains poorly understood, particularly the relationship between distant metastasis and primary site-derived cells. Here, we modified the classical MMTV–PyMT breast carcinoma model to trace the fate of mammary-derived carcinoma cells. We show that within the lung, when the metastatic breast carcinoma cells are conditionally depleted, transformed lung epithelial cells generate new metastases. Metastatic breast carcinoma cells transmit H19 long noncoding (lnc) RNA to lung epithelial cells through exosomes. SF3B1 bearing mutations at arginine-625 alternatively splices H19 lncRNA in lung epithelial cells, which selectively acts like a molecular sponge to sequester let-7a and induces Myc upregulation. Under the conditional elimination of primary site-derived breast carcinoma cells, lung malignant cells expressing the mutated SF3B1 splice variant dominate the newly created tumors. Our study suggests that these new carcinoma cells originating from within the colonized organ can replace the primary site-derived malignant cells whenever their expansion is abrogated using an inducible diphtheria toxin receptor in our designed system. These findings should call for a better understanding of metastatic tumors with the specific origin during cancer metastasis.