Expressing Cre Recombinase Research Articles

Lsh/HELLS is required for B lymphocyte development and immunoglobulin class switch recombination

Mutation of HELLS (Helicase, Lymphoid-Specific)/Lsh in human DNA causes a severe immunodeficiency syndrome, but the nature of the defect remains unknown. We assessed here the role of Lsh in hematopoiesis using conditional Lsh knockout mice with expression of Mx1 or Vav Cre-recombinase. Bone marrow transplantation studies revealed that Lsh depletion in hematopoietic stem cells severely reduced B cell numbers and impaired B cell development in a hematopoietic cell-autonomous manner. Lsh-deficient mice without bone marrow transplantation exhibited lower Ig levels in vivo compared to controls despite normal peripheral B cell numbers. Purified B lymphocytes proliferated normally but produced less immunoglobulins in response to in vitro stimulation, indicating a reduced capacity to undergo class switch recombination (CSR). Analysis of germline transcripts, examination of double-stranded breaks using biotin-labeling DNA break assay, and End-seq analysis indicated that the initiation of the recombination process was unscathed. In contrast, digestion-circularization PCR analysis and high-throughput sequencing analyses of CSR junctions and a chromosomal break repair assay indicated an impaired ability of the canonical end-joining pathway in Lsh-deficient B cells. Our data suggest a hematopoietic cell-intrinsic role of Lsh in B cell development and in CSR providing a potential target for immunodeficiency therapy.

Notch activation in the mouse mammary luminal lineage leads to ductal hyperplasia and altered partitioning of luminal cell subtypes

Hyperactivated Notch signalling has been implicated in breast cancer, but how elevated levels of Notch signalling contribute to mammary dysplasia and tumorigenesis is not fully understood. In this study, we express an activated form of Notch1 in the mouse mammary luminal lineage and analyse the consequences for tumour formation and the transcriptomic landscape in the luminal lineage. Simultaneous conditional activation of a Notch1 intracellular domain (Notch1 ICD) and EGFP in the luminal lineage was achieved by removal of a stop cassette by CRE-recombinase expression from the whey acidic protein (WAP) promoter. Mice in which Notch1 ICD was activated in the luminal lineage (WAP-CRE;R26-N1ICD mice) exhibit ductal hyperplasia after lactation with an increase in branching frequency and in the number of side-branch ends in the ductal tree. A subset of the mice developed mammary tumours and the majority of the tumour cells expressed EGFP (as a proxy for Notch1 ICD), indicating that the tumours originate from the Notch1 ICD-expressing cells. Single-cell transcriptome analysis of the EGFP-positive mammary cells identified six subtypes of luminal cells. The same six subtypes were found in control mice (WAP-CRE;R26-tdTomato mice expressing the tdTomato reporter from WAP-CRE-mediated activation), but the proportion of cells in the various subtypes differed between the WAP-CRE;R26-N1ICD and control WAP-CRE;R26-tdTomato mice. In conclusion, we show that Notch1 ICD expression in the luminal lineage produces a ductal hyperplasia and branching phenotype accompanied by altered luminal cell subtype partitioning.

Slc6a3-dependent expression of a CAPS-associated Nlrp3 allele results in progressive behavioral abnormalities and neuroinflammation in aging mice

BackgroundAn association between neuroinflammation and age-related neurologic disorders has been established but the molecular mechanisms and cell types involved have not been thoroughly characterized. Activity of the proinflammatory NLRP3 inflammasome is implicated in Alzheimer’s and Parkinson’s disease and our recent studies in patients suggest that dopaminergic neurons within the degenerating mesencephalon express NLRP3 throughout the progression of PD. Here, we directly test the impact of enhanced inflammasome activity in mesencephalic neurons by characterizing motor function, tissue integrity, and neuroinflammation in aging mice harboring hyperactivating mutations within the endogenous murine Nlrp3 locus, enabled only in cells expressing the dopaminergic neuron-specific Slc6a3 promoter.MethodsWe compared mice harboring inducible alleles encoding the cryopyrin-associated periodic syndrome activating mutations Nlrp3A350V and Nlrp3L351P inserted into the endogenous mouse Nlrp3 locus. Tissue specific expression was driven by breeding these animals with mice expressing Cre recombinase under the control of the dopaminergic neuron-specific Slc6a3 promoter. The experimental mice, designed to express hyperactive NLRP3 only when the endogenous mouse Nlrp3 promotor is active in dopaminergic neurons, were analyzed throughout 18 months of aging using longitudinal motor function assessments. Biochemical and histologic analyses of mesencephalic tissues were conducted in 1- and 18-month-old animals.ResultsWe observed progressive and significant deficits in motor function in animals expressing Nlrp3L351P, compared with animals expressing Nlrp3WT and Nlrp3A350V. Age-dependent neuroinflammatory changes in the mesencephalon were noted in all animals. Analysis of GFAP-immunoreactive astrocytes in the substantia nigra revealed a significant increase in astrocyte number in animals expressing Nlrp3L351P compared with Nlrp3WT and Nlrp3A350V. Further analysis of Nlrp3L351P striatal tissues indicated genotype specific gliosis, elevated Il1b expression, and both morphologic and gene expression indicators of proinflammatory A1 astrocytes.ConclusionsDopaminergic neurons have the potential to accumulate NLRP3 inflammasome activators with age, including reactive oxygen species, dopamine metabolites, and misfolded proteins. Results indicate the Nlrp3 locus is active in dopaminergic neurons in aging mice, and that the hyperactive Nlrp3L351P allele can drive neuroinflammatory changes in association with progressive behavioral deficits. Findings suggest neuronal NLRP3 inflammasome activity may contribute to neuroinflammation observed during normal aging and the progression of neurologic disorders.

Generation of a novel mouse strain with fibroblast-specific expression of Cre recombinase

Cell-specific expression of genes offers the possibility to use their promoters to drive expression of Cre-recombinase, thereby allowing for detailed expression analysis using reporter gene systems, cell lineage tracing, conditional gene deletion, and cell ablation. In this context, current data suggest that the integrin α11 subunit has the potential to serve as a fibroblast biomarker in tissue regeneration and pathology, in particular in wound healing and in tissue- and tumor fibrosis. The mesenchyme-restricted expression pattern of integrin α11 thus prompted us to generate a novel ITGA11-driver Cre mouse strain using a ϕC31 integrase-mediated knock-in approach. In this transgenic mouse, the Cre recombinase is driven by regulatory promoter elements within the 3 kb segment of the human ITGA11 gene. β-Galactosidase staining of embryonic tissues obtained from a transgenic ITGA11-Cre mouse line crossed with Rosa 26R reporter mice (ITGA11-Cre;R26R) revealed ITGA11-driven Cre expression and activity in mesenchymal cells in a variety of mesenchymal tissues in a pattern reminiscent of endogenous α11 protein expression in mouse embryos. Interestingly, X-gal staining of mouse embryonic fibroblasts (MEFs) isolated from the ITGA11-Cre;R26R mice indicated heterogeneity in the MEF population. ITGA11-driven Cre activity was shown in approximately 60% of the MEFs, suggesting that the expression of integrin α11 could be exploited for isolation of different fibroblast populations. ITGA11-driven Cre expression was found to be low in adult mouse tissues but was induced in granulation tissue of excisional wounds and in fibrotic hearts following aortic banding. We predict that the ITGA11-Cre transgenic mouse strain described in this report will be a useful tool in matrix research for the deletion of genes in subsets of fibroblasts in the developing mouse and for determining the function of subsets of pro-fibrotic fibroblasts in tissue fibrosis and in different subsets of cancer-associated fibroblasts in the tumor microenvironment.

Liver fibrosis is driven by protease‐activated receptor‐1 expressed by hepatic stellate cells in experimental chronic liver injury

BackgroundBlood coagulation protease activity is proposed to drive hepatic fibrosis through activation of protease‐activated receptors (PARs). Whole‐body PAR‐1 deficiency reduces experimental hepatic fibrosis, and in vitro studies suggest a potential contribution by PAR‐1 expressed by hepatic stellate cells. However, owing to a lack of specific tools, the cell‐specific role of PAR‐1 in experimental hepatic fibrosis has never been formally investigated. Using a novel mouse expressing a conditional PAR‐1 allele, we tested the hypothesis that PAR‐1 expressed by hepatic stellate cells contributes to hepatic fibrosis. MethodsPAR‐1flox/flox mice were crossed with mice expressing Cre recombinase controlled by the lecithin retinol acyltransferase (LRAT) promoter, which induces recombination in hepatic stellate cells. Male PAR‐1flox/flox/LRATCre and PAR‐1flox/flox mice were challenged twice weekly with carbon tetrachloride (CCl4, 1 mL/kg i.p.) for 6 weeks to induce liver fibrosis. ResultsPAR‐1 mRNA levels were reduced (>95%) in hepatic stellate cells isolated from PAR‐1flox/flox/LRATCre mice. Hepatic stellate cell activation was evident in CCl4‐challenged PAR‐1flox/flox mice, indicated by increased α‐smooth muscle actin labeling and induction of several profibrogenic genes. CCl4‐challenged PAR‐1flox/flox mice displayed robust hepatic collagen deposition, indicated by picrosirius red staining and type I collagen immunolabeling. Notably, stellate cell activation and collagen deposition were significantly reduced (>30%) in PAR‐1flox/flox/LRATCre mice. Importantly, the reduction in liver fibrosis was not a consequence of reduced acute CCl4 hepatotoxicity in PAR‐1flox/flox/LRATCre mice. ConclusionsThe results constitute the first direct experimental evidence that PAR‐1 expressed by stellate cells directly promotes their profibrogenic phenotype and hepatic fibrosis in vivo.

Electrophysiological Properties of Genetically Identified Histaminergic Neurons

Histaminergic neurons of the tuberomammillary nucleus (TMN) are important regulators of behavioral and homeostatic processes. Previous work suggested that histaminergic neurons exhibit a characteristic electrophysiological signature, allowing for their identification in brain slice preparations. However, these previous investigations focused on neurons in the ventral subregion of the TMN of rats. Consequently, it remains unclear whether such electrophysiological properties extend to mice, including other subregions of the TMN, and the potential for differences between males and females. To further characterize the electrophysiological properties of histaminergic neurons, we performed whole-cell patch-clamp recordings on transgenic mice expressing Cre recombinase in histidine decarboxylase (HDC)-expressing cells; the sole enzyme for histamine synthesis (Hdc-cre::tdTomato). Despite similarities with the electrophysiological properties reported in rats, we observed considerable variability in mouse HDC neuron passive membrane properties, action potential firing, and intrinsic subthreshold active membrane properties. Overall, the electrophysiological properties of HDC neurons appeared similar across subregions of the TMN, consistent with a lack of topographical organization in this nucleus. Moreover, we found no obvious sex differences in the electrical excitability of HDC neurons. However, our data reveal a diversity in the electrophysiological properties of genetically identified histaminergic neurons from mice not previously appreciated from rat studies. Thus, these data highlight the utility of mouse genetics to target the widespread histaminergic neuronal population within the TMN and support the idea that histaminergic neurons are a heterogeneous neuronal population.

Optogenetic Activation of Type III Taste Cells Modulates Taste Responses.

Studies have suggested that communication between taste cells shapes the gustatory signal before transmission to the brain. To further explore the possibility of intragemmal signal modulation, we adopted an optogenetic approach to stimulate sour-sensitive (Type III) taste cells using mice expressing Cre recombinase under a specific Type III cell promoter, Pkd2l1 (polycystic kidney disease-2-like 1), crossed with mice expressing Cre-dependent channelrhodopsin (ChR2). The application of blue light onto the tongue allowed for the specific stimulation of Type III cells and circumvented the nonspecific effects of chemical stimulation. To understand whether taste modality information is preprocessed in the taste bud before transmission to the sensory nerves, we recorded chorda tympani nerve activity during light and/or chemical tastant application to the tongue. To assess intragemmal modulation, we compared nerve responses to various tastants with or without concurrent light-induced activation of the Type III cells. Our results show that light significantly decreased taste responses to sweet, bitter, salty, and acidic stimuli. On the contrary, the light response was not consistently affected by sweet or bitter stimuli, suggesting that activation of Type II cells does not affect nerve responses to stimuli that activate Type III cells.

296 Recombination efficiency and expression of fibroblast-specific Cre drivers in mouse skin

Cell-type specific genetic recombination is a powerful strategy for advancing understanding of cellular function in genetically-modified mouse models. This approach relies on cell-type specific gene promoters to drive genetic recombination in target cells. Although the utility of many cell-type specific promoters has been well characterized, thorough characterization of fibroblast-specific promoters is lacking. Therefore, we employed the mTmG two-color fluorescent reporter system to examine the genetic recombination efficiency and expression patterns of three fibroblast-specific promoters in mouse skin. mTmG mice express the red fluorescent protein tdTomato (mT) constitutively and ubiquitously. Genetic recombination catalyzed by Cre recombinase silences mT expression and induces expression of enhanced green fluorescent protein (mG). Thus, cells that express Cre recombinase appear green, while all other cells appear red. We generated mTmG mice that express Cre recombinase under the control of promoter elements in three fibroblast-specific genes 1) fibroblast specific protein-1 (FSP-1), 2) type Iα2 collagen (COL1A2), or 3) platelet-derived growth factor receptor-α (PDGFRα). Dorsal skin from mTmG/Cre positive and control mice were analyzed by immunofluorescence microscopy. Surprisingly, mTmG/FSP-1 Cre mice exhibited recombination largely in keratinocytes throughout all the layers of the epidermis and follicular epithelium. Recombination was also observed in scattered fibroblast-like cells in the dermis. mTmG/COL1A2 Cre mice displayed recombination that was specific for dermal cells with fibroblast-like morphology throughout the interstitial dermis and in dermal papillae. mTmG/PDGFRα Cre mice displayed recombination that was similar to that of COL1A2-driven recombination, although the number of positive dermal cells and the intensity of mG expression was approximately five-fold greater. These data provide a foundation for the use of Cre drivers to study fibroblast gene expression in genetically-modified mouse models.

Club Cell Heme Oxygenase-1 Deletion: Effects in Hyperoxia-Exposed Adult Mice.

Thioredoxin reductase-1 (TXNRD1) inhibition activates nuclear factor (erythroid-derived 2)-like 2 (Nrf2) responses and prevents acute lung injury (ALI). Heme oxygenase-1 (HO-1) induction following TXNRD1 inhibition is Nrf2-dependent in airway epithelial (club) cells in vitro. The influence of club cell HO-1 on lung development and lung injury responses is poorly understood. The present studies characterized the effects of hyperoxia on club cell-specific HO-1 knockout (KO) mice. These mice were generated by crossing Hmox1 flox mice with transgenic mice expressing cre recombinase under control of the club cell-specific Scgb1a1 promoter. Baseline analyses of lung architecture and function performed in age-matched adult wild-type and KO mice indicated an increased alveolar size and airway resistance in HO-1 KO mice. In subsequent experiments, adult wild-type and HO-1 KO mice were either continuously exposed to >95% hyperoxia or room air for 72 h or exposed to >95 hyperoxia for 48 h followed by recovery in room air for 48 h. Injury was quantitatively assessed by calculating right lung/body weight ratios (g/kg). Analyses indicated an independent effect of hyperoxia but not genotype on right lung/body weight ratios in both wild-type and HO-1 KO mice. The magnitude of increases in right lung/body weight ratios was similar in mice of both genotypes. In the recovery model, an independent effect of hyperoxia but not genotype was also detected. In contrast to the continuous exposure model, right lung/body weight ratio mice were significantly elevated in HO-1 KO but not wild-type mice. Though club cell HO-1 does not alter hyperoxic sensitivity in adult mice, it significantly influences lung development and resolution of lung injury following acute hyperoxic exposure.

Chronic brain histamine depletion in adult mice induced depression-like behaviours and impaired sleep-wake cycle

Histamine acts as a neurotransmitter to regulate various physiological processes. Brain histamine is synthesized from an essential amino acid histidine in a reaction catalysed by histidine decarboxylase (Hdc). Hdc-positive neurons exist mainly in the tuberomammillary nucleus (TMN) of the posterior hypothalamus and project their axons to the entire brain. Recent studies have reported that a chronic decrease in histamine levels in the adult human brain was observed in several neurological disorders. However, it is poorly understood whether lower histamine levels play a causative role in those disorders. In the present study, we induced chronic histamine deficiency in the brains of adult mice to allow direct interpretation of the relationship between an impaired histaminergic nervous system and the resultant phenotype. To induce chronic brain histamine deficiency starting in adulthood, adeno-associated virus expressing Cre recombinase was microinjected into the TMN of Hdc flox mice (cKO mice) at the age of 8 weeks. Immunohistochemical analysis showed expression of Cre recombinase in the TMN of cKO mice. The reduction of histamine contents with the decreased Hdc expression in cKO brain was also confirmed. Behavioural studies revealed that chronic histamine depletion in cKO mice induced depression-like behaviour, decreased locomotor activity in the home cage, and impaired aversive memory. Sleep analysis showed that cKO mice exhibited a decrease in wakefulness and increase in non-rapid eye movement sleep throughout the day. Taken together, this study clearly demonstrates that chronic histamine depletion in the adult mouse brain plays a causative role in brain dysfunction.

A novel Cre-enabled tetracycline-inducible transgenic system for tissue-specific cytokine expression in the zebrafish: CETI-PIC3.

ABSTRACTMaladaptive signaling by pro-inflammatory cytokines (PICs), such as TNFα, IL1β and IFNɣ, can activate downstream signaling cascades that are implicated in the development and progression of multiple inflammatory diseases. Despite playing critical roles in pathogenesis, the availability of in vivo models in which to model tissue-specific induction of PICs is limited. To bridge this gap, we have developed a novel multi-gene expression system dubbed Cre-enabled and tetracycline-inducible transgenic system for conditional, tissue-specific expression of pro-inflammatory cytokines (CETI-PIC3). This binary transgenic system permits the stoichiometric co-expression of proteins Tumor necrosis factor a (Tnfa), Interleukin-1 beta (Il1b) and Interferon gamma (Ifng1), and H2B-GFP fluorescent reporter in a dose-dependent manner. Furthermore, cytokine misexpression is enabled only in tissue domains that can be defined by Cre recombinase expression. We have validated this system in zebrafish using an insulin:cre line. In doubly transgenic fish, quantitative real-time polymerase chain reaction demonstrated increased expression levels of tnfa, il1b and ifng1 mRNA. Moreover, specific expression in pancreatic β cells was demonstrated by both Tnfa immunofluorescence and GFP fluorescence. Cytokine-overexpressing islets elicited specific responses: β cells exhibited increased expression of genes associated with reactive oxidative species-mediated stress and endoplasmic reticulum stress, surveilling and infiltrating macrophages were increased, and β cell death was promoted. This powerful and versatile model system can be used for modeling, analysis and therapy development of diseases with an underlying inflammatory etiology.This article has an associated First Person interview with the first author of the paper.

254-LB: Characterization of Ghrelin Receptor Expression in Mouse Islets Reveals Pancreatic Polypeptide Cells as a Key Ghrelin Target

Objective: While expression of the ghrelin receptor, GHSR (growth hormone secretagogue receptor), by pancreatic islets has been investigated, data regarding the specific islet cell types that express GHSR are inconsistent and incomplete. For instance, transcriptomic studies indicate marked GHSR expression by somatostatin (SST)-secreting delta cells vs. little to no GHSR expression by alpha cells and beta cells, which contrasts studies suggesting direct actions of ghrelin on GHSRs expressed by alpha cells and beta cells. Also, to our knowledge, GHSR expression by pancreatic polypeptide (PP) cells has not previously been directly investigated. The aims of this study were to determine GHSR expression within the four principal islet endocrine cell types and the effects of ghrelin on circulating PP. Methods: Ghsr-IRES-Cre mice, which express Cre recombinase directed by the GHSR promoter, crossed to Cre-dependent ROSA26-YFP reporter mice were used to histologically identify GHSR expression in islets. Double and triple labeling for YFP-immunoreactivity (IR) with insulin-IR, glucagon-IR, SST-IR, and/or PP-IR was performed to determine GHSR co-expression with islet hormones. Pharmacological manipulations were used to examine in vivo effects of acyl-ghrelin on plasma PP. Results: Eighty-five % of YFP-IR cells expressed PP-IR, 50% expressed SST-IR, and none expressed insulin-IR or glucagon-IR. GHSR antagonist raised plasma PP in overnight-fasted C57BL/6N mice. However, acyl-ghrelin administration had no effect on plasma PP in C57BL/6N mice. Conclusions: These results suggest that within mouse pancreatic islets, GHSR expression occurs most abundantly in PP cells and that pharmacologic blockade of ghrelin action raises plasma PP. Disclosure D. Gupta: None. B.K. Mani: None. K. Shankar: None. S. Osborne-Lawrence: None. N. Metzger: None. J.M. Zigman: Stock/Shareholder; Self; Medtronic. Funding Diana and Richard C. Strauss Professorship in Biomedical Research; Mr. and Mrs. Bruce G. Brookshire Professorship in Medicine; Kent and Jodi Foster Distinguished Chair in Endocrinology; University of Texas Southwestern Medical Center (to J.M.Z.)

51-OR: Loss of Critical ER Chaperone GRP78 Leads to Beta-Cell Death by a JNK Dependent Mechanism

Endoplasmic reticulum (ER) health is an important determinant of pancreatic beta cell function or failure. Adaptive ER stress leads to expansion of beta cell number through proliferation, but decompensated ER stress results in beta cell loss and contributes to both T1D and T2D. Therefore, understanding the mechanisms of ER-stress-induced beta cell death could help identify novel strategies for the prevention or treatment of diabetes. Glucose responsive protein GRP78/HSPA5 is a key ER chaperone. Loss of GRP78 causes cell death in many cell types; including, in our own prior work, the beta cell. To study the mechanism by which GRP78 loss leads to beta cell death we devised an ex vivo model of GRP78 knockdown by transducing Grp78-flox mouse islets with adenovirus expressing Cre recombinase. To study human islets, we used adenovirus expressing shRNA against GRP78. Using these tools, we successfully reduced GRP78 RNA and protein in primary mouse and human islet cells. Loss of GRP78 strongly activated the ATF6, IRE1 and PERK pathways of the unfolded protein response (UPR). GRP78 knockdown decreased beta cell number due to increased cell death as measured by Annexin V and TUNEL staining. Probing the mechanisms of cell loss, we found that loss of GRP78 activated multiple death-related pathways, notably autophagy and apoptosis; cell death effectors Chop, caspase 9 and cleaved caspase 3 were increased. Additionally, GRP78 deletion increased stress kinase JNK (c-Jun-N-terminal kinase) which has been implicated in cellular proliferation as well as cell death. Inhibition of JNK kinase using JNK-IN-8 inhibitor during GRP78 loss conditions decreased phosphorylation of JNK and rescued beta cells from cell death as measured with Annexin V and TUNEL. Therefore, we conclude that ER stress related cell death induced by loss of GRP78 (ER stress) involves JNK activation, and that modulation of JNK under certain circumstances may help maintain beta cell mass to protect against diabetes. Disclosure R.B. Sharma: None. C.O. Darko: None. B. Gablaski: None. A.S. Lee: None. L.C. Alonso: Consultant; Self; Fairbanks Pharmaceuticals Inc. Research Support; Self; American Diabetes Association. Funding National Institute of Diabetes and Digestive and Kidney Diseases (5R01DK113300-02)

323-OR: Synergism between Protein Quality Control Pathways to Maintain Alpha-Cell Mass and Function

Endocrine cells rely on efficient prohormone synthesis, folding, and elimination of misfolded peptides for proper function. Endoplasmic reticulum-associated degradation (ERAD) and autophagy are two primary mechanisms of cellular protein quality control, and each are required for normal beta cell function. However, their role in alpha cells has largely been unexplored. ERAD is mediated by two highly conserved ER membrane proteins: Hrd1, a ubiquitin ligase that retrotranslocates and ubiquitinates misfolded ER proteins for proteasomal degradation, and Sel1L, an adaptor protein required for Hrd1 stability and function. Autophagy is a conserved mechanism for recycling ER and cytosolic products via lysosomal degradation and requires a precise signaling sequence for activation, involving key autophagosome protein Atg7. To investigate the role of ERAD and autophagy in alpha cells, we targeted deletion of Sel1L and/or Atg7 with the Cre-lox system, using mice expressing Cre recombinase under the proglucagon promoter. Sel1LΔGcg, Atg7ΔGcg, and Sel1L;Atg7ΔGcg mice all showed normal growth and oral glucose tolerance. Sel1LΔGcg mice had dilated ER and reduced glucagon content in alpha cells following ERAD inactivation, but glucagon secretory capacity in vivo was largely maintained. In contrast, disruption of autophagy in Atg7ΔGcg mice led to alpha cell hypertrophy and vacuolization, and reduced glucagon secretion in vivo following insulin-induced hypoglycemia and 2-deoxyglucose-mediated glucoprivation. These secretory defects were exacerbated in Sel1L;Atg7ΔGcg mice, which also had reduced glucagon secretion after a 24 hour fast and a 77% reduction in pancreatic glucagon content. By immunohistochemistry, very few alpha cells were present in islets of adult Sel1L;Atg7ΔGcg mice. Our data demonstrate that alpha cells are more susceptible to defective autophagy rather than defective Sel1L-Hrd1 ERAD, but that these pathways act synergistically to maintain functional alpha cell mass. Disclosure R.B. Reinert: None. X. Cui: None. N. Shrestha: None. L. Qi: None. Funding National Institutes of Health (T32DK007245)

P0045GENETIC ABLATION OF NFAT5 IN THE PRINCIPAL CELLS OF THE COLLECTING DUCT LEADS TO A WATER HANDLING DEFECT

Abstract Background and Aims Nuclear Factor of Activated T-Cells 5 (NFAT5), also called TonEBP/OREBP, helps protecting kidney cells from the stress of extracellular hypertonic challenge. In response to hypertonicity, NFAT5 rapidly translocates into the nucleus and enhances the transcription of osmoprotective genes. Mice lacking NFAT5 have embryonic lethality, with those surviving having severe renal atrophy and hydronephrosis. Overexpression of a dominant-negative NFAT5 in all epithelial cells of the thick ascending limb, distal tubule and collecting duct (CD) leads to an impairment in the urine concentration, with reduced expression of AQP2 and urea transporters UT-A1 and UT-A2. Here, we generated a novel mouse model to assess the role of NFAT5 in the water transporting principal cells of the CD. Method The mouse model was generated by breeding floxed NFAT5 mice with mice specifically expressing Cre recombinase in AQP2 expressing cells (NFAT5f/f-AQP2cre+/-). AQP2cre+/- mice served as controls. For renal function analysis, mice were kept in metabolic cages for 5 days and water intake, urinary volume, osmolality, and serum and urine electrolytes were evaluated. Mice were euthanized and the kidneys were used for western blot analysis. Results NFAT5f/f-AQP2cre+/- mice show a significant increase of 24 hour urine output compared to the control (KO: 6.267 ± 0.9367 µl/h/g BW vs WT: 0.9593 ± 0.0796 µl/h/g BW), a decrease in the urine osmolality (KO: 505.6 ± 107.6 mOsm/Kg H2O vs WT: 6205 ± 752.7 mOsm/Kg H2O) and urea concentration. After 3h and half intraperitoneal injection of dDAVP (1ug/kg) the osmolality was still significantly low in NFAT5f/f-AQP2cre+/- mice (KO: 743.7 ± 81.77 vs WT: 3342 ± 80.14 mOsm/Kg H2O), as well as after 8h water restriction (KO: 732.8 ± 24.90 vs WT: 3784 ± 450.9 mOsm/Kg H2O). Immunoblotting demonstrated a significant decrease in AQP2, AQP3 and AQP4 abundance in cortical kidney fractions and inner medulla. There were no differences in vasopressin V2 receptor (V2R) and AVP-regulated transporters NKCC2, ROMK and alpha-ENaC. Conclusion We demonstrate that NFAT5 ablation from AQP2-expressing cells results in a urinary concentration defect. NFAT5f/f-AQP2cre+/- mice have a hypoosmotic polyuria, without major effects on the AVP-V2R axis, suggesting Nephrogenic Diabetes Insipidus.

Sex-Specific Difference in Adipose Tissue and Blood Pressure in a New Mouse Model Expressing Human Soluble Prorenin Receptor in Adipocytes

Abstract Objectives Sex differences exist in obesity associated with cardiovascular disease; however, underlying mechanisms are not completely understood. Previous studies from our laboratory demonstrated that the prorenin receptor (PRR) and its soluble form (sPRR) contribute to adipogenesis and blood pressure control. The present study aimed to determine whether adipose-sPRR stimulated obesity is associated with hypertension and whether it is sex-dependent. Methods Transgenic mice on the C57BL/6 J background were generated expressing the human form of the soluble prorenin receptor (HsPRR) in a Cre-inducible manner. Male mice expressing Cre recombinase under the control of the adiponectin promotor were bred to heterozygote HsPRR/+ female mice to generate mice over-secreting sPRR (adi-HsPRR) and control littermate mice (CTL). The secretion of sPRR in the media doubled in primary adipocytes of adi-HsPRR mice compared to control mice (sPRR. CTL: 3729 ± 805 pg/ml; adi-HsPRR: 6170 ± 1237 pg/ml, P < 0.05) validating the mouse model. Male (CTL = 4; adi-HsPRR = 8) and female mice (CTL = 10; adi-HsPRR = 10) were fed a low-fat (LF) diet or a high-fat diet (HF) for 20 weeks. Body weight was assessed weekly and EchoMRI was examined monthly. Results After 20 weeks on LF diet, adi-HsPRR male mice gained significantly more weight than CTL male mice (CTL: 25.1 ± 0.8 g; adi-HsPRR: 29.0 ± 0.8 g P < 0.05), whereas no significant differences in body weights were observed in female mice. The body composition revealed a significant increase of fat mass, specifically in the epidydimal fat (CTL: 0.35 ± 0.04 g; adi-HsPRR: 0.61 ± 0.07 g, P < 0.05), and lean mass of HsPRR male mice compared to CTL male mice. In contrast, female mice exhibited similar body weights (CTL: 20.6 ± 0.3 g; adi-HsPRR: 20.4 ± 0.4 g) and there was no differences of fat mass or lean mass between CTL and adi-HsPRR female mice. The sex-specific mechanism of sPRR on adipogenesis and blood pressure (by radiotelemetry) with LF and HF diet is currently under investigation. Conclusions Overall, sPRR stimulated body weight gain and fat mass expansion in male mice but not in female mice suggesting that female mice are protected from sPRR induced-hypertrophic effect. Funding Sources R01_HL142969–01 Yiannikouris, PI 07/15/2018–06/30/2022 NIH/NHLBI Title: The role of soluble prorenin receptor in hypertension associated with obesity Role: Ph.D Graduate Student.

Loss of Histone Deacetylase 4 in Hepatocytes Increases De Novo Lipogenesis in Diet-Induced Obesity Mice

Abstract Objectives Evidence suggests that histone deacetylase 4 (HDAC4) is downregulated in adipose tissue of obese subjects. We determined the role of HDAC4 in the regulation of energy metabolism of metabolically active tissues, such as the liver and adipose tissue. Methods Hepatocyte-specific (Hdac4HKO) and adipocyte-specific (Hdac4AKO) Hdac4 knockout mice were generated by crossing homozygous Hdac4 floxed (Hdac4fl/fl) mice with mice expressing Cre recombinase under the control of the enhancer/promoter of Albumin or Adipoq gene, respectively. Hdac4fl/fl and Hdac4HKO mice were fed a high fat/high sucrose (HF/HS; 57%/28% energy from fat/sucrose) with 2% cholesterol (w/w) diet for 16 weeks. Hdac4fl/fl and Hdac4AKO mice were fed an obesogenic HF/HS diet for 16 weeks. The final serum was collected by cardiopuncture for blood analysis. Tissues were snap frozen for mRNA and protein analysis. Results Both Hdac4HKO and Hdac4AKO mice did not show differences in body weight compared to Hdac4fl/fl mice following the 16-week of experimental diet. However, loss of hepatic HDAC4 increased serum alanine transaminase levels, a marker for liver injury. Also, Hdac4HKO mice had exacerbated hepatic steatosis with higher liver weights and triglyceride levels than Hdac4fl/fl mice. Consistently, hepatic expression of genes for de novo lipogenesis, including Srebf1c and its target genes, fatty acid synthase and acetyl CoA carboxylase 1, was significantly higher in Hdac4HKO mice compared with control mice. Interestingly, the loss of hepatocyte HDAC4 aggravated inflammation and fibrosis in white adipose tissue. Serum cytokine array indicated increases in fibroblast growth factor 1, pentraxin 3, tissue inhibitor of metalloproteinases 1, and decrease in endocan, which may contribute to the crosstalk between the liver and adipose tissue in Hdac4HKO. On the other hand, the loss of adipocyte HDAC4 elicited minimal changes in mRNA levels of lipogenic, inflammatory, and fibrogenic genes in adipose tissue and the liver. Conclusions The lack of functional hepatocyte HDAC4 increased lipid accumulation in the liver of obesity mice via increasing hepatic de novo lipogenesis, and also aggravated adipose tissue inflammation and fibrosis. Further investigation is warranted to elucidate the crosstalk between the liver and adipose tissue in Hdac4HKO. Funding Sources This study was supported by NIH.

NBCe2 (Slc4a5) Is Expressed in the Renal Connecting Tubules and Cortical Collecting Ducts and Mediates Base Extrusion.

Arterial hypertension, is a common disorder with multiple and variable etiologies. Single nucleotide polymorphism analyses have detected an association between variants in the gene encoding the electrogenic Na+:HCO3– cotransporter NBCe2 (Slc4a5), and salt-sensitive hypertension. Mice with genetic deletion of NBCe2 are hypertensive, and the cause of the blood pressure (BP) increase is believed to arise from a lack of renal NBCe2 function. The exact cellular expression of NBCe2 in the kidney tubular system is, however, not determined. Here, we find NBCe2 to be expressed predominantly in isolated connecting tubules (CNT) and cortical collecting ducts (CD) by RT-PCR. In isolated renal CNT and CCD, genetic deletion of NBCe2 leads to decreased net base extrusion. To determine the role of renal NBCe2 in the development of hypertension, we generated CNT and intercalated cell NBCe2 knockout mice by crossing an Slc4a5 lox mouse with mice expressing cre recombinase under the V-ATPase B1 subunit promotor. Although the mice displayed changes in the expression of renal membrane transporters, we did not detect hypertension in these mice by tail cuff recordings. In conclusion, while global NBCe2 deletion certainly causes hypertension this study cannot confirm the role of renal NBCe2 expression in blood pressure regulation.

PTPσ Controls Presynaptic Organization of Neurotransmitter Release Machinery at Excitatory Synapses

SummaryLeukocyte common antigen-related receptor tyrosine phosphatases (LAR-RPTPs) are evolutionarily conserved presynaptic organizers. The synaptic role of vertebrate LAR-RPTPs in vivo, however, remains unclear. In the current study, we analyzed the synaptic role of PTPσ using newly generated, single conditional knockout (cKO) mice targeting PTPσ. We found that the number of synapses was reduced in PTPσ cKO cultured neurons in association with impaired excitatory synaptic transmission, abnormal vesicle localization, and abnormal synaptic ultrastructure. Strikingly, loss of presynaptic PTPσ reduced neurotransmitter release prominently at excitatory synapses, concomitant with drastic reductions in excitatory innervations onto postsynaptic target areas in vivo. Furthermore, loss of presynaptic PTPσ in hippocampal CA1 pyramidal neurons had no impact on postsynaptic glutamate receptor responses in subicular pyramidal neurons. Postsynaptic PTPσ deletion had no effect on excitatory synaptic strength. Taken together, these results demonstrate that PTPσ is a bona fide presynaptic adhesion molecule that controls neurotransmitter release and excitatory inputs.

Systemic but not MDSC-specific IRF4 deficiency promotes an immunosuppressed tumor microenvironment in a murine pancreatic cancer model

Pancreatic ductal adenocarcinoma is characterized by a strong immunosuppressive network with a dense infiltration of myeloid cells including myeloid-derived suppressor cells (MDSC). Two distinct populations of MDSC have been defined: polymorphonuclear MDSC (PMN-MDSC) and monocytic MDSC (M-MDSC). Several factors influence the development and function of MDSC including the transcription factor interferon regulatory factor 4 (IRF4). Here, we show that IRF4 deficiency accelerates tumor growth and reduces survival, accompanied with a dense tumor infiltration with PMN-MDSC and reduced numbers of CD8+ T cells. As IRF4 has been described to modulate myeloid cell development and function, particularly of PMN-MDSC, we analyzed its role using MDSC-specific IRF4 knockout mice with the Ly6G or LysM knock-in allele expressing Cre recombinase and Irf4flox. In GM-CSF-driven bone marrow cultures, IRF4 deficiency increased the frequency of MDSC-like cells with a strong T cell suppressive capacity. Myeloid (LysM)-specific depletion of IRF4 led to increased tumor weight and a moderate splenic M-MDSC expansion in tumor-bearing mice. PMN cell (Ly6G)-specific depletion of IRF4, however, did not influence tumor progression or MDSC accumulation in vivo in accordance with our finding that IRF4 is not expressed in PMN-MDSC. This study demonstrates a critical role of IRF4 in the generation of an immunosuppressive tumor microenvironment in pancreatic cancer, which is independent of IRF4 expression in PMN-MDSC.