Biology subjects

Turner, J. R.

Publications and source records attributed to Turner, J. R..

17 recordsLinked to original sources

bioRxiv2026

EcoHIV Treated Primary Microglia Exhibit a Highly Specialized and Sex-Dependent Transcriptional State

The pathogenesis of HIV-1-associated neurocognitive disorders (HAND) is partly driven by microglial dysregulation. Indeed, HIV-1-induced microglial dyshomeostasis is characterized by a sustained pro-inflammatory state and exacerbated microglia proliferation. Herein, in vitro studies were conducted in primary microglia to elucidate the transcriptomic alterations underlying EcoHIV induced microglia hyperproliferation. Consistent with, and expanding upon, prior studies, treatment with EcoHIV exacerbated microglia proliferation evidenced by the upregulation of Pcna and Mki67. With regards to microglial receptors and adapter proteins, the transcriptomic expression of Trem2 and Dap12 was suppressed and increased, respectively, in microglia treated with EcoHIV. Crucially, dysregulation of the Trem2/Dap12 signaling complex explained 51.4% of the variance in microglial proliferation in EcoHIV treated microglia. A preliminary investigation of the Trem2-mediated microglial survival and immunoregulation pathway affords additional support for a unique EcoHIV-induced transcriptomic profile. Specifically, independent of biological sex, EcoHIV treatment upregulated Syk and downregulated Ctnnb1 mRNA expression. Notably, biological sex regulates precisely how EcoHIV treated microglia respond to the suppression of Trem2 and compensatory hyperproliferative state. Collectively, primary microglia treated with EcoHIV exhibit a highly specialized and sex-dependent transcriptional state. Enhancing our understanding of how HIV-1 alters microglial homeostasis is crucial for the identification of novel therapeutic targets for HAND.

neuroscience↗

bioRxiv2026

Rapid imaging of lysyl oxidase activity and fibrogenesis with a turn-on fluorophore

Fibrogenesis is essential to wound healing, but aberrant fibrogenesis is a driver of many chronic diseases and cancers. Lysyl oxidases (LOX) play a pivotal role in fibrogenesis by catalyzing the oxidation of lysine residues to reactive aldehydes (allysine) in collagens and elastin, resulting in the crosslinking and excessive deposition of these extracellular matrix components. Currently, rapid and robust histological assays to visualize the spatial distribution of LOX activity are lacking, hindering the precise validation of anti-fibrotic therapies. Here, we present a histological fluorescent staining method to visualize fibrogenesis (active fibrosis) and LOX activity in tissue sections utilizing a bioorthogonal tag and a click reaction with a turn-on fluorophore. Notably, requiring only two commercial reagents, this protocol can be completed in under two hours and is compatible with other imaging modalities, including second-harmonic generation and immunofluorescence staining. We validated this method across various healthy and fibrotic mouse and human tissue specimens.

bioengineering↗

bioRxiv2026

A HISTORY OF ADOLESCENT OXYCODONE SELF-ADMINISTRATION: NEURAL MECHANISMS UNDERLYING NEUROCOGNITIVE IMPAIRMENTS

Maturation of the prefrontal cortex and higher-order cognitive processes is ongoing through adolescence and young adulthood; developmental periods associated with the nonmedical use of prescription opioids. Initiation of oxycodone (OXY) self-administration during adolescence induces long-term dose-dependent impairments in neurobehavioral and neurocognitive development. To date, however, the neural mechanisms underlying neurocognitive impairments induced by adolescent prescription opioid use disorder (APOUD) have not been systematically evaluated. A DiOlistic labeling technique, in concert with sophisticated neuronal reconstruction software, revealed a prominent sex- and dose-dependent population shift in the morphological parameters of dendritic spines in pyramidal neurons in the anterior cingulate cortex (ACC) of rodents with a history of adolescent OXY self-administration relative to their control counterparts. Similarly, the biochemical mechanisms underlying dendritic spine dysmorphology, which were investigated using quantitative polymerase chain reaction, were dependent upon biological sex. In male rodents, a dose-dependent decrease in mRNA expression of GRIN1, a N-methyl-D-aspartate receptor, was observed in animals with a history of adolescent OXY self-administration relative to controls. In contrast, female rats with a history of adolescent OXY self-administration exhibited increased mRNA expression of cdk5, a protein kinase involved in the regulation of pre- and post-synaptic function; a transcriptional alteration that may result from excessive glutamate accumulation. Fundamentally, transcriptional dysregulation and dendritic spine dysmorphology mechanistically underlie, at least partly, APOUD-induced neurocognitive impairments. Collectively, results support that sex-dependent glutamatergic dysregulation may underlie dendritic spine dysmorphology in the ACC resulting in APOUD-induced neurocognitive impairments. Glutamatergic dysregulation, therefore, may afford a key target for the development of novel therapeutics. SIGNIFICANCE STATEMENTIndividuals with a history of adolescent prescription opioid use disorder (APOUD) are afflicted with profound neurocognitive impairments. Herein, sex-dependent glutamatergic dysregulation may underlie dendritic spine dysmorphology in the ACC resulting in APOUD-induced neurocognitive impairments, thereby affording a key target for the development of novel therapeutics.

neuroscience↗

bioRxiv2025

Colitis-primed circulating T cells drive small intestinal barrier function through Nod2, microbiota and myosin light chain kinase-dependent mechanisms

Inflammatory bowel diseases (IBD), including Crohns disease (CD) and ulcerative colitis (UC), are characterized by intestinal inflammation and barrier dysfunction. While disruption of the intestinal barrier contributes to the pathogenesis of IBD, yet how colonic inflammation alters small intestinal homeostasis remains poorly defined. Here, we demonstrate that three models of colitis, including 2,4,6-Trinitrobenzenesulfonic acid (TNBS), Dextran Sulfate Sodium (DSS), and oxazolone, induce paracellular barrier dysfunction in the small intestine, with model-specific immune profiles. Th1/Th17-skewed responses (TNBS and DSS) were associated with microbiota-dependent upregulation of myosin light chain kinase (MLCK), ROR{gamma}t CD4 T cell expansion, and elevated expression of Nod2, which was not observed under Th2-dominant (oxazolone) conditions. Inhibition of MLCK restored barrier function and suppressed inflammation in a CD4 T cell-dependent manner. Using Nod2-deficient and 2939insC mutant mice, we showed Nod2 as a critical regulator of small intestinal permeability during colitis. Bone marrow chimeras revealed compartment-specific roles of Nod2, with non-hematopoietic Nod2 sufficient to preserve epithelial integrity, while hematopoietic Nod2 expression is required to limit cytokine-mediated inflammation. Moreover, MLCK inhibition ameliorated intestinal lesions only in mice with Nod2 deficiency in the hematopoietic compartment. Finally, microbiota transfer experiments ruled out a causal role for dysbiosis in driving small intestinal permeability defects in Nod2-deficient mice. These findings uncover a Nod2-MLCK-CD4 T cell axis linking colonic inflammation to small intestinal barrier dysfunction and highlight distinct immune-epithelial-microbial mechanisms shaping intestinal homeostasis during colitis. Graphical AbstractCirculating ROR{gamma}t Foxp3- CD4 T cells from mice with TNBS- or DSS-induced colitis increase small intestinal permeability by promoting cytokine secretion that enhances MLCK activity. This effect is attenuated by Nod2 activation. Specifically, Nod2 expression in hematopoietic cells reduces cytokine secretion and permeability, while activation of Nod2 in non-hematopoietic cells alone is sufficient to prevent increased permeability by directly regulating MLCK activity, independently of cytokine influence. Moreover, the absence of Nod2 is associated with inflammatory lesions in the small intestine of TNBS-treated mice.

immunology↗

bioRxiv2024

Behavioral and genetic markers of susceptibility to escalate fentanyl intake.

BackgroundThe "loss of control" over drug consumption, present in opioid use disorder (OUD) and known as escalation of intake, is well-established in preclinical rodent models. However, little is known about how antecedent behavioral characteristics, such as valuation of hedonic reinforcers prior to drug use, may impact the trajectory of fentanyl intake over time. Moreover, it is unclear if distinct escalation phenotypes may be driven by genetic markers predictive of OUD susceptibility. MethodsMale and female Sprague-Dawley rats (n=63) were trained in a sucrose reinforcement task using a progressive ratio schedule. Individual differences in responsivity to sucrose were hypothesized to predict escalation of fentanyl intake. Rats underwent daily 1-h acquisition sessions for i.v. fentanyl self-administration (2.5 {micro}g/kg; FR1) for 7 days, followed by 21 6-h escalation sessions, then tissue from prefrontal cortex was collected for RNA sequencing and qPCR. Latent growth curve and group-based trajectory modeling were used, respectively, to evaluate the association between sucrose reinforcement and fentanyl self-administration and to identify whether distinct escalation phenotypes can be linked to gene expression patterns. ResultsSucrose breakpoints were not predictive of fentanyl acquisition nor change during escalation, but did predict fentanyl intake on the first day of extended access to fentanyl. Permutation analyses did not identify associations between behavior and single gene expression when evaluated overall, or between our ascertained phenotypes. However, weighted genome correlation network analysis (WGCNA) and gene set enrichment analysis (GSEA) determined several gene modules linked to escalated fentanyl intake, including genes coding for voltage-gated potassium channels, calcium channels, and genes involved in excitatory synaptic signaling. Transcription factor analyses identified EZH2 and JARID2 as potential transcriptional regulators associated with escalated fentanyl intake. Genome-wide association study (GWAS) term categories were also generated and positively associated with terms relating to substance use disorders. DiscussionEscalation of opioid intake is largely distinct from motivation for natural reward, such as sucrose. Further, the gene networks associated with fentanyl escalation suggest that engagement of select molecular pathways distinguish individuals with "addiction prone" behavioral endophenotypes, potentially representing druggable targets for opioid use disorder. Our extended in silico identification of SNPs and transcription factors associated with the "addiction prone" high escalating rats highlights the importance of integrating findings from translational preclinical models. Through a precision medicine approach, our results may aid in the development of patient-centered treatment options for those with OUD.

neuroscience↗

bioRxiv2023

WNT2B Deficiency Causes Increased Susceptibility to Colitis in Mice and Impairs Intestinal Epithelial Development in Humans

Background and aimsWNT2B is a canonical Wnt ligand previously thought to be fully redundant with other Wnts in the intestinal epithelium. However, humans with WNT2B deficiency have severe intestinal disease, highlighting a critical role for WNT2B. We sought to understand how WNT2B contributes to intestinal homeostasis. MethodsWe investigated the intestinal health of Wnt2b knock out (KO) mice. We assessed the impact of inflammatory challenge to the small intestine, using anti-CD3{chi} antibody, and to the colon, using dextran sodium sulfate (DSS). In addition, we generated human intestinal organoids (HIOs) from WNT2B-deficient human iPSCs for transcriptional and histological analyses. ResultsMice with WNT2B deficiency had significantly decreased Lgr5 expression in the small intestine and profoundly decreased expression in the colon, but normal baseline histology. The small intestinal response to anti-CD3{chi} antibody was similar in Wnt2b KO and wild type (WT) mice. In contrast, the colonic response to DSS in Wnt2b KO mice showed an accelerated rate of injury, featuring earlier immune cell infiltration and loss of differentiated epithelium compared to WT. WNT2B-deficient HIOs showed abnormal epithelial organization and an increased mesenchymal gene signature. ConclusionWNT2B contributes to maintenance of the intestinal stem cell pool in mice and humans. WNT2B deficient mice, which do not have a developmental phenotype, show increased susceptibility to colonic injury but not small intestinal injury, potentially due to a higher reliance on WNT2B in the colon compared to the small intestine. WNT2B deficiency causes a developmental phenotype in human intestine with HIOs showing a decrease in their mesenchymal component and WNT2B-deficient patients showing epithelial disorganization. Data Transparency StatementAll RNA-Seq data will be available through online repository as indicated in Transcript profiling. Any other data will be made available upon request by emailing the study authors.

molecular biology↗

bioRxiv2023

Dynamic Effects of Ventral Hippocampal NRG3/ERBB4 Signaling on Nicotine Withdrawal-Induced Responses

Tobacco smoking remains a leading cause of preventable death in the United States, with a less than 5% success rate for smokers attempting to quit. High relapse rates have been linked to several genetic factors, indicating that the mechanistic relationship between genes and drugs of abuse is a valuable avenue for the development of novel smoking cessation therapies. For example, various single nucleotide polymorphisms (SNPs) in the gene for neuregulin 3 (NRG3) and its cognate receptor, the receptor tyrosine-protein kinase erbB-4 (ERBB4), have been linked to nicotine addiction. Our lab has previously shown that ERBB4 plays a role in anxiety-like behavior during nicotine withdrawal (WD); however, the neuronal mechanisms and circuit-specific effects of NRG3-ERBB4 signaling during nicotine and WD are unknown. The present study utilizes genetic, biochemical, and functional approaches to examine the anxiety-related behavioral and functional role of NRG3-ERBB4 signaling, specifically in the ventral hippocampus (VH). We report that 24hWD from nicotine is associated with altered synaptic expression of VH NRG3 and ERBB4, and genetic disruption of VH ErbB4 leads to an elimination of anxiety-like behaviors induced during 24hWD. Moreover, we observed attenuation of GABAergic transmission as well as alterations in Ca2+-dependent network activity in the ventral CA1 area of VH ErbB4 knock-down mice during 24hWD. Our findings further highlight contributions of the NRG3-ERBB4 signaling pathway to anxiety-related behaviors seen during nicotine WD.

neuroscience↗

bioRxiv2022

Selective perijunctional MLCK1 recruitment in Crohn's disease: Identification of essential structural domains

Intestinal epithelia express two long myosin light chain kinase (MLCK) splice variants, MLCK1 and MLCK2. Unlike MLCK2, MLCK1 is concentrated at the perijunctional actomyosin ring and this localization is enhanced by tumor necrosis factor (TNF) signaling. Here we sought to identify and characterize the domain(s) that direct basal and TNF-induced MLCK1 subcellular localization. Quantitative morphometry demonstrated specific increases in MLCK1 expression and perijunctional localization in Crohns disease patient biopsies, relative to controls. TNF induced perijunctional recruitment of MLCK1-EGFP but did not affect localization of MLCK2-EGFP, which was predominantly associated with basal stress fibers. Recombinant N-terminal MLCK1 and MLCK2 regions accelerated actin polymerization in vitro but were not different from one another. In contrast, the affinity of N-terminal MLCK1 binding to F-actin was greater than that of MLCK2. Perijunctional MLCK1 and MLCK2 domain recruitment in intestinal epithelial cells paralleled in vitro F-actin binding. The unique MLCK1 Ig3 domain was necessary, but not sufficient, for both F-actin binding and perijunctional recruitment, but, nevertheless, displaced perijunctional MLCK1, enhanced steady-state barrier function, and limited TNF-induced MLCK1 recruitment and barrier loss. These data demonstrate selective perijunctional MLCK1 recruitment in Crohns disease, suggest that F-actin binding contributes to perijunctional recruitment, and show that Ig3 can act as a dominant negative effector that limits TNF-induced MLCK1 recruitment and barrier loss. These results data provide key mechanistic detail that will enable development of therapeutics that target Ig3, or its intercellular binding partners, to reverse inflammation-induced barrier loss and limit disease progression.

cell biology↗

bioRxiv2022

The impact of dose on the pathogen population that initiates infection defines the protection provided by host bottlenecks

Host bottlenecks prevent many infections before the onset of disease by eliminating invading pathogens. Monitoring the diversity of a barcoded population of the diarrhea causing bacterium Citrobacter rodentium during colonization of its natural host, mice, allowed us to determine the number of cells that found the infection by establishing a replicative niche. The size of the pathogens founding population scaled with dose and was controlled by a severe yet slow-acting bottleneck. Reducing stomach acid or changing host genotype modestly relaxed the bottleneck without breaking the fractional relationship between dose and founders. In contrast, disrupting the microbiota caused the founding population to no longer scale with the size of the inoculum and allowed the pathogen to infect at almost any dose, indicating that the microbiota creates the dominant bottleneck. Further, in the absence of competition with the microbiota, the diversity of the pathogen population slowly contracted as the population was overtaken by bacteria that lost the critical virulence island, the locus of enterocyte effacement (LEE). Collectively, our findings reveal that the mechanisms of protection by colonization bottlenecks are reflected in and can be generally defined by the impact of dose on the pathogens founding population.

microbiology↗

bioRxiv2022

Target-agnostic discovery of Rett Syndrome therapeutics by coupling computational network analysis and CRISPR-enabled in vivo disease modeling

Many neurodevelopmental genetic disorders, such as Rett syndrome, are caused by a single gene mutation but trigger changes in expression and regulation of numerous other genes. This severely impair functions of multiple organs and organ systems beyond the central nervous system (CNS), adding to the challenge of developing broadly effective treatments based on a single drug target. This challenge is further complicated by the lack of sufficiently broad and biologically relevant drug screens, and the inherent complexity in identifying clinically relevant targets responsible for diverse phenotypes. Here, we combined human gene regulatory network-based computational drug prediction with in vivo screening in a population-level diversity, CRISPR-edited, Xenopus laevis tadpole model of Rett syndrome to carry out target-agnostic drug discovery, which rapidly led to the identification of the FDA-approved drug vorinostat as a potential repurposing candidate. Vorinostat broadly improved both CNS and non-CNS (e.g., gastrointestinal, respiratory, inflammatory) abnormalities in a pre-clinical mouse model of Rett syndrome. This is the first Rett syndrome treatment to demonstrate pre-clinical efficacy across multiple organ systems when dosed after the onset of symptoms, and network analysis revealed a putative therapeutic mechanism for its cross-organ normalizing effects based on its impact on acetylation metabolism and post-translational modifications of microtubules. Although traditionally considered an inhibitor of histone deacetylases (HDAC), vorinostat unexpectedly restored protein acetylation across both hypo- and hyperacetylated tissues, suggesting non-HDAC-mediated therapeutic mechanisms supported by proteomic analysis.

neuroscience↗

bioRxiv2022

Epithelial monolayer development and tight junction assembly on nanopillar arrays

Nanostructured materials provide an outstanding opportunity to both stimulate and measure cellular processes. In the context of tight junctions, it was previously reported that transient application of a nanotopographic surface over the apical brush border membrane of epithelial monolayers triggers redistribution of ZO-1, claudins, and F-actin that increases paracellular macromolecular flux. In excitable tissues, nanomaterials have been used to apply and measure electrical signals, such action potentials. As a first step towards translating these technologies for use in analysis of epithelial function, we sought to culture monolayers composed of transporting epithelia over nanopillar arrays without perturbing cellular structure or function. Madin-Darby Canine kidney I (MDCK I) cells were cultured on collagen-coated silicon chips with [~]1 m diameter nanopillar arrays. Fluorescence and scanning electron microscopy were used to assess the impact of height on nanopillar-epithelial interactions. Monolayers formed over and were largely unaffected by short nanopillars. These nanopillars were located beneath basal epithelial surfaces and were not preferentially located within lateral intercellular spaces or beneath ZO-1-containing junctions. In contrast, tall nanopillars that exceeded cell height disrupted MDCK I monolayer growth. Cells interacted with, encircled, and extended cytoplasm over the top of tall nanopillars, and dense ZO-1 and F-actin accumulations occasionally surrounded apical membranes adjacent to nanopillars. Finally, when grown over arrays composed of nanopillars 1 - 2 m shorter than cells, MDCK I frequently grew between nanopillars. As a result, nanopillars were more commonly present within lateral intercellular spaces beneath junctions. Apical complex structure was intact, as assessed by fluorescence microscopy of ZO-1, occludin, claudin-2, F-actin, and E-cadherin. Apical microvilli were also unaffected. We therefore show that conditions can be defined to allow growth of mature, correctly assembled epithelial monolayers with nanopillars localized to lateral intercellular spaces. This sets the stage for application of nanotechnologies for perturbation and analysis of epithelial biology.

cell biology↗

bioRxiv2021

The epithelial-specific ER stress sensor IRE1β enables host-microbiota crosstalk to affect colon goblet cell development

Epithelial cells lining mucosal surfaces of the gastrointestinal and respiratory tracts uniquely express IRE1{beta} (Ern2), a paralogue of the most evolutionarily conserved endoplasmic reticulum stress sensor IRE1. How IRE1{beta} functions at the host-environment interface and why a second IRE1 paralogue evolved remain incompletely understood. Using conventionally raised and germ-free Ern2-/- mice, we found that IRE1{beta} was required for microbiota-induced goblet cell maturation and mucus barrier assembly in the colon. This occurred only after colonization of the alimentary tract with normal gut microflora, which induced IRE1{beta} expression. IRE1{beta} acted by splicing Xbp1 mRNA to expand ER function and prevent ER stress in goblet cells. Although IRE1 can also splice Xbp1 mRNA, it did not act redundantly to IRE1{beta} in this context. By regulating assembly of the colon mucus layer, IRE1{beta} further shaped the composition of the gut microbiota. Mice lacking IRE1{beta} had a dysbiotic microbial community that failed to induce goblet cell development when transferred into germ-free wild type mice. These results show that IRE1{beta} evolved at mucosal surfaces to mediate crosstalk between gut microbes and the colonic epithelium required for normal homeostasis and host defense.

cell biology↗

bioRxiv2019

Human Rotavirus Diarrhea Is Associated with Altered Trafficking and Expression of Apical Membrane Transport Proteins

BackgroundRotavirus (RV) is the 5th leading cause of death in children <5 years old but the leading cause of diarrhea related deaths in this age group. The mechanism of RV diarrhea involves decreased activity of Na+-dependent solute transporters with increased luminal secretion of Cl- in the absence of significant histologic damage. While our understanding of RV diarrhea has come from studies in animal models and cancer cell lines, the mechanism of the diarrhea and the transport proteins affected in human RV disease remains only partially understood. This understanding is likely to impact drug development therapy for RV diarrhea.\n\nMethodsFormalin-fixed paraffin-embedded small intestinal specimens from patients diagnosed with RV diarrhea (confirmed by anti-RV antibodies) were analyzed by immunofluorescence for changes in apical/basolateral ion/nutrient transporters/channels as well as tight junctional and cytoskeletal proteins. Proximal small intestinal enteroids generated from biopsies obtained from healthy human subjects were grown as monolayers, differentiated to resemble villus epithelial cells, and infected with human RV.\n\nResultsRV diarrhea was associated with reduced expression and intracellular localization of transport proteins normally found in the brush border membranes, including SGLT1, NHE3, NHE2, the Na+-dependent amino acid transporter SLC6A19, and CFTR. In contrast, basolateral proteins, including Na+/K+-ATPase, NKCC1, and {beta}-catenin, the brush border marker ezrin, as well as the tight junction protein, ZO-1, were expressed and localized normally. RV-induced mislocalization of NHE3, SGLT1, SLC6A19 and CFTR was also seen when human small intestinal enteroids were infected with RV.\n\nConclusionsThese data demonstrate a new pathophysiologic mechanism of acute diarrhea in which expression of multiple apical transport proteins are reduced. This acute diarrhea is likely to be caused by an effect on a common apical trafficking pathway, as exemplified by RV diarrhea, and its contribution to other enteric pathogen-induced diarrheal diseases should be determined.

cell biology↗

bioRxiv2019

Inhibition of the DDX3 prevents HIV-1 Tat and cocaine-induced neurotoxicity by targeting microglia activation

HIV-1 Associated Neurocognitive Disorder (HAND) is commonly seen in HIV-infected patients. Viral proteins including Tat cause neuronal toxicity and is worsened by drugs of abuse. To uncover potential targets for anti-HAND therapy, we employed a literature mining system, MOLIERE. Here, we validated Dead Box RNA Helicase 3 (DDX3) as a target to treat HAND via a selective DDX3 inhibitor, RK-33. The combined neurotoxicity of Tat protein and cocaine was blocked by RK-33 in rat and mouse cortical cultures. Transcriptome analysis showed that Tat-activated transcripts include makers and regulators of microglial activation, and RK-33 blocked Tat-induced activation of these mRNAs. Elevated production of proinflammatory cytokines was also inhibited by RK-33. These findings show that DDX3 contributes to microglial activation triggered by Tat and cocaine, and DDX3 inhibition shows promise as a therapy for HAND. Moreover, DDX3 may contribute to the pathology of other neurodegenerative diseases with pathological activation of microglia.

neuroscience↗

bioRxiv2019

Nod2 protects remote small intestinal sites in case of colonic inflammation.

NOD2 mutations are key risk factors for Crohns disease (CD). NOD2 contributes to intestinal homeostasis by regulating innate and adaptive immunity together with intestinal epithelial function. However, the roles of NOD2 during gut inflammation is not known. We initially observed that NOD2 expression was increased in epithelial cells remote from inflamed areas in CD patients. To explore this finding, Nod2 mRNA expression, inflammation and gut permeability were examined in the small bowel of wild-type (WT), Nod2 knockout and Nod2 mutant mice after rectal instillation of 2,4,6-trinitrobenzene sulfonic acid (TNBS). In WT mice, Nod2 upregulation remote to rectal injury was associated with pro-inflammatory cytokine expression, recirculating CD4+ T-cells, increased paracellular permeability and myosin like chain kinase activity. Nod2 knockout or mutation led to duodenitis and ileitis demonstrating the remote protective role of Nod2. Bone morrow stem cell (BMSC) transplantations indicated that the small intestinal inflammation was due to NOD2 loss in both hematopoietic and non-hematopoietic compartments. As a whole, WT but not mutant NOD2 prevents disease extension at sites remote from the initial intestinal injury.

immunology↗

bioRxiv2018

cAMP stimulates SLC26A3 activity in human colon by a CFTR-dependent mechanism that does not require CFTR activity

Background & AimsDRA (SLC26A3) is an electroneutral Cl-/HCO3- exchanger that is present in the apical domain of multiple intestinal segments. An area that has continued to be poorly understood is related to DRA regulation in acute cAMP-related diarrheas, in which DRA appears to be both inhibited as part of NaCl absorption and stimulated to contribute to increased HCO3- secretion. Different cell models expressing DRA have shown that cAMP inhibits, stimulates or does not affect its activity.\n\nMethodsThis study reevaluated cAMP regulation of DRA using new \"tools\" including a successful knockout cell model, a specific DRA inhibitor (DRAinh-A250), specific antibodies, and a transport assay that did not rely on non-specific inhibitors. The studies compared DRA regulation in colonoids made from normal human colon with regulation in the colon cancer cell line, Caco-2.\n\nResultsDRA is an apical protein in human proximal colon, differentiated colonoid monolayers and Caco-2 cells. It is glycosylated and appears as two bands. cAMp(forskolin) acutely stimulated DRA activity in human colonoids and Caco-2 cells. In these cells, DRA is the predominant apical Cl-/HCO3- exchanger and is inhibited by DRAinh-A250 with IC50 of 0.5 mol/L and 0.2 {micro}mol/L, respectively. However, there was no effect of cAMP in HEK293/DRA cells that lacked CFTR. When CFTR was expressed in HEK293/DRA cells, cAMP also stimulated DRA activity. In all cases, cAMP stimulation of DRA was not inhibited by CFTRinh-172.\n\nConclusionsDRA is acutely stimulated by cAMP by a process that is CFTR-dependent but appears to be one of multiple regulatory effects of CFTR that does not require CFTR activity.

physiology↗

bioRxiv2017

An essential post-developmental role for Lis1 in mice

LIS1 mutations cause lissencephaly (LIS), a severe developmental brain malformation. Much less is known about its role in the mature nervous system. LIS1 regulates the microtubule motor cytoplasmic dynein 1 (dynein), and as LIS1 and dynein are both expressed in the adult nervous system, Lis1 could potentially regulate dynein-dependent processes such as axonal transport. We therefore knocked out Lis1 in adult mice using tamoxifen-induced, Cre-ER-mediated recombination. When an actin promoter was used to drive Cre-ER expression (Act-Cre-ER), heterozygous Lis1 KO caused no obvious change in viability or behavior, despite evidence of widespread recombination by a Cre reporter three weeks after tamoxifen exposure. In contrast, homozygous Lis1 KO caused the rapid onset of neurological symptoms in both male and female mice. One tamoxifen-dosing regimen caused prominent recombination in the midbrain/hindbrain, PNS, and cardiac/skeletal muscle within a week; these mice developed severe symptoms in that time frame and were euthanized. A different tamoxifen regimen resulted in delayed recombination in midbrain/hindbrain, but not in other tissues, and also delayed the onset of symptoms. This indicates that Lis1 loss in the midbrain/hindbrain causes the severe phenotype. In support of this, brainstem regions known to house cardiorespiratory centers showed signs of axonal dysfunction in KO animals. Transport defects, neurofilament alterations, and varicosities were observed in axons in cultured DRG neurons from KO animals. Because no symptoms were observed when a cardiac specific Cre-ER promoter was used, we propose a vital role for Lis1 in autonomic neurons and implicate defective axonal transport in the KO phenotype.\n\nSIGNIFICANCE STATEMENTMammalian Lis1 is best known for its role in brain development. Lis1 binds to and regulates the microtubule motor, cytoplasmic dynein. We show that Lis1 function is needed post-developmentally and provide evidence that loss of Lis1 in the hindbrain leads to death. The effect is dose dependent in mice, as loss of only one allele does not produce an overt phenotype. However, since LIS1 haploinsufficiency causes lissencephaly (LIS) in humans, our study raises the possibility that post-developmental axonal transport defects could contribute to worsening symptoms in children with LIS1 mutations. Our data are consistent with the hypothesis is that Lis1 regulates dynein-dependent axon transport in the mature nervous system.

neuroscience↗