Cantύ Syndrome (CS), caused by gain-of-function (GOF) mutations in pore-forming (Kir6.1, KCNJ8) and accessory (SUR2, ABCC9) ATP-sensitive potassium (KATP) channel subunit genes, is frequently accompanied by gastrointestinal (GI) dysmotility, and we describe one CS patient who required an implanted intestinal irrigation system for successful stooling. We used gene-modified mice to assess the underlying KATP channel subunits in gut smooth muscle, and to model the consequences of altered KATP channels in CS gut. We show that Kir6.1/SUR2 subunits underlie smooth muscle KATP channels throughout the small intestine and colon. Knock-in mice, carrying human KCNJ8 and ABCC9 CS mutations in the endogenous loci, exhibit reduced intrinsic contractility throughout the intestine, resulting in death when weaned onto solid food in the most severely affected animals. Death is avoided by weaning onto a liquid gel diet, implicating intestinal insufficiency and bowel impaction as the underlying cause, and GI transit is normalized by treatment with the KATP inhibitor glibenclamide. We thus define the molecular basis of intestinal KATP channel activity, the mechanism by which overactivity results in GI insufficiency, and a viable approach to therapy.
Nathaniel W. York, Helen Parker, Zili Xie, David Tyus, Maham A. Waheed, Zihan Yan, Dorothy K. Grange, Maria S. Remedi, Sarah K. England, Hongzhen Hu, Colin G. Nichols
The habenula (Hb) is a bilateral, evolutionarily conserved epithalamic structure connecting forebrain and midbrain structures that has gained attention for its roles in depression,(1) addiction,(2-5) rewards processing,(6) and motivation (7,8). Of its two major subdivisions, the medial (MHb) and lateral Hb (LHb), MHb circuitry and function is poorly understood relative to LHb (9). Prkar2a codes for cAMP-dependent protein kinase (PKA) regulatory subunit IIα (RIIα), a component of the PKA holoenzyme at the center of one of the major cell-signaling pathways conserved across systems and species. Type 2 regulatory subunits (RIIα, RIIβ) determine the subcellular localization of PKA, and unlike other PKA subunits, Prkar2a has minimal brain expression except in the MHb (10). We previously showed that RIIα knockout (RIIαKO) mice resist diet-induced obesity (DIO) (11). In the present study, we report that RIIαKO mice have decreased consumption of palatable, “rewarding” foods and increased motivation for voluntary exercise. Prkar2a deficiency led to decreased habenular PKA enzymatic activity and impaired dendritic localization of PKA catalytic subunits in MHb neurons. Re-expression of Prkar2a in the Hb rescued this phenotype confirming differential roles for Prkar2a in regulating the drives for palatable foods and voluntary exercise. Our findings show that in the MHb decreased PKA signaling and dendritic PKA activity decrease motivation for food rewards while enhancing the motivation for exercise, a desirable combination of behaviors.
Edra London, Jason C. Wester, Michelle S. Bloyd, Shelby Bettencourt, Chris J. McBain, Constantine A. Stratakis
Ongoing societal changes in views on medical and recreational roles of cannabis increased the use of concentrated plant extracts with a Δ9-tetrahydrocannabinol (THC) content of >90%. Even though prenatal THC exposure is widely considered adverse for neuronal development, equivalent experimental data for young age cohorts are largely lacking. Here, we administered plant-derived THC (1 or 5 mg/kg) to mice daily during postnatal days (P)5-16 and P5-35 and monitored its effects on hippocampal neuronal survival and specification by high resolution imaging and the hippocampal proteome by iTRAQ proteomics, respectively. We find that THC indiscriminately affects pyramidal cells and both cannabinoid receptor 1 (CB1R)+ and CB1R- interneurons by P16. THC particularly disrupted the expression of mitochondrial proteins (complexes I-IV), a change that had persisted even 4 months after the end of drug exposure. This was reflected by a THC-induced loss of membrane integrity occluding mitochondrial respiration and could be partially or completely rescued by pH stabilization, antioxidants, bypassed glycolysis, and targeting either mitochondrial soluble adenylyl cyclase or the mitochondrial voltage-dependent anion channel. Overall, THC exposure during infancy induces significant and long-lasting reorganization of neuronal circuits through mechanisms that, in a large part, render cellular bioenergetics insufficient to sustain key developmental processes in otherwise healthy neurons.
Johannes Beiersdorf, Zsofia Hevesi, Daniela Calvigioni, Jakob Pyszkowski, Roman A. Romanov, Edit Szodorai, Gert Lubec, Sally L. Shirran, Catherine H. Botting, Siegfried Kasper, Geoffrey W. Guy, Roy A. Gray, Vincenzo Di Marzo, Tibor Harkany, Erik Keimpema
Loss of functional small bowel surface area following surgical resection for disorders such as Crohn’s disease, intestinal ischemic injury, radiation enteritis, and in children, necrotizing enterocolitis, atresia and gastroschisis, may result in short bowel syndrome (SBS) with attendant high morbidity, mortality and health care costs in the U.S. Following resection, the remaining small bowel epithelium mounts an adaptive response resulting in increased crypt cell proliferation, increased villus height, crypt depth and enhanced nutrient and electrolyte absorption. Although these morphologic and functional changes are well-described in animal models, the adaptive response in humans is less well understood, and clinically the response is unpredictable and often inadequate. Here we address the hypotheses that human intestinal stem cell populations are expanded and the stem cell niche is regulated following massive gut resection in short bowel syndrome. We use intestinal enteroid cultures from SBS patients to show that the magnitude and phenotype of the adaptive stem cell response is regulated by stromal niche cells including intestinal subepithelial myofibroblasts, which are activated by intestinal resection to enhance epithelial stem and proliferative cell responses. Our data suggest that myofibroblast regulation of bone morphogenetic protein signaling pathways plays a role in the gut adaptive response post resection.
Vered Gazit, Elzbieta A. Swietlicki, Miranda U. Liang, Adam Surti, Raechel McDaniel, Mackenzie Geisman, David M. Alvarado, Matthew A. Ciorba, Grant V. Bochicchio, Obeid Ilahi, John Kirby, William J. Symons, Nicholas O. Davidson, Marc S. Levin, Deborah C. Rubin
Alveolar macrophages (AM) are differentially regulated by human surfactant protein-A (SP-A)1 or SP-A2. However, AM are very heterogeneous and differences are difficult to characterize in intact cells. Using the Toponome Imaging System (TIS), an imaging technique that uses sequential immunostaining to identity patterns of biomarker expression or combinatorial molecular phenotypes (CMP), we studied individual single cells and identified subgroups of AM (n=168) from SP-A knockout (KO) mice and mice expressing either SP-A1 or SP-A2. The effects, as shown by CMPs, of SP-A1 and SP-A2 on AM were significant and differed. SP-A1 AM were the most diverse and shared the fewest CMPs with KO and SP-A2. Clustering analysis of each group showed three clusters where the CMP-based phenotype was distinct in each cluster. Moreover, a clustering analysis of all 168 AM revealed ten clusters, many dominated by one group. Some CMP, overlap among groups was observed with SP-A2 AM sharing the most CMPs and SP-A1 AM the fewest. The CMP-based patterns identified here provide a basis for not only understanding AM diversity, but, most importantly, the molecular basis for the diversity of functional differences in mouse models where the impact of genetics of innate immune molecules on AM has been studied.
David S. Phelps, Vernon M. Chinchilli, Judith Weisz, Lili Yang, Debra Shearer, Xuesheng Zhang, Joanna Floros
Atrial fibrillation (AF) commonly occurs after surgery and is associated with atrial remodeling. TRPV4 is functionally expressed in the heart, and its activation affects cardiac structure and functions. We hypothesized that TRPV4 blockade alleviates atrial remodeling and reduces AF induction in sterile pericarditis (SP) rats. TRPV4 antagonist GSK2193874 or vehicle was orally administered 1 d before pericardiotomy. AF susceptibility and atrial function were assessed using in vivo electrophysiology, ex vivo optical mapping, patch-clamp, and molecular biology on day 3 after surgery. TRPV4 expression increased in the atria of SP rats and patients with AF. GSK2193874 significantly reduced AF vulnerability in vivo and the frequency of atrial ectopy and AF with a reentrant pattern ex vivo. Mechanistically, GSK2193874 reversed the abnormal action potential duration (APD) prolongation in atrial myocytes through the regulation of voltage-gated K+ currents (IK), reduced the activation of atrial fibroblasts by inhibiting P38, AKT, and STAT3 pathways, and alleviated the infiltration of immune cells. Our results reveal that TRPV4 blockade prevents abnormal changes in atrial myocyte electrophysiology and ameliorated atrial fibrosis and inflammation in SP rats, and, therefore, might be a promising strategy to treat AF, particularly post-operative AF.
Jie Liao, Qiongfeng Wu, Cheng Qian, Ning Zhao, Zhaoyang Zhao, Kai Lu, Shaoshao Zhang, Qian Dong, Lei Chen, Qince Li, Yimei Du
Resident vascular adventitial SCA1(+) progenitor (AdvSca1) cells are essential in vascular development and injury. However, the heterogeneity of AdvSca1 cells presents a unique challenge in understanding signaling pathways orchestrating their behavior in homeostasis and injury responses. Using smooth muscle cell (SMC) lineage tracing models, we identified a subpopulation of AdvSca1 cells (AdvSca1-SM) originating from mature SMCs that undergo reprogramming in situ and exhibit a multipotent phenotype. Here we employed lineage tracing and RNA sequencing to define the signaling pathways regulating SMC-to-AdvSca1-SM cell reprogramming and AdvSca1-SM progenitor cell phenotype. Unbiased hierarchical clustering revealed that genes related to hedgehog/WNT/beta-catenin signaling are significantly enriched in AdvSca1-SM cells, emphasizing the importance of this signaling axis in the reprogramming event. Leveraging AdvSca1-SM-specific expression of Gli1, we generated Gli1-CreERT2-ROSA26-YFP reporter mice to selectively track AdvSca1-SM cells. We demonstrated that physiologically relevant vascular injury or AdvSca1-SM cell-specific Klf4 depletion facilitated the proliferation and differentiation of AdvSca1-SM cells to a pro-fibrotic myofibroblast phenotype rather than macrophages. Surprisingly, AdvSca1-SM cells selectively contributed to adventitial remodeling and fibrosis, but little to neointima formation. Together, these findings strongly support therapeutics aimed at preserving the AdvSca1-SM cell phenotype as a viable anti-fibrotic approach.
Sizhao Lu, Austin J. Jolly, Keith A. Strand, Allison M. Dubner, Marie F. Mutryn, Karen S. Moulton, Raphael A. Nemenoff, Mark W. Majesky, Mary C.M. Weiser-Evans
Proprotein convertase subtilisin/kexin type 9 (PCSK9) affects cholesterol homeostasis by targeting hepatic low-density lipoprotein receptor (LDLR) for lysosomal degradation. Clinically, PCSK9 inhibitors effectively reduce LDL cholesterol (LDL-C) level and the incidence of cardiovascular events. Because microRNAs (miRs) are integral regulators of cholesterol homeostasis, we investigated the involvement of miR-483 in regulating LDL-C metabolism. Using in silico analysis, we predicted that miR-483-5p targets the 3’UTR of PCSK9 mRNA. In HepG2 cells, miR-483-5p targeted the PCSK9 3’UTR, leading to decreased PCSK9 protein and mRNA expression, increased LDLR expression and enhanced LDL-C uptake. In hyperlipidemic mice and humans, serum levels of total cholesterol and LDL-C were inversely correlated with miR-483-5p level. In mice, hepatic miR-483 overexpression increased LDLR level by targeting Pcsk9, with a significant reduction in plasma total cholesterol and LDL-C levels. Mechanistically, the cholesterol-lowering effect of miR-483-5p was significant in mice receiving AAV8 PCSK9-3’UTR but not Ldlr-knockout mice or mice receiving AAV8 PCSK9-3’UTR (deltaBS) with the miR-483-5p targeting site deleted. Thus, exogenously administered miR-483 or similarly optimized compounds have potential to ameliorate hypercholesterolemia.
Jianjie Dong, Ming He, Jie Li, Ariane R. Pessentheiner, Chen Wang, Jin Zhang, Yameng Sun, Wei-Ting Wang, Yuqing Zhang, Junhui Liu, Shen-Chih Wang, Po-Hsun Huang, Philip L.S.M. Gordts, Zu-Yi Yuan, Sotirios Tsimikas, John Y-J Shyy
Cardiac ischemia is associated with arrhythmias, but effective therapies are limited. The cardiac voltage gated sodium channel α-subunit (SCN5A), encoding the Nav1.5 current, plays a key role in the cardiac electrical conduction and arrhythmic risk. Here, we show that hypoxia reduces Nav1.5 through effects on a microRNA (miR), miR-448. The expression of miR-448 is increased in ischemic cardiomyopathy. miR-448 has a conserved binding site in 3’-UTR of SCN5A. miR-448 binding to this site suppressed SCN5A expression and sodium currents. Hypoxia-induced HIF1α and NF-κB were major transcriptional regulators for MIR448. Hypoxia also relieved MIR448 transcriptional suppression by RE1 silencing transcription factor (REST). Inhibition of miR-448 reduced arrhythmic risk after myocardial infarction. These results indicated that ischemia drove miR-448 expression, reduced Nav1.5 current and increased arrhythmic risk. Arrhythmic risk was improved by preventing Nav1.5 downregulation, suggesting a new approach to antiarrhythmic therapy.
Gyeoung-Jin Kang, An Xie, Hong Liu, Samuel C. Dudley, Jr.
A possible etiological link between the onset of endemic pemphigus in Tunisia and bites of Phlebotomus (P). papatasi, the vector of zoonotic cutaneous leishmaniasis has been previously suggested. We hypothesized that the immunodominant P. papatasi salivary protein PpSP32 binds to desmogleins (Dsg1 and Dsg3), triggering loss of tolerance to these pemphigus target autoantigens. We here show by Far-Western blot that the recombinant PpSP32 protein (rPpSP32) binds to epidermal proteins with a molecular weight of about 170kDa. Co-immunoprecipitation revealed the interaction of rPpSP32 with either Dsg1 or Dsg3. A specific interaction between PpSP32 and Dsg (1 and 3) was further demonstrated by ELISA assays. Finally, mice immunized with rPpSP32 twice a week exhibited significantly increased levels of anti-Dsg1 and anti-Dsg3 antibodies from day 75 to day 120. Such antibodies were specific for Dsg1 and Dsg3 and were not the result of crossreactivity to PpSP32. Herein, we demonstrated for the first time a specific binding between the PpSP32 and the dsg1 and 3, which might underlie the triggering of anti-Dsg antibodies in patients exposed to sand fly bites. We also confirmed the development of specific anti-Dsg1 and -Dsg3 antibodies in vivo after PpSP32 immunization in mice. Collectively, our results provide evidence that environmental factors, such the exposure to P. papatasi bites, can trigger the development of autoimmune antibodies.
Soumaya Marzouki, Ines Zaraa, Maha Abdeladhim, Chaouki Ben Abdessalem, Fabiano Oliveira, Shaden Kamhawi, Mourad Mokni, Hechmi Louzir, Jesus Valenzuela, Mélika Ben Ahmed
Caspase 8 (CASP8) is one of the most frequently mutated genes in head and neck squamous carcinomas (HNSCC), and CASP8 mutations are associated with poor survival. The distribution of these mutations in HNSCC suggests that they are likely to be inactivating. Inhibition of CASP8 has been reported to sensitize cancer cells to necroptosis, a regulated cell death mechanism. Here, we show that knockdown of CASP8 renders HNSCCs susceptible to necroptosis by a second mitochondria-derived activator of caspase (SMAC) mimetic, Birinapant, in combination with pan-caspase inhibitors zVAD FMK or Emricasan and radiation. In a syngeneic mouse model of oral cancer, Birinapant, particularly when combined with radiation delayed tumor growth and enhanced survival under CASP8 loss. Exploration of molecular underpinnings of necroptosis sensitivity confirmed that the level of functional receptor-interacting serine/threonine-protein kinase 3 (RIP3) determines susceptibility to this mode of death. Although an in vitro screen revealed that low RIP3 levels render many HNSCC cell lines resistant to necroptosis, patient tumors maintain RIP3 expression and should therefore remain sensitive. Collectively, these results suggest that targeting the necroptosis pathway with SMAC mimetics, especially in combination with radiation, may be relevant therapeutically in HNSCC with compromised CASP8 status, provided that RIP3 function is maintained.
Burak Uzunparmak, Meng Gao, Antje Lindemann, Kelly Erikson, Li Wang, Eric Lin, Steven J. Frank, Frederico O. Gleber-Netto, Mei Zhao, Heath D. Skinner, Jared M. Newton, Andrew G. Sikora, Jeffrey N. Myers, Curtis R. Pickering
Pituitary developmental defects lead to partial or complete hormone deficiency and significant health problems. The majority of cases are sporadic and of unknown cause. We screened 28 patients with pituitary stalk interruption syndrome (PSIS) for mutations in the FAT/DCHS family of protocadherins that have high functional redundancy. We identified seven variants, four of which putatively damaging, in FAT2 and DCHS2 in six patients with pituitary developmental defects recruited through a cohort of patients with mostly ectopic posterior pituitary gland and/or pituitary stalk interruption. All patients had growth hormone deficiency and two presented with multiple hormone deficiencies and small glands. FAT2 and DCHS2 were strongly expressed in the mesenchyme surrounding the normal developing human pituitary. We analyzed Dchs2-/- mouse mutants and identified anterior pituitary hypoplasia and partially penetrant infundibular defects. Overlapping infundibular abnormalities and distinct anterior pituitary morphogenesis defects were observed in Fat4-/- and Dchs1-/- mouse mutants but all animal models displayed normal commitment to the anterior pituitary cell type. Together our data implicate FAT/DCHS protocadherins in normal hypothalamic-pituitary development and identify FAT2 and DCHS2 as candidates underlying pituitary gland developmental defects such as ectopic pituitary gland and/or pituitary stalk interruption.
Emily J. Lodge, Paraskevi Xekouki, Tatiane S. Silva, Cristiane Kochi, Carlos A. Longui, Fabio R. Faucz, Alice Santambrogio, James L. Mills, Nathan Pankratz, John Lane, Dominika Sosnowska, Tina Hodgson, Amanda L. Patist, Philippa Francis-West, Francoise Helmbacher, Constantine Stratakis, Cynthia L. Andoniadou
We remain largely without effective prophylactic/therapeutic interventions for COVID-19. Although many human COVID-19 clinical trials are ongoing, there remains a deficiency of supportive preclinical drug efficacy studies to help guide decisions. Here we assessed the prophylactic/therapeutic efficacy of hydroxychloroquine (HCQ), a drug of interest for COVID-19 management, in two animal disease models. The standard human malaria HCQ prophylaxis (6.5 mg/kg given weekly) and treatment (6.5 mg/kg) did not significantly benefit clinical outcome nor reduce SARS-CoV-2 replication/shedding in the upper and lower respiratory tract in the rhesus macaque disease model. Similarly, when used for prophylaxis or treatment neither the standard human malaria dose (6.5 mg/kg) nor a high dose (50 mg/kg) of HCQ had any beneficial effect on clinical disease or SARS-CoV-2 kinetics (replication/shedding) in the Syrian hamster disease model. Results from these two preclinical animal models may prove helpful in guiding clinical use of HCQ for prophylaxis/treatment of COVID-19.
Kyle Rosenke, Michael A. Jarvis, Friederike Feldmann, Benjamin Schwarz, Atsushi Okumura, Jamie Lovaglio, Greg Saturday, Patrick W. Hanley, Kimberly Meade-White, Brandi N. Williamson, Frederick A. Hansen, Lizette Pérez-Pérez, Shanna Leventhal, Tsing-Lee Tang-Huau, Julie Callison, Elaine Haddock, Kaitlin A. Stromberg, Graham Sewell, Dana Scott, Catharine M. Bosio, David W. Hawman, Emmie de Wit, Heinz Feldmann