Cell biology
Abstract
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.
Authors
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
Abstract
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.
Authors
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
Abstract
Although congenital heart defects (CHDs) represent the most common birth defect, a comprehensive understanding of disease etiology remains unknown. This is further complicated since CHDs can occur in isolation or as a feature of another disorder. Analyzing disorders with associated CHDs provides a powerful platform to identify primary pathogenic mechanisms driving disease. Aberrant localization and expression of cathepsin proteases can perpetuate later-stage heart diseases, but their contribution toward CHDs is unclear. To investigate the contribution of cathepsins during cardiovascular development and congenital disease, we analyzed the pathogenesis of cardiac defects in zebrafish models of the lysosomal storage disorder mucolipidosis II (MLII). MLII is caused by mutations in the GlcNAc-1-phosphotransferase enzyme (Gnptab) that disrupt carbohydrate-dependent sorting of lysosomal enzymes. Without Gnptab, lysosomal hydrolases, including cathepsin proteases, are inappropriately secreted. Analyses of heart development in gnptab-deficient zebrafish show cathepsin K secretion increases its activity, disrupts TGF-β–related signaling, and alters myocardial and valvular formation. Importantly, cathepsin K inhibition restored normal heart and valve development in MLII embryos. Collectively, these data identify mislocalized cathepsin K as an initiator of cardiac disease in this lysosomal disorder and establish cathepsin inhibition as a viable therapeutic strategy.
Authors
Po-Nien Lu, Trevor Moreland, Courtney J. Christian, Troy C. Lund, Richard A. Steet, Heather Flanagan-Steet
Abstract
Actin-associated nonmuscle myosin II (NM2) motor proteins play critical roles in a myriad of cellular functions including endocytosis and organelle transport pathways. Cell type-specific expression and unique subcellular localization of the NM2 proteins, encoded by the Myh9 and Myh10 genes, in the mouse kidney tubules led us to hypothesize that these proteins have specialized functional roles within the renal epithelium. Inducible, conditional knockout (cKO) of Myh9 and Myh10 in the renal tubules of adult mice resulted in progressive kidney disease. Prior to overt renal tubular injury, we observed intracellular accumulation of the GPI-anchored protein uromodulin and gradual loss of Na+ K+ 2Cl- cotransporter from the apical membrane of the thick ascending limb (TAL) epithelia. The UMOD accumulation coincided with expansion of endoplasmic reticulum (ER) tubules, activation of ER stress and unfolded protein response pathways in Myh9&10 cKO kidneys. We conclude that NM2 proteins are required for localization and transport of UMOD and loss of function results in accumulation of UMOD and ER stress mediated progressive renal tubulointerstitial disease. These observations establish cell type-specific role(s) for NM2 proteins in regulation of specialized renal epithelial transport pathways and reveal the possibility that human kidney disease associated with MYH9 mutations could be of renal epithelial origin..
Authors
Karla L. Otterpohl, Brook W. Busselman, Ishara Ratnayake, Ryan G. Hart, Kimberly Hart, Claire Evans, Carrie L. Phillips, Jordan R. Beach, Phil Ahrenkiel, Bruce Molitoris, Kameswaran Surendran, Indra Chandrasekar
Abstract
The ability of HDL to inhibit inflammation in adipocytes and adipose tissue is reduced when HDL contains serum amyloid A (SAA) due to trapping of SAA in HDL by proteoglycans at the adipocyte surface. Since we recently found that the major extracellular matrix proteoglycan produced by hypertrophic adipocytes is versican, whereas activated adipose tissue macrophages produce mainly biglycan, the role of proteoglycans in determining the anti-inflammatory properties of HDL was further investigated. The distribution of versican, biglycan, apolipoprotein A-I (the major apolipoprotein of HDL) and SAA were similar in adipose tissue from obese mice and obese human subjects. Co-localization of SAA-enriched HDL with versican and biglycan at the cell surface of adipocyte and peritoneal macrophages, respectively, was blocked by silencing these proteoglycans, which also restored the anti-inflammatory property of SAA-enriched HDL despite the presence of SAA. Similar to adipocytes, normal HDL exerts its anti-inflammatory function in macrophages by reducing lipid rafts, reactive oxygen species generation and translocation of toll like receptor 4 and NADPH oxidase 2 into lipid rafts, effects that are not observed with SAA-enriched HDL. These findings imply that SAA present in HDL can be trapped by adipocyte-derived versican and macrophage-derived biglycan, thereby blunting HDL’s anti-inflammatory properties.
Authors
Chang Yeop Han, Inkyung Kang, Mohamed Omer, Shari Wang, Tomasz Wietecha, Thomas N. Wight, Alan Chait
Abstract
Ischemia-reperfusion-induced edema (IRE) one of the most significant causes of mortality after lung transplantation can be mimicked ex-vivo in isolated perfused mouse lungs (IPL). Transient receptor potential vanilloid 4 (TRPV4) is a non-selective cation channel studied in endothelium, while its role in the lung epithelium remains elusive. Here we show enhanced IRE in TRPV4-deficient (TRPV4–/–) IPL compared to wild-type (WT) controls, indicating a protective role of TRPV4 to maintain the alveolar epithelial barrier. By immunohistochemistry, mRNA profiling and electrophysiological characterization, we detected TRPV4 in bronchial epithelium, alveolar type I (ATI) and alveolar type II (ATII) cells. Genetic ablation of TRPV4 resulted in reduced expression of the water conducting aquaporin-5 (AQP-5) channel in ATI cells. Migration of TRPV4–/– ATI cells was reduced and cell barrier function was impaired. Analysis of isolated primary TRPV4-deficient ATII cells revealed a reduced expression of surfactant protein C (SP-C) and the TRPV4 activator GSK1016790A induced increases in current densities only in WT ATII cells. Moreover, TRPV4–/– lungs of adult mice developed significantly larger mean chord lengths and altered lung function compared to WT lungs. Therefore, our data discover essential functions of TRPV4 channels in alveolar epithelial cells and in the protection from edema formation.
Authors
Jonas Weber, Suhasini Rajan, Christian Schremmer, Yu-Kai Chao, Gabriela Krasteva-Christ, Martina Kannler, Ali Önder Yildirim, Monika Brosien, Johann Schredelseker, Norbert Weissmann, Christian Grimm, Thomas Gudermann, Alexander Dietrich
Abstract
Focal adhesion kinase (FAK) is an important mediator of extracellular matrix-integrin mechano-signal transduction that regulates cell motility, survival, and proliferation. As such, FAK is being investigated as a potential therapeutic target for malignant and fibrotic diseases, and numerous clinical trials of FAK inhibitors are underway. The function of FAK in non-malignant non-motile epithelial cells is not well understood. We previously showed that hepatocytes demonstrated activated FAK near stiff collagen tracts in fibrotic liver. In this study, we examined the role of liver epithelial FAK by inducing fibrotic liver disease in mice with liver epithelial FAK deficiency. We found that mice that lack FAK in liver epithelial cells develop more severe liver injury and worse fibrosis as compared to controls. Increased fibrosis in liver epithelial FAK-deficient mice is linked to the activation of several pro-fibrotic pathways, including the hedgehog-smoothened pathway. FAK-deficient hepatocytes produce increased Indian hedgehog in a manner dependent on matrix stiffness. Furthermore, expression of the hedgehog receptor, smoothened, is increased in macrophages and biliary cells of hepatocyte-specific FAK-deficient fibrotic liver. These results indicate that liver epithelial FAK has important regulatory roles in the response to liver injury and progression of fibrosis.
Authors
Yun Weng, Tyler J. Lieberthal, Vivian X. Zhou, Maya Lopez-Ichikawa, Manuel Armas-Phan, Tristan K. Bond, Miya C. Yoshida, Won-Tak Choi, Tammy T. Chang
Abstract
Huntington’s disease (HD) is a progressive autosomal dominant neurodegenerative disorder affecting striatal neurons beginning in young adults with loss of muscle coordination and cognitive decline. Less appreciated is the fact that HD patients also exhibit cardiac and respiratory dysfunction including pulmonary insufficiency and cardiac arrhythmias. The underlying mechanism for these symptoms is poorly understood. In the present study we provide insight into the cause of cardiorespiratory dysfunction in HD and identify a novel therapeutic target. We now show that intracellular calcium (Ca2+) leak via post-translationally modified ryanodine receptor/intracellular calcium release (RyR) channels plays an important role in HD pathology. RyR channels were oxidized, PKA phosphorylated and leaky in brain, heart and diaphragm in both HD patients and in a murine model of HD (Q175). HD mice (Q175) with endoplasmic reticulum (ER) Ca2+ leak exhibited cognitive dysfunction, decreased parasympathetic tone associated with cardiac arrhythmias, and reduced diaphragmatic contractile function resulting in impaired respiratory function. Defects in cognitive, motor and respiratory functions were ameliorated by treatment with a novel Rycal small molecule drug (S107) that fixes leaky RyR. Thus, leaky RyRs likely play a role in neuronal, cardiac and diaphragmatic pathophysiology in HD and identify RyRs as a potential novel therapeutic target.
Authors
Haikel Dridi, Xiaoping Liu, Qi Yuan, Steve Reiken, Yehya Mohamad, Leah R. Sittenfeld, Panagiota Apostolou, Julie Buron, Pierre Sicard, Stefan Matecki, Jérôme Thireau, Clement Menuet, Alain Lacampagne, Andrew R. Marks
Abstract
Prader-Willi syndrome (PWS) is a developmental disorder caused by loss of maternally imprinted genes on 15q11-q13, including melanoma antigen gene family member L2 (MAGEL2). The clinical phenotypes of PWS suggest impaired hypothalamic neuroendocrine function; however, the exact cellular defects are unknown. Here, we report deficits in secretory granule (SG) abundance and bioactive neuropeptide production upon loss of MAGEL2 in humans and mice. Unbiased proteomic analysis of Magel2pΔ/m+ mice revealed a reduction in components of SG in the hypothalamus that was confirmed in 2 PWS patient–derived neuronal cell models. Mechanistically, we show that proper endosomal trafficking by the MAGEL2-regulated WASH complex is required to prevent aberrant lysosomal degradation of SG proteins and reduction of mature SG abundance. Importantly, loss of MAGEL2 in mice, NGN2-induced neurons, and human patients led to reduced neuropeptide production. Thus, MAGEL2 plays an important role in hypothalamic neuroendocrine function, and cellular defects in this pathway may contribute to PWS disease etiology. Moreover, these findings suggest unanticipated approaches for therapeutic intervention.
Authors
Helen Chen, A. Kaitlyn Victor, Jonathon Klein, Klementina Fon Tacer, Derek J.C. Tai, Celine de Esch, Alexander Nuttle, Jamshid Temirov, Lisa C. Burnett, Michael Rosenbaum, Yiying Zhang, Li Ding, James J. Moresco, Jolene K. Diedrich, John R. Yates III, Heather S. Tillman, Rudolph L. Leibel, Michael E. Talkowski, Daniel D. Billadeau, Lawrence T. Reiter, Patrick Ryan Potts
Abstract
Based on its clinical benefits, Trikafta, the combination of folding correctors VX-661 (tezacaftor), VX-445 (elexacaftor), and the gating potentiator VX-770 (ivacaftor) was FDA-approved for treatment of cystic fibrosis (CF) patients carrying deletion of phenylalanine 508 (F508del) of the CF Transmembrane Conductance Regulator (CFTR) on at least one allele. Neither the mechanism of action of VX-445, nor the susceptibility of rare CF folding mutants to Trikafta are known. Here we show that in human bronchial epithelial cells, VX-445 synergistically restores F508del-CFTR processing in combination with type I or II correctors that target the nucleotide binding domain 1 (NBD1)-membrane spanning domains (MSDs) interface and NBD2, respectively, consistent with a type III corrector mechanism. This inference was supported by the VX-445 binding to and unfolding suppression of the isolated F508del-NBD1 of CFTR. The VX-661+VX-445 treatment restored F508del-CFTR chloride channel function in the presence of VX-770 to ~62% of wild-type CFTR in homozygous nasal epithelia. Substantial rescue of rare misprocessing mutations (S13F, R31C, G85E, E92K, V520F, M1101K and N1303K), confined to MSD1, MSD2, NBD1 and NBD2 of CFTR, was also observed in airway epithelia, suggesting an allosteric correction mechanism and the possible application of Trikafta for patients with rare misfolding mutants of CFTR.
Authors
Guido Veit, Ariel Roldan, Mark A. Hancock, Dillon F. Da Fonte, Haijin Xu, Maytham Hussein, Saul Frenkiel, Elias Matouk, Tony Velkov, Gergely L. Lukacs
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