Life Sciences Commons^™

Dsarm/Sarm1 Is Required For Activation Of An Injury-Induced Axon Death Pathway, Jeannette Osterloh, Jing Yang, Timothy Rooney, A. Fox, Robert Adalbert, Eric Powell, Amy Sheehan, Michelle Avery, Rachel Hackett, Mary Logan, Jennifer Macdonald, Jennifer Ziegenfuss, Stefan Milde, Ying-Ju Hou, Carl Nathan, Aihao Ding, Robert Brown, Laura Comforti, Michael Coleman, Marc Tessier-Lavigne, Stephan Zuchner, Marc Freeman

Dr Robert Brown

Axonal and synaptic degeneration is a hallmark of peripheral neuropathy, brain injury, and neurodegenerative disease. Axonal degeneration has been proposed to be mediated by an active autodestruction program, akin to apoptotic cell death; however, loss-of-function mutations capable of potently blocking axon self-destruction have not been described. Here, we show that loss of the Drosophila Toll receptor adaptor dSarm (sterile alpha/Armadillo/Toll-Interleukin receptor homology domain protein) cell-autonomously suppresses Wallerian degeneration for weeks after axotomy. Severed mouse Sarm1 null axons exhibit remarkable long-term survival both in vivo and in vitro, indicating that Sarm1 prodegenerative signaling is conserved in mammals. Our results provide direct …

Targeted Mutation Of Mouse Skeletal Muscle Sodium Channel Produces Myotonia And Potassium-Sensitive Weakness, Lawrence Hayward, Joanna Kim, Ming-Yang Lee, Hongru Zhou, Ji Kim, Kumudini Misra, Mohammad Salajegheh, Fen-Fen Wu, Shinji Matsuda, Valerie Reid, Didier Cros, Eric Hoffman, Jean-Marc Renaud, Stephen Cannon, Robert Brown

Dr Robert Brown

Hyperkalemic periodic paralysis (HyperKPP) produces myotonia and attacks of muscle weakness triggered by rest after exercise or by K+ ingestion. We introduced a missense substitution corresponding to a human familial HyperKPP mutation (Met1592Val) into the mouse gene encoding the skeletal muscle voltage-gated Na+ channel NaV1.4. Mice heterozygous for this mutation exhibited prominent myotonia at rest and muscle fiber-type switching to a more oxidative phenotype compared with controls. Isolated mutant extensor digitorum longus muscles were abnormally sensitive to the Na+/K+ pump inhibitor ouabain and exhibited age-dependent changes, including delayed relaxation and altered generation of tetanic force. Moreover, rapid and sustained weakness …

Decreased Metallation And Activity In Subsets Of Mutant Superoxide Dismutases Associated With Familial Amyotrophic Lateral Sclerosis, Lawrence Hayward, Jorge Rodriguez, Ji Kim, Ashutosh Tiwari, Joy Goto, Diane Cabelli, Joan Valentine, Robert Brown

Dr Robert Brown

Over 90 different mutations in the gene encoding copper/zinc superoxide dismutase (SOD1) cause approximately 2% of amyotrophic lateral sclerosis (ALS) cases by an unknown mechanism. We engineered 14 different human ALS-related SOD1 mutants and obtained high yields of biologically metallated proteins from an Sf21 insect cell expression system. Both the wild type and mutant "as isolated" SOD1 variants were deficient in copper and were heterogeneous by native gel electrophoresis. By contrast, although three mutant SOD1s with substitutions near the metal binding sites (H46R, G85R, and D124V) were severely deficient in both copper and zinc ions, zinc deficiency was not a …

Dendritic Cells In Hepatitis C Infection: Can They (Help) Win The Battle, Angela Dolganiuc, Gyongyi Szabo

Gyongyi Szabo

Infection with hepatitis C virus (HCV) is a public health problem; it establishes a chronic course in ~85% of infected patients and increases their risk for developing liver cirrhosis, hepatocellular carcinoma, and significant extrahepatic manifestations. The mechanisms of HCV persistence remain elusive and are largely related to inefficient clearance of the virus by the host immune system. Dendritic cells (DCs) are the most efficient inducers of immune responses; they are capable of triggering productive immunity and maintaining the state of tolerance to self- and non-self antigens. During the past decade, multiple research groups have focused on DCs, in hopes of …

Hypoxia And Hypoxia Inducible Factors: Diverse Roles In Liver Diseases, Bharath Nath, Gyongyi Szabo

Gyongyi Szabo

Hypoxia has been shown to have a role in the pathogenesis of several forms of liver disease. The hypoxia inducible factors (HIFs) are a family of evolutionarily conserved transcriptional regulators that affect a homeostatic response to low oxygen tension and have been identified as key mediators of angiogenesis, inflammation, and metabolism. In this review we summarize the evidence for a role of HIFs across a range of hepatic pathophysiology. We describe regulation of the HIFs and review investigations that demonstrate a role for HIFs in the development of liver fibrosis, activation of innate immune pathways, hepatocellular carcinoma, as well as …

Mitochondrial Antiviral Signaling Protein Defect Links Impaired Antiviral Response And Liver Injury In Steatohepatitis In Mice, Timea Csak, Angela Dolganiuc, Karen Kodys, Bharath Nath, Jan Petrasek, Shashi Bala, Dora Lippai, Gyongyi Szabo

Gyongyi Szabo

Mitochondrial dysfunction is a pathogenic feature of nonalcoholic steatohepatitis (NASH). NASH complicates hepatotropic viral disease. The mitochondrial antiviral signaling protein (MAVS) is the adapter of helicase receptors involved in sensing double-stranded RNA (dsRNA). We hypothesized that impaired MAVS function may contribute to insufficient antiviral response and liver damage in steatohepatitis. We identified reduced MAVS protein levels and increased MAVS association with the proteasome subunit alpha type 7 (PSMA7) in livers from mice given a methionine-choline-deficient (MCD) diet. Decreased association of MAVS with mitochondria and increased cytosolic cytochrome c indicated mitochondrial damage in steatohepatitis. In vivo administration of the synthetic dsRNA …

An Essential Role For Monocyte Chemoattractant Protein-1 In Alcoholic Liver Injury: Regulation Of Proinflammatory Cytokines And Hepatic Steatosis In Mice, Pranoti Mandrekar, Aditya Ambade, Arlene Lim, Gyongyi Szabo, Donna Catalano

Gyongyi Szabo

The importance of chemokines in alcoholic liver injury has been implicated. The role of the chemokine, monocyte chemoattractant protein-1 (MCP-1), elevated in patients with alcoholic liver disease is not yet understood. Here, we evaluated the pathophysiological significance of MCP-1 and its receptor, chemokine (C-C motif) receptor 2 (CCR2), in alcoholic liver injury. The Leiber-DeCarli diet containing alcohol or isocaloric control diets were fed to wild-type (WT) and MCP-1-deficient knockout (KO) mice for 6 weeks. In vivo and in vitro assays were performed to study the role of MCP-1 in alcoholic liver injury. MCP-1 was increased in Kupffer cells (KCs) as …

Fatty Acid And Endotoxin Activate Inflammasomes In Mouse Hepatocytes That Release Danger Signals To Stimulate Immune Cells, Timea Csak, Michal Ganz, Justin Pespisa, Karen Kodys, Angela Dolganiuc, Gyongyi Szabo

Gyongyi Szabo

The pathogenesis of nonalcoholic steatohepatitis (NASH) and inflammasome activation involves sequential hits. The inflammasome, which cleaves pro-interleukin-1beta (pro-IL-1beta) into secreted IL-1beta, is induced by endogenous and exogenous danger signals. Lipopolysaccharide (LPS), a toll-like receptor 4 ligand, plays a role in NASH and also activates the inflammasome. In this study, we hypothesized that the inflammasome is activated in NASH by multiple hits involving endogenous and exogenous danger signals. Using mouse models of methionine choline-deficient (MCD) diet-induced NASH and high-fat diet-induced NASH, we found up-regulation of the inflammasome [including NACHT, LRR, and PYD domains-containing protein 3 (NALP3; cryopyrin), apoptosis-associated speck-like CARD-domain containing …

A Dna Vaccine Prime Followed By A Liposome-Encapsulated Protein Boost Confers Enhanced Mucosal Immune Responses And Protection, Kejian Yang, Barbara Whalen, Rebecca S Tirabassi, Liisa Selin, Tatyana Levchenko, Vladimir Torchilin, Edward Kislauskis, Dennis Guberski

Vladimir Torchilin

A variety of DNA vaccine prime and recombinant viral boost immunization strategies have been developed to enhance immune responses in humans, but inherent limitations to these strategies exist. There is still an overwhelming need to develop safe and effective approaches that raise broad humoral and T cell-mediated immune responses systemically and on mucosal surfaces. We have developed a novel mucosal immunization regimen that precludes the use of viral vectors yet induces potent T cell responses. Using hepatitis B surface Ag (HBsAg), we observed that vaccination of BALB/c mice with an i.m. HBsAg-DNA vaccine prime followed by an intranasal boost with …

A Dna Vaccine Prime Followed By A Liposome-Encapsulated Protein Boost Confers Enhanced Mucosal Immune Responses And Protection, Kejian Yang, Barbara Whalen, Rebecca S Tirabassi, Liisa Selin, Tatyana Levchenko, Vladimir Torchilin, Edward Kislauskis, Dennis Guberski

Tatyana Levchenko

A variety of DNA vaccine prime and recombinant viral boost immunization strategies have been developed to enhance immune responses in humans, but inherent limitations to these strategies exist. There is still an overwhelming need to develop safe and effective approaches that raise broad humoral and T cell-mediated immune responses systemically and on mucosal surfaces. We have developed a novel mucosal immunization regimen that precludes the use of viral vectors yet induces potent T cell responses. Using hepatitis B surface Ag (HBsAg), we observed that vaccination of BALB/c mice with an i.m. HBsAg-DNA vaccine prime followed by an intranasal boost with …

Chop Mediates Endoplasmic Reticulum Stress-Induced Apoptosis In Gimap5-Deficient T Cells, Steven Pino, Bryan O'Sullivan-Murphy, Erich Lidstone, Chaoxing Yang, Kathryn Lipson, Agata Jurczyk, Philip Diiorio, Michael Brehm, John Mordes, Dale Greiner, Aldo Rossini, Rita Bortell

Philip J diIorio Jr

Gimap5 (GTPase of the immunity-associated protein 5) has been linked to the regulation of T cell survival, and polymorphisms in the human GIMAP5 gene associate with autoimmune disorders. The BioBreeding diabetes-prone (BBDP) rat has a mutation in the Gimap5 gene that leads to spontaneous apoptosis of peripheral T cells by an unknown mechanism. Because Gimap5 localizes to the endoplasmic reticulum (ER), we hypothesized that absence of functional Gimap5 protein initiates T cell death through disruptions in ER homeostasis. We observed increases in ER stress-associated chaperones in T cells but not thymocytes or B cells from Gimap5(-/-) BBDP rats. We then …

Failure Of Alpha-Galactosylceramide To Prevent Diabetes In Virus-Inducible Models Of Type 1 Diabetes In The Rat, Prerna Chopra, Philip Diiorio, Steven Pino, S. Brian Wilson, Nancy Phillips, John Mordes, Aldo Rossini, Dale Greiner, Leonard Shultz, Rita Bortell

Philip J diIorio Jr

BACKGROUND: Alpha-galactosylceramide (alpha-GalCer) is an invariant natural killer T (iNKT) cell ligand that prevents type 1 diabetes in NOD mice. However, alpha-GalCer can activate or suppress immune responses, raising concern about its potential use in human diabetes.

MATERIALS AND METHODS: To evaluate this therapeutic issue further, BBDR and LEW.1WR1 rats were treated with Kilham rat virus (KRV) plus polyinosinic-polycytidylic acid, with or without alpha-GalCer, and followed for onset of diabetes.

RESULTS: alpha-GalCer did not prevent diabetes in inducible rat models. To investigate this discrepancy, we analyzed iNKT cell function. Splenocytes stimulated with alpha-GalCer produced similar levels of IFNgamma in all …

A Zebrafish Unc-45-Related Gene Expressed During Muscle Development, Letitiah Etheridge, Philip Diiorio, Charles Sagerstrom

Philip J diIorio Jr

We report the isolation and expression pattern of zebrafish unc45r, a gene related to Caenorhabditis elegans unc-45. UNC-45 is a muscle-specific protein thought to interact with myosin and promote the assembly of muscle thick filaments during C. elegans development. Zebrafish Unc45r shares sequence features with C. elegans UNC-45, including three tetratricopeptide repeats and a CRO1/She4p homology domain. unc45r is expressed in mesoderm adjacent to the dorsal midline during late gastrula stages and is coexpressed with muscle specific genes in somitic mesoderm during development of trunk skeletal muscle. unc45r is also expressed in cranial skeletal muscle as well as in cardiac …

Role Of Sec61alpha In The Regulated Transfer Of The Ribosome-Nascent Chain Complex From The Signal Recognition Particle To The Translocation Channel, Weiqun Song, David Raden, Elisabet Mandon, Reid Gilmore

Elisabet Mandon

Targeting of ribosome-nascent chain complexes to the translocon in the endoplasmic reticulum is mediated by the concerted action of the signal recognition particle (SRP) and the SRP receptor (SR). Ribosome-stripped microsomes were digested with proteases to sever cytoplasmic domains of SRalpha, SRbeta, TRAM, and the Sec61 complex. We characterized protein translocation intermediates that accumulate when Sec61alpha or SRbeta is inactivated by proteolysis. In the absence of a functional Sec61 complex, dissociation of SRP54 from the signal sequence is blocked. Experiments using SR proteoliposomes confirmed the assembly of a membrane-bound posttargeting intermediate. These results strongly suggest that the Sec61 complex regulates …

A Monomeric Protein In The Golgi Membrane Catalyzes Both N-Deacetylation And N-Sulfation Of Heparan Sulfate, Elisabet Mandon, Ellis Kempner, Masayuki Ishihara, Carlos Hirschberg

Elisabet Mandon

Recent studies have shown that the rat liver heparan sulfate N-deacetylase/N-sulfotransferase is a glycoprotein encoded by a single polypeptide chain of 882 amino acids. Using radiation inactivation analyses, we have now determined that in rat liver Golgi vesicles the target size for the N-deacetylase is 88 +/- 14 kDa, whereas that of the N-sulfotransferase is 92 +/- 8 kDa. These results, together with previous biochemical and molecular cloning approaches, demonstrate that 1) in rat liver Golgi membranes there exists only on population of molecules expressing both activities, 2) the active protein in the Golgi membrane functions as a monomer, and …