Research Article
Abstract
Numerous studies have suggested that muscle atrophy is accompanied by apoptotic loss of myonuclei and therefore recovery would require replenishment by muscle stem cells. We used in vivo time-lapse microscopy to observe the loss and replenishment of myonuclei in murine muscle fibers following induced muscle atrophy. To our surprise, imaging of single fibers for up to 28 days did not support the concept of nuclear loss during atrophy. Muscles were inactivated by denervation, nerve impulse block, or mechanical unloading. Nuclei were stained in vivo either acutely by intracellular injection of fluorescent oligonucleotides or in time-lapse studies after transfection with a plasmid encoding GFP with a nuclear localization signal. We observed no loss of myonuclei in fast- or slow-twitch muscle fibers despite a greater than 50% reduction in fiber cross-sectional area. TUNEL labeling of fragmented DNA on histological sections revealed high levels of apoptotic nuclei in inactive muscles. However, when costained for laminin and dystrophin, virtually none of the TUNEL-positive nuclei could be classified as myonuclei; apoptosis was confined to stromal and satellite cells. We conclude that disuse atrophy is not a degenerative process, but is rather a change in the balance between protein synthesis and proteolysis in a permanent cell syncytium.
Authors
Jo C. Bruusgaard, Kristian Gundersen
Abstract
The hormone estradiol affects the auditory system both by itself and by its interaction with neuroprotective factors. In this study, we examined the role of estrogen receptors (ERs) in response to auditory trauma. We found a ligand-dependent protective role for ERβ in the auditory system by investigating mice deficient in ERα (ERKO mice), ERβ (BERKO mice), and aromatase (ARKO mice). Basal auditory brainstem response (ABR) thresholds were similar in all animals. An acoustic trauma causing a temporary hearing loss raised ABR thresholds in male and female BERKO and ARKO mice compared with WT and ERKO mice. The ERα-selective agonist, propyl(1H) pyrazole-1,3,5-triyl-trisphenol (PPT), partially protected ARKO mice from trauma, while the ERβ-selective agonist, 2,3-bis (4-hydroxyphenyl)-propionitrile (DPN), protected WT and ARKO mice. Immunohistochemistry and western blotting confirmed the expression of ERβ in cochlea of WT males and females. Levels of brain-derived neurotrophic factor (BDNF), a neuroprotective peptide that can be induced by estrogen, was lower in BERKO and ARKO mice compared with WT. DPN treatment increased BDNF expression in ARKO mice. These data indicate ERβ-mediated neuroprotection involving BDNF in the auditory system of males and females.
Authors
Inna Meltser, Yeasmin Tahera, Evan Simpson, Malou Hultcrantz, Konstantina Charitidi, Jan-Åke Gustafsson, Barbara Canlon
Abstract
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 of isolated mutant muscles was induced when the extracellular K+ concentration was increased from 4 mM to 10 mM, a level observed in the muscle interstitium of humans during exercise. Mutant muscle recovered from stimulation-induced fatigue more slowly than did control muscle, and the extent of recovery was decreased in the presence of high extracellular K+ levels. These findings demonstrate that expression of the Met1592Val Na+ channel in mouse muscle is sufficient to produce important features of HyperKPP, including myotonia, K+-sensitive paralysis, and susceptibility to delayed weakness during recovery from fatigue.
Authors
Lawrence J. Hayward, Joanna S. Kim, Ming-Yang Lee, Hongru Zhou, Ji W. Kim, Kumudini Misra, Mohammad Salajegheh, Fen-fen Wu, Chie Matsuda, Valerie Reid, Didier Cros, Eric P. Hoffman, Jean-Marc Renaud, Stephen C. Cannon, Robert H. Brown Jr.
IFN-γ– and TNF-dependent bystander eradication of antigen-loss variants in established mouse cancers
Abstract
Tumors elicit antitumor immune responses, but over time they evolve and can escape immune control through various mechanisms, including the loss of the antigen to which the response is directed. The escape of antigen-loss variants (ALVs) is a major obstacle to T cell–based immunotherapy for cancer. However, cancers can be cured if both the number of CTLs and the expression of antigen are high enough to allow targeting of not only tumor cells, but also the tumor stroma. Here, we showed that IFN-γ and TNF produced by CTLs were crucial for the elimination of established mouse tumors, including ALVs. In addition, both BM- and non-BM–derived stromal cells were required to express TNF receptors and IFN-γ receptors for the elimination of ALVs. Although IFN-γ and TNF were not required by CTLs for perforin-mediated killing of antigen-expressing tumor cells, the strong inference is that tumor antigen–specific CTLs must secrete IFN-γ and TNF for destruction of tumor stroma. Therefore, bystander killing of ALVs may result from IFN-γ and TNF acting on tumor stroma.
Authors
Bin Zhang, Theodore Karrison, Donald A. Rowley, Hans Schreiber
Abstract
13-cis retinoic acid (13-cis RA; also known as isotretinoin), is the most potent agent available for treatment of acne. It is known that the drug induces apoptosis in cells cultured from human sebaceous glands, but its mechanism of action has not been determined. In this study, skin biopsies were taken from 7 patients with acne prior to and at 1 week of treatment with 13-cis RA. TUNEL staining confirmed that 13-cis RA induced apoptosis in sebaceous glands. Transcriptional profiling of patient skin and cultured human sebaceous gland cells (SEB-1 sebocytes) indicated that lipocalin 2 was among the genes most highly upregulated by 13-cis RA. Lipocalin 2 encodes neutrophil gelatinase–associated lipocalin (NGAL), which functions in innate immune defense and induces apoptosis of murine B lymphocytes. Increased immunolocalization of NGAL was noted in patients’ sebaceous glands following treatment with 13-cis RA, and recombinant NGAL induced apoptosis in SEB-1 sebocytes. Furthermore, apoptosis in response to 13-cis RA was inhibited in the presence of siRNA to lipocalin 2. These data indicate that NGAL mediates the apoptotic effect of 13-cis RA and suggest that agents that selectively induce NGAL expression in sebaceous glands might represent therapeutic alternatives to the use of 13-cis RA to treat individuals with acne.
Authors
Amanda M. Nelson, Wei Zhao, Kathryn L. Gilliland, Andrea L. Zaenglein, Wenlei Liu, Diane M. Thiboutot
Abstract
Dominant mutations in the gene encoding the mRNA splicing factor PRPF31 cause retinitis pigmentosa, a hereditary form of retinal degeneration. Most of these mutations are characterized by DNA changes that lead to premature termination codons. We investigated 6 different PRPF31 mutations, represented by single-base substitutions or microdeletions, in cell lines derived from 9 patients with dominant retinitis pigmentosa. Five of these mutations lead to premature termination codons, and 1 leads to the skipping of exon 2. Allele-specific measurement of PRPF31 transcripts revealed a strong reduction in the expression of mutant alleles. As a consequence, total PRPF31 protein abundance was decreased, and no truncated proteins were detected. Subnuclear localization of the full-length PRPF31 that was present remained unaffected. Blocking nonsense-mediated mRNA decay significantly restored the amount of mutant PRPF31 mRNA but did not restore the synthesis of mutant proteins, even in conjunction with inhibitors of protein degradation pathways. Our results indicate that most PRPF31 mutations ultimately result in null alleles through the activation of surveillance mechanisms that inactivate mutant mRNA and, possibly, proteins. Furthermore, these data provide compelling evidence that the pathogenic effect of PRPF31 mutations is likely due to haploinsufficiency rather than to gain of function.
Authors
Thomas Rio Frio, Nicholas M. Wade, Adriana Ransijn, Eliot L. Berson, Jacques S. Beckmann, Carlo Rivolta
Abstract
Bardet-Biedl syndrome (BBS) is a heterogeneous genetic disorder characterized by many features, including obesity and cardiovascular disease. We previously developed knockout mouse models of 3 BBS genes: BBS2, BBS4, and BBS6. To dissect the mechanisms involved in the metabolic disorders associated with BBS, we assessed the development of obesity in these mouse models and found that BBS-null mice were hyperphagic, had low locomotor activity, and had elevated circulating levels of the hormone leptin. The effect of exogenous leptin on body weight and food intake was attenuated in BBS mice, which suggests that leptin resistance may contribute to hyperleptinemia. In other mouse models of obesity, leptin resistance may be selective rather than systemic; although mice became resistant to leptin’s anorectic effects, the ability to increase renal sympathetic nerve activity (SNA) was preserved. Although all 3 of the BBS mouse models were similarly resistant to leptin, the sensitivity of renal SNA to leptin was maintained in Bbs4–/– and Bbs6–/– mice, but not in Bbs2–/– mice. Consequently, Bbs4–/– and Bbs6–/– mice had higher baseline renal SNA and arterial pressure and a greater reduction in arterial pressure in response to ganglionic blockade. Furthermore, we found that BBS mice had a decreased hypothalamic expression of proopiomelanocortin, which suggests that BBS genes play an important role in maintaining leptin sensitivity in proopiomelanocortin neurons.
Authors
Kamal Rahmouni, Melissa A. Fath, Seongjin Seo, Daniel R. Thedens, Christopher J. Berry, Robert Weiss, Darryl Y. Nishimura, Val C. Sheffield
Abstract
The essential contribution of the antidepressant-sensitive serotonin (5-HT) transporter SERT (which is encoded by the SLC6A4 gene) to platelet 5-HT stores suggests an important role of this transporter in platelet function. Here, using SERT-deficient mice, we have established a role for constitutive SERT expression in efficient ADP- and thrombin-triggered platelet aggregation. Additionally, using pharmacological blockers of SERT and the vesicular monoamine transporter (VMAT), we have identified a role for ongoing 5-HT release and SERT activity in efficient human platelet aggregation. We have also demonstrated that fibrinogen, an activator of integrin αIIbβ3, enhances SERT activity in human platelets and that integrin αIIbβ3 interacts directly with the C terminus of SERT. Consistent with these findings, knockout mice lacking integrin β3 displayed diminished platelet SERT activity. Conversely, HEK293 cells engineered to express human SERT and an activated form of integrin β3 exhibited enhanced SERT function that coincided with elevated SERT surface expression. Our results support an unsuspected role of αIIbβ3/SERT associations as well as αIIbβ3 activation in control of SERT activity in vivo that may have broad implications for hyperserotonemia, cardiovascular disorders, and autism.
Authors
Ana Marin D. Carneiro, Edwin H. Cook, Dennis L. Murphy, Randy D. Blakely
Abstract
A fundamental property of leukemic stem cells is clonal dominance of the bone marrow microenvironment. Truncation mutations of CSF3R, which encodes the G-CSF receptor (G-CSFR), are implicated in leukemic progression in patients with severe congenital neutropenia. Here we show that expression of a truncated mutant Csf3r in mice confers a strong clonal advantage at the HSC level that is dependent upon exogenous G-CSF. G-CSF–induced proliferation, phosphorylation of Stat5, and transcription of Stat5 target genes were increased in HSCs isolated from mice expressing the mutant Csf3r. Conversely, the proliferative advantage conferred by the mutant Csf3r was abrogated in myeloid progenitors lacking both Stat5A and Stat5B, and HSC function was reduced in mice expressing a truncated mutant Csf3r engineered to have impaired Stat5 activation. These data indicate that in mice, inappropriate Stat5 activation plays a key role in establishing clonal dominance by stem cells expressing mutant Csf3r.
Authors
Fulu Liu, Ghada Kunter, Maxwell M. Krem, William C. Eades, Jennifer A. Cain, Michael H. Tomasson, Lothar Hennighausen, Daniel C. Link
Abstract
Growth hormone (GH) is an underappreciated but important regulator of T cell development that can reverse age-related declines in thymopoiesis in rodents. Here, we report findings of a prospective randomized study examining the effects of GH on the immune system of HIV-1–infected adults. GH treatment was associated with increased thymic mass. In addition, GH treatment enhanced thymic output, as measured by both the frequency of T cell receptor rearrangement excision circles in circulating T cells and the numbers of circulating naive and total CD4+ T cells. These findings provide compelling evidence that GH induces de novo T cell production and may, accordingly, facilitate CD4+ T cell recovery in HIV-1–infected adults. Further, these randomized, prospective data have shown that thymic involution can be pharmacologically reversed in humans, suggesting that immune-based therapies could be used to enhance thymopoiesis in immunodeficient individuals.
Authors
Laura A. Napolitano, Diane Schmidt, Michael B. Gotway, Niloufar Ameli, Erin L. Filbert, Myra M. Ng, Julie L. Clor, Lorrie Epling, Elizabeth Sinclair, Paul D. Baum, Kai Li, Marisela Lua Killian, Peter Bacchetti, Joseph M. McCune
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