Research Article
Abstract
It is unclear whether neurogenesis occurs in the adult mammalian enteric nervous system (ENS). Neural crest–derived cells capable of forming multilineage colonies in culture, and neurons and glia upon transplantation into chick embryos, persist throughout adult life in the mammalian ENS. In this study we sought to determine the physiological function of these cells. We discovered that these cells could be identified based on CD49b expression and that they had characteristics of enteric glia, including p75, GFAP, S100B, and SOX10 expression. To test whether new neurons or glia arise in the adult gut under physiological conditions, we marked dividing progenitors with a thymidine analog in rodents under steady-state conditions, or during aging, pregnancy, dietary changes, hyperglycemia, or exercise. We also tested gut injuries including inflammation, irradiation, benzalkonium chloride treatment, partial gut stenosis, and glial ablation. We readily observed neurogenesis in a neurogenic region of the central nervous system, but not reproducibly in the adult ENS. Lineage tracing of glial cells with GFAP-Cre and GFAP-CreERT2 also detected little or no adult ENS neurogenesis. Neurogenesis in the adult gut is therefore very limited under the conditions we studied. In contrast, ENS gliogenesis was readily observed under steady-state conditions and after injury. Adult enteric glia thus have the potential to form neurons and glia in culture but are fated to form mainly glia under physiological conditions and after the injuries we studied.
Authors
Nancy M. Joseph, Shenghui He, Elsa Quintana, Yun-Gi Kim, Gabriel Núñez, Sean J. Morrison
Abstract
In rodents, acute brain insulin action reduces blood glucose levels by suppressing the expression of enzymes in the hepatic gluconeogenic pathway, thereby reducing gluconeogenesis and endogenous glucose production (EGP). Whether a similar mechanism is functional in large animals, including humans, is unknown. Here, we demonstrated that in canines, physiologic brain hyperinsulinemia brought about by infusion of insulin into the head arteries (during a pancreatic clamp to maintain basal hepatic insulin and glucagon levels) activated hypothalamic Akt, altered STAT3 signaling in the liver, and suppressed hepatic gluconeogenic gene expression without altering EGP or gluconeogenesis. Rather, brain hyperinsulinemia slowly caused a modest reduction in net hepatic glucose output (NHGO) that was attributable to increased net hepatic glucose uptake and glycogen synthesis. This was associated with decreased levels of glycogen synthase kinase 3β (GSK3β) protein and mRNA and with decreased glycogen synthase phosphorylation, changes that were blocked by hypothalamic PI3K inhibition. Therefore, we conclude that the canine brain senses physiologic elevations in plasma insulin, and that this in turn regulates genetic events in the liver. In the context of basal insulin and glucagon levels at the liver, this input augments hepatic glucose uptake and glycogen synthesis, reducing NHGO without altering EGP.
Authors
Christopher J. Ramnanan, Viswanathan Saraswathi, Marta S. Smith, E. Patrick Donahue, Ben Farmer, Tiffany D. Farmer, Doss Neal, Philip E. Williams, Margaret Lautz, Andrea Mari, Alan D. Cherrington, Dale S. Edgerton
Abstract
Spermatogonial stem cells (SSCs) capable of self-renewal and differentiation are the foundation for spermatogenesis. Although several factors important for these processes have been identified, the fundamental mechanisms regulating SSC self-renewal and differentiation remain unknown. Here, we investigated a role for the Foxo transcription factors in mouse spermatogenesis and found that Foxo1 specifically marks mouse gonocytes and a subset of spermatogonia with stem cell potential. Genetic analyses showed that Foxo1 was required for both SSC homeostasis and the initiation of spermatogenesis. Combined deficiency of Foxo1, Foxo3, and Foxo4 resulted in a severe impairment of SSC self-renewal and a complete block of differentiation, indicating that Foxo3 and Foxo4, although dispensable for male fertility, contribute to SSC function. By conditional inactivation of 3-phosphoinositide–dependent protein kinase 1 (Pdk1) and phosphatase and tensin homolog (Pten) in the male germ line, we found that PI3K signaling regulates Foxo1 stability and subcellular localization, revealing that the Foxos are pivotal effectors of PI3K-Akt signaling in SSCs. We also identified a network of Foxo gene targets — most notably Ret — that rationalized the maintenance of SSCs by the Foxos. These studies demonstrate that Foxo1 expression in the spermatogenic lineage is intimately associated with the stem cell state and revealed what we believe to be novel Foxo-dependent mechanisms underlying SSC self-renewal and differentiation, with implications for common diseases, including male infertility and testicular cancer, due to abnormalities in SSC function.
Authors
Meredith J. Goertz, Zhuoru Wu, Teresa D. Gallardo, F. Kent Hamra, Diego H. Castrillon
Abstract
It has long been hypothesized that abnormalities in lipid biology contribute to degenerative brain diseases. Consistent with this, emerging epidemiologic evidence links lipid alterations with Parkinson disease (PD), and disruption of lipid metabolism has been found to predispose to α-synuclein toxicity. We therefore investigated whether Parkin, an E3 ubiquitin ligase found to be defective in patients with early onset PD, regulates systemic lipid metabolism. We perturbed lipid levels by exposing Parkin+/+ and Parkin–/– mice to a high-fat and -cholesterol diet (HFD). Parkin–/– mice resisted weight gain, steatohepatitis, and insulin resistance. In wild-type mice, the HFD markedly increased hepatic Parkin levels in parallel with lipid transport proteins, including CD36, Sr-B1, and FABP. These lipid transport proteins were not induced in Parkin–/– mice. The role of Parkin in fat uptake was confirmed by increased oleate accumulation in hepatocytes overexpressing Parkin and decreased uptake in Parkin–/– mouse embryonic fibroblasts and patient cells harboring complex heterozygous mutations in the Parkin-encoding gene PARK2. Parkin conferred this effect, in part, via ubiquitin-mediated stabilization of the lipid transporter CD36. Reconstitution of Parkin restored hepatic fat uptake and CD36 levels in Parkin–/– mice, and Parkin augmented fat accumulation during adipocyte differentiation. These results demonstrate that Parkin is regulated in a lipid-dependent manner and modulates systemic fat uptake via ubiquitin ligase–dependent effects. Whether this metabolic regulation contributes to premature Parkinsonism warrants investigation.
Authors
Kye-Young Kim, Mark V. Stevens, M. Hasina Akter, Sarah E. Rusk, Robert J. Huang, Alexandra Cohen, Audrey Noguchi, Danielle Springer, Alexander V. Bocharov, Tomas L. Eggerman, Der-Fen Suen, Richard J. Youle, Marcelo Amar, Alan T. Remaley, Michael N. Sack
Abstract
The enteric nervous system (ENS) in mammals forms from neural crest cells during embryogenesis and early postnatal life. Nevertheless, multipotent progenitors of the ENS can be identified in the adult intestine using clonal cultures and in vivo transplantation assays. The identity of these neurogenic precursors in the adult gut and their relationship to the embryonic progenitors of the ENS are currently unknown. Using genetic fate mapping, we here demonstrate that mouse neural crest cells marked by SRY box–containing gene 10 (Sox10) generate the neuronal and glial lineages of enteric ganglia. Most neurons originated from progenitors residing in the gut during mid-gestation. Afterward, enteric neurogenesis was reduced, and it ceased between 1 and 3 months of postnatal life. Sox10-expressing cells present in the myenteric plexus of adult mice expressed glial markers, and we found no evidence that these cells participated in neurogenesis under steady-state conditions. However, they retained neurogenic potential, as they were capable of generating neurons with characteristics of enteric neurons in culture. Furthermore, enteric glia gave rise to neurons in vivo in response to chemical injury to the enteric ganglia. Our results indicate that despite the absence of constitutive neurogenesis in the adult gut, enteric glia maintain limited neurogenic potential, which can be activated by tissue dissociation or injury.
Authors
Catia Laranjeira, Katarina Sandgren, Nicoletta Kessaris, William Richardson, Alexandre Potocnik, Pieter Vanden Berghe, Vassilis Pachnis
Abstract
Streptococcus pneumoniae colonizes the mucosal surface of the human upper respiratory tract. A colonization event is gradually cleared through phagocytosis by monocytes/macrophages that are recruited to the airway lumen. Here, we sought to define the bacterial and host factors that promote monocyte/macrophage influx and S. pneumoniae clearance using intranasal bacterial challenge in mice. We found that the recruitment of monocytes/macrophages required their expression of the chemokine receptor CCR2 and correlated with expression of the CCR2 ligand CCL2. Production of CCL2 and monocyte/macrophage recruitment were deficient in mice lacking digestion of peptidoglycan by lysozyme (LysM) and cytosolic sensing of the products of digestion by Nod2. Ex vivo macrophages produced CCL2 following bacterial uptake, digestion by LysM, and sensing of peptidoglycan by Nod2. Sensing of digested peptidoglycan by Nod2 also required the pore-forming toxin pneumolysin. The generation of an adaptive immune response, as measured by anti-pneumococcal antibody titers, was also LysM- and Nod2-dependent. Together, our data suggest that bacterial uptake by professional phagocytes is followed by LysM-mediated digestion of S. pneumoniae–derived peptidoglycan, sensing of the resulting products by Nod2, release of the chemokine CCL2, and CCR2-dependent recruitment of the additional monocytes/macrophages required for the clearance of an S. pneumoniae colonization event.
Authors
Kimberly M. Davis, Shigeki Nakamura, Jeffrey N. Weiser
Abstract
Pneumococcal infection of the respiratory tract is often secondary to recent influenza virus infection and accounts for much of the morbidity and mortality during seasonal and pandemic influenza. Here, we show that coinfection of the upper respiratory tract of mice with influenza virus and pneumococcus leads to synergistic stimulation of type I IFNs and that this impairs the recruitment of macrophages, which are required for pneumococcal clearance, due to decreased production of the chemokine CCL2. Type I IFN expression was induced by pneumococcal colonization alone. Colonization followed by influenza coinfection led to a synergistic type I IFN response, resulting in increased density of colonizing bacteria and susceptibility to invasive infection. This enhanced type I IFN response inhibited production of the chemokine CCL2, which promotes the recruitment of macrophages and bacterial clearance. Stimulation of CCL2 by macrophages upon pneumococcal infection alone required the pattern recognition receptor Nod2 and expression of the pore-forming toxin pneumolysin. Indeed, the increased colonization associated with concurrent influenza virus infection was not observed in mice lacking Nod2 or the type I IFN receptor, or in mice challenged with pneumococci lacking pneumolysin. We therefore propose that the synergistic stimulation of type I IFN production during concurrent influenza virus and pneumococcal infection leads to increased bacterial colonization and suggest that this may contribute to the higher rates of disease associated with coinfection in humans.
Authors
Shigeki Nakamura, Kimberly M. Davis, Jeffrey N. Weiser
OSCAR is a collagen receptor that costimulates osteoclastogenesis in DAP12-deficient humans and mice
Abstract
Osteoclasts are terminally differentiated leukocytes that erode the mineralized bone matrix. Osteoclastogenesis requires costimulatory receptor signaling through adaptors containing immunoreceptor tyrosine-based activation motifs (ITAMs), such as Fc receptor common γ (FcRγ) and DNAX-activating protein of 12 kDa. Identification of these ITAM-containing receptors and their ligands remains a high research priority, since the stimuli for osteoclastogenesis are only partly defined. Osteoclast-associated receptor (OSCAR) was proposed to be a potent FcRγ-associated costimulatory receptor expressed by preosteoclasts in vitro, but OSCAR lacks a cognate ligand and its role in vivo has been unclear. Using samples from mice and patients deficient in various ITAM signaling pathways, we show here that OSCAR costimulates one of the major FcRγ-associated pathways required for osteoclastogenesis in vivo. Furthermore, we found that OSCAR binds to specific motifs within fibrillar collagens in the ECM that become revealed on nonquiescent bone surfaces in which osteoclasts undergo maturation and terminal differentiation in vivo. OSCAR promoted osteoclastogenesis in vivo, and OSCAR binding to its collagen motif led to signaling that increased numbers of osteoclasts in culture. Thus, our results suggest that ITAM-containing receptors can respond to exposed ligands in collagen, leading to the functional differentiation of leukocytes, which provides what we believe to be a new concept for ITAM regulation of cytokine receptors in different tissue microenvironments.
Authors
Alexander David Barrow, Nicolas Raynal, Thomas Levin Andersen, David A. Slatter, Dominique Bihan, Nicholas Pugh, Marina Cella, Taesoo Kim, Jaerang Rho, Takako Negishi-Koga, Jean-Marie Delaisse, Hiroshi Takayanagi, Joseph Lorenzo, Marco Colonna, Richard W. Farndale, Yongwon Choi, John Trowsdale
Abstract
The ubiquitin-proteasome system degrades most intracellular proteins, including misfolded proteins. Proteasome functional insufficiency (PFI) has been observed in proteinopathies, such as desmin-related cardiomyopathy, and implicated in many common diseases, including dilated cardiomyopathy and ischemic heart disease. However, the pathogenic role of PFI has not been established. Here we created inducible Tg mice with cardiomyocyte-restricted overexpression of proteasome 28 subunit α (CR-PA28αOE) to investigate whether upregulation of the 11S proteasome enhances the proteolytic function of the proteasome in mice and, if so, whether the enhancement can rescue a bona fide proteinopathy and protect against ischemia/reperfusion (I/R) injury. We found that CR-PA28αOE did not alter the homeostasis of normal proteins and cardiac function, but did facilitate the degradation of a surrogate misfolded protein in the heart. By breeding mice with CR-PA28αOE with mice representing a well-established model of desmin-related cardiomyopathy, we demonstrated that CR-PA28αOE markedly reduced aberrant protein aggregation. Cardiac hypertrophy was decreased, and the lifespan of the animals was increased. Furthermore, PA28α knockdown promoted, whereas PA28α overexpression attenuated, accumulation of the mutant protein associated with desmin-related cardiomyopathy in cultured cardiomyocytes. Moreover, CR-PA28αOE limited infarct size and prevented postreperfusion cardiac dysfunction in mice with myocardial I/R injury. We therefore conclude that benign enhancement of cardiac proteasome proteolytic function can be achieved by CR-PA28αOE and that PFI plays a major pathogenic role in cardiac proteinopathy and myocardial I/R injury.
Authors
Jie Li, Kathleen M. Horak, Huabo Su, Atsushi Sanbe, Jeffrey Robbins, Xuejun Wang
Abstract
Merkel cell polyomavirus (MCV) is the recently discovered cause of most Merkel cell carcinomas (MCCs), an aggressive form of nonmelanoma skin cancer. Although MCV is known to integrate into the tumor cell genome and to undergo mutation, the molecular mechanisms used by this virus to cause cancer are unknown. Here, we show that MCV small T (sT) antigen is expressed in most MCC tumors, where it is required for tumor cell growth. Unlike the closely related SV40 sT, MCV sT transformed rodent fibroblasts to anchorage- and contact-independent growth and promoted serum-free proliferation of human cells. These effects did not involve protein phosphatase 2A (PP2A) inhibition. MCV sT was found to act downstream in the mammalian target of rapamycin (mTOR) signaling pathway to preserve eukaryotic translation initiation factor 4E–binding protein 1 (4E-BP1) hyperphosphorylation, resulting in dysregulated cap-dependent translation. MCV sT–associated 4E-BP1 serine 65 hyperphosphorylation was resistant to mTOR complex (mTORC1) and mTORC2 inhibitors. Steady-state phosphorylation of other downstream Akt-mTOR targets, including S6K and 4E-BP2, was also increased by MCV sT. Expression of a constitutively active 4E-BP1 that could not be phosphorylated antagonized the cell transformation activity of MCV sT. Taken together, these experiments showed that 4E-BP1 inhibition is required for MCV transformation. Thus, MCV sT is an oncoprotein, and its effects on dysregulated cap-dependent translation have clinical implications for the prevention, diagnosis, and treatment of MCV-related cancers.
Authors
Masahiro Shuda, Hyun Jin Kwun, Huichen Feng, Yuan Chang, Patrick S. Moore
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ISSN: 0021-9738 (print), 1558-8238 (online)