GLUT4, AMP kinase, but not the insulin receptor, are required for hepatoportal glucose sensor-stimulated muscle glucose utilization.

Recent evidence suggests the existence of a hepatoportal vein glucose sensor, whose activation leads to enhanced glucose use in skeletal muscle, heart, and brown adipose tissue. The mechanism leading to this increase in whole body glucose clearance is not known, but previous data suggest that it is insulin independent. Here, we sought to further determine the portal sensor signaling pathway by selectively evaluating its dependence on muscle GLUT4, insulin receptor, and the evolutionarily conserved sensor of metabolic stress, AMP-activated protein kinase (AMPK). We demonstrate that the increase in muscle glucose use was suppressed in mice lacking the expression of GLUT4 in the organ muscle. In contrast, glucose use was stimulated normally in mice with muscle-specific inactivation of the insulin receptor gene, confirming independence from insulin-signaling pathways. Most importantly, the muscle glucose use in response to activation of the hepatoportal vein glucose sensor was completely dependent on the activity of AMPK, because enhanced hexose disposal was prevented by expression of a dominant negative AMPK in muscle. These data demonstrate that the portal sensor induces glucose use and development of hypoglycemia independently of insulin action, but by a mechanism that requires activation of the AMPK and the presence of GLUT4.

Basal and antigen-induced exposure of the proline-rich sequence in CD3ε.

The CD3ε cytoplasmic tail contains a conserved proline-rich sequence (PRS) that influences TCR-CD3 expression and signaling. Although the PRS can bind the SH3.1 domain of the cytosolic adapter Nck, whether the PRS is constitutively available for Nck binding or instead represents a cryptic motif that is exposed via conformational change upon TCR-CD3 engagement (CD3Δc) is currently unresolved. Furthermore, the extent to which a cis-acting CD3ε basic amino acid-rich stretch (BRS), with its unique phosphoinositide-binding capability, might impact PRS accessibility is not clear. In this study, we found that freshly harvested primary thymocytes expressed low to moderate basal levels of Nck-accessible PRS ("open-CD3"), although most TCR-CD3 complexes were inaccessible to Nck ("closed-CD3"). Ag presentation in vivo induced open-CD3, accounting for half of the basal level found in thymocytes from MHC(+) mice. Additional stimulation with either anti-CD3 Abs or peptide-MHC ligands further elevated open-CD3 above basal levels, consistent with a model wherein antigenic engagement induces maximum PRS exposure. We also found that the open-CD3 conformation induced by APCs outlasted the time of ligand occupancy, marking receptors that had been engaged. Finally, CD3ε BRS-phosphoinositide interactions played no role in either adoption of the initial closed-CD3 conformation or induction of open-CD3 by Ab stimulation. Thus, a basal level of open-CD3 is succeeded by a higher, induced level upon TCR-CD3 engagement, involving CD3Δc and prolonged accessibility of the CD3ε PRS to Nck.

TACI, unlike BAFF-R, is solely activated by oligomeric BAFF and APRIL to support survival of activated B cells and plasmablasts.

The cytokine BAFF binds to the receptors TACI, BCMA, and BAFF-R on B cells, whereas APRIL binds to TACI and BCMA only. The signaling properties of soluble trimeric BAFF (BAFF 3-mer) were compared with those of higher-order BAFF oligomers. All forms of BAFF bound BAFF-R and TACI, and elicited BAFF-R-dependent signals in primary B cells. In contrast, signaling through TACI in mature B cells or plasmablasts was only achieved by higher-order BAFF and APRIL oligomers, all of which were also po-tent activators of a multimerization-dependent reporter signaling pathway. These results indicate that, although BAFF-R and TACI can provide B cells with similar signals, only BAFF-R, but not TACI, can respond to soluble BAFF 3-mer, which is the main form of BAFF found in circulation. BAFF 60-mer, an efficient TACI agonist, was also detected in plasma of BAFF transgenic and nontransgenic mice and was more than 100-fold more active than BAFF 3-mer for the activation of multimerization-dependent signals. TACI supported survival of activated B cells and plasmablasts in vitro, providing a rational basis to explain the immunoglobulin deficiency reported in TACI-deficient persons.

Sodium and potassium balance depends on αENaC expression in connecting tubule.

Mutations in α, β, or γ subunits of the epithelial sodium channel (ENaC) can downregulate ENaC activity and cause a severe salt-losing syndrome with hyperkalemia and metabolic acidosis, designated pseudohypoaldosteronism type 1 in humans. In contrast, mice with selective inactivation of αENaC in the collecting duct (CD) maintain sodium and potassium balance, suggesting that the late distal convoluted tubule (DCT2) and/or the connecting tubule (CNT) participates in sodium homeostasis. To investigate the relative importance of ENaC-mediated sodium absorption in the CNT, we used Cre-lox technology to generate mice lacking αENaC in the aquaporin 2-expressing CNT and CD. Western blot analysis of microdissected cortical CD (CCD) and CNT revealed absence of αENaC in the CCD and weak αENaC expression in the CNT. These mice exhibited a significantly higher urinary sodium excretion, a lower urine osmolality, and an increased urine volume compared with control mice. Furthermore, serum sodium was lower and potassium levels were higher in the genetically modified mice. With dietary sodium restriction, these mice experienced significant weight loss, increased urinary sodium excretion, and hyperkalemia. Plasma aldosterone levels were significantly elevated under both standard and sodium-restricted diets. In summary, αENaC expression within the CNT/CD is crucial for sodium and potassium homeostasis and causes signs and symptoms of pseudohypoaldosteronism type 1 if missing.

Dysregulated ADAM10-Mediated Processing of APP during a Critical Time Window Leads to Synaptic Deficits in Fragile X Syndrome.

The Fragile X mental retardation protein (FMRP) regulates neuronal RNA metabolism, and its absence or mutations leads to the Fragile X syndrome (FXS). The β-amyloid precursor protein (APP) is involved in Alzheimer's disease, plays a role in synapse formation, and is upregulated in intellectual disabilities. Here, we show that during mouse synaptogenesis and in human FXS fibroblasts, a dual dysregulation of APP and the α-secretase ADAM10 leads to the production of an excess of soluble APPα (sAPPα). In FXS, sAPPα signals through the metabotropic receptor that, activating the MAP kinase pathway, leads to synaptic and behavioral deficits. Modulation of ADAM10 activity in FXS reduces sAPPα levels, restoring translational control, synaptic morphology, and behavioral plasticity. Thus, proper control of ADAM10-mediated APP processing during a specific developmental postnatal stage is crucial for healthy spine formation and function(s). Downregulation of ADAM10 activity at synapses may be an effective strategy for ameliorating FXS phenotypes.

Synaptic Integration of Adult-Born Hippocampal Neurons Is Locally Controlled by Astrocytes.

Adult neurogenesis is regulated by the neurogenic niche, through mechanisms that remain poorly defined. Here, we investigated whether niche-constituting astrocytes influence the maturation of adult-born hippocampal neurons using two independent transgenic approaches to block vesicular release from astrocytes. In these models, adult-born neurons but not mature neurons showed reduced glutamatergic synaptic input and dendritic spine density that was accompanied with lower functional integration and cell survival. By taking advantage of the mosaic expression of transgenes in astrocytes, we found that spine density was reduced exclusively in segments intersecting blocked astrocytes, revealing an extrinsic, local control of spine formation. Defects in NMDA receptor (NMDAR)-mediated synaptic transmission and dendrite maturation were partially restored by exogenous D-serine, whose extracellular level was decreased in transgenic models. Together, these results reveal a critical role for adult astrocytes in local dendritic spine maturation, which is necessary for the NMDAR-dependent functional integration of newborn neurons.

Pre-existing astrocytes form functional perisynaptic processes on neurons generated in the adult hippocampus.

The adult dentate gyrus produces new neurons that morphologically and functionally integrate into the hippocampal network. In the adult brain, most excitatory synapses are ensheathed by astrocytic perisynaptic processes that regulate synaptic structure and function. However, these processes are formed during embryonic or early postnatal development and it is unknown whether astrocytes can also ensheathe synapses of neurons born during adulthood and, if so, whether they play a role in their synaptic transmission. Here, we used a combination of serial-section immuno-electron microscopy, confocal microscopy, and electrophysiology to examine the formation of perisynaptic processes on adult-born neurons. We found that the afferent and efferent synapses of newborn neurons are ensheathed by astrocytic processes, irrespective of the age of the neurons or the size of their synapses. The quantification of gliogenesis and the distribution of astrocytic processes on synapses formed by adult-born neurons suggest that the majority of these processes are recruited from pre-existing astrocytes. Furthermore, the inhibition of astrocytic glutamate re-uptake significantly reduced postsynaptic currents and increased paired-pulse facilitation in adult-born neurons, suggesting that perisynaptic processes modulate synaptic transmission on these cells. Finally, some processes were found intercalated between newly formed dendritic spines and potential presynaptic partners, suggesting that they may also play a structural role in the connectivity of new spines. Together, these results indicate that pre-existing astrocytes remodel their processes to ensheathe synapses of adult-born neurons and participate to the functional and structural integration of these cells into the hippocampal network.

Étude d'une population de lymphocytes T associée à la résistance au diabète auto-immun

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Influenza A virus infection impairs mycobacteria-specific T cell responses and mycobacterial clearance in the lung during pulmonary coinfection.

Individuals infected with mycobacteria are likely to experience episodes of concurrent infections with unrelated respiratory pathogens, including the seasonal or pandemic circulating influenza A virus strains. We analyzed the impact of influenza A virus and mycobacterial respiratory coinfection on the development of CD8 T cell responses to each pathogen. Coinfected mice exhibited reduced frequency and numbers of CD8 T cells specific to Mycobacterium bovis bacille Calmette-Guérin (BCG) in the lungs, and the IFN-γ CD8 T cell response to BCG-encoded OVA was decreased in the lungs of coinfected mice, when compared with mice infected with BCG alone. Moreover, after 2 wk of infection, mice coinfected with both pathogens showed a significant increase in the number of mycobacteria present in the lung compared with mice infected with BCG only. Following adoptive transfer into coinfected mice, transgenic CD8 T cells specific for OVA257–264 failed to proliferate as extensively in the mediastinal lymph nodes as in mice infected only with BCG-OVA. Also noted was a reduction in the proliferation of BCG-specific CD4 transgenic T cells in mice coinfected with influenza compared with mice infected with BCG alone. Furthermore, phenotypic analysis of CD11c+ dendritic cells from mediastinal lymph nodes of the infected mice showed that coinfection was associated with decreased surface expression of MHC class II and class I. Thus, concurrent pulmonary infection with influenza A virus is associated with decreased MHC expression on dendritic cells, reduced activation of BCG-specific CD4 and CD8 T cells, and impaired clearance of mycobacteria.

N-Acetylcysteine improves mitochondrial function and ameliorates behavioral deficits in the R6/1 mouse model of Huntington's disease

Huntington's disease (HD) is a neurodegenerative disorder, involving psychiatric, cognitive and motor symptoms, caused by a CAG-repeat expansion encoding an extended polyglutamine tract in the huntingtin protein. Oxidative stress and excitotoxicity have previously been implicated in the pathogenesis of HD. We hypothesized that N-acetylcysteine (NAC) may reduce both excitotoxicity and oxidative stress through its actions on glutamate reuptake and antioxidant capacity. The R6/1 transgenic mouse model of HD was used to investigate the effects of NAC on HD pathology. It was found that chronic NAC administration delayed the onset and progression of motor deficits in R6/1 mice, while having an antidepressant-like effect on both R6/1 and wild-type mice. A deficit in the astrocytic glutamate transporter protein, GLT-1, was found in R6/1 mice. However, this deficit was not ameliorated by NAC, implying that the therapeutic effect of NAC is not due to rescue of the GLT-1 deficit and associated glutamate-induced excitotoxicity. Assessment of mitochondrial function in the striatum and cortex revealed that R6/1 mice show reduced mitochondrial respiratory capacity specific to the striatum. This deficit was rescued by chronic treatment with NAC. There was a selective increase in markers of oxidative damage in mitochondria, which was rescued by NAC. In conclusion, NAC is able to delay the onset of motor deficits in the R6/1 model of Huntington's disease and it may do so by ameliorating mitochondrial dysfunction. Thus, NAC shows promise as a potential therapeutic agent in HD. Furthermore, our data suggest that NAC may also have broader antidepressant efficacy.

Cell Therapy with Bone Marrow Mononuclear Cells in Elastase-Induced Pulmonary Emphysema

Emphysema is characterized by destruction of alveolar walls with loss of gas exchange surface and consequent progressive dyspnea. This study aimed to evaluate the efficiency of cell therapy with bone marrow mononuclear cells (BMMC) in an animal model of elastase-induced pulmonary emphysema. Emphysema was induced in C57Bl/J6 female mice by intranasal instillation of elastase. After 21 days, the mice received bone marrow mononuclear cells from EGFP male mice with C57Bl/J6 background. The groups were assessed by comparison and statistically significant differences (p & 0. 05) were observed among the groups treated with BMMC and evaluated after 7, 14 and 21 days. Analysis of the mean linear intercept (Lm) values for the different groups allowed to observe that the group treated with BMMC and evaluated after 21 days showed the most significant result. The group that received no treatment showed a statistically significant difference when compared to other groups, except the group treated and evaluated after 21 days, evidencing the efficacy of cell therapy with BMMC in pulmonary emphysema. © 2012 Springer Science+Business Media New York.

Mechanismen der Aktivierung und Detoxifizierung von Aflatoxin B 1 in verschiedenen Leberzelltypen von Ratten, Mäusen und Waldmurmeltieren

Zusammenfassung: Die Applikation des Mykotoxins Aflatoxin B1 (AFB1) führt in der Ratte zu Lebertumoren hepatozellulären Ursprungs, während bisher keine transformierende Wirkung dieses Mykotoxins auf Kupffer- und Endothelzellen (Nichtparenchymzellen, NPC) nachgewiesen werden konnte. Diese Resistenzmechanismen der NPC gegenüber AFB1 wurden im ersten Teil dieser Arbeit untersucht. AFB1 ist per se inaktiv, wird jedoch durch Verstoffwechselung in den chemisch reaktiven, an DNA bindenden Metaboliten AFB1-8,9-Epoxid überführt. Daneben stellt die enzymatische Hydroxylierung von AFB1 am Kohlenstoff-9a zum Aflatoxin M1 eine Detoxifizierung dar. Durch HPLC-Analyse der AFB1-Metabolite konnte gezeigt werden, daß in Nichtparenchymzellen (NPC) das Verhältnis von 9a-Hydroxylierung zu 8,9-Epoxidierung höher als in Parenchymzellen (PC) ist. Die AFB1-9a-hydroxylase fördert insbesondere in den NPC der Leber die Bildung des weniger gentoxischen Metaboliten AFM1 und konkurriert daher um die Aktivierung von AFB1 zum mutagenen und kanzerogenen 8,9-Epoxid. Dieser metabolische Unterschied scheint also einen Beitrag zur Resistenz der NPC der Leber gegenüber der hepatokanzerogenen Wirkung von AFB1 zu leisten. Da ein Synergismus zwischen der AFB1-Exposition und einer Infektion mit dem Hepatitis B-Virus (HBV) beim Menschen bezüglich des Auftretens von hepatozellulären Karzinomen zu bestehen scheint, wurde im zweiten Teil dieser Arbeit untersucht, ob die metabolische Aktivierung von AFB1 durch eine HBV-Infektion verstärkt wird. In einem Vergleich der Biotransformation von AFB1 mit mikrosomalen Leberfraktionen von transgenen HBV-Mäusen und Kontrollmäusen wurde keine signifikanten Unterschiede festgestellt. Dagegen wurde bei Virus-infizierten Waldmurmeltieren eine deutlich reduzierte Bildung des AFB1-8,9-Epoxids beobachtet. Es konnte z.T. ein Zusammenhang zwischen den verschiedenen Stadien der Leberschädigung und den Metabolismusraten festgestellt werden, wobei die metabolische Aktivierung mit zunehmender Leberschädigung abzunehmen scheint. Auch hinsichtlich der Aktivitäten verschiedener Cytochrom P450 abhängiger Monooxygenasen wurde eine weitgehende Übereinstimmung mit den durch HPLC ermittelten Metabolitenprofilen des AFB1 beobachtet. Diese Studien mit subzellulären Leberfraktion der transgenen HBV-Mäusen und der Waldmurmeltieren zeigen, daß die Interaktion zwischen Hepatitis und AFB1 nicht mit der verstärkten metabolischer Aktivierung von AFB1 zu erklären ist. TGF-ß1, aus der Gruppe der Cytokine, wird als Mediator bei Entzündungsprozessen in der Leber so z.B. im Verlauf einer Virushepatitis freigesetzt. Aufgrund der besonderen Bedeutung des murinen CYP2A5 (ortholog zum humanen CYP2A6) bei der Aktivierung von AFB1 wurde der Einfluß von TGF-ß1 auf CYP2A5 in Primärkulturen von Maushepatozyten untersucht. Durch Messung der Aktivität der Cumarin-7-hydroxylase sowie durch Bestimmung der Proteinmenge von CYP2A5 mittels Western Blotting konnte zunächst die Induzierbarkeit des CYP2A5-Isoenzyms durch Phenobarbital in kultivierten Hepatozyten der Maus gezeigt werden. Nur bei einer niedrigen TGF-ß1-Konzentration wurde eine leicht erhöhte Expression von CYP2A5 festgestellt, ansonsten führte die Behandlung der kultivierten Maushepatozyten mit TGF-ß1 zu einer dosisabhängigen Verminderung der Expression von CYP2A5.

Activation of Src kinase Lyn by the Kaposi sarcoma-associated herpesvirus K1 protein: implications for lymphomagenesis.

The K1 gene of Kaposi sarcoma-associated herpesvirus (KSHV) encodes a transmembrane glycoprotein bearing a functional immunoreceptor tyrosine-based activation motif (ITAM). Previously, we reported that the K1 protein induced plasmablastic lymphomas in K1 transgenic mice, and that these lymphomas showed enhanced Lyn kinase activity. Here, we report that systemic administration of the nuclear factor kappa B (NF-kappaB) inhibitor Bay 11-7085 or an anti-vascular endothelial growth factor (VEGF) antibody significantly reduced K1 lymphoma growth in nude mice. Furthermore, in KVL-1 cells, a cell line derived from a K1 lymphoma, inhibition of Lyn kinase activity by the Src kinase inhibitor PP2 decreased VEGF induction, NF-kappaB activity, and the cell proliferation index by 50% to 75%. In contrast, human B-cell lymphoma BJAB cells expressing K1, but not the ITAM sequence-deleted mutant K1, showed a marked increase in Lyn kinase activity with concomitant VEGF induction and NF-kappaB activation, indicating that ITAM sequences were required for the Lyn kinase-mediated activation of these factors. Our results suggested that K1-mediated constitutive Lyn kinase activation in K1 lymphoma cells is crucial for the production of VEGF and NF-kappaB activation, both strongly implicated in the development of KSHV-induced lymphoproliferative disorders.

Dicer is required for female reproductive tract development and fertility in the mouse.

Dicer encodes a riboendonuclease required for microRNA biosynthesis. Dicer was inactivated in Müllerian duct mesenchyme-derived tissues of the reproductive tract of the mouse, using an Amhr2-Cre allele. Although Amhr2-Cre; Dicer conditional mutant males appeared normal and were fertile, mutant females were infertile. In adult mutant females, there was a reduction in the size of the oviducts and uterine horns. The oviducts were less coiled compared to controls and cysts formed at the isthmus near the uterotubal junction. Unfertilized, degenerate oocytes were commonly found within these cysts, indicating a defect in embryo transit. Beads transferred into the mutant oviduct failed to migrate into the uterus. In addition, blastocysts transferred directly into the mutant uterus did not result in pregnancy. Histological analysis demonstrated that the mutant uterus contained less glandular tissue and often the few glands that remained were found within the myometrium, an abnormal condition known as adenomyosis. In adult mutants, there was ectopic expression of Wnt4 and Wnt5a in the luminal epithelium (LE) and glandular epithelium (GE) of the uterus, and Wnt11 was ectopically expressed in GE. These results demonstrate that Dicer is necessary for postnatal differentiation of Müllerian duct mesenchyme-derived tissues of the female reproductive tract, suggesting that microRNAs are important regulators of female reproductive tract development and fertility.

Loss of DNA polymerase zeta enhances spontaneous tumorigenesis.

Mammalian genomes encode at least 15 distinct DNA polymerases, functioning as specialists in DNA replication, DNA repair, recombination, or bypass of DNA damage. Although the DNA polymerase zeta (polzeta) catalytic subunit REV3L is important in defense against genotoxins, little is known of its biological function. This is because REV3L is essential during embryogenesis, unlike other translesion DNA polymerases. Outstanding questions include whether any adult cells are viable in the absence of polzeta and whether polzeta status influences tumorigenesis. REV3L-deficient cells have properties that could influence the development of neoplasia in opposing ways: markedly reduced damage-induced point mutagenesis and extensive chromosome instability. To answer these questions, Rev3L was conditionally deleted from tissues of adult mice using MMTV-Cre. Loss of REV3L was tolerated in epithelial tissues but not in the hematopoietic lineage. Thymic lymphomas in Tp53(-/-) Rev3L conditional mice occurred with decreased latency and higher incidence. The lymphomas were populated predominantly by Rev3L-null T cells, showing that loss of Rev3L can promote tumorigenesis. Remarkably, the tumors were frequently oligoclonal, consistent with accelerated genetic changes in the absence of Rev3L. Mammary tumors could also arise from Rev3L-deleted cells in both Tp53(+/+) and Tp53(+/-) backgrounds. Mammary tumors in Tp53(+/-) mice deleting Rev3L formed months earlier than mammary tumors in Tp53(+/-) control mice. Prominent preneoplastic changes in glandular tissue adjacent to these tumors occurred only in mice deleting Rev3L and were associated with increased tumor multiplicity. Polzeta is the only specialized DNA polymerase yet identified that inhibits spontaneous tumor development.