Role of the p63-FoxN1 regulatory axis in thymic epithelial cell homeostasis during aging

The p63 gene regulates thymic epithelial cell (TEC) proliferation, whereas FoxN1 regulates their differentiation. However, their collaborative role in the regulation of TEC homeostasis during thymic aging is largely unknown. In murine models, the proportion of TAp63⁺, but not ΔNp63⁺, TECs was increased with age, which was associated with an age-related increase in senescent cell clusters, characterized by SA-β-Gal⁺ and p21⁺ cells. Intrathymic infusion of exogenous TAp63 cDNA into young wild-type (WT) mice led to an increase in senescent cell clusters. Blockade of TEC differentiation via conditional FoxN1 gene knockout accelerated the appearance of this phenotype to early middle age, whereas intrathymic infusion of exogenous FoxN1 cDNA into aged WT mice brought only a modest reduction in the proportion of TAp63⁺ TECs, but an increase in ΔNp63⁺ TECs in the partially rejuvenated thymus. Meanwhile, we found that the increased TAp63⁺ population contained a high proportion of phosphorylated-p53 TECs, which may be involved in the induction of cellular senescence. Thus, TAp63 levels are positively correlated with TEC senescence but inversely correlated with expression of FoxN1 and FoxN1-regulated TEC differentiation. Thereby, the p63-FoxN1 regulatory axis in regulation of postnatal TEC homeostasis has been revealed.     

Type A Francisella tularensis Acid Phosphatases Contribute to Pathogenesis

Different Francisella spp. produce five or six predicted acid phosphatases (AcpA, AcpB, AcpC, AcpD, HapA and HapB). The genes encoding the histidine acid phosphatases (hapA, hapB) and acpD of F. tularensis subsp. Schu S4 strain are truncated or disrupted. However, deletion of HapA (FTT1064) in F. tularensis Schu S4 resulted in a 33% reduction in acid phosphatase activity and loss of the four functional acid phosphatases in F. tularensis Schu S4 (ΔABCH) resulted in a>99% reduction in acid phosphatase activity compared to the wild type strain. All single, double and triple mutants tested, demonstrated a moderate decrease in mouse virulence and survival and growth within human and murine phagocytes, whereas the ΔABCH mutant showed >3.5-fold decrease in intramacrophage survival and 100% attenuation of virulence in mouse. While the Schu S4 ΔABCH strain was attenuated in the mouse model, it showed only limited protection against wild type challenge. F. tularensis Schu S4 failed to stimulate reactive oxygen species production in phagocytes, whereas infection by the ΔABCH strain stimulated 5- and 56-fold increase in reactive oxygen species production in neutrophils and human monocyte-derived macrophages, respectively. The ΔABCH mutant but not the wild type strain strongly co-localized with p47^phox and replicated in macrophages isolated from p47^phox knockout mice. Thus, F. tularensis Schu S4 acid phosphatases, including the truncated HapA, play a major role in intramacrophage survival and virulence of this human pathogen.     

Block of Death-Receptor Apoptosis Protects Mouse Cytomegalovirus from Macrophages and Is a Determinant of Virulence in Immunodeficient Hosts

The inhibition of death-receptor apoptosis is a conserved viral function. The murine cytomegalovirus (MCMV) gene M36 is a sequence and functional homologue of the human cytomegalovirus gene UL36, and it encodes an inhibitor of apoptosis that binds to caspase-8, blocks downstream signaling and thus contributes to viral fitness in macrophages and in vivo. Here we show a direct link between the inability of mutants lacking the M36 gene (ΔM36) to inhibit apoptosis, poor viral growth in macrophage cell cultures and viral in vivo fitness and virulence. ΔM36 grew poorly in RAG1 knockout mice and in RAG/IL-2-receptor common gamma chain double knockout mice (RAGγC^−/−), but the depletion of macrophages in either mouse strain rescued the growth of ΔM36 to almost wild-type levels. This was consistent with the observation that activated macrophages were sufficient to impair ΔM36 growth in vitro. Namely, spiking fibroblast cell cultures with activated macrophages had a suppressive effect on ΔM36 growth, which could be reverted by z-VAD-fmk, a chemical apoptosis inhibitor. TNFα from activated macrophages synergized with IFNγ in target cells to inhibit ΔM36 growth. Hence, our data show that poor ΔM36 growth in macrophages does not reflect a defect in tropism, but rather a defect in the suppression of antiviral mediators secreted by macrophages. To the best of our knowledge, this shows for the first time an immune evasion mechanism that protects MCMV selectively from the antiviral activity of macrophages, and thus critically contributes to viral pathogenicity in the immunocompromised host devoid of the adaptive immune system.     

The carboxyl-terminal region of SDCCAG8 comprises a functional module essential for cilia formation as well as organ development and homeostasis

In humans, ciliary dysfunction causes ciliopathies, which present as multiple organ defects, including developmental and sensory abnormalities. Sdccag8 is a centrosomal/basal body protein essential for proper cilia formation. Gene mutations in SDCCAG8 have been found in patients with ciliopathies manifesting a broad spectrum of symptoms, including hypogonadism. Among these mutations, several that are predicted to truncate the SDCCAG8 carboxyl (C) terminus are also associated with such symptoms; however, the underlying mechanisms are poorly understood. In the present study, we identified the Sdccag8 C-terminal region (Sdccag8-C) as a module that interacts with the ciliopathy proteins, Ick/Cilk1 and Mak, which were shown to be essential for the regulation of ciliary protein trafficking and cilia length in mammals in our previous studies. We found that Sdccag8-C is essential for Sdccag8 localization to centrosomes and cilia formation in cultured cells. We then generated a mouse mutant in which Sdccag8-C was truncated (Sdccag8^ΔC/ΔC mice) using a CRISPR-mediated stop codon knock-in strategy. In Sdccag8^ΔC/ΔC mice, we observed abnormalities in cilia formation and ciliopathy-like organ phenotypes, including cleft palate, polydactyly, retinal degeneration, and cystic kidney, which partially overlapped with those previously observed in Ick- and Mak-deficient mice. Furthermore, Sdccag8^ΔC/ΔC mice exhibited a defect in spermatogenesis, which was a previously uncharacterized phenotype of Sdccag8 dysfunction. Together, these results shed light on the molecular and pathological mechanisms underlying ciliopathies observed in patients with SDCCAG8 mutations and may advance our understanding of protein–protein interaction networks involved in cilia development.     

The Mycobacterium tuberculosis Proteasome Active Site Threonine Is Essential for Persistence Yet Dispensable for Replication and Resistance to Nitric Oxide

Previous work revealed that conditional depletion of the core proteasome subunits PrcB and PrcA impaired growth of Mycobacterium tuberculosis in vitro and in mouse lungs, caused hypersusceptibility to nitric oxide (NO) and impaired persistence of the bacilli during chronic mouse infections. Here, we show that genetic deletion of prcBA led to similar phenotypes. Surprisingly, however, an active site mutant proteasome complemented the in vitro and in vivo growth defects of the prcBA knockout (ΔprcBA) as well as its NO hypersensitivity. In contrast, long-term survival of M. tuberculosis in stationary phase and during starvation in vitro and in the chronic phase of mouse infection required a proteolytically active proteasome. Inhibition of inducible nitric oxide synthase did not rescue survival of ΔprcBA, revealing a function beyond NO defense, by which the proteasome contributes to M. tuberculosis fitness during chronic mouse infections. These findings suggest that proteasomal proteolysis facilitates mycobacterial persistence, that M. tuberculosis faces starvation during chronic mouse infections and that the proteasome serves a proteolysis-independent function.     

Generation of a Tph2 Conditional Knockout Mouse Line for Time- and Tissue-Specific Depletion of Brain Serotonin

Serotonin has been gaining increasing attention during the last two decades due to the dual function of this monoamine as key regulator during critical developmental events and as neurotransmitter. Importantly, unbalanced serotonergic levels during critical temporal phases might contribute to the onset of neuropsychiatric disorders, such as schizophrenia and autism. Despite increasing evidences from both animal models and human genetic studies have underpinned the importance of serotonin homeostasis maintenance during central nervous system development and adulthood, the precise role of this molecule in time-specific activities is only beginning to be elucidated. Serotonin synthesis is a 2-step process, the first step of which is mediated by the rate-limiting activity of Tph enzymes, belonging to the family of aromatic amino acid hydroxylases and existing in two isoforms, Tph1 and Tph2, responsible for the production of peripheral and brain serotonin, respectively. In the present study, we generated and validated a conditional knockout mouse line, Tph2 ^flox/flox, in which brain serotonin can be effectively ablated with time specificity. We demonstrated that the Cre-mediated excision of the third exon of Tph2 gene results in the production of a Tph2 ^null allele in which we observed the near-complete loss of brain serotonin, as well as the growth defects and perinatal lethality observed in serotonin conventional knockouts. We also revealed that in mice harbouring the Tph2 ^null allele, but not in wild-types, two distinct Tph2 mRNA isoforms are present, namely Tph2Δ3 and Tph2Δ3Δ4, with the latter showing an in-frame deletion of amino acids 84–178 and coding a protein that could potentially retain non-negligible enzymatic activity. As we could not detect Tph1 expression in the raphe, we made the hypothesis that the Tph2Δ3Δ4 isoform can be at the origin of the residual, sub-threshold amount of serotonin detected in the brain of Tph2 ^null/null mice. Finally, we set up a tamoxifen administration protocol that allows an efficient, time-specific inactivation of brain serotonin synthesis. On the whole, we generated a suitable genetic tool to investigate how serotonin depletion impacts on time-specific events during central nervous system development and adulthood life.     

Proton motive force during growth of Streptococcus lactis cells

Experiments with the aerotolerant anaerobe Streptococcus lactis provide the opportunity for determining the proton motive force (Δp) in dividing cells. The two components of Δp, ΔΨ (the transmembrane potential) and ΔpH (the chemical gradient of H⁺), were determined by the accumulation of radiolabeled tetraphenylphosphonium (TPP⁺) and benzoate ions. The ΔΨ was calibrated with the K⁺ diffusion potential in starved, valinomycin-treated cells. With resting, glycolyzing cells, the Δp was measured also by the accumulation of the non-metabolizable sugar thiomethyl-β-galactoside (TMG). In resting cells the Δp, calculated either by adding ΔΨ and ZΔpH or from the levels of TMG, was relatively constant between pH 5 to 7, decreasing from 160 to 150 mV and decreasing further to 100 mV at pH 8.0. With the TPP⁺ probe for ΔΨ, we confirmed our previous finding that the K⁺ ions dissipate ΔΨ and increase ΔpH, whereas Na⁺ ions have little effect on ΔΨ and no effect on ΔpH. [³H]TPP⁺ and [¹⁴C]benzoate were added during exponential phase to S. lactis cells growing at pH 5 to 7 at 28°C in a defined medium with glucose as energy source. As with resting cells, the ΔpH and ΔΨ were dependent on the pH of the medium. At pH 5.1, the ΔpH was equivalent to 60 mV (alkaline inside) and decreased to 25 mV at pH 6.8. The ΔΨ increased from 83 mV (negative inside) at pH 5.1 to 108 mV at pH 6.8. The Δp, therefore, was fairly constant between pH 5 and 7, decreasing from 143 to 133 mV. The values for Δp in growing cells, just as in resting cells, are consistent with a system in which the net efflux of H⁺ ions is effected by a membrane-bound adenosine triphosphatase and glycolytically generated adenosine triphosphate. The data suggest that in both growing and resting cells the pH of the medium and its K⁺ concentration are the two principal factors that determine the relative contribution of ΔpH and ΔΨ to the proton motive force.     

Generation and characterization of a conditional deletion allele for Lmna in mice

Extensive efforts have been devoted to study A-type lamins because mutations in their gene, LMNA in humans, are associated with a number of diseases. The mouse germline mutations in the A-type lamins (encoded by Lmna) exhibit postnatal lethality at either 4–8 postnatal (P) weeks or P16–18 days, depending on the deletion alleles. These mice exhibit defects in several tissues including hearts and skeletal muscles. Despite numerous studies, how the germline mutation of Lmna, which is expressed in many postnatal tissues, affects only selected tissues remains poorly understood. Addressing the tissue specific functions of Lmna requires the generation and careful characterization of conditional Lmna null alleles. Here we report the creation of a conditional Lmna knockout allele in mice by introducing loxP sites flanking the second exon of Lmna. The Lmna^flox/flox mice are phenotypically normal and fertile. We show that Lmna homozygous mutants (Lmna^Δ/Δ) generated by germline Cre expression display postnatal lethality at P16–18 days with defects similar to a recently reported germline Lmna knockout mouse that exhibits the earliest lethality compared to other germline knockout alleles. This conditional knockout mouse strain should serve as an important genetic tool to study the tissue specific roles of Lmna, which would contribute toward the understanding of various human diseases associated with A-type lamins.     

Adipocyte-specific Disruption of Fat-specific Protein 27 Causes Hepatosteatosis and Insulin Resistance in High-fat Diet-fed Mice

White adipose tissue (WAT) functions as an energy reservoir where excess circulating fatty acids are transported to WAT, converted to triglycerides, and stored as unilocular lipid droplets. Fat-specific protein 27 (FSP27, CIDEC in humans) is a lipid-coating protein highly expressed in mature white adipocytes that contributes to unilocular lipid droplet formation. However, the influence of FSP27 in adipose tissue on whole-body energy homeostasis remains unclear. Mice with adipocyte-specific disruption of the Fsp27 gene (Fsp27^ΔAd) were generated using an aP2-Cre transgene with the Cre/LoxP system. Upon high-fat diet feeding, Fsp27^ΔAd mice were resistant to weight gain. In the small WAT of these mice, small adipocytes containing multilocular lipid droplets were dispersed. The expression levels of the genes associated with mitochondrial abundance and brown adipocyte identity were increased, and basal lipolytic activities were significantly augmented in adipocytes isolated from Fsp27^ΔAd mice compared with the Fsp27^F/F counterparts. The impaired fat-storing function in Fsp27^ΔAd adipocytes and the resultant lipid overflow from WAT led to marked hepatosteatosis, dyslipidemia, and systemic insulin resistance in high-fat diet-treated Fsp27^ΔAd mice. These results demonstrate a critical role for FSP27 in the storage of excess fat in WAT with minimizing ectopic fat accumulation that causes insulin-resistant diabetes and non-alcoholic fatty liver disease. This mouse model may be useful for understanding the significance of fat-storing properties of white adipocytes and the role of local FSP27 in whole-body metabolism and estimating the pathogenesis of human partial lipodystrophy caused by CIDEC mutations.     

The novel proteins Rng8 and Rng9 regulate the myosin-V Myo51 during fission yeast cytokinesis

The myosin-V family of molecular motors is known to be under sophisticated regulation, but our knowledge of the roles and regulation of myosin-Vs in cytokinesis is limited. Here, we report that the myosin-V Myo51 affects contractile ring assembly and stability during fission yeast cytokinesis, and is regulated by two novel coiled-coil proteins, Rng8 and Rng9. Both rng8Δ and rng9Δ cells display similar defects as myo51Δ in cytokinesis. Rng8 and Rng9 are required for Myo51’s localizations to cytoplasmic puncta, actin cables, and the contractile ring. Myo51 puncta contain multiple Myo51 molecules and walk continuously on actin filaments in rng8⁺ cells, whereas Myo51 forms speckles containing only one dimer and does not move efficiently on actin tracks in rng8Δ. Consistently, Myo51 transports artificial cargos efficiently in vivo, and this activity is regulated by Rng8. Purified Rng8 and Rng9 form stable higher-order complexes. Collectively, we propose that Rng8 and Rng9 form oligomers and cluster multiple Myo51 dimers to regulate Myo51 localization and functions.