Thymic Alterations in GM2 Gangliosidoses Model Mice

BACKGROUND: Sandhoff disease is a lysosomal storage disorder characterized by the absence of β-hexosaminidase and storage of GM2 ganglioside and related glycolipids. We have previously found that the progressive neurologic disease induced in Hexb(-/-) mice, an animal model for Sandhoff disease, is associated with the production of pathogenic anti-glycolipid autoantibodies. METHODOLOGY/PRINCIPAL FINDINGS: In our current study, we report on the alterations in the thymus during the development of mild to severe progressive neurologic disease. The thymus from Hexb(-/-) mice of greater than 15 weeks of age showed a marked decrease in the percentage of immature CD4(+)/CD8(+) T cells and a significantly increased number of CD4(+)/CD8(-) T cells. During involution, the levels of both apoptotic thymic cells and IgG deposits to T cells were found to have increased, whilst swollen macrophages were prominently observed, particularly in the cortex. We employed cDNA microarray analysis to monitor gene expression during the involution process and found that genes associated with the immune responses were upregulated, particularly those expressed in macrophages. CXCL13 was one of these upregulated genes and is expressed specifically in the thymus. B1 cells were also found to have increased in the thy mus. It is significant that these alterations in the thymus were reduced in FcRγ additionally disrupted Hexb(-/-) mice. CONCLUSIONS/SIGNIFICANCE: These results suggest that the FcRγ chain may render the usually poorly immunogenic thymus into an organ prone to autoimmune responses, including the chemotaxis of B1 cells toward CXCL13

A Large Scale shRNA Barcode Screen Identifies the Circadian Clock Component ARNTL as Putative Regulator of the p53 Tumor Suppressor Pathway

BACKGROUND: The p53 tumor suppressor gene is mutated in about half of human cancers, but the p53 pathway is thought to be functionally inactivated in the vast majority of cancer. Understanding how tumor cells can become insensitive to p53 activation is therefore of major importance. Using an RNAi-based genetic screen, we have identified three novel genes that regulate p53 function. RESULTS: We have screened the NKI shRNA library targeting 8,000 human genes to identify modulators of p53 function. Using the shRNA barcode technique we were able to quickly identify active shRNA vectors from a complex mixture. Validation of the screening results indicates that the shRNA barcode technique can reliable identify active shRNA vectors from a complex pool. Using this approach we have identified three genes, ARNTL, RBCK1 and TNIP1, previously unknown to regulate p53 function. Importantly, ARNTL (BMAL1) is an established component of the circadian regulatory network. The latter finding adds to recent observations that link circadian rhythm to the cell cycle and cancer. We show that cells having suppressed ARNTL are unable to arrest upon p53 activation associated with an inability to activate the p53 target gene p21(CIP1). CONCLUSIONS: We identified three new regulators of the p53 pathway through a functional genetic screen. The identification of the circadian core component ARNTL strengthens the link between circadian rhythm and cancer

Sox2 Is Essential for Formation of Trophectoderm in the Preimplantation Embryo

In preimplantation mammalian development the transcription factor Sox2 (SRY-related HMG-box gene 2) forms a complex with Oct4 and functions in maintenance of self-renewal of the pluripotent inner cell mass (ICM). Previously it was shown that Sox2-/- embryos die soon after implantation. However, maternal Sox2 transcripts may mask an earlier phenotype. We investigated whether Sox2 is involved in controlling cell fate decisions at an earlier stage.We addressed the question of an earlier role for Sox2 using RNAi, which removes both maternal and embryonic Sox2 mRNA present during the preimplantation period. By depleting both maternal and embryonic Sox2 mRNA at the 2-cell stage and monitoring embryo development in vitro we show that, in the absence of Sox2, embryos arrest at the morula stage and fail to form trophectoderm (TE) or cavitate. Following knock-down of Sox2 via three different short interfering RNA (siRNA) constructs in 2-cell stage mouse embryos, we have shown that the majority of embryos (76%) arrest at the morula stage or slightly earlier and only 18.7-21% form blastocysts compared to 76.2-83% in control groups. In Sox2 siRNA-treated embryos expression of pluripotency associated markers Oct4 and Nanog remained unaffected, whereas TE associated markers Tead4, Yap, Cdx2, Eomes, Fgfr2, as well as Fgf4, were downregulated in the absence of Sox2. Apoptosis was also increased in Sox2 knock-down embryos. Rescue experiments using cell-permeant Sox2 protein resulted in increased blastocyst formation from 18.7% to 62.6% and restoration of Sox2, Oct4, Cdx2 and Yap protein levels in the rescued Sox2-siRNA blastocysts.We conclude that the first essential function of Sox2 in the preimplantation mouse embryo is to facilitate establishment of the trophectoderm lineage. Our findings provide a novel insight into the first differentiation event within the preimplantation embryo, namely the segregation of the ICM and TE lineages

Autophagy Induced by HIF1α Overexpression Supports Trophoblast Invasion by Supplying Cellular Energy

<div><p>Extravillous trophoblasts (EVTs) characterize the invasion of the maternal decidua under low oxygen and poor nutrition at the early feto-maternal interface to establish a successful pregnancy. We previously reported that autophagy in EVTs was activated under 2% O₂<i>in vitro</i>, and autophagy activation was also observed in EVTs at the early feto-maternal interface <i>in vivo</i>. Here, we show that autophagy is an energy source for the invasion of EVTs. Cobalt chloride (CoCl₂), which induces hypoxia inducible factor 1α (HIF1α) overexpression, activated autophagy in HTR8/SVneo cells, an EVT cell line. The number of invading HTR8-ATG4B^C74A cells, an autophagy-deficient EVT cell line, was markedly reduced by 81 percent with the CoCl₂ treatment through the suppression of MMP9 level, although CoCl₂ did not affect the cellular invasion of HTR8-mStrawberry cells, a control cell line. HTR8-ATG4B^C74A cells treated with CoCl₂ showed a decrease in cellular adenosine triphosphate (ATP) levels and a compensatory increase in the expression of purinergic receptor P2X ligand-gated ion channel 7 (P2RX7), which is stimulated with ATP, whereas HTR8-mStrawberry cells maintained cellular ATP levels and did not affect P2RX7 expression. Furthermore, the decreased invasiveness of HTR8-ATG4B^C74A cells treated with CoCl₂ was neutralized by ATP supplementation to the level of HTR8-ATG4B^C74A cells treated without CoCl₂. These results suggest that autophagy plays a role in maintaining homeostasis by countervailing HIF1α-mediated cellular energy consumption in EVTs.</p></div

CoCl₂ decreased cellular ATP levels, but increased P2RX7 expression in autophagy-deficient HTR8/SVneo cells.

<p>a) Cellular ATP levels in HTR8-Atg4B^C74A mutant cells, the autophagy-deficient EVT cell line, or HTR8-mStrawberry cells, the control cell line, were determined after the treatment with DMSO (control: white bars) or 250 µM CoCl₂ (black bars) for 48 h. b) P2RX7 expression in HTR8-Atg4B^C74A mutant cells (upper panel) or HTR8-mStrawberry cells (lower panel) treated with DMSO (red lines) or 250 µM CoCl₂ (black solids) were determined by flow cytometry for 48 h. Isotype controls are shown as black lines. c) P2RX7 expression was estimated by the mean fluorescent intensity in HTR8-Atg4B^C74A mutant cells or HTR8-mStrawberry cells treated with DMSO (control: white bars) or 250 µM CoCl₂ (black bars) for 48 h. These experiments were independently performed at least three times. N.S.: not significant</p

Correlation chart between autophagy and HIF1α for the invasion of EVTs.

<p>(a) In an intact invasion of EVTs, HIF1α activates autophagy via the PI3K pathway. Autophagy then supplies cellular energy to enhance the invasion of EVTs. (b) In an invasion failure, severe hypoxia or long term hypoxia may accelerate HIF1α overexpression in EVTs. EVTs with an impaired autophagy status by soluble endoglin did not produce energy for the invasion of EVTs with HIF1α overexpression, resulting in the inhibition of EVT invasion. Compensatory P2RX7 expression was enhanced by reacting to the decrease in cellular energy in EVTs observed with preeclampsia.</p

ATP supplementation recovered the suppression of invasion in autophagy-deficient HTR8/SVneo cells.

<p>a) Invasion assays were performed with HTR8-Atg4B^C74A mutant cells (upper panel), the autophagy-deficient EVT cell line, or HTR8-mStrawberry cells (lower panel), the control cell line, under DMSO (control) or 250 µM CoCl₂ in the presence (black bars) or absence (white bars) of 100 µM ATP for 48 h. The <i>Y</i>-axis indicates the number of invading cells. b) Invasion assays were performed with HTR8-Atg4B^C74A mutant cells under 250 µM CoCl₂ in the presence of increasing concentrations of ATP, as indicated for 48 h. The <i>Y</i>-axis indicates the number of invading cells. These experiments were independently performed at least three times.</p

Estimation of autophagy in HTR8/SVneo cells, when CoCl₂ induced HIF1α expression.

<p>a) Western blots in HTR8/SVneo cells under 250 µM CoCl₂, 2% oxygen tension, or DMSO (control) for 24 h were shown as follows: HIF1α and α-tubulin. b) The expression of MAP1LC3B-I and -II (LC3-I and LC3-II) in HTR8/SVneo cells was examined in the presence of E64d and pepstatin under 250 µM CoCl₂ for the indicated times. The expression of α-tubulin was used as an internal control. c) Representative panels show anti-LC3 staining in HTR8/SVneo cells under DMSO (control) or 250 µM CoCl₂ for 24 h. Negative control (Nega cont) was treated with rabbit serum instead of anti-LC3 antibody and stained with DAPI (4′, 6-diamidino-2-phenylindole). Scale bar: 30 µm. d) The graph indicates the number of LC3 puncta in HTR8/SVneo cells under DMSO (control), rapamycin (500 nM), 250 µM CoCl₂, or 250 µM CoCl₂ with 5 mM 3-MA for 24 h. These experiments were independently performed at least three times.</p