Stefania Senger

APC/CFzr/Cdh1 promotes cell cycle progression during the Drosophila endocycle

The endocycle is a commonly observed variant cell cycle in which cells undergo repeated rounds of DNA replication with no intervening mitosis. How the cell cycle machinery is modified to transform a mitotic cycle into endocycle has long been a matter of interest. In both plants and animals, the transition from the mitotic cycle to the endocycle requires Fzr/Cdh1, a positive regulator of the Anaphase-Promoting Complex/Cyclosome (APC/C). However, because many of its targets are transcriptionally downregulated upon entry into the endocycle, it remains unclear whether the APC/C functions beyond the mitotic/endocycle boundary. Here, we report that APC/CFzr/Cdh1 activity is required to promote the G/S oscillation of the Drosophila endocycle. We demonstrate that compromising APC/C activity, after cells have entered the endocycle, inhibits DNA replication and results in the accumulation of multiple APC/C targets, including the mitotic cyclins and Geminin. Notably, our data suggest that the activity of APC/CFzr/Cdh1 during the endocycle is not continuous but is cyclic, as demonstrated by the APC/C-dependent oscillation of the pre-replication complex component Orc1. Taken together, our data suggest a model in which the cyclic activity of APC/CFzr/Cdh1 during the Drosophila endocycle is driven by the periodic inhibition of Fzr/Cdh1 by Cyclin E/Cdk2. We propose that, as is observed in mitotic cycles, during endocycles, APC/CFzr/Cdh1 functions to reduce the levels of the mitotic cyclins and Geminin in order to facilitate the relicensing of DNA replication origins and cell cycle progression.

Celiac Disease Association with CD8+ T Cell Responses: Identification of a Novel Gliadin-Derived HLA-A2-Restricted Epitope

One of the diagnostic hallmarks of the histological lesions associated with celiac disease is the extensive infiltration of the small intestinal epithelium by CD8+ T cells of unknown Ag specificity. In this study, we report recognition of the gliadin-derived peptide (A-gliadin 123–132) by CD8+ T lymphocytes from celiac patients. A-gliadin 123–132-specific IFN-γ production and cytotoxic activity were detected in PBMCs derived from patients on gluten-free diet, but not from either celiac patients on gluten-containing diet or healthy controls. In contrast, A-gliadin 123–132-specific cells were isolated from small intestine biopsies of patients on either gluten-free or gluten-containing diets. Short-term T cell lines derived from the small intestinal mucosa and specific for the 123–132 epitope recognized human APC pulsed with either whole recombinant α-gliadin or a partial pepsin-trypsin gliadin digest. Finally, we speculate on a possible mechanism leading to processing and presentation of class I-restricted gliadin-derived epitopes in celiac disease patients.

Intranasal administration of a recombinant α-gliadin down-regulates the immune response to wheat gliadin in DQ8 transgenic mice

The mucosal lesion in coeliac disease (CD) is an immune-mediated injury triggered by gliadin and associated with HLA-DQ2 and HLA-DQ8. In view of this, an approach that re-induces tolerance to this antigen should be considered as possible alternative to a strict gluten-free diet in treating CD. However, T-cell activation to multiple antigens, as a consequence of the chemical complexity shown by the antigen gliadin, could hamper the efforts to identify single component(s) useful for tolerance induction. To address this issue, a recombinant alpha-gliadin was tested in tolerance experiments in HLA-DQ8 transgenic mice. As tissue transglutaminase (tTG) treatment of gliadin, previously reported to enhance its stimulatory activity in CD, did not increase its immunogenicity when parenterally administered in these mice, untreated gliadin was used as immunogen. A decrease in systemic T cell responses to the recombinant alpha-gliadin was found after nasal administration of antigen, reflected by lymphocytes proliferation assay. Interestingly, while the immunisation protocol induced transcription of both Th1 (IFN-gamma) and Th2 (IL-4 and IL-10) cytokines, the tolerisation protocol down-regulated significantly only the IFN-gamma mRNA expression. More important, the recombinant alpha-gliadin induced a similar down-regulatory effect in mice immunised with a commercial preparation of wheat gliadin, that is a mixture of many different gliadin components. As the Th1 phenotype and the HLA-DQ8 molecule play a role in the pathogenesis of CD, our data underlined the potential usefulness of this recombinant protein for the immunomodulation of this disease.

Identification of Immunodominant Epitopes of α-Gliadin in HLA-DQ8 Transgenic Mice following Oral Immunization

Celiac disease, triggered by wheat gliadin and related prolamins from barley and rye, is characterized by a strong association with HLA-DQ2 and HLA-DQ8 genes. Gliadin is a mixture of many proteins that makes difficult the identification of major immunodominant epitopes. To address this issue, we expressed in Escherichia coli a recombinant α-gliadin (r-α-gliadin) showing the most conserved sequence among the fraction of α-gliadins. HLA-DQ8 mice, on a gluten-free diet, were intragastrically immunized with a chymotryptic digest of r-α-gliadin along with cholera toxin as adjuvant. Spleen and mesenteric lymph node T cell responses were analyzed for in vitro proliferative assay using a panel of synthetic peptides encompassing the entire sequence of r-α-gliadin. Two immunodominant epitopes corresponding to peptide p13 (aa 120–139) and p23 (aa 220–239) were identified. The response was restricted to DQ and mediated by CD4+ T cells. In vitro tissue transglutaminase deamidation of both peptides did not increase the response; furthermore, tissue transglutaminase catalyzed extensive deamidation in vitro along the entire r-α-gliadin molecule, but failed to elicit new immunogenic determinants. Surprisingly, the analysis of the cytokine profile showed that both deamidated and native peptides induced preferentially IFN-γ secretion, despite the use of cholera toxin, a mucosal adjuvant that normally induces a Th2 response to bystander Ags. Taken together, these data suggest that, in this model of gluten hypersensitivity, deamidation is not a prerequisite for the initiation of gluten responses.

Blood–brain barrier and intestinal epithelial barrier alterations in autism spectrum disorders

BackgroundAutism spectrum disorders (ASD) are complex conditions whose pathogenesis may be attributed to gene–environment interactions. There are no definitive mechanisms explaining how environmental triggers can lead to ASD although the involvement of inflammation and immunity has been suggested. Inappropriate antigen trafficking through an impaired intestinal barrier, followed by passage of these antigens or immune-activated complexes through a permissive blood–brain barrier (BBB), can be part of the chain of events leading to these disorders. Our goal was to investigate whether an altered BBB and gut permeability is part of the pathophysiology of ASD.MethodsPostmortem cerebral cortex and cerebellum tissues from ASD, schizophrenia (SCZ), and healthy subjects (HC) and duodenal biopsies from ASD and HC were analyzed for gene and protein expression profiles. Tight junctions and other key molecules associated with the neurovascular unit integrity and function and neuroinflammation were investigated.ResultsClaudin (CLDN)-5 and -12 were increased in the ASD cortex and cerebellum. CLDN-3, tricellulin, and MMP-9 were higher in the ASD cortex. IL-8, tPA, and IBA-1 were downregulated in SCZ cortex; IL-1b was increased in the SCZ cerebellum. Differences between SCZ and ASD were observed for most of the genes analyzed in both brain areas. CLDN-5 protein was increased in ASD cortex and cerebellum, while CLDN-12 appeared reduced in both ASD and SCZ cortexes. In the intestine, 75% of the ASD samples analyzed had reduced expression of barrier-forming TJ components (CLDN-1, OCLN, TRIC), whereas 66% had increased pore-forming CLDNs (CLDN-2, -10, -15) compared to controls.ConclusionsIn the ASD brain, there is an altered expression of genes associated with BBB integrity coupled with increased neuroinflammation and possibly impaired gut barrier integrity. While these findings seem to be specific for ASD, the possibility of more distinct SCZ subgroups should be explored with additional studies.

The nucleoporin Seh1 forms a complex with Mio and serves an essential tissue-specific function in Drosophila oogenesis

The nuclear pore complex (NPC) mediates the transport of macromolecules between the nucleus and cytoplasm. Recent evidence indicates that structural nucleoporins, the building blocks of the NPC, have a variety of unanticipated cellular functions. Here, we report an unexpected tissue-specific requirement for the structural nucleoporin Seh1 during Drosophila oogenesis. Seh1 is a component of the Nup107-160 complex, the major structural subcomplex of the NPC. We demonstrate that Seh1 associates with the product of the missing oocyte (mio) gene. In Drosophila, mio regulates nuclear architecture and meiotic progression in early ovarian cysts. Like mio, seh1 has a crucial germline function during oogenesis. In both mio and seh1 mutant ovaries, a fraction of oocytes fail to maintain the meiotic cycle and develop as pseudo-nurse cells. Moreover, the accumulation of Mio protein is greatly diminished in the seh1 mutant background. Surprisingly, our characterization of a seh1 null allele indicates that, although required in the female germline, seh1 is dispensable for the development of somatic tissues. Our work represents the first examination of seh1 function within the context of a multicellular organism. In summary, our studies demonstrate that Mio is a novel interacting partner of the conserved nucleoporin Seh1 and add to the growing body of evidence that structural nucleoporins can have novel tissue-specific roles.

Major signaling pathways in intestinal stem cells

BACKGROUND The discovery of markers to identify the intestinal stem cell population and the generation of powerful transgenic mouse models to study stem cell physiology have led to seminal discoveries in stem cell biology. SCOPE OF REVIEW In this review we give an overview of the current knowledge in the field of intestinal stem cells (ISCs) highlighting the most recent progress on markers defining the ISC population and pathways governing intestinal stem cell maintenance and differentiation. Furthermore we review their interaction with other stem cell related pathways. Finally we give an overview of alteration of these pathways in human inflammatory gastrointestinal diseases. MAJOR CONCLUSIONS We highlight the complex network of interactions occurring among different pathways and put in perspective the many layers of regulation that occur in maintaining the intestinal homeostasis. GENERAL SIGNIFICANCE Understanding the involvement of ISCs in inflammatory diseases can potentially lead to new therapeutic approaches to treat inflammatory GI pathologies such as IBD and celiac disease and could reveal the molecular mechanisms leading to the pathogenesis of dysplasia and cancer in inflammatory chronic conditions. This article is part of a Special Issue entitled Biochemistry of Stem Cells.

Identification of transglutaminase-mediated deamidation sites in a recombinant α-gliadin by advanced mass-spectrometric methodologies

Celiac disease is a permanent immune‐mediated food intolerance triggered by ingestion of wheat gliadins in genetically susceptible individuals. It has been reported that tissue transglutaminase plays an important role in the onset of celiac disease by converting specific glutamine residues within gliadin fragments into glutamic acid residues. This process increases binding affinity of gliadin peptides to HLA‐DQ2/DQ8 molecules, thus enhancing the immune response. The aim of the present study was to achieve a detailed structural characterization of modifications induced by transglutaminase on gliadin peptides. Therefore, structural analyses were carried out on a recombinant α‐gliadin and on a panel of 26 synthetic peptides, overlapping the complete protein sequence. Modified glutamine residues were identified by means of advanced mass‐spectrometric methodologies on the basis of MALDI‐TOF‐MS and tandem mass spectrometry. Results led to the identification of 19 of 94 glutamine residues present in the recombinant α‐gliadin, which were converted into glutamic acid residues by a transglutaminase‐mediated reaction. This allowed us to achieve a global view of the modifications induced by the enzyme on this protein. Furthermore, results gathered could likely be utilized as relevant information for a better understanding of processes leading to T‐cell recognition of gliadin peptides involved in celiac disease.

TORC1 regulators Iml1/GATOR1 and GATOR2 control meiotic entry and oocyte development in Drosophila

Significance The target of rapamycin complex 1 (TORC1) promotes cell growth and anabolic metabolism. In yeast, entry into meiosis is contingent on the down-regulation of TORC1 activity by the increased minichromosome loss 1/GTPase-activating proteins toward Rags 1 (Iml1/GATOR1) complex in response to amino acid starvation. Here we define the developmental requirements for the TORC1 regulators Iml1/GATOR1 and GATOR2 during Drosophila oogenesis. We demonstrate that, as is observed in yeast, the Iml1/GATOR1 complex down-regulates TORC1 activity to facilitate the mitotic/meiotic transition in Drosophila ovarian cysts. Later in oogenesis, components of the GATOR2 complex oppose the activity of GATOR1 to enable a rise in TORC1 activity that drives oocyte development and growth. Thus, a conserved nutrient stress pathway has been incorporated into a developmental program that regulates meiotic progression in Drosophila. In single-cell eukaryotes the pathways that monitor nutrient availability are central to initiating the meiotic program and gametogenesis. In Saccharomyces cerevisiae an essential step in the transition to the meiotic cycle is the down-regulation of the nutrient-sensitive target of rapamycin complex 1 (TORC1) by the increased minichromosome loss 1/ GTPase-activating proteins toward Rags 1 (Iml1/GATOR1) complex in response to amino acid starvation. How metabolic inputs influence early meiotic progression and gametogenesis remains poorly understood in metazoans. Here we define opposing functions for the TORC1 regulatory complexes Iml1/GATOR1 and GATOR2 during Drosophila oogenesis. We demonstrate that, as is observed in yeast, the Iml1/GATOR1 complex inhibits TORC1 activity to slow cellular metabolism and drive the mitotic/meiotic transition in developing ovarian cysts. In iml1 germline depletions, ovarian cysts undergo an extra mitotic division before meiotic entry. The TORC1 inhibitor rapamycin can suppress this extra mitotic division. Thus, high TORC1 activity delays the mitotic/meiotic transition. Conversely, mutations in Tor, which encodes the catalytic subunit of the TORC1 complex, result in premature meiotic entry. Later in oogenesis, the GATOR2 components Mio and Seh1 are required to oppose Iml1/GATOR1 activity to prevent the constitutive inhibition of TORC1 and a block to oocyte growth and development. To our knowledge, these studies represent the first examination of the regulatory relationship between the Iml1/GATOR1 and GATOR2 complexes within the context of a multicellular organism. Our data imply that the central role of the Iml1/GATOR1 complex in the regulation of TORC1 activity in the early meiotic cycle has been conserved from single cell to multicellular organisms.

Celiac Disease Histopathology Recapitulates Hedgehog Downregulation, Consistent with Wound Healing Processes Activation

Background In celiac disease (CD), intestinal epithelium damage occurs secondary to an immune insult and is characterized by blunting of the villi and crypt hyperplasia. Similarities between Hedgehog (Hh)/BMP4 downregulation, as reported in a mouse model, and CD histopathology, suggest mechanistic involvement of Hh/BMP4/WNT pathways in proliferation and differentiation of immature epithelial cells in the context of human intestinal homeostasis and regeneration after damage. Herein we examined the nature of intestinal crypt hyperplasia and involvement of Hh/BMP4 in CD histopathology. Methods and Findings Immunohistochemistry, qPCR and in situ hybridization were used to study a cohort of 24 healthy controls (HC) and 24 patients with diagnosed acute celiac disease (A-CD) intestinal biopsies. In A-CD we observed an increase in cells positive for Leucin-rich repeat-containing G protein-coupled receptor 5 (LGR5), an epithelial stem cell specific marker and expansion of WNT responding compartment. Further, we observed alteration in number and distribution of mesenchymal cells, predicted to be part of the intestinal stem cells niche. At the molecular level we found downregulation of indian hedgehog (IHH) and other components of the Hh pathway, but we did not observe a concurrent downregulation of BMP4. However, we observed upregulation of BMPs antagonists, gremlin 1 and gremlin 2. Conclusions Our data suggest that acute CD histopathology partially recapitulates the phenotype reported in Hh knockdown models. Specifically, Hh/BMP4 paradigm appears to be decoupled in CD, as the expansion of the immature cell population does not occur consequent to downregulation of BMP4. Instead, we provide evidence that upregulation of BMP antagonists play a key role in intestinal crypt hyperplasia. This study sheds light on the molecular mechanisms underlying CD histopathology and the limitations in the use of mouse models for celiac disease.