Novel acid phosphatase in Candida glabrata suggests selective pressure and niche specialization in the phosphate signal transduction pathway.

Evolution through natural selection suggests unnecessary genes are lost. We observed that the yeast Candida glabrata lost the gene encoding a phosphate-repressible acid phosphatase (PHO5) present in many yeasts including Saccharomyces cerevisiae. However, C. glabrata still had phosphate starvation-inducible phosphatase activity. Screening a C. glabrata genomic library, we identified CgPMU2, a member of a three-gene family that contains a phosphomutase-like domain. This small-scale gene duplication event could allow for sub- or neofunctionalization. On the basis of phylogenetic and biochemical characterizations, CgPMU2 has neofunctionalized to become a broad range, phosphate starvation-regulated acid phosphatase, which functionally replaces PHO5 in this pathogenic yeast. We determined that CgPmu2, unlike ScPho5, is not able to hydrolyze phytic acid (inositol hexakisphosphate). Phytic acid is present in fruits and seeds where S. cerevisiae grows, but is not abundant in mammalian tissues where C. glabrata grows. We demonstrated that C. glabrata is limited from an environment where phytic acid is the only source of phosphate. Our work suggests that during evolutionary time, the selection for the ancestral PHO5 was lost and that C. glabrata neofunctionalized a weak phosphatase to replace PHO5. Convergent evolution of a phosphate starvation-inducible acid phosphatase in C. glabrata relative to most yeast species provides an example of how small changes in signal transduction pathways can mediate genetic isolation and uncovers a potential speciation gene.

Germ cell-specific targeting of DICER or DGCR8 reveals a novel role for endo-siRNAs in the progression of mammalian spermatogenesis and male fertility.

Small non-coding RNAs act as critical regulators of gene expression and are essential for male germ cell development and spermatogenesis. Previously, we showed that germ cell-specific inactivation of Dicer1, an endonuclease essential for the biogenesis of micro-RNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs), led to complete male infertility due to alterations in meiotic progression, increased spermatocyte apoptosis and defects in the maturation of spermatozoa. To dissect the distinct physiological roles of miRNAs and endo-siRNAs in spermatogenesis, we compared the testicular phenotype of mice with Dicer1 or Dgcr8 depletion in male germ cells. Dgcr8 mutant mice, which have a defective miRNA pathway while retaining an intact endo-siRNA pathway, were also infertile and displayed similar defects, although less severe, to Dicer1 mutant mice. These included cumulative defects in meiotic and haploid phases of spermatogenesis, resulting in oligo-, terato-, and azoospermia. In addition, we found by RNA sequencing of purified spermatocytes that inactivation of Dicer1 and the resulting absence of miRNAs affected the fine tuning of protein-coding gene expression by increasing low level gene expression. Overall, these results emphasize the essential role of miRNAs in the progression of spermatogenesis, but also indicate a role for endo-siRNAs in this process.

Atopic dermatitis-like disease and associated lethal myeloproliferative disorder arise from loss of Notch signaling in the murine skin.

BACKGROUND: The Notch pathway is essential for proper epidermal differentiation during embryonic skin development. Moreover, skin specific loss of Notch signaling in the embryo results in skin barrier defects accompanied by a B-lymphoproliferative disease. However, much less is known about the consequences of loss of Notch signaling after birth. METHODOLOGY AND PRINCIPAL FINDINGS: To study the function of Notch signaling in the skin of adult mice, we made use of a series of conditional gene targeted mice that allow inactivation of several components of the Notch signaling pathway specifically in the skin. We demonstrate that skin-specific inactivation of Notch1 and Notch2 simultaneously, or RBP-J, induces the development of a severe form of atopic dermatitis (AD), characterized by acanthosis, spongiosis and hyperkeratosis, as well as a massive dermal infiltration of eosinophils and mast cells. Likewise, patients suffering from AD, but not psoriasis or lichen planus, have a marked reduction of Notch receptor expression in the skin. Loss of Notch in keratinocytes induces the production of thymic stromal lymphopoietin (TSLP), a cytokine deeply implicated in the pathogenesis of AD. The AD-like associated inflammation is accompanied by a myeloproliferative disorder (MPD) characterized by an increase in immature myeloid populations in the bone marrow and spleen. Transplantation studies revealed that the MPD is cell non-autonomous and caused by dramatic microenvironmental alterations. Genetic studies demontrated that G-CSF mediates the MPD as well as changes in the bone marrow microenvironment leading to osteopenia. SIGNIFICANCE: Our data demonstrate a critical role for Notch in repressing TSLP production in keratinocytes, thereby maintaining integrity of the skin and the hematopoietic system.

Pathogenesis of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumours worldwide. The major aetiologies and risk factors for the development of HCC are well defined and some of the multiple steps involved in hepatocarcinogenesis have been elucidated in recent years. However, no clear picture of how and in what sequence these factors interact at the molecular level has emerged yet. Malignant transformation of hepatocytes may occur as a consequence of various aetiologies, such as chronic viral hepatitis, alcohol, and metabolic disorders, in the context of increased cellular turnover induced by chronic liver injury, regeneration and cirrhosis. Activation of cellular oncogenes, inactivation of tumour suppressor genes, genomic instability, including DNA mismatch repair defects and impaired chromosomal segregation, overexpression of growth and angiogenic factors, and telomerase activation may contribute to the development of HCC. Overall, HCCs are genetically very heterogeneous tumours. New technologies, including gene expression profiling and proteomic analyses, should allow us to further elucidate the molecular events underlying HCC development and identify novel diagnostic markers as well as therapeutic targets.

Jagged1-dependent Notch signaling is dispensable for hematopoietic stem cell self-renewal and differentiation.

Jagged1-mediated Notch signaling has been suggested to be critically involved in hematopoietic stem cell (HSC) self-renewal. Unexpectedly, we report here that inducible Cre-loxP-mediated inactivation of the Jagged1 gene in bone marrow progenitors and/or bone marrow (BM) stromal cells does not impair HSC self-renewal or differentiation in all blood lineages. Mice with simultaneous inactivation of Jagged1 and Notch1 in the BM compartment survived normally following a 5FU-based in vivo challenge. In addition, Notch1-deficient HSCs were able to reconstitute mice with inactivated Jagged1 in the BM stroma even under competitive conditions. In contrast to earlier reports, these data exclude an essential role for Jagged1-mediated Notch signaling during hematopoiesis.

Physiology of GLP-1--lessons from glucoincretin receptor knockout mice.

GLP-1 has both peripheral and central actions, as this hormone is secreted by gut endocrine cells and brainstem neurons projecting into the hypothalamus and other brain regions. GLP-1 has multiple regulatory functions participating in the control of glucose homeostasis, beta-cell proliferation and differentiation, food intake, heart rate and even learning. GLP-1 action depends on binding to a specific G-coupled receptor linked to activation of the adenylyl cyclase pathway. Analysis of mice with inactivation of the GLP-1 receptor gene has provided evidence that absence of GLP-1 action in the mouse, despite this hormone potent physiological effects when administered in vivo, only leads to mild abnormalities in glucose homeostasis without any change in body weight. However, a critical role for this hormone and its receptor was demonstrated in the function of the hepatoportal vein glucose sensor, in contrast to that of the pancreatic beta-cells, although absence of both GLP-1 and GIP receptors leads to a more severe phenotype characterized by a beta-cell-autonomous defect in glucose-stimulated insulin secretion. Together, the studies of these glucoincretin receptor knockout mice provide evidence that these hormones are part of complex regulatory systems where multiple redundant signals are involved.

Astrocytic αVβ3 integrin inhibits neurite outgrowth and promotes retraction of neuronal processes by clustering Thy-1.

Thy-1 is a membrane glycoprotein suggested to stabilize or inhibit growth of neuronal processes. However, its precise function has remained obscure, because its endogenous ligand is unknown. We previously showed that Thy-1 binds directly to α(V)β(3) integrin in trans eliciting responses in astrocytes. Nonetheless, whether α(V)β(3) integrin might also serve as a Thy-1-ligand triggering a neuronal response has not been explored. Thus, utilizing primary neurons and a neuron-derived cell line CAD, Thy-1-mediated effects of α(V)β(3) integrin on growth and retraction of neuronal processes were tested. In astrocyte-neuron co-cultures, endogenous α(V)β(3) integrin restricted neurite outgrowth. Likewise, α(V)β(3)-Fc was sufficient to suppress neurite extension in Thy-1(+), but not in Thy-1(-) CAD cells. In differentiating primary neurons exposed to α(V)β(3)-Fc, fewer and shorter dendrites were detected. This effect was abolished by cleavage of Thy-1 from the neuronal surface using phosphoinositide-specific phospholipase C (PI-PLC). Moreover, α(V)β(3)-Fc also induced retraction of already extended Thy-1(+)-axon-like neurites in differentiated CAD cells as well as of axonal terminals in differentiated primary neurons. Axonal retraction occurred when redistribution and clustering of Thy-1 molecules in the plasma membrane was induced by α(V)β(3) integrin. Binding of α(V)β(3)-Fc was detected in Thy-1 clusters during axon retraction of primary neurons. Moreover, α(V)β(3)-Fc-induced Thy-1 clustering correlated in time and space with redistribution and inactivation of Src kinase. Thus, our data indicates that α(V)β(3) integrin is a ligand for Thy-1 that upon binding not only restricts the growth of neurites, but also induces retraction of already existing processes by inducing Thy-1 clustering. We propose that these events participate in bi-directional astrocyte-neuron communication relevant to axonal repair after neuronal damage.

WNT Signaling in Drosophila Neuromuscular Junctions

The Wnt -Wingless (Wg) in Drosophila- signaling is an evolutionary conserved, fundamental signal transduction pathway in animals, having a crucial role in early developmental processes. In the adult animal the Wnt cascade is mainly shut off; aberrant activation leads to cancer. One physiological exception in the adult animal is the activation of Wnt signaling in the nervous system. In the present work, we investigated Wg signaling in the Drosophila neuromuscular junctions (NMJs). The fly NMJs closely resemble the glutamatergic synapses in the mammalian central nervous system and serves as a model system to investigate the mechanism of synapse formation and stability. We demonstrate that the trimeric G-protein Go has a fundamental role in the presynaptic cell in the NMJ. It is implicated in the presynaptic Wg pathway, acting downstream of the ligand Wg and its receptor Frizzled2 (Fz2). Furthermore, we prove that the presynaptic Wg-Fz2-Gαo pathway is essential for correct NMJ formation. The neuronal protein Ankyrin2 (Ank2) localizes to the NMJ and has so far been considered to be a static player in NMJ formation, linking the plasma membrane to the cytoskeleton. We identify Ank2 as a direct target of Gαo. The physical and genetic interaction of Gαo with Ank2 represents a novel branch of the presynaptic Wg pathway, regulating the microtubule cytoskeleton in NMJ formation, jointly with the previously established Futsch-dependent branch, which controls microtubule stability downstream of the kinase Sgg (the homolog of GSK3ß). We moreover demonstrate that the Gαo-Ankyrin interaction to regulate the cytoskeleton is conserved in mammalian neuronal cells. Our findings therefore provide a novel, universally valid regulation of the cytoskeleton in the nervous system. Aberrant inactivation of the neuronal Wnt pathway is believed to be involved in the pathogenesis of the Aß peptide in Alzheimer's disease (AD). We modeled AD in Drosophila by expressing Aß42 in the nervous system and in the eye. Neuronal expression drastically shortens the life span of the flies. We prove that this effect depends on the expression specifically in glutamatergic neurons. However, Aß42 does not induce any morphological changes in the NMJ; therefore this synapse is not suitable to study the mechanism of Aß42 induced neurotoxicity. We furthermore demonstrate that genetic activation of the Wnt pathway does not rescue the Aß42 induced phenotypes - in opposition to the dominating view in the field. These results advice caution when interpreting data on the potential interaction of Wnt signaling and AD in other models. -- La voie de signalisation Wnt (Wingless (Wg) chez la drosophile) est conservée dans l'évolution et fondamentale pour le développement des animaux. Cette signalisation est normalement inactive chez l'animal adulte; une activation anormale peut provoquer le cancer. Or, ceci n'est pas le cas dans le système nerveux des adultes. La présente thèse avait pour but d'analyser le rôle de la voie de signalisation Wingless dans la plaque motrice de Drosophila melanogaster. En effet, cette plaque ressemble fortement aux synapses glutaminergiques du système nerveux central des mammifères et procure ainsi un bon modèle pour l'étude des mécanismes impliqués dans la formation et la stabilisation des synapses. Nos résultats montrent que la protéine trimérique Go joue un rôle fondamental dans la fonction de la cellule présynaptique de la plaque motrice. Go est en effet impliqué dans la voie de signalisation Wg, opérant en aval du ligand Wg et de son récepteur Frizzled2. Nous avons pu démontrer que cette voie de signalisation Wg-Fz2-Gαo est essentielle pour le bon développement et le fonctionnement de la plaque motrice. Fait intéressant, nous avons montré que la protéine neuronale Ankyrin2 (Ank2), qui est connue pour jouer un rôle statique en liant la membrane plasmique au cytosquelette dans la plaque motrice, est une cible directe de Gαo. L'interaction physique et génétique entre Gαo et Ank2 constitue ainsi une bifurcation de la voie de signalisation présynaptique Wg. Cette voie régule le cytosquelette des microtubules en coopération avec la branche liée à la protéine Futsch. Cette protéine est l'homologue de la protéine liant les microtubules MAP1B des mammifères et contrôle la stabilité des microtubules opérant en aval de la kinase Sgg (l'homologue de GSK3ß). De plus, la régulation du cytosquelette par l'interaction entre Gαo et Ankyrin est conservée chez les mammifères. Dans leur ensemble, nos résultats ont permis d'identifier un nouveau mode de régulation du cytosquelette dans le système nerveux, probablement valable de manière universelle. La voie de signalisation Wnt est soupçonnée d'être impliquée dans la toxicité provoquée par le peptide Aß dans le cadre de la maladie d'Alzheimer. Nous avons tenté de modéliser la maladie chez la drosophile en exprimant Aß42 spécifiquement dans le cerveau. Cette expérience a montré que l'expression neuronale d'Aß42 réduit la durée de vie des mouches de manière significative par un mécanisme impliquant les cellules glutamatergiques. Par contre, aucune modification morphologique n'est provoquée par Aß42 dans les plaques motrices glutamatergiques. Ces résultats montrent que ce modèle de Drosophile n'est pas adéquat pour l'étude de la maladie d'Alzheimer. De plus, l'activation génétique de la voie de signalisation Wg n'a pas réussi à restaurer les phénotypes de survie ou ceux des yeux causés par Aß42. Ces résultats indiquent que l'implication de la voie de signalisation Wg dans la maladie d'Alzheimer doit être considérée avec prudence.

Clinical trial substantiates the predictive value of O-6-methylguanine-DNA methyltransferase promoter methylation in glioblastoma patients treated with temozolomide.

PURPOSE: In the setting of a prospective clinical trial, we determined the predictive value of the methylation status of the O-6-methylguanine-DNA methyltransferase (MGMT) promoter for outcome in glioblastoma patients treated with the alkylating agent temozolomide. Expression of this excision repair enzyme has been associated with resistance to alkylating chemotherapy. EXPERIMENTAL DESIGN: The methylation status of MGMT in the tumor biopsies was evaluated in 38 patients undergoing resection for newly diagnosed glioblastoma and enrolled in a Phase II trial testing concomitant and adjuvant temozolomide and radiation. The epigenetic silencing of the MGMT gene was determined using methylation-specific PCR. RESULTS: Inactivation of the MGMT gene by promoter methylation was associated with longer survival (P = 0.0051; Log-rank test). At 18 months, survival was 62% (16 of 26) for patients testing positive for a methylated MGMT promoter but reached only 8% (1 of 12) in absence of methylation (P = 0.002; Fisher's exact test). In the presence of other clinically relevant factors, methylation of the MGMT promoter remains the only significant predictor (P = 0.017; Cox regression). CONCLUSIONS: This prospective clinical trial identifies MGMT-methylation status as an independent predictor for glioblastoma patients treated with a methylating agent. The association of the epigenetic inactivation of the DNA repair gene MGMT with better outcome in this homogenous cohort may have important implications for the design of future trials and supports efforts to deplete MGMT by O-6-benzylguanine, a noncytotoxic substrate of this enzyme.

The contact region between three domains of the extracellular loop of ASIC1a is critical for channel function.

Acid-sensing ion channels are members of the epithelial Na(+) channel/degenerin family. They are neuronal nonvoltage-gated Na(+) channels that are activated by extracellular acidification. In this study, we investigated the role of a highly conserved region of the extracellular part of ASIC1a that forms the contact between the finger domain, the adjacent beta-ball, and the upper palm domain in ASIC1a. The finger domain contributes to the pH-dependent gating and is linked via this contact zone to the rest of the protein. We found that mutation to Cys of residues in this region led to decreased channel expression and current amplitudes. Exposure of the engineered Cys residues to Cd(2+) or to charged methane thiosulfonate sulfhydryl reagents further reduced current amplitudes. This current inhibition was not due to changes in acid-sensing ion channel pH dependence or unitary conductance and was likely due to a decrease of the probability of channel opening. For some mutants, the effect of sulfhydryl reagents depended on the pH of exposure in the range 7.4 to 6.8, suggesting that this zone undergoes conformational changes during inactivation. Our study identifies a region in ASIC1a whose integrity is required for normal channel function.

Trypsin cleaves acid-sensing ion channel 1a in a domain that is critical for channel gating.

Acid-sensing ion channels (ASICs) are neuronal Na(+) channels that are members of the epithelial Na(+) channel/degenerin family and are transiently activated by extracellular acidification. ASICs in the central nervous system have a modulatory role in synaptic transmission and are involved in cell injury induced by acidosis. We have recently demonstrated that ASIC function is regulated by serine proteases. We provide here evidence that this regulation of ASIC function is tightly linked to channel cleavage. Trypsin cleaves ASIC1a with a similar time course as it changes ASIC1a function, whereas ASIC1b, whose function is not modified by trypsin, is not cleaved. Trypsin cleaves ASIC1a at Arg-145, in the N-terminal part of the extracellular loop, between a highly conserved sequence and a sequence that is critical for ASIC1a inhibition by the venom of the tarantula Psalmopoeus cambridgei. This channel domain controls the inactivation kinetics and co-determines the pH dependence of ASIC gating. It undergoes a conformational change during inactivation, which renders the cleavage site inaccessible to trypsin in inactivated channels.

PKCθ/β and CYLD are antagonistic partners in the NFκB and NFAT transactivation pathways in primary mouse CD3+ T lymphocytes.

In T cells PKCθ mediates the activation of critical signals downstream of TCR/CD28 stimulation. We investigated the molecular mechanisms by which PKCθ regulates NFκB transactivation by examining PKCθ/β single and double knockout mice and observed a redundant involvement of PKCθ and PKCβ in this signaling pathway. Mechanistically, we define a PKCθ-CYLD protein complex and an interaction between the positive PKCθ/β and the negative CYLD signaling pathways that both converge at the level of TAK1/IKK/I-κBα/NFκB and NFAT transactivation. In Jurkat leukemic T cells, CYLD is endoproteolytically processed in the initial minutes of stimulation by the paracaspase MALT1 in a PKC-dependent fashion, which is required for robust IL-2 transcription. However, in primary T cells, CYLD processing occurs with different kinetics and an altered dependence on PKC. The formation of a direct PKCθ/CYLD complex appears to regulate the short-term spatial distribution of CYLD, subsequently affecting NFκB and NFAT repressional activity of CYLD prior to its MALT1-dependent inactivation. Taken together, our study establishes CYLD as a new and critical PKCθ interactor in T cells and reveals that antagonistic PKCθ/β-CYLD crosstalk is crucial for the adjustment of immune thresholds in primary mouse CD3(+) T cells.

Scnn1 sodium channel gene family in genetically engineered mice.

The amiloride-sensitive epithelial sodium channel is the limiting step in salt absorption. In mice, this channel is composed of three subunits (alpha, beta, and gamma), which are encoded by different genes (Scnn1a, Scnn1b, and Scnn1c, respectively). The functions of these genes were recently investigated in transgenic (knockout) experiments, and the absence of any subunit led to perinatal lethality. More defined phenotypes have been obtained by introducing specific mutations or using transgenic rescue experiments. In this report, these approaches are summarized and a current gene-targeting strategy to obtain conditional inactivation of the channel is illustrated. This latter approach will be indispensable for the investigation of channel function in a wide variety of organ systems.

Nervous glucose sensing regulates postnatal β cell proliferation and glucose homeostasis.

How glucose sensing by the nervous system impacts the regulation of β cell mass and function during postnatal development and throughout adulthood is incompletely understood. Here, we studied mice with inactivation of glucose transporter 2 (Glut2) in the nervous system (NG2KO mice). These mice displayed normal energy homeostasis but developed late-onset glucose intolerance due to reduced insulin secretion, which was precipitated by high-fat diet feeding. The β cell mass of adult NG2KO mice was reduced compared with that of WT mice due to lower β cell proliferation rates in NG2KO mice during the early postnatal period. The difference in proliferation between NG2KO and control islets was abolished by ganglionic blockade or by weaning the mice on a carbohydrate-free diet. In adult NG2KO mice, first-phase insulin secretion was lost, and these glucose-intolerant mice developed impaired glucagon secretion when fed a high-fat diet. Electrophysiological recordings showed reduced parasympathetic nerve activity in the basal state and no stimulation by glucose. Furthermore, sympathetic activity was also insensitive to glucose. Collectively, our data show that GLUT2-dependent control of parasympathetic activity defines a nervous system/endocrine pancreas axis that is critical for β cell mass establishment in the postnatal period and for long-term maintenance of β cell function.

c-Myc controls the development of CD8alphaalpha TCRalphabeta intestinal intraepithelial lymphocytes from thymic precursors by regulating IL-15-dependent survival.

The murine gut epithelium contains a large population of thymus-derived intraepithelial lymphocytes (IELs), including both conventional CD4(+) and CD8alphabeta(+) T cells (expressing T-cell receptor alphabeta [TCRalphabeta]) and unconventional CD8alphaalpha(+) T cells (expressing either TCRalphabeta or TCRgammadelta). Whereas conventional IELs are widely accepted to arise from recirculation of activated CD4(+) and CD8alphabeta(+) T cells from the secondary lymphoid organs to the gut, the origin and developmental pathway of unconventional CD8alphaalpha IELs remain controversial. We show here that CD4-Cre-mediated inactivation of c-Myc, a broadly expressed transcription factor with a wide range of biologic activities, selectively impairs the development of CD8alphaalpha TCRalphabeta IELs. In the absence of c-Myc, CD4(-) CD8(-) TCRalphabeta(+) thymic precursors of CD8alphaalpha TCRalphabeta IELs are present but fail to develop on adoptive transfer in immunoincompetent hosts. Residual c-Myc-deficient CD8alphaalpha TCRalphabeta IEL display reduced proliferation and increased apoptosis, which correlate with significantly decreased expression of interleukin-15 receptor subunits and lower levels of the antiapoptotic protein Bcl-2. Transgenic overexpression of human BCL-2 resulted in a pronounced rescue of CD8alphaalpha TCRalphabeta IEL in c-Myc-deficient mice. Taken together, our data support a model in which c-Myc controls the development of CD8alphaalpha TCRalphabeta IELs from thymic precursors by regulating interleukin-15 receptor expression and consequently Bcl-2-dependent survival.