Defective tumor necrosis factor-alpha-dependent control of astrocyte glutamate release in a transgenic mouse model of Alzheimer disease.

The cytokine tumor necrosis factor-alpha (TNFalpha) induces Ca2+-dependent glutamate release from astrocytes via the downstream action of prostaglandin (PG) E2. By this process, astrocytes may participate in intercellular communication and neuromodulation. Acute inflammation in vitro, induced by adding reactive microglia to astrocyte cultures, enhances TNFalpha production and amplifies glutamate release, switching the pathway into a neurodamaging cascade (Bezzi, P., Domercq, M., Brambilla, L., Galli, R., Schols, D., De Clercq, E., Vescovi, A., Bagetta, G., Kollias, G., Meldolesi, J., and Volterra, A. (2001) Nat. Neurosci. 4, 702-710). Because glial inflammation is a component of Alzheimer disease (AD) and TNFalpha is overexpressed in AD brains, we investigated possible alterations of the cytokine-dependent pathway in PDAPP mice, a transgenic model of AD. Glutamate release was measured in acute hippocampal and cerebellar slices from mice at early (4-month-old) and late (12-month-old) disease stages in comparison with age-matched controls. Surprisingly, TNFalpha-evoked glutamate release, normal in 4-month-old PDAPP mice, was dramatically reduced in the hippocampus of 12-month-old animals. This defect correlated with the presence of numerous beta-amyloid deposits and hypertrophic astrocytes. In contrast, release was normal in cerebellum, a region devoid of beta-amyloid deposition and astrocytosis. The Ca2+-dependent process by which TNFalpha evokes glutamate release in acute slices is distinct from synaptic release and displays properties identical to those observed in cultured astrocytes, notably PG dependence. However, prostaglandin E2 induced normal glutamate release responses in 12-month-old PDAPP mice, suggesting that the pathology-associated defect involves the TNFalpha-dependent control of secretion rather than the secretory process itself. Reduced expression of DENN/MADD, a mediator of TNFalpha-PG coupling, might account for the defect. Alteration of this neuromodulatory astrocytic pathway is described here for the first time in relation to Alzheimer disease.

Autoimmunity-Associated LYP-W620 Does Not Impair Thymic Negative Selection of Autoreactive T Cells.

A C1858T (R620W) variation in the PTPN22 gene encoding the tyrosine phosphatase LYP is a major risk factor for human autoimmunity. LYP is a known negative regulator of signaling through the T cell receptor (TCR), and murine Ptpn22 plays a role in thymic selection. However, the mechanism of action of the R620W variant in autoimmunity remains unclear. One model holds that LYP-W620 is a gain-of-function phosphatase that causes alterations in thymic negative selection and/or thymic output of regulatory T cells (Treg) through inhibition of thymic TCR signaling. To test this model, we generated mice in which the human LYP-W620 variant or its phosphatase-inactive mutant are expressed in developing thymocytes under control of the proximal Lck promoter. We found that LYP-W620 expression results in diminished thymocyte TCR signaling, thus modeling a "gain-of-function" of LYP at the signaling level. However, LYP-W620 transgenic mice display no alterations of thymic negative selection and no anomalies in thymic output of CD4(+)Foxp3(+) Treg were detected in these mice. Lck promoter-directed expression of the human transgene also causes no alteration in thymic repertoire or increase in disease severity in a model of rheumatoid arthritis, which depends on skewed thymic selection of CD4(+) T cells. Our data suggest that a gain-of-function of LYP is unlikely to increase risk of autoimmunity through alterations of thymic selection and that LYP likely acts in the periphery perhaps selectively in regulatory T cells or in another cell type to increase risk of autoimmunity.

Looking for factors involved in the light-dependent degradation of phytochrome A

SUMMARY : Phytochromes constitute a family of red/far-red photoreceptors regulating all the major transitions during the life cycle of plants. In Arabidopsis, five members: phyA,_ B, C, D and E, were identified. Phytochromes are synthesized in their inactive red-light absorbing form called Pr. Upon light absorbance they convert to the far-red light absorbing Pfr form. The Pfr form is the active conformer which converts back to the Pr form either rapidly upon far-red perception or in a slower process called dark reversion. ph~A represents an exception, in that it does not significantly dark-revert and two specific processes have been developed by the plants to decrease the amount of biologically active phyA. The first one is alight-dependent repression of the PHYA gene expression and the second one is alight-dependent degradation of the phyA protein. The latter is the most efficient process to rapidly decrease the level of active phyA. The ability of plants to regulate the amount of active phyA is critical in a far-red rich environment, a situation observed under a canopy. In these conditions, phyA is essential to induce the germination and the deetiolation of the young seedling. Later in the development the ability of phyA to repress growth counteracts the shade avoidance response. Therefore decreasing the amount of phyA allows stem growth and to compete with neighbours for the light. In this thesis, I investigate the light-dependent degradation of phyA. I developed a reverse genetic approach based on the systematic analysis of the light-dependent accumulation of phyA in the different cullin mutant cull, cul3a; cul3b and cul4. This analysis allowed me to show that CUL1 and CUL3A-based E3 ligase complexes are involved in the regulation of phyA degradation. Surprisingly, our results also demonstrate that cu14 is not affected in the degradation of phyA whereas constitutive Photomorphogenic 1 (COP1) a subunit of one CUL4based E3 complex was reported to be involved. Further investigations showed that the phenotype of cop1 is conditional, the mutant being defective in phyA degradation only in the presence of metabolisable sugars. I also showed that phyA is degraded by a proteasome-dependent mechanism both in the cytoplasm and in the nucleus using mutants and transgenic lines affected in the localization of phyA. Interestingly, I observed that phyA degradation was faster in the nucleus than in the cytosol and that rapid degradation of Pr also occurred in the nucleus suggesting that cytosolic accumulation of phyA in the dark is a way to regulate its proteolysis. Finally, we identify a short region similar to a PEST sequence required for phyA stability and we developed a unbiased genetic screen to identify new components involved in the regulation of the light-dependent degradation of phyA. The significance of these results are discussed. RESUME : Les phytochromes (phy) constituent une famille de photorécepteurs absorbant la lumière rouge et rouge lointaine et régulant toutes les étapes de transitions majeures dans la vie des plantes. Chez Arabidopsis, cinq membres : phyA, B, C, D et E ont été identifiés. Les phytochromes sont synthétisés sous une forme inactive appelée Pr absorbant la lumière rouge. Après perception de lumière ils passent sous une forme active Pfr absorbant dans le rouge lointain. La forme Pfr peut retourner sous la forme Pr après absorption de lumiëre rouge lointaine ou dans un processus lent appelé «réversion à l'obscurité ». phyA représente une exception à cette règle car il ne retoune pas significativement sous sa forme inactive dans le noir. Deux processus spécifiques ont donc été développés pour diminuer le taux de phyA actif. Le premier consiste en la répression du gène PHYA en condition de lumière et le second en une dégradation induite par la lumière de la protéine phyA. Ce dernier processus est le plus efficace pour diminuer rapidement le niveau de phyA. La capacité des plantes à réguler le taux de phyA actifs est critique dans un environnement riche en lumière rouge lointaine, une situation observée sous une canopée. Sous une canopée, phyA est essentiel pour induire la germination et la dé-étiolation de la jeune pousse. Plus tard dans le développement la capacité de phyA de réprimer la croissance freine la «réponse à l'évitement de l'ombre ». Par conséquent diminuer le taux de phyA permet la croissance de la tige et donc de rentrer en compétition pour la lumière avec les plantes avoisinantes. Dans cette thèse, j'ai étudié la dégradation de phyA. J'ai développé une approche génétique inverse basée sur l'analyse systématique de l'accumulation de phyA en condition de lumière dans les différents mutants cullin, cul1, cul3a, cul3b et cul4. Ces analyses nous ont permis d'identifier qu'un complexe E3 ligase CUL1 et un complexe E3 ligase CUL3A sont impliqués dans la régulation de la dégradation de phyA. Mes résultats démontrent aussi que le mutant cul4 n'est pas affecté dans la dégradation de phyA alors que Çonstitutive Photomorphogenic 1 (COPI) une sous unité d'un complexe CUL4 à été identifier dans la régulation de cette dégradation. Des analyses supplémentaires suggèrent que l'effet de la mutation cop1 est dépendante dë la présence de sucres métabolisables. J'ai aussi montré que phyA est dégradé dans le noyau et dans le cytoplasme par un mécanisme dépendant du protéasome et que la dégradation dans le.noyau est non seulement aspécifique de la forme Pr ou Pfr mais aussi est plus rapide que dans le cytoplasme. Ceci suggère que l'accumulation de phyA dans le cytoplasme permet son accumulation à des niveaux élevés à l'obscurité. Enfin j'ai identifié une région similaire à un motif PEST requise pour la stabilité de phyA et j'ai aussi développé un criblage génétique non biaisé pour identifier de nouveaux composants impliqués dans la régulation de la dégradation de phyA. L'importance de ces résultats est discutée dans le dernier chapitre de cette thèse.

The δ-Opioid Receptor Affects Epidermal Homeostasis via ERK-Dependent Inhibition of Transcription Factor POU2F3.

Neuropeptides and their receptors are present in human skin, and their importance for cutaneous homeostasis and during wound healing is increasingly appreciated. However, there is currently a lack of understanding of the molecular mechanisms by which their signaling modulates keratinocyte function. Here, we show that δ-opioid receptor (DOPr) activation inhibits proliferation of human keratinocytes, resulting in decreased epidermal thickness in an organotypic skin model. DOPr signaling markedly delayed induction of keratin intermediate filament (KRT10) during in vitro differentiation and abolished its induction in the organotypic skin model. This was accompanied by deregulation of involucrin (IVL), loricrin, and filaggrin. Analysis of the transcription factor POU2F3, which is involved in regulation of KRT10, IVL, and profilaggrin expression, revealed a DOPr-mediated extracellular signal-regulated kinase (ERK)-dependent downregulation of this factor. We propose that DOPr signaling specifically activates the ERK 1/2 mitogen-activated protein kinase pathway to regulate keratinocyte functions. Complementing our earlier studies in DOPr-deficient mice, these data suggest that DOPr activation in human keratinocytes profoundly influences epidermal morphogenesis and homeostasis.

Activity-dependent phosphorylation of SNAP-25 in hippocampal organotypic cultures.

Synaptosomal-associated protein of 25 kDa (SNAP-25) is thought to play a key role in vesicle exocytosis and in the control of transmitter release. However, the precise mechanisms of action as well as the regulation of SNAP-25 remain unclear. Here we show by immunoprecipitation that activation of protein kinase C (PKC) by phorbol esters results in an increase in SNAP-25 phosphorylation. In addition, immunochemical analysis of two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels shows that SNAP-25 focuses as three or four distinct spots in the expected range of molecular weight and isoelectric point. Changing the phosphorylation level of the protein by incubating the slices in the presence of either a PKC agonist (phorbol 12,13-dibutyrate) or antagonist (chelerythrine) modified the distribution of SNAP-25 among these spots. Phorbol 12,13-dibutyrate increased the intensity of the spots with higher molecular weight and lower isoelectric point, whereas chelerythrine produced the opposite effect. This effect was specific for regulators of PKC, as agonists of other kinases did not produce similar changes. Induction of long-term potentiation, a property involved in learning mechanisms, and production of seizures with a GABA(A) receptor antagonist also increased the intensity of the spots with higher molecular weight and lower isoelectric point. This effect was prevented by the PKC inhibitor chelerythrine. We conclude that SNAP-25 can be phosphorylated in situ by PKC in an activity-dependent manner.

A combined computational and functional approach identifies new residues involved in pH-dependent gating of ASIC1a.

Acid-sensing ion channels (ASICs) are key receptors for extracellular protons. These neuronal nonvoltage-gated Na(+) channels are involved in learning, the expression of fear, neurodegeneration after ischemia, and pain sensation. We have applied a systematic approach to identify potential pH sensors in ASIC1a and to elucidate the mechanisms by which pH variations govern ASIC gating. We first calculated the pK(a) value of all extracellular His, Glu, and Asp residues using a Poisson-Boltzmann continuum approach, based on the ASIC three-dimensional structure, to identify candidate pH-sensing residues. The role of these residues was then assessed by site-directed mutagenesis and chemical modification, combined with functional analysis. The localization of putative pH-sensing residues suggests that pH changes control ASIC gating by protonation/deprotonation of many residues per subunit in different channel domains. Analysis of the function of residues in the palm domain close to the central vertical axis of the channel allowed for prediction of conformational changes of this region during gating. Our study provides a basis for the intrinsic ASIC pH dependence and describes an approach that can also be applied to the investigation of the mechanisms of the pH dependence of other proteins.

Time-Dependent Specificity of Immunopathologic (C4d-CD68) and Histologic Criteria of Antibody-Mediated Rejection for Donor-Specific Antibodies and Allograft Dysfunction in Heart Transplantation.

BACKGROUND: In heart transplantation, antibody-mediated rejection (AMR) is diagnosed and graded on the basis of immunopathologic (C4d-CD68) and histopathologic criteria found on endomyocardial biopsies (EMB). Because some pathologic AMR (pAMR) grades may be associated with clinical AMR, and because humoral responses may be affected by the intensity of immunosuppression during the first posttransplantation year, we investigated the incidence and positive predictive values (PPV) of C4d-CD68 and pAMR grades for clinical AMR as a function of time. METHODS: All 564 EMB from 40 adult heart recipients were graded for pAMR during the first posttransplantation year. Clinical AMR was diagnosed by simultaneous occurrence of pAMR on EMB, donor specific antibodies and allograft dysfunction. RESULTS: One patient demonstrated clinical AMR at postoperative day 7 and one at 6 months (1-year incidence 5%). C4d-CD68 was found on 4,7% EMB with a "decrescendo" pattern over time (7% during the first 4 months vs. 1.2% during the last 8 months; P < 0.05). Histopathologic criteria of AMR occurred on 10.3% EMB with no particular time pattern. Only the infrequent (1.4%) pAMR2 grade (simultaneous histopathologic and immunopathologic markers) was predictive for clinical AMR, particularly after the initial postoperative period (first 4 months and last 8 months PPV = 33%-100%; P < 0.05). CONCLUSION: In the first posttransplantation year, AMR immunopathologic and histopathologic markers were relatively frequent, but only their simultaneous occurrence (pAMR2) was predictive of clinical AMR. Furthermore, posttransplantation time may modulate the occurrence of C4d-CD68 on EMB and thus the incidence of pAMR2 and its relevance to the diagnosis of clinical AMR.

In vitro negative selection of viral superantigen-reactive thymocytes by thymic dendritic cells.

Intrathymic expression of endogenous mouse mammary tumor virus (MMTV)-encoded superantigens (SAg) induces the clonal deletion of T cells bearing SAg-reactive T-cell receptor (TCR) Vbeta elements. However, the identity of the thymic antigen-presenting cells (APC) involved in the induction of SAg tolerance remains to be defined. We have analyzed the potential of dendritic cells (DC) to mediate the clonal deletion of Mtv-7-reactive TCR alphabeta P14 transgenic thymocytes in an in vitro assay. Our results show that both thymic and splenic DC induced the deletion of TCR transgenic double positive (DP) thymocytes. DC appear to be more efficient than splenic B cells as negatively selecting APC in this experimental system. Interestingly, thymic and splenic DC display a differential ability to induce CD4+ SP thymocyte proliferation. These observations suggest that thymic DC may have an important role in the induction of SAg tolerance in vivo.

Age-Dependent Defects of Regulatory B Cells in Wiskott-Aldrich Syndrome Gene Knockout Mice.

The Wiskott-Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency characterized by recurrent infections, thrombocytopenia, eczema, and high incidence of malignancy and autoimmunity. The cellular mechanisms underlying autoimmune complications in WAS have been extensively studied; however, they remain incompletely defined. We investigated the characteristics of IL-10-producing CD19+CD1dhighCD5+ B cells (CD1dhighCD5+ Breg) obtained from Was gene knockout (WKO) mice and found that their numbers were significantly lower in these mice compared to wild type (WT) controls. Moreover, we found a significant age-dependent reduction of the percentage of IL-10-expressing cells in WKO CD1dhighCD5+ Breg cells as compared to age-matched WT control mice. CD1dhighCD5+ Breg cells from older WKO mice did not suppress the in vitro production of inflammatory cytokines from activated CD4+ T cells. Interestingly, CD1dhighCD5+ Breg cells from older WKO mice displayed a basal activated phenotype which may prevent normal cellular responses, among which is the expression of IL-10. These defects may contribute to the susceptibility to autoimmunity with age in patients with WAS.

Inducible nitric oxide synthase-dependent stimulation of PKGI and phosphorylation of VASP in human embryonic kidney cells.

Inducible nitric oxide synthase (iNOS) production of nitric oxide (NO) has been mostly associated with so-called nitrosative stress or interaction with superoxide anion. However, recent investigations have indicated that, as for the other isoenzymes producing NO, guanylyl cyclase (GC) is a very sensitive target of iNOS activity. To further investigate this less explored signaling, the NO-cyclic guanosine 3'-5'-monophosphate (NO-cGMP)-induced vasodilator-stimulated phosphoprotein (VASP) phosphorylation on serine 239 was investigated in human embryonic kidney 293 cells (HEK cells). First, the expression and activity of alpha2 and beta1 NO-sensitive GC subunits was determined by Western blot analysis, reverse transcription-polymerase chain reaction and NO donors administration. Then, the expression of a functional cGMP-dependent protein kinase I (PKGI) was verified by addition of 8-Br-cGMP followed by determination of phosphorylation of VASP on serine 239. Finally, iNOS activation of this signaling pathway was characterized after transfection of HEK cells with human iNOS cDNA. Altogether our data show that iNOS-derived NO activates endogenous NO-sensitive GC and leads to VASP phosphorylation in HEK cells.

Flagellin promotes myeloid differentiation factor 88-dependent development of Th2-type response.

Activation of dendritic cells (DC) by microbial products via Toll-like receptors (TLR) is instrumental in the induction of immunity. In particular, TLR signaling plays a major role in the instruction of Th1 responses. The development of Th2 responses has been proposed to be independent of the adapter molecule myeloid differentiation factor 88 (MyD88) involved in signal transduction by TLRs. In this study we show that flagellin, the bacterial stimulus for TLR5, drives MyD88-dependent Th2-type immunity in mice. Flagellin promotes the secretion of IL-4 and IL-13 by Ag-specific CD4(+) T cells as well as IgG1 responses. The Th2-biased responses are associated with the maturation of DCs, which are shown to express TLR5. Flagellin-mediated DC activation requires MyD88 and induces NF-kappaB-dependent transcription and the production of low levels of proinflammatory cytokines. In addition, the flagellin-specific response is characterized by the lack of secretion of the Th1-promoting cytokine IL-12 p70. In conclusion, this study suggests that flagellin and, more generally, TLR ligands can control Th2 responses in a MyD88-dependent manner.

Selection of patients with germline MLH1 mutated Lynch syndrome by determination of MLH1 methylation and BRAF mutation.

Lynch syndrome is one of the most common hereditary colorectal cancer (CRC) syndrome and is caused by germline mutations of MLH1, MSH2 and more rarely MSH6, PMS2, MLH3 genes. Whereas the absence of MSH2 protein is predictive of Lynch syndrome, it is not the case for the absence of MLH1 protein. The purpose of this study was to develop a sensitive and cost effective algorithm to select Lynch syndrome cases among patients with MLH1 immunohistochemical silencing. Eleven sporadic CRC and 16 Lynch syndrome cases with MLH1 protein abnormalities were selected. The BRAF c.1799T> A mutation (p.Val600Glu) was analyzed by direct sequencing after PCR amplification of exon 15. Methylation of MLH1 promoter was determined by Methylation-Sensitive Single-Strand Conformation Analysis. In patients with Lynch syndrome, there was no BRAF mutation and only one case showed MLH1 methylation (6%). In sporadic CRC, all cases were MLH1 methylated (100%) and 8 out of 11 cases carried the above BRAF mutation (73%) whereas only 3 cases were BRAF wild type (27%). We propose the following algorithm: (1) no further molecular analysis should be performed for CRC exhibiting MLH1 methylation and BRAF mutation, and these cases should be considered as sporadic CRC; (2) CRC with unmethylated MLH1 and negative for BRAF mutation should be considered as Lynch syndrome; and (3) only a small fraction of CRC with MLH1 promoter methylation but negative for BRAF mutation should be true Lynch syndrome patients. These potentially Lynch syndrome patients should be offered genetic counselling before searching for MLH1 gene mutations.

Aging and motor programming: differential effects according to the selection of action

There are controversial reports about the effect of aging on movement preparation, and it is unclear to which extent cognitive and/or motor related cerebral processes may be affected. This study examines the age effects on electro-cortical oscillatory patterns during various motor programming tasks, in order to assess potential differences according to the mode of action selection. Twenty elderly (EP, 60-84 years) and 20 young (YP, 20-29 years) participants with normal cognition underwent 3 pre-cued response tasks (S1-S2 paradigm). S1 carried either complete information on response side (Full; stimulus-driven motor preparation), no information (None; general motor alertness), or required free response side selection (Free; internally-driven motor preparation). Electroencephalogram (EEG) was recorded using 64 surface electrodes. Alpha (8-12 Hz) desynchronization (ERD)/synchronization (ERS) and motor-related amplitude asymmetries (MRAA) were analyzed during the S1-S2 interval. Reaction times (RTs) to S2 were slower in EP than YP, and in None than in the other 2 tasks. There was an Age x Task interaction due to increased RTs in Free compared to Full in EP only. Central bilateral and midline activation (alpha ERD) was smaller in EP than YP in None. In Full just before S2, readiness to move was reflected by posterior midline inhibition (alpha ERS) in both groups. In Free, such inhibition was present only in YP. Moreover, MRAA showed motor activity lateralization in both groups in Full, but only in YP in Free. The results indicate reduced recruitment of motor regions for motor alertness in the elderly. They further show less efficient cerebral processes subtending free selection of movement in elders, suggesting reduced capacity for internally-driven action with age.

Context-dependent interpretation of the prognostic value of BRAF and KRAS mutations in colorectal cancer.

BACKGROUND: The mutation status of the BRAF and KRAS genes has been proposed as prognostic biomarker in colorectal cancer. Of them, only the BRAF V600E mutation has been validated independently as prognostic for overall survival and survival after relapse, while the prognostic value of KRAS mutation is still unclear. We investigated the prognostic value of BRAF and KRAS mutations in various contexts defined by stratifications of the patient population. METHODS: We retrospectively analyzed a cohort of patients with stage II and III colorectal cancer from the PETACC-3 clinical trial (N = 1,423), by assessing the prognostic value of the BRAF and KRAS mutations in subpopulations defined by all possible combinations of the following clinico-pathological variables: T stage, N stage, tumor site, tumor grade and microsatellite instability status. In each such subpopulation, the prognostic value was assessed by log rank test for three endpoints: overall survival, relapse-free survival, and survival after relapse. The significance level was set to 0.01 for Bonferroni-adjusted p-values, and a second threshold for a trend towards statistical significance was set at 0.05 for unadjusted p-values. The significance of the interactions was tested by Wald test, with significance level of 0.05. RESULTS: In stage II-III colorectal cancer, BRAF mutation was confirmed a marker of poor survival only in subpopulations involving microsatellite stable and left-sided tumors, with higher effects than in the whole population. There was no evidence for prognostic value in microsatellite instable or right-sided tumor groups. We found that BRAF was also prognostic for relapse-free survival in some subpopulations. We found no evidence that KRAS mutations had prognostic value, although a trend was observed in some stratifications. We also show evidence of heterogeneity in survival of patients with BRAF V600E mutation. CONCLUSIONS: The BRAF mutation represents an additional risk factor only in some subpopulations of colorectal cancers, in others having limited prognostic value. However, in the subpopulations where it is prognostic, it represents a marker of much higher risk than previously considered. KRAS mutation status does not seem to represent a strong prognostic variable.