Neuroserpine et ischémie cérébrale / par Nathalie Lebeurrier (rôles et régulation transcriptionnelle)

CAEN-BU Sciences et STAPS (141182103) / SudocSudocFranceF

Tissue-type plasminogen activator rescues neurones from serum deprivation-induced apoptosis through a mechanism independent of its proteolytic activity

International audienceAlthough the mechanism of action of tissue-type plasmino-gen activator (tPA) in excitotoxic necrosis is well documented , whether this serine protease can influence the apoptotic cascade remains a subject of debate. Here, we report that tPA protects cultured cortical neurones against apoptotic cell death induced by serum deprivation, an effect associated with a reduction of caspase-3 activation. Interestingly , blocking tPA proteolytic activity by either tPA stop or neuroserpin did not prevent this neuroprotection. Similarly , prevention of the interaction between tPA and its receptor low-density lipoprotein receptor-related protein (LRP) could not alter tPA anti-apoptotic activity. Interestingly , the survival-promoting effect of tPA was blocked by the phosphatidylinositol-3 (PI-3) kinase inhibitor, LY294002, but not by the mitogen-activated protein (MAP) kinase inhibitor, U0126. In conclusion, the present demonstration of an anti-apoptotic effect of tPA, independent of its enzymatic activity, reveals an additional level of complexity in our understanding of this critical mediator of brain physiology and pathology. Keywords: apoptosis, serine protease, serum deprivation, tissue-type plasminogen activator

Human central nervous system astrocytes support survival and activation of B cells: implications for MS pathogenesis

Abstract Background The success of clinical trials of selective B cell depletion in patients with relapsing multiple sclerosis (MS) indicates B cells are important contributors to peripheral immune responses involved in the development of new relapses. Such B cell contribution to peripheral inflammation likely involves antibody-independent mechanisms. Of growing interest is the potential that B cells, within the MS central nervous system (CNS), may also contribute to the propagation of CNS-compartmentalized inflammation in progressive (non-relapsing) disease. B cells are known to persist in the inflamed MS CNS and are more recently described as concentrated in meningeal immune-cell aggregates, adjacent to the subpial cortical injury which has been associated with progressive disease. How B cells are fostered within the MS CNS and how they may contribute locally to the propagation of CNS-compartmentalized inflammation remain to be elucidated. Methods We considered whether activated human astrocytes might contribute to B cell survival and function through soluble factors. B cells from healthy controls (HC) and untreated MS patients were exposed to primary human astrocytes that were either maintained under basal culture conditions (non-activated) or pre-activated with standard inflammatory signals. B cell exposure to astrocytes included direct co-culture, co-culture in transwells, or exposure to astrocyte-conditioned medium. Following the different exposures, B cell survival and expression of T cell co-stimulatory molecules were assessed by flow cytometry, as was the ability of differentially exposed B cells to induce activation of allogeneic T cells. Results Secreted factors from both non-activated and activated human astrocytes robustly supported human B cell survival. Soluble products of pre-activated astrocytes also induced B cell upregulation of antigen-presenting cell machinery, and these B cells, in turn, were more efficient activators of T cells. Astrocyte-soluble factors could support survival and activation of B cell subsets implicated in MS, including memory B cells from patients with both relapsing and progressive forms of disease. Conclusions Our findings point to a potential mechanism whereby activated astrocytes in the inflamed MS CNS not only promote a B cell fostering environment, but also actively support the ability of B cells to contribute to the propagation of CNS-compartmentalized inflammation, now thought to play key roles in progressive disease

Additional file 5: of Human central nervous system astrocytes support survival and activation of B cells: implications for MS pathogenesis

Figure S4. Astrocyte-secreted factors also support survival and activation of relapsing remitting MS (RRMS) B cells. B cells derived from patients with RRMS were cultured in transwell either with unstimulated human astrocytes or with astrocytes that had previously been stimulated as described above. (a) B-cell viability was assessed after 48 h of transwell co-culture using 7AAD and Annexin V staining; (b) CD86 MFI was determined by flow cytometry following 48 h of transwell co-culture. Data were analyzed using one way ANOVA test (n = 6 independent experiments; n.s.: not significant; *: p < 0.05; **: p < 0.01; ***: p < 0.001). (TIFF 2647 kb

Sp1 and Smad transcription factors co-operate to mediate TGF-β-dependent activation of amyloid-β precursor protein gene transcription

Abnormal deposition of Aβ (amyloid-β peptide) is one of the hallmarks of AD (Alzheimer's disease). This peptide results from the processing and cleavage of its precursor protein, APP (amyloid-β precursor protein). We have demonstrated previously that TGF-β (transforming growth factor-β), which is overexpressed in AD patients, is capable of enhancing the synthesis of APP by astrocytes by a transcriptional mechanism leading to the accumulation of Aβ. In the present study, we aimed at further characterization of the molecular mechanisms sustaining this TGF-β-dependent transcriptional activity. We report the following findings: first, TGF-β is capable of inducing the transcriptional activity of a reporter gene construct corresponding to the +54/+74 region of the APP promoter, named APP(TRE) (APP TGF-β-responsive element); secondly, although this effect is mediated by a transduction pathway involving Smad3 (signalling mother against decapentaplegic peptide 3) and Smad4, Smad2 or other Smads failed to induce the activity of APP(TRE). We also observed that the APP(TRE) sequence not only responds to the Smad3 transcription factor, but also the Sp1 (signal protein 1) transcription factor co-operates with Smads to potentiate the TGF-β-dependent activation of APP. TGF-β signalling induces the formation of nuclear complexes composed of Sp1, Smad3 and Smad4. Overall, the present study gives new insights for a better understanding of the fine molecular mechanisms occurring at the transcriptional level and regulating TGF-β-dependent transcription. In the context of AD, our results provide additional evidence for a key role for TGF-β in the regulation of Aβ production

Additional file 4: of Human central nervous system astrocytes support survival and activation of B cells: implications for MS pathogenesis

Figure S3. IFNγ production by proliferative T-cells. Human B cells were cultured in transwell as described previously, either alone or with stimulated or unstimulated astrocytes. Following 2 days in culture, B cells were harvested, thoroughly washed and co-cultured with human T cells from allogeneic donors at a B-cell:T-cell ratio of 1:4. Conditioned media of B-cell:T-cell co-culure was collected and IFNγ was measured using ELISA (representative experiment). (TIFF 7670 kb

Additional file 2: of Human central nervous system astrocytes support survival and activation of B cells: implications for MS pathogenesis

Figure S1. Confirming activation of human astrocytes. Astrocytes were cultured for 24 h and were either left unstimulated or were stimulated with IFNγ (10 ng/ml) and IL-1β (10 ng/ml). After 24 h, the astrocytes were washed thoroughly and fresh medium was added. After an additional 24 h in culture, at which time cultures were imaged and supernatants were collected for subsequent measurement of astrocyte-secreted IL-6 by ELISA. Compared to unstimulated astrocytes (a), stimulated astrocytes exhibited activated morphology (b) and significantly-enhanced production of IL-6 (c; p = 0.0016; paired t-test). (TIFF 3951 kb