Optimizing filter trap assay for the detection of aggregated alpha-synuclein in brain samples

No abstract availabl

Tau expression and phosphorylation in enteroendocrine cells

Background and objectiveThere is mounting evidence to suggest that the gut-brain axis is involved in the development of Parkinson’s disease (PD). In this regard, the enteroendocrine cells (EEC), which faces the gut lumen and are connected with both enteric neurons and glial cells have received growing attention. The recent observation showing that these cells express alpha-synuclein, a presynaptic neuronal protein genetically and neuropathologically linked to PD came to reinforce the assumption that EEC might be a key component of the neural circuit between the gut lumen and the brain for the bottom-up propagation of PD pathology. Besides alpha-synuclein, tau is another key protein involved in neurodegeneration and converging evidences indicate that there is an interplay between these two proteins at both molecular and pathological levels. There are no existing studies on tau in EEC and therefore we set out to examine the isoform profile and phosphorylation state of tau in these cells.MethodsSurgical specimens of human colon from control subjects were analyzed by immunohistochemistry using a panel of anti-tau antibodies together with chromogranin A and Glucagon-like peptide-1 (two EEC markers) antibodies. To investigate tau expression further, two EEC lines, namely GLUTag and NCI-H716 were analyzed by Western blot with pan-tau and tau isoform specific antibodies and by RT-PCR. Lambda phosphatase treatment was used to study tau phosphorylation in both cell lines. Eventually, GLUTag were treated with propionate and butyrate, two short chain fatty acids known to sense EEC, and analyzed at different time points by Western blot with an antibody specific for tau phosphorylated at Thr205.ResultsWe found that tau is expressed and phosphorylated in EEC in adult human colon and that both EEC lines mainly express two tau isoforms that are phosphorylated under basal condition. Both propionate and butyrate regulated tau phosphorylation state by decreasing its phosphorylation at Thr205.Conclusion and inferenceOur study is the first to characterize tau in human EEC and in EEC lines. As a whole, our findings provide a basis to unravel the functions of tau in EEC and to further investigate the possibility of pathological changes in tauopathies and synucleinopathies

Combined chemotherapy and allogeneic human Vγ9Vδ2 T lymphocyte-immunotherapies efficiently control the development of human epithelial ovarian cancer cells in vivo

International audienceEpithelial ovarian cancer (EOC) represents 5% of human gynecologic cancers in the world, is hetero- geneous and highly invasive with a dismal prognosis (5 year-survival rate <35%). Diagnosis of EOC is frequently made at advanced stages and, despite aggressive treatments combining surgery and che- motherapy, fatal relapse rapidly occurs and is accompanied by a peritoneal carcinosis. In this context, novel therapeutical advances are urgently required. Adoptive transfer(s) of immune effector cells, including allogeneic human Vγ9Vδ2 T lymphocytes, represent attractive targets for efficiently and safely tracking tissue-invading tumor cells and controlling tumor dissemination in the organism. Our study describes the establishment of robust and physiological orthotopic model of human EOC in mouse, that includes surgical resection (ovariectomy) and chemotherapy, which are ineluctably accompanied by a fatal peritoneal carcinosis recurrence. Through a complementary set of in vitro and in vivo experiments, we provide here a preclinical proof of interest of the antitumor efficiency of adoptive transfers of allogeneic human Vγ9Vδ2 T lymphocytes against EOC, in association with surgical debulking and standard chemotherapies (i.e., taxanes and platinum salts). Moreover, our results indicate that chemo- and immunotherapies can be combined to improve the antitumor efficiency of immunotherapeutic lines. Altogether, these results further pave the way for next-generation antitumor immunotherapies, based on local administrations of human allogeneic human Vγ9Vδ2 T lymphocytes, in association with standard treatments

Characterisation of Mitochondria-associated ER membranes in the enteric nervous system under physiological and pathological conditions

International audienceAlterations in endoplasmic reticulum-mitochondria associations and in mitochondria-associated ER membrane (MAM) behaviour have been reported in the brain in several neurodegenerative diseases. Despite the emerging role of the gut-brain axis in neurodegenerative disorders, the biology of MAM in the enteric nervous system (ENS) has not previously been studied. Therefore, we set out to characterise the MAM in the distal colon of wild type C57BL/6J mice and in senescence accelerated mouse prone 8 (SAMP8), a mouse model of age-related neurodegeneration. We showed for the first time that MAM are widely present in enteric neurons and that their association is altered in SAMP8 mice. We then examined the functions of MAM in a primary culture model of enteric neurons and showed that calcium homeostasis was altered in SAMP8 mice when compared to control animals. These findings provide the first detailed characterisation of MAM in the ENS under physiological conditions and during age-associated neurodegeneration. Further investigation of MAM modifications in the ENS in disease may provide valuable information about the possible role of enteric MAM in neurodegenerative diseases

Psychological stress induces an increase in cholinergic enteric neuromuscular pathways mediated by glucocorticoid receptors

Introduction Repeated acute stress (RASt) is known to be associated with gastrointestinal dysfunctions. However, the mechanisms underlying these effects have not yet been fully understood. While glucocorticoids are clearly identified as stress hormones, their involvement in RASt-induced gut dysfunctions remains unclear, as does the function of glucocorticoid receptors (GR). The aim of our study was to evaluate the involvement of GR on RASt-induced changes in gut motility, particularly through the enteric nervous system (ENS). Methods Using a murine water avoidance stress (WAS) model, we characterized the impact of RASt upon the ENS phenotype and colonic motility. We then evaluated the expression of glucocorticoid receptors in the ENS and their functional impact upon RASt-induced changes in ENS phenotype and motor response. Results We showed that GR were expressed in myenteric neurons in the distal colon under basal conditions, and that RASt enhanced their nuclear translocation. RASt increased the proportion of ChAT-immunoreactive neurons, the tissue concentration of acetylcholine and enhanced cholinergic neuromuscular transmission as compared to controls. Finally, we showed that a GR-specific antagonist (CORT108297) prevented the increase of acetylcholine colonic tissue level and in vivo colonic motility. Discussion Our study suggests that RASt-induced functional changes in motility are, at least partly, due to a GR-dependent enhanced cholinergic component in the ENS

PRIMA-1Met induces myeloma cell death independent of p53 by impairing the GSH/ROS balance

International audienceThe aim of this study was to assess the efficiency of p53 reactivation and induction of massive apoptosis (PRIMA-1(Met)) in inducing myeloma cell death, using 27 human myeloma cell lines (HMCLs) and 23 primary samples. Measuring the lethal dose (LD50) of HMCLs revealed that HMCLs displayed heterogeneous sensitivity, with an LD50 ranging from 4 μM to more than 200 μM. The sensitivity of HMCLs did not correlate with myeloma genomic heterogeneity or TP53 status, and PRIMA-1(Met) did not induce or increase expression of the p53 target genes CDKN1A or TNFRSF10B/DR5. However, PRIMA-1(Met) increased expression of NOXA in a p53-independent manner, and NOXA silencing decreased PRIMA1(Met)-induced cell death. PRIMA-1(Met) depleted glutathione (GSH) content and induced reactive oxygen species production. The expression of GSH synthetase correlated with PRIMA-1(Met) LD50 values, and we showed that a GSH decrease mediated by GSH synthetase silencing or by and L-buthionine sulphoximine, an irreversible inhibitor of γ-glutamylcysteine synthetase, increased PRIMA-1(Met)-induced cell death and overcame PRIMA-1(Met) resistance. PRIMA-1(Met) (10 μM) induced cell death in 65% of primary cells independent of the presence of del17p; did not increase DR5 expression, arguing against an activation of p53 pathway; and synergized with L-buthionine sulphoximine in all samples. Finally, we showed in mouse TP53(neg) JJN3-xenograft model that PRIMA-1(Met) inhibited myeloma growth and synergized with L-buthionine sulphoximine in vivo

Improved Functionality of the Vasculature during Conventionally Fractionated Radiation Therapy of Prostate Cancer

<div><p>Although endothelial cell apoptosis participates in the tumor shrinkage after single high-dose radiotherapy, little is known regarding the vascular response after conventionally fractionated radiation therapy. Therefore, we evaluated hypoxia, perfusion and vascular microenvironment changes in an orthotopic prostate cancer model of conventionally fractionated radiation therapy at clinically relevant doses (2 Gy fractions, 5 fractions/week). First, conventionally fractionated radiation therapy decreased tumor cell proliferation and increased cell death with kinetics comparable to human prostate cancer radiotherapy. Secondly, the injection of Hoechst 33342 or fluorescent-dextrans showed an increased tumor perfusion within 14 days in irradiated tumors, which was correlated with a clear reduction of hypoxia. Improved perfusion and decreased hypoxia were not explained by increased blood vessel density, size or network morphology. However, a tumor vascular maturation defined by perivascular desmin+/SMA+ cells coverage was clearly observed along with an increase in endothelial, zonula occludens (ZO)-1 positive, intercellular junctions. Our results show that, in addition to tumor cell killing, vascular maturation plays an uncovered role in tumor reoxygenation during fractionated radiation therapy.</p></div

LRRK2 is reduced in Parkinson’s disease gut

International audienceNo abstract availabl

Fractionated irradiation reduces hypoxia and increases tumor perfusion.

<p>(<b>A</b>) Pseudo-confocal images of tumors during CFRT, stained for hypoxia (EF5) and endothelial cells (CD31). (<b>B</b>) Image quantification of EF5+ surface in tumors during CFRT. Values represent the average of n≥13 per point ± sem. (<b>C</b>) Pseudo-confocal images of tumors perfused with Hoechst 33342 and 10 kDa/2 MDa dextrans before (t0) or after 2 weeks of CFRT (t14). SYBR green was used as a counterstain of total cell nuclei. (<b>D,E,F</b>) Image quantification of Hoechst+ (<b>D</b>), and medium (<b>E</b>) and large (<b>F</b>) dextran+ surfaces in tumors during CFRT (n  =  6). (<b>B</b>,<b>D</b>,<b>E</b>,<b>F</b>). Statistical comparisons vs. t0.</p

Maintenance of vascular density and distribution during fractionated irradiation.

<p>(<b>A</b>) Microvessel density in tumors during CFRT. Values represent the average of n≥13 per point ± sem. (<b>B</b>) Distance profile between cells and the closest blood vessel, from tumors during CFRT. Profiles are based on n≥13. Statistical comparisons vs. t0. (<b>C</b>) Pseudo-confocal images of tumor-associated blood vessels (CD31+) stained for TUNEL during CFRT. Arrows: TUNEL+/CD31+ cells. (<b>D</b>) Image quantification of CD31+/TUNEL+ surface. Values represent the average of n≥13 per point ± sem. (<b>E</b>) Representative Z-stack images of 100 µm-thick tumor sections before (t0) or after 2 weeks of CFRT (t14) and stained for blood vessels (CD31+/Fli-1+). (<b>F</b>) Image analysis of blood vessel network from 100 µm-thick tumor sections. Values represent the average of n  =  9 per point ± sem.</p