OPA1 functions in mitochondria and dysfunctions in optic nerve

OPA1 is the major gene responsible for Dominant Optic Atrophy (DOA), a blinding disease that affects specifically the retinal ganglion cells (RGCs), which function consists in connecting the neuro-retina to the brain. OPA1 encodes an intra-mitochondrial dynamin, involved in inner membrane structures and ubiquitously expressed, raising the critical question of the origin of the disease pathophysiology. Here, we review the fundamental knowledge on OPA1 functions and regulations, highlighting their involvements in mitochondrial respiration, membrane dynamic and apoptosis. In light of these functions, we then describe the remarkable RGC mitochondrial network physiology and analyse data collected from animal models expressing OPA1 mutations. If, to date RGC mitochondria does not present any peculiarity at the molecular level, they represent possible targets of numerous assaults, like light, pressure, oxidative stress and energetic impairment, which jeopardize their function and survival, as observed in OPA1 mouse models. Although fascinating fields of investigation are still to be addressed on OPA1 functions and on DOA pathophysiology, we have reached a conspicuous state of knowledge with pertinent cell and animal models, from which therapeutic trials can be initiated and deeply evaluated

The Effect of OPA1 on Mitochondrial Ca2+ Signaling

The dynamin-related GTPase protein OPA1, localized in the intermembrane space and tethered to the inner membrane of mitochondria, participates in the fusion of these organelles. Its mutation is the most prevalent cause of Autosomal Dominant Optic Atrophy. OPA1 controls the diameter of the junctions between the boundary part of the inner membrane and the membrane of cristae and reduces the diffusibility of cytochrome c through these junctions. We postulated that if significant Ca2+ uptake into the matrix occurs from the lumen of the cristae, reduced expression of OPA1 would increase the access of Ca2+ to the transporters in the crista membrane and thus would enhance Ca2+ uptake. In intact H295R adrenocortical and HeLa cells cytosolic Ca2+ signals evoked with K+ and histamine, respectively, were transferred into the mitochondria. The rate and amplitude of mitochondrial [Ca2+] rise (followed with confocal laser scanning microscopy and FRET measurements with fluorescent wide-field microscopy) were increased after knockdown of OPA1, as compared with cells transfected with control RNA or mitofusin1 siRNA. Ca2+ uptake was enhanced despite reduced mitochondrial membrane potential. In permeabilized cells the rate of Ca2+ uptake by depolarized mitochondria was also increased in OPA1-silenced cells. The participation of Na+/Ca2+ and Ca2+/H+ antiporters in this transport process is indicated by pharmacological data. Altogether, our observations reveal the significance of OPA1 in the control of mitochondrial Ca2+ metabolism

Primary intestinal lymphangiectasia (Waldmann's disease)

Primary intestinal lymphangiectasia (PIL) is a rare disorder characterized by dilated intestinal lacteals resulting in lymph leakage into the small bowel lumen and responsible for protein-losing enteropathy leading to lymphopenia, hypoalbuminemia and hypogammaglobulinemia. PIL is generally diagnosed before 3 years of age but may be diagnosed in older patients. Prevalence is unknown. The main symptom is predominantly bilateral lower limb edema. Edema may be moderate to severe with anasarca and includes pleural effusion, pericarditis or chylous ascites. Fatigue, abdominal pain, weight loss, inability to gain weight, moderate diarrhea or fat-soluble vitamin deficiencies due to malabsorption may also be present. In some patients, limb lymphedema is associated with PIL and is difficult to distinguish lymphedema from edema. Exsudative enteropathy is confirmed by the elevated 24-h stool α1-antitrypsin clearance. Etiology remains unknown. Very rare familial cases of PIL have been reported. Diagnosis is confirmed by endoscopic observation of intestinal lymphangiectasia with the corresponding histology of intestinal biopsy specimens. Videocapsule endoscopy may be useful when endoscopic findings are not contributive. Differential diagnosis includes constrictive pericarditis, intestinal lymphoma, Whipple's disease, Crohn's disease, intestinal tuberculosis, sarcoidosis or systemic sclerosis. Several B-cell lymphomas confined to the gastrointestinal tract (stomach, jejunum, midgut, ileum) or with extra-intestinal localizations were reported in PIL patients. A low-fat diet associated with medium-chain triglyceride supplementation is the cornerstone of PIL medical management. The absence of fat in the diet prevents chyle engorgement of the intestinal lymphatic vessels thereby preventing their rupture with its ensuing lymph loss. Medium-chain triglycerides are absorbed directly into the portal venous circulation and avoid lacteal overloading. Other inconsistently effective treatments have been proposed for PIL patients, such as antiplasmin, octreotide or corticosteroids. Surgical small-bowel resection is useful in the rare cases with segmental and localized intestinal lymphangiectasia. The need for dietary control appears to be permanent, because clinical and biochemical findings reappear after low-fat diet withdrawal. PIL outcome may be severe even life-threatening when malignant complications or serous effusion(s) occur

Inhibitory Action Of A New Lectin From Xerocomus Chrysenteron On Cell-Substrate Adhesion

Lectins are carbohydrate-binding proteins which potentially link to cell surface glycoconjugates and affect cell proliferation. We investigated the effect of a new lectin from the mushroom Xerocomus chrysenteron (XCL) on cell proliferation using adherent and suspension cell lines. XCL caused a dose-dependent inhibition of proliferation of the adherent cell lines NIH-3T3 and HeLa. Several experiments suggest that disruption of cell-substrate adhesion is the main factor affecting cell growth inhibition. (i) No antiproliferative effect was observed on the SF9 cell line, which does not require to be attached to grow. (ii) XCL was shown to affect the adherence of cells following their suspension by trypsin treatment. (iii) XCL was localized on the cell surface where it would act as a coating agent. (iv) XCL induced morphological changes from well spread to rounded cells and disrupted the actin cytoskeleton. By contrast, flow cytometric analysis showed that XCL does not interfere with the cell cycle, and does not induce apoptosis

Rapid Sequestration of DPP IV/CD26 and Other Cell Surface Proteins in an Autophagic-Like Compartment in Caco-2 Cells Treated with Forskolin

Contains fulltext : 28050___.PDF (publisher's version ) (Open Access

Rapid sequestration of DPPIV/CD26 and other cell surface in an autophagic-like compartment in Caco-2 cells treated with forsfokolin

Contains fulltext : 20994.PDF (publisher's version ) (Open Access

Decrease of mRNA levels and biosynthesis of sucrase-isomaltase but not dipeptidylpeptidase IV in forskolin or monensin-treated Caco-2 cells.

International audienceTreatment for 48 h of differentiated, confluent Caco-2 cells with 2.5 10(-5) M forskolin or 10(-6) M monensin, which produces a significant decrease of the de novo biosynthesis of sucrase-isomaltase, does not change quantitatively the de novo biosynthesis of dipeptidylpeptidase IV. Western blot analysis and silver nitrate staining indicate that neither drug induces any modification in the steady state expression of these two brush border hydrolases. Northern blot analysis shows that the level of dipeptidylpeptidase IV mRNA does not change in treated as compared to control Caco-2 cells. In contrast, forskolin and monensin dramatically decrease the level of sucrase-isomaltase mRNA. These observations suggest a separate regulation of biosynthesis for sucrase-isomaltase and dipeptidylpeptidase IV in intestinal cells. The mechanisms responsible for such a difference are discussed. Among them, the role of glucose metabolism, which is perturbed by both drugs, appears to be of crucial importance

Use of HT-29, a cultured human colon cancer cell line, to study the effect of fermented milks on colon cancer cell growth and differentiation.

International audienceEpidemiological and in vivo and in vitro experimental studies have suggested that fermented milks may interfere with the emergence and/or the development of colon cancer. The results, however, remain inconclusive. This prompted us to develop a new approach based on the use of HT-29, a cultured human colon cancer cell line, to study at the cellular level the effect of fermented milks on colon cancer cell growth and differentiation characteristics. Undifferentiated HT-29 cells have been grown in the continuous presence of milks fermented by one of the following bacterial populations: Lactobacillus helveticus, Bifidobacterium, L.acidophilus or a mix of Streptococcus thermophilus and L. bulgaricus. Penicillin G was added to the cell culture medium, resulting in a complete blockade of bacterial growth without significant effect on bacterial viability. One out of the four bacteria species studied, namely L.acidophilus, was without effect on both cell growth and differentiation. The three other bacterial strains induced a significant, although variable, reduction in the growth rate of HT-29 cells, which resulted in a 10-50% decrease in the cell number at steady-state (i.e. at cell confluency). The most efficient strains in lowering the HT-29 growth rate were L. helveticus and Bifidobacterium. Concomitantly, the specific activities of dipeptidyl peptidase IV (DPP IV), a sensitive and specific marker of HT-29 cell differentiation, and that of three other brush border enzymes (sucrase, aminopeptidase N and alkaline phosphatase) were significantly increased, thus suggesting that these cells may have entered a differentiation process. Altogether, these results indicate that the use of cultured colon cancer cells may be a useful tool to further study the effect of fermented milks on colon cancer and that bacterial strains may exert a different and specific effect on cancer cell growth and differentiation when used in fermented milk products

Forskolin blocks the apical expression of dipeptidyl peptidase IV in Caco-2 cells and induces its retention in lamp-1-containing vesicles.

International audienceIn a previous work, we showed that the differentiation-dependent expression of dipeptidyl peptidase IV (DPP IV) in Caco-2 cells, a human colon adenocarcinoma cell line, was controlled at the mRNA level (D. Darmoul et al. J. Biol. Chem., 1992, 267, 4824-4833). Whether post-translational events may contribute to the final control of DPP IV cell surface expression was explored here by studying the potential effect of forskolin (FK), a drug known to permanently stimulates adenylyl cyclase and to strongly perturbs glucose metabolism in fully differentiated Caco-2 cells. FK treatment reduces by about 50% the amount of active DPP IV present at the brush border membrane, whereas the total amount of active DPP IV remains unchanged. Biosynthesis and maturation of DPP IV were measured using [35S]methionine labeling and were shown to be essentially unaffected by FK treatment. Pulse-chase experiments demonstrate that up to 50% of the neosynthesized DPP IV do not appear at the apical membrane after FK treatment. To get further insight into this phenomenon, we have used confocal laser scanning microscopy. We demonstrate that the blockade of DPP IV transport is associated with the accumulation of this protein in intracellular vesicles. Double-staining experiments demonstrate that these vesicles are not labeled with a monoclonal antibody directed against the Golgi apparatus but display a strong staining with a polyclonal antibody raised against lamp-1, a lysosomal membrane protein. Using a newly developed image analysis procedure, we have been able to quantitate the relative distribution of lamp-1 and DPP IV labels in both control and forskolin-treated cells. We show that the overlap of the two labels dramatically increases in FK-treated Caco-2 cells. These results suggest that, beside the transcriptional level, post-translational events may be involved in the final control of the apical expression of a differentiation-dependent hydrolase