Autophagy Defect Is Associated with Low Glucose-Induced Apoptosis in 661W Photoreceptor Cells

Glucose is an important metabolic substrate of the retina and diabetic patients have to maintain a strict normoglycemia to avoid diabetes secondary effects, including cardiovascular disease, nephropathy, neuropathy and retinopathy. Others and we recently demonstrated the potential role of hypoglycemia in diabetic retinopathy. We showed acute hypoglycemia to induce retinal cell death both in vivo during an hyperinsulinemic/hypoglycemic clamp and in vitro in 661W photoreceptor cells cultured at low glucose concentration. In the present study, we showed low glucose to induce a decrease of BCL2 and BCL-XL anti-apoptotic proteins expression, leading to an increase of free pro-apoptotic BAX. In parallel, we showed that, in retinal cells, low glucose-induced apoptosis is involved in the process of autophagosomes formation through the AMPK/RAPTOR/mTOR pathway. Moreover, the decrease of LAMP2a expression led to a defect in the autophagosome/lysosome fusion process. Specific inhibition of autophagy, either by 3-methyladenine or by down-regulation of ATG5 or ATG7 proteins expression, increased caspase 3 activation and 661W cell death. We show that low glucose modifies the delicate equilibrium between apoptosis and autophagy. Cells struggled against low nutrient condition-induced apoptosis by starting an autophagic process, which led to cell death when inhibited. We conclude that autophagy defect is associated with low glucose-induced 661W cells death that could play a role in diabetic retinopathy. These results could modify the way of addressing negative effects of hypoglycemia. Short-term modulation of autophagy could be envisioned to treat diabetic patients in order to avoid secondary complications of the disease.

A critical role of autophagy in antileukemia/lymphoma effects of APO866, an inhibitor of NAD biosynthesis

Somi Reddy Majjigapu, Bernard Sordat, Pierre Vogel

APO866, an inhibitor of NAD biosynthesis, exhibits potent antitumor properties in various malignancies. Recently, it has been shown that APO866 induces apoptosis and autophagy in human hematological cancer cells, but the role of autophagy in APO866-induced cell death remains unclear. Here, we report studies on the molecular mechanisms underlying APO866-induced cell death with emphasis on autophagy. Treatment of leukemia and lymphoma cells with APO866 induced both autophagy, as evidenced by an increase in autophagosome formation and in SQSTM1/p62 degradation, but also increased caspase activation as revealed by CASP3/caspase 3 cleavage. As an underlying mechanism, APO866-mediated autophagy was found to deplete CAT/catalase, a reactive oxygen species (ROS) scavenger, thus promoting ROS production and cell death. Inhibition of autophagy by ATG5 or ATG7 silencing prevented CAT degradation, ROS production, caspase activation, and APO866-induced cell death. Finally, supplementation with exogenous CAT also abolished APO866 cytotoxic activity. Altogether, our results indicated that autophagy is essential for APO866 cytotoxic activity on cells from hematological malignancies and also indicate an autophagy-dependent CAT degradation, a novel mechanism for APO866-mediated cell killing. Autophagy-modulating approaches could be a new way to enhance the antitumor activity of APO866 and related agents.

Landes Bioscience2014

Role of Nimrod receptors in the Drosophila melanogaster cellular immune response

Claudia Melcarne

The use of modern molecular biology tools and simple but powerful tractable model organisms such as Drosophila has contributed significantly to our recent advances in the innate immunity field, notably phagocytosis. Since 2001, many phagocytic transmembrane receptors, potential opsonins, and in some cases their ligands, have been discovered. Among them, the Nimrod family, which contains key phagocytic receptors and potential opsonins, deserves special interest as accumulating genetic and biochemical evidence points to their critical role in phagocytosis of both bacteria and apoptotic cells. In this PhD thesis we provided a deeper characterization of two Nimrod transmembrane receptors, Eater and NimC1. In particular, by using single or combined mutations in eater and NimC1, we aimed to further elucidate i) the involvement of each receptor in bacterial phagocytosis and ii) the role of Eater in hemocytes sessility and adhesion. Both receptors have been shown to be specifically expressed in hemocytes, the insect blood cells, and to mediate Gram-positive but not Gram-negative bacteria phagocytosis. In this work we generated a novel NimC1 mutant and demonstrated that the single NimC1 deletion does not affect phagocytosis of bacteria. However, by using the compound NimC1;eater mutant we were able to show that these receptors contribute in a synergistic way to microbes engulfment, but that Eater can bypass the requirement for NimC1. Moreover, we discovered that NimC1, but not Eater, is required for phagocytosis of non-immunogenic particles, namely latex beads and yeast zymosan. Therefore, this work provides evidences of Eater and NimC1 being the main receptors for bacteria phagocytosis, and suggests that those proteins likely play distinct roles in microbial uptake, as tethering and docking receptors. The second part of this thesis re-addresses the role of Eater in hemocyte sessility and adhesion. A previous study showed that this phagocytic receptor is essential to mediate blood cell sessility to the animal integument during larval stages. However, a series of questions about Eater-mediated sessility remains still open. For example, is Eater mediating hemocyte adhesion also in adult flies? And yet, does Eater mediate hemocyte sessility by regulating the expression of cell adhesion genes, therefore functioning as a signalling receptor? By using eater1 mutant adult flies, we showed that, as in larval stages, Eater is required in adult hemocytes to confer adhesion and sessility. Interestingly, we uncovered a potential role of sessility in hemocyte survival during adulthood, since eater1 flies have a decreased number of blood cells. Preliminary transcriptomics analysis led us to hypothesise that this receptor might not work as a signalling molecule, but rather as an adhesion protein providing the initial hemocyte contact to its target surface. In line with Eaterâs role as an adhesion molecule, its over-expression in hemocytes leads to the formation of enlarged cells compared to control. To conclude, this thesis extended our knowledge on the Drosophila cellular immune response, by providing new insights on two Nimrod phagocytic receptors.

EPFL2020

The role of TIF1 gamma during adult murine hematopoiesis

Konstantin Shakhbazov

Transforming Growth Factor beta (TGFβ) signaling plays an important role in a variety of cellular processes during embryonic development, adult tissue homeostasis and cancer. TGFβI ligand is a potent inhibitor of early hematopoietic progenitor [1] cells in vitro, however the exact role and mechanisms of this pathway during adult hematopoietic homeostasis remains poorly understood. The Transcriptional Intermediary Factor 1 gamma (Tif1γ) gene encodes a recently uncovered nuclear protein in the TGFβ transduction pathway. Tif1γ is defective in the zebrafish moonshine mutant, which exhibits primitive and definitive hematopoiesis defects [2]. In addition, Tif1γ has been shown to influence differentiation of human erythroid cells in vitro [3]. Recent work in our laboratory has provided genetic evidence that Tif1γ is required for the generation and survival of hematopoietic stem/progenitor cells during murine development (C.Adolphe, K.Shakhbazov et al., manuscript under submission). Given that Tif1γ function is required for embryonic development and null mutants are embryonic lethal [4] (R.Losson unpublished data), we took advantage of a conditional Tif1γ flox allele (R.Losson unpublished data) and combined it with the inducible MxCre line [5] to generate mice in which Tif1γ function can be specifically ablated during adult hematopoiesis. MxCre:Tif1γKO/flox mutants exhibit a decrease in erythroblasts and dramatic increase in granulocytes within the bone marrow. This phenotype is attributable to a massive increase in granulocyte-monocyte progenitors (GMPs) at the expense of common myeloid progenitors (CMPs) and megakaryocyte- erythrocyte progenitors (MEPs), which are severely decreased in Tif1γ mutant bone marrow. In addition, pre-B cells were dramatically decreased in numbers, attributable to an increase in cell death, indicating a pro-survival role for Tif1γ in B lymphocytes. In addition, we performed deletion of Tif1γ in non-competitive and competitive bone marrow transplantation settings, which indicated that only the CMP/MEP phenotype is a non-cell autonomous effect. Two distinct models have been reported suggesting how Tif1γ functions within the TGFβ pathway: one indicating that Tif1γ binds and acts through Smad2/3 and the other that Tif1γ functions as a Smad4 mono-ubiquitin ligase [3, 6, 7]. To genetically address this apparent discrepancy, we generated double Tif1γ/Smad4 hematopoietic specific mutant mice, whose phenotype was surprisingly similar to that observed in Tif1γ single mutants. These data suggest that Tif1γ function is independent of Smad4 in hematopoietic cells. To address the mechanism by which Tif1γ functions, we performed microarray profiling of normal and mutant progenitors, which revealed prostaglandin D2 synthase as a hub gene linking Tif1γ and TGFβ signaling. Finally, we were able to mimic the hematopoietic-Tif1γ null phenotype by providing 16,16-dimethyl prostaglandin D2 to wild-type hematopoietic cells in vitro. In summary, our data provide genetic evidence for a key role of Tif1γ during early myeloid development as well as pre-B cell survival. In addition, we have identified that the majority of Tif1γ function is Smad4 independent. Finally, we identified a novel link between Tif1γ function and prostaglandin metabolism/signaling as a likely mechanism by which Tif1γ regulates hematopoiesis.