Refractory epilepsy and mitochondrial dysfunction due to GM3 synthase deficiency.

We report two children, born from consanguineous parents, who presented with early-onset refractory epilepsy associated with psychomotor delay, failure to thrive, blindness and deafness. Polarographic and spectrophotometric analyses in fibroblasts and liver revealed a respiratory chain (RC) dysfunction. Surprisingly, we identified a homozygous nonsense mutation in the GM3 synthase gene by using exome sequencing. GM3 synthase catalyzes the formation of GM3 ganglioside from lactosylceramide, which is the first step in the synthesis of complex ganglioside species. Mass spectrometry analysis revealed that the complete absence of GM3 ganglioside and its biosynthetic derivatives was associated with an upregulation of the alternative globoside pathway in fibroblasts. The accumulation of Gb3 and Gb4 globosides likely has a role in RC dysfunction and in the decrease of mitochondrial membrane potential leading to apoptosis, which we observed in fibroblasts. We show for the first time that GM3 synthase deficiency, responsible for early-onset epilepsy syndrome, leads to a secondary RC dysfunction. Our study highlights the role of secondary mitochondrial disorders that can interfere with the diagnosis and the evolution of other metabolic diseases.

Combination of glycolysis inhibition with chemotherapy results in an antitumor immune response.

Most DNA-damaging agents are weak inducers of an anticancer immune response. Increased glycolysis is one of the best-described hallmarks of tumor cells; therefore, we investigated the impact of glycolysis inhibition, using 2-deoxyglucose (2DG), in combination with cytotoxic agents on the induction of immunogenic cell death. We demonstrated that 2DG synergized with etoposide-induced cytotoxicity and significantly increased the life span of immunocompetent mice but not immunodeficient mice. We then established that only cotreated cells induced an efficient tumor-specific T-cell activation ex vivo and that tumor antigen-specific T cells could only be isolated from cotreated animals. In addition, only when mice were immunized with cotreated dead tumor cells could they be protected (vaccinated) from a subsequent challenge using the same tumor in viable form. Finally, we demonstrated that this effect was at least partially mediated through ERp57/calreticulin exposure on the plasma membrane. These data identify that the targeting of glycolysis can convert conventional tolerogenic cancer cell death stimuli into immunogenic ones, thus creating new strategies for immunogenic chemotherapy.

Severe thymic atrophy in a mouse model of skin inflammation accounts for impaired TNFR1 signaling.

Transgenic mice expressing the caspase-cleaved form of the tyrosine kinase Lyn (LynΔN) develop a TNFα-dependent skin disease that accurately recapitulates human psoriasis. Participation of lymphocytes in this disease was confirmed by backcrossing LynΔN mice on a Rag-1 deficient background. The present study was therefore conducted to analyze whether modification of lymphocyte homeostasis does occur and participate in the phenotype of LynΔN mice. We show here that LynΔN mice consistently exhibit thymic atrophy that correlates with both a net decrease in the CD4+/CD8+ Double Positive (DP) and an increase in Single Positive (SP) thymocyte sub-populations, but also display an increase of splenic mature B cell. Interestingly, a normal immune phenotype was rescued in a TNFR1 deficient background. Finally, none of these immune alterations was detected in newborn mice before the onset of inflammation. Therefore, we conclude that chronic inflammation can induce thymic atrophy and perturb spleen homeostasis in LynΔN mice through the increased production of TNFα, LTß and TNFR1 signaling.

Mitochondrial control of caspase-dependent and -independent cell death.

Mitochondria control whether a cell lives or dies. The role mitochondria play in deciding the fate of a cell was first identified in the mid-1990s, because mitochondria-enriched fractions were found to be necessary for activation of death proteases, the caspases, in a cell-free model of apoptotic cell death. Mitochondrial involvement in apoptosis was subsequently shown to be regulated by Bcl-2, a protein that was known to contribute to cancer in specific circumstances. The important role of mitochondria in promoting caspase activation has therefore been a major focus of apoptosis research; however, it is also clear that mitochondria contribute to cell death by caspase-independent mechanisms. In this review, we will highlight recent findings and discuss the mechanism underlying the mitochondrial control of apoptosis and caspase-independent cell death.

Modulation of caspase-independent cell death leads to resensitization of imatinib mesylate-resistant cells.

Imatinib mesylate is widely used for the treatment of patients with chronic myelogenous leukemia (CML). This compound is very efficient in killing Bcr-Abl-positive cells in a caspase-dependent manner. Nevertheless, several lines of evidence indicated that caspase-mediated cell death (i.e., apoptosis) is not the only type of death induced by imatinib. The goal of our study was to evaluate the importance of the newly described caspase-independent cell death (CID) in Bcr-Abl-positive cells. We established in several CML cell lines that imatinib, in conjunction with apoptosis, also induced CID. CID was shown to be as efficient as apoptosis in preventing CML cell proliferation and survival. We next investigated the potential implication of a recently identified mechanism used by cancer cells to escape CID through overexpression of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We showed here, in several CML cell lines, that GAPDH overexpression was sufficient to induce protection from CID. Furthermore, imatinib-resistant Bcr-Abl-positive cell lines were found to spontaneously overexpress GAPDH. Finally, we showed that a GAPDH partial knockdown, using specific short hairpin RNAs, was sufficient to resensitize those resistant cells to imatinib-induced cell death. Taken together, our results indicate that CID is an important effector of imatinib-mediated cell death. We also established that GAPDH overexpression can be found in imatinib-resistant Bcr-Abl-positive cells and that its down-regulation can resensitize those resistant cells to imatinib-induced death. Therefore, drugs able to modulate GAPDH administered together with imatinib could find some therapeutic benefits in CML patients.

Localization of Fas/CD95 into the lipid rafts on down-modulation of the phosphatidylinositol 3-kinase signaling pathway.

Activation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway is known to protect tumor cells from apoptosis and more specifically from the Fas-mediated apoptotic signal. The antitumoral agent edelfosine sensitizes leukemic cells to death by inducing the redistribution of the apoptotic receptor Fas into plasma membrane subdomains called lipid rafts. Herein, we show that inhibition of the PI3K signal by edelfosine triggers a Fas-mediated apoptotic signal independently of the Fas/FasL interaction. Furthermore, similarly to edelfosine, blockade of the PI3K activity, using specific inhibitors LY294002 and wortmannin, leads to the clustering of Fas whose supramolecular complex is colocalized within the lipid rafts. These findings indicate that the antitumoral agent edelfosine down-modulates the PI3K signal to sensitize tumor cells to death through the redistribution of Fas into large platform of membrane rafts.

Dominant-negative Fas mutation is reversed by down-expression of c-FLIP.

Fas triggering by agonistic antibodies or by its cognate ligand, FasL, induces apoptotic cell death, whereas mutation in the Fas death domain is associated with lymphoma progression. On prolonged culture in the presence of an agonistic anti-Fas antibody, we raised a Jurkat cell line resistant to agonistic antibodies but still sensitive to soluble FasL, which carried at the heterozygous state, a point mutation into the Fas death domain. Down-modulation of c-FLIP expression reversed the blockade of the Fas pathway. We show that the activation threshold for the Fas receptor is more easily overcome by multimeric FasL than by agonistic antibodies and that the increase of this threshold due to mutation in the Fas death domain can be overcome by acting on a downstream effector of the Fas signal, c-FLIP. These findings put forward a new approach to eradicate Fas-resistant tumor cells.

Cutting edge: SDS-stable Fas microaggregates: an early event of Fas activation occurring with agonistic anti-Fas antibody but not with Fas ligand.

The 45 kDa Fas or CD95 receptor triggers apoptosis via the caspase cascade when stimulated by its ligand FasL or by agonistic Abs. Activated Fas receptors seem to oligomerize very early into SDS-stable and reducing agent-resistant microaggregates of 200-250 kDa on SDS-PAGE. However, these microaggregates have so far only been reported using agonistic anti-Fas Abs, and no results have been reported using FasL. Here, we demonstrate that the microaggregates do not form in response to FasL, while they always appear in response to the agonistic Ab, in four different cell lines and in normal lymphocytes from human blood. Therefore, the Fas microaggregates are not required for the induction of apoptosis via FasL. These results also suggest that subtle differences exist in the apoptotic pathways triggered by anti-Fas agonistic Abs and by FasL.