Mislocalization of the exitatory amino-acid transporters (EAATs) in human astrocytoma and non-astrocytoma cancer cells: effect of the cell confluence

<p>Abstract</p> <p>Background</p> <p>Astrocytomas are cancers of the brain in which high levels of extracellular glutamate plays a critical role in tumor growth and resistance to conventional treatments. This is due for part to a decrease in the activity of the glutamate transporters, i.e. the Excitatory Amino Acid Transporters or EAATs, in relation to their nuclear mislocalization in astrocytoma cells. Although non-astrocytoma cancers express EAATs, the localization of EAATs and the handling of L-glutamate in that case have not been investigated.</p> <p>Methods</p> <p>We looked at the cellular localization and activity of EAATs in human astrocytoma and non-astrocytoma cancer cells by immunofluorescence, cell fractionation and L-glutamate transport studies.</p> <p>Results</p> <p>We demonstrated that the nuclear mislocalization of EAATs was not restricted to astrocytoma and happened in all sub-confluent non-astrocytoma cancer cells we tested. In addition, we found that cell-cell contact caused the relocalization of EAATs from the nuclei to the plasma membrane in all human cancer cells tested, except astrocytoma.</p> <p>Conclusions</p> <p>Taken together, our results demonstrated that the mislocalization of the EAATs and its associated altered handling of glutamate are not restricted to astrocytomas but were also found in human non-astrocytoma cancers. Importantly, we found that a cell contact-dependent signal caused the relocalization of EAATs at the plasma membrane at least in human non-astrocytoma cancer cells, resulting in the correction of the altered transport of glutamate in such cancer cells but not in astrocytoma.</p

Intracellular trafficking of peptide-vectors that target the LDL receptor for the delivery of imaging or therapeutic agents across the blood brain barrier

La plupart des médicaments développés pour les maladies du SNC n’atteignent pas leur cible en raison des propriétés uniques de la BHE, nécessitant la mise en place de stratégies de délivrance comme l'utilisation d'un processus physiologique, le RMT. Des peptides ciblant le LDLR (exprimé à la BHE et impliqué dans ces processus) ont été développés. Les objectifs de cette thèse ont été de caractériser le trafic intracellulaire et la capacité de transport de différentes formes de ces peptides dans différents modèles in vitro y compris dans un modèle de BHE.Les résultats obtenus dans une lignée cellulaire surexprimant le LDLR tagué GFP par imagerie en fluorescence montrent que les différentes formes de ces peptides lient le LDLR à la membrane plasmique d’où ils sont internalisés et adressés aux lysosomes sans interférer avec l’endocytose des LDL. Ils permettent l’adressage aux lysosomes de petites molécules (fluorochrome) et de protéines qui leur sont fusionnées, ces résultats indiquent qu’ils pourraient être utilisés pour cibler des molécules thérapeutiques aux lysosomes de cellules exprimant les LDLR. Dans le modèle in vitro de BHE, les peptides sont internalisés via le LDLR à partir du pôle apical et suivent un transport intracellulaire similaire aux LDL, étant déroutés de la voie de dégradation vers les lysosomes pour être transportés jusqu’au compartiment abluminal comme précédemment décrit pour le LDL et la transferrine. Ces données indiquent donc que les peptides ciblant le LDLR sont des candidats vecteurs intéressants pour compléter/améliorer le panel de peptide/anticorps existant et permettre le ciblage et le transport de molécules thérapeutiques à travers la BHE.Many drugs are ineffective in treating CNS diseases due in part to unique properties of the BBB, requiring the establishment of delivery strategies such as the use of a physiological process, as the RMT. Peptides targeting the LDLR (expressed in the BBB and involved in these processes) have been developed. The objectives of this thesis were to characterize the intracellular traffic and transport capacity of different shapes of these peptides in various in vitro models including a model of BBB.The results obtained in a cell line overexpressing the LDLR tagged GFP by fluorescence imaging shows that the various forms of these peptides bind plasma membrane LDLR, where they are internalized and sent to lysosomes without interfering with LDL endocytosis. They allow lysosomal targeting of small molecules (fluorochrome) and proteins that are fused to them. These results indicate that it might be used to target therapeutic compounds to cells expressing LDLR lysosomes. In the in vitro BBB model, the peptides are internalized via the LDLR from the apical pole and follow a similar intracellular transport than LDL, being diverted from the lysosomal degradation pathway to be transported to the abluminal compartment as previously described for LDL and transferrin. These data indicate that the LDLR-targeting peptides seems useful vectors candidates to complete/improve the existing peptide/antibodies panel and allow the targeting and the transport of therapeutic molecules through the BBB

Anandamide-ceramide interactions in a membrane environment: Molecular dynamic simulations data

Anandamide is a lipid neurotransmitter that interacts with various plasma membrane lipids. The data here consists of molecular dynamics simulations of anandamide, C18-ceramide and cholesterol performed in vacuo and within a hydrated palmitoyl-oleoyl-phosphatidylcholine (POPC)/cholesterol membrane. Several models of anandamide/cholesterol and anandamide/ceramide complexes are presented. The energy of interaction and the nature of the intermolecular forces involved in each of these complexes are detailed. The impact of water molecules hydrating the POPC/cholesterol membrane for the stability of the anandamide/cholesterol and anandamide/ceramide complexes is also analyzed. From a total number of 1920 water molecules stochatiscally merged with the lipid matrix, 48 were eventually redistributed around the polar head groups of the anandamide/ceramide complex, whereas only 15 reached with the anandamide/cholesterol complex. The interpretation of this dataset is presented in the accompanying article “Ceramide binding to anandamide increases its half-life and potentiates its cytotoxicity in human neuroblastoma cells” [1]

The food-associated fungal neurotoxin ochratoxin A inhibits the absorption of glutamate by astrocytes through a decrease in cell surface expression of the excitatory amino-acid transporters GLAST and GLT-1.

International audienceThe food-associated mycotoxin ochratoxin A (OTA) has been demonstrated to be deleterious to numerous cell types including brain cells. Although OTA has been proved to be toxic to astrocytes, no other investigation has been conducted on the impact of OTA on astrocytic functions. In the present study, we evaluated the effect of OTA on one of the major astrocytic functions, i.e. the reabsorption of extracellular glutamate. We found that OTA suppressed glutamate absorption by rat cortical astrocytes with a half inhibitory concentration of 1.3 and 10.1 microM in the absence and presence of fetal calf serum. Although OTA inhibits glutamine synthetase activity, this effect was not involved in OTA-mediated alteration of glutamate absorption since decrease in enzyme activity only occurred at high cytotoxic concentrations of toxin (100 microM). Similarly, alterations in the expression of the excitatory amino-acid transporters were not involved since OTA failed to modify total expression level of GLAST and GLT-1. We found that inhibition of glutamate absorption by OTA was due to a decrease in the expression of GLAST and GLT-1 at the cell surface. We propose that, in addition to being directly toxic to neurons and astrocytes, OTA could also cause the death of brain cells through inhibition of glutamate uptake by astrocytes, leading to the accumulation of extracellular glutamate and ultimately to excitotoxicity

The ribotoxin deoxynivalenol affects the viability and functions of glial cells.

International audienceGlial cells are responsible for maintaining brain homeostasis. Modification of the viability and functions of glial cells, including astrocytes and microglia, are associated with neuronal death and neurological diseases. Many toxins (heavy metals, pesticides, bacterial or viral toxins) are known to impact on brain cell viability and functions. Although recent publications suggest a potential link between environmental exposure of humans to mycotoxins and neurological diseases, data regarding the effects of fungal toxins on brain cells are scarce. In the present study, we looked at the impact of deoxynivalenol (DON), a fungal ribotoxin, on glial cells from animal and human origin. We found that DON decreased the viability of glial cells with a higher toxicity against microglial cells compared with astrocytes. In addition to cellular toxicity, DON affected key functions of glial cells. Thus, DON caused a biphasic effect on the neuroinflammatory response of microglia to lipopolysaccharide (LPS), while sublethal doses of DON increased the LPS-induced secretion of TNF-α and nitric oxide, toxic doses inhibited it. In addition to affecting microglial functions, sublethal doses of DON also suppressed the uptake of L-glutamate by astrocytes. This inhibition was associated with a modification of the expression of the glutamate transporters at the plasma membrane. Our results suggest that environmental ribotoxins such as DON could, at low doses, cause modifications of brain homeostasis and possibly participate in the etiology of neurological diseases in which alterations of the glia are involved. © 2011 Wiley-Liss, Inc

Identification and characterization of highly versatile peptide-vectors that bind non-competitively to the low-density lipoprotein receptor for in vivo targeting and delivery of small molecules and protein cargos

International audienceInsufficient membrane penetration of drugs, in particular biotherapeutics and/or low target specificity remain a major drawback in their efficacy. We propose here the rational characterization and optimization of peptides to be developed as vectors that target cells expressing specific receptors involved in endocytosis or transcytosis. Among receptors involved in receptor-mediated transport is the LDL receptor. Screening complex phage-displayed peptide libraries on the human LDLR (hLDLR) stably expressed in cell lines led to the characterization of a family of cyclic and linear peptides that specifically bind the hLDLR. The VH41 1 lead cyclic peptide allowed endocytosis of payloads such as the S-Tag peptide or antibodies into cells expressing the hLDLR. Size reduction and chemical optimization of this lead peptide-vector led to improved receptor affinity. The optimized peptide-vectors were successfully conjugated to cargos of different nature and size including small organic molecules, siRNAs, peptides or a protein moiety such as an Fc fragment. We show that in all cases, the peptide-vectors retain their binding affinity to the hLDLR and potential for endocytosis. Following i.v. administration in wild type or Idlr-!-mice, an Fc fragment chemically conjugated or fused in C-terminal to peptide-vectors showed significant biodistribution in LDLR-enriched organs. We have thus developed highly versatile peptide-vectors endowed with good affinity for the LDLR as a target receptor. These peptide-vectors have the potential to be further developed for efficient transport of therapeutic or imaging agents into cells-including pathological cells-or organs that express the LDLR

Use of LDL receptor-targeting peptide vectors for in vitro and in vivo cargo transport across the blood-brain barrier.

International audienceThe blood-brain barrier (BBB) prevents the entry of many drugs into the brain and, thus, is a major obstacle in the treatment of CNS diseases. There is some evidence that the LDL receptor (LDLR) is expressed at the BBB and may participate in the transport of endogenous ligands from blood to brain, a process referred to as receptor-mediated transcytosis. We previously described a family of peptide vectors that were developed to target the LDLR. In the present study, in vitro BBB models that were derived from either wild-type and LDLR knockout animals (ldlr(-/-)) were used to validate the specific LDLR-dependent transcytosis of LDL via a nondegradative route. We next showed that LDLR-targeting peptide vectors, whether in fusion or chemically conjugated to an Ab Fc fragment, promote binding to apical LDLR and transendothelial transfer of the Fc fragment across BBB monolayers via the same route as LDL. Finally, we demonstrated in vivo that LDLR significantly contributes to the brain uptake of vectorized Fc. We thus provide further evidence that LDLR is a relevant receptor for CNS drug delivery via receptor-mediated transcytosis and that the peptide vectors we developed have the potential to transport drugs, including proteins or Ab based, across the BBB.-Molino, Y., David, M., Varini, K., Jabès, F., Gaudin, N., Fortoul, A., Bakloul, K., Masse, M., Bernard, A., Drobecq, L., Lécorché, P., Temsamani, J., Jacquot, G., Khrestchatisky, M. Use of LDL receptor-targeting peptide vectors for in vitro and in vivo cargo transport across the blood-brain barrier