AMPA Receptors Commandeer an Ancient Cargo Exporter for Use as an Auxiliary Subunit for Signaling

Fast excitatory neurotransmission in the mammalian central nervous system is mainly mediated by ionotropic glutamate receptors of the AMPA subtype (AMPARs). AMPARs are protein complexes of the pore-lining α-subunits GluA1-4 and auxiliary β-subunits modulating their trafficking and gating. By a proteomic approach, two homologues of the cargo exporter cornichon, CNIH-2 and CNIH-3, have recently been identified as constituents of native AMPARs in mammalian brain. In heterologous reconstitution experiments, CNIH-2 promotes surface expression of GluAs and modulates their biophysical properties. However, its relevance in native AMPAR physiology remains controversial. Here, we have studied the role of CNIH-2 in GluA processing both in heterologous cells and primary rat neurons. Our data demonstrate that CNIH-2 serves an evolutionarily conserved role as a cargo exporter from the endoplasmic reticulum (ER). CNIH-2 cycles continuously between ER and Golgi complex to pick up cargo protein in the ER and then to mediate its preferential export in a coat protein complex (COP) II dependent manner. Interaction with GluA subunits breaks with this ancestral role of CNIH-2 confined to the early secretory pathway. While still taking advantage of being exported preferentially from the ER, GluAs recruit CNIH-2 to the cell surface. Thus, mammalian AMPARs commandeer CNIH-2 for use as a bona fide auxiliary subunit that is able to modify receptor signaling

THE IMMUNOSUPPRESSIVE EFFECT OF MESENCHYMAL STROMAL CELLS ON B LYMPHOCYTES IS MEDIATED BY MEMBRANE VESICLES.

The immunomodulatory properties of mesenchymal stromal cells are the subject of increasing interest and of widening clinical applications, but the reproducibility of their effects is controversial and the underlying mechanisms have not been fully clarified. We investigated the transfer of membrane vesicles, a recently recognized pathway of intercellular communication, as possible mediator of the interaction between mesenchymal stromal cells and B lymphocytes. Mesenchymal stromal cells exhibited a strong dose-dependent inhibition of B cell proliferation and differentiation in a CpG-stimulated peripheral blood mononuclear cell co-culture system. We observed that these effects could be fully reproduced by membrane vesicles isolated from mesenchymal stromal cell culture supernatants, in a dose-dependent fashion. Next, we evaluated the localization of fluorescent labeled membrane vesicles within specific cell subtypes both by flow cytometry and by confocal microscopy analysis. Membrane vesicles were found to be associated with stimulated B lymphocytes, but not with other cell phenotypes (T lymphocytes, dendritic cells, NK cells), in peripheral blood mononuclear cell culture. These results suggest that membrane vesicles derived from mesenchymal stromal cells are the conveyors of the immunosuppressive effect on B lymphocytes. These particles should be further evaluated as immunosuppressive agents in place of the parent cells, with possible advantages in term of standardization, safety and feasibility

Stable High-Level Expression of Heterologous Genes In Vitro and In Vivo by Noncytopathic DNA-Based Kunjin Virus Replicon Vectors

Primary features of the flavivirus Kunjin (KUN) subgenomic replicons include continuous noncytopathic replication in host cell cytoplasm and the ability to be encapsidated into secreted virus-like particles (VLPs). Previously we reported preparation of RNA-based KUN replicon vectors and expression of heterologous genes (HG) in cell culture after RNA transfection or after infection with recombinant KUN VLPs (A. N. Varnavski and A. A. Khromykh, Virology 255:366–375, 1999). In this study we describe the development of the next generation of KUN replicon vectors, which allow synthesis of replicon RNA in vivo from corresponding plasmid DNAs. These DNA-based vectors were able to direct stable expression of β-galactosidase (β-Gal) in several mammalian cell lines, and expression remained high (∼150 pg per cell) throughout cell passaging. The applicability of these vectors in vivo was demonstrated by β-Gal expression in the mouse lung epithelium for at least 8 weeks after intranasal inoculation and induction of anti-β-Gal antibody response after intramuscular inoculation of the β-Gal-encoding KUN replicon DNA. The noncytopathic nature of DNA-based KUN replicon vectors combined with high-level and stability of HG expression in a broad range of host cells should prove them to be useful in a variety of applications in vitro and in vivo