Gap junctional communication is required to maintain mouse cortical neural progenitor cells in a proliferative state

The mechanisms that determine whether neural stem cells remain in a proliferative state or differentiate into neurons or glia are largely unknown. Here we establish a pivotal role for gap junction-mediated intercellular communication in determining the proliferation and survival of mouse neural progenitor cells (NPCs). When cultured in the presence of basic fibroblast growth factor (bFGF), NPCs express the gap junction protein connexin 43 and are dye-coupled. Upon withdrawal of bFGF, levels of connexin 43 and dye coupling decrease, and the cells cease proliferating and differentiate into neurons; the induction of gap junctions by bFGF is mediated by p42/p44 mitogen-activated protein kinases. Inhibition of gap junctions abolishes the ability of bFGF to maintain NPCs in a proliferative state resulting in cell differentiation or cell death, while overexpression of connexin 43 promotes NPC self-renewal in the absence of bFGF. In addition to promoting their proliferation, gap junctions are required for the survival of NPCs. Gap junctional communication is therefore both necessary and sufficient to maintain NPCs in a self-renewing state.     

Temporal regulation of cerebellar EGL migration through a switch in cellular responsiveness to the meninges

We have studied the temporal and spatial control of cell migration from the external germinal layer (EGL) in the mammalian cerebellum as a model for cortical migration. Our results have demonstrated that embryonic EGL cells do not migrate into internal layers because they respond to a diffusible attractant in the meninges, the nonneural tissues covering the nervous system, and to a repellent in the neuroepithelium. Two developmental changes are important for postnatal EGL migration: the disappearance of the repellent in the inner layers and a switch in cellular responsiveness of EGL cells so that the postnatal EGL cells respond to the repellent, but not the attractant in the meninges. Besides revealing the signaling role of meninges in cortical development, our study suggests that an active mechanism is required to prevent cell migration, and that mechanisms of cell migration should be studied even in the absence of apparent changes in cell positions. We propose a model for the developmental control of neuronal migration in the cerebellar cortex.     

In vitro resistance studies of hepatitis C virus serine protease inhibitors, VX-950 and BILN 2061: structural analysis indicates different resistance mechanisms

We have used a structure-based drug design approach to identify small molecule inhibitors of the hepatitis C virus (HCV) NS3.4A protease as potential candidates for new anti-HCV therapies. VX-950 is a potent NS3.4A protease inhibitor that was recently selected as a clinical development candidate for hepatitis C treatment. In this report, we describe in vitro resistance studies using a subgenomic replicon system to compare VX-950 with another HCV NS3.4A protease inhibitor, BILN 2061, for which the Phase I clinical trial results were reported recently. Distinct drug-resistant substitutions of a single amino acid were identified in the HCV NS3 serine protease domain for both inhibitors. The resistance conferred by these mutations was confirmed by characterization of the mutant enzymes and replicon cells that contain the single amino acid substitutions. The major BILN 2061-resistant mutations at Asp(168) are fully susceptible to VX-950, and the dominant resistant mutation against VX-950 at Ala(156) remains sensitive to BILN 2061. Modeling analysis suggests that there are different mechanisms of resistance to VX-950 and BILN 2061.     

Inhibition of sodium/proton exchange by a Rab-GTPase-activating protein regulates endosomal traffic in yeast

Endosomal Na+/H+ exchangers are important for salt and osmotolerance, vacuolar pH regulation, and endosomal trafficking. We show that the C terminus of yeast Nhx1 interacts with Gyp6, a GTPase-activating protein for the Ypt/Rab family of GTPases, and that Gyp6 colocalizes with Nhx1 in the endosomal/prevacuolar compartment (PVC). The gyp6 null mutant exhibits novel phenotypes consistent with loss of negative regulation of Nhx1, including increased tolerance to hygromycin, increased vacuolar pH, and decreased plasma membrane potential. In contrast, overexpression of Gyp6 increases sensitivity to hygromycin, decreases vacuolar pH, and results in a slight missorting of vacuolar carboxypeptidase Y to the cell surface. We conclude that Gyp6 is a negative regulator of Nhx1-dependent trafficking out of the PVC. Taken together with its GTPase-activating protein-dependent role as a negative regulator of Ypt6-mediated retrograde traffic to the Golgi, we propose that Gyp6 coordinates upstream and downstream events in the PVC to Golgi pathway. Our findings provide a possible molecular link between intraendosomal pH and regulation of vesicular trafficking.     

The short stature homeodomain protein SHOX induces cellular growth arrest and apoptosis and is expressed in human growth plate chondrocytes

Mutations in the homeobox gene SHOX cause growth retardation and the skeletal abnormalities associated with Léri-Weill, Langer, and Turner syndromes. Little is known about the mechanism underlying these SHOX-related inherited disorders of bone formation. Here we demonstrate that SHOX expression in osteogenic stable cell lines, primary oral fibroblasts, and primary chondrocytes leads to cell cycle arrest and apoptosis. These events are associated with alterations in the expression of several cellular genes, including pRB, p53, and the cyclin kinase inhibitors p21(Cip1) and p27(Kip1). A SHOX mutant, such as seen in Léri-Weill syndrome patients, does not display these activities of the wild type protein. We have also shown that endogenous SHOX is mainly expressed in hypertrophic/apoptotic chondrocytes of the growth plate, strongly suggesting that the protein plays a direct role in regulating the differentiation of these cells. This study provides the first insight into the biological function of SHOX as regulator of cellular proliferation and viability and relates these cellular events to the phenotypic consequences of SHOX deficiency.     

NAK is recruited to the TNFR1 complex in a TNFalpha-dependent manner and mediates the production of RANTES: identification of endogenous TNFR-interacting proteins by a proteomic approach

Tumor necrosis factor alpha (TNFalpha) is a proinflammatory cytokine with pleiotropic immunological and biological activities. TNFalpha signaling is triggered by the engagement of soluble TNFalpha to two types of cell surface receptors, TNFR1 and TNFR2. This recruits cytosolic proteins to the intracellular domains of the receptors and initiates signaling to downstream effectors. In this study, we used a proteomic approach to identify these cytosolic proteins from affinity-purified, endogenous TNFalpha.TNFR complexes in human myelomonocytic U937 cells. Seven proteins were identified, including TRADD, TRAP2, and TRAF2, which are three proteins known to be recruited to TNFalpha receptors. NAK, RasGAP3, TRCP1, and TRCP2 were also identified. We further showed that NAK is recruited to TNFR1 in a temporally regulated and TNFalpha-dependent manner and that it mediates the TNFalpha-induced production of the chemokine RANTES (regulated on activation normal T cell expressed and secreted). These data demonstrate that NAK is a component of the TNFalpha.TNFR1 signaling complex and confirm the physiological role of NAK in the TNFalpha-mediated response.     

Oligomeric Hsp33 with enhanced chaperone activity: gel filtration, cross-linking, and small angle x-ray scattering (SAXS) analysis

Hsp33, an Escherichia coli cytosolic chaperone, is inactive under normal conditions but becomes active upon oxidative stress. It was previously shown to dimerize upon activation in a concentration- and temperature-dependent manner. This dimer was thought to bind to aggregation-prone target proteins, preventing their aggregation. In the present study, we report small angle x-ray scattering (SAXS), steady state and time-resolved fluorescence, gel filtration, and glutaraldehyde cross-linking analysis of full-length Hsp33. Our circular dichroism and fluorescence results show that there are significant structural changes in oxidized Hsp33 at different temperatures. SAXS, gel filtration, and glutaraldehyde cross-linking results indicate, in addition to the dimers, the presence of oligomeric species. Oxidation in the presence of physiological salt concentration leads to significant increases in the oligomer population. Our results further show that under conditions that mimic the crowded milieu of the cytosol, oxidized Hsp33 exists predominantly as an oligomeric species. Interestingly, chaperone activity studies show that the oligomeric species is much more efficient compared with the dimers in preventing aggregation of target proteins. Taken together, these results indicate that in the cell, Hsp33 undergoes conformational and quaternary structural changes leading to the formation of oligomeric species in response to oxidative stress. Oligomeric Hsp33 thus might be physiologically relevant under oxidative stress.     

Manganese induces the mitochondrial permeability transition in cultured astrocytes

Manganese is known to cause central nervous system injury leading to parkinsonism and to contribute to the pathogenesis of hepatic encephalopathy. Although mechanisms of manganese neurotoxicity are not completely understood, chronic exposure of various cell types to manganese has shown oxidative stress and mitochondrial energy failure, factors that are often implicated in the induction of the mitochondrial permeability transition (MPT). In this study, we examined whether exposure of cultured neurons and astrocytes to manganese induces the MPT. Cells were treated with manganese acetate (10-100 microM), and the MPT was assessed by changes in the mitochondrial membrane potential and in mitochondrial calcein fluorescence. In astrocytes, manganese caused a dissipation of the mitochondrial membrane potential and decreased the mitochondrial calcein fluorescence in a concentration- and time-dependent manner. These changes were completely blocked by pretreatment with cyclosporin A, consistent with induction of the MPT. On the other hand, similarly treated cultured cortical neurons had a delayed or reduced MPT as compared with astrocytes. The manganese-induced MPT in astrocytes was blocked by pretreatment with antioxidants, suggesting the potential involvement of oxidative stress in this process. Induction of the MPT by manganese and associated mitochondrial dysfunction in astrocytes may represent key mechanisms in manganese neurotoxicity.