GLUT1 deficiency links nutrient availability and apoptosis during embryonic development

GLUT1 is essential for human brain development and function, as evidenced by the severe epileptic encephalopathy observed in children with GLUT1 deficiency syndrome resulting from inherited loss-of-function mutations in the gene encoding this facilitative glucose transporter. To further elucidate the pathophysiology of this disorder, the zebrafish orthologue of human GLUT1 was identified, and expression of this gene was abrogated during early embryonic development, resulting in a phenotype of aberrant brain organogenesis consistent with the observed expression of Glut1 in the embryonic tectum and specifically rescued by human GLUT1 mRNA. Affected embryos displayed impaired glucose uptake concomitant with increased neural cell apoptosis and subsequent ventricle enlargement, trigeminal ganglion cell loss, and abnormal hindbrain architecture. Strikingly, inhibiting expression of the zebrafish orthologue of the proapoptotic protein Bad resulted in complete rescue of this phenotype, and this occurred even in the absence of restoration of apparent glucose uptake. Taken together, these studies describe a tractable system for elucidating the cellular and molecular mechanisms of Glut1 deficiency and provide compelling in vivo genetic evidence directly linking nutrient availability and activation of mitochondria-dependent apoptotic mechanisms during embryonic brain development.     

Biochemical analysis of a missense mutation in aceruloplasminemia.

Aceruloplasminemia is an inherited neurodegenerative disease characterized by parenchymal iron accumulation secondary to loss-of-function mutations in the ceruloplasmin gene. To elucidate the molecular pathogenesis of aceruloplasminemia, the biosynthesis of a missense mutant ceruloplasmin (P177R) occurring in an affected patient was examined. Chinese hamster ovary cells transfected with cDNAs encoding secreted and glycosylphosphatidylinositol (GPI)-linked wild-type or P177R human ceruloplasmin were examined by pulse-chase metabolic labeling. These experiments, as well as immunofluorescent analysis and N-linked glycosylation studies, indicate that both the secreted and GPI-linked forms of the P177R mutant are retained in the endoplasmic reticulum (ER). The P177R mutation resides within a novel motif, which is repeated six times in human ceruloplasmin and is conserved in the homologous proteins hephaestin and factor VIII. Analysis of additional mutations in these motifs suggests a critical role for this region in ceruloplasmin trafficking and indicates that substitution of the arginine residue is critical to the ER retention of the P177R mutant. Metabolic labeling of transfected Chinese hamster ovary cells with (64)Cu indicates that the P177R mutant is retained in the ER as an apoprotein and that copper is incorporated into both secreted and GPI-linked ceruloplasmin as a late event in the secretory pathway. Taken together, these studies reveal new insights into the determinants of holoceruloplasmin biosynthesis and indicate that aceruloplasminemia can result from retention of mutant ceruloplasmin within the early secretory pathway.     

Nucleopolyhedrovirus Detection and Distribution in Terrestrial,Freshwater, and Marine Habitats of Appledore Island,Gulf of Maine

Viruses in aquatic ecosystems comprise those produced by both autochthonous and allochthonous host taxa. However, there is little information on the diversity and abundance of viruses of allochthonous origin, particularly from non-anthropogenic sources, in freshwater and marine ecosystems. We investigated the presence of nucleopolyhedroviruses (NPV) (Baculovirus), which commonly infect terrestrial lepidopteran taxa, across the landscape of Appledore Island, Gulf of Maine. PCR and qPCR primers were developed around a 294-bp fragment of the polyhedrin (polH) gene, which is the major constituent protein of NPV multivirion polyhedral occlusion bodies. polH was successfully amplified from several aquatic habitats, and recovered polH sequences were most similar to known lepidopteran NPV. Using quantitative PCR designed around a cluster of detected sequences, we detected polH in Appledore Island soils, supratidal freshwater ponds, nearshore sediments, near- and offshore plankton, and in floatsam. This diverse set of locations suggests that NPVs are widely dispersed along the terrestrial—marine continuum and that free polyhedra may be washed into ponds and eventually to sea. The putative hosts of detected NPVs were webworms (Hyphantria sp.) which form dense nests in late summer on the dominant Appledore Island vegetation (Prunus virginiana). Our data indicate that viruses of terrestrial origin (i.e., allochthonous viruses) may be dispersed widely in coastal marine habitats. The dispersal of NPV polH and detection within offshore net plankton (>64 μm) demonstrates that terrestrial viruses may interact with larger particles and plankton of coastal marine ecosystem, which further suggests that viral genomic information may be transported between biomes.