AB3254 Sigma-AldrichAnti-HA Epitope Tag (Influenza Hemaglutinin) Antibody

The ability to map the functional connectivity of discrete cell types in the intact mammalian brain during behavior is crucial for advancing our understanding of brain function in normal and disease states. We combined designer receptor exclusively activated by designer drug (DREADD) technology and behavioral imaging with μPET and [18F]fluorodeoxyglucose (FDG) to generate whole-brain metabolic maps of cell-specific functional circuits during the awake, freely moving state. We have termed this approach DREADD-assisted metabolic mapping (DREAMM) and documented its ability in rats to map whole-brain functional anatomy. We applied this strategy to evaluating changes in the brain associated with inhibition of prodynorphin-expressing (Pdyn-expressing) and of proenkephalin-expressing (Penk-expressing) medium spiny neurons (MSNs) of the nucleus accumbens shell (NAcSh), which have been implicated in neuropsychiatric disorders. DREAMM revealed discrete behavioral manifestations and concurrent engagement of distinct corticolimbic networks associated with dysregulation of Pdyn and Penk in MSNs of the NAcSh. Furthermore, distinct neuronal networks were recruited in awake versus anesthetized conditions. These data demonstrate that DREAMM is a highly sensitive, molecular, high-resolution quantitative imaging approach.

Proteolipid protein (PLP) and its alternatively spliced isoform DM20 comprise ∼50% of central nervous system (CNS) myelin protein. The two proteins are identical in sequence except for the presence of a 35 amino sequence within the intracellular loop of PLP that is absent in DM20. In this work, we compared the expression of PLP/DM20 in transfected cells, oligodendrocytes and brain. In all 3 tissues, PLP exists as both a monomer and a disulfide-linked dimer; in contrast, DM20 is found mainly as a monomer. PLP dimers were increased by both chemical crosslinking and incubation with hydrogen peroxide, and were mediated by a cysteine at amino acid 108, located within the proximal intracellular loop of both PLP and DM20. The PLP-specific sequence thus influences the accessibility of this cysteine to chemical modification, perhaps as a result of altering protein structure. Consistent with these findings, several mutant PLPs known to cause Pelizaeus-Merzbacher disease form predominantly disulfide-linked, high molecular weight aggregates in transfected COS7 cells that are arrested in the ER and are associated with increased expression of CHOP, a part of the cellular response to unfolded proteins. In contrast, the same mutations in DM20 accumulate fewer high molecular weight disulfide-linked species that are expressed at the cell surface, and are not associated with increased CHOP. Taken together, these data suggest that mutant PLP multimerization, mediated in part by way of cysteine 108, may be part of the pathogenesis of Pelizaeus-Merzbacher disease.