In the process of screening cell-type-specific genes, we identified juxtanodin (JN), an oligodendroglial protein featuring a putative C-terminal actin-binding domain. At the cellular level, JN in the rat CNS colocalized with 2 ,3 -cyclic nucleotide-3 -phosphodiesterase (CNPase), a cytoskeleton-related oligodendroglial protein. In the myelin sheath, JN was found mainly in the abaxon and the lateral few terminal loops. Its apposition to the myelinated axon, through the latter, defined an axonal subregion, herewith termed juxtanode, at the Ranvier node-paranode junction. During forebrain ontogenesis, JN expression paralleled that of MBPs but lagged behind CNPase. Juxtanodin transfection promoted arborization of cultured OLN-93 cells and augmented endogenous CNPase expression and transport to the process arbors of cultured primary oligodendrocyte precursors. These results reveal JN as a cytoskeleton-related oligodendroglial protein that delineates the juxtanode and might serve oligodendrocyte motility, differentiation, or myelin-axon signaling. Functionally, JN may be involved in CNS myelination and͞or specialization of the node of Ranvier.myelin-axon interaction ͉ oligodendrocyte ͉ node of Ranvier ͉ cytoskeleton O ligodendroglia are highly specialized myelin-forming cells of the CNS. Adequate myelination serves to insulate and accelerate the propagation of action potentials. Abnormalities in myelin formation͞maintenance may underlie diverse neurological disorders, ranging from multiple sclerosis to schizophrenia (1, 2). Matched with highly specialized functions and unique architecture, various oligodendrocyte͞myelin-selective molecules, such as myelin basic protein (MBP), myelin-associated glycoprotein, and 2Ј,3Ј-cyclic nucleotide-3Ј-phosphodiesterase (CNPase), have been isolated and characterized (1, 3). It is likely that many more are yet to be revealed. The identification and characterization of such molecules will probably help elucidate molecular mechanisms of myelination and dys-or demyelinating diseases.The unique architecture of the myelin sheath entails specialized, yet elusive, cytoskeletal mechanisms of myelin-forming cells. Here, we report the molecular features, cellular expression, and functional roles of juxtanodin (JN), a previously unidentified cytoskeleton-related oligodendrocyte-specific protein. Materials and MethodsIdentification and Characterization of the mRNA. Cell-type-specific CNS genes were sought out by in situ hybridization histochemistry (ISH). Briefly, cDNA clones (n ϭ 1,500) from a rat brain cDNA library (Invitrogen) were sequenced and compared by BLAST searches against National Center for Biotechnology Information nucleotide and protein nonredundant databases (4). For the unannotated cDNA sequences (n ϭ 274), expression of the mRNAs in the CNS was mapped by ISH using digoxigeninlabeled riboprobes. A 3.6-kb cDNA clone with a predicted ORF encoding 282 amino acid residues was thereby identified, and the gene was subsequently named juxtanodin (JN). A search of EST databases revealed an...

The gene mutated in the mouse open brain (opb) phenotype antagonizes sonic hedgehog-mediated signaling and encodes a small GTPase of the Rab family, Rab23. To date, the brain expression profile and exact mechanism of function of the Rab23 protein has remained unknown. Specific antibodies generated against Rab23 showed that the protein is highly enriched in the adult rodent brain and present in low levels in multiple tissues of the adult rodent. Rab23 is found in the cytosol as well as being associated with the plasma and endosomal membranes. In the adult mouse brain, Rab23 is found in betaIII tubulin (TuJ) positive neuronal cell bodies and are most prominent in the cortex, hypothalamus and the cerebellum. It is, however, absent from glial fibrillary acidic protein (GFAP) positive astrocytes or CNPase positive oligodendrocytes. Despite the plasma membrane/endosomal membrane localization of Rab23, neither overexpression of the GTP-restricted nor the GDP-bound mutant forms affect internalization of transferrin or epidermal growth factor. Exogenous overexpression of Rab23 or its mutants also did not affect the morphological differentiation of thalamic neurons in culture. Expression of Rab23 in the adult brain is suggestive, however, of having a postnatal function beyond its role in embryonic development.

To identify suitable cell lines for a mimetic system of in vivo blood-brain barrier (BBB) for drug permeability assessment, we characterized two immortalized cell lines, ECV304 and bEnd3 in the respect of the tightness, tight junction proteins, P-glycoprotein (P-gp) function and discriminative brain penetration. The ECV304 monoculture achieved higher transendothelial electrical resistance (TEER) and lower permeability to Lucifer yellow than bEnd3. However, co-culture with rat glioma C6 cells impaired the integrity of ECV304 and bEnd3 cell layers perhaps due to the heterogeneity among C6 cells in inducing BBB characteristics. The immunostaining of ZO-1 delivered distinct bands along cell borders on both cell lines while those of occludin and claudin-5 were diffused and weak. P-gp functionality was only proved in bEnd3 by Rhodamine 123 (R123) uptake assay. A permeability test of reference compounds displayed a similar rank order (digoxin < R123 < quinidine, verapamil < propranolol) in ECV304 and bEnd3 cells. In comparison with bEnd3, ECV304 developed tighter barrier for the passage of reference compounds and higher discrimination between transcellular and paracellular transport. However, the monoculture models of ECV304 and bEnd3 fail to achieve the sufficient tightness of in vitro BBB permeability models with high TEER and evident immunostaining of tight junction proteins. Further strategies to enhance the paracellular tightness of both cell lines to mimic in vivo BBB tight barrier deserve to be conducted.