Background:The structure and function of myelin P0-related 36-kDa protein are totally unknown. Results: A novel isoform of P0, L-MPZ, contains an extra C terminus derived from 3Ј-UTR of P0 mRNA and is expressed in peripheral myelin. Conclusion: L-MPZ is produced by stop codon readthrough and probably related with peripheral myelinogenesis. Significance: Analyses of L-MPZ are crucial for understanding readthrough mechanism in mammals and myelinogenesis.

BackgroundPhospholipase D (PLD) catalyzes conversion of phosphatidylcholine into choline and phosphatidic acid, leading to a variety of intracellular signal transduction events. Two classical PLDs, PLD1 and PLD2, contain phosphatidylinositide-binding PX and PH domains and two conserved His-x-Lys-(x)4-Asp (HKD) motifs, which are critical for PLD activity. PLD4 officially belongs to the PLD family, because it possesses two HKD motifs. However, it lacks PX and PH domains and has a putative transmembrane domain instead. Nevertheless, little is known regarding expression, structure, and function of PLD4.Methodology/Principal FindingsPLD4 was analyzed in terms of expression, structure, and function. Expression was analyzed in developing mouse brains and non-neuronal tissues using microarray, in situ hybridization, immunohistochemistry, and immunocytochemistry. Structure was evaluated using bioinformatics analysis of protein domains, biochemical analyses of transmembrane property, and enzymatic deglycosylation. PLD activity was examined by choline release and transphosphatidylation assays. Results demonstrated low to modest, but characteristic, PLD4 mRNA expression in a subset of cells preferentially localized around white matter regions, including the corpus callosum and cerebellar white matter, during the first postnatal week. These PLD4 mRNA-expressing cells were identified as Iba1-positive microglia. In non-neuronal tissues, PLD4 mRNA expression was widespread, but predominantly distributed in the spleen. Intense PLD4 expression was detected around the marginal zone of the splenic red pulp, and splenic PLD4 protein recovered from subcellular membrane fractions was highly N-glycosylated. PLD4 was heterologously expressed in cell lines and localized in the endoplasmic reticulum and Golgi apparatus. Moreover, heterologously expressed PLD4 proteins did not exhibit PLD enzymatic activity.Conclusions/SignificanceResults showed that PLD4 is a non-PLD, HKD motif-carrying, transmembrane glycoprotein localized in the endoplasmic reticulum and Golgi apparatus. The spatiotemporally restricted expression patterns suggested that PLD4 might play a role in common function(s) among microglia during early postnatal brain development and splenic marginal zone cells.

Estrogen receptor (ER)-beta is a member of the nuclear receptor superfamily and mediates various estrogenic actions. Changes in ER-alpha mRNA expression induced by estrogen have been well documented, whereas those with regard to ER-beta have only been reported for a part of the hypothalamus. In the present study, we examined the effect of estrogen on ER-beta mRNA expression in the female rat brain. Detection of ER-beta mRNA using the in situ hybridization method with a digoxigenin-labeled RNA probe was performed in two groups of female rats: ovariectomized (OVX) and estrogen (E2)-treated. A wide distribution of ER-beta mRNA-containing cells was demonstrated in both groups. In the E2-treated group compared with the OVX group, the number of ER-beta mRNA-containing cells was significantly reduced in the external plexiform layer of the olfactory bulb, entorhinal cortex, intermediate part of the lateral septal nucleus, nucleus of the horizontal limb of the diagonal band, amygdala (lateral, medial and basolateral part), thalamus (anteroventral, laterodorsal and lateral posterior part), medial geniculate nucleus, suprachiasmatic nucleus and Purkinje cells in the cerebellum. These results reveal that ER-beta mRNA-containing cells were decreased by estrogen in several brain regions in the female rat brain, suggesting that ER-beta mRNA is downregulated by the physiological level of estrogen in a region-specific manner.

Galactocerebroside and sulfatide are two major glycolipids in myelin; however, their independent functions are not fully understood. The absence of these glycolipids causes disruption of paranodal junctions, which separate voltage-gated Na(+) and Shaker-type K(+) channels in the node and juxtaparanode, respectively. In contrast to glial cells in the central nervous system (CNS), myelinating Schwann cells in the peripheral nervous system (PNS) possess characteristic structures, including microvilli and Schmidt-Lanterman incisures, in addition to paranodal loops. All of these regions are involved in axo-glial interactions. In the present study, we examined cerebroside sulfotransferase-deficient mice to determine whether sulfatide is essential for axo-glial interactions in these PNS regions. Interestingly, marked axonal protrusions were observed in some of the nodal segments, which often contained abnormally enlarged vesicles, like degenerated mitochondria. Moreover, many transversely cut ends of microvilli surrounded the mutant nodes, suggesting that alignments of the microvilli were disordered. The mutant PNS showed mild elongation of nodal Na(+) channel clusters. Even though Caspr and NF155 were completely absent in half of the paranodes, short clusters of these molecules remained in the rest of the paranodal regions. Ultrastructural analysis indicated the presence of transverse bands in some paranodal regions and detachment of the outermost several loops. Furthermore, the numbers of incisures were remarkably increased in the mutant internode. Therefore, these results indicate that sulfatide may play an important role in the PNS, especially in the regions where myelin-axon interactions occur.

Phospholipase D4 (PLD4) is a recently identified protein that is mainly expressed in the ionized calcium binding adapter molecule 1 (Iba1)-positive microglia in the early postnatal mouse cerebellar white matter. Unlike PLD1 and PLD2, PLD4 exhibits no enzymatic activity for conversion of phosphatidylcholine into choline and phosphatidic acid, and its function is completely unknown. In the present study, we examined the distribution of PLD4 in mouse cerebellar white matter during development and under pathological conditions. Immunohistochemical analysis revealed that PLD4 expression was associated with microglial activation under such two different circumstances. A primary cultured microglia and microglial cell line (MG6) showed that PLD4 was mainly present in the nucleus, except the nucleolus, and expression of PLD4 was upregulated by lipopolysaccharide (LPS) stimulation. In the analysis of phagocytosis of LPS-stimulated microglia, PLD4 was co-localized with phagosomes that contained BioParticles. Inhibition of PLD4 expression using PLD4 specific small interfering RNA (siRNA) in MG6 cells significantly reduced the ratio of phagocytotic cell numbers. These results suggest that the increased PLD4 in the activation process is involved in phagocytosis of activated microglia in the developmental stages and pathological conditions of white matter.

No abstract

No abstract

The ability to analyze proteins in developing and damaged myelin will be crucial to improve our understanding of the mechanisms of myelinogenesis, dysmyelination, and demyelination. Comparative two-dimensional electrophoresis (2-DE) is a powerful approach to analyze these proteins. In part 1 of this study (see accompanying paper), a method for the 2-DE analysis of myelin proteins using the cationic detergents benzyldimethyl-n-hexadecylammonium chloride (16-BAC) and hexadecyltrimethylammonium bromide (cetyltrimethylammonium bromide; CTAB) was described. We obtained improved separation and found that 16-BAC is the most effective agent for separation in 2-DE of myelin proteins and that CTAB is the most effective agent for solubilization of myelin proteins. Here in part 2, major myelin proteins as well as membrane proteins with multitransmembrane domains were identified by mass spectrometry after 16-BAC/SDS-PAGE and CTAB/SDS-PAGE. In addition, a high-molecular-weight protein enriched in myelin fraction was identified as unconventional myosin ID using 16-BAC/SDS-PAGE, which had previously not been detected using immobilized pH gradient isoelectric focusing (IPG)/SDS-PAGE. From these results, we concluded that combinational analysis using IPG/SDS-PAGE, 16-BAC/SDS-PAGE, and CTAB/SDS-PAGE provides a powerful technique facilitating the proteomic analysis of myelin proteins in either developmental or pathological changes.