Yoshihide Yamaguchi

MYELIN PROTEOLIPID PROTEIN (PLP), BUT NOT DM-20, IS AN INOSITOL HEXAKISPHOSPHATE-BINDING PROTEIN

Myelin proteolipid protein (PLP) and its alternatively spliced isoform, DM-20, are the major integral membrane proteins of central nervous system myelin. It is known that PLP and DM-20 are delivered to myelin by a finely regulated vesicular transport system in oligodendrocytes. Evolutionarily, it is believed that ancestral DM-20 acquired a PLP-specific exon to create PLP, after which PLP/DM-20 became a major component of central nervous system myelin. We purified PLP as an inositol 1,3,4,5-tetrakisphosphate-binding protein after solubilization in a non-organic solvent. However, under the isotonic condition, PLP binds inositol hexakisphosphate (InsP6) significantly, not inositol 1,3,4,5-tetrakisphosphate. Most of the InsP6-binding proteins are involved in vesicular transport, suggesting the involvement of PLP in vesicular transport. We separated DM-20 from PLP by CM-52 chromatography and showed that DM-20 has no InsP6 binding activity. These findings indicate that the PLP-specific domain confers the InsP6 binding activity and this interaction may be important for directing PLP transport to central nervous system myelin.

Nodal protrusions, increased Schmidt-Lanterman incisures, and paranodal disorganization are characteristic features of sulfatide-deficient peripheral nerves.

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 D family member 4, a transmembrane glycoprotein with no phospholipase D activity, expression in spleen and early postnatal microglia.

Background Phospholipase 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 Findings PLD4 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/Significance Results 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.

Two-Dimensional Electrophoresis With Cationic Detergents : a Powerful Tool for the Proteomic Analysis of Myelin Proteins. Part 2: Analytical Aspects

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.

PLD4 Is Involved in Phagocytosis of Microglia: Expression and Localization Changes of PLD4 Are Correlated with Activation State of Microglia

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.

L-MPZ, a Novel Isoform of Myelin P0, Is Produced by Stop Codon Readthrough

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 3′-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. Myelin protein zero (P0 or MPZ) is a major myelin protein (∼30 kDa) expressed in the peripheral nervous system (PNS) in terrestrial vertebrates. Several groups have detected a P0-related 36-kDa (or 35-kDa) protein that is expressed in the PNS as an antigen for the serum IgG of patients with neuropathy. The molecular structure and function of this 36-kDa protein are, however, still unknown. We hypothesized that the 36-kDa protein may be derived from P0 mRNA by stop codon readthrough. We found a highly conserved region after the regular stop codon in predicted sequences from the 3′-UTR of P0 in higher animals. MS of the 36-kDa protein revealed that both P0 peptides and peptides deduced from the P0 3′-UTR sequence were found among the tryptic fragments. In transfected cells and in an in vitro transcription/translation system, the 36-kDa molecule was also produced from the identical mRNA that produced P0. We designated this 36-kDa molecule as large myelin protein zero (L-MPZ), a novel isoform of P0 that contains an additional domain at the C terminus. In the PNS, L-MPZ was localized in compact myelin. In transfected cells, just like P0, L-MPZ was localized at cell-cell adhesion sites in the plasma membrane. These results suggest that L-MPZ produced by the stop codon readthrough mechanism is potentially involved in myelination. Since this is the first finding of stop codon readthrough in a common mammalian protein, detailed analysis of L-MPZ expression will help to understand the mechanism of stop codon readthrough in mammals.

Two-dimensional electrophoresis with cationic detergents, a powerful tool for the proteomic analysis of myelin proteins. Part 1: Technical aspects of electrophoresis

The analysis of proteins in damaged myelin is crucial to clarify the mechanisms of dysmyelination and demyelination. In the present study, proteomic analysis of myelin using a modified two‐dimensional electrophoresis (2‐DE) method was carried out to obtain a better understanding of myelin biology. Although standard 2‐DE (immobilized pH gradient isoelectric focusing/sodium dodecyl sulfate‐polyacrylamide gel electrophoresis; IPG/SDS‐PAGE) methods of analysis provide high resolutions of soluble proteins with isoelectric focusing points in the pH range of 4–8, major myelin components include highly basic proteins are compacted at the basic edge of the 2‐DE gels and are not sufficiently separated for satisfactory analysis. In an attempt to improve the separation of these proteins, an alternative 2‐DE method using the cationic detergents was applied. In part 1 of this study, we describe technical aspects of conditioning 2‐DE using cationic detergent. In the accompanying paper (part 2), practical 2‐DE analysis using cationic detergents is described to identify proteins in the purified CNS myelin fraction. We carried out benzyldimethyl‐n‐hexadecylammonium chloride (16‐BAC)/SDS‐PAGE 2‐DE and tested 2‐DE with four other cationic detergents. We found that 16‐BAC was the most effective agent for separation of myelin proteins and that hexadecyltrimethylammonium bromide (cetyltrimethylammonium bromide; CTAB) was the most effective agent for solubilization of myelin proteins. The combination of 16‐BAC/SDS‐PAGE and CTAB/SDS‐PAGE is a powerful tool for the analysis of myelin proteins, including highly basic, high‐MW (MW > 100K), and integral membrane proteins.

Paranodal axoglial junction is required for the maintenance of the Nav1.6‐type sodium channel in the node of Ranvier in the optic nerves but not in peripheral nerve fibers in the sulfatide‐deficient mice

In myelinated axons, voltage‐gated sodium channels specifically cluster at the nodes of Ranvier, while voltage‐gated potassium channels are located at the juxtaparanodes. These characteristic localizations are influenced by myelination. During development, Nav1.2 first appears in the predicted nodes during myelination, and Nav1.6 replaces it in the mature nodes. Such replacements may be important physiologically. We examined the influence of the paranodal junction on switching of sodium channel subunits using the sulfatide‐deficient mouse. This mutant displayed disruption of paranodal axoglial junctions and altered nodal lengths and channel distributions. The initial switching of Nav1.2 to Nav1.6 occurred in the mutant optic nerves; however, the number of Nav1.2‐positive clusters was significantly higher than in wild‐type mice. Although no signs of demyelination were observed at least up to 36 weeks of age, sodium channel clusters decreased markedly with age. Interestingly, Nav1.2 stayed in some of the nodal regions, especially where the nodal lengths were elongated, while Nav1.6 tended to remain in the normal‐length nodes. The results in the mutant optic nerves suggested that paranodal junction formation may be necessary for complete replacement of nodal Nav1.2 to Nav1.6 during development as well as maintenance of Nav1.6 clusters at the nodes. Such subtype abnormality was not observed in the sciatic nerve, where paranodal disruption was observed. Thus, the paranodal junction significantly influences the retention of Nav1.6 in the node, which is followed by disorganization of nodal structures. However, its importance may differ between the central and peripheral nervous system.

Unconventional myosin ID is expressed in myelinating oligodendrocytes

Myelin is a dynamic multilamellar structure that ensheathes axons and is crucial for normal neuronal function. In the central nervous system (CNS), myelin is produced by oligodendrocytes that wrap many layers of plasma membrane around axons. The dynamic membrane trafficking system, which relies on motor proteins, is required for myelin formation and maintenance. Previously, we found that myosin ID (Myo1d), a class I myosin, is enriched in the rat CNS myelin fraction. Myo1d is an unconventional myosin and has been shown to be involved in membrane trafficking in the recycling pathway in an epithelial cell line. Western blotting revealed that Myo1d expression begins early in myelinogenesis and continues to increase into adulthood. The localization of Myo1d in CNS myelin has not been reported, and the function of Myo1d in vivo remains unknown. To demonstrate the expression of Myo1d in CNS myelin and to begin to explore the function of Myo1d in myelination, we produced a new antibody against Myo1d that has a high titer and specificity for rat Myo1d. By using this antibody, we demonstrated that Myo1d is expressed in rat CNS myelin and is especially abundant in abaxonal and adaxonal regions (the outer and inner cytoplasm‐containing loops, respectively), but that expression is low in peripheral nervous system myelin. In culture, Myo1d was expressed in mature rat oligodendrocytes. Furthermore, an increase in expression of Myo1d during maturation of CNS white matter (cerebellum and corpus callosum) was demonstrated by histological analysis. These results suggest that Myo1d may be involved in the formation and/or maintenance of CNS myelin.

Knockdown of Unconventional Myosin ID Expression Induced Morphological Change in Oligodendrocytes.

Myelin is a special multilamellar structure involved in various functions in the nervous system. In the central nervous system, the oligodendrocyte (OL) produces myelin and has a unique morphology. OLs have a dynamic membrane sorting system associated with cytoskeletal organization, which aids in the production of myelin. Recently, it was reported that the assembly and disassembly of actin filaments is crucial for myelination. However, the partner myosin molecule which associates with actin filaments during the myelination process has not yet been identified. One candidate myosin is unconventional myosin ID (Myo1d) which is distributed throughout central nervous system myelin; however, its function is still unclear. We report here that Myo1d is expressed during later stages of OL differentiation, together with myelin proteolipid protein (PLP). In addition, Myo1d is distributed at the leading edge of the myelin-like membrane in cultured OL, colocalizing mainly with actin filaments, 2′,3′-cyclic nucleotide phosphodiesterase and partially with PLP. Myo1d-knockdown with specific siRNA induces significant morphological changes such as the retraction of processes and degeneration of myelin-like membrane, and finally apoptosis. Furthermore, loss of Myo1d by siRNA results in the impairment of intracellular PLP transport. Together, these results suggest that Myo1d may contribute to membrane dynamics either in wrapping or transporting of myelin membrane proteins during formation and maintenance of myelin.