Hiroko Baba

Anti-GM1 Antibodies Cause Complement-Mediated Disruption of Sodium Channel Clusters in Peripheral Motor Nerve Fibers

Voltage-gated Na+ (Nav) channels are highly concentrated at nodes of Ranvier in myelinated axons and facilitate rapid action potential conduction. Autoantibodies to gangliosides such as GM1 have been proposed to disrupt nodal Nav channels and lead to Guillain-Barré syndrome, an autoimmune neuropathy characterized by acute limb weakness. To test this hypothesis, we examined the molecular organization of nodes in a disease model caused by immunization with gangliosides. At the acute phase with progressing limb weakness, Nav channel clusters were disrupted or disappeared at abnormally lengthened nodes concomitant with deposition of IgG and complement products. Paranodal axoglial junctions, the nodal cytoskeleton, and Schwann cell microvilli, all of which stabilize Nav channel clusters, were also disrupted. The nodal molecules disappeared in lesions with complement deposition but no localization of macrophages. During recovery, complement deposition at nodes decreased, and Nav channels redistributed on both sides of affected nodes. These results suggest that Nav channel alterations occur as a consequence of complement-mediated disruption of interactions between axons and Schwann cells. Our findings support the idea that acute motor axonal neuropathy is a disease that specifically disrupts the nodes of Ranvier.

A Myelin Galactolipid, Sulfatide, Is Essential for Maintenance of Ion Channels on Myelinated Axon But Not Essential for Initial Cluster Formation

Myelinated axons are divided into four distinct regions: the node of Ranvier, paranode, juxtaparanode, and internode, each of which is characterized by a specific set of axonal proteins. Voltage-gated Na+ channels are clustered at high densities at the nodes, whereas shaker-type K+ channels are concentrated at juxtaparanodal regions. These channels are separated by the paranodal regions, where septate-like junctions are formed between the axon and the myelinating glial cells. Although oligodendrocytes and myelin sheaths are believed to play an instructive role in the local differentiation of the axon to distinct domains, the molecular mechanisms involved are poorly understood. In the present study, we have examined the distribution of axonal components in mice incapable of synthesizing sulfatide by disruption of the galactosylceramide sulfotransferase gene. These mice displayed abnormal paranodal junctions in the CNS and PNS, whereas their compact myelin was preserved. Immunohistochemical analysis demonstrated a decrease in Na+ and K+ channel clusters, altered nodal length, abnormal localization of K+channel clusters appearing primarily in the presumptive paranodal regions, and diffuse distribution of contactin-associated protein along the internode. Similar abnormalities have been reported previously in mice lacking both galactocerebroside and sulfatide. Interestingly, although no demyelination was observed, these channel clusters decreased markedly with age. The initial timing and the number of Na+ channel clusters formed were normal during development. These results indicate a critical role for sulfatide in proper localization and maintenance of ion channels clusters, whereas they do not appear to be essential for initial cluster formation of Na+ channels.

Gangliosides contribute to stability of paranodal junctions and ion channel clusters in myelinated nerve fibers

Paranodal axo‐glial junctions are important for ion channel clustering and rapid action potential propagation in myelinated nerve fibers. Paranode formation depends on the cell adhesion molecules neurofascin (NF) 155 in glia, and a Caspr and contactin heterodimer in axons. We found that antibody to ganglioside GM1 labels paranodal regions. Autoantibodies to the gangliosides GM1 and GD1a are thought to disrupt nodes of Ranvier in peripheral motor nerves and cause Guillain‐Barré syndrome, an autoimmune neuropathy characterized by acute limb weakness. To elucidate ganglioside function at and near nodes of Ranvier, we examined nodes in mice lacking gangliosides including GM1 and GD1a. In both peripheral and central nervous systems, some paranodal loops failed to attach to the axolemma, and immunostaining of Caspr and NF155 was attenuated. K+ channels at juxtaparanodes were mislocalized to paranodes, and nodal Na+ channel clusters were broadened. Abnormal immunostaining at paranodes became more prominent with age. Moreover, the defects were more prevalent in ventral than dorsal roots, and less frequent in mutant mice lacking the b‐series gangliosides but with excess GM1 and GD1a. Electrophysiological studies revealed nerve conduction slowing and reduced nodal Na+ current in mutant peripheral motor nerves. The amounts of Caspr and NF155 in low density, detergent insoluble membrane fractions were reduced in mutant brains. These results indicate that gangliosides are lipid raft components that contribute to stability and maintenance of neuron‐glia interactions at paranodes.

Cloning of a G‐protein‐coupled receptor that shows an activity to transform NIH3T3 cells and is expressed in gastric cancer cells

The present study was directed towards the identification of novel factors involved in the transformation process leading to the formation of gastric cancer. A cDNA library from human gastric cancer cells was constructed using a retroviral vector. Functional cloning was performed by screening for transformation activity in transduced NIH3T3 cells. Six cDNA clones were isolated, including one encoding the elongation factor 1asubunit, which was already known to play a role in tumorigenesis. One cDNA (clone 56.2), which was repeatedly isolated during the course of screening, encoded a protein identical to a G‐protein‐coupled receptor protein, GPR35. In addition, another cDNA clone (72.3) was found to be an alternatively spliced product of the GPR35 gene, whereby 31 amino acids were added to the N‐terminus of GPR35. Hence, the proteins encoded by clones 56.2 and 72.3 were designated GPR35a and GPR35b, respectively. RT‐PCR experiments revealed that GPR35 gene expression is low or absent in surrounding non‐cancerous regions, while both mRNAs were present in all of the gastric cancers examined. The level of 72.3‐encoded mRNA was consistently significantly higher than that of 56.2 encoded mRNA. An expression pattern similar to that observed in gastric cancers was detected in normal intestinal mucosa. Based on the apparent transformation activities of the two GPR35 clones in NIH3T3 cells, and the marked up‐regulation of their expression levels in cancer tissues, it is speculated that these two novel isoforms of GPR35 are involved in the course of gastric cancer formation.

Tetraspanin Protein CD9 Is a Novel Paranodal Component Regulating Paranodal Junctional Formation

The axoglial paranodal junction is essential for the proper localization of ion channels around the node of Ranvier. The integrity of this junction is important for nerve conduction. Although recent studies have made significant progress in understanding the molecular composition of the paranodal junction, it is not known how these membrane components are distributed to the appropriate sites and interact with each other. Here we show that CD9, a member of the tetraspanin family, is present at the paranode. CD9 is concentrated in the paranode as myelination proceeds, but CD9 clusters become diffuse, associated with disruption of the paranode, in cerebroside sulfotransferase-deficient mice. Immunohistochemical and Western blot analysis showed that CD9 is distributed predominantly in the PNS. Ablation of CD9 in mutant mice disrupts junctional attachment at the paranode and alters the paranodal components contactin-associated protein (also known as Paranodin) and neurofascin 155, although the frequency of such abnormalities varies among individuals and individual axons even in the same mouse. Electron micrographs demonstrated that compact myelin sheaths were also affected in the PNS. Therefore, CD9 is a myelin protein important for the formation of paranodal junctions. CD9 also plays a role in the formation of compact myelin in the PNS.

Mice with Altered Myelin Proteolipid Protein Gene Expression Display Cognitive Deficits Accompanied by Abnormal Neuron–Glia Interactions and Decreased Conduction Velocities

Conduction velocity (CV) of myelinated axons has been shown to be regulated by oligodendrocytes even after myelination has been completed. However, how myelinating oligodendrocytes regulate CV, and what the significance of this regulation is for normal brain function remain unknown. To address these questions, we analyzed a transgenic mouse line harboring extra copies of the myelin proteolipid protein 1 (plp1) gene (plp1 tg/− mice) at 2 months of age. At this stage, the plp1 tg/− mice have an unaffected myelin structure with a normally appearing ion channel distribution, but the CV in all axonal tracts tested in the CNS is greatly reduced. We also found decreased axonal diameters and slightly abnormal paranodal structures, both of which can be a cause for the reduced CV. Interestingly the plp1 tg/− mice showed altered anxiety-like behaviors, reduced prepulse inhibitions, spatial learning deficits and working memory deficit, all of which are schizophrenia-related behaviors. Our results implicate that abnormalities in the neuron-glia interactions at the paranodal junctions can result in reduced CV in the CNS, which then induces behavioral abnormalities related to schizophrenia.

Leukemia inhibitory factor regulates the timing of oligodendrocyte development and myelination in the postnatal optic nerve.

Leukemia inhibitory factor (LIF) promotes the survival of oligodendrocytes both in vitro and in an animal model of multiple sclerosis, but the possible role of LIF signaling in myelination during normal development has not been investigated. We find that LIF−/− mice have a pronounced myelination defect in optic nerve at postnatal day 10. Myelin basic protein (MBP)‐ and proteolipid protein (PLP)‐positive myelin was evident throughout the optic nerve in the wild‐type mice, but staining was present only at the chiasmal region in LIF−/− mice of the same age. Further experiments suggest that the myelination defect was a consequence of a delay in maturation of oligodendrocyte precursor cell (OPC) population. The number of Olig2‐positive cells was dramatically decreased in optic nerve of LIF−/− mice, and the distribution of Olig2‐positive cells was restricted to the chiasmal region of the nerve in a steep gradient toward the retina. Gene expression profiling and cell culture experiments revealed that OPCs from P10 optic nerve of LIF−/− mice remained in a highly proliferative immature stage compared with littermate controls. Interestingly, by postnatal day 14, MBP immunostaining in the LIF−/− optic nerve was comparable to that of LIF+/+ mice. These results suggest that, during normal development of mouse optic nerve, there is a defined developmental time window when LIF is required for correct myelination. Myelination seems to recover by postnatal day 14, so LIF is not necessary for the completion of myelination during postnatal development.

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.

Opalin, a Transmembrane Sialylglycoprotein Located in the Central Nervous System Myelin Paranodal Loop Membrane

In contrast to compact myelin, the series of paranodal loops located in the outermost lateral region of myelin is non-compact; the intracellular space is filled by a continuous channel of cytoplasm, the extracellular surfaces between neighboring loops keep a definite distance, but the loop membranes have junctional specializations. Although the proteins that form compact myelin have been well studied, the protein components of paranodal loop membranes are not fully understood. This report describes the biochemical characterization and expression of Opalin as a novel membrane protein in paranodal loops. Mouse Opalin is composed of a short N-terminal extracellular domain (amino acid residues 1–30), a transmembrane domain (residues 31–53), and a long C-terminal intracellular domain (residues 54–143). Opalin is enriched in myelin of the central nervous system, but not that of the peripheral nervous system of mice. Enzymatic deglycosylation showed that myelin Opalin contained N- and O-glycans, and that the O-glycans, at least, had negatively charged sialic acids. We identified two N-glycan sites at Asn-6 and Asn-12 and an O-glycan site at Thr-14 in the extracellular domain. Site-directed mutations at the glycan sites impaired the cell surface localization of Opalin. In addition to the somata and processes of oligodendrocytes, Opalin immunoreactivity was observed in myelinated axons in a spiral fashion, and was concentrated in the paranodal loop region. Immunogold electron microscopy demonstrated that Opalin was localized at particular sites in the paranodal loop membrane. These results suggest a role for highly sialylglycosylated Opalin in an intermembranous function of the myelin paranodal loops in the central nervous system.

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.