Phosphacan acts as a repulsive cue in murine and rat cerebellar granule cells in a TAG-1/GD3 rafts-dependent manner.

Phosphacan, a chondroitin sulfate proteoglycan, is a repulsive cue of cerebellar granule cells. This study aims to explore the molecular mechanism. The glycosylphosphatidylinositol-anchored neural adhesion molecule TAG-1 is a binding partner of phosphacan, suggesting that the repulsive effect of phosphacan is possibly because of its interaction with TAG-1. The repulsive effect was greatly reduced on primary cerebellar granule cells of TAG-1-deficient mice. Surface plasmon resonance analysis confirmed the direct interaction of TAG-1 with chondroitin sulfate C. On postnatal days 1, 4, 7, 11, 15, and 20 and in adulthood, phosphacan was present in the molecular layer and internal granular layer, but not in the external granular layer. In contrast, transient TAG-1 expression was observed exclusively within the premigratory zone of the external granular layer on postnatal days 1, 4, 7, and 11. Boyden chamber cell migration assay demonstrated that phosphacan exerted its repulsive effect on the spontaneous and brain-derived neurotrophic factor (BDNF)-induced migration of cerebellar granule cells. The BDNF-induced migration was inhibited by MK-2206, an Akt inhibitor. The pre-treatment with a raft-disrupting agent, methyl-ß-cyclodextrin, also inhibited the BDNF-induced migration, suggesting that lipid rafts are involved in the migration of cerebellar granule cells. In primary cerebellar granule cells obtained on postnatal day 7 and cultured for 7 days, the ganglioside GD3 and TAG-1 preferentially localized in the cell body, whereas the ganglioside GD1b and NB-3 localized in not only the cell body but also neurites. Pre-treatment with the anti-GD3 antibody R24, but not the anti-GD1b antibody GGR12, inhibited the spontaneous and BDNF-induced migration, and attenuated BDNF-induced Akt activation. These findings suggest that phosphacan is responsible for the repulsion of TAG-1-expressing cerebellar granule cells via GD3 rafts to attenuate BDNF-induced migration signaling.

Histochemical Analysis of Heparan Sulfate 3-O-sulfotransferase Expression in Mouse Brain.

In situ hybridization provides information for understanding the localization of gene expression in various tissues. The relative expression levels of mRNAs in a single cell can be sensitively visualized by this technique. Furthermore, since in situ hybridization is a histological technique, tissue structure is maintained after fixation, and it is possible to accurately identify the cell types. We have examined the expression of heparan sulfate sulfotransferases by in situ hybridization to better understand the functions of heparan sulfate in the development of mouse nervous system. This chapter describes methods of in situ hybridization analyses using cRNA probes labeled with non-radioactive nucleotides.

Glypicans and Heparan Sulfate in Synaptic Development, Neural Plasticity, and Neurological Disorders.

Heparan sulfate proteoglycans (HSPGs) are components of the cell surface and extracellular matrix, which bear long polysaccharides called heparan sulfate (HS) attached to the core proteins. HSPGs interact with a variety of ligand proteins through the HS chains, and mutations in HSPG-related genes influence many biological processes and cause various diseases. In particular, recent findings from vertebrate and invertebrate studies have raised the importance of glycosylphosphatidylinositol-anchored HSPGs, glypicans, as central players in the development and functions of synapses. Glypicans are important components of the synapse-organizing protein complexes and serve as ligands for leucine-rich repeat transmembrane neuronal proteins (LRRTMs), leukocyte common antigen-related (LAR) family receptor protein tyrosine phosphatases (RPTPs), and G-protein-coupled receptor 158 (GPR158), regulating synapse formation. Many of these interactions are mediated by the HS chains of glypicans. Neurexins (Nrxs) are also synthesized as HSPGs and bind to some ligands in common with glypicans through HS chains. Therefore, glypicans and Nrxs may act competitively at the synapses. Furthermore, glypicans regulate the postsynaptic expression levels of ionotropic glutamate receptors, controlling the electrophysiological properties and non-canonical BMP signaling of synapses. Dysfunctions of glypicans lead to failures in neuronal network formation, malfunction of synapses, and abnormal behaviors that are characteristic of neurodevelopmental disorders. Recent human genetics revealed that glypicans and HS are associated with autism spectrum disorder, neuroticism, and schizophrenia. In this review, we introduce the studies showing the roles of glypicans and HS in synapse formation, neural plasticity, and neurological disorders, especially focusing on the mouse and Drosophila as potential models for human diseases.

The HSPG Glypican Regulates Experience-Dependent Synaptic and Behavioral Plasticity by Modulating the Non-Canonical BMP Pathway.

Under food deprivation conditions, Drosophila larvae exhibit increases in locomotor speed and synaptic bouton numbers at neuromuscular junctions (NMJs). Octopamine, the invertebrate counterpart of noradrenaline, plays critical roles in this process; however, the underlying mechanisms remain unclear. We show here that a glypican (Dlp) negatively regulates type I synaptic bouton formation, postsynaptic expression of GluRIIA, and larval locomotor speed. Starvation-induced octopaminergic signaling decreases Dlp expression, leading to increases in synapse formation and locomotion. Dlp is expressed by postsynaptic muscle cells and suppresses the non-canonical BMP pathway, which is composed of the presynaptic BMP receptor Wit and postsynaptic GluRIIA-containing ionotropic glutamate receptor. We find that during starvation, decreases in Dlp increase non-canonical BMP signaling, leading to increases in GluRIIA expression, type I bouton number, and locomotor speed. Our results demonstrate that octopamine controls starvation-induced neural plasticity by regulating Dlp and provides insights into how proteoglycans can influence behavioral and synaptic plasticity.

Synaptic transmission from subplate neurons controls radial migration of neocortical neurons.

The neocortex exhibits a six-layered structure that is formed by radial migration of excitatory neurons, for which the multipolar-to-bipolar transition of immature migrating multipolar neurons is required. Here, we report that subplate neurons, one of the first neuron types born in the neocortex, manage the multipolar-to-bipolar transition of migrating neurons. By histochemical, imaging, and microarray analyses on the mouse embryonic cortex, we found that subplate neurons extend neurites toward the ventricular side of the subplate and form transient glutamatergic synapses on the multipolar neurons just below the subplate. NMDAR (N-methyl-d-aspartate receptor)-mediated synaptic transmission from subplate neurons to multipolar neurons induces the multipolar-to-bipolar transition, leading to a change in migration mode from slow multipolar migration to faster radial glial-guided locomotion. Our data suggested that transient synapses formed on early immature neurons regulate radial migration.

Heparan sulfate proteoglycans in Drosophila neuromuscular development.

Heparan sulfate proteoglycans (HSPGs) are glycoconjugates bearing heparan sulfate (HS) chains covalently attached to core proteins, which are ubiquitously distributed on the cell surface and in the extracellular matrix. HSPGs interact with a number of molecules mainly through HS chains, which play critical roles in diverse physiological and disease processes. Among these, recent vertebrate studies showed that HSPGs are closely involved in synapse development and function. However, the detailed molecular mechanisms remain elusive. Genetic studies from fruit flies, Drosophila melanogaster, have begun to reveal the molecular mechanisms by which HSPGs regulate synapse formation at neuromuscular junctions (NMJs). In this review, we introduce Drosophila studies showing how HSPGs regulate various signaling pathways in developing NMJs. This article is part of a Special Issue entitled Neuro-glycoscience, edited by Kenji Kadomatsu and Hiroshi Kitagawa.

The Strip-Hippo Pathway Regulates Synaptic Terminal Formation by Modulating Actin Organization at the Drosophila Neuromuscular Synapses.

Synapse formation requires the precise coordination of axon elongation, cytoskeletal stability, and diverse modes of cell signaling. The underlying mechanisms of this interplay, however, remain unclear. Here, we demonstrate that Strip, a component of the striatin-interacting phosphatase and kinase (STRIPAK) complex that regulates these processes, is required to ensure the proper development of synaptic boutons at the Drosophila neuromuscular junction. In doing so, Strip negatively regulates the activity of the Hippo (Hpo) pathway, an evolutionarily conserved regulator of organ size whose role in synapse formation is currently unappreciated. Strip functions genetically with Enabled, an actin assembly/elongation factor and the presumptive downstream target of Hpo signaling, to modulate local actin organization at synaptic termini. This regulation occurs independently of the transcriptional co-activator Yorkie, the canonical downstream target of the Hpo pathway. Our study identifies a previously unanticipated role of the Strip-Hippo pathway in synaptic development, linking cell signaling to actin organization.

Proteoglycans and neuronal migration in the cerebral cortex during development and disease.

Chondroitin sulfate proteoglycans and heparan sulfate proteoglycans are major constituents of the extracellular matrix and the cell surface in the brain. Proteoglycans bind with many proteins including growth factors, chemokines, axon guidance molecules, and cell adhesion molecules through both the glycosaminoglycan and the core protein portions. The functions of proteoglycans are flexibly regulated due to the structural variability of glycosaminoglycans, which are generated by multiple glycosaminoglycan synthesis and modifying enzymes. Neuronal cell surface proteoglycans such as PTPζ, neuroglycan C and syndecan-3 function as direct receptors for heparin-binding growth factors that induce neuronal migration. The lectican family, secreted chondroitin sulfate proteoglycans, forms large aggregates with hyaluronic acid and tenascins, in which many signaling molecules and enzymes including matrix proteases are preserved. In the developing cerebrum, secreted chondroitin sulfate proteoglycans such as neurocan, versican and phosphacan are richly expressed in the areas that are strategically important for neuronal migration such as the striatum, marginal zone, subplate and subventricular zone in the neocortex. These proteoglycans may anchor various attractive and/or repulsive cues, regulating the migration routes of inhibitory neurons. Recent studies demonstrated that the genes encoding proteoglycan core proteins and glycosaminoglycan synthesis and modifying enzymes are associated with various psychiatric and intellectual disorders, which may be related to the defects of neuronal migration.

Histochemical analysis of heparan sulfate 3-O-sulfotransferase expression in mouse brain.

In situ hybridization provides information for understanding the localization of gene expression in various tissues. The relative expression levels of mRNAs in a single cell can be sensitively visualized by this technique. Furthermore, since in situ hybridization is a histological technique, tissue structure is maintained after fixation, and it is possible to accurately identify cell types. We have examined the expression of heparan sulfate sulfotransferases by in situ hybridization to better understand the functions of heparan sulfate in the development of mouse nervous system. This chapter describes methods of in situ hybridization analyses using cRNA probes labeled with nonradioactive nucleotides.

Structural and biochemical characterization of O-mannose-linked human natural killer-1 glycan expressed on phosphacan in developing mouse brains.

The human natural killer-1 (HNK-1) carbohydrate comprising a sulfated trisaccharide (HSO3-3GlcAß1-3Galß1-4GlcNAc-) is expressed on N-linked and O-mannose-linked glycans in the nervous system and involved in learning and memory functions. Although whole/core glycan structures and carrier glycoproteins for the N-linked HNK-1 epitope have been studied, carrier glycoproteins and the biosynthetic pathway of the O-mannose-linked HNK-1 epitope have not been fully characterized. Here, using mass spectrometric analyses, we identified the major carrier glycoprotein of the O-linked HNK-1 as phosphacan in developing mouse brains and determined the major O-glycan structures having the terminal HNK-1 epitope from partially purified phosphacan. The O-linked HNK-1 epitope on phosphacan almost disappeared due to the knockout of protein O-mannose ß1,2-N-acetylglucosaminyltransferase 1, an N-acetylglucosaminyltransferase essential for O-mannose-linked glycan synthesis, indicating that the reducing terminal of the O-linked HNK-1 is mannose. We also showed that glucuronyltransferase-P (GlcAT-P) was involved in the biosynthesis of O-mannose-linked HNK-1 using the gene-deficient mice of GlcAT-P, one of the glucuronyltransferases for HNK-1 synthesis. Consistent with this result, we revealed that GlcAT-P specifically synthesized O-linked HNK-1 onto phosphacan using cultured cells. Furthermore, we characterized the as-yet-unknown epitope of the 6B4 monoclonal antibody (mAb), which was thought to recognize a unique phosphacan glycoform. The reactivity of the 6B4 mAb almost completely disappeared in GlcAT-P-deficient mice, and exogenously expressed phosphacan was selectively recognized by the 6B4 mAb when co-expressed with GlcAT-P, suggesting that the 6B4 mAb preferentially recognizes O-mannose-linked HNK-1 on phosphacan. This is the first study to show that 6B4 mAb-reactive O-mannose-linked HNK-1 in the brain is mainly carried by phosphacan.

Perlecan regulates bidirectional Wnt signaling at the Drosophila neuromuscular junction.

Heparan sulfate proteoglycans (HSPGs) play pivotal roles in the regulation of Wnt signaling activity in several tissues. At the Drosophila melanogaster neuromuscular junction (NMJ), Wnt/Wingless (Wg) regulates the formation of both pre- and postsynaptic structures; however, the mechanism balancing such bidirectional signaling remains elusive. In this paper, we demonstrate that mutations in the gene of a secreted HSPG, perlecan/trol, resulted in diverse postsynaptic defects and overproduction of synaptic boutons at NMJ. The postsynaptic defects, such as reduction in subsynaptic reticulum (SSR), were rescued by the postsynaptic activation of the Frizzled nuclear import Wg pathway. In contrast, overproduction of synaptic boutons was suppressed by the presynaptic down-regulation of the canonical Wg pathway. We also show that Trol was localized in the SSR and promoted postsynaptic accumulation of extracellular Wg proteins. These results suggest that Trol bidirectionally regulates both pre- and postsynaptic activities of Wg by precisely distributing Wg at the NMJ.

In vivo manipulation of heparan sulfate structure and its effect on Drosophila development.

Heparan sulfate proteoglycans (HSPGs) participate in a wide range of biological processes through interactions with a number of ligand proteins. The nature of these interactions largely depends on the heparan sulfate (HS) moiety of HSPGs, which undergoes a series of modifications by various HS-modifying enzymes (HSMEs). Although the effects of alterations in a single HSME on physiological processes have started to be studied, it remains elusive how a combination of these molecules control the structure and function of HS. Here we systematically manipulated the HS structures and analyzed their effect on morphogenesis and signaling, using the genetically tractable model organism, Drosophila. We generated transgenic fly strains overexpressing HSMEs alone or in combination. Unsaturated disaccharide analyses of HS showed that expression of various HSMEs generates distinct HS structures, and the enzymatic activities of HSMEs are influenced by coexpression of other HSMEs. Furthermore, these transgenic HSME animals showed a different extent of lethality, and a subset of HSMEs caused specific morphological defects due to defective activities of Wnt and bone morphogenetic protein signaling. There is no obvious relationship between HS unsaturated disaccharide composition and developmental defects in HSME animals, suggesting that other structural factors, such as domain organization or sulfation sequence, might regulate the function of HS.

Structural variation of chondroitin sulfate and its roles in the central nervous system.

Chondroitin sulfate is popular in the field of neuroscience, because the treatment of nervous tissues with chondroitinase ABC, which degrades chondroitin sulfate up to unsaturated disaccharides, causes severe changes in various aspects of neural development and functions. Chondroitinase ABC treatments of developing nervous tissue impair the growth and differentiation of neural progenitor cells, and cause various pathfinding errors of axons. After injury to the adult central nervous system, axon regeneration fails at scar regions expressing large amounts of chondroitin sulfate proteoglycans. However, after chondroitinase ABC treatment, many axons regenerate and traverse the damaged areas. Furthermore, it was revealed that chondroitin sulfate proteoglycans are involved in neural plasticity. These observations indicated that chondroitin sulfate proteoglycans as major components of the extracellular matrix and cell surface play pivotal roles in the development, regeneration, and plasticity of neuronal networks. Chondroitin sulfate shows highly diverse structural variation, and recent studies indicated that this glycosaminoglycan binds with various growth factors, chemokines and axon guidance molecules in a structure-dependent manner and regulates their activities. Notably, oversulfated structures such as D (GlcA(2-O-sulfate)beta 1-3GalNAc(6-O-sulfate)) and E (GlcAbeta1-3GalNAc(4,6-O-disulfate)) units constitute the binding sites for many proteins, and play important roles in regulation of the growth of neural progenitors, neurite extension, and neuronal migration. The synthesis of these structures is strictly regulated by the chondroitin sulfate synthase family and many sulfotransferases, which should be useful therapeutic targets in neurological disorders.

Chondroitin sulfate proteoglycans in neural development and plasticity.

PTPzeta and lectican family members are major chondroitin sulfate proteoglycans (CS-PGs) in the brain, which bind with many proteins via core protein and CS portions. Recent studies revealed that the oversulfated structures in CS constitute high affinity binding sites for various growth factors and axon guidance molecules, and play important roles in the proliferation of neural progenitor cells, neurite extension and neuronal migration. PTPzeta uses pleiotrophin as a ligand. The CS portion of PTPzeta constitutes a part of the pleiotrophin-binding site, and oversulfated D unit increases the binding affinity. Pleiotrophin-PTPzeta signaling regulates the morphogenesis of Purkinje cell by controlling the tyrosine phosphorylation of a Notch-related transmembrane protein, DNER. In the brain of adult animals, a subset of neurons are surrounded by CS-PG-rich extracellular matrix called perineuronal net, in which lecticans form complexes with hyaluronic acid and tenascin-R. CS-PGs in the perineuronal net regulate ocular dominance plasticity in the visual cortex by enhancing the uptake of Otx2 homeoprotein by parvalbumin-positive interneurons in a CS-dependent manner. These studies revealed unexpectedly complex mechanisms of CS-PG functions.

Oversulfated chondroitin sulfate plays critical roles in the neuronal migration in the cerebral cortex.

Chondroitin sulfate (CS) proteoglycans bind with various proteins through CS chains in a CS structure-dependent manner, in which oversulfated structures, such as iB (IdoA(2-O-sulfate)alpha1-3GalNAc(4-O-sulfate)), D (GlcA(2-O-sulfate)beta1-3GalNAc(6-O-sulfate)), and E (GlcAbeta1-3GalNAc(4,6-O-disulfate)) units constitute the critical functional module. In this study, we examined the expression and function of three CS sulfotransferases in the developing neocortex: uronyl 2-O-sulfotransferase (UST), N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (4,6-ST) and dermatan 4-O-sulfotransferase-1 (D4-ST), which are responsible for the synthesis of oversulfated structures. The CS chains of the neocortex of mouse embryos contained significant amounts of D and E units that are generated by UST and 4,6-ST, respectively. UST and 4,6-ST mRNAs were expressed in the ventricular and subventricular zones, and their expression increased during late embryonic development. In utero electroporation experiments indicated that knockdown of UST and 4,6-ST resulted in the disturbed migration of cortical neurons. The neurons electroporated with the short hairpin RNA constructs of UST and 4,6-ST accumulated in the lower intermediate zone and in the subventricular zone, showing a multipolar morphology. The cDNA constructs of UST and 4,6-ST rescued the defects caused by the RNA interference, and the neurons were able to migrate radially. On the other hand, knockdown of D4-ST, which is involved in the biosynthesis of the iB unit, caused no migratory defects. These results revealed that specific oversulfated structures in CS chains play critical roles in the migration of neuronal precursors during cortical development.

Spatiotemporal expression of chondroitin sulfate sulfotransferases in the postnatal developing mouse cerebellum.

Chondroitin sulfate (CS) proteoglycans are major components of the cell surface and the extracellular matrix in the developing brain and bind to various proteins via CS chains in a CS structure-dependent manner. This study demonstrated the expression pattern of three CS sulfotransferase genes, dermatan 4-O-sulfotransferase (D4ST), uronyl 2-O-sulfotransferase (UST), and N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST), in the mouse postnatal cerebellum. These sulfotransferases are responsible for the biosynthesis of oversulfated structures in CS chains such as B, D, and E units, which constitute the binding sites for various heparin-binding proteins. Real-time reverse transcription-polymerase chain reaction analysis indicated that the expression of UST increased remarkably during cerebellar development. The amounts of B and D units, which are generated by UST activity, in the cerebellar CS chains also increased during development. In contrast, the expression of GalNAc4S-6ST and its biosynthetic product, E unit, decreased during postnatal development. In situ hybridization experiments revealed the levels of UST and GalNAc4S-6ST mRNAs to correlate inversely in many cells including Purkinje cells, granule cells in the external granular layer, and inhibitory interneurons. In these neurons, the expression of UST increased and that of GalNAc4S-6ST decreased during development and/or maturation. D4ST was also expressed by many neurons, but its expression was not simply correlated with development, which might contribute to the diversification of CS structures expressed by distinct neurons. These results suggest that the CS structures of various cerebellar neurons change during development and such changes of CS are involved in the regulation of various signaling pathways.

Receptor type protein tyrosine phosphatase zeta-pleiotrophin signaling controls endocytic trafficking of DNER that regulates neuritogenesis.

Protein tyrosine phosphatase zeta (PTPzeta) is a receptor type protein tyrosine phosphatase that uses pleiotrophin as a ligand. Pleiotrophin inactivates the phosphatase activity of PTPzeta, resulting in the increase of tyrosine phosphorylation levels of its substrates. We studied the functional interaction between PTPzeta and DNER, a Notch-related transmembrane protein highly expressed in cerebellar Purkinje cells. PTPzeta and DNER displayed patchy colocalization in the dendrites of Purkinje cells, and immunoprecipitation experiments indicated that these proteins formed complexes. Several tyrosine residues in and adjacent to the tyrosine-based and the second C-terminal sorting motifs of DNER were phosphorylated and were dephosphorylated by PTPzeta, and phosphorylation of these tyrosine residues resulted in the accumulation of DNER on the plasma membrane. DNER mutants lacking sorting motifs accumulated on the plasma membrane of Purkinje cells and Neuro-2A cells and induced their process extension. While normal DNER was actively endocytosed and inhibited the retinoic-acid-induced neurite outgrowth of Neuro-2A cells, pleiotrophin stimulation increased the tyrosine phosphorylation level of DNER and suppressed the endocytosis of this protein, which led to the reversal of this inhibition, thus allowing neurite extension. These observations suggest that pleiotrophin-PTPzeta signaling controls subcellular localization of DNER and thereby regulates neuritogenesis.

Reduced expression of MAb6B4 epitopes on chondroitin sulfate proteoglycan aggrecan in perineuronal nets from cerebral cortices of SAMP10 mice: a model for age-dependent neurodegeneration.

The accelerated senescence-prone SAMP10 mouse strain is a model for age-dependent neurodegeneration and is characterized by brain atrophy and deficits in learning and memory. Because perineuronal nets play an important role in the synaptic plasticity of adult brains, we examined the distributions of molecules that constitute perineuronal nets in SAMP10 mouse brain samples and compared them with those in control SAMR1 mouse samples. Proteoglycan-related monoclonal antibody 6B4 (MAb6B4) clearly immunostained perineuronal nets in SAMR1 mice cortices, but the corresponding immunostaining in SAMP10 mice was very faint. MAb6B4 recognizes phosphacan/PTPzeta in immature brains. However, this antibody recognized several protein bands, including a 400-kDa core glycoprotein from chondroitin sulfate proteoglycan in homogenates of mature cortices from SAMR1 mice. The 400-kDa band was also recognized by antiaggrecan antibodies. The aggrecan core glycoprotein band was also detectable in samples from SAMP10 mice, but this glycoprotein was faintly immunostained by MAb6B4. Because MAb6B4 recognized the same set of protein bands that the monoclonal antibody Cat-315 recognized in mature cerebral cortices of SAMR1 mice, the MAb6B4 epitope appears to be closely related to that of Cat-315 and presumably represents a novel type of oligosaccharide that attaches to aggrecans. The Cat-315 epitope colocalized with aggrecan in perineuronal nets from SAMR1 mouse brain samples, whereas its expression was prominently reduced in SAMP10 mouse brain samples. The biological significance of the MAb6B4/Cat-315 epitope in brain function and its relationship to the neurodegeneration and learning disabilities observed in SAMP10 mice remain to be elucidated.