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.

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.

Solo/Trio8, a membrane-associated short isoform of Trio, modulates endosome dynamics and neurite elongation.

With DNA microarrays, we identified a gene, termed Solo, that is downregulated in the cerebellum of Purkinje cell degeneration mutant mice. Solo is a mouse homologue of rat Trio8-one of multiple Trio isoforms recently identified in rat brain. Solo/Trio8 contains N-terminal sec14-like and spectrin-like repeat domains followed by a single guanine nucleotide exchange factor 1 (GEF1) domain, but it lacks the C-terminal GEF2, immunoglobulin-like, and kinase domains that are typical of Trio. Solo/Trio8 is predominantly expressed in Purkinje neurons of the mouse brain, and expression begins following birth and increases during Purkinje neuron maturation. We identified a novel C-terminal membrane-anchoring domain in Solo/Trio8 that is required for enhanced green fluorescent protein-Solo/Trio8 localization to early endosomes (positive for both early-endosome antigen 1 [EEA1] and Rab5) in COS-7 cells and primary cultured neurons. Solo/Trio8 overexpression in COS-7 cells augmented the EEA1-positive early-endosome pool, and this effect was abolished via mutation and inactivation of the GEF domain or deletion of the C-terminal membrane-anchoring domain. Moreover, primary cultured neurons transfected with Solo/Trio8 showed increased neurite elongation that was dependent on these domains. These results suggest that Solo/Trio8 acts as an early-endosome-specific upstream activator of Rho family GTPases for neurite elongation of developing Purkinje neurons.

Identification and functional characterization of mouse TPO1 as a myelin membrane protein.

TPO1 is a member of the AIGP family, a unique group of proteins that contains 11 putative transmembrane domains. Expression of the rat TPO1 gene is upregulated in cultured oligodendrocytes (OLs) during development from pro-oligodendroblasts to postmitotic OLs. However, the distribution of native TPO1 protein in cultured OLs and in the brain has not been elucidated. We investigated the distribution and cellular function of TPO1 in myelinating cells of the nervous system. In mice, TPO1 gene expression was detected in the central (CNS) and peripheral (PNS) nervous systems and was markedly upregulated at postnatal days 10-20, an early phase of myelination in the mouse brain. To investigate TPO1 localization, we generated affinity-purified antibodies to synthetic peptides derived from mouse TPO1. Immunohistochemical analysis showed that TPO1 was expressed in OLs and Schwann cells but not in neurons and astrocytes. Schwann cells from trembler mice, which lack PNS myelin, had significantly decreased TPO1 expression and an altered localization pattern, suggesting that TPO1 is a functional myelin membrane protein. In OL lineage cell cultures, TPO1 was detected in A2B5+ bipolar early progenitors, A2B5+ multipolar Pro-OLs, GalC+ immature OLs and MBP+ mature OLs. The subcellular localization of TPO1 in OL lineage cells was mapped to the GM130+ Golgi in cell bodies and Fyn+ cell processes and myelin-like sheets. Furthermore, TPO1 selectively colocalized with non-phosphorylated Fyn and promoted Fyn autophosphorylation in COS7 cells, suggesting that TPO1 may play a role in myelin formation via Fyn kinase activation in the PNS and CNS.

The binding of chondroitin sulfate to pleiotrophin/heparin-binding growth-associated molecule is regulated by chain length and oversulfated structures.

Pleiotrophin is an 18-kDa heparin-binding growth factor, which uses chondroitin sulfate (CS) proteoglycan, PTPzeta as a receptor. It has been suggested that the D-type structure (GlcA(2S)beta1-3GalNAc(6S)) in CS contributes to the high affinity binding between PTPzeta and pleiotrophin. Here, we analyzed the interaction of shark cartilage CS-D with pleiotrophin using a surface plasmon resonance biosensor to reveal the importance of D-type structure. CS-D was partially digested with chondroitinase ABC, and fractionated using a Superdex 75pg column. The > or =18-mer CS fractions showed significant binding to pleiotrophin, and the longer fractions had stronger affinity for pleiotrophin than the shorter ones. The approximately 46-mer CS fraction bound to densely immobilized pleiotrophin with high affinity (K(D) = approximately 30 nM), and the binding reactions fitted the bivalent analyte model. However, when the density of the immobilized pleiotrophin was lowered, the strength of affinity remarkably decreased (K(D) = approximately 2.5 microM), and the reactions no longer fitted the model and were considered to be monovalent binding. The 20 approximately 24-mer fractions showed low affinity binding to densely immobilized pleiotrophin (K(D) = 3 approximately 20 microM), which seemed to be monovalent. When approximately 22-mer CS oligosaccharides were fractionated by strong anion exchange HPLC, each fraction differed in affinity for pleiotrophin (K(D) = 0.36 approximately >10 microM), and the affinity correlated with the amounts of D- and E- (GlcAbeta1-3GalNAc(4S,6S)) type oversulfated structures. These results suggest that the binding of pleiotrophin to CS is regulated by multivalency with CS approximately 20 mer as a unit and by the amounts of oversulfated structures.