Protein tyrosine phosphatase receptor type Z dephosphorylates TrkA receptors and attenuates NGF-dependent neurite outgrowth of PC12 cells

Protein tyrosine phosphatase receptor type Z (Ptprz/Ptpzeta / RPTPbeta) is a receptor-like protein tyrosine phosphatase (RPTP) which is predominantly expressed in the central nervous system. Tropomyosin-related kinases (Trks) are single-pass transmembrane molecules that are highly expressed in the developing nervous system. Upon the ligand binding of neurotrophins, Trk receptors are activated through autophosphorylation of tyrosine residues; however, the PTPs responsible for the negative regulation of Trk receptors have not been fully elucidated. Here, we identified Ptprz as a specific PTP that efficiently dephosphorylates TrkA as a substrate. Co-expression of Ptprz with Trk receptors in 293T cells showed that Ptprz suppresses the ligand-independent tyrosine phosphorylation of TrkA, but not of TrkB or TrkC, and that Ptprz attenuates TrkA activation induced by nerve growth factor (NGF). Co-expression analyses with TrkA mutants revealed that Ptprz dephosphorylates phosphotyrosine residues in the activation loop of the kinase domain, which are requisite for activation of the TrkA receptor. Consistent with these findings, forced expression of Ptprz in PC12D cells markedly inhibited neurite extension induced by a low dose of NGF. In addition, an increment in the tyrosine phosphorylation of TrkA was observed in the brain of Ptprz-deficient mice. Ptprz thus appears to be one of the PTPs which regulate the activation and signalling of TrkA receptors.     

NCAM-dependent neurite outgrowth is inhibited in neurons from Fyn-minus mice

Src-related nonreceptor protein tyrosine kinases in nerve growth cones (p59fyn, pp60c-src, and pp62c-yes) are potential intracellular signaling molecules for cell adhesion molecule-directed axonal growth. To determine whether src-related tyrosine kinases mediate NCAM- dependent neurite outgrowth, cultures of cerebellar and sensory neurons from fyn-, src-, and yes- minus mice were analyzed for neurite outgrowth on monolayers of NCAM140-transfected L fibroblasts. NCAM- dependent neurite outgrowth was selectively inhibited in cultures of cerebellar and dorsal root ganglion neurons from fyn-, but not src- or yes- mice. Neurite outgrowth by fyn-, src-, or yes- neurons on untransfected fibroblast monolayers was unaffected, indicating that these kinases do not contribute significantly to axon growth on at least some integrins or other adhesive substrates present on fibroblasts. This study demonstrates that p59fyn is an essential component of the NCAM signaling pathway leading to axonal growth.     

Neural cells in the esophagus respond to glial cell line-derived neurotrophic factor and neurturin, and are RET-dependent

Glial cell line-derived neurotrophic factor (GDNF) is expressed in the gastrointestinal tract of the developing mouse and appears to play an important role in the migration of enteric neuron precursors into and along the small and large intestines. Two other GDNF family members, neurturin and artemin, are also expressed in the developing gut although artemin is only expressed in the esophagus. We examined the effects of GDNF, neurturin, and artemin on neural crest cell migration and neurite outgrowth in explants of mouse esophagus, midgut, and hindgut. Both GDNF and neurturin induced neural crest cell migration and neurite outgrowth in all regions examined. In the esophagus, the effect of GDNF on migration and neurite outgrowth declined with age between E11.5 and E14.5, but neurturin still had a strong neurite outgrowth effect at E14.5. Artemin did not promote neural migration or neurite outgrowth in any region investigated. The effects of GDNF family ligands are mediated by the Ret tyrosine kinase. We examined the density of neurons in the esophagus of Ret-/- mice, which lack neurons in the small and large intestines. The density of esophageal neurons in Ret-/- mice was only about 4% of the density of esophageal neurons in Ret+/- and Ret+/+ mice. These results show that GDNF and neurturin promote migration and neurite outgrowth of crest-derived cells in the esophagus as well as the intestine. Moreover, like intestinal neurons, the development of esophageal neurons is largely Ret-dependent.     

p190 RhoGAP is the principal Src substrate in brain and regulates axon outgrowth, guidance and fasciculation

The Src tyrosine kinases have been implicated in several aspects of neural development and nervous system function; however, their relevant substrates in brain and their mechanism of action in neurons remain to be established clearly. Here we identify the potent Rho regulatory protein, p190 RhoGAP (GTPase-activating protein), as the principal Src substrate detected in the developing and mature nervous system. We also find that mice lacking functional p190 RhoGAP exhibit defects in axon guidance and fasciculation. p190 RhoGAP is co-enriched with F-actin in the distal tips of axons, and overexpressing p190 RhoGAP in neuroblastoma cells promotes extensive neurite outgrowth, indicating that p190 RhoGAP may be an important regulator of Rho-mediated actin reorganization in neuronal growth cones. p190 RhoGAP transduces signals downstream of cell-surface adhesion molecules, and we find that p190-RhoGAP-mediated neurite outgrowth is promoted by the extracellular matrix protein laminin. Together with the fact that mice lacking neural adhesion molecules or Src kinases also exhibit defects in axon outgrowth, guidance and fasciculation, our results suggest that p190 RhoGAP mediates a Src-dependent adhesion signal for neuritogenesis to the actin cytoskeleton through the Rho GTPase.     

Identification of neurite outgrowth-promoting domains of neuroglycan C, a brain-specific chondroitin sulfate proteoglycan, and involvement of phosphatidylinositol 3-kinase and protein kinase C signaling pathways in neuritogenesis

Neuroglycan C (NGC) is a transmembrane-type chondroitin sulfate proteoglycan that is exclusively expressed in the central nervous system. We report that the recombinant ectodomain of NGC core protein enhances neurite outgrowth from rat neocortical neurons in culture. Both protein kinase C (PKC) inhibitors and phosphatidylinositol 3-kinase (PI3K) inhibitors attenuated the NGC-mediated neurite outgrowth in a dose-dependent manner, suggesting that NGC promotes neurite outgrowth via PI3K and PKC pathways. The active sites of NGC for neurite outgrowth existed in the epidermal growth factor (EGF)-like domain and acidic amino acid (AA)-domain of the NGC ectodomain. The EGF-domain caused cells to extend preferentially one neurite from a soma, whereas the AA-domain caused several neurites to develop. The EGF-domain also enhanced neurite outgrowth from GABA-positive neurons, but the AA-domain did not. These results suggest that the EGF-domain and AA-domain have distinct functions in terms of neuritogenesis. From these findings, NGC can be considered to be involved in neuritogenesis in the developing central nervous system.     

CNS neurotrophins are biologically active and expressed by multiple cell types

Neutrotrophins are increasingly appreciated as potential modulators of neuronal function in the adult central nervous system (CNS). To describe the neurotrophin environment within the adult CNS, mRNA and protein expression patterns of neurotrophins-3 and –4 and of brain-derived neurotrophin were investigated in adult rat spinal cord and brain. Co-localization studies with CNS cell type-specific markers demonstrates that multiple cell types, including both neurons and glia, express these neurotrophins in the normal adult CNS. Although widely implicated in important CNS functions such as synaptic plasticity, biological activity of endogenous CNS neurotrophins has not been directly demonstrated. With a sensitive neurite outgrowth bioassay we demonstrate that CNS neurotrophins elicit neurite outgrowth and are biologically active. Moreover, antibody-blocking studies suggest that these three neurotrophins may comprise the bulk of adult CNS neurotrophic activity.     

Th1 cells promote neurite outgrowth from cortical neurons via a mechanism dependent on semaphorins

The roles of T lymphocytes in the central nervous system (CNS) are diverse; their roles in the injured CNS have been reported to be both detrimental and advantageous. Hence, an investigation of the effects of specific subsets of T cells on neurons may provide an insight into the interaction between the nervous system and the immune system. In the present study, we demonstrate that a specific subset of T lymphocytes enhanced neurite outgrowth in vitro. When cultured T helper type 1 (Th1) cells were co-cultured with cortical neurons, neurite outgrowth from neurons was enhanced; however, the same was not observed when Th2 or naïve T cells were used. We observed that the promotion of neurite outgrowth by Th1 cells was completely inhibited by anti-interferon γ (IFN-γ) neutralizing antibody, but that IFN-γ did not directly promote neurite growth. Furthermore, experiments using knockout mice revealed that semaphorin 4A (Sema4A) but not Sema7A was required for the effect produced by Th1 cells. These results demonstrate that Sema4A and IFN-γ expressed in Th1 cells play a critical role in enhancing neurite outgrowth from cortical neurons.     

The neurite growth promoting protease nexin 1 in glial cells of the olfactory bulb of the gerbil: An ultrastructural study

The glia-derived serine protease inhibitor and neurite outgrowth promotor protease nexin-1 (PN-1) is expressed in Schwann cell precursors and astroblasts during embryogenesis. In the adult nervous system, PN-1 persists in the Schwann cells and olfactory glia only. Light-microscopic immunohistochemistry has revealed the presence of PN-1 in the olfactory mucosa and in the nerve fiber layer of the olfactory bulb. The present electron-microscopic study of the gerbil olfactory bulb confirms the occurrence of PN-1 in ensheathing cells of the olfactory nerve fiber layer, a special type of glia which envelops olfactory axons. In addition, PN-1 is contained in typical astrocytes of the nerve fiber layer and of the glomerular layer. It is inferred that synthesis of PN-1 in the olfactory bulbs is maintained throughout adulthood because its neurite outgrowth promoting action is required for the continuous renewal of olfactory receptor neurons.     

PTPmu regulates N-cadherin-dependent neurite outgrowth

Cell adhesion is critical to the establishment of proper connections in the nervous system. Some receptor-type protein tyrosine phosphatases (RPTPs) have adhesion molecule-like extracellular segments with intracellular tyrosine phosphatase domains that may transduce signals in response to adhesion. PTPmu is a RPTP that mediates cell aggregation and is expressed at high levels in the nervous system. In this study, we demonstrate that PTPmu promotes neurite outgrowth of retinal ganglion cells when used as a culture substrate. In addition, PTPmu was found in a complex with N-cadherin in retinal cells. To determine the physiological significance of the association between PTPmu and N-cadherin, the expression level and enzymatic activity of PTPmu were perturbed in retinal explant cultures. Downregulation of PTPmu expression through antisense techniques resulted in a significant decrease in neurite outgrowth on an N-cadherin substrate, whereas there was no effect on laminin or L1-dependent neurite outgrowth. The overexpression of a catalytically inactive form of PTPmu significantly decreased neurite outgrowth on N-cadherin. These data indicate that PTPmu specifically regulates signals required for neurites to extend on an N-cadherin substrate, implicating reversible tyrosine phosphorylation in the control of N-cadherin function. Together, these results suggest that PTPmu plays a dual role in the regulation of neurite outgrowth.     

HNK-1 sulfotransferase null mice express glucuronyl glycoconjugates and show normal cerebellar granule neuron migration in vivo and in vitro

Sulfoglucuronyl carbohydrate (SGC), reactive with antibody against human natural killer cell antigen, is expressed in several glycolipids, glycoproteins and proteoglycans of the nervous system and has been implicated in cell-cell recognition, neurite outgrowth and neuronal migration during development, through its interaction with SGC-binding protein (SBP) 1. However, sulfotransferase (ST) null mutant mice, which lack SGC, were shown to have normal development with usual gross anatomy of the nervous system and other organs. Failure to observe a severe phenotype in the ST null mice prompted us to determine the compensatory molecular replacement of SGC by analyzing the carbohydrate of glycolipids and glycoproteins of the ST mutant nervous system. In the ST null mice, SGC-containing molecules were absent; instead the precursor glucuronyl carbohydrate (GC)-containing molecules accumulated. Other relevant glycolipids and proteins were not affected. The GC molecules in the mutant were localized at the same anatomical sites in the nervous system as the SGC molecules in the wild type. In vitro binding studies showed that, similar to sulfoglucuronyl glycolipids, glucuronyl glycolipids interacted with SBP-1, but with a lower binding capacity. In vitro studies with explant cultures of cerebellum indicated that neurite outgrowth and cell migration were not significantly affected in the mutant, possibly owing to interaction of SBP-1 with GC molecules. The results suggested that in vivo SBP-1-GC interaction was sufficient to allow normal neurite outgrowth and cell migration in the mutant, giving rise to a wild-type phenotype. However, the role of other compensatory molecules involved in these processes cannot be completely ruled out.     

Binding of the Receptor Tyrosine Kinase TrkB to the Neural Cell Adhesion Molecule (NCAM) Regulates Phosphorylation of NCAM and NCAM-dependent Neurite Outgrowth

Recognition molecules and neurotrophins play important roles during development and maintenance of nervous system functions. In this study, we provide evidence that the neural cell adhesion molecule (NCAM) and the neurotrophin receptor TrkB directly interact via sequences in their intracellular domains. Stimulation of TrkB by brain-derived neurotrophic factor leads to tyrosine phosphorylation of NCAM at position 734. Mutation of this tyrosine to phenylalanine completely abolishes tyrosine phosphorylation of NCAM by TrkB. Moreover, the knockdown of TrkB in hippocampal neurons leads to a reduction of NCAM-induced neurite outgrowth. Transfection of NCAM-deficient hippocampal neurons with mutated NCAM carrying an exchange of tyrosine by phenylalanine at position 734 leads to promotion of NCAM-induced neurite outgrowth in comparison with that observed after transfection with wild-type NCAM, whereas a reduction of neurite outgrowth was observed after transfection with mutated NCAM, which carries an exchange of tyrosine by glutamate that mimics the phosphorylated tyrosine. Our observations indicate a functional relationship between TrkB and NCAM.     

Sodium channel beta1 subunits promote neurite outgrowth in cerebellar granule neurons

Many immunoglobulin superfamily members are integral in development through regulation of processes such as growth cone guidance, cell migration, and neurite outgrowth. We demonstrate that homophilic interactions between voltage-gated sodium channel beta1 subunits promote neurite extension in cerebellar granule neurons. Neurons isolated from wild-type or beta1(-/-) mice were plated on top of parental, mock-, or beta1-transfected fibroblasts. Wild-type neurons consistently showed increased neurite length when grown on beta1-transfected monolayers, whereas beta1(-/-) neurons showed no increase compared with control conditions. beta1-mediated neurite extension was mimicked using a soluble beta1 extracellular domain and was blocked by antibodies directed against the beta1 extracellular domain. Immunohistochemical analysis suggests that the beta1 and beta4 subunits, but not beta2 and beta3, are expressed in cerebellar Bergmann glia as well as granule neurons. These results suggest a novel role for beta1 during neuronal development and are the first demonstration of a functional role for sodium channel beta subunit-mediated cell adhesive interactions.     

Nogo-66 receptor at cerebellar cortical glia gap junctions in the rat

Nogo-A is a myelin inhibitor of neurite outgrowth that accounts for the difficulty in fiber regeneration in the central nervous system. Its 66-amino-acid extracellular domain (Nogo-66) contributes to the inhibitory activity of Nogo-A. The Nogo-66 receptor is widely distributed in neurons of the central nervous system, including the cerebellum. In our study on the distribution of Nogo-66 receptor in the cerebellar cortex in the rat, we unexpectedly found Nogo-66 receptor immunoreactivity in the glia cells, particularly abundant beneath the Purkinje cells. The presence of Nogo-66 receptor in glia cells has not been reported before. A detailed study was thus conducted. Immunoelectron microscopic investigation clearly demonstrated that the Nogo-66 receptor immunoreactivity could be ascertained at the gap junction between glia cells, indicating that the Nogo-66 receptor may modulate the communication between glia cells through gap junctions.     

CRYP-2: A receptor-type tyrosine phosphatase selectively expressed by developing vertebrate neurons

Axonal growth and guidance, like other aspects of neuronal differentiation, can be regulated by changes in tyrosine phosphorylation. Although much is known concerning the role of tyrosine kinases in these processes, relatively little is known about the nature and function of protein tyrosine phosphatases (PTPs) that may be involved. To identify the PTPs expressed in the embryonic chicken CNS at the time of axon growth, we performed a polymerase chain reaction based “screen” using degenerate primers directed against conserved regions of the PTP catalytic domain. We obtained five distinct PTP-related cDNAs, two of which code for novel PTPs. One, designated CRYP-2, is selectively expressed in the CNS. Full-length cloning of CRYP-2 revealed that it is a receptor-type PTP with an adhesion molecule-like extracellular region comprising fibronectin (FN) type III repeats and a single catalytic domain in the intracellular region. It is alternatively spliced in the juxtamembrane region, similar to other PTPs recently cloned. CRYP-2 mRNA is strongly expressed in the brain during the time of axon growth; it is downregulated toward the end of embryo-genesis. Western blot analysis identifies a 330-kDa glycoprotein as CRYP-2 and confirms that the protein is downregulated after hatching. Immunostaining of cerebellar neurons in vitro reveals that CRYP-2 is expressed on neuronal cell bodies and processes, but not on glia. The CAM-like structure, developmental pattern of expression, and neuron-specific localization of the CRYP-2 PTP suggest that it is involved in neuronal differentiation, particularly axon growth. © 1996 John Wiley & Sons, Inc.     

Myelin-associated glycoprotein interacts with ganglioside GT1b. A mechanism for neurite outgrowth inhibition

Myelin-associated glycoprotein (MAG) is expressed on myelinating glia and inhibits neurite outgrowth from post-natal neurons. MAG has a sialic acid binding site in its N-terminal domain and binds to specific sialylated glycans and gangliosides present on the surface of neurons, but the significance of these interactions in the effect of MAG on neurite outgrowth is unclear. Here we present evidence to suggest that recognition of sialylated glycans is essential for inhibition of neurite outgrowth by MAG. Arginine 118 on MAG is known to make a key contact with sialic acid. We show that mutation of this residue reduces the potency of MAG inhibitory activity but that residual activity is also a result of carbohydrate recognition. We then go on to investigate gangliosides GT1b and GD1a as candidate MAG receptors. We show that MAG specifically binds both gangliosides and that both are expressed on the surface of MAG-responsive neurons. Furthermore, antibody cross-linking of cell surface GT1b, but not GD1a, mimics the effect of MAG, in that neurite outgrowth is inhibited through activation of Rho kinase. These data strongly suggest that interaction with GT1b on the neuronal cell surface is a potential mechanism for inhibition of neurite outgrowth by MAG.     

Phosphorylation at Tyr-694 of Nogo-A by Src-family kinases

Nogo-A is a neurite outgrowth inhibitor protein associated with myelin in the central nervous system. Unexpectedly, targeted disruption of Nogo-A in mice results in little or no improvement of axonal regeneration, suggesting that Nogo-A has other functions and/or receives complex regulations to exert its inhibitory functions. Here, we have found that Nogo-A becomes phosphorylated at Tyr-694 in the N-terminal region. The phosphorylation is mediated co-operatively by Src-family tyrosine kinases, which play many important roles in the nervous system. Levels of tyrosine phosphorylation of Nogo-A seem to be irrelevant to developmental stages of oligodendrocytes, and might be regulated by specific extracellular stimuli. Identification of tyrosine phosphorylation of Nogo-A will introduce an additional level of complexity into Nogo-A functions.     

Neurite formation by neurons derived from adult rat hippocampal progenitor cells is susceptible to myelin inhibition

Myelin-associated inhibitors expressed following injury to the adult central nervous system (CNS) induce growth cone collapse and retraction of the axonal cytoskeleton. Myelin-associated glycoprotein (MAG) is a bi-functional molecule that promotes neuritogenesis in some immature neurons during development then becomes inhibitory to neurite outgrowth as neurons mature. Progress is being made towards the elucidation of the downstream events that regulate myelin inhibition of regeneration in neuronal populations. However it is not known how adult-derived neural stem cells or progenitors respond to myelin during neuronal differentiation and neuritogenesis. Here we examine the effect of MAG on neurons derived from an adult rat hippocampal progenitor cell line (AHPCs). We show that, unlike their developmental counterparts, AHPC-derived neurons are susceptible to MAG inhibition of neuritogenesis during differentiation and display a 57% reduction in neurite outgrowth when compared with controls. We demonstrate that this effect can be overcome (by up to 69%) by activation of the neurotrophin, cyclic AMP and protein kinase A pathways or by Rho-kinase suppression. We also demonstrate that combination of these factors enhanced neurite outgrowth from differentiating neurons in the presence of MAG. This work provides important information for the successful generation of new neurons from adult neural stem cell populations within compromised adult circuitry and is thus directly relevant to endogenous repair and regeneration of the adult CNS.