GIT1 enhances neurite outgrowth by stimulating microtubule assembly

GIT1, a G-protein-coupled receptor kinase interacting protein, has been reported to be involved in neurite outgrowth. However, the neu-robiological functions of the protein remain unclear. In this study, we found that GIT1 was highly expressed in the nervous system, and its expression was maintained throughout all stages of neuritogenesis in the brain. In primary cultured mouse hippocampal neurons from GIT1 knockout mice, there was a signiifcant reduction in total neurite length per neuron, as well as in the average length of axon-like struc-tures, which could not be prevented by nerve growth factor treatment. Overexpression of GIT1 signiifcantly promoted axon growth and fully rescued the axon outgrowth defect in the primary hippocampal neuron cultures from GIT1 knockout mice. The GIT1 N terminal region, including the ADP ribosylation factor-GTPase activating protein domain, the ankyrin domains and the Spa2 homology domain, were sufifcient to enhance axonal extension. Importantly, GIT1 bound to many tubulin proteins and microtubule-associated proteins, and it accelerated microtubule assemblyin vitro. Collectively, our ifndings suggest that GIT1 promotes neurite outgrowth, at least partially by stimulating microtubule assembly. This study provides new insight into the cellular and molecular pathogenesis of GIT1-associated neuro-logical diseases.     

蛋白激酶C调节脊髓神经元突起的生长(英文)

目的关于蛋白激酶C(PKC)在神经元突起生长和神经再生中的作用,目前仍存有争议。本研究主要观察PKC对离体培养的脊髓神经元生长的调节作用,旨在阐明PKC对突起生长的调节作用。方法分离纯化胎龄14天(E14)的SD胎鼠的脊髓前角神经元,进行原代培养,并检测不同时相点膜/浆PKC活性(m/c-PKCactivity)的比值。结果神经元培养3-11d期间,神经元内m/c-PKC比值以及PKC-βII在突起中的表达水平均与突起生长呈显著相关关系(r=0.95,P<0.01;r=0.73,P<0.01)。此外,PKC激动剂PMA能显著提高m/c-PKC比值,且与神经突起的生长一致(r=0.99,P<0.01)。而PKC抑制剂GF109203X则能显著抑制突起生长,且不被PMA作用所逆转。结论PKC的活性在脊髓神经元突起生长调节中具有重要作用,其中βII亚型可能扮演重要角色。     

Antibodies to the retina N-acetylgalactosaminylphosphotransferase inhibit neurite outgrowth.

The neural retina N-acetylgalactosaminylphosphotransferase (GalNAcPTase) is a cell surface molecule (Balsamo and Lilien, 1980, 1983; Balsamo et al., 1986a) that is tightly associated with, and glycosylates, the calcium-dependent, cell-cell adhesion molecule, N-cadherin (Balsamo and Lilien, 1990). N-cadherin has been implicated in neuronal attachment and neurite outgrowth when at the surface of cells (Bixby et al., 1987, 1988; Matsunaga et al., 1988; Neugebauer et al., 1988; Tomaselli et al., 1988). The intimate association of the GalNAcPTase and N-cadherin prompted us to test the possibility that the GalNAcPTase is also involved in the process of neurite outgrowth. We tested the effect of one polyclonal and two monoclonal anti-GalNAcPTase antibodies in cultures of chick neural retina cells extending neurites on substrates requiring N-cadherin, beta integrin receptors, or the chicken homologue of L1, G4. The length and number of neurites produced were dramatically reduced on all of these substrates by the polyclonal and one of the monoclonal anti-GalNAcPTase antibodies. The second monoclonal antibody bound to the cell surface but was not inhibitory, indicating that it reacts with a different epitope. The mechanism through which the retina cell surface GalNAcPTase may modulate neurite outgrowth on many substrates is discussed.     

VASE-encoded peptide modifies NCAM- and L1-mediated neurite outgrowth

Interactions between the neural cell adhesion molecule (NCAM) with NCAM-expressing neurons (trans-interaction) stimulate outgrowth of neurites. The extent of NCAM-triggered neurite outgrowth depends on the presence of 10 amino acids derived from the variable alternatively spliced exon (VASE or π-exon) in the fourth immunoglobulin-like domain of NCAM (Ig4): NCAM with VASE reduces and without VASE enhances neurite outgrowth in cis- or trans-interaction. We have investigated the role of VASE in neurite outgrowth by characterizing the receptors at the cell surface of cultured cerebellar neurons. Results from experiments with L1 and NCAM antibodies and with cerebellar neurons derived from wild-type or NCAM-deficient mice show that substrate-coated Ig4 with VASE (Ig4+) or without VASE (Ig4−) stimulates neurite outgrowth by a trans-interaction with L1 and that Ig4− promotes neurite outgrowth more strongly than Ig4+ by a transinteraction with NCAM. J. Neurosci. Res. 50:62–68, 1997. © 1997 Wiley-Liss, Inc.     

Cytoplasmic domain of NCAM 180 reduces NCAM-mediated neurite outgrowth

The neural cell adhesion molecule (NCAM) is one of the best-characterized cell adhesion molecules of the immunoglobulin superfamily. In the nervous system, NCAM is involved in cell migration, axon fasciculation and in neurite outgrowth. Neurite outgrowth is mediated by homophilic NCAM-NCAM interactions. Alternative splicing generates three major isoforms of NCAM differing in their intracellular portion. Two of them, NCAM 180 and NCAM 140, are transmembrane proteins with large intracellular domains. The present study is concerned with novel details of the intracellular domains of NCAM 140 and NCAM 180. We expressed these NCAM isoforms consisting only of the transmembrane and intracellular domains (without extracellular domains) in PC12 cells and elaborated their function in neurite outgrowth assays. Our data demonstrate that membrane-associated NCAM 180 interferes with neurite outgrowth, whereas membrane-associated NCAM 140 promotes neurite outgrowth.     

Opposing influences of protein kinase activities on neurite outgrowth in human neuroblastoma cells: initiation by kinase A and restriction by kinase C.

The respective roles of cAMP-dependent protein kinase (protein kinase A [PKA]) and protein kinase C (PKC) in the early stages of neurite outgrowth were examined in SH-SY-5Y human neuroblastoma cells. Forskolin or dbcAMP, agents that increase intracellular cAMP levels, and intracellular delivery of PKA catalytic subunit induced neurite outgrowth. The PKA inhibitor, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA 1004), prevented the increases, and decreased further the percentage of cells possessing short, filopodia-like neurites in the absence of inducers. In contrast to effects on PKA activation, PKC activation by 12-0-tetradecanoylphorbol-13-acetate (TPA) reduced the percentage of filopodia-like neurites elaborated by otherwise untreated cells, and prevented neurite outgrowth induced by PKA activators. PKC inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7), staurosporine, and sphingosine induced neurite outgrowth. Neurites induced by PKA activation contained higher levels of tubulin immunoreactivity than those induced by PKC inhibition. Furthermore, PKA-induced neurites rapidly retracted in the presence of colchicine, while those elaborated following PKC inhibition were more resistant. These data suggest that neurites elaborated in response to PKA activation are dependent upon microtubule polymerization, and that neurite induction following PKC inhibition is mediated by a different mechanism. PKA activators and PKC inhibitors exerted additive effects on neurite outgrowth, suggesting that the distinct pathways regulated by these two kinases function cooperatively during neuritogenesis.     

Staurosporin induces neurite outgrowth through ROS generation in HN33 hippocampal cell lines

Summary. Staurosporin, a specific inhibitor of PKC, is widely used in studies of signal transduction pathways. Previous studies have shown that staurosporin induces neurite outgrowth, but the underlying mechanisms remain unclear. Here we report that staurosporin induces neurite outgrowth in HN33 hippocampal cells. Two other PKC inhibitors, Go 6976 (specific for α- and β-isoforms) and rotterlin (a selective inhibitor of PKC δ), have no neuritogenic effect. In addition, staurosporin specifically increases ROS generation. NAC, which inhibits the generation of ROS, suppresses the staurosporin-induced neurite outgrowth in HN33 cells. Further, H₂O₂ causes neurite outgrowth. Taken together, these results confirm a neuritogenic effect of staurosporin and point to ROS as the signal mediator of staurosporin-induced neurite outgrowth in HN33 hippocampal cells. Theme: Development and regeneration Topic: Neurotrophic factors: receptors and cellular mechanisms     

Phosphatase and tensin homolog deletion enhances neurite outgrowth during neural stem cell differentiation

Neural stem cell (NSC) transplantation has emerged as a promising approach for the treatment of neurological disorders such as cerebral ischemia. As the majority of newly generated cells from exogenous NSCs fail to integrate into the ischemic brain and establish functional synaptic networks, NSC transplantation for ischemic stroke experiences limited neurological function recovery. Augment of endogenous neurite growth in the process of NSC differentiation is an avenue to promote synaptic networks. Phosphatase and tensin homolog (PTEN), a tumor suppressor, has been established to regulate axon growth in the adult central nervous system. The aim of this study was to explore the role of PTEN on neurite growth during NSC differentiation. Our results revealed that the protein expression of PTEN was significantly increased during NSC differentiation, whereas the expression of phosphorylated S6 ribosomal (p-S6R) was markedly decreased. Small interfering RNA knockdown of PTEN in NSCs can accelerate neurite outgrowth during NSC differentiation. These results indicated a remarkable effect of PTEN inhibition on neuronal process after NSC differentiation, and identified a novel route to promote endogenous neurite growth in differentiated NSCs, which may facilitate the application of NSC transplantation in ischemic stroke.     

Mitogen-activated protein kinase mediates purinergic-enhanced nerve growth factor-induced neurite outgrowth in PC12 cells

In 1999, we reported new observations that several compounds, including ATP, enhance neurite expression in PC12 cells when coapplied with nerve growth factor (NGF). Because purinergic and NGF signaling have several potential interfaces in PC12 cells, a series of experiments was conducted to elucidate the signal mediators contributing to the enhancement. Activities of selected kinases were measured and Western blots evaluated mitogen-activated protein kinase (MAPK) active and nonactive isoforms in lysates of the treated PC12 cells. In terms of purinergic potency, ATP and beta,gamma-methylene ATP elicited the greatest neurite-enhancing effect, whereas adenosine and alpha,beta-methylene ATP elicited the smallest. The effectiveness of a nonhydrolyzable analog such as beta,gamma-methylene ATP indicates that a nonmetabolic process is responsible. In response to ATP, NGF, or NGF + ATP, MAPK activity (measured by 32P incorporation) was maximal within 2 hr and remained statistically elevated over control levels throughout the 24 hr monitored. At maximal 32P incorporation, MAPK activity in response to ATP, NGF, and NGF + ATP was two-, four-, and sixfold higher, respectively, than control values; the observed increase was qualitatively confirmed using Western blots. Short-term inhibition experiments with protein kinase C and MAPK indicated that MAPK transduces the enhancing signal. We conclude from these experiments that ATP coapplied with NGF increases PC12 neurite expression by elevation of MAPK activity, likely by P2 receptor activation, and suggest that combination therapies with NGF and its enhancing adjunct compounds may be plausible for certain degenerative neurological disorders.     

Microglia-derived plasminogen enhances neurite outgrowth from explant cultures of rat brain

Recently, we reported the production and secretion of plasminogen (Pg) in cultured rat brain microglia [Nakajima et al., (1992) Fedn. Eur. Biochem. Socs Lett.308, 179–182]. To investigate the physiological significance of Pg, we determined the effect of Pg on neurite outgrowth of cultured neocortical explants of an embryonic rat brain in serum-free chemically defined medium. Pg markedly enhanced the neurite outgrowth. Although plasmin, which is derived from Pg by activation by urokinase (UK), had a similar effect in this explant culture system, UK itself did not show any effect. Furthermore, we studied the characteristics of Pg binding to cultured neocortical neurons dissociated from an embryonic 16-day-old rat brain by using ¹²⁵I-Pg. Specific binding of Pg to neocortical neurons was detected and Scatchard plot analysis revealed high- and low-affinity binding sites on the neurons. The estimated dissociation constants of high- and low-affinity binding sites were approximately 16.1 and 124.2 nM, respectively. These results suggest that microglia-derived Pg plays certain roles in the regulation of neurite extension through binding to the surface of neocortical neurons.     

Alternative acetylcholinesterase molecular forms exhibit similar ability to induce neurite outgrowth

Several groups have reported that acetylcholinesterase (AChE), through a mechanism not involving its catalytic activity, may have a role in fiber elongation. These observations were performed on experimental systems in which acetylcholine synthesis was active. Because neurite outgrowth can be modulated by neurotransmitters, we used the N18TG2 neuroblastoma line, which is defective for neurotransmitter production, to evaluate whether AChE may modulate neurite sprouting in nonenzymatic ways. To avoid the possibility that differences between transfected and mock-transfected clones may be due to the selection procedure, N18TG2 cells were previously subcloned, and the FB5 subclone was used for transfections. We performed transfections of FB5 cells with three distinct constructs encoding for the glycosylphosphoinositol-anchored AChE form, the tetrameric AChE form, and a soluble monomeric AChE form truncated in its C-terminus. A morphometric analysis of retinoic acid-differentiated clones was also undertaken. The results revealed that higher AChE expression following transfection brings about a greater ability of the clones to grow fibers with respect to nontransfected or mock-transfected cells irrespective of the used construct. Having observed no differences between the morphology of the transfected clones, we tested the possibility that the culture substrate can affect the capability of the clones to extend fibers. Also in this case we revealed no differences between the clones cultured on uncoated or collagen-pretreated dishes. These data indicate that alternative AChE molecular forms that differ in their C-teminal region exhibit similar ability to induce fiber outgrowth and suggest that the protein region responsible for this role is located in the invariant portion of the AChE molecule.     

Adipocyte-derived angiopoietin-1 supports neurite outgrowth and synaptogenesis of sensory neurons

Sensory and sympathetic innervation of the white fat tissue (WAT) contributes to lipolysis. In addition, both fiber types adapt in density to weight gain and loss. Because these findings are indicative for a tight control of nerve fiber plasticity by adipokines, we tested whether adipocytes control neurite growth of sensory neurons through angiopoietin-1 (Ang-1). We further considered initial hints that Ang-1-induced neuritogenesis involves transactivation of the high-affinity nerve growth factor (NGF) receptor trkA. Coculturing dorsal root ganglion (DRG) cells with 3T3-L1 adipocytes supported neurite outgrowth. These neurotrophic effects were associated with the increased expression of Ang-1 (presumably in adipocytes) as well as of trkA. The effects were abolished upon inactivating Ang-1 in culture with selective antibodies. Likewise, neurite outgrowth was impaired in the presence of inactivating NGF antibodies as well as upon inhibition of the NGF high-affinity trkA receptor with the antagonist K252a, indicating a tight cooperation of Ang-1 and NGF in the control of neuritogenesis. DRG-adipipocyte cocultures were further used to establish whether sensory neurons would form synaptic contacts with adipocytes. Electron microscopy demonstrated that cultured sensory neurons develop predominantly neuroneuronal synapses but seem to affect adipocytes by synapses en passant. Comparably to the case for neuritogenesis, expression of the presynaptic protein synaptophysin as well of the postsynaptic protein PSD-95 correlated with Ang-1 levels in culture. It is concluded that adipocyte-secreted Ang-1 supports neurite outgrowth, which is involved in synaptogenesis. The novel function of Ang-1 appears to play a physiological role in WAT plasticity.     

Galanin and galanin-like peptide modulate neurite outgrowth via protein kinase C-mediated activation of extracellular signal-related kinase

The neuropeptide galanin is widely distributed in the central nervous system and plays a role in a number of processes in the adult brain. Galanin also has neurotrophic effects in the developing nervous system and after nerve injury. The current study investigated the mechanism by which galanin promotes neurite outgrowth in the neuronal cell line PC12 and in neurospheres derived from adult hippocampal progenitor cells. We demonstrated that galanin can induce extracellular signal-related kinase (ERK) phosphorylation transiently in a concentration-dependent manner in neurons. Galanin-like peptide, which is thought to signal primarily through the GalR2 receptor subtype, induced ERK phosphorylation with similar kinetics to galanin. In functional studies, the ability of galanin and galanin-like peptide to induce neurite outgrowth was dependent on activation of both protein kinase C and ERK. This study identified a novel physiological role for galanin-induced ERK phosphorylation and identified ERK and protein kinase C as important signaling components in the galanin-mediated modulation of neurite outgrowth.