The ubiquitously expressed plasma membrane Na⁺/H⁺ exchanger isoform 1 (NHE1) plays an important role in directed cell migration in non-neuronal cells, an effect which requires both the ion translocation and actin cytoskeleton anchoring functions of the protein. In the present study, an analogous role for NHE1 as a modulator of neurite outgrowth was evaluated in vitro utilizing NGF-differentiated PC12 cells as well as mouse neocortical neurons in primary culture. Examined at 3 d.i.v., endogenous NHE1 was found to be expressed in growth cones, where it gave rise to an elevated intracellular pH in actively-extending neurites. Application of the NHE inhibitor cariporide at an NHE1-selective concentration (1 μM) resulted in reductions in neurite extension and elaboration while application of 100 μM cariporide, to inhibit all known plasmalemmal NHE isoforms, failed to exert additional inhibitory effects, suggesting a dominant role for the NHE1 isoform in modulating neurite outgrowth. In addition, whereas transient overexpression of full-length NHE1 enhanced neurite outgrowth in a cariporide-sensitive manner in both NGF-differentiated PC12 cells and WT neocortical neurons, neurite outgrowth was reduced in NGF-differentiated PC12 cells overexpressing NHE1 mutants deficient in either ion translocation activity or actin cytoskeleton anchoring, suggesting that both functional domains of NHE1 are important for modulating neurite elaboration. A role for NHE1 in modulating neurite outgrowth was confirmed in neocortical neurons obtained from NHE1-/- mice which displayed reduced neurite outgrowth when compared to neurons obtained from their NHE1⁺/⁺ littermates. Further, neurite outgrowth in NHE1-/- neurons was rescued by transient overexpression of full-length NHE1 but not with mutant NHE1 constructs again suggesting that both functional domains of NHE1 are important for modulating neurite outgrowth. Finally, the growth promoting effects of netrin-1 but not BDNF or IGF-1 were abolished by cariporide in WT neocortical neurons and while both BDNF and IGF-1 were able to promote neurite outgrowth in NHE1-/- neurons, netrin-1 was unable to elicit this effect. Taken together, these results indicate that NHE1 is a permissive regulator of early neurite morphogenesis and also plays a novel role in netrin-1-stimulated neurite outgrowth. / Medicine, Faculty of / Graduate

During neuronal development, the axonal and dendritic projections are polarized and oriented along specific body axis. To further explore the molecular basis of neuritogenesis in vivo, we used the nematode Caenorhabditis elegans as a developmental model and performed a forward genetic screen to identify genes that specify the polarity of neurite outgrowth. We examined the VC4 and VC5 neurons, members of the six VC motor neurons using the Pcat-1

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22338号 / 医博第4579号 / 新制||医||1041(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 渡邉 大, 教授 渡邊 直樹, 教授 髙橋 良輔 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23095号 / 医博第4722号 / 新制||医||1050(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 伊佐 正, 教授 井上 治久, 教授 渡邉 大 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Kappa opioid receptor (KOPR) is involved in many physiological functions and pharmacological responses such as analgesia, anti-pruritic effect, sedation, motor incoordination and aversion (Simonin et al., 1998; Liu-Chen, 2004). The cellular mechanisms following activation of KOPR involve in part Gi/o protein-dependent pathways (Law et al., 2000). Following KOPR activation, the receptor is phosphorylated and arrestins are recruited. Arrestins mediate agonist-dependent KOPR desensitization, internalization and down-regulation (Liu-Chen, 2004). In recent years, arrestins were found to initiate arrestin-dependent downstream signaling. Thus, agonist-promoted KOPR phosphorylation plays a pivotal role in KOPR regulation and signaling. Previous studies from our lab showed that in Chinese hamster ovary (CHO) cells stably transfected with the human KOPR (hKOPR), U50,488H induced phosphorylation (Li et al., 2002a); however, sites of phosphorylation were not determined. Using LC-MS/MS, our lab recently identified four residues (S356, T357, T363 and S369) to be the sites of U50,488H-promoted phosphorylation in the mouse KOPR (mKOPR) stably expressed in N2A cells (Chen et al., 2016). Antibodies were generated against phosphopeptides and purified and three antibodies were found to have high specificity for the mKOPR phosphorylated at S356/T357, T363 and S369, respectively (Chen et al., 2016). Our lab previously showed that while U50,488H promoted robust hKOPR phosphorylation and internalization, etorphine induced little phosphorylation and internalization, although both were potent full agonists in enhancing [35S]GTPγS (Li et al., 2002a; Zhang et al., 2002; Li et al., 2003). Etorphine caused lower levels of KOPR phosphorylation at all the four residues than U50,488H by immunoblotting with the phospho-specific antibodies (Chen et al., 2016). Using the SILAC (stable isotope labeling by amino acids in cell culture) approach, we have found that compared to etorphine, U50,488H promoted higher levels of single phosphorylation at T363 and S369 and double phosphorylation at T363+S369 and T357+S369 as well as triple phosphorylation at S356+T357+S369 (Chen et al., 2016). These results indicate that an above-threshold phosphorylation is required for KOPR internalization. It has been reported that KOPR is involved in neuronal differentiation and neurogenesis. In the first chapter, I focused on whether there are differences in the mechanisms underlying neurite outgrowth induced by U50,488H and etorphine. In the chapter 2, mechanisms of KOPR phosphorylation were characterized in detail using phospho-specific KOPR antibodies. Protein kinase C was found, for the first time, to be involved in agonist-promoted KOPR phosphorylation. The roles of PKC in behavioral effects induced by KOPR agonists in mice were examined. For the chapter 1, in Neuro2a mouse neuroblastoma cells stably transfected with the hKOPR (N2A-3HA-hKOPR), U50,488H robustly induced neurite outgrowth, but etorphine caused outgrowth to a much lower extent. G protein-dependent pathway was found to be involved in the actions of both agonists, but β-arrestin-dependent pathway was not. Inhibition of ERK1/2 phosphorylation decreased neurite outgrowth promoted by both agonists, indicating the roles of MAP kinase cascades in KOPR agonist-induced neuritogenesis. In contrast, β-arrestin2, 14-3-3ζ, GEC1 and Rap1 are not involved in U50,488H- or etorphine-promoted neurite outgrowth. Thus, the two agonists appear to share the same signaling pathways and the difference between two agonists is likely due to the lower efficacy of etorphine. For the chapter 2, U50,488H caused phosphorylation of the mKOPR at S356, T357, T363 and S369 in N2A cells stably transfected with FmK6H (FmK6H-N2A cells). NorBNI abolished U50,488H-induced KOPR phosphorylation at all four residues. GRKs (GRKs2, 3, 5 and 6) and PKCs were involved in U50,488H-mediated KOPR phosphorylation. In addition, PKC also participated in agonist-independent KOPR phosphorylation. This is the first time that PKC was shown to be involved in agonist-induced KOPR phosphorylation. We found that U50,488H caused KOPR phosphorylation at T363 and S369 in the mouse brain and PKC participated in phosphorylation of S369, but not T363, by using the PKC inhibitor chelerythrine (CHL). Thus, we further characterized effects of PKC inhibition on KOPR-mediated behaviors in CD1 mice. PKC was involved in KOPR-mediated sedation, motor incoordination and conditioned place aversion, but not analgesia and anti-scratching effect in mice. Studies in this thesis revealed the mechanisms of KOPR-mediated neurite outgrowth and KOPR-mediated phosphorylation and the involvement of PKC in KOPR-mediated pharmacological effects in vivo. These studies push the frontier of molecular pharmacology of the KOPR, which may be useful for development of KOPR agonists for therapeutic use. / Pharmacology

<p>Neuroblastoma is a malignant childhood cancer, originating from sympathetic neuroblasts of the peripheral nervous system. Neuroblastoma is a heterogenous group of tumours, while some are highly malignant others can spontaneosly mature into a more benign form or regress. Less than half of the patients survive and this statistics has improved only modestly over the past 20 years. </p><p>SH-SY5Y is a human neuroblastoma cell line established from a highly malignant tumour. The cells have retained a capacity to differentiate <i>in vitro</i> in response to low concentrations of the phorbolester 12-O-tetradecanoylphorbol-13-acetate (TPA) in the presence of serum or defined growth factors. Differentiated cells are characterised by neurite formation and upregulation of neuronal marker genes. SH-SY5Y are unresponsive to nerve growth factor (NGF), but when transfected to express the NGF-receptor TrkA, they differentiate in response to NGF. Protein kinase C (PKC) is pivotal for the differentiation response to take place.</p><p>We have investigated the role of signaling through the Ras-MAPK pathway in differentiating SH-SY5Y, with respect to neurite formation, expression of neuronal marker genes and growth control. Our results show that differentiation-promoting treatment induced a sustained activation and nuclear accumulation of the MAPK ERK in SH-SY5Y. The nuclear accumulation of ERK was PKC-dependent. However, nuclear accumulation of ERK was not sufficient for a differentiation response to take place in these cells, but ERK activity was needed for the characteristic upregulation of <i>NPY</i> and <i>GAP-43</i> induced by TPA. ERK activity did not induce neurite formation, neither was it necessary for TPA-induced neurite formation. Instead, stimulation of a pathway distinct from MEK/ERK, but downstream of Ras, was needed for morphological differentiation. We could also show that differentiated cells still entered S-phase and that there was no correlation between expression of the CKI p21_cip1 (an ERK target), BrdU-incorporation or neurite formation. </p>

<p>Proteins of the band 4.1 superfamily are characterized by their sequence similarity to the ERM proteins ezrin, radixin and moesin, which are involved in cell motility, adhesion of cells, and signal transduction events. Little is however known of the function of ERM proteins in the nervous system, though an essential role for radixin and moesin in neuronal growth cone motility has been suggested. </p><p> This thesis is focused on the cloning, functional characterization and description of the tissue distribution in rat brain of MIR, a novel member of the band 4.1 superfamily. </p><p> The cDNA of MIR encods a protein of 445 amino acids which is composed of an ERM-homology domain and a RING finger, separated by an interregion. To reveal the cellular function of MIR, PC12 cell lines overexpressing MIR was generated and observed to inhibit NGF stimulated neurite outgrowth. </p><p> To elucidate the signal transduction of MIR by which it exerts its physiological activity, the yeast two-hybrid system was employed to screen for proteins that interact with MIR. A number of interactors known to regulate the cytoskeleton was obtained - among them myosin regulatory light chain-B which controls the actomyosin complex - and a novel type 2 membrane protein denoted NSAP for its similarity to saposin A-D. Overexpressed NSAP induced neurite outgrowth in PC12 cells and enhanced cell adhesion in fibroblasts. </p><p> The tissue distribution of MIR in rat brain, as determined by immunohistochemistry studies, showed that MIR is localized especially to neurons in hippocampus and cerebellum. The chromosomal localization of the MIR gene was assessed to 6p22.3-23, a region lost in the 6p23 deletion syndrome.</p><p> These results suggests that MIR is expressed in neurons in discrete regions of rat brain where it may regulate neurite outgrowth by modulating the cytoskeleton.</p>

Neuroblastoma is a malignant childhood cancer, originating from sympathetic neuroblasts of the peripheral nervous system. Neuroblastoma is a heterogenous group of tumours, while some are highly malignant others can spontaneosly mature into a more benign form or regress. Less than half of the patients survive and this statistics has improved only modestly over the past 20 years. SH-SY5Y is a human neuroblastoma cell line established from a highly malignant tumour. The cells have retained a capacity to differentiate in vitro in response to low concentrations of the phorbolester 12-O-tetradecanoylphorbol-13-acetate (TPA) in the presence of serum or defined growth factors. Differentiated cells are characterised by neurite formation and upregulation of neuronal marker genes. SH-SY5Y are unresponsive to nerve growth factor (NGF), but when transfected to express the NGF-receptor TrkA, they differentiate in response to NGF. Protein kinase C (PKC) is pivotal for the differentiation response to take place. We have investigated the role of signaling through the Ras-MAPK pathway in differentiating SH-SY5Y, with respect to neurite formation, expression of neuronal marker genes and growth control. Our results show that differentiation-promoting treatment induced a sustained activation and nuclear accumulation of the MAPK ERK in SH-SY5Y. The nuclear accumulation of ERK was PKC-dependent. However, nuclear accumulation of ERK was not sufficient for a differentiation response to take place in these cells, but ERK activity was needed for the characteristic upregulation of NPY and GAP-43 induced by TPA. ERK activity did not induce neurite formation, neither was it necessary for TPA-induced neurite formation. Instead, stimulation of a pathway distinct from MEK/ERK, but downstream of Ras, was needed for morphological differentiation. We could also show that differentiated cells still entered S-phase and that there was no correlation between expression of the CKI p21cip1 (an ERK target), BrdU-incorporation or neurite formation.

Proteins of the band 4.1 superfamily are characterized by their sequence similarity to the ERM proteins ezrin, radixin and moesin, which are involved in cell motility, adhesion of cells, and signal transduction events. Little is however known of the function of ERM proteins in the nervous system, though an essential role for radixin and moesin in neuronal growth cone motility has been suggested. This thesis is focused on the cloning, functional characterization and description of the tissue distribution in rat brain of MIR, a novel member of the band 4.1 superfamily. The cDNA of MIR encods a protein of 445 amino acids which is composed of an ERM-homology domain and a RING finger, separated by an interregion. To reveal the cellular function of MIR, PC12 cell lines overexpressing MIR was generated and observed to inhibit NGF stimulated neurite outgrowth. To elucidate the signal transduction of MIR by which it exerts its physiological activity, the yeast two-hybrid system was employed to screen for proteins that interact with MIR. A number of interactors known to regulate the cytoskeleton was obtained - among them myosin regulatory light chain-B which controls the actomyosin complex - and a novel type 2 membrane protein denoted NSAP for its similarity to saposin A-D. Overexpressed NSAP induced neurite outgrowth in PC12 cells and enhanced cell adhesion in fibroblasts. The tissue distribution of MIR in rat brain, as determined by immunohistochemistry studies, showed that MIR is localized especially to neurons in hippocampus and cerebellum. The chromosomal localization of the MIR gene was assessed to 6p22.3-23, a region lost in the 6p23 deletion syndrome. These results suggests that MIR is expressed in neurons in discrete regions of rat brain where it may regulate neurite outgrowth by modulating the cytoskeleton.