Reorganization of the actin cytoskeleton is an early cellular response to various extracellular signals. Sema3A, a repulsive axon guidance molecule, induces the reorganization of actin cytoskeleton in the growth cones. Collapsin response mediator protein 1 (CRMP1) mediates the intracellular Sema3A signalling through its Ser522 phosphorylation. Here we show that UNC-33, CRMP1 C. elegans homologue, interacts with FLN-1, an actin-binding Filamin-A orthologue. In nematodes, this interaction participates in the projection of DD/VD motor neurons. CRMP1 binds both the actin-binding domain and the last immunoglobulin-like repeat of Filamin-A. The alanine mutants of Filamin-A or CRMP1 in their interacting residues suppress the Sema3A repulsion in neurons. Conversely, a phosphor-mimicking mutant CRMP1(Ser522Asp) enhances the Sema3A response. Atomic-force microscopy analysis reveals that the V-shaped Filamin-A changes to a condensed form with CRMP1(Ser522Asp). CRMP1(Ser522Asp) weakens the F-actin gelation crosslinked by Filamin-A. Thus, phosphorylated CRMP1 may remove Filamin-A from the actin cytoskeleton to facilitate its remodelling.
l-3,4-Dihydroxyphenylalanine (DOPA) is the metabolic precursor of dopamine, and the single most effective agent in the treatment of Parkinson's disease. One problem with DOPA therapy for Parkinson's disease is its cardiovascular side effects including hypotension and syncope, the underlying mechanisms of which are largely unknown. We proposed that DOPA is a neurotransmitter in the central nervous system, but specific receptors for DOPA had not been identified. Recently, the gene product of ocular albinism 1 (OA1) was shown to possess DOPA-binding activity. It was unknown, however, whether or not OA1 is responsible for the actions of DOPA itself. Immunohistochemical examination revealed that OA1 was expressed in the nucleus tractus solitarii (NTS). OA1-positive cells adjacent to tyrosine hydroxylase-positive cell bodies and nerve fibers were detected in the depressor sites of the NTS. OA1 knockdown using oa1-specific shRNA-adenovirus vectors in the NTS reduced the expression levels of OA1 in the NTS. The prior injection of the shRNA against OA1 suppressed the depressor and bradycardic responses to DOPA but not to glutamate in the NTS of anesthetized rats. Thus OA-1 is a functional receptor of DOPA in the NTS, which warrants reexamination of the mechanisms for the therapeutic and untoward actions of DOPA.
l-3,4-Dihydroxyphenylalanine (DOPA) has been believed to be a precursor of dopamine, and itself being an inert amino acid. Previously, we have proposed DOPA as a neurotransmitter candidate in the central nervous system (CNS). Recent findings have suggested DOPA as an endogenous agonist of a G-protein coupled receptor, ocular albinism 1 gene product (OA1), which is highly expressed in the retinal pigmental epithelium. However, whether OA1 functions as a receptor for DOPA in vivo, and whether this receptor-ligand interaction is responsible for a wide variety of DOPA actions have not been determined yet. To gain insight into the functional implication of OA1, we perform immunohistochemical examination with anti-OA1 antibody to localize OA1 in the adult rat brain. We observed OA1 immunoreactive cells in the hippocampus, cerebral cortex, cerebellum cortex, striatum, substantia nigra, hypothalamic median eminence and supraoptic nucleus, nucleus tractus solitarii and caudal ventrolateral medulla and rostral ventrolateral medulla, medial habenular nucleus and olfactory bulb. This study reveals, for the first time, the unique distribution pattern of OA1-immunoreactive neurons and/or cells in the rat CNS.
In the 'Millennium Genome Project', we identified ATP2B1 as a gene responsible for hypertension through single-nucleotide polymorphism analysis. The ATP2B1 gene encodes the plasma membrane calcium ATPase isoform 1, which contributes to the maintenance of intracellular calcium homeostasis by removing calcium ions.
Axon growth inhibitors such as Nogo proteins, myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgp), and B lymphocyte stimulator (BLyS) commonly bind to Nogo receptor-1 (NgR1), leading to enormous restriction of functional recovery after damage to the adult central nervous system. Recently, we found that lateral olfactory tract usher substance (LOTUS) antagonizes NgR1-mediated Nogo signaling. However, whether LOTUS exerts antagonism of NgR1 when bound by the other three ligands has not been determined. Overexpression of LOTUS together with NgR1 in COS7 cells blocked the binding of MAG, OMgp, and BLyS to NgR1. In cultured dorsal root ganglion neurons in which endogenous LOTUS is only weakly expressed, overexpression of LOTUS suppressed growth cone collapse and neurite outgrowth inhibition induced by these three NgR1 ligands. LOTUS suppressed NgR1 ligand-induced growth cone collapse in cultured olfactory bulb neurons, which endogenously express LOTUS. Growth cone collapse was induced by NgR1 ligands in lotus-deficient mice. These data suggest that LOTUS functions as a potent endogenous antagonist for NgR1 when bound by all four known NgR1 ligands, raising the possibility that LOTUS may protect neurons from NgR1-mediated axonal growth inhibition and thereby may be useful for promoting neuronal regeneration as a potent inhibitor of NgR1.
The dendritic targeting of neurotransmitter receptors is vital for dendritic development and function. However, how such localization is established remains unclear. Here we show that semaphorin 3A (Sema3A) signalling at the axonal growth cone is propagated towards the cell body by retrograde axonal transport and drives AMPA receptor GluA2 to the distal dendrites, which regulates dendritic development. Sema3A enhances glutamate receptor interacting protein 1-dependent localization of GluA2 in dendrites, which is blocked by knockdown of cytoplasmic dynein heavy chain. PlexinA (PlexA), a receptor component for Sema3A, interacts with GluA2 at the immunoglobulin-like Plexin-transcription-factor domain (PlexA-IPT) in somatodendritic regions. Overexpression of PlexA-IPT suppresses dendritic localization of GluA2 and induces aproximal bifurcation phenotype in the apical dendrites of CA1 hippocampal neurons. Thus, we propose a control mechanism by which retrograde Sema3A signalling regulates the glutamate receptor localization through trafficking of cis-interacting PlexA with GluA2 along dendrites.
The glucokinase-induced up-regulation of insulin receptor substrate 2 (IRS-2) plays an important role in ?-cell adaptive proliferation in response to high-fat diet-induced insulin resistance. This study aimed to investigate the role of IRS-2 in the proliferation of ?-cells after a 60% partial pancreatectomy. IRS-2-deficient (IRS-2(-/-)) mice or wild-type mice were subjected to a pancreatectomy (60% partial pancreatectomy) or a sham operation (Sham). The ?-cell proliferation and gene expression profiles of the islets were then assessed. Gene expression in islets from pancreatectomized and Sham C57BL/6J male mice was analyzed using a cDNA microarray analysis. To compare with ?-cell proliferation induced by a high-fat diet, Gck(+/-) mice subjected to a pancreatectomy were also analyzed. The IRS-2(-/-) mice exhibited ?-cell expansion and a significant increase in ?-cell proliferation after the pancreatectomy, compared with the Sham group. Although glucose-stimulated insulin secretion from islets was not impaired, IRS-2(-/-) mice manifested severe hyperglycemia after the pancreatectomy. The expression levels of Aurora kinase B, Cyclin A, and Cyclin B1 in the pancreatectomized islets were also enhanced in the IRS-2(-/-) mice. A gene set enrichment analysis suggested an association between the genes that were up-regulated in the pancreatectomized islets and those involved in M phase progression in the cell cycle. ?-Cell proliferation after a pancreatectomy was observed even in the Gck(+/-) mice. In conclusion, IRS-2 was not required for ?-cell proliferation but might be needed for functional ?-cell mass, after a pancreatectomy. A partial pancreatectomy in mice may be an attractive model for the development of new strategy for exploring the unique nature of ?-cell proliferation.
Alzheimers disease (AD) is characterized by amyloid-? (A?) protein and tau deposition in the brain. Numerous studies have reported a central role of A? in the development of AD, but the pathogenesis is not well understood. Collapsin response mediator protein 2 (CRMP2), an intracellular protein mediating a repulsive axon guidance molecule, Semaphorin3A, is also accumulated in neurofibrillary tangles in AD brains. To gain insight into the role of CRMP2 phosphorylation in AD pathogenesis, we investigated the effects of A? neurotoxicity in CRMP2 phosphorylation-deficient knock-in (crmp2(ki/ki)) mice, in which the serine residue at 522 was replaced with alanine. Intracerebroventricular (i.c.v.) injection of A?25-35 peptide, a neurotoxic fragment of A? protein, to wild-type (wt) mice increased hippocampal phosphorylation of CRMP2. Behavioral assessment revealed that i.c.v. injection of A?25-35 peptide caused impairment of novel object recognition in wt mice, while the same peptide did not in crmp2(ki/ki) mice. In electrophysiological recording, wt and crmp2(ki/ki) mice have similar input-output basal synaptic transmission and paired-pulse ratios. However, long-term potentiation was impaired in hippocampal slices of A?25-35 peptide-treated wt but not those of crmp2(ki/ki). Our findings indicate that CRMP2 phosphorylation is required for A?-induced impairment of cognitive memory and synaptic plasticity.
Biosensors selectively detecting a very small amount of biomarker protein in human blood are desired for early and reliable diagnoses of severe diseases. This paper reports the detection of protein (streptavidin: SA) in ultra-low concentration, with an ultra-high selectivity against contaminants, using photonic crystal nanolasers. For biotin-modified nanolasers in pure water with SA, an extremely-low detection limit of 16 zM is evaluated. Even in a mixture with 1 ?M bovine serum albumin as the contaminant, 100 zM SA is detected, meaning a selectivity of 10(13). These are remarkable capabilities that are promising for practical biosensing in the medical applications mentioned above.
In the sexually dimorphic anteroventral periventricular nucleus (AVPV) of the hypothalamus, females have a greater number of tyrosine hydroxylase-immunoreactive (TH-ir) and kisspeptin-immunoreactive (kisspeptin-ir) neurons than males. In this study, we used proteomics analysis and gene-deficient mice to identify proteins that regulate the number of TH-ir and kisspeptin-ir neurons in the AVPV. Analysis of protein expressions in the rat AVPV on postnatal day 1 (PD1; the early phase of sex differentiation) using two-dimensional fluorescence difference gel electrophoresis followed by MALDI-TOF-MS identified collapsin response mediator protein 4 (CRMP4) as a protein exhibiting sexually dimorphic expression. Interestingly, this sexually differential expressions of CRMP4 protein and mRNA in the AVPV was not detected on PD6. Prenatal testosterone exposure canceled the sexual difference in the expression of Crmp4 mRNA in the rat AVPV. Next, we used CRMP4-knockout (CRMP4-KO) mice to determine the in vivo function of CRMP4 in the AVPV. Crmp4 knockout did not change the number of kisspeptin-ir neurons in the adult AVPV in either sex. However, the number of TH-ir neurons was increased in the AVPV of adult female CRMP4-KO mice as compared with the adult female wild-type mice. During development, no significant difference in the number of TH-ir neurons was detected between sexes or genotypes on embryonic day 15, but a female-specific increase in TH-ir neurons was observed in CRMP4-KO mice on PD1, when the sex difference was not yet apparent in wild-type mice. These results indicate that CRMP4 regulates the number of TH-ir cell number in the female AVPV.
Growth cone motility and morphology, which are critical for axon guidance, are controlled through intracellular events such as actin cytoskeletal reorganization and vesicular trafficking. The membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P?] has been implicated in regulation of these cellular processes in a diverse range of cell types. The main kinases involved in the production of PI(4,5)P? are the type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K) family, which consist of three isozymes, ?, ? and ?. Here, we demonstrate the involvement of PIP5K? in growth cone dynamics. Overexpression of a lipid kinase-deficient mutant of PIP5K? (PIP5K?-KD) in mouse dorsal root ganglion (DRG) neurons stimulated axon elongation and increased growth cone size, whereas wild-type PIP5K? tended to show opposite effects. Furthermore, PIP5K?-KD inhibited growth cone collapse of DRG neurons induced by semaphorin 3A (Sema3A). These results provide evidence that PIP5K? negatively regulates axon elongation and growth cone size and is involved in the cellular signaling pathway for Sema3A-triggered repulsion in DRG neurons.
BACKGROUND: Altered expression of collapsin response mediator proteins (CRMPs) has been reported in several malignant tumors, including downregulation of CRMP1 in lung cancer and upregulation of CRMP2 in colorectal cancer. This study aimed to investigate the relationship between CRMP expression and clinicopathological characteristics in patients with breast cancer. METHODS: Twenty-two breast cancer and four normal breast tissues were used to assess CRMP mRNA expression. The average expression level of each CRMP (CRMP1-5) mRNA was analyzed in a subset of breast cancer specimens and compared with that in normal breast tissue by real-time quantitative reverse-transcription polymerase chain reaction. Furthermore, 173 breast cancer specimens and matching normal breast controls were used for immunohistochemistry based on the tissue microarray technique. Levels of CRMP2 and phosphorylated CRMP2 protein were assessed, and possible correlations between the clinicopathological characteristics were evaluated. RESULTS: The expression of CRMP2 mRNA was significantly decreased in breast cancer tissues, while that of the other CRMPs was similar between normal and breast cancer tissues. Immunohistochemistry revealed that CRMP2 protein expression was also decreased in breast cancer tissues (P < 0.001). Phosphorylated CRMP2 was observed in the nuclei of breast cancer cells but not in normal mammary cells (P < 0.001). Furthermore, nuclear phosphorylated CRMP2 expression was increased in proportion to the histological grade and triple-negative subtype. CONCLUSIONS: Reduced CRMP2 expression and elevated expression of nuclear phosphorylated CRMP2 may be associated with breast cancer progression.
Rheumatoid arthritis (RA) is an autoimmune disease of which the pathogenetic mechanisms are not fully understood. Semaphorin3A (Sema3A) has an immune regulatory role. Neuropilin1 (NRP1), the primary receptor for Sema3A, is also a receptor for vascular endothelial growth factor 165 (VEGF 165). It has been shown that Sema3A competitively antagonizes VEGF 165 signaling. This study investigated whether Sema3A is expressed in synovial tissues, and is associated with disease activity and the histological features of synovial tissues from RA patients.
The precise role of AMP-activated protein kinase (AMPK), a target of metformin, in pancreatic ? cells remains controversial, even though metformin was recently shown to enhance the expression of incretin receptors (GLP-1 and GIP receptors) in pancreatic ? cells. In this study, we investigated the effect of AMPK in the regulation of incretin receptors expression in pancreatic islets. The phosphorylation of AMPK in the mouse islets was decreased by increasing glucose concentrations. We showed the expression of incretin receptors in bell-shaped response to glucose. Expression of the incretin receptors in the isolated islets showed higher levels under a medium glucose concentration (11.1 mM) than that under a low glucose concentration (2.8 mM), but was suppressed under a high glucose concentration (22.2 mM). Both treatment with an AMPK inhibitor and DN-AMPK expression produced a significant increase of the incretin receptors expression under a low glucose concentration. By contrast, in hyperglycemic db/db islets, the enhancing effect of the AMPK inhibitor on the expression of incretin receptors was diminished under a low glucose concentration. Taken together, AMPK is involved in the regulation of incretin receptors expression in pancreatic islets under a low glucose concentration.
The ability to detect harmful chemicals rapidly is essential for the survival of all animals. In Caenorhabditis elegans (C. elegans), repellents trigger an avoidance response, causing animals to move away from repellents. Dihydrocaffeic acid (DHCA) is a water-soluble repellent and nonflavonoid catecholic compound that can be found in plant products. Using a Xenopus laevis (X. laevis) oocyte expression system, we identified a candidate dihydrocaffeic acid receptor (DCAR), DCAR-1. DCAR-1 is a novel seven-transmembrane protein that is expressed in the ASH avoidance sensory neurons of C. elegans. dcar-1 mutant animals are defective in avoidance response to DHCA, and cell-specific expression of dcar-1 in the ASH neurons of dcar-1 mutant animals rescued the defect in avoidance response to DHCA. Our findings identify DCAR-1 as the first seven-transmembrane receptor required for avoidance of a water-soluble repellent, DHCA, in C. elegans.
Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant tumor, for which the development of new biomarkers and therapeutic targets has become critical. The main cause of poor prognosis in PDAC patients is the high invasive and metastatic potential of the cancer. In the present study, we report a new signaling pathway that was found to mediate the enhanced tumor cell motility in pancreatic cancer. Semaphorin 4D (Sema4D) is a ligand known to be expressed on different cell types, and has been reported to be involved in the regulation of immune functions, epithelial morphogenesis, and tumor growth and metastasis. In this study, we revealed for the first time that the cancer tissue cells expressing Sema4D in PDAC are tumor-infiltrating lymphocytes. The overexpression of Sema4D and of its receptor, plexinB1, was found to be significantly correlated with clinical factors, such as lymph node metastasis, distant metastasis, and poor prognosis in patients with PDAC. Through in vitro analysis, we demonstrated that Sema4D can potentiate the invasiveness of pancreatic cancer cells and we identified the downstream molecules. The binding of Sema4D to plexinB1 induced small GTPase Ras homolog gene family, member A activation and resulted in the phosphorylation of MAPK and Akt. In addition, in terms of potential therapeutic application, we clearly demonstrated that the enhanced-cell invasiveness induced by Sema4D could be inhibited by knockdown of plexinB1, suggesting that blockade of plexinB1 might diminish the invasive potential of pancreatic cancer cells. Our findings provide new insight into possible prognostic biomarkers and therapeutic targets in PDAC patients.
Sneezing and persistent itching of the nasal mucosa are distressing symptoms of allergic rhinitis (AR). Recent studies have revealed that hyperinnervation of sensory neurons in the nasal turbinate is one of the underlying causes of sneezing and itching. Since Semaphorin-3A (Sema3A) has been previously shown to restrict innervation of sensory neurons, it is presumed that reduced Sema3A expression in the nasal mucosa might contribute to the hypersensitivity. Analysis of the mouse model of ovalbumin-sensitized AR demonstrated a decreased expression of Sema3A in the nasal epithelium, which was accompanied by an increased nerve fiber density in the lamina propria of the turbinate. In rescue experiments, intranasal administration of recombinant Sema3A in the AR model mice alleviated sneezing and nasal rubbing symptoms. In addition, histological examinations also revealed that nerve fiber density was decreased in the lamina propria of the Sema3A-treated nasal turbinate. These results suggest that the nasal hypersensitivity of AR may be attributed to reduction of Sema3A expression and intranasal administration of Sema3A may provide a novel approach to alleviate the allergic symptoms for AR treatment.
Neural circuitry formation depends on the molecular control of axonal projection during development. By screening with fluorophore-assisted light inactivation in the developing mouse brain, we identified cartilage acidic protein-1B as a key molecule for lateral olfactory tract (LOT) formation and named it LOT usher substance (LOTUS). We further identified Nogo receptor-1 (NgR1) as a LOTUS-binding protein. NgR1 is a receptor of myelin-derived axon growth inhibitors, such as Nogo, which prevent neural regeneration in the adult. LOTUS suppressed Nogo-NgR1 binding and Nogo-induced growth cone collapse. A defasciculated LOT was present in lotus-deficient mice but not in mice lacking both lotus- and ngr1. These findings suggest that endogenous antagonism of NgR1 by LOTUS is crucial for normal LOT formation.
Semaphorin3A (Sema3A) is a repulsive guidance molecule for axons, which acts by inducing growth cone collapse through phosphorylation of CRMP2 (collapsin response mediator protein 2). Here, we show a role for CRMP2 oxidation and thioredoxin (TRX) in the regulation of CRMP2 phosphorylation and growth cone collapse. Sema3A stimulation generated hydrogen peroxide (H2O2) through MICAL (molecule interacting with CasL) and oxidized CRMP2, enabling it to form a disulfide-linked homodimer through cysteine-504. Oxidized CRMP2 then formed a transient disulfide-linked complex with TRX, which stimulated CRMP2 phosphorylation by glycogen synthase kinase-3, leading to growth cone collapse. We also reconstituted oxidation-dependent phosphorylation of CRMP2 in vitro, using a limited set of purified proteins. Our results not only clarify the importance of H2O2 and CRMP2 oxidation in Sema3A-induced growth cone collapse but also indicate an unappreciated role for TRX in linking CRMP2 oxidation to phosphorylation.
Polarized neurites (axons and dendrites) form the functional circuitry of the nervous system. Secreted guidance cues often control the polarity of neuron migration and neurite outgrowth by regulating ion channels. Here, we show that secreted semaphorin 3A (Sema3A) induces the neurite identity of Xenopus spinal commissural interneurons (xSCINs) by activating Ca(V)2.3 channels (Ca(V)2.3). Sema3A treatment converted the identity of axons of cultured xSCINs to that of dendrites by recruiting functional Ca(V)2.3. Inhibition of Sema3A signalling prevented both the expression of Ca(V)2.3 and acquisition of the dendrite identity, and inhibition of Ca(V)2.3 function resulted in multiple axon-like neurites of xSCINs in the spinal cord. Furthermore, Sema3A-triggered cGMP production and PKG activity induced, respectively, the expression of functional Ca(V)2.3 and the dendrite identity. These results reveal a mechanism by which a guidance cue controls the identity of neurites during nervous system development.
Collapsin response mediator protein 5 (CRMP5) is one of the CRMP members that expresses abundantly in the developing brain. To examine the in vivo function of CRMP5, we generated crmp5-deficient (crmp5(-/-)) mice. Anti-calbindin immunofluorescence studies of crmp5(-/-) mice revealed aberrant dendrite morphology; specifically, a decrease in the size of soma and diameter of primary dendrite of the cerebellar Purkinje cells at postnatal day 21 (P21) and P28, but not at P14. Coincidentally, CRMP5 is detected in Purkinje cells at P21 and P28 from crmp5(+/-) mice. In cerebellar slices of crmp5(-/-) mice, the induction of long-term depression of excitatory synaptic transmission between parallel fibers and Purkinje cells was deficient. Given that brain-derived neurotrophic factor (BDNF) plays major roles in dendritic development, we tried to elucidate the possible roles of CRMP5 in BDNF signaling. The effect of BDNF to induce dendritic branching was markedly attenuated in cultured crmp5(-/-) neurons. Furthermore, CRMP5 was tyrosine phosphorylated when coexpressed with neurotrophic tyrosine kinase receptor type 2 (TrkB), a receptor for BDNF, in HEK293T cells. These findings suggest that CRMP5 is involved in the development, maintenance and synaptic plasticity of Purkinje cells.
Oxygen is essential for animals, but high concentrations of oxygen are toxic to them probably because of an increase in reactive oxygen species (ROS). Many genes are involved in the reactions from which ROS are generated, but not much attention has been focused on them. To identify these genes, we screened for mutants with an altered sensitivity to oxidative stress in the nematode Caenorhabditis elegans and isolated a mutant, oxy-5(qa5002). oxy-5 showed an increased sensitivity to oxygen and decreased longevity. The decreased life span in oxy-5 was probably due to increased oxidative stress because it was recovered to a normal level when oxy-5 was cultured under hypoxic conditions. Our genetic analysis has revealed that the responsible gene for oxy-5 encodes a protein similar to mitochondrial ribosomal protein S36. The OXY-5 protein was highly expressed in the neurons, pharynx, and intestine, and expression of oxy-5 from the pan-neuronal H20 promoter efficiently suppressed the increased sensitivity to oxygen in oxy-5. These findings suggested that oxy-5 played an important role in the regulation of the sensitivity to oxygen in neuronal cells in C. elegans.
Axonal transport plays a crucial role in neuronal morphogenesis, survival, and function. Despite its importance, however, the molecular mechanisms of axonal transport remain mostly unknown because a simple and quantitative assay system for axonal transport has been lacking. In order to better characterize the molecular mechanisms involved in axonal transport, we here developed a computer-assisted monitoring system. Using lipophilic fluorochrome chloromethylbenzamido dialkylcarbocyanine (CM-DiI) as a labeling dye, we have successfully labeled membranous organelles in cultured chick dorsal root ganglia neurons. We confirmed that sodium azide, an ATPase inhibitor, and nocodazole, a microtubule-destabilizing agent, markedly suppressed anterograde and retrograde axonal transport of CM-DiI-labeled particles. We further tested the effects of several anti-neoplastic drugs on axonal transport. Paclitaxel, vincristine, cisplatin, and oxaliplatin, all of which are known to be neurotoxic and to cause neurological symptoms, suppressed anterograde and retrograde axonal transport. Another series of anti-neoplastic drugs, including methotrexate and 5-fluorouracil, did not affect the axonal transport. This is the first report of an automated monitoring system for axonal transport. This system will be useful for toxicity assays, characterizing axonal transport, or screening drugs that may modify neuronal functions.
Semaphorins and their receptor plexins constitute a pleiotropic cell-signalling system that is used in a wide variety of biological processes, and both protein families have been implicated in numerous human diseases. The binding of soluble or membrane-anchored semaphorins to the membrane-distal region of the plexin ectodomain activates plexins intrinsic GTPase-activating protein (GAP) at the cytoplasmic region, ultimately modulating cellular adhesion behaviour. However, the structural mechanism underlying the receptor activation remains largely unknown. Here we report the crystal structures of the semaphorin 6A (Sema6A) receptor-binding fragment and the plexin A2 (PlxnA2) ligand-binding fragment in both their pre-signalling (that is, before binding) and signalling (after complex formation) states. Before binding, the Sema6A ectodomain was in the expected face-to-face homodimer arrangement, similar to that adopted by Sema3A and Sema4D, whereas PlxnA2 was in an unexpected head-on homodimer arrangement. In contrast, the structure of the Sema6A-PlxnA2 signalling complex revealed a 2:2 heterotetramer in which the two PlxnA2 monomers dissociated from one another and docked onto the top face of the Sema6A homodimer using the same interface as the head-on homodimer, indicating that plexins undergo partner exchange. Cell-based activity measurements using mutant ligands/receptors confirmed that the Sema6A face-to-face dimer arrangement is physiologically relevant and is maintained throughout signalling events. Thus, homodimer-to-heterodimer transitions of cell-surface plexin that result in a specific orientation of its molecular axis relative to the membrane may constitute the structural mechanism by which the ligand-binding signal is transmitted to the cytoplasmic region, inducing GAP domain rearrangements and activation.
The localization of specific mRNAs and their local translation in growth cones of developing axons has been shown to play an important mechanism to regulate growth cone turning responses to attractive or repulsive cues. However, the mechanism whereby local translation and growth cone turning may be controlled by specific mRNA-binding proteins is unknown. Here we demonstrate that brain-derived neurotrophic factor (BDNF) signals the Src-dependent phosphorylation of the beta-actin mRNA zipcode binding protein 1 (ZBP1), which is necessary for beta-actin synthesis and growth cone turning. We raised a phospho-specific ZBP1 antibody to Tyr396, which is a Src phosphorylation site, and immunofluorescence revealed BDNF-induced phosphorylation of ZBP1 within growth cones. The BDNF-induced increase in fluorescent signal of a green fluorescent protein translation reporter with the 3 untranslated region of beta-actin was attenuated with the Src family kinase-specific inhibitor PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine]. Furthermore, a nonphosphorylatable mutant, ZBP1 Y396F, suppressed the BDNF-induced and protein synthesis-dependent increase in beta-actin localization in growth cones. Last, the ZBP1 Y396F mutant blocked BDNF-induced attractive growth cone turning. These results indicate that phosphorylation of ZBP1 at Tyr396 within growth cones has a critical role to regulate local protein synthesis and growth cone turning. Our findings provide new insight into how the regulated phosphorylation of mRNA-binding proteins influences local translation underlying growth cone motility and axon guidance.
Semaphorins, one of the repulsive axonal guidance factors during development, are produced under pathological conditions in adult animals. In the neuropathic pain state associated with peripheral nerve injury, synaptic reorganization occurs in spinal cord dorsal horn. In the present study, we investigated the roles of intrathecal administration of Sema3A, a secreted semaphorin, in the spinal cord of chronic constriction injury (CCI) model rat. Neuropilin 1 (NPR1) and Plexin A (PlexA), co-receptors of Sema3A, were expressed in the dorsal horn of naïve rats. NPR1, and not PlexA, protein expression increased in the dorsal spinal cord of CCI rats. Recombinant Sema3A protein attenuated mechanical allodynia and heat hyperalgesia in CCI rats, whereas heat-inactivated Sema3A had no effect. Immunohistochemistry revealed that Sema3A partially restored the decrease of isolectin B4-positive unmyelinated nerve terminals in lamina II of the ipsilateral dorsal horn of CCI rats. Contrary to our expectations, Sema3A did not change the distribution of myelinated fibers in lamina II at 7 days after CCI. Those results suggested that the suppressive role for Sema3A in the development of neuropathic pain associated with peripheral nerve injury in adult rats, which seemed to be independent from prevention of the myelinated fiber sprouting into lamina II.
UNC-51 is a serine/threonine protein kinase conserved from yeast to humans. The yeast homolog Atg1 regulates autophagy (catabolic membrane trafficking) required for surviving starvation. In C. elegans, UNC-51 regulates the axon guidance of many neurons by a different mechanism than it and its homologs use for autophagy. UNC-51 regulates the subcellular localization (trafficking) of UNC-5, a receptor for the axon guidance molecule UNC-6/Netrin; however, the molecular details of the role for UNC-51 are largely unknown. Here, we report that UNC-51 physically interacts with LET-92, the catalytic subunit of serine/threonine protein phosphatase 2A (PP2A-C), which plays important roles in many cellular functions. A low allelic dose of LET-92 partially suppressed axon guidance defects of weak, but not severe, unc-51 mutants, and a low allelic dose of PP2A regulatory subunits A (PAA-1/PP2A-A) and B (SUR-6/PP2A-B) partially enhanced the weak unc-51 mutants. We also found that LET-92 can work cell-non-autonomously on axon guidance in neurons, and that LET-92 colocalized with UNC-51 in neurons. In addition, PP2A dephosphorylated phosphoproteins that had been phosphorylated by UNC-51. These results suggest that, by forming a complex, PP2A cooperates with UNC-51 to regulate axon guidance by regulating phosphorylation. This is the first report of a serine/threonine protein phosphatase functioning in axon guidance in vivo.
UNC-6/Netrin is an evolutionarily conserved, secretory axon guidance molecule. In Caenorhabditis elegans, UNC-6 provides positional information to the axons of developing neurons, probably by establishing a concentration gradient from the ventral to the dorsal side of the animal. Although the proper localization of UNC-6 is important for accurate neuronal network formation, little is known about how its localization is regulated. Here, to examine the localization mechanism for UNC-6, we generated C. elegans expressing UNC-6 tagged with the fluorescent protein Venus and identified 13 genes, which are involved in the cellular localization of VenusUNC-6. For example, in unc-51, unc-14, and unc-104 mutants, the neurons showed an abnormal accumulation of VenusUNC-6 in the cell body and less than normal level of VenusUNC-6 in the axon. An aberrant accumulation of VenusUNC-6 in muscle cells was seen in unc-18 and unc-68 mutants. unc-51, unc-14, and unc-104 mutants also showed defects in the guidance of dorso-ventral axons, suggesting that the abnormal localization of UNC-6 disturbed the positional information it provides. We propose that these genes regulate the process of UNC-6 secretion: expression, maturation, sorting, transport, or exocytosis. Our findings provide novel insight into the localization mechanism of the axon guidance molecule UNC-6/Netrin.
Semaphorin-3A (Sema3A) is an attractive guidance molecule for cortical apical dendrites. To elucidate the role of Sema3A in hippocampal dendritic formation, we examined the Sema3A expression pattern in the perinatal hippocampal formation and analyzed hippocampal dendrites of the brains from young adult sema3A mutant mice. Sema3A protein was predominantly expressed in the hippocampal plate and the inner marginal zone at the initial period of apical dendritic growth. Neuropilin-1 and plexin-A, the receptor components for Sema3A, were also localized in the same regions. The Golgi impregnation method revealed that in wildtype mice more than 90% of hippocampal CA1 pyramidal neurons extended a single trunk or apical trunks bifurcated in stratum radiatum. Seven percent of the pyramidal neurons showed proximal bifurcation of apical trunks in stratum pyramidale or at the border of the stratum pyramidale and stratum radiatum. In sema3A mutant mice, proximally bifurcated apical dendrites were increased to 32%, while the single apical dendritic pyramidal neurons were decreased. We designate this phenotype in sema3A mutant mice as "proximal bifurcation." In the dissociated culture system, approximately half of the hippocampal neurons from wildtype mice resembled pyramidal neurons, which possess a long, thick, and tapered dendrite. In contrast, only 30% of the neurons from sema3A mutants exhibited pyramidal-like morphology. Proximal bifurcation of CA1 pyramidal neurons was also increased in the mutant mice of p35, an activator of cyclin-dependent kinase 5 (Cdk5). Thus, Sema3A may facilitate the initial growth of CA1 apical dendrites via the activation of p35/Cdk5, which may in turn signal hippocampal development.
Collapsin response mediator protein 2 (CRMP2) is an intracellular protein that mediates signaling of Semaphorin3A (Sema3A), a repulsive axon guidance molecule. Fyn, a Src-type tyrosine kinase, is involved in the Sema3A signaling. However, the relationship between CRMP2 and Fyn in this signaling pathway is still unknown. In our research, we demonstrated that Fyn phosphorylated CRMP2 at Tyr(32) residues in HEK293T cells. Immunohistochemical analysis using a phospho-specific antibody at Tyr(32) of CRMP showed that Tyr(32)-phosphorylated CRMP was abundant in the nervous system, including dorsal root ganglion neurons, the molecular and Purkinje cell layer of adult cerebellum, and hippocampal fimbria. Overexpression of a nonphosphorylated mutant (Tyr(32) to Phe(32)) of CRMP2 in dorsal root ganglion neurons interfered with Sema3A-induced growth cone collapse response. These results suggest that Fyn-dependent phosphorylation of CRMP2 at Tyr(32) is involved in Sema3A signaling.
Semaphorin-4D (Sema4D), a member of class 4 membrane-bound Semaphorins, acts as a chemorepellant to the axons of retinal ganglion cells and hippocampal neurons. Plexin-B1, a neuronal Sema4D receptor, associates with either one of receptor tyrosine kinases, c-Met or ErbB2, to mediate Sema4D-signaling. In contrast to this significance, the involvement of protein tyrosine phosphatases in Semaphorin-signaling remains unknown. We here show that Src homology 2-containing protein-tyrosine phosphatase 2 (SHP2) participates in Sema4D-signaling. SHP2 was localized in the growth cones of chick embryonic retinal ganglion neurons. Phenylarsine oxide, a protein tyrosine phosphatase inhibitor, suppressed Sema4D-induced contractile response in COS-7 cells expressing Plexin-B1. Ectopic expression of a phosphatase-inactive mutant of SHP2 in the retinal ganglion cells attenuated Sema4D-induced growth cone collapse response. A SHP1/2 specific inhibitor, 8-hydroxy-7-(6-sulfonaphthalen-2-yl)diazenyl-quinoline-5-sulfonic acid (NSC-87877), also suppressed this collapse response. These results suggest that SHP2-mediated tyrosine dephosphorylation is an important step in Sema4D-induced axon repulsion.
Intracellular calcium ions (Ca(2+)) have an essential role in the regulation of neurite outgrowth, but how outgrowth is controlled remains largely unknown. In this study, we examined how the mechanisms of neurite outgrowth change during development in chick and mouse dorsal root ganglion neurons. 2APB, a potent inhibitor of inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)R), inhibited neurite outgrowth at early developmental stages, but not at later stages. In contrast, pharmacological inhibition with Ni(2+), Cd(2+), or dantrolene revealed that ryanodine receptor (RyR)-mediated Ca(2+)-induced Ca(2+) release (CICR) was involved in neurite outgrowth at later stage, but not at early stages. The distribution of IP(3)R and RyR in growth cones also changed during development. Furthermore, pharmacological inhibition of the Ca(2+)-calmodulin-dependent phosphatase calcineurin with FK506 reduced neurite outgrowth only at early stages. These data suggest that the calcium signaling that regulates neurite outgrowth may change during development from an IP(3)R-mediated pathway to a RyR-mediated pathway.
To establish the neurotransmitter role(s) of L-3,4-dihydroxyphenylalanine (DOPA) in its own right, we attempted to clarify whether i.p. injection of a DOPA antagonist, DOPA cyclohexyl ester (CHE), would antagonize the behavioral responses of conscious rats to DOPA in the presence of 3-hydroxybenzylhydrazine (NSD-1015) (100 mg/kg i.p.), a central aromatic L-amino acid decarboxylase (AADC) inhibitor. DOPA-CHE (40, 60 and 100 mg/kg) elicited a dose-dependent partial antagonism against the increase in locomotor activity induced by DOPA (100 mg/kg i.p.). A low dose of DOPA-CHE (10 mg/kg) elicited full antagonism against the potentiating effect of a non-effective dose of DOPA (20 mg/kg) on the increase in locomotor activity induced by a dopamine D(2) agonist quinpirole (0.3 mg/kg s.c.). DOPA-CHE (100 mg/kg) elicited full antagonism against licking behavior induced by DOPA (100 mg/kg). We confirmed that DOPA (100 mg/kg) increased the striatal dopamine content but elicited no effect on locomotor activity in the presence of benserazide (50 mg/kg i.p.), a peripheral AADC inhibitor. DOPA also increased the dopamine content in the presence of NSD-1015 to a maximal degree similar to that in the presence of benserazide. Thus, we conclude that DOPA-CHE is a suitable DOPA antagonist that would be available under in vivo experimental conditions. DOPA plays a role in the neuromodulation of behavior.
Decreased ?-cell mass is a hallmark of type 2 diabetes, and therapeutic approaches to increase the pancreatic ?-cell mass have been expected. In recent years, gastrointestinal incretin peptides have been shown to exert a cell-proliferative effect in pancreatic ?-cells. Trefoil factor 2 (TFF2), which is predominantly expressed in the surface epithelium of the stomach, plays a role in antiapoptosis, migration, and proliferation. The TFF family is expressed in pancreatic ?-cells, whereas the role of TFF2 in pancreatic ?-cells has been obscure. In this study, we investigated the mechanism by which TFF2 enhances pancreatic ?-cell proliferation. The effects of TFF2 on cell proliferation were evaluated in INS-1 cells, MIN6 cells, and mouse islets using an adenovirus vector containing TFF2 or a recombinant TFF2 peptide. The forced expression of TFF2 led to an increase in bromodeoxyuridine (BrdU) incorporation in both INS-1 cells and islets, without any alteration in insulin secretion. TFF2 significantly increased the mRNA expression of cyclin A2, D1, D2, D3, and E1 in islets. TFF2 peptide increased ERK1/2 phosphorylation and BrdU incorporation in MIN6 cells. A MAPK kinase inhibitor (U0126) abrogated the TFF2 peptide-mediated proliferation of MIN6 cells. A CX-chemokine receptor-4 antagonist also prevented the TFF2 peptide-mediated increase in ERK1/2 phosphorylation and BrdU incorporation in MIN6 cells. These results indicated that TFF2 is involved in ?-cell proliferation at least partially via CX-chemokine receptor-4-mediated ERK1/2 phosphorylation, suggesting TFF2 may be a novel target for inducing ?-cell proliferation.
Nerve growth cones contain mRNA and its translational machinery and thereby synthesize protein locally. The regulatory mechanisms in the growth cone, however, remain largely unknown. We previously found that the calcium entry-induced increase of phosphorylation of eukaryotic elongation factor-2 (eEF2), a key component of mRNA translation, within growth cones showed growth arrest of neurites. Because dephosphorylated eEF2 and phosphorylated eEF2 are known to promote and inhibit mRNA translation, respectively, the data led to the hypothesis that eEF2-mediating mRNA translation may regulate neurite outgrowth. Here, we validated the hypothesis by using a chromophore-assisted light inactivation (CALI) technique to examine the roles of localized eEF2 and eEF2 kinase (EF2K), a specific calcium calmodulin-dependent enzyme for eEF2 phosphorylation, in advancing growth cones of cultured chick dorsal root ganglion (DRG) neurons. The phosphorylated eEF2 was weakly distributed in advancing growth cones, whereas eEF2 phosphorylation was increased by extracellular adenosine triphosphate (ATP)-evoked calcium transient through P2 purinoceptors in growth cones and resulted in growth arrest of neurites. The increase of eEF2 phosphorylation within growth cones by inhibition of protein phosphatase 2A known to dephosphorylate eEF2 also showed growth arrest of neurites. CALI of eEF2 within growth cones resulted in retardation of neurite outgrowth, whereas CALI of EF2K enhanced neurite outgrowth temporally. Moreover, CALI of EF2K abolished the ATP-induced retardation of neurite outgrowth. These findings suggest that an eEF2 phosphorylation state localized to the growth cone regulates neurite outgrowth.
Semaphorin3A (Sema3A) exerts a wide variety of biological functions by regulating reorganization of actin and tubulin cytoskeletal proteins through signaling pathways including sequential phosphorylation of collapsin response mediator protein 1 (CRMP1) and CRMP2 by cyclin-dependent kinase-5 and glycogen synthase kinase-3? (GSK3?). To delineate how GSK3? mediates Sema3A signaling, we here determined the substrates of GSK3? involved. Introduction of either GSK3? mutants, GSK3?-R96A, L128A, or K85M into chick dorsal root ganglion (DRG) neurons suppressed Sema3A-induced growth cone collapse, thereby suggesting that unprimed as well as primed substrates are involved in Sema3A signaling. Axin-1, a key player in Wnt signaling, is an unprimed substrate of GSK3?. The phosphorylation of Axin-1 by GSK3? accelerates the association of Axin-1 with ?-catenin. Immunocytochemical studies revealed that Sema3A induced an increase in the intensity levels of ?-catenin in the DRG growth cones. Axin-1 siRNA knockdown suppressed Sema3A-induced growth cone collapse. The reintroduction of RNAi-resistant Axin-1 (rAxin-1)-wt rescued the responsiveness to Sema3A, while that of nonphosphorylated mutants, rAxin S322A/S326A/S330A and T485A/S490A/S497A, did not. Sema3A also enhanced the colocalization of GSK3?, Axin-1, and ?-catenin in the growth cones. The increase of ?-catenin in the growth cones was suppressed by the siRNA knockdown of Axin-1. Furthermore, either Axin-1 or ?-catenin RNAi knockdown suppressed the internalization of Sema3A. These results suggest that Sema3A induces the formation of GSK3?/Axin-1/?-catenin complex, which regulates signaling cascade of Sema3A via an endocytotic mechanism. This finding should provide clue for understanding of mechanisms of a wide variety of biological functions of Sema3A.
The semaphorins were initially described as axon guidance molecules that play important roles in the development of nervous system. Recent studies suggest that semaphorins and their receptors also exert such diverse functions as immune response, control of vascular endothelial cell motility and invasion of many types of cancer cells.
The neural circuit in the hippocampus is important for higher brain functions. Dendrites of CA1 pyramidal neurons mainly receive input from the axons of CA3 pyramidal neurons in this neural circuit. A CA1 pyramidal neuron has a single apical dendrite and multiple basal dendrites. In wild-type mice, most of CA1 pyramidal neurons extend a single trunk, or alternatively, the apical dendrite bifurcates into two daughter trunks at the stratum radiatum layer. We previously reported the proximal bifurcation phenotype in Sema3A-/-, p35-/-, and CRMP4-/- mice. Cdk5/p35 phosphorylates CRMP2 at Ser522, and inhibition of this phosphorylation suppressed Sema3A-induced growth cone collapse. In this study, we analyzed the bifurcation points of the apical dendrites of hippocampal CA1 pyramidal neurons in CRMP2KI/KI mice in which the Cdk5/p35-phosphorylation site Ser522 was mutated into an Ala residue. The proximal bifurcation phenotype was not observed in CRMP2KI/KI mice; however, severe proximal bifurcation of apical dendrites was found in CRMP2KI/KI;CRMP4-/- mice. Cultured hippocampal neurons from CRMP2KI/KI and CRMP2KI/KI;CRMP4-/- embryos showed an increased number of dendritic branching points compared to those from wild-type embryos. Sema3A increased the number of branching points and the total length of dendrites in wild-type hippocampal neurons, but these effects of Sema3A for dendrites were not observed in CRMP2KI/KI and CRMP2KI/KI;CRMP4-/-hippocampal neurons. Binding of CRMP2 to tubulin increased in both CRMP2KI/KI and CRMP2KI/KI:CRMP4-/- brain lysates. These results suggest that CRMP2 and CRMP4 synergistically regulate dendritic development, and CRMP2 phosphorylation is critical for proper bifurcation of apical dendrite of CA1 pyramidal neurons.
Pancreatic cancer is an aggressive malignancy with one of the worst mortality rates of all cancers. Recently, collapsin response mediator proteins (CRMPs) were reported to be associated with proliferation, apoptosis, differentiation, and invasion in several cancers. However, CRMP expression and their role in pancreatic cancer have not been investigated. This study aimed to clarify the clinical significance of CRMPs in pancreatic cancer.
Suppression of inhibition of axonal outgrowth and promotion of axonal protection from progressive axonal degeneration are both therapeutic strategies for the treatment of neuronal diseases characterized by axonal loss. Myelin-associated inhibitors (MAIs) have been shown to suppress axonal outgrowth, but a specific MAI, myelin-associated glycoprotein (MAG), has also been shown to protect neurons from axonal degeneration through activation of the small GTPase protein RhoA. Recent in vitro studies have shown that collapsin response mediator protein 4 (CRMP4) interacts with RhoA and that the CRMP4b/RhoA complex mediates MAG-induced inhibitory signaling against axonal outgrowth. However, whether CRMP4 is involved in MAG-mediated axon protection signaling remains unclear. Here, we show involvement of CRMP4 in MAG-induced inhibition of axonal outgrowth and axonal protection using the CRMP4-/- mouse model. In dorsal root ganglion (DRG) neurons, loss of CRMP4 prevents MAG-induced inhibition of axonal outgrowth and growth cone collapse and increases sensitivity to microtubule destabilizing factor Vincristine (VNC)-induced axonal degeneration. MAG-mediated axon protection against VNC is suppressed in CRMP4-/- DRG neurons. Understanding the molecular mechanism of MAG-mediated inhibition and protection via CRMP4 may provide novel opportunities to control axonal degeneration and regeneration.
Semaphorin3A (Sema3A), a secreted factor that navigates axons and dendrites of developing neurons, facilitates axonal transport. However, little is known about the mechanism underlying Sema3A-induced facilitation and its functional implications. Here we show that Sema3A induces facilitation of axonal transport via local calcium signaling in growth cone. The facilitation of axonal transport was blocked by inhibitors of voltage-gated sodium channels (tetrodotoxin, TTX), L-type voltage-gated calcium channel, and ryanodine receptor (RyR). Sema3A evoked intracellular Ca(2+) elevation in growth cone by local application of Sema3A to growth cone. Sema3A also activated RyR in growth cone as well as cell body. Notably, TTX suppressed Sema3A-induced RyR activation in cell body but not in growth cone. Our results identify a novel mechanism of Sema3A-induced axonal transport, and further suggest that Sema3A-induced local calcium signaling in growth cone is propagated to cell body in a TTX-sensitive manner.
The glucagon-like peptide-1 receptor agonist liraglutide is used to treat diabetes. A hallmark of liraglutide is the glucose-dependent facilitation of insulin secretion from pancreatic ?-cells. In ?-cells, the glycolytic enzyme glucokinase plays a pivotal role as a glucose sensor. However, the role of glucokinase in the glucose-dependent action of liraglutide remains unknown. We first examined the effects of liraglutide on glucokinase haploinsufficient (Gck(+/-)) mice. Single administration of liraglutide significantly improved glucose tolerance in Gck(+/-) mice without increase of insulin secretion. We also assessed the effects of liraglutide on the survival rates, metabolic parameters, and histology of liver or pancreas of ?-cell-specific glucokinase-deficient (Gck(-/-)) newborn mice. Liraglutide reduced the blood glucose levels in Gck(-/-) neonates but failed to prolong survival, and all the mice died within 1 wk. Furthermore, liraglutide did not improve glucose-induced insulin secretion in isolated islets from Gck(-/-) neonates. Liraglutide initially prevented increases in alanine aminotransferase, free fatty acids, and triglycerides in Gck(-/-) neonates but not at 4 d after birth. Liraglutide transiently prevented liver steatosis, with reduced triglyceride contents and elevated glycogen contents in Gck(-/-) neonate livers at 2 d after birth. Liraglutide also protected against reductions in ?-cells in Gck(-/-) neonates at 4 d after birth. Taken together, ?-cell glucokinase appears to be essential for liraglutide-mediated insulin secretion, but liraglutide may improve glycemic control, steatosis, and ?-cell death in a glucokinase-independent fashion.
Pruritus is a common symptom of psoriasis, which affects quality of life. This symptom accompanies the hyper-innervation of sensory C-fibres in psoriatic lesions. Two extracellular molecules, nerve growth factor (NGF) and semaphorin-3A, regulate C-fibre extension. In this study, the expression levels of these 2 molecules in biopsy specimens from psoriatic and healthy skin were quantified by immunohistochemistry and quantitative reverse-transcription PCR. Semaphorin-3A expression was lower in the psoriatic samples compared with the healthy samples, whereas NGF was higher. C-fibre innervation in the epidermis was also increased in psoriatic skin. Semaphorin-3A mRNA expression was negatively correlated with itch intensity and severity of psoriasis. We propose that decreased semaphorin-3A and increased NGF expression levels may trigger the outgrowth of C-fibres, leading to pruritus.
Netrin is an evolutionarily conserved, secretory axon guidance molecule. Netrins receptors, UNC-5 and UNC-40/DCC, are single trans-membrane proteins with immunoglobulin domains at their extra-cellular regions. Netrin is thought to provide its positional information by establishing a concentration gradient. UNC-5 and UNC-40 act at growth cones, which are specialized axonal tip structures that are generally located at a long distance from the neural cell body. Thus, the proper localization of both Netrin and its receptors is critical for their function. This review addresses the localization mechanisms of UNC-6/Netrin and its receptors in Caenorhabditis elegans, focusing on our recent reports. These findings include novel insights on cytoplasmic proteins that function upstream of the receptors.
We investigated a possible association between serum plasminogen activator inhibitor-1 (PAI-1) levels and renal dysfunction in 124 type 2 diabetes patients. Multiple linear regression analyses indicated that the PAI-1 levels were significantly inversely correlated with estimated glomerular filtration rate (eGFR) independent of albuminuria, BMI, LDL-C, and triglyceride.
Axonal transport plays a crucial role in neuronal morphogenesis, survival and function. Despite its importance, however, the molecular mechanisms of axonal transport remain mostly unknown because a simple and quantitative assay system for monitoring this cellular process has been lacking. In order to better characterize the mechanisms involved in axonal transport, we formulate a novel computer-assisted monitoring system of axonal transport. Potential uses of this system and implications for future studies will be discussed.
Collapsin response mediator protein 1 (CRMP1) and CRMP2 have been known as mediators of extracellular guidance cues such as semaphorin 3A and contribute to cytoskeletal reorganization in the axonal pathfinding process. To date, how CRMP1 and CRMP2 focally regulate axonal pathfinding in the growth cone has not been elucidated. To delineate the local functions of these CRMPs, we carried out microscale-chromophore-assisted light inactivation (micro-CALI), which enables investigation of localized molecular functions with highly spatial and temporal resolutions. Inactivation of either CRMP1 or CRMP2 in the neurite shaft led to arrested neurite outgrowth. Micro-CALI of CRMP2 in the central domain of the growth cones consistently arrested neurite outgrowth, whereas micro-CALI of CRMP1 in the same region caused significant lamellipodial retraction, followed by retardation of neurite outgrowth. Focal inactivation of CRMP1 in its half region of the growth cone resulted in the growth cone turning away from the irradiated site. Conversely, focal inactivation of CRMP2 resulted in the growth cone turning toward the irradiated site. These findings suggest different functions for CRMP1 and CRMP2 in growth cone behavior and neurite outgrowth.
Collapsin response mediator protein (CRMP) was originally identified as a molecule involved in semaphorin3A signaling. CRMPs are now known to consist of five homologous cytosolic proteins, CRMP1-5. All of them are phosphorylated and highly expressed in the developing and adult nervous system. In vitro experiments have clearly demonstrated that CRMPs play important roles in neuronal development and maturation through the regulation of their phosphorylation. Several recent knockout mice studies have revealed in vivo roles of CRMPs in neuronal migration, neuronal network formation, synapse formation, synaptic plasticity, and neuronal diseases. Dynamic spatiotemporal regulation of phosphorylation status of CRMPs is involved in many aspects of neuronal development.
We reported previously that ATP2B1 was one of the genes for hypertension receptivity in a large-scale Japanese population, which has been replicated recently in Europeans and Koreans. ATP2B1 encodes the plasma membrane calcium ATPase isoform 1, which plays a critical role in intracellular calcium homeostasis. In addition, it is suggested that ATP2B1 plays a major role in vascular smooth muscle contraction. Because the ATP2B1 knockout (KO) mouse is embryo-lethal, we generated mice with vascular smooth muscle cell-specific KO of ATP2B1 using the Cre-loxP system to clarify the relationship between ATP2B1 and hypertension. The KO mice expressed significantly lower levels of ATP2B1 mRNA and protein in the aorta compared with control mice. KO mice showed significantly higher systolic blood pressure as measured by tail-cuff method and radiotelemetric method. Similar to ATP2B1, the expression of the Na(+)-Ca(2+) exchanger isoform 1 mRNA was decreased in vascular smooth muscle cells of KO mice. However, ATP2B4 expression was increased in KO mice. The cultured vascular smooth muscle cells of KO mice showed increased intracellular calcium concentration not only in basal condition but also in phenylephrine-stimulated condition. Furthermore, phenylephrine-induced vasoconstriction was significantly increased in vascular rings of the femoral artery of KO mice. These results suggest that ATP2B1 plays important roles in the regulation of blood pressure through alteration of calcium handling and vasoconstriction in vascular smooth muscle cells.
Myelin-derived axon growth inhibitors, such as Nogo, bind to Nogo receptor-1 (NgR1) and thereby limit the action of axonal regeneration after injury in the adult central nervous system. Recently, we have found that cartilage acidic protein-1B (Crtac1B)/lateral olfactory tract usher substance (LOTUS) binds to NgR1 and functions as an endogenous NgR1 antagonist. To examine the functional domain of LOTUS in the antagonism to NgR1, analysis using the deletion mutants of LOTUS was performed and revealed that the carboxyl-terminal region (UA/EC domain) of LOTUS bound to NgR1. The UA/EC fragment of LOTUS overexpressed together with NgR1 in COS7 cells abolished the binding of Nogo66 to NgR1. Overexpression of the UA/EC fragment in cultured chick dorsal root ganglion neurons suppressed Nogo66-induced growth cone collapse. These findings suggest that the UA/EC region is a functional domain of LOTUS serving for an antagonistic action to NgR1.
Collapsin response mediator proteins (CRMPs) are intracellular proteins that mediate signals for several extracellular molecules, such as Semaphorin3A and neurotrophins. The phosphorylation of CRMP1 and CRMP2 by Cdk5 at Ser522 is involved in axonal guidance and spine development. Here, we found that the Ser522-phosphorylated CRMP1 and/or CRMP2 are enriched in the dendrites of cultured cortical neurons and P7 cortical section. To determine the physiological role of CRMPs in dendritic development, we generated CRMP2 knock-in mutant mice (crmp2ki/ki) in which the Ser residue at 522 was replaced with Ala. Strikingly, the cortical basal dendrites of double mutant crmp2ki/ki and crmp1-/- mice exhibited severe abnormal dendritic patterning, which we defined as "curling phenotype." These findings demonstrate that the function of CRMP1 and CRMP2 synergistically control dendritic projection, and the phosphorylation of CRMP2 at Ser522 is essential for proper dendritic field organization in vivo.
Collapsin response mediator proteins (CRMPs) are a family of cytosolic phosphoproteins that consist of 5 members (CRMP 1-5). CRMP2 and CRMP4 regulate neurite outgrowth by binding to tubulin heterodimers, resulting in the assembly of microtubules. CRMP2 also mediates the growth cone collapse response to the repulsive guidance molecule semaphorin-3A (Sema3A). However, the role of CRMP4 in Sema3A signaling and its function in the developing mouse brain remain unclear. We generated CRMP4-/- mice in order to study the in vivo function of CRMP4 and identified a phenotype of proximal bifurcation of apical dendrites in the CA1 pyramidal neurons of CRMP4-/- mice. We also observed increased dendritic branching in cultured CRMP4-/- hippocampal neurons as well as in cultured cortical neurons treated with CRMP4 shRNA. Sema3A induces extension and branching of the dendrites of hippocampal neurons; however, these inductions were compromised in the CRMP4-/- hippocampal neurons. These results suggest that CRMP4 suppresses apical dendrite bifurcation of CA1 pyramidal neurons in the mouse hippocampus and that this is partly dependent on Sema3A signaling.
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