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The semaphorin family is a well‐characterized family of secreted or membrane‐bound proteins that are involved in activity‐independent neurodevelopmental processes, such as axon guidance, cell migration, and immune functions. Although semaphorins have recently been demonstrated to regulate activity‐dependent synaptic scaling, their roles in Hebbian synaptic plasticity as well as learning and memory remain poorly understood. Here, using a rodent model, we found that an inhibitory avoidance task, a hippocampus‐dependent contextual learning paradigm, increased secretion of semaphorin 3A in the hippocampus. Furthermore, the secreted semaphorin 3A in the hippocampus mediated contextual memory formation likely by driving AMPA receptors into hippocampal synapses via the neuropilin1–plexin A4–semaphorin receptor complex. This signaling process involves alteration of the phosphorylation status of collapsin response mediator protein 2, which has been characterized as a downstream molecule in semaphorin signaling. These findings implicate semaphorin family as a regulator of Hebbian synaptic plasticity and learning., Contextual fear learning task increased the secretion of semaphorin 3A, a secreted type of semaphorin, in the hippocampus. Furthermore, the secreted semaphorin 3A in the hippocampus mediated contextual memory formation by driving AMPA receptors into hippocampal synapses via the neuropilin 1/plexin A4 semaphorin receptor complex. Thus, the semaphorin family regulates both Hebbian synaptic plasticity and learning.

Recent studies have shown that mutation at Ser522 causes inhibition of collapsin response mediator protein 2 (CRMP2) phosphorylation and induces axon elongation and partial recovery of the lost sensorimotor function after spinal cord injury (SCI). We aimed to reveal the intracellular mechanism in axotomized neurons in the CRMP2 knock-in (CRMP2KI) mouse model by performing transcriptome analysis in mouse sensorimotor cortex using micro-dissection punching system. Prior to that, we analyzed the structural pathophysiology in axotomized or neighboring neurons after SCI and found that somatic atrophy and dendritic spine reduction in sensorimotor cortex were suppressed in CRMP2KI mice. Further analysis of the transcriptome has aided in the identification of four hemoglobin genes Hba-a1, Hba-a2, Hbb-bs, and Hbb-bt that are significantly upregulated in wild-type mice with concomitant upregulation of genes involved in the oxidative phosphorylation and ribosomal pathways after SCI. However, we observed substantial upregulation in channel activity genes and downregulation of genes regulating vesicles, synaptic function, glial cell differentiation in CRMP2KI mice. Moreover, the transcriptome profile of CRMP2KI mice has been discussed wherein energy metabolism and neuronal pathways were found to be differentially regulated. Our results showed that CRMP2KI mice displayed improved SCI pathophysiology not only via microtubule stabilization in neurons, but also possibly via the whole metabolic system in the central nervous system, response changes in glial cells, and synapses. Taken together, we reveal new insights on SCI pathophysiology and the regenerative mechanism of central nervous system by the inhibition of CRMP2 phosphorylation at Ser522. All these experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee at Waseda University, Japan (2017-A027 approved on March 21, 2017; 2018-A003 approved on March 25, 2018; 2019-A026 approved on March 25, 2019).

Collapsin response mediator proteins (CRMPs), which consist of five homologous cytosolic proteins, are one of the major phosphoproteins in the developing nervous system. The prominent feature of the CRMP family proteins is a new class of microtubule-associated proteins that play important roles in the whole process of developing the nervous system, such as axon guidance, synapse maturation, cell migration, and even in adult brain function. The CRMP C-terminal region is subjected to posttranslational modifications such as phosphorylation, which, in turn, regulates the interaction between the CRMPs and various kinds of proteins including receptors, ion channels, cytoskeletal proteins, and motor proteins. The gene-knockout of the CRMP family proteins produces different phenotypes, thereby showing distinct roles of all CRMP family proteins. Also, the phenotypic analysis of a non-phosphorylated form of CRMP2-knockin mouse model, and studies of pharmacological responses to CRMP-related drugs suggest that the phosphorylation/dephosphorylation process plays a pivotal role in pathophysiology in neuronal development, regeneration, and neurodegenerative disorders, thus showing CRMPs as promising target molecules for therapeutic intervention.

Pancreatic intraepithelial neoplasia (PanIN), the most common premalignant lesion of the pancreas, is a histologically well-defined precursor to invasive pancreatic ductal adenocarcinoma (PDAC). However, the molecular mechanisms underlying the progression of PanINs have not been fully elucidated. Previously, we demonstrated that the expression of collapsin response mediator protein 4 (CRMP4) in PDAC was associated with poor prognosis. The expression of CRMP4 was also augmented in a pancreatitis mouse model. However, the role of CRMP4 in the progression of PanIN lesions remains uncertain. In the present study, we examined the relationship between CRMP4 expression and progression of PanIN lesions using genetically engineered mouse models. PanIN lesions were induced by peritoneal injection of the cholecystokinin analog caerulein in LSL-KRASG12D; Pdx1-Cre (KC-Crmp4 wild-type, WT) mice and LSL-KRASG12D; Pdx1-Cre; Crmp4-/- (KC-Crmp4 knockout, KO) mice. We analyzed pancreatic tissue sections from these mice and evaluated PanIN grade by hematoxylin and eosin staining. CRMP4 expression was examined and the cellular components assessed by immunohistochemistry using antibodies against CRMP4, CD3, and α-smooth muscle actin (SMA). The incidence of high-grade PanIN in KC-Crmp4 WT mice was higher than that in KC-Crmp4 KO animals. CRMP4 was expressed not only in epithelial cells but also in αSMA-positive cells in stromal areas of PanIN lesions. The CRMP4 expression in stromal areas correlated with PanIN grade in WT mice. These results suggested that the expression of CRMP4 in stromal cells may underlie the incidence or progression of PanIN.

Fragile X mental retardation protein (FMRP) is an RNA-binding protein that regulates local translation in dendrites and spines for synaptic plasticity. In axons, FMRP is implicated in axonal extension and axon guidance. We previously demonstrated the involvement of FMRP in growth cone collapse via a translation-dependent response to Semaphorin-3A (Sema3A), a repulsive axon guidance factor. In the case of attractive axon guidance factors, RNA-binding proteins such as zipcode binding protein 1 (ZBP1) accumulate towards the stimulated side of growth cones for local translation. However, it remains unclear how Sema3A effects FMRP localization in growth cones. Here, we show that levels of FMRP in growth cones of hippocampal neurons decreased after Sema3A stimulation. This decrease in FMRP was suppressed by the ubiquitin-activating enzyme E1 enzyme inhibitor PYR-41 and proteasome inhibitor MG132, suggesting that the ubiquitin-proteasome pathway is involved in Sema3A-induced FMRP degradation in growth cones. Moreover, the E1 enzyme or proteasome inhibitor suppressed Sema3A-induced increases in microtubule-associated protein 1B (MAP1B) in growth cones, suggesting that the ubiquitin-proteasome pathway promotes local translation of MAP1B, whose translation is mediated by FMRP. These inhibitors also blocked the Sema3A-induced growth cone collapse. Collectively, our results suggest that Sema3A promotes degradation of FMRP in growth cones through the ubiquitin-proteasome pathway, leading to growth cone collapse via local translation of MAP1B. These findings reveal a new mechanism of axon guidance regulation: degradation of the translational suppressor FMRP via the ubiquitin-proteasome pathway.

CRMP2, alternatively designated as DPYSL2, was the first CRMP family member to be identified as an intracellular molecule mediating the signaling of the axon guidance molecule Semaphorin 3A (Sema3A). In Sema3A signaling, cyclin-dependent kinase 5 (Cdk5) primarily phosphorylates CRMP2 at Ser522. Glycogen synthase kinase-3β (GSK-3β) subsequently phosphorylates the residues of Thr509 and Thr514 of CRMP2. Previous studies showed that CRMP2 is involved in pathogenesis of neurological disorders such as Alzheimer's disease. In Alzheimer's disease, hyper-phosphorylated forms of CRMP2 are accumulated in the paired helical filaments. To get insight into the possible involvement of the phosphorylation of CRMP2 in pathogenesis of neurological disorders, we previously created CRMP2 S522A knock-in (crmp2ki/ki) mice and demonstrated that the phosphorylation of CRMP2 at Ser522 is involved in normal dendrite patterning in cortical neurons. However, the behavioral impact and in vivo signaling network of the CRMP2 phosphorylation are not fully understood. In this study, we performed behavioral and proteomics analysis of crmp2ki/ki mice. The crmp2ki/ki mice appeared healthy and showed no obvious differences in physical characteristics compared to wild-type mice, but they showed impaired emotional behavior, reduced sociality, and low sensitivity to pain stimulation. Through mass-spectrometry-based proteomic analysis, we found that 59 proteins were increased and 77 proteins were decreased in the prefrontal cortex of crmp2ki/ki mice. Notably, CRMP3, CRMP4, and CRMP5, the other CRMP family proteins, were increased in crmp2ki/ki mice. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyses identified 14 pathways in increased total proteins and 13 pathways in decreased total proteins which are associated with the pathogenesis of Parkinson's, Alzheimer's, and Huntington's diseases. We also detected 20 pathways in increased phosphopeptides and 16 pathways in decreased phosphopeptides including "inflammatory mediator regulation of TRP channels" in crmp2ki/ki mice. Our study suggests that the phosphorylation of CRMP2 at Ser522 is involved in the signaling pathways that may be related to neuropsychiatric and neurodegenerative diseases and pain.

Leukocyte common antigen-related (LAR) class protein tyrosine phosphatases (PTPs) are critical for axonal guidance; however, their relation to specific guidance cues is poorly defined. We here show that PTP-3, a LAR homolog in Caenorhabditis elegans, is involved in axon guidance regulated by Semaphorin-2A-signaling. PTPδ, one of the vertebrate LAR class PTPs, participates in the Semaphorin-3A (Sema3A)-induced growth cone collapse response of primary cultured dorsal root ganglion neurons from Mus musculus embryos. In vivo, however, the contribution of PTPδ in Sema3A-regualted axon guidance was minimal. Instead, PTPδ played a major role in Sema3A-dependent cortical dendritic growth. Ptpδ(−/−) and Sema3a(−/−) mutant mice exhibited poor arborization of basal dendrites of cortical layer V neurons. This phenotype was observed in both male and female mutants. The double-heterozygous mutants, Ptpδ(+/−); Sema3a(+/−), also showed a similar phenotype, indicating the genetic interaction. In Ptpδ(−/−) brains, Fyn and Src kinases were hyperphosphorylated at their C-terminal Tyr527 residues. Sema3A-stimulation induced dephosphorylation of Tyr527 in the dendrites of wild-type cortical neurons but not of Ptpδ(−/−). Arborization of cortical basal dendrites was reduced in Fyn(−/−) as well as in Ptpδ(+/−); Fyn(+/−) double-heterozygous mutants. Collectively, PTPδ mediates Sema3A-signaling through the activation of Fyn by C-terminal dephosphorylation. SIGNIFICANCE STATEMENT The relation of leukocyte common antigen-related (LAR) class protein tyrosine phosphatases (PTPs) and specific axon guidance cues is poorly defined. We show that PTP-3, a LAR homolog in Caenorhabditis elegans, participates in Sema2A-regulated axon guidance. PTPδ, a member of vertebrate LAR class PTPs, is involved in Sema3A-regulated cortical dendritic growth. In Sema3A signaling, PTPδ activates Fyn and Src kinases by dephosphorylating their C-terminal Tyr residues. This is the first evidence showing that LAR class PTPs participate in Semaphorin signaling in vivo.

We identified the neuroprotein collapsing response mediator protein-4 (CRMP4) as a noncanonical osteogenic factor that regulates the differentiation of mouse bone marrow skeletal stem cells (bone marrow stromal stem cells [mBMSCs]) into osteoblastic cells. CRMP4 is the only member of the CRMP1-CRMP5 family to be expressed by mBMSCs and in osteoprogenitors of both adult mouse and human bones. In vitro gain-of-function and loss-of-function of CRMP4 in murine stromal cells revealed its inhibitory effect on osteoblast differentiation. In addition, Crmp4-deficient mice (Crmp4-/- ) displayed a 40% increase in bone mass, increased mineral apposition rate, and bone formation rate, compared to wild-type controls. Increased bone mass in Crmp4-/- mice was associated with enhanced BMP2 signaling and BMP2-induced osteoblast differentiation in Crmp4-/- osteoblasts (OBs). Furthermore, Crmp4-/- OBs exhibited enhanced activation of RhoA/focal adhesion kinase (FAK) signaling that led to cytoskeletal changes with increased cell spreading. In addition, Crmp4-/- OBs exhibited increased cell proliferation that was mediated via inhibiting cyclin-dependent kinase inhibitor 1B, p27Kip1 and upregulating cyclin D1 expression which are targets of RhoA signaling pathway. Our findings identify CRMP4 as a novel negative regulator of osteoblast differentiation. © 2016 American Society for Bone and Mineral Research.

De novo variants (DNVs) cause many genetic diseases. When DNVs are examined in the whole coding regions of genes in next-generation sequencing analyses, pathogenic DNVs often cluster in a specific region. One such region is the last exon and the last 50 bp of the penultimate exon, where truncating DNVs cause escape from nonsense-mediated mRNA decay [NMD(−) region]. Such variants can have dominant-negative or gain-of-function effects. Here, we first developed a resource of rates of truncating DNVs in NMD(−) regions under the null model of DNVs. Utilizing this resource, we performed enrichment analysis of truncating DNVs in NMD(−) regions in 346 developmental and epileptic encephalopathy (DEE) trios. We observed statistically significant enrichment of truncating DNVs in semaphorin 6B (SEMA6B) (p value: 2.8 × 10(−8); exome-wide threshold: 2.5 × 10(−6)). The initial analysis of the 346 individuals and additional screening of 1,406 and 4,293 independent individuals affected by DEE and developmental disorders collectively identified four truncating DNVs in the SEMA6B NMD(−) region in five individuals who came from unrelated families (p value: 1.9 × 10(−13)) and consistently showed progressive myoclonic epilepsy. RNA analysis of lymphoblastoid cells established from an affected individual showed that the mutant allele escaped NMD, indicating stable production of the truncated protein. Importantly, heterozygous truncating variants in the NMD(+) region of SEMA6B are observed in general populations, and SEMA6B is most likely loss-of-function tolerant. Zebrafish expressing truncating variants in the NMD(−) region of SEMA6B orthologs displayed defective development of brain neurons and enhanced pentylenetetrazole-induced seizure behavior. In summary, we show that truncating DNVs in the final exon of SEMA6B cause progressive myoclonic epilepsy.

l-3,4-dihydroxyphenylalanine (l-DOPA) is a candidate neurotransmitter. l-DOPA is released by nicotine through nicotinic receptors. Recently, G-protein coupled receptor GPR143, was identified as a receptor for l-DOPA. In this study, genetic association studies between GPR143 genetic polymorphisms and smoking behaviors revealed that the single-nucleotide polymorphism rs6640499, in the GPR143 gene, was associated with traits of smoking behaviors in Japanese individuals. In Gpr143 gene-deficient mice, nicotine-induced hypolocomotion and rewarding effect were attenuated compared to those in wild-type mice. Our findings suggest the involvement of GPR143 in the smoking behaviors.

Axonal degeneration occurs in various neurological diseases and traumatic nerve injury, and axonal regeneration is restricted by inhibitory factors in the central nervous system. Cyclin-dependent kinase 5 and glycogen synthase kinase 3β (GSK3β) are activated by one of those inhibitors, and collapsin response mediator protein 2 (CRMP2) is phosphorylated by both kinases. We previously developed a CRMP2 knock-in (CRMP2 KI) mouse line, in which CRMP2 phosphorylation at Ser 522 is inhibited. Because CRMP2 KI mice showed promotion of axonal regeneration after spinal cord injury, we hypothesized that CRMP2 KI mice would show higher axonal regeneration after optic nerve injury. In this study, we first show that depolymerization of microtubules after optic nerve crush (ONC) injury was suppressed in CRMP2 KI mice. Loss of retinal ganglia cells was also reduced after ONC. We found that protein level of GAP43, a marker of regenerative axons, was higher in the optic nerve from CRMP2KI than that from wild type 4 weeks after of ONC. We further observed increased numbers of axons labeled by tracer in the optic nerve after ONC in CRMP2 KI mice. These results suggest that inhibition of phosphorylation of CRMP2 suppresses axonal degeneration and promotes axonal regeneration after optic nerve injury.

The capacity for regeneration in the injured adult mammalian central nervous system (CNS) is largely limited by potent inhibitory barriers. Chondroitin sulfate proteoglycans (CSPGs) are major inhibitors of axonal regeneration/sprouting and accumulate at lesion sites after CNS trauma. Despite extensive research during the two decades since their discovery, the molecular mechanisms remain elusive, including intracellular phosphorylation events. Collapsin response mediator protein 4 (CRMP4) is known to directly regulate cytoskeletal dynamics and neurite extension, while phosphorylated CRMP4 loses its binding affinity for cytoskeletal proteins. We have previously found that spinal cord injury (SCI) induces CRMP4 upregulation and phosphorylation and that CRMP4 knockout (Crmp4-/-) mice show behavioral recovery of locomotor function after SCI. However, the role of CRMP4 in the recovery of other forms of physiological function such as sensation remains largely unknown. We here have demonstrated CRMP4 involvement in CSPG-induced inhibitory signaling and nociceptive recovery in Crmp4-/- mice after SCI. We cultured dorsal root ganglion (DRG) neurons on CSPG-coated dishes; Crmp4 deletion overrode CSPG-induced inhibition of axon growth in vitro. CRMP4 levels were increased in DRGs in vivo after SCI. Crmp4-/- mice exhibited axonal growth of sensory neurons and recovery of nociceptive function after spinal transection. These results support Crmp4 deletion as a therapeutic target in the treatment of SCI.

Protein tyrosine phosphate δ (PTPδ), one of the receptor type IIa protein tyrosine phosphates, is known for its roles in axon guidance, synapse formation, cell adhesion, and tumor suppression. Alternative splicing of this gene generates at least four (A-D) isoforms; however, the major isoform in vivo is yet to be determined. The protein localization has neither been revealed. We have generated anti-mouse PTPδ-specific monoclonal antibody and analyzed the protein expression in wild-type and Ptpδ knockout mice. Immunoblot analysis of various organs revealed that neuronal tissues express both C-and D-isoforms of PTPδ, whereas non-neuronal tissues express only C-isoform. Immunohistochemistry of wild-type or Ptpδ heterozygous sections showed that olfactory bulb, cerebral cortex, hippocampus, cerebellum, and several nuclei in brain stem exhibit moderate to strong positive signals. These signals were absent in Ptpδ knockout specimens. Higher magnification revealed differences between expression patterns of PTPδ mRNA and its protein product. In hippocampus, weak mRNA expression in CA1 stratum pyramidale but strong immunostaining in the stratum lacunosum moleculare was observed, suggesting the axonal expression of PTPδ in the entorhinal cortical afferents. Olfactory mitral cells exhibited mRNA expression in cell bodies and protein localization in their dendritic fields, glomerular and external plexiform layers. Nissl staining showed that the external plexiform layer was reduced in Ptpδ knockout mice. Golgi-impregnation confirmed the poor dendritic growth of homozygous mitral cells. These results suggest that PTPδ may localize in axons as well as in dendrites to regulate their elaboration in the central nervous system.

Growing axons rely on local signaling at the growth cone for guidance cues. Semaphorin3A (Sema3A), a secreted repulsive axon guidance molecule, regulates synapse maturation and dendritic branching. We previously showed that local Sema3A signaling in the growth cones elicits retrograde retrograde signaling via PlexinA4 (PlexA4), one component of the Sema3A receptor, thereby regulating dendritic localization of AMPA receptor GluA2 and proper dendritic development. In present study, we found that nimodipine (voltage-gated L-type Ca(2+) channel blocker) and tetrodotoxin (TTX; voltage-gated Na(+) channel blocker) suppress Sema3A-induced dendritic localization of GluA2 and dendritic branch formation in cultured hippocampal neurons. The local application of nimodipine or TTX to distal axons suppresses retrograde transport of Venus-Sema3A that has been exogenously applied to the distal axons. Sema3A facilitates axonal transport of PlexA4, which is also suppressed in neurons treated with either TTX or nimodipine. These data suggest that voltage-gated calcium and sodium channels mediate Sema3A retrograde signaling that regulates dendritic GluA2 localization and branch formation.

Oa1 is the casual gene for ocular albinism-1 in humans. The gene product OA1, alternatively designated as GPR143, belongs to G-protein coupled receptors. It has been reported that OA1 is a specific receptor for 3, 4-dihydroxy- L-phenylalanine (DOPA) in retinal pigmental epithelium where DOPA facilitates the pigmentation via OA1 stimulation. We have recently shown that OA1 mediates DOPA-induced depressor response in rat nucleus tractus solitarii. However, the distribution and function of OA1 in other regions are largely unknown. We have generated oa1 knockout mice and examined OA1 expression in both neuronal and non-neuronal tissues by immunohistochemical analyses using anti-mouse OA1 monoclonal antibodies. In the telencephalon, OA1 was expressed in cerebral cortex and hippocampus. Predominant expression of OA1 was observed in the pyramidal neurons in these regions. OA1 was also expressed in habenular nucleus, hypothalamus, substantia nigra, and medulla oblongata. The expression of OA1 in the nucleus tractus solitarii of medulla oblongata may support the reduction of blood pressure by the microinjection of DOPA into this region. Outside of the nervous system, OA1 was expressed in heart, lung, liver, kidney and spleen. Abundant expression was observed in the renal tubules and the splenic capsules. These peripheral regions are innervated by numerous sympathetic nerve endings. In addition, substantia nigra contains a large population of dopaminergic neurons. Thus, the immunohistochemical analyses suggest that OA1 may modulate the monoaminergic functions in both peripheral and central nervous systems.

Axonal regeneration in the adult mammalian central nervous system is limited in part by the non-permissive environment, including axonal growth inhibitors such as the Nogo-A protein. How the functions of these inhibitors can be blocked remains unclear. Here, we examined the role of LOTUS, an endogenous Nogo receptor antagonist, in promoting functional recovery and neural repair after spinal cord injury (SCI), as well as axonal regeneration after optic nerve crush. Wild-type untreated mice show incomplete but substantial intrinsic motor recovery after SCI. The genetic deletion of LOTUS delays and decreases the extent of motor recovery, suggesting that LOTUS is required for spontaneous neural repair. The neuronal overexpression of LOTUS in transgenic mice promotes motor recovery after SCI, and recombinant viral overexpression of LOTUS enhances retinal ganglion cell axonal regeneration after optic nerve crush. Thus, the level of LOTUS function titrates axonal regeneration.

Microtubule associated protein Collapsin response mediator protein 2 (CRMP2) regulates neuronal polarity in developing neurons through interactions with tubulins or microtubules. However, how CRMP2 promotes axonal formation by affecting microtubule behavior remains unknown. This study aimed to obtain the structural basis for CRMP2–tubulin/microtubule interaction in the course of axonogenesis. The X-ray structural studies indicated that the main interface to the soluble tubulin-dimer is the last helix H19 of CRMP2 that is distinct from the known C-terminal tail-mediated interaction with assembled microtubules. In vitro structural and functional studies also suggested that the H19-mediated interaction promoted the rapid formation of GTP-state microtubules directly, which is an important feature of the axon. Consistently, the H19 mutants disturbed axon elongation in chick neurons, and failed to authorize the structural features for axonal microtubules in Caenorhabditis elegans. Thus, CRMP2 induces effective axonal microtubule formation through H19-mediated interactions with a soluble tubulin-dimer allowing axonogenesis to proceed.

Autism spectrum disorders (ASD) are more common among boys than girls. The mechanisms responsible for ASD symptoms and their sex differences remain mostly unclear. We previously identified collapsin response mediator protein 4 (CRMP4) as a protein exhibiting sex-different expression during sexual differentiation of the hypothalamic sexually dimorphic nucleus. This study investigated the relationship between the sex-different development of autistic features and CRMP4 deficiency. Whole-exome sequencing detected a de novo variant (S541Y) of CRMP4 in a male ASD patient. The expression of mutated mouse CRMP4S540Y, which is homologous to human CRMP4S541Y, in cultured hippocampal neurons derived from Crmp4-knockout (KO) mice had increased dendritic branching, compared to those transfected with wild-type (WT) Crmp4, indicating that this mutation results in altered CRMP4 function in neurons. Crmp4-KO mice showed decreased social interaction and several alterations of sensory responses. Most of these changes were more severe in male Crmp4-KO mice than in females. The mRNA expression levels of some genes related to neurotransmission and cell adhesion were altered in the brain of Crmp4-KO mice, mostly in a gender-dependent manner. These results indicate a functional link between a case-specific, rare variant of one gene, Crmp4, and several characteristics of ASD, including sexual differences.

The molecular pathogenesis of bipolar disorder (BPD) is poorly understood. Using human-induced pluripotent stem cells (hiPSCs) to unravel such mechanisms in polygenic diseases is generally challenging. However, hiPSCs from BPD patients responsive to lithium offered unique opportunities to discern lithium's target and hence gain molecular insight into BPD. By profiling the proteomics of BDP-hiPSC-derived neurons, we found that lithium alters the phosphorylation state of collapsin response mediator protein-2 (CRMP2). Active non-phosphorylated CRMP2, which binds cytoskeleton, is present throughout the neuron; inactive phosphorylated CRMP2, which dissociates from cytoskeleton, exits dendritic spines. CRMP2 elimination yields aberrant dendritogenesis with diminished spine density and lost lithium responsiveness (LiR). The "set-point" for the ratio of pCRMP2: CRMP2 is elevated uniquely in hiPSC-derived neurons from LiR BPD patients, but not with other psychiatric (including lithium-nonresponsive BPD) and neurological disorders. Lithium (and other pathway modulators) lowers pCRMP2, increasing spine area and density. Human BPD brains show similarly elevated ratios and diminished spine densities; lithium therapy normalizes the ratios and spines. Consistent with such "spine-opathies," human LiR BPD neurons with abnormal ratios evince abnormally steep slopes for calcium flux; lithium normalizes both. Behaviorally, transgenic mice that reproduce lithium's postulated site-of-action in dephosphorylating CRMP2 emulate LiR in BPD. These data suggest that the " lithium response pathway" in BPD governs CRMP2's phosphorylation, which regulates cytoskeletal organization, particularly in spines, modulating neural networks. Aberrations in the posttranslational regulation of this developmentally critical molecule may underlie LiR BPD pathogenesis. Instructively, examining the proteomic profile in hiPSCs of a functional agent-even one whose mechanism-of-action is unknown-might reveal otherwise inscrutable intracellular pathogenic pathways., Funding Agencies:NIH's Library of Integrated Network-based Cellular Signatures Program Viterbi Foundation Neuroscience Initiative Stanley Medical Research Institute R21MH093958 R33MH087896 R01MH095088 Tau Consortium California Institute of Regenerative Medicine training grants University of California, San Diego T32 training grant in psychiatry California Bipolar Foundation International Bipolar Foundation Creation of Innovation Centers for Advanced Interdisciplinary Research Areas Program in the Project for Developing Innovation Systems from the Ministry of Education, Science, Sports and Culture in Japan 42890001 RC2MH090011

Semaphorin3A (Sema3A) is a secreted type of axon guidance molecule that regulates axon wiring through complexes of neuropilin-1 (NRP1) with PlexinA protein receptors. Sema3A regulates the dendritic branching through tetrodotoxin (TTX)-sensitive retrograde axonal transport of PlexA proteins and tropomyosin-related kinase A (TrkA) complex. We here demonstrate that Nav1.7 (encoded by SCN9A), a TTX-sensitive Na+ channel, by coupling with collapsin response mediator protein 1 (CRMP1), mediates the Sema3A-induced retrograde transport. In mouse dorsal root ganglion (DRG) neurons, Sema3A increased co-localization of PlexA4 and TrkA in the growth cones and axons. TTX treatment and RNAi knockdown of Nav1.7 sustained Sema3A-induced colocalized signals of PlexA4 and TrkA in growth cones and suppressed the subsequent localization of PlexA4 and TrkA in distal axons. A similar localization phenotype was observed in crmp1-/- DRG neurons. Sema3A induced colocalization of CRMP1 and Nav1.7 in the growth cones. The half maximal voltage was increased in crmp1-/- neurons when compared to that in wild type. In HEK293 cells, introduction of CRMP1 lowered the threshold of co-expressed exogenous Nav1.7. These results suggest that Nav1.7, by coupling with CRMP1, mediates the axonal retrograde signaling of Sema3A.

Alzheimer’s disease (AD) is an incurable neurodegenerative disease characterized by memory loss and neurotoxic amyloid beta (Aβ) plaques accumulation. Numerous pharmacological interventions targeting Aβ plaques accumulation have failed to alleviate AD. Also, the pathological alterations in AD start years before the onset of clinical symptoms. To identify proteins at play during the early stage of AD, we conducted proteomic analysis of the hippocampus of young AppNL-F mice model of AD at the preclinical phase of the disease. This was followed by interactome ranking of the proteome into hubs that were further validated in vivo using immunoblot analysis. We also performed double-immunolabeling of these hub proteins and Aβ to quantify colocalization. Behavioral analysis revealed no significant difference in memory performance between 8-month-old AppNL-F and control mice. The upregulation and downregulation of several proteins were observed in the AppNL-F mice compared to control. These proteins corresponded to pathways and processes related to Aβ clearance, inflammatory-immune response, transport, mitochondrial metabolism, and glial cell proliferation. Interactome analysis revealed several proteins including DLGP5, DDX49, CCDC85A, ADCY6, HEPACAM, HCN3, PPT1 and TNPO1 as essential proteins in the AppNL-F interactome. Validation by immunoblot confirmed the over-expression of these proteins except HCN3 in the early-stage AD mice hippocampus. Immunolabeling revealed a significant increase in ADCY6/Aβ and HEPACAM/Aβ colocalized puncta in AppNL-F mice compared to WT. These data suggest that these proteins may be involved in the early stage of AD. Our work suggests new targets and biomarkers for AD diagnosis and therapeutic intervention.

Background and Purpose L-DOPA is generally considered to alleviate the symptoms of Parkinson's disease by its conversion to dopamine. We have proposed that DOPA is itself a neurotransmitter in the CNS. However, specific receptors for DOPA have not been identified. Recently, the gene product of ocular albinism 1 (OA1) was found to exhibit DOPA-binding activity. Here, we have investigated whether OA1 is a functional receptor of DOPA in the nucleus tractus solitarii (NTS). Experimental Approach We examined immunohistochemical expression of OA1 in the NTS, and the effects of DOPA microinjected into the depressor sites of NTS on blood pressure and heart rate in anaesthetized rats, with or without prior knock-down of OA1 in the NTS, using shRNA against OA1. Key Results Using a specific OA1 antibody, OA1-positive cells and nerve fibres were found in the depressor sites of the NTS. OA1 expression in the NTS was markedly suppressed by microinjection into the NTS of adenovirus vectors carrying the relevant shRNA sequences against OA1. In animals treated with OA1 shRNA, depressor and bradycardic responses to DOPA, but not those to glutamate, microinjected into the NTS were blocked. Bilateral injections into the NTS of DOPA cyclohexyl ester, a competitive antagonist against OA1, suppressed phenylephrine-induced bradycardic responses without affecting blood pressure responses. Conclusion and Implications OA1 acted as a functional receptor for DOPA in the NTS, mediating depressor and bradycardic responses. Our results add to the evidence for a central neurotransmitter role for DOPA, without conversion to dopamine.

Alzheimer's disease (AD) is characterized by amyloid-β (Aβ) protein and tau deposition in the brain. Numerous studies have reported a central role of Aβ 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 Aβ 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 Aβ₂₅₋₃₅ peptide, a neurotoxic fragment of Aβ protein, to wild-type (wt) mice increased hippocampal phosphorylation of CRMP2. Behavioral assessment revealed that i.c.v. injection of Aβ₂₅₋₃₅ 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 Aβ₂₅₋₃₅ peptide-treated wt but not those of crmp2(ki/ki). Our findings indicate that CRMP2 phosphorylation is required for Aβ-induced impairment of cognitive memory and synaptic plasticity.

There is increasing evidence that localized mRNAs in axons and growth cones play an important role in axon extension and pathfinding via local translation. A few studies have revealed the presence of microRNAs (miRNAs) in axons, which may control local protein synthesis during axon development. However, so far, there has been no attempt to screen for axon-enriched miRNAs and to validate their possible localization to growth cones of developing axons from neurons of the central nervous system. In this study, the localization of miRNAs in axons and growth cones in cortical neurons was examined using a "neuron ball" culture method that is suitable to prepare axonal miRNAs with high yield and purity. Axonal miRNAs prepared from the neuron ball cultures of mouse cortical neurons were analyzed by quantitative real-time RT-PCR. Among 375 miRNAs that were analyzed, 105 miRNAs were detected in axons, and six miRNAs were significantly enriched in axonal fractions when compared with cell body fractions. Fluorescence in situ hybridization revealed that two axon-enriched miRNAs, miR-181a-1* and miR-532, localized as distinct granules in distal axons and growth cones. The association of these miRNAs with the RNA-induced silencing complex further supported their function to regulate mRNA levels or translation in the brain. These results suggest a mechanism to localize specific miRNAs to distal axons and growth cones, where they could be involved in local mRNA regulation. These findings provide new insight into the presence of axonal miRNAs and motivate further analysis of their function in local protein synthesis underlying axon guidance.

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)P2] 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)P2 are the type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K) family, which consist of three isozymes, α, β and γ. Here, we demonstrate the involvement of PIP5Kβ in growth cone dynamics. Overexpression of a lipid kinase-deficient mutant of PIP5Kβ (PIP5Kβ-KD) in mouse dorsal root ganglion (DRG) neurons stimulated axon elongation and increased growth cone size, whereas wild-type PIP5Kβ tended to show opposite effects. Furthermore, PIP5Kβ-KD inhibited growth cone collapse of DRG neurons induced by semaphorin 3A (Sema3A). These results provide evidence that PIP5Kβ negatively regulates axon elongation and growth cone size and is involved in the cellular signaling pathway for Sema3A-triggered repulsion in DRG neurons.

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.

Mitral cells are major projection neurons of the olfactory bulb (OB) that form an axonal bundle known as the lateral olfactory tract (LOT). After axonal bundle formation, collateral branches sprout from primary axons of the LOT. Recently, we identified LOT usher substance (LOTUS) as an endogenous Nogo receptor-1 (NgR1) antagonist and demonstrated that LOTUS contributes to the formation of the LOT axonal bundle. Immunoblots revealed that the expression level of Nogo-A in the OB developmentally increased during axonal collateral formation. Next, we found that the axonal collateral branches were increased in cultured OB neurons from LOTUS-knockout (KO) mice, whereas they were decreased in cultured OB neurons from NgR1-KO mice. Knockdown of Nogo-A in cultured OB neurons reduced the number of axonal collateral branches, suggesting that endogenous Nogo-A induces axonal branching. Finally, the collateral branches of the LOT were increased in LOTUS-KO mice, whereas those in NgR1-KO mice were decreased. Moreover, the abnormal increase of axonal branching observed in LOTUS-KO mice was rescued in the double mutant of LOTUS- and NgR1-KO mice. These findings suggest that Nogo-A and NgR1 interactions may contribute to axonal branching in LOT development.

Elucidating the local function of proteins is essential for understanding not only the individual proteins but also the organization of the cell or even tissue as a whole. However, until now, few attempts have been made to understand local proteins function in cells because of a lack of acute inactivation technique of local proteins with high versatility. Here we describe the application of the chromophore-assisted light inactivation (CALI) method to elucidate the role of the semaphorin signaling component CRMP located within the growth cone area in axon growth and growth cone turning.

Collapsin response mediator protein 2 (CRMP2) plays a key role in axon guidance, dendritic morphogenesis and cell polarization. CRMP2 is implicated in various neurological and psychiatric disorders. However, in vivo functions of CRMP2 remain unknown. We generated CRMP2 gene-deficient (crmp2-/- ) mice and examined their behavioral phenotypes. During 24-h home cage monitoring, the activity level during the dark phase of crmp2-/- mice was significantly higher than that of wild-type (WT) mice. Moreover, the time during the open arm of an elevated plus maze was longer for crmp2-/- mice than for WT mice. The duration of social interaction was shorter for crmp2-/- mice than for WT mice. Crmp2-/- mice also showed mild impaired contextual learning. We then examined the methamphetamine-induced behavioral change of crmp2-/- mice. Crmp2-/- mice showed increased methamphetamine-induced ambulatory activity and serotonin release. Crmp2-/- mice also showed altered expression of proteins involved in GABAergic synapse, glutamatergic synapse and neurotrophin signaling pathways. In addition, SNAP25, RAB18, FABP5, ARF5 and LDHA, which are related genes to schizophrenia and methamphetamine sensitization, are also decreased in crmp2-/- mice. Our study implies that dysregulation of CRMP2 may be involved in pathophysiology of neuropsychiatric disorders.

Numerous cell adhesion molecules, extracellular matrix proteins and axon guidance molecules participate in neuronal network formation through local effects at axo-dendritic, axo-axonic or dendro-dendritic contact sites. In contrast, neurotrophins and their receptors play crucial roles in neural wiring by sending retrograde signals to remote cell bodies. Semaphorin 3A (Sema3A), a prototype of secreted type 3 semaphorins, is implicated in axon repulsion, dendritic branching and synapse formation via binding protein neuropilin-1 (NRP1) and the signal transducing protein PlexinAs (PlexAs) complex. This review focuses on Sema3A retrograde signaling that regulates dendritic localization of AMPA-type glutamate receptor GluA2 and dendritic patterning. This signaling is elicited by activation of NRP1 in growth cones and is propagated to cell bodies by dynein-dependent retrograde axonal transport of PlexAs. It also requires interaction between PlexAs and a high-affinity receptor for nerve growth factor, toropomyosin receptor kinase A. We propose a control mechanism by which retrograde Sema3A signaling regulates the glutamate receptor localization through trafficking of cis-interacting PlexAs with GluA2 along dendrites; this remote signaling may be an alternative mechanism to local adhesive contacts for neural network formation.

Collapsin response mediator protein 2, CRMP2, has been identified as an intracellular signaling mediator for Semaphorin 3A (Sema3A). CRMP2 plays a key role in axon guidance, dendritic morphogenesis, and cell polarization. It has been also implicated in a variety of neurological and psychiatric disorders. However, the in vivo functions of CRMP2 remain unknown. We generated CRMP2 gene-deficient (crmp2(-/-) ) mice. The crmp2(-/-) mice showed irregular development of dendritic spines in cortical neurons. The density of dendritic spines was reduced in the cortical layer V pyramidal neurons of crmp2(-/-) mice as well as in those of sema3A(-/-) and crmp1(-/-) mice. However, no abnormality was found in dendritic patterning in crmp2(-/-) compared to wild-type (WT) neurons. The level of CRMP1 was increased in crmp2(-/-) , but the level of CRMP2 was not altered in crmp1(-/-) compared to WT cortical brain lysates. Dendritic spine density and branching were reduced in double-heterozygous sema3A(+/-) ;crmp2(+/-) and sema3A(+/-) ;crmp1(+/-) mice. The phenotypic defects had no genetic interaction between crmp1 and crmp2. These findings suggest that both CRMP1 and CRMP2 mediate Sema3A signaling to regulate dendritic spine maturation and patterning, but through overlapping and distinct signaling pathways.

Neural circuit formation is a critical process in brain development. Axon guidance molecules, their receptors, and intracellular mediators are important to establish neural circuits. Collapsin response mediator proteins (CRMPs) are known intercellular mediators of a number of repulsive guidance molecules. Studies of mutant mice suggest roles of CRMPs in dendrite development. However, molecular mechanisms of CRMP-mediated dendritic development remain to elucidate. In this study, we show abnormal orientation of basal dendrites (extension to deeper side) of layer V pyramidal neurons in the cerebral cortex of CRMP4-/- mice. Moreover, we observed severe abnormality in orientation of the basal dendrites of these neurons in double knockout of CRMP1 and 4, suggesting redundant functions of these two genes. Redundant gene functions were also observed in proximal bifurcation phenotype in apical dendrites of hippocampal CA1 pyramidal neurons. These results indicate that CRMP1 and CRMP4 regulate proper orientation of the basal dendrites of layer V neurons in the cerebral cortex.

Semaphorin 3A (Sema3A), a secretory semaphorin, exerts various biological actions through a complex between neuropilin-1 and plexin-As (PlexAs). Sema3A induces retrograde signaling, which is involved in regulating dendritic localization of GluA2 (also known as GRIA2), an AMPA receptor subunit. Here, we investigated a possible interaction between retrograde signaling pathways for Sema3A and nerve growth factor (NGF). Sema3A induces colocalization of PlexA4 (also known as PLXNA4) signals with those of tropomyosin-related kinase A (TrkA, also known as NTRK1) in growth cones, and these colocalized signals were then observed along the axons. The time-lapse imaging of PlexA4 and several TrkA mutants showed that the kinase and dynein-binding activity of TrkA were required for Sema3A-induced retrograde transport of the PlexA4-TrkA complex along the axons. The inhibition of the phosphoinositide 3-kinase (PI3K)-Akt signal, a downstream signaling pathway of TrkA, in the distal axon suppressed Sema3A-induced dendritic localization of GluA2. The knockdown of TrkA suppressed Sema3A-induced dendritic localization of GluA2 and that suppressed Sema3A-regulated dendritic branching both in vitro and in vivo These findings suggest that by interacting with PlexA4, TrkA plays a crucial role in redirecting local Sema3A signaling to retrograde axonal transport, thereby regulating dendritic GluA2 localization and patterning.

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.

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. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2012

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.

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.

The ability to detect harmful chemicals rapidly is essential for the survival of all animals. InCaenorhabditis 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 aXenopus 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 ofC. elegans. dcar-1mutant animals are defective in avoidance response to DHCA, and cell-specific expression ofdcar-1in the ASH neurons ofdcar-1mutant 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, inC. 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.

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.

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.

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 plexin's 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.

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 Venus∷UNC-6. For example, in unc-51, unc-14, and unc-104 mutants, the neurons showed an abnormal accumulation of Venus∷UNC-6 in the cell body and less than normal level of Venus∷UNC-6 in the axon. An aberrant accumulation of Venus∷UNC-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.

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.

Calcium acts as an important second messenger in the intracellular signal pathways in a variety of cell functions. Strictly controlled intracellular calcium is required for proper neurite outgrowth of developing neurons. However, the molecular mechanisms of this process are still largely unknown. Neuronal calcium sensor-1 (NCS-1) is a high-affinity and low-capacity calcium binding protein, which is specifically expressed in the nervous system. NCS-1 was distributed throughout the entire region of growth cones located at a distal tip of neurite in cultured chick dorsal root ganglion neurons. In the central domain of the growth cone, however, NCS-1 was distributed in a clustered specific pattern and co-localized with the type 1 inositol 1,4,5-trisphosphate receptor (InsP(3)R1). The pharmacological inhibition of InsP(3) receptors decreased the clustered specific distribution of NCS-1 in the growth cones and inhibited neurite outgrowth but did not change the growth cone morphology. The acute and localized loss of NCS-1 function in the growth cone induced by chromophore-assisted laser inactivation (CALI) resulted in the growth arrest of neurites and lamellipodial and filopodial retractions. These findings suggest that NCS-1 is involved in the regulation of both neurite outgrowth and growth cone morphology. In addition, NCS-1 is functionally linked to InsP(3)R1, which may play an important role in the regulation of neurite outgrowth.

Collapsin response mediator protein 1 (CRMP1) is one of the CRMP family members that mediates signal transduction of axonal guidance and neuronal migration. We show here evidence that CRMP1 is involved in semaphorin3A (Sema3A)-induced spine development in the cerebral cortex. In the cultured cortical neurons fromcrmp1+/−mice, Sema3A increased the density of clusters of synapsin I and postsynaptic density-95, but this increase was markedly attenuated incrmp1−/−mice. This attenuation was also seen incyclin-dependent kinase 5(cdk5)−/−neurons. Furthermore, the introduction of wild-type CRMP1 but not CRMP1-T509A/S522A, (Thr 509 and Ser 522 were replaced by Ala), a mutant that cannot be phosphorylated by Cdk5, intocrmp1−/−neurons rescued the defect in Sema3A responsiveness. The Golgi-impregnation method showed that thecrmp1−/−layer V cortical neurons showed a lower density of synaptic bouton-like structures and that this phenotype had genetic interaction withsema3A. These findings suggest that Sema3A-induced spine development is regulated by phosphorylation of CRMP1 by Cdk5.

Netrin is an evolutionarily conserved axon guidance molecule that has both axonal attraction and repulsion activities. In Caenorhabditis elegans, Netrin/UNC-6 is secreted by ventral cells, attracting some axons ventrally and repelling some axons, which extend dorsally. One axon guided by UNC-6 is that of the HSN neuron. The axon guidance process for HSN neurons is complex, consisting of ventral growth, dorsal growth, branching, second ventral growth, fasciculation with ventral nerve cords, and then anterior growth. The vulval precursor cells (VPC) and the PVP and PVQ neurons are required for the HSN axon guidance; however, the molecular mechanisms involved are completely unknown. In this study, we found that the VPC strongly expressed UNC-6 during HSN axon growth. Silencing of UNC-6 expression in only the VPC, using a novel tissue-specific RNAi technique, resulted in abnormal HSN axon guidance. The expression of Netrin/UNC-6 by only the VPC in unc-6 null mutants partially rescued the HSN ventral axon guidance. Furthermore, the expression of Netrin/UNC-6 by the VPC and the ventral nerve cord (VNC) in unc-6 null mutants restored the complex HSN axon guidance. These results suggest that UNC-6 expressed by the VPC and the VNC cooperatively regulates the complex HSN axon guidance.