Clinical evidence that a dysregulated master neural network modulator may aid in diagnosing schizophrenia.

There are no validated biomarkers for schizophrenia (SCZ), a disorder linked to neural network dysfunction. We demonstrate that collapsin response mediator protein-2 (CRMP2), a master regulator of cytoskeleton and, hence, neural circuitry, may form the basis for a biomarker because its activity is uniquely imbalanced in SCZ patients. CRMP2's activity depends upon its phosphorylation state. While an equilibrium between inactive (phosphorylated) and active (nonphosphorylated) CRMP2 is present in unaffected individuals, we show that SCZ patients are characterized by excess active CRMP2. We examined CRMP2 levels first in postmortem brains (correlated with neuronal morphometrics) and then, because CRMP2 is expressed in lymphocytes as well, in the peripheral blood of SCZ patients versus age-matched unaffected controls. In the brains and, more starkly, in the lymphocytes of SCZ patients <40 y old, we observed that nonphosphorylated CRMP2 was higher than in controls, while phosphorylated CRMP2 remained unchanged from control. In the brain, these changes were associated with dendritic structural abnormalities. The abundance of active CRMP2 with insufficient opposing inactive p-CRMP2 yielded a unique lowering of the p-CRMP2:CRMP2 ratio in SCZ patients, implying a disruption in the normal equilibrium between active and inactive CRMP2. These clinical data suggest that measuring CRMP2 and p-CRMP2 in peripheral blood might reflect intracerebral processes and suggest a rapid, minimally invasive, sensitive, and specific adjunctive diagnostic aid for early SCZ: increased CRMP2 or a decreased p-CRMP2:CRMP2 ratio may help cinch the diagnosis in a newly presenting young patient suspected of SCZ (versus such mimics as mania in bipolar disorder, where the ratio is high).

De Novo Truncating Variants in the Last Exon of SEMA6B Cause Progressive Myoclonic Epilepsy.

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.

Right ventricular overloading is attenuated in monocrotaline-induced pulmonary hypertension model rats with a disrupted Gpr143 gene, the gene that encodes the 3,4-l-dihydroxyphenyalanine (l-DOPA) receptor.

Pulmonary hypertension (PH) is a severe and progressive disease that causes elevated right ventricular systolic pressure, right ventricular hypertrophy and ultimately right heart failure. However, the underlying pathophysiologic mechanisms are poorly understood. We previously showed that 3,4-l-dihydroxylphenyalanine (DOPA) sensitizes vasomotor response to sympathetic tone via coupling between the adrenergic receptor alpha1 (ADRA1) and a G protein-coupled receptor 143 (GPR143), a DOPA receptor. We investigated whether DOPA similarly enhances ADRA1-mediated contraction in pulmonary arteries isolated from rats, and whether GPR143 is involved in the PH pathogenesis. Pretreating the isolated pulmonary arteries with DOPA 1 µM enhanced vasoconstriction in response to phenylephrine, an ADRA1 agonist, but not to U-46619, a thromboxane A2 agonist or endothelin-1. We generated Gpr143 gene-deficient (Gpr143-/y) rats, and confirmed that DOPA did not augment phenylephrine-induced contractile response in Gpr143-/y rat pulmonary arteries. We utilized a rat model of monocrotaline (MCT)-induced PH. In the MCT model, the right ventricular systolic pressure was attenuated in the Gpr143-/y rats than in WT rats. Phenylephrine-induced cell migration and proliferation were also suppressed in Gpr143-/y pulmonary artery smooth muscle cells than in WT cells. Our result suggests that GPR143 is involved in the PH pathogenesis in the rat models of PH.

Storage-Related Changes in Autologous Whole Blood and Irradiated Allogeneic Red Blood Cells and Their Ex Vivo Effects on Deformability, Indices, and Density of Circulating Erythrocytes in Patients Undergoing Cardiac Surgery With Cardiopulmonary Bypass.

OBJECTIVES: Blood-processing techniques and preservation conditions cause storage lesions, possibly leading to adverse outcomes after transfusion. The authors investigated the metabolic changes and deformability of red blood cells (RBCs) during storage and determined the effect of storage lesions on circulating RBCs during cardiac surgery. DESIGN: Prospective study. SETTING: Tertiary care center affiliated with a university hospital. PARTICIPANTS: Adults who underwent elective cardiac surgery requiring cardiopulmonary bypass. INTERVENTIONS: The authors collected aliquots of autologous and irradiated allogeneic RBCs and blood samples from seven patients who received autologous whole blood and nine patients who received irradiated allogeneic RBCs before incision (baseline), at the start and end of cardiopulmonary bypass, and at completion of surgery. MEASUREMENTS AND MAIN RESULTS: The authors analyzed RBC deformability, erythrocyte indices, and density distribution to evaluate blood banking-induced alterations of autologous and allogeneic RBCs and changes in circulating RBCs in recipients, after blood transfusion. Time-dependent biochemical changes and significant decreases in deformability during storage occurred in both groups; however, homologous RBCs had significantly lower deformability than autologous RBCs. Trends in mean corpuscular volume and mean corpuscular hemoglobin concentration differed in both groups. In the homologous transfusion group, during cardiac surgery, RBC deformability, mean corpuscular volume, and mean corpuscular hemoglobin concentration showed significant changes compared with baseline values, and a greater number of denser subpopulations was observed at surgery completion. CONCLUSIONS: Blood-processing techniques contribute to storage lesions, suggesting that transfusion of autologous whole blood, rather than allogeneic RBCs, could maintain the ability of circulating RBCs to deform and lead to potentially better transfusion outcomes.

The Sympathetic Nervous System Contributes to the Establishment of Pre-Metastatic Pulmonary Microenvironments.

Emerging evidence suggests that neural activity contributes to tumor initiation and its acquisition of metastatic properties. More specifically, it has been reported that the sympathetic nervous system regulates tumor angiogenesis, tumor growth, and metastasis. The function of the sympathetic nervous system in primary tumors has been gradually elucidated. However, its functions in pre-metastatic environments and/or the preparation of metastatic environments far from the primary sites are still unknown. To investigate the role of the sympathetic nervous system in pre-metastatic environments, we performed chemical sympathectomy using 6-OHDA in mice and observed a decrease in lung metastasis by attenuating the recruitment of myeloid-derived suppressor cells. Furthermore, we note that neuro-immune cell interactions could be observed in tumor-bearing mouse lungs in conjunction with the decreased expression of Sema3A. These data indicate that the sympathetic nervous system contributes to the preparation of pre-metastatic microenvironments in the lungs, which are mediated by neuro-immune cell interactions.

Phosphorylation of Collapsin Response Mediator Protein 1 (CRMP1) at Tyrosine 504 residue regulates Semaphorin 3A-induced cortical dendritic growth.

Collapsin response mediator proteins (CRMPs) have been identified as mediating proteins of repulsive axon guidance cue Semaphorin-3A (Sema3A). Phosphorylation of CRMPs plays a crucial role in the Sema3A signaling cascade. It has been shown that Fyn phosphorylates CRMP1 at Tyrosine 504 residue (Tyr504); however, the physiological role of this phosphorylation has not been examined. We found that CRMP1 was the most strongly phosphorylated by Fyn among the five members of CRMPs. We confirmed Tyr504 phosphorylation of CRMP1 by Fyn. Immunocytochemistry of mouse dorsal root ganglion (DRG) neurons showed that phosphotyrosine signal in the growth cones was transiently increased in the growth cones upon Sema3A stimulation. Tyr504-phosphorylated CRMP1 also tended to increase after Sema3A simulation. Ectopic expression of a single amino acid mutant of CRMP1 replacing Tyr504 with phenylalanine (CRMP1-Tyr504Phe) suppressed Sema3A-induced growth cone collapse response in chick DRG neurons. CRMP1-Tyr504Phe expression in mouse hippocampal neurons also suppressed Sema3A but not Sema3F-induced growth cone collapse response. Immunohistochemistry showed that Tyr504-phosphorylated CRMP1 was present in the cell bodies and in the dendritic processes of mouse cortical neurons. CRMP1-Tyr504Phe suppressed Sema3A-induced dendritic growth of primary cultured mouse cortical neurons as well as the dendritic development of cortical pyramidal neurons in vivo. Fyn± ; Crmp1± double heterozygous mutant mice exhibited poor development of cortical layer V basal dendrites, which was the similar phenotype observed in Sema3a-/- , Fyn-/- , and Crmp1-/- mice. These findings demonstrate that Tyr504 phosphorylation of CRMP1 by Fyn is an essential step of Sema3A-regulated dendritic development of cortical pyramidal neurons. (247 words).

Activity-induced secretion of semaphorin 3A mediates learning.

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.

Reduction in flippase activity contributes to surface presentation of phosphatidylserine in human senescent erythrocytes.

Mature human erythrocytes circulate in blood for approximately 120 days, and senescent erythrocytes are removed by splenic macrophages. During this process, the cell membranes of senescent erythrocytes express phosphatidylserine, which is recognized as a signal for phagocytosis by macrophages. However, the mechanisms underlying phosphatidylserine exposure in senescent erythrocytes remain unclear. To clarify these mechanisms, we isolated senescent erythrocytes using density gradient centrifugation and applied fluorescence-labelled lipids to investigate the flippase and scramblase activities. Senescent erythrocytes showed a decrease in flippase activity but not scramblase activity. Intracellular ATP and K+ , the known influential factors on flippase activity, were altered in senescent erythrocytes. Furthermore, quantification by immunoblotting showed that the main flippase molecule in erythrocytes, ATP11C, was partially lost in the senescent cells. Collectively, these results suggest that multiple factors, including altered intracellular substances and reduced ATP11C levels, contribute to decreased flippase activity in senescent erythrocytes in turn to, present phosphatidylserine on their cell membrane. The present study may enable the identification of novel therapeutic approaches for anaemic states, such as those in inflammatory diseases, rheumatoid arthritis, or renal anaemia, resulting from the abnormally shortened lifespan of erythrocytes.

Probing the lithium-response pathway in hiPSCs implicates the phosphoregulatory set-point for a cytoskeletal modulator in bipolar pathogenesis.

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 nonphosphorylated 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.

Network-guided analysis of hippocampal proteome identifies novel proteins that colocalize with Aß in a mice model of early-stage Alzheimer's disease.

Alzheimer's disease (AD) is an incurable neurodegenerative disease characterized by memory loss and neurotoxic amyloid beta (Aß) plaques accumulation. Numerous pharmacological interventions targeting Aß 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 Aß 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 Aß 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/Aß and HEPACAM/Aß 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.

A functional coupling between CRMP1 and Nav1.7 for retrograde propagation of Semaphorin3A signaling.

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.

ATP11C T418N, a gene mutation causing congenital hemolytic anemia, reduces flippase activity due to improper membrane trafficking.

Distribution of phosphatidylserine (PS) in the erythrocyte membrane is essential for its activity. Flippase transports phospholipids from the outer to the inner leaflet of the lipid bilayer and maintains asymmetric distribution of phospholipids in the plasma membrane. ATP11C, a flippase, catalyzes PS flipping at the plasma membrane in association with cell cycle control protein 50A (CDC50A). ATP11C T418 N mutation causes 90% decrease in erythrocyte PS-flippase activity. However, the mechanism of the activity reduction remains unknown. To study the endogenous expression of ATP11C in erythrocytes, we produced a monoclonal antibody against human ATP11C. Immunoblotting analyses with this antibody revealed the absence of ATP11C in erythrocyte membranes derived from a patient with the T418 N mutation. Transiently expressed ATP11C wild-type in cultured cells localized in the cell membranes in the presence of CDC50A. Contrastingly, ATP11C T418 N mutants stacked at the endoplasmic reticulum (ER) even in the presence of CDC50A, suggesting improper intracellular trafficking. Expression of the T418 N mutant in cultured cells was lower than that in the wild-type. However, reduced expression of the T418 N mutant was partially restored by treatment with proteasome inhibitors, suggesting ER-associated degradation of the mutant protein. Cells expressing T418 N did not show flippase activity at the plasma membrane. These data show that the loss of PS-flippase activity in erythrocytes carrying ATP11C T418 N mutation is due to impaired enzymatic activity, improper membrane trafficking, and increased proteasome degradation.

Changes in Erythrocyte Morphology at Initiation of Cardiopulmonary Bypass Without Blood Transfusion Were Not Associated With Less Deformability During Cardiac Surgery.

OBJECTIVES: During cardiac surgery, circulating red blood cells (RBCs) are at risk of exposure to environmental factors during extracorporeal circulation and transfusion of stored RBCs. For this study, the authors observed morphological differences, deformability, density distribution, and erythrocyte indices of RBCs during cardiac surgery with cardiopulmonary bypass (CPB). DESIGN: Prospective study. SETTING: Tertiary care center affiliated with a university hospital. PARTICIPANTS: Adults who underwent elective cardiac surgery requiring CPB. INTERVENTIONS: Blood samples were obtained from 13 patients before incision (baseline), at initiation of CPB, after separation from CPB, and at completion of surgery. MEASUREMENTS AND MAIN RESULTS: The morphological index (MI) in RBCs using light microscopy and the maximum deformability index (DImax) using an ektacytometer were evaluated. In addition, the fractionation of RBCs and erythrocyte indices were measured. The MI at initiation of CPB was significantly higher without blood transfusion compared with baseline, although the DImax did not significantly decrease simultaneously. The DImax after separation from CPB and at completion of surgery were significantly lower than that at baseline. This lowered DImax was accompanied by a significantly reduced mean corpuscular volume and elevated mean corpuscular hemoglobin concentration compared with baseline. Dense RBC subpopulations increased after initiating CPB. The MI after separation from CPB and at completion of surgery partially recovered. Administered stored RBCs showed a high MI and the lowest DImax. CONCLUSIONS: Morphological changes at initiation of CPB are considered potentially reversible transformations without loss of the membrane surface area and do not have a significant effect on the DImax. A decrease in deformability likely is due to transfusion of stored RBCs.

Protein Tyrosine Phosphatase δ Mediates the Sema3A-Induced Cortical Basal Dendritic Arborization through the Activation of Fyn Tyrosine Kinase.

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.

CRMP1 and CRMP2 have synergistic but distinct roles in dendritic development.

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.

Comprehensive behavioral study and proteomic analyses of CRMP2-deficient mice.

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.

Perlecan Diversely Regulates the Migration and Proliferation of Distinct Cell Types in vitro.

Perlecan is a multifunctional component of the extracellular matrix. It shows different effects on distinct cell types, and therefore it is thought to show potential for therapies targeting multiple cell types. However, the full range of multifunctionality of perlecan remains to be elucidated. We cultured various cell types, which were derived from epithelial/endothelial, connective and muscle tissues, in the presence of either antiserum against perlecan or exogenous perlecan, and examined the effects of perlecan on cell migration and proliferation. Cell migration was determined using a scratch assay. Blocking of perlecan by anti-perlecan antiserum inhibited the migration of vascular endothelial cells (VECs) and bone marrow-derived mesenchymal stem cells, and exogenous perlecan added to the culture medium promoted the migration of these cell types. The migration of other cell types was inhibited or was not promoted by exogenous perlecan. Cell proliferation was measured using a water-soluble tetrazolium dye. When cells were cultured at low densities, perlecan blocking inhibited the proliferation of VECs, and exogenous perlecan promoted the proliferation of keratinocytes. In contrast, the proliferation of fibroblasts, pre-adipocytes and vascular smooth muscle cells cultured at low densities was inhibited by exogenous perlecan. When cells were cultured at high densities, perlecan blocking promoted the proliferation of most cell types, with the exception of skeletal system-derived cells (chondrocytes and osteoblasts), which were inhibited by exogenous perlecan. Our results provide an overview of the multiple functions of perlecan in various cell types, and implicate a potential role of perlecan to inhibit undesirable activities, such as fibrosis, obesity and intimal hyperplasia.

Cardiovascular actions of DOPA mediated by the gene product of ocular albinism 1.

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.

Phosphorylation of CRMP2 (collapsin response mediator protein 2) is involved in proper dendritic field organization.

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.

GSK3ß/axin-1/ß-catenin complex is involved in semaphorin3A signaling.

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-3ß (GSK3ß). To delineate how GSK3ß mediates Sema3A signaling, we here determined the substrates of GSK3ß involved. Introduction of either GSK3ß mutants, GSK3ß-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 GSK3ß. The phosphorylation of Axin-1 by GSK3ß accelerates the association of Axin-1 with ß-catenin. Immunocytochemical studies revealed that Sema3A induced an increase in the intensity levels of ß-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 GSK3ß, Axin-1, and ß-catenin in the growth cones. The increase of ß-catenin in the growth cones was suppressed by the siRNA knockdown of Axin-1. Furthermore, either Axin-1 or ß-catenin RNAi knockdown suppressed the internalization of Sema3A. These results suggest that Sema3A induces the formation of GSK3ß/Axin-1/ß-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.