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. PTP delta, 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 PTP delta in Sema3A-regualted axon guidance was minimal. Instead, PTP delta played a major role in Sema3A-dependent cortical dendritic growth. Ptp delta(-/-) 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, Ptp delta(+/-); Sema3a(+/-), also showed a similar phenotype, indicating the genetic interaction. In Ptp delta(-/-) 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 Ptp delta(-/-) Arborization of cortical basal dendrites was reduced in Fyn(-/-) as well as in Ptp delta(+/-); Fyn(+/-) double-heterozygous mutants. Collectively, PTP? mediates Sema3A-signaling through the activation of Fyn by C-terminal dephosphorylation.

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.

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, p27(Kip1) and upregulating cyclin D1 expression which are targets of RhoA signaling pathway. Our findings identify CRMP4 as a novel negative regulator of osteoblast differentiation. (c) 2016 American Society for Bone and Mineral Research.

Adult neurogenesis attracts broad attention as a possible cure for neurological disorders. However, its regulatory mechanism is still unclear. Therefore, they have been studying the cell proliferation mechanisms of neural stem cells (NSCs) using zebrafish, which have high regenerative potential in the adult brain. The presence of neuroepithelial-type NSCs in the optic tectum of adult zebrafish has been previously reported. In the present study, it was first confirmed that NSCs in the optic tectum decrease or increase in proportion to projection of the optic nerves from the retina. At 4 days after optic nerve crush (ONC), BrdU-positive cells decreased in the optic tectum's operation side. In contrast, at 3 weeks after ONC, BrdU-positive cells increased in the optic tectum's operation side. To study the regulatory mechanisms, they focused on the BDNF/TrkB system as a regulatory factor in the ONC model. It was found that bdnf was mainly expressed in the periventricular gray zone (PGZ) of the optic tectum by using in situ hybridization. Interestingly, expression level of bdnf significantly decreased in the optic tectum at 4 days after ONC, and its expression level tended to increase at 3 weeks after ONC. They conducted rescue experiments using a TrkB agonist and confirmed that decrease of NSC proliferation in the optic tectum by ONC was rescued by TrkB signal activation, suggesting stimuli-dependent regulation of NSC proliferation in the optic tectum of adult zebrafish. (C) 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 474-482, 2017.

Alzheimer's disease (AD) is the most common neurodegenerative disorder characterized by cognitive impairment with neuronal loss. The number of patients suffering from AD has increased, but none of the present therapies stops the progressive symptoms in patients with AD. It has been reported that the activation of microglial cells induces harmful chronic inflammation, leading to neuronal death. Furthermore, the impairment of adult neurogenesis in the hippocampus has been observed earlier than amyloid plaque formation. Inflammatory response may lead to impaired adult neurogenesis in patients with AD. This study examines the relationship between adult neurogenesis and neuroinflammation using APPswe/PS1M146V/tauP301L (3xTg) mice. We observed a decline in the proliferation of neural stem cells and the occurrence of severe inflammation in the hippocampus of 3xTg mouse brains at 12 months of age. Previously, our research had shown an anti-inflammatory effect of all-trans retinoic acid (ATRA) in the 3xTg mouse brain. We found that ATRA has effects on the recovery of proliferative cells along with suppression of activated microglia in the hippocampus. These results suggest that the inhibition of microglial activation by ATRA leads to recovery of adult neurogenesis in the hippocampus in an AD mouse model. (c) 2016 Wiley Periodicals, Inc.

The mammalian central nervous system (CNS) has limited regenerative ability after injury, largely due to scar formation and axonal growth inhibitors. Experimental suppression of neuroinflammation encourages recovery from spinal cord injury (SCI), yet practical means for pharmacologically treating SCI have remained elusive. Lanthionine ketimine (LK) is a natural brain sulfur amino acid metabolite with demonstrated anti-neuroinflammatory and neurotrophic activities. LK and its synthetic brainpenetrating ethyl ester (LKE) promote growth factor-dependent neurite extension in cultured cell and suppress microglial activation in animal models of neurodegeneration. Thus far however, LKE has not been explored as a potential therapy for SCI. The present study investigated the hypothesis that systemic LKE could improve motor functional recovery after SCI in a mouse model. Intraperitoneal administration of LKE (100 mg/kg/d) after near-complete transect of spinal cord at the T7 level significantly improved motor function over a 4-week time course. Vehicle-treated mice, in contrast, demonstrated negligible functional recovery. In terms of histology, LKE treatment reduced pro-neuroinflammatory microglia/ macrophage activation evidenced by quantitative Iba1 labeling and shifted the microglial phenotype toward a more neurotrophic M2 character evidenced by changes in the M2 marker arginase-1. This was correlated with less dense scar formation and more extensive axonal regrowth across the transection site demonstrated by 5-hydroxytryptamine (5HT) immunolabeling of raphespinal tract axons. These data provide evidence that LKE or similar compounds have potential therapeutic value for recovery after certain forms of SCI. (C) 2016 Elsevier Inc. All rights reserved.

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.

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. (C) 2016 Elsevier Inc. All rights reserved.

© 2016 Springer Science+Business Media New YorkNeurodegeneration in the adult mammalian central nervous system (CNS) is fundamentally accelerated by its intrinsic neuronal mechanisms, including its poor regenerative capacity and potent extrinsic inhibitory factors. Thus, the treatment of neurodegenerative diseases faces many obstacles. The degenerative processes, consisting of axonal/dendritic structural disruption, abnormal axonal transport, release of extracellular factors, and inflammation, are often controlled by the cytoskeleton. From this perspective, regulators of the cytoskeleton could potentially be a therapeutic target for neurodegenerative diseases and CNS injury. Collapsin response mediator proteins (CRMPs) are known to regulate the assembly of cytoskeletal proteins in neurons, as well as control axonal growth and neural circuit formation. Recent studies have provided some novel insights into the roles of CRMPs in several inhibitory signaling pathways of neurodegeneration, in addition to its functions in neurological disorders and CNS repair. Here, we summarize the roles of CRMPs in axon regeneration and its emerging functions in non-neuronal cells, especially in inflammatory responses. We also discuss the direct and indirect targeting of CRMPs as a novel therapeutic strategy for neurological diseases.

Parkinson's disease (PD) is a progressive neurodegenerative disorder that is characterized by the selective loss of dopaminergic neurons in the substantia nigra pars compacta (SNc). Several lines of evidence suggest that neurodegeneration in PD is accelerated by a vicious cycle in which apoptosis in dopaminergic neurons triggers the activation of microglia and harmful inflammatory processes that further amplify neuronal death. Recently, we demonstrated that the deletion of collapsin response mediator protein 4 (CRMP4) suppresses inflammatory responses and cell death in a mouse model of spinal cord injury, leading to improved functional recovery. We thus hypothesized that Crmp4-1 mice may have limited inflammatory responses and a decrease in the loss of SNc dopaminergic neurons in an 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. We observed CRMP4 expression in neurons, astrocytes, and microglia/macrophages following the injection of 25 mg/kg MPTP. We compared the number of dopaminergic neurons and the inflammatory response in SNc between Crmp4+I+ and Crmp4 l mice after MPTP injection. Limited loss of SNc dopaminergic neurons and decreased activations of microglia and astrocytes were observed in Crrnp4-1 mice. These results suggest that CRMP4 is a novel therapeutic target in the treatment of PD patients.

Alzheimer's disease (AD) is one of the best known neurodegenerative diseases; it causes dementia and its pathological features include accumulation of amyloid beta (A beta) and neurofibrillary tangles (NFTs) in the brain. Elevated Cdk5 activity and CRMP2 phosphorylation have been reported in the brains of AD model mice at the early stage of the disease, but the significance thereof in human AD remains unelucidated. We have recently reported that A beta accumulation in the cerebellum of AD model APPswe/PS1dE9 (APP/PS1) mice, and cerebellar dysfunctions, such as impairment of motor coordination ability and long-term depression (LTD) induction, at the pre-A beta accumulation stage.
In the present study, we found increased phosphorylation levels of CRMP2 as well as increased p35 protein levels in the cerebellum of APP/PS1 mice. Interestingly, we show that pioglitazone, an agonist of peroxisome proliferator-activated receptor gamma, normalized the p35 protein and CRMP2 phosphorylation levels in the cerebellum. Impaired motor coordination ability and LTD in APP/PS1 mice were ameliorated by pioglitazone treatment at the pre-A beta accumulation stage. These results suggest a correlation between CRMP2 phosphorylation and AD pathophysiology, and indicate the effectiveness of pioglitazone treatment at the pre-A beta accumulation stage in AD model mice. (C) 2016 Elsevier Inc. All rights reserved.

Cyclin-dependent kinase 5 (Cdk5) activity is dependent on its association with 1 of 2 neuron-specific activators, p35 or p39. Cdk5 and its activators play an important role in brain development as well as higher functions like synaptic plasticity, learning, and memory. Reduction in p35 was reported in postmortem schizophrenia brain, in which reduced dendritic spine density was observed. Previous in vitro experiments have shown that Cdk5 is involved in dendritic spine formation, although in vivo evidence is limited. We examined dendritic spine formation in inducible-p35 conditional knockout (p35 cKO); p39 KO mice. When we deleted the p35 gene either during early postnatal days or at adult stage, we observed reduced spine densities of layer V neurons in the cerebral cortex and CA1 pyramidal neurons in the hippocampus. We further generated CA1-specific p35 conditional knockout (CA1-p35 cKO) mice and also CA1-p35 cKO; p39 KO mice in which have specific deletion of p35 in the CA1 region of hippocampus. We found a greater reduction in spine densities in CA1 pyramidal neurons in CA1-p35 cKO; p39 KO mice than in CA1-p35 cKO mice. These results indicate that dendritic spine formation and neuronal maintenance are dependent on Cdk5 activity.

Central nervous system (CNS) regeneration is restricted by both the lack of neurotrophic responses and the presence of inhibitory factors. As of yet, a common mediator of these two pathways has not been identified. Microtubule dynamics is responsible for several key processes after CNS injuries: intracellular trafficking of receptors for neurotrophic factors, axonal retraction by inhibitory factors, and secondary tissue damages by inflammation and scarring. Kinases regulating microtubule organization, such as Cdk5 or GSK3 beta, may play pivotal roles during CNS recovery, but the molecular mechanisms remain to be elucidated. Collapsin response mediator protein 2 (CRMP2) stabilizes cytoskeletal polymerization, while CRMP2 phosphorylation by Cdk5 and GSK3 beta loses its affinity for cytoskeleton proteins, leading to the inhibition of axonal growth. Here, we characterized CRMP2 phosphorylation as the first crucial factor regulating neurotrophic and inhibitory responses after spinal cord injury (SCI). We found that pharmacological inhibition of GSK3 beta enhanced brain-derived neurotrophic factor (BDNF)-induced axonal growth response in cultured dorsal root ganglion (DRG) neurons. DRG neurons from CRMP2 knock-in (Crmp2KI/KI) mice, where CRMP2 phosphorylation was eliminated, showed elevated sensitivity to BDNF as well. Additionally, cultured Crmp2KI/KI neurons exhibited suppressed axonal growth inhibition by chondroitin sulfate proteoglycan (CSPG). These data suggest a couple of new molecular insights: the BDNF/GSK3 beta/CRMP2 and CSPG/GSK3 beta/CRMP2 pathways. Next we tested the significance of CRMP2 phosphorylation after CNS injury in vivo. The phosphorylation level of CRMP2 was enhanced in the injured spinal cord. Crmp2KI/KI mice exhibited prominent recovery of locomotive and nociceptive functions after SCI, which correlated with the enhanced axonal growth of the motor and sensory neurons. Neuroprotective effects against SCI, such as microtubule stabilization, reduced inflammation, and suppressed scarring were also observed by inhibiting CRMP2 phosphorylation. Therefore, inhibition of CRMP2 phosphorylation demonstrates the unique potential to repair SCI by both enhancing sensitivity to BDNF and reducing inhibitory responses. (C) 2016 Elsevier Inc. All rights reserved.

Alzheimer's disease (AD) is the most common type of dementia among the elderly. Neurofibrillary tangles (NFTs), a major pathological hallmark of AD, are composed of tau protein that is hyperphosphorylated by cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3 beta (GSK3 beta). NFTs also contain Wiskott-Aldrich syndrome protein family verprolin-homologous protein 1 (WAVE1) and collapsin response-mediator protein 2 (CRMP2). Although Cdk5 is known to phosphorylate tau, WAVE1, and CRMP2, the significance of this with respect to NFT formation remains to be elucidated. This study examines the involvement of phosphorylated (p-) CRMP2 and WAVE1 in p-tau aggregates using a triple-transgenic (3xTg; APPswe/PS1M146V/tauP301L) AD mouse model. First, we verified the colocalization of p- WAVE1 and p-CRMP2 with aggregated hyperphosphorylated tau in the hippocampus at 23 months of age. Biochemical analysis revealed the inclusion of p-WAVE1, p-CRMP2, and tau in the sarkosyl-insoluble fractions of hippocampal homogenates. To test the significance of phosphorylation of these proteins further, we administered all-trans-retinoic acid (ATRA) to the 3xTg mice, which downregulates Cdk5 and GSK3b activity. In ATRA-treated mice, fewer and smaller tau aggregates were observed compared with non-ATRA-treated mice. These results suggest the possibility of novel therapeutic target molecules for preventing tau pathology. (C) 2015 Wiley Periodicals, Inc.

Proper density and morphology of dendritic spines are important for higher brain functions such as learning and memory. However, our knowledge about molecular mechanisms that regulate the development and maintenance of dendritic spines is limited. We recently reported that cyclin-dependent kinase 5 (Cdk5) is required for the development and maintenance of dendritic spines of cortical neurons in the mouse brain. Previous in vitro studies have suggested the involvement of Cdk5 substrates in the formation of dendritic spines; however, their role in spine development has not been tested in vivo. Here, we demonstrate that Cdk5 phosphorylates collapsin response mediator protein 2 (CRMP2) in the dendritic spines of cultured hippocampal neurons and in vivo in the mouse brain. When we eliminated CRMP2 phosphorylation in CRMP2(KI/KI) mice, the densities of dendritic spines significantly decreased in hippocampal CA1 pyramidal neurons in the mouse brain. These results indicate that phosphorylation of CRMP2 by Cdk5 is important for dendritic spine development in cortical neurons in the mouse hippocampus.

Alzheimer's disease (AD) is the most common type of dementia among the elderly. Neurofibrillary tangles (NFTs), a major pathological hallmark of AD, are composed of tau protein that is hyperphosphorylated by cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3 beta (GSK3 beta). NFTs also contain Wiskott-Aldrich syndrome protein family verprolin-homologous protein 1 (WAVE1) and collapsin response-mediator protein 2 (CRMP2). Although Cdk5 is known to phosphorylate tau, WAVE1, and CRMP2, the significance of this with respect to NFT formation remains to be elucidated. This study examines the involvement of phosphorylated (p-) CRMP2 and WAVE1 in p-tau aggregates using a triple-transgenic (3xTg; APPswe/PS1M146V/tauP301L) AD mouse model. First, we verified the colocalization of p- WAVE1 and p-CRMP2 with aggregated hyperphosphorylated tau in the hippocampus at 23 months of age. Biochemical analysis revealed the inclusion of p-WAVE1, p-CRMP2, and tau in the sarkosyl-insoluble fractions of hippocampal homogenates. To test the significance of phosphorylation of these proteins further, we administered all-trans-retinoic acid (ATRA) to the 3xTg mice, which downregulates Cdk5 and GSK3b activity. In ATRA-treated mice, fewer and smaller tau aggregates were observed compared with non-ATRA-treated mice. These results suggest the possibility of novel therapeutic target molecules for preventing tau pathology. (C) 2015 Wiley Periodicals, Inc.

The zebrafish brain can continue to produce new neurons in widespread neurogenic brain regions throughout life. In contrast, neurogenesis in the adult mammalian brain is restricted to the subventricular zone (SVZ) and dentate gyrus (DG). In neurogenic regions in the adult brain, radial glial cells (RGCs) are considered to function as neural stem cells (NSCs). We generated a Tg(gfap:Gal4FF) transgenic zebrafish line, which enabled us to express specific genes in RGCs. To study the function of RGCs in neurogenesis in the adult zebrafish brain, we also generated a Tg(gfap: Gal4FF
UAS:nfsB-mcherry) transgenic zebrafish line, which allowed us to induce cell death exclusively within RGCs upon addition of metronidazole (Mtz) to the media. RGCs expressing nitroreductase were specifically ablated by the Mtz treatment, decreasing the number of proliferative RGCs. Using the Tg(gfap:Gal4FF
UAS:nfsB-mcherry) transgenic zebrafish line, we found that RGCs were specifically ablated in the adult zebrafish telencephalon. The Tg(gfap:Gal4FF) line could be useful to study the function of RGCs.

:Axonal outgrowth inhibitors and scar formation are two major obstacles to central nervous system (CNS) repair. No target molecule that regulates both axonal growth and scarring has been identified. Here we identified collapsin response mediator protein 4 (CRMP4), a common mediator of inhibitory signals after neural injury, as a crucial factor that contributes to both axonal growth inhibition and scarring after spinal cord injury (SCI). We found increases in the inhibitory and toxic forms of CRMP4 in injured spinal cord. Notably, CRMP4 expression was evident in inflammatory cells as well as in neurons after spinal cord transection. Crmp4-/- mice displayed neuroprotection against SCI and reductions in inflammatory response and scar formation. This permissive environment for axonal growth due to CRMP4 deletion restored locomotor activity at an unusually early phase of healing. These results suggest that deletion of CRMP4 is a unique therapeutic strategy that overcomes two obstacles to CNS repair after SCI.

The formation of the six-layered structure of the mammalian cortex via the inside-out pattern of neuronal migration is fundamental to neocortical functions. Extracellular cues such as Reelin induce intracellular signaling cascades through the protein phosphorylation. Migrating neurons also have intrinsic machineries to regulatecy to skeletal proteins and adhesion properties. Protein phosphorylation regulates these processes. Moreover, the balance between phosphorylation and dephosphorylation is modified by extracellular cues. Multipolar-bipolar transition, radial glia-guided locomotion and terminal translocation are critical steps of radial migration of cortical pyramidal neurons. Protein kinases such as Cyclin-dependent kinase 5 (Cdk5) and c-Jun N-terminal kinases (JNKs) involve these steps. In this review, I shall give an overview the roles of protein kinases in neuronal migration.

Background: Valproic acid (VPA) has been used to treat epilepsy and bipolar disorder. Several reports have demonstrated that VPA functions as a histone deacetylase (HDAC) inhibitor. While VPA is known to cause teratogenic changes in the embryonic zebrafish brain, its effects on neural stem cells (NSCs) in both the embryonic and adult zebrafish are not well understood. Results: In this study, we observed a proliferative effect of VPA on NSCs in the embryonic hindbrain. In contrast, VPA reduced cell proliferation in the adult zebrafish optic tectum. Treatment with HDAC inhibitors showed a similar inhibitory effect on cell proliferation in the adult zebrafish optic tectum, suggesting that VPA reduces cell proliferation through HDAC inhibition. Cell cycle progression was also suppressed in the optic tectum of the adult zebrafish brain because of HDAC inhibition. Recent studies have demonstrated that HDAC inhibits the Notch signaling pathway
hence, adult zebrafish were treated with a Notch inhibitor. This increased the number of proliferating cells in the adult zebrafish optic tectum with down-regulated expression of her4, a target of Notch signaling. Conclusions: These results suggest that VPA inhibits HDAC activity and upregulates Notch signaling to reduce cell proliferation in the optic tectum of adult zebrafish.

Previous studies have implicated the role of Purkinje cells in motor learning and the underlying mechanisms have also been identified in great detail during the last decades. Here we report that cyclin-dependent kinase 5 (Cdk5)/p35 in Purkinje cell also contributes to synaptic plasticity. We previously showed that p35(-/-) (p35 KO) mice exhibited a subtle abnormality in brain structure and impaired spatial learning and memory. Further behavioral analysis showed that p35 KO mice had a motor coordination defect, suggesting that p35, one of the activators of Cdk5, together with Cdk5 may play an important role in cerebellar motor learning. Therefore, we created Purkinje cell-specific conditional Cdk5/p35 knockout (L7-p35 cKO) mice, analyzed the cerebellar histology and Purkinje cell morphology of these mice, evaluated their performance with balance beam and rota-rod test, and performed electrophysiological recordings to assess long-term synaptic plasticity. Our analyses showed that Purkinje cell-specific deletion of Cdk5/p35 resulted in no changes in Purkinje cell morphology but severely impaired motor coordination. Furthermore, disrupted cerebellar long-term synaptic plasticity was observed at the parallel fiber-Purkinje cell synapse in L7-p35 cKO mice. These results indicate that Cdk5/p35 is required for motor learning and involved in long-term synaptic plasticity.

Neuronal migration is crucial for development of the mammalian-specific six-layered cerebral cortex. Migrating neurons are known to exhibit distinct features; they form a cytoplasmic dilation, a structure specific to migrating neurons, at the proximal region of the leading process, followed by nuclear elongation and forward movement. However, the molecular mechanisms of dilation formation and nuclear elongation remain unclear. Using ex vivo chemical inhibitor experiments, we show here that rottlerin, which is widely used as a specific inhibitor for PKCδ, suppresses the formation of a cytoplasmic dilation and nuclear elongation in cortical migrating neurons. Although our previous study showed that cortical neuronal migration depends on Jnk, another downstream target of rottlerin, Jnk inhibition disturbs only the nuclear elongation and forward movement, but not the dilation formation. We found that an unconventional cyclin-dependent kinase, Cdk5, is a novel downstream target of rottlerin, and that pharmacological or knockdown-mediated inhibition of Cdk5 suppresses both the dilation formation and nuclear elongation. We also show that Cdk5 inhibition perturbs endocytic trafficking as well as microtubule organization, both of which have been shown to be required for dilation formation. Furthermore, knockdown of Dcx, a Cdk5 substrate involved in microtubule organization and membrane trafficking, or p27(kip1), another Cdk5 substrate involved in actin and microtubule organization, disturbs the dilation formation and nuclear elongation. These data suggest that Cdk5 and its substrates, Dcx and p27(kip1), characterize migrating neuron-specific features, cytoplasmic dilation formation and nuclear elongation in the mouse cerebral cortex, possibly through the regulation of microtubule organization and an endocytic pathway.

Cdk5 is a member of the cyclin-dependent kinase (Cdk) family. In contrast to other Cdks that promote cell proliferation, Cdk5 plays a role in regulating various neuronal functions, including neuronal migration, synaptic activity, and neuron death. Cdks responsible for cell proliferation need phosphorylation in the activation loop for activation in addition to binding a regulatory subunit cyclin. Cdk5, however, is activated only by binding to its activator, p35 or p39. Furthermore, in contrast to Cdk1 and Cdk2, which are inhibited by phosphorylation at Tyr-15, the kinase activity of Cdk5 is reported to be stimulated when phosphorylated at Tyr-15 by Src family kinases or receptor-type tyrosine kinases. We investigated the activation mechanism of Cdk5 by phosphorylation at Tyr-15. Unexpectedly, however, it was found that Tyr-15 phosphorylation occurred only on monomeric Cdk5, and the coexpression of activators, p35/p25, p39, or Cyclin I, inhibited the phosphorylation. In neuron cultures, too, the activation of Fyn tyrosine kinase did not increase Tyr-15 phosphorylation of Cdk5. Further, phospho-Cdk5 at Tyr-15 was not detected in the p35-bound Cdk5. In contrast, expression of active Fyn increased p35 in neurons. These results indicate that phosphorylation at Tyr-15 is not an activation mechanism of Cdk5 but, rather, indicate that tyrosine kinases could activate Cdk5 by increasing the protein amount of p35. These results call for reinvestigation of how Cdk5 is regulated downstream of Src family kinases or receptor tyrosine kinases in neurons, which is an important signaling cascade in a variety of neuronal activities. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

The cyclin-dependent kinase 5 (Cdk5) is a neuron-specific Ser/Thr kinase that is activated by the regulatory subunit p35. Overactivation of Cdk5, which is induced by the cleavage of p35 by calpain, is implicated in neuronal death in various neurodegenerative diseases. In contrast, depletion of the Cdk5 activity renders neurons vulnerable to stresses. Recent reports suggest the involvement of Cdk5 in mental disorders. We hypothesized that perturbation of Cdk5 activity is related to mental conditions. To verify this hypothesis, we investigated the effect of valproic acid (VPA), which is a drug of choice for psychiatric disorders, on Cdk5 activity. VPA decreased the expression of p35 at both the protein and mRNA levels in cultured neurons, resulting in a decrease of Cdk5 activity. VPA decreased the p35 mRNA via histone deacetylase inhibition. The chronic administration of VPA also downregulated p35 in mouse brains. These results indicate that VPA regulates Cdk5 activity in neurons via p35 transcription mediated by HDAC inhibition. © 2014 Elsevier Inc. All rights reserved.

Background: Cyclin-dependent kinase 5 (Cdk5) is essential for brain development and function, and its deregulated expression is implicated in some of neurodegenerative diseases. We reported earlier that the forebrain-specific Cdk5 conditional knockout (cKO) mice displayed an early lethality associated with neuroinflammation, increased expression of the neuronal tissue-type plasminogen activator (tPA), and neuronal migration defects.
Methods: In order to suppress neuroinflammation in the cKO mice, we first treated these mice with pioglitazone, a PPAR gamma agonist, and analyzed its effects on neuronal loss and longevity. In a second approach, to delineate the precise role of tPA in neuroinflammation in these mice, we generated Cdk5 cKO; tPA double knockout (dKO) mice.
Results: We found that pioglitazone treatment significantly reduced astrogliosis, microgliosis, neuronal loss and behavioral deficit in Cdk5 cKO mice. Interestingly, the dKO mice displayed a partial reversal in astrogliosis, but they still died at early age, suggesting that the increased expression of tPA in the cKO mice does not contribute significantly to the pathological process leading to neuroinflammation, neuronal loss and early lethality.
Conclusion: The suppression of neuroinflammation in Cdk5 cKO mice ameliorates gliosis and neuronal loss, thus suggesting the potential beneficial effects of the PPAR gamma agonist pioglitazone for the treatment for neurodegenerative diseases.

:Cyclin-dependent kinase 5 (Cdk5) activity is dependent on its association with 1 of 2 neuron-specific activators, p35 or p39. Cdk5 and its activators play an important role in brain development as well as higher functions like synaptic plasticity, learning, and memory. Reduction in p35 was reported in postmortem schizophrenia brain, in which reduced dendritic spine density was observed. Previous in vitro experiments have shown that Cdk5 is involved in dendritic spine formation, although in vivo evidence is limited. We examined dendritic spine formation in inducible-p35 conditional knockout (p35 cKO); p39 KO mice. When we deleted the p35 gene either during early postnatal days or at adult stage, we observed reduced spine densities of layer V neurons in the cerebral cortex and CA1 pyramidal neurons in the hippocampus. We further generated CA1-specific p35 conditional knockout (CA1-p35 cKO) mice and also CA1-p35 cKO; p39 KO mice in which have specific deletion of p35 in the CA1 region of hippocampus. We found a greater reduction in spine densities in CA1 pyramidal neurons in CA1-p35 cKO; p39 KO mice than in CA1-p35 cKO mice. These results indicate that dendritic spine formation and neuronal maintenance are dependent on Cdk5 activity.

BACKGROUND:Cyclin-dependent kinase 5 (Cdk5), which is activated by binding to p35 or p39, is involved in synaptic plasticity and affects learning and memory formation. In Cdk5 knockout (KO) mice and p35 KO mice, brain development is severely impaired because neuronal migration is impaired and lamination is disrupted. To avoid these developmental confounders, we generated inducible CreER-p35 conditional (cKO) mice to study the role of Cdk5/p35 in higher brain function.;RESULTS:CreER-p35 cKO mice exhibited spatial learning and memory impairments and reduced anxiety-like behavior. These phenotypes resulted from a decrease in the dendritic spine density of CA1 pyramidal neurons and defective long-term depression induction in the hippocampus.;CONCLUSIONS:Taken together, our findings reveal that Cdk5/p35 regulates spatial learning and memory, implicating Cdk5/p35 as a therapeutic target in neurological disorders.

Background: Patent ductus arteriosus (PDA) is one of the most common congenital cardiovascular defects in children. The Brown-Norway (BN) inbred rat presents a higher frequency of PDA. A previous study reported that 2 different quantitative trait loci on chromosomes 8 and 9 were significantly linked to PDA in this strain. Nevertheless, the genetic or molecular mechanisms underlying PDA phenotypes in BN rats have not been fully investigated yet. Methods and Results: It was found that the elastic fibers were abundant in the subendothelial area but scarce in the media even in the closed ductus arteriosus (DA) of full-term BN neonates. DNA microarray analysis identified 52 upregulated genes (fold difference &gt
2.5) and 23 downregulated genes (fold difference &lt
0.4) when compared with those of F344 control neonates. Among these genes, 8 (Tbx20, Scn3b, Stac, Sphkap, Trpm8, Rup2, Slc37a2, and RGD1561216) are located in chromosomes 8 and 9. Interestingly, it was also suggested that the significant decrease in the expression levels of the PGE2-specfic receptor, EP4, plays a critical role in elastogenesis in the DA. Conclusions: BN rats exhibited dysregulation of elastogenesis in the DA. DNA microarray analysis identified the candidate genes including EP4 involved in the DNA phenotype. Further investigation of these newly identified genes will hopefully clarify the molecular mechanisms underlying the irregular formation of elastic fibers in PDA.

:Cyclin-dependent kinase 5 (Cdk5)-p35 is a proline-directed Ser/Thr kinase which plays a key role in neuronal migration, neurite outgrowth, and spine formation during brain development. Dynamic remodeling of cytoskeletons is required for all of these processes. Cdk5-p35 phosphorylates many cytoskeletal proteins, but it is not fully understood how Cdk5-p35 regulates cytoskeletal reorganization associated with neuronal migration. Since actin filaments are critical for the neuronal movement and process formation, we aimed to find Cdk5 substrates among actin-binding proteins. In this study, we isolated actin gels from mouse brain extracts, which contain many actin-binding proteins, and phosphorylated them by Cdk5-p35 in vitro. Drebrin, a side binding protein of actin filaments and well known for spine formation, was identified as a phosphorylated protein. Drebrin has two isoforms, an embryonic form drebrin E and an adult type long isoform drebrin A. Ser142 was identified as a common phosphorylation site to drebrin E and A and Ser342 as a drebrin A-specific site. Phosphorylated drebrin is localized at the distal area of total drebrin in the growth cone of cultured primary neurons. By expressing nonphosphorylatable or phosphorylation mimicking mutants in developing neurons in utero, the reversible phosphorylation/dephosphorylation reaction of drebrin was shown to be involved in radial migration of cortical neurons. These results suggest that Cdk5-p35 regulates neuronal migration through phosphorylation of drebrin in growth cone processes.

Alzheimer's disease (AD) is a neurodegenerative disorder that represents the most common type of dementia among elderly people. Amyloid beta (Aβ) peptides in extracellular Aβ plaques, produced from the amyloid precursor protein (APP) via sequential processing by β- and γ-secretases, impair hippocampal synaptic plasticity, and cause cognitive dysfunction in AD patients. Here, we report that Aβ peptides also impair another form of synaptic plasticity
cerebellar long-term depression (LTD). In the cerebellum of commonly used AD mouse model, APPswe/PS1dE9 mice, Aβ plaques were detected from 8 months and profound accumulation of Aβ plaques was observed at 18 months of age. Biochemical analysis revealed relatively high levels of APP protein and Aβ in the cerebellum of APPswe/PS1dE9 mice. At pre-Aβ accumulation stage, LTD induction, and motor coordination are disturbed. These results indicate that soluble Aβ oligomers disturb LTD induction and cerebellar function in AD mouse model. © 2014 International Society for Neurochemistry.

Circadian rhythm is a biological rhythm governing physiology and behavior with a period of approximate to 24 h. At the molecular level, circadian output is controlled by a molecular clock composed of positive and negative feedback loops in transcriptional and post-translational processes. CLOCK is a transcription factor known as a central component of the molecular clock feedback loops generating circadian oscillation. Although CLOCK is known to undergo multiple post-translational modifications, the knowledge of their entities remains limited. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine-threonine kinase that is involved in various neuronal processes. Here, we report that Cdk5 is a novel regulator of CLOCK protein. Cdk5 phosphorylates CLOCK at the Thr-451 and Thr-461 residues in association with transcriptional activation of CLOCK. The Cdk5-dependent regulation of CLOCK function is mediated by alterations of its stability and subcellular distribution. These results suggest that Cdk5 is a novel regulatory component of the core molecular clock machinery.

Alzheimer's disease (AD) is characterized by amyloid-beta (A beta) protein and tau deposition in the brain. Numerous studies have reported a central role of A beta in the development of AD, but the pathogenesis is not well understood. Collapsin response mediator protein 2 (CRMP2), an intracellular protein mediating a repulsive axon guidance molecule, Semaphorin3A, is also accumulated in neurofibrillary tangles in AD brains. To gain insight into the role of CRMP2 phosphorylation in AD pathogenesis, we investigated the effects of A beta neurotoxicity in CRMP2 phosphorylation-deficient knock-in (crmp2(ki/ki)) mice, in which the serine residue at 522 was replaced with alanine. Intracerebroventricular (i.c.v.) injection of A beta(25-35) peptide, a neurotoxic fragment of A beta protein, to wild-type (wt) mice increased hippocampal phosphorylation of CRMP2. Behavioral assessment revealed that i.c.v. injection of A beta(25-35) peptide caused impairment of novel object recognition in wt mice, while the same peptide did not in crmp2(ki/ki) mice. In electrophysiological recording, wt and crmp2(ki/ki) mice have similar input-output basal synaptic transmission and paired-pulse ratios. However, long-term potentiation was impaired in hippocampal slices of A beta(25-35) peptide-treated wt but not those of crmp2(ki/ki). Our findings indicate that CRMP2 phosphorylation is required for A beta-induced impairment of cognitive memory and synaptic plasticity. (C) 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Alzheimer's disease (AD) is characterized by amyloid-beta (A beta) protein and tau deposition in the brain. Numerous studies have reported a central role of A beta in the development of AD, but the pathogenesis is not well understood. Collapsin response mediator protein 2 (CRMP2), an intracellular protein mediating a repulsive axon guidance molecule, Semaphorin3A, is also accumulated in neurofibrillary tangles in AD brains. To gain insight into the role of CRMP2 phosphorylation in AD pathogenesis, we investigated the effects of A beta neurotoxicity in CRMP2 phosphorylation-deficient knock-in (crmp2(ki/ki)) mice, in which the serine residue at 522 was replaced with alanine. Intracerebroventricular (i.c.v.) injection of A beta(25-35) peptide, a neurotoxic fragment of A beta protein, to wild-type (wt) mice increased hippocampal phosphorylation of CRMP2. Behavioral assessment revealed that i.c.v. injection of A beta(25-35) peptide caused impairment of novel object recognition in wt mice, while the same peptide did not in crmp2(ki/ki) mice. In electrophysiological recording, wt and crmp2(ki/ki) mice have similar input-output basal synaptic transmission and paired-pulse ratios. However, long-term potentiation was impaired in hippocampal slices of A beta(25-35) peptide-treated wt but not those of crmp2(ki/ki). Our findings indicate that CRMP2 phosphorylation is required for A beta-induced impairment of cognitive memory and synaptic plasticity. (C) 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

The type 1 inositol 1,4,5- trisphosphate receptor (IP(3)R1) is a Ca2+ channel on the endoplasmic reticulum and is a predominant isoform in the brain among the three types of IP(3)Rs. Mice lacking IP(3)R1 show seizure-like behavior; however the cellular and neural circuit mechanism by which IP(3)R1 deletion causes the abnormal movements is unknown. Here, we found that the conditional knockout mice lacking IP(3)R1 specifically in the cerebellum and brainstem experience dystonia and show that cerebellar Purkinje cell (PC) firing patterns were coupled to specific dystonic movements. Recordings in freely behaving mice revealed epochs of low and high frequency PC complex spikes linked to body extension and rigidity, respectively. Remarkably, dystonic symptoms were independent of the basal ganglia, and could be rescued by inactivation of the cerebellum, inferior olive or in the absence of PCs. These findings implicate IP(3)R1-dependent PC firing patterns in cerebellum in motor coordination and the expression of dystonia through the olivo-cerebellar pathway.

The accessory olfactory bulb (AOB) is a critical olfactory structure that has been implicated in mediating social behavior. It receives input from the vomeronasal organ and projects to targets in the amygdaloid complex. Its anterior and posterior components (aAOB and pAOB) display molecular, connectional and functional segregation in processing reproductive and defensive and aggressive behaviors, respectively. We observed a dichotomy in the development of the projection neurons of the aAOB and pAOB in mice. We found that they had distinct sites of origin and that different regulatory molecules were required for their specification and migration. aAOB neurons arose locally in the rostral telencephalon, similar to main olfactory bulb neurons. In contrast, pAOB neurons arose caudally, from the neuroepithelium of the diencephalic-telencephalic boundary, from which they migrated rostrally to reach their destination. This unusual origin and migration is conserved in Xenopus, providing an insight into the origin of a key component of this system in evolution.

Background: Sphingolipids represent a major class of lipids which both serve as structural components of membranes and as bioactive molecules involved in lipid signaling. Ceramide synthases (cers) reside in the center of sphingolipid metabolism by producing ceramide through de novo synthesis or degradative pathways. While the six mammalian cers family members have been extensively studied in cell culture and in adult tissues, a systematic analysis of cers expression and function during embryogenesis is still lacking. Results: Using bioinformatic and phylogenetic analysis, we identified nine highly conserved homologs of the vertebrate cers gene family in the zebrafish genome. A systematic expression analysis throughout five developmental stages indicates that, whereas until 48 hours post fertilization most zebrafish cers homologs are expressed in distinct patterns, e.g., in the intermediate cell mass and the pronephric duct, they show a highly overlapping expression during later stages of embryonic development, mostprominently in the developing brain. Conclusions: In this study, the expression of the cers gene homologs is comprehensively analyzed for the first time during vertebrate embryogenesis. Our data indicate that each embryonic tissue has a unique profile of cers expression during zebrafish embryogenesis suggesting tissue-specific profiles of ceramides and their derivatives. © 2012 Wiley Periodicals, Inc.

Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase, and its kinase activity is dependent upon its association with either of the activating subunits p35 or p39, which are mainly expressed in neurons. We previously reported that Cdk5 knockout (KO) mice exhibit perinatal lethality, defective neuronal migration, and abnormal positioning of neurons in the facial motor nucleus and inferior olive in the hindbrain and Purkinje cells (PCs) in the cerebellum. In this study, we focused on the analysis of the role of Cdk5 in cerebellar development. For this purpose we generated midbrain-hindbrain-specific Cdk5 conditional knockout (MHB-Cdk5 KO) mice because the cerebellum develops postnatally, whereas Cdk5 KO mice die perinatally. Histological analysis of the MHB-Cdk5 KO mice revealed a significant size reduction of the cerebellum. In addition, profound disturbance of inward migration of granule cells (GC) was observed in the developing cerebellum. A normal dendritic development of the Purkinje cells (PCs) was disturbed in MHB-Cdk5 KO mice. Cultured Cdk5-null PCs showed similar dendritic abnormalities. These results indicate that Cdk5/p35 plays an important role in neuronal migration of PCs and GCs and dendrite formation of PCs in cerebellar development. (c) 2012 Elsevier Inc. All rights reserved.

: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.

:Dpysls (CRMPs) that were initially identified as mediator proteins of Semaphorin3a (Sema3a) signaling are involved in neuronal polarity and axon elongation in cultured neurons. Previous studies have shown that knockdown of neuropilin1a, one of the sema3a receptors, exhibited ectopic primary motor neurons (PMNs) outside of the spinal cord in zebrafish. However, downstream molecules of sema3a signaling involved in the positioning of motor neurons are largely unknown. Here, we addressed the role of Dpysl2 (CRMP2) and Dpysl3 (CRMP4) in the positioning of PMNs in the zebrafish spinal cord. We found that the knockdown of dpysls by antisense morpholino oligonucleotides (AMO) causes abnormal positioning of caudal primary (CaP) motor neurons outside the spinal cord. The knockdown of cdk5 and dyrk2 by AMO also caused similar phenotype in the positioning of CaP motor neurons, and this phenotype was rescued by co-injection of phosphorylation-mimic type dpysl2 mRNA. These results suggest that the phosphorylation of Dpysl2 and Dpysl3 by Cdk5 and Dyrk2 is required for correct positioning of CaP motor neurons in the zebrafish spinal cord.

The activities of CDK5 and p35 are thought to be important in the pathogenesis of neurodegenerative diseases, including Alzheimer's disease (AD). We studied the effect of p35 deletion in Tg2576 mice, which is an AD animal model. To obtain the desired mice, we crossed p35-/- with Tg2576 mice. The resulting p35-/-/Tg2576 (KO/Tg) mice displayed higher mortality rates and exhibited impaired spatial learning and memory at 6 months of age. Using immunohistochemical and biochemical approaches, we observed a reduction in the expression of pre- and post-synaptic markers such as NMDAR1, synaptophysin and GluR1. In addition, the intensity of MAP-2-positive dendrites extending from neuronal cell bodies was significantly decreased in KO/Tg mice compared with KO/WT and WT/Tg mice. We also detected increased neuronal cell death in the hippocampus, along with thinned and collapsed morphological changes in the alveus region and a dramatic increase in the number of microglial cells. Microglial infiltration in the hippocampus could result in the increased secretion of the soluble high mobility group box-1 protein (HMGB-1). The secretion of HMGB-1 is increased by Aβ, and secretion of HMGB-1 promotes neuronal cell death. Moreover, we found that HMGB-1 secretion induced by Aβ in KO/Tg mice gave rise to ER-mediated cell death. In summary, during the stages of KO/Tg mice model, the microglial infiltration and secretion of soluble HMGB-1 were significantly increased in the hippocampus. These conditions promote neuronal death, synaptic destruction and behavioral deficits. © 2013 Bentham Science Publishers.

Dpysl2 (CRMP2) and Dpysl3 (CRMP4) are involved in neuronal polarity and axon elongation in cultured neurons. These proteins are expressed in various regions of the developing nervous system, but their roles in vivo are largely unknown. In dpysl2 and dpysl3 double morphants, Rohon-Beard (RB) primary sensory neurons that were originally located bilaterally along the midline shifted their position to a more medial location in the dorsal-most part of spinal cord. A similar phenotype was observed in the cdk5 and dyrk2 double morphants. Dpysl2 and Dpysl3 phosphorylation mimics recovered this phenotype. Cell transplantation analysis demonstrated that this ectopic RB cell positioning was non-cell autonomous and correlated with the abnormal position of neural crest cells (NCCs), which also occupied the dorsal-most part of the spinal cord during the neural rod formation stage. The cell position of other interneuron and motor neurons within the central nervous system was normal in these morphants. These results suggest that the phosphorylation of Dpysl2 and Dpysl3 by Cdk5 and DYRK2 is required for the proper positioning of RB neurons and NCCs during neurulation in zebrafish embryos. © 2012 Elsevier Inc.

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. © 2012 Elsevier Ireland Ltd.

Kif26b, a member of the kinesin superfamily proteins (KIFs), is essential for kidney development. Kif26b expression is restricted to the metanephric mesenchyme, and its transcription is regulated by a zinc finger transcriptional regulator Sall1. However, the mechanism(s) by which Kif26b protein is regulated remain unknown. Here, we demonstrate phosphorylation and subsequent polyubiquitination of Kif26b in the developing kidney. We find that Kif26b interacts with an E3 ubiquitin ligase, neural precursor cell expressed developmentally down-regulated protein 4 (Nedd4) in developing kidney. Phosphorylation of Kif26b at Thr-1859 and Ser-1962 by the cyclin-dependent kinases (CDKs) enhances the interaction of Kif26b with Nedd4. Nedd4 polyubiquitinates Kif26b and thereby promotes degradation of Kif26b via the ubiquitin-proteasome pathway. Furthermore, Kif26b lacks ATPase activity but does associate with microtubules. Nocodazole treatment not only disrupts the localization of Kif26b to microtubules but also promotes phosphorylation and polyubiquitination of Kif26b. These results suggest that the function of Kif26b is microtubule-based and that Kif26b degradation in the metanephric mesenchyme via the ubiquitin-proteasome pathway may be important for proper kidney development.

:Collapsin response mediator proteins (CRMPs) are a family of cytosolic phosphoproteins that consist of 5 members (CRMP 1-5). CRMP2 and CRMP4 regulate neurite outgrowth by binding to tubulin heterodimers, resulting in the assembly of microtubules. CRMP2 also mediates the growth cone collapse response to the repulsive guidance molecule semaphorin-3A (Sema3A). However, the role of CRMP4 in Sema3A signaling and its function in the developing mouse brain remain unclear. We generated CRMP4-/- mice in order to study the in vivo function of CRMP4 and identified a phenotype of proximal bifurcation of apical dendrites in the CA1 pyramidal neurons of CRMP4-/- mice. We also observed increased dendritic branching in cultured CRMP4-/- hippocampal neurons as well as in cultured cortical neurons treated with CRMP4 shRNA. Sema3A induces extension and branching of the dendrites of hippocampal neurons; however, these inductions were compromised in the CRMP4-/- hippocampal neurons. These results suggest that CRMP4 suppresses apical dendrite bifurcation of CA1 pyramidal neurons in the mouse hippocampus and that this is partly dependent on Sema3A signaling.

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 (crmp2(ki/ki)) in which the Ser residue at 522 was replaced with Ala. Strikingly, the cortical basal dendrites of double mutant crmp2(ki/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.

Cyclin-dependent kinase 5 (Cdk5) plays a pivotal role in neuronal migration and differentiation, and in axonal elongation. Although many studies have been conducted to analyze neuronal functions of Cdk5, its kinase activity has also been reported during oligodendrocyte differentiation, which suggests Cdk5 may play an important role in oligodendrocytes. Here, we describe a hypomyelination phenotype observed in Emx1-cre mediated Cdk5 conditional knockout (cKO) mice (Emx1-cKO), in which the Cdk5 gene was deleted in neurons, astrocytes and oligodendrocyte -lineage cells. In contrast, the Cdk5 gene in CaMKII cKO mice was deleted only in neurons. Because the development of mature oligodendrocytes from oligodendrocyte precursor cells is a complex process, we performed in situ hybridization using markers for the oligodendrocyte precursor cell and for the differentiated oligodendrocyte. Our results indicate that hypomyelination in Emx1-cKO is due to the impaired differentiation of oligodendrocytes, rather than to the proliferation or migration of their precursors. The present study confirmed the in vivo role of Cdk5 in oligodendrocyte differentiation. © 2011 Springer Science+Business Media, LLC.

We isolated a novel zebrafish mutant, lullaby (llb), and showed that the llb locus encodes the zebrafish orthologue of isl1. Rohon-Beard (RB) primary sensory neurons are multipolar neurons that extend their central axons longitudinally within the spinal cord and also extend their peripheral axons under the skin. In llb embryos, the outgrowth of the peripheral axons of RB neurons was selectively impaired, which correlated with down-regulation of the expression of dihydropyrimidinase-like 3 (dpysl3, also known as collapsin response mediator protein 4, crmp4). Antisense morpholino oligonucleotide (AMO)-mediated knockdown of dpysl3 inhibited the outgrowth of the peripheral axons of RB neurons, and semaphorin 3d (sema3d) AMO enhanced this effect. These data indicate that Dpysl3 is cooperating with Sema3d in the peripheral axon outgrowth, and Isl1 is required for the selective outgrowth of the peripheral axons of RB neurons by maintaining the expression of dpysl3. Developmental Dynamics 240: 9-22, 2011. (C) 2010 Wiley-Liss, Inc.

In the adult teleost brain, proliferating cells are observed in a broad area, while these cells have a restricted distribution in adult mammalian brains. In the adult teleost optic tectum, most of the proliferating cells are distributed in the caudal margin of the periventricular gray zone (PGZ). We found that the PGZ is largely divided into 3 regions: 1 mitotic region and 2 post-mitotic regions-the superficial and deep layers. These regions are distinguished by the differential expression of several marker genes: pcna, sox2, msi1, elavl3, gfap, fabp7a, and s100 beta. Using transgenic zebrafish Tg (gfap:GFP), we found that the deep layer cells specifically express gfap:GFP and have a radial glial morphology. We noted that bromodeoxyuridine (BrdU)-positive cells in the mitotic region did not exhibit glial properties, but maintained neuroepithelial characteristics. Pulse chase experiments with BrdU-positive cells revealed the presence of self-renewing stem cells within the mitotic region. BrdU-positive cells differentiate into glutamatergic or GABAergic neurons and oligodendrocytes in the superficial layer and into radial glial cells in the deep layer. These results demonstrate that the proliferating cells in the PGZ contribute to neuronal and glial lineages to maintain the structure of the optic tectum in adult zebrafish. (C) 2010 Elsevier Inc. All rights reserved.

Apoptosis-associated tyrosine kinase 1 (AATYK1), a novel serine/threonine kinase that is highly expressed in the brain, is involved in neurite extension and apoptosis of cerebellar granule neurons; however, its precise function remains unknown. In this study, we investigated the interaction of AATYK1A with Cyclin-dependent kinase 5 (Cdk5)/p35, a proline-directed protein kinase that is predominantly expressed in neurons. AATYK1A bound to the p35 activation subunit of Cdk5 in cultured cells and in mouse brains and colocalized with p35 on endosomes in COS-7 cells. AATYK1A was phosphorylated at Ser34 by Cdk5/p35 in vitro, in cultured neurons and in mouse brain. In PC12D cells, Ser34 phosphorylation increased after treatment with nerve growth factor and phosphorylated AATYK1A accumulated in growth cones of PC12D cells. Ser34 phosphorylation suppressed the tyrosine phosphorylation of AATYK1A by Src family kinases. These results suggest a possibility that AATYK1A plays a role in early to recycling endosomes and its function is regulated by phosphorylation with Cdk5 or Src-family kinases.

Neuronal migration disorders are often identified in patients with epilepsy refractory to medical treatment. The prolonged or repeated seizures are known to cause neuronal death; however, the mechanism underlying seizure-induced neuronal death remains to be elucidated. An essential role of cyclin-dependent kinase 5 (Cdk5) in brain development has been demonstrated in Cdk5(-/-) mice, which show neuronal migration defects and perinatal lethality. Here, we show the consequences of Cdk5 deficiency in the postnatal brain by generating Cdk5 conditional knockout mice, in which Cdk5 is selectively eliminated from neurons in the developing forebrain. The conditional mutant mice were viable, but exhibited complex neurological deficits including seizures, tremors, and growth retardation. The forebrain not only showed disruption of layering, but also neurodegenerative changes accompanied by neuronal loss and microglial activation. The neurodegenerative changes progressed with age and were accompanied by up-regulation of the neuronal tissue-type plasminogen activator, a serine protease known to mediate microglial activation. Thus age-dependent neurodegeneration in the Cdk5 conditional knockout mouse brain invoked a massive inflammatory reaction. These findings indicate an important role of Cdk5 in inflammation, and also provide a mouse model to examine the possible involvement of inflammation in the pathogenesis of progressive cognitive decline in patients with neuronal migration disorders. (Am J Pathol 2010, 176:320-329; DOI: 10.2353/ajpath.2010.081158)

:In the adult teleost brain, proliferating cells are observed in a broad area, while these cells have a restricted distribution in adult mammalian brains. In the adult teleost optic tectum, most of the proliferating cells are distributed in the caudal margin of the periventricular gray zone (PGZ). We found that the PGZ is largely divided into 3 regions: 1 mitotic region and 2 post-mitotic regions-the superficial and deep layers. These regions are distinguished by the differential expression of several marker genes: pcna, sox2, msi1, elavl3, gfap, fabp7a, and s100beta. Using transgenic zebrafish Tg (gfap:GFP), we found that the deep layer cells specifically express gfap:GFP and have a radial glial morphology. We noted that bromodeoxyuridine (BrdU)-positive cells in the mitotic region did not exhibit glial properties, but maintained neuroepithelial characteristics. Pulse chase experiments with BrdU-positive cells revealed the presence of self-renewing stem cells within the mitotic region. BrdU-positive cells differentiate into glutamatergic or GABAergic neurons and oligodendrocytes in the superficial layer and into radial glial cells in the deep layer. These results demonstrate that the proliferating cells in the PGZ contribute to neuronal and glial lineages to maintain the structure of the optic tectum in adult zebrafish.

:Doublecortin (DCX) is expressed in young neurons and functions as a microtubule-associated protein. DCX is essential for neuronal migration because humans with mutations in the DCX gene exhibit cortical lamination defects known as lissencephaly in males and subcortical laminar heterotopia (or double cortex syndrome) in females. Phosphorylation of DCX alters its affinity for tubulin and may modulate neurite extension and neuronal migration. Previous in vitro phosphorylation experiments revealed that cyclin-dependent kinase 5 (Cdk5) phosphorylates multiple sites of DCX, including Ser332, (S332). However, phosphorylation at only Ser297 has been shown in vivo. In the present study, we examined phosphorylation of S332 of DCX in the Cdk5-/- mouse brain and results found, unexpectedly, indicate an increased DCX phosphorylation at S332. We found that JNK, not Cdk5, phosphorylates DCX at S332 in vivo. To examine the physiological significance of S332 phosphorylation of DCX in neuronal cells, we transfected cells with either GFP, GFP-DCX-WT, or GFP-DCX-S332A and analyzed neurite extension and migration. Introduction of GFP-DCX-WT enhanced neurite extension and migration. These effects of DCX introduction were suppressed when we used GFP-DCX-S332A. Treatment of neurons with JNK inhibitor increased the amount of DCX that bound to tubulin. Interestingly, amount of DCX that bound to tubulin decreased in Cdk5-/- brain homogenates, which indicates that phosphorylation of DCX by JNK is critical for the regulation of DCX binding to tubulin. These results suggest the physiological importance of phosphorylation of DCX for its function.

The mammalian cerebral cortex consists of six layers that are generated via coordinated neuronal migration during the embryonic period. Recent studies identified specific phases of radial migration of cortical neurons. After the final division, neurons transform from a multipolar to a bipolar shape within the subventricular zone-intermediate zone (SVZ-IZ) and then migrate along radial glial fibres. Mice lacking Cdk5 exhibit abnormal corticogenesis owing to neuronal migration defects. When we introduced GFP into migrating neurons at E14.5 by in utero electroporation, we observed migrating neurons in wild-type but not in Cdk5(-/-) embryos after 3-4 days. Introduction of the dominant-negative form of Cdk5 into the wild-type migrating neurons confirmed specific impairment of the multipolar-to-bipolar transition within the SVZ-IZ in a cell-autonomous manner. Cortex-specific Cdk5 conditional knockout mice showed inverted layering of the cerebral cortex and the layer V and callosal neurons, but not layer VI neurons, had severely impaired dendritic morphology. The amount of the dendritic protein Map2 was decreased in the cerebral cortex of Cdk5-deficient mice, and the axonal trajectory of cortical neurons within the cortex was also abnormal. These results indicate that Cdk5 is required for proper multipolar-to-bipolar transition, and a deficiency of Cdk5 results in abnormal morphology of pyramidal neurons. In addition, proper radial neuronal migration generates an inside-out pattern of cerebral cortex formation and normal axonal trajectories of cortical pyramidal neurons.

The serine/threonine kinase p21-activated kinase 1 (Pak1) modulates actin and microtubule dynamics. The neuronal functions of Pak1, despite its abundant expression in the brain, have not yet been fully delineated. Previously, we reported that Pak1 mediates initiation of dendrite formation. In the present study, the role of Pak1 in dendritogenesis, spine formation and maintenance was examined in detail. Overexpression of constitutively active-Pak1 in immature cortical neurons increased not only the number of the primary branching on apical dendrites but also the number of basal dendrites. In contrast, introduction of dominant negative-Pak caused a reduction in both of these morphological features. The length and the number of secondary apical branch points of dendrites were not significantly different in cultured neurons expressing these mutant forms, suggesting that Pak1 plays a role in dendritogenesis. Pak1 also plays a role in the formation and maintenance of spines, as evidenced by the altered spine morphology, resulting from overexpression of mutant forms of Pak1 in immature and mature hippocampal neurons. Thus, our results provide further evidence of the key role of Pak1 in the regulation of dendritogenesis, dendritic arborization, the spine formation, and maintenance. (c) 2007 Wiley Periodicals, Inc.

The Reelin signaling and Cyclin-dependent kinase 5 (Cdk5) both regulate neuronal positioning in the developing brain. Using double-transgenic mice, we have previously shown that these two signaling pathways lie in parallel fashion and have a genetic interaction. Disabled-1 (Dab1), an adapter protein, mediates Reelin signaling and becomes tyrosine-phosphorylated on the binding of Reelin to its receptors. Several isoforms of Dab1 are expressed in embryonic mouse brain, and p80 [Dab1(S55)] is the major protein translated. In the present study, we investigated whether CdkS-mediated phosphorylation of Dab1 modulates Reelin signaling. Cdk5 phosphorylates p80 Dabl at multiple sites in its carboxyl terminal region, and tyrosine phosphorylation of p80 Dabl by Fyn tyrosine kinase is attenuated by this Cdk5-mediated phosphorylation in vitro. Tyrosine phosphorylation. of p80 Dab1 induced by exogenous Reelin is enhanced in Cdk5-deficient neurons, corroborating the inhibitory effect of Cdk5-mediated Ser/Thr phosphorylation on tyrosine phosphorylation of p80 Dabl. Another isoform, p45 Dabl [Dabl(271)], however, is phosphorylated by CdkS at one serine residue within a unique carboxyl-terminal region, and its serine phosphorylation enhances tyrosine phosphorylation. by Fyn and results in progressive degradation of p45 Dabl. These results indicate that Cdk5 modulates Reelin signaling through the Ser/Thr phosphorylation. of Dabl differently in an isoform-specific manner. (c) 2006 Elsevier B.V. All rights reserved.

Regulation of cytoskeletal dynamics is essential to neuronal plasticity during development and adulthood. Dysregulation of these mechanisms may contribute to neuropsychiatric and neurodegenerative diseases. The neuronal protein kinase, cyclin-dependent kinase 5 (Cdk5), is involved in multiple aspects of neuronal function, including regulation of cytoskeleton. A neuroproteomic search identified the tubulin-binding protein, stathmin, as a novel Cdk5 substrate. Stathmin was phosphorylated by Cdk5 in vitro at Ser25 and Ser38, previously identified as mitogen-activated protein kinase (MAPK) and p38 MAPK delta sites. Cdk5 predominantly phosphorylated Ser38, while MAPK and p38 MAPK delta predominantly phosphorylated Ser25. Stathmin was phosphorylated at both sites in mouse brain, with higher levels in cortex and striatum. Cdk5 knockout mice exhibited decreased phospho-Ser38 levels. During development, phospho-Ser25 and -Ser38 levels peaked at post-natal day 7, followed by reduction in total stathmin. Inhibition of protein phosphatases in striatal slices caused an increase in phospho-Ser25 and a decrease in total stathmin. Interestingly, the prefrontal cortex of schizophrenic patients had increased phospho-Ser25 levels. In contrast, total and phospho-Ser25 stoichiometries were decreased in the hippocampus of Alzheimer's patients. Thus, microtubule regulatory mechanisms involving the phosphorylation of stathmin may contribute to developmental synaptic pruning and structural plasticity, and may be involved in neuropsychiatric and neurodegenerative disorders.

In developing brain, axon and dendritic guidance are regulated by repulsive and attractive axon guidance molecules such as semaphorin3A (Sema3A) and netrin. Collpapsin response mediator protein (CRMP) has originally been identified as an intracellular protein that mediates Sema3A. We found that Sema3A elicits axoplasmic transport that may be involved in regulating the localization of AMPA type glutamate receptors in hippocampal neurons. To elucidate in vivo role of CRMPs, we generated several crmp1 and other crmp family gene-deficient mice and performed phenotypic analysis of these mice. For instance, in crmp1-deficient mice, the cell migration of cortical neurons at early embryonic stages is retarded. CRMP1 is colocalized with disabled-1 (Dab1), an adaptor protein in Reln signaling. In the Relnrl/rl cortex, CRMP1 and Dab1 are expressed at a higher level, yet tyrosine phosphorylated at a lower level. Loss of crmp1 in a dab1 heterozygous background lead to the disruption of hippocampal lamination, a Reeler-like phenotype. CRMP1 is also involved in Sema3A-induced localization of AMPA receptors and spine development in the cerebral cortex. In the cultured cortical neurons from crmp1 mice, Sema3A increases the density of clusters of synapsin I and postsynaptic density-95, but this increase is markedly attenuated in crmp1-deficient mice. In our study of C. elegans, we identified several mutant alleles which show aberrant localization of netrin/UNC-6 and axon guidance defects. We therefore conclude that the regulation of the glutamate receptor and the axon guidance molecule localization may play an important role in a wide variety of developmental processes from cell migration to neural network formation

Purpose and Method : In this proposal, I attempt to understand the mechanism of brain development by analyzing protein phosphorylations which are important for such processes. Comparison of phospho-proteins between controls and mutant mice which have defective brain development will provide us new findings about molecular mechanism of brain development. For this purpose, we conducted 2D-DIGE method followed by MS analysis to identify the proteins.Results: In 2006, we analyzed the cerebral cortex samples from Cdk5 KO mice and their littermate controls at E 18.5. Cdk5 is a neuron-specific Ser/Thr protein kinase and Cdk5 KO mice exhibit defective brain development particularly in layer formation of cortical structures. We conducted protetomics of phospho-proteins by 2D-DIGE method, and identified CRMP1, 2 and 4 among 23 differential spots. We previously reported that Cdk5 phosphorylates CRMP2 at Ser522. We confirmed that Cdk5 phosphorylates CRMP1 at Thr 509 in vivo using phosphor-specific antibody for pThr509 CRMP1. However, we couldn't detect the reduction of phosphorylation of CRMP4 Ser522, indicating the redundant phosphorylation by other kinase (s). We also found the reduced phosphorylation of stathmin Ser38 (Hayashi, et. al., 2006).In 2007, we analyzed cerebella from Dab 1 mutant yotari and cerebellum-specific Cdk5 KO mice along with their littermate controls. We identified several differential spots and further analysis of these spots is now on going in our laboratory. We also reported that Cdk5 inhibits Reelin signaling through the phosphorylation of Dabl (Ohshima, et. al., 2007)

Cyclin-dependent kinase 5 (Cdk5)は、他のCdkが分裂中の細胞に活性が高いのに対して、activating subunit p35, p39が神経細胞に特異的に発現しているため、神経細胞に活性を認めるユニークなCdkである。我々のp35の欠損マウスの解析から、空間学習・記憶にCdk5が必須であり、LTDの誘導に何らかの関与がある可能性が示唆された(Ohshima, et. al., 2005)。Cdk5の誘導型コンディショナルKOのライン用いて、Cdk5の記憶・学習などの脳機能における役割を明らかにする事を計画していたが、米国のBibb博士らの研究グループから、Cdk5の誘導型コンディショナルノックアウトを解析した結果の報告がなされた(Nat. Neurosci. 10:880-886, 2007)。こうした現状を踏まえて、領域特異的なコンディショナルKOの解析を行なう事とし、Emx1Creマウスを用いたコンディショナルKO (ECKO)マウスを作成した。まず、Cdk5欠損神経細胞の放射状神経細胞の移動を解析したところ、細胞移動中に観察される双極性(bipolar)の形態から多極性(multipolar)への変換が障害されている事が明らかとなった。さらに、ECKOの解析により、こうした異常が生後脳において、結果的に大脳皮質の錐体神経細胞の形態異常を引き起こす事が明らかとなった(Development134:2273-2282, 2007)。また、共同研究により、Cdk5がamygdaloidのnLot細胞(Nat. Neurosci. 10, 1038-1047, 2007)や、RMSの細胞移動(J. Neurosci. 27:12829-12838, 2007)に必須なキナーゼである事を示した

The purpose of this study is to analyze the function of neuron-specific serine/threonine kinase Cyclin-dependent kinase 5(Cdk5) by using conditional KO mice of its activating subunit p35 trough the behavioral analysis such as spatial learning and fear conditioning test, and electrophysiological analysis and biochemical analysis. In this study we generated p35-flox mice in which two loxP sequences were inserted in p35 gene allele. We generated inducible p35 conditional KO by crossing p35-flox and CreER mice in which Cre is activated by Tamoxifen(TM) and p35 gene was destructed after brain formed. We confirmed decrease of p35 protein by p. o. administration of 2 mg TM for three days. We also generated CA1-specific p35 conditional KO mice by crossing CaMKII-Cre mice, and confirmed decreased level of p35 protein in hippocampus. We observed decreased numbers of dendritic spines in the hippocampal CA1 pyramidal neurons in CA1-p35 cKO mice. Using these p35 conditional KO mice, we currently conduct behavioral analysis of p35 cKO mice

成体脳において神経新生が行われる現象は、ヒトを含む哺乳類では限られた脳領域で行なわれているが、ゼブラフィッシュなどの硬骨魚類では、比較的広範囲の脳領域で行なわれている。本研究では、神経幹細胞であるラジアルグリア細胞の増殖・分化のプロセスと、Ca動態を可視化することにより、グリア内細胞シグナルと幹細胞増殖・分化の関連を明らかにすることを目的とした。バイオリソースセンターよりGFAP:GCaMP6a導入して、解析を開始した。また、Gal4-UAS系を用いて、GFAP:GalFF Tg fish(Shimizuら2015)とUAS:GCaMP7を交配する予定である。

統合失調症患者脳の解析からCdk5活性化サブユニットp35タンパク質量の減少が報告された。また、統合失調症患者の神経細胞のスパイン密度が減少していることが報告されている（Gareyら1993, 1995,1998; Glantz&Lewis 1995, 1996）。本研究では、条件付けp35遺伝子改変マウス（p35cKOマウス）におけるスパイン密度を解析した。その結果、発達期と成熟期のいずれにおいてもスパイン密度が減少していること、さらにその減少が大脳皮質と海馬の神経細胞で観察し、学術誌に報告した（MitaらCerebral cortex2016）。

【研究目的】中枢神経系損傷に対する決定的な治療法は未だない。そのため、中枢神経系の限られた再生能力の分子機構解明が急がれている。Collapsin-response mediator protein (CRMP)は軸索ガイダンス分子であるセマファリンの細胞内シグナル伝達物質として同定され、その後の研究で、軸索伸長などへの関与が示された。CRMPはファミリータンパク質でCRMP1-5が別々の遺伝子にコードされている。これらはヘテロ４量体を形成して機能していると考えられているが、生体内での役割は不明な点が多い。本研究は、CRMP遺伝子の改変マウスを用いて、中枢神経損傷後のCRMPの機能解明を目的とする。我々はこれまでに、CRMP4欠損マウス由来の後根神経節細胞の培養実験では中枢神経系神経再生を阻害する物質に対する反応が減弱していることを見出して報告している（Nagaiら2012）。従って本研究では特にCRMP4欠損（Crmp4-/-）マウスを用いた研究を行なった。【研究成果】野生型マウスの損傷した脊髄において再生阻害活性あるいは細胞毒性のあるCRMP4 のいくつかのformが上昇していることが確認された。こうした結果を踏まえて、本研究ではCrmp4-/-マウスを用いることにより、脊髄損傷後の再生過程におけるCRMP4の機能解明を試みた。脊髄損傷4週間後、Crmp4+/+マウスにおいては損傷部位以降の脊髄神経軸索消失、半身不随が観察されたが、Crmp4-/-マウスの脊髄では神経軸索が長距離にわたり再生し、後肢に加重ができるほど運動機能が回復していることが確認された。組織学的にも、再生する軸索のマーカーであるGAP43の増加が、生化学的および組織学的に示された。従って、CRMP4の機能を阻害することで、脊髄損傷の回復が改善される可能性がin vivoで示された。これらの結果から、CRMP4を標的とした中枢神経系損傷に対する新規治療開発の可能性が示唆された。

＜研究目的＞サイクリン依存性キナーゼ（Cdk）は増殖細胞で細胞周期を制御する最も重要な因子である。Cdk5はCdkのメンバーでありながら、最終分化した神経細胞で機能するユニークなCdkである。Cdk5の活性化サブユニットであるp35欠損マウスでの空間学習記憶の障害を報告した（Ohshima et al., JNC 2005）が、p35欠損マウスには脳形成の異常があり、空間学習記憶の障害が脳形成の異常に起因する可能性がある。H21-23文科省基盤研究Ｃ「活性化サブユニットp35コンディショナルKOを用いたCdk5の機能解析」で確立したp35コンディショナルKO(p35cKO)の行動解析を行なうとともに、Cdk5のもう一つのサブユニットであるp39欠損も併せ持つp35KO;p39KOマウスを作製し、脳高次機能を解析することで、Cdk5のキナーゼとしての機能を明らかにすることを目的とした。　＜研究結果＞p35コンディショナルKOマウスは、タモキシフェン投与により誘導されるタイプのCreERマウスとの交配により確立した。このマウスラインを用いて研究を行なった。タモキシフェンを３日間経口投与し、１週後に脳各部位のp35タンパク質量をコントロールと比較し、p35タンパク質量が顕著に減少していることを確認した。３ヶ月齢以降でタモキシフェンを投与し、p35遺伝子欠損を誘導した。これにより脳構造に異常を有しない状態でのp35遺伝子欠損による機能的変化を調べることが可能となった。これまでにモリス水迷路試験を行ない、空間記憶・学習機能の障害が明らかになっている。現在、その障害の原因となる海馬のシナプス可塑性についての電気生理学的な検討と、生化学的な解析を行なっている。さらに、p39KOマウスと交配することで、誘導型p35cKO;p39KOマウスラインを確立した。今後このマウスラインを用いた研究を行なって行く予定である。

成体脳での神経新生は、失われた神経機能を回復させる神経再生医療の面で注目されている。主に神経前駆細胞を補う細胞療法と、神経新生に係わる因子を用いて、内在性の神経新生機能を促進することで神経再生を成し遂げようとする、神経再生因子療法の２つに大別される。後者の場合、どの様な因子が有用であるかの研究が盛んに行われている。本研究では、生物モデルであるゼブラフィッシュ成魚で神経新生をモニタリング出来るラインを確立し、成体での神経新生促進因子を個体が生きたままスクリーニング出来るシステムを構築することを目的とする。具体的には、神経幹細胞特異的にルシフェラーゼと蛍光タンパク質を発現させ、ルシフェリンを化学発光させ、検出器IVISを用いて生きたままで成体脳での神経新生を経時的にモニタリングする系を確立することを目指した。手法としてはGal4-UASシステムを応用し、神経幹細胞特異的Gal4を発現しているTgゼブラフュッシュとUSAによりルシフェラーゼと蛍光タンパク質の発現が調整されているTgゼブラフィッシュの両系統を交配して得る。神経幹細胞のマーカーであるGFAPの遺伝子発現制御領域下でGal4の改良型であるGFFを発現させるTgライン(GFAP:GFF)を作成した。 このGFAP:GFF Tg fishとUAS-GFP fishと交配したF1を生後３日、１ヵ月、３か月齢でGFPの発現がGFAPの発現と同様であるかを検討した。方法として、1) GFAP-GFP fishでのGFP発現と比較を行う。2) GFAP陽性細胞の別のマーカーであるBLBPの免疫染色で確認した。1),2)の方法により、内在性のGFAP陽性の神経幹細胞と同様の発現を示すTg fishラインが得られたことが確認できた。さらにUAS下にルシフェラーゼと蛍光タンパク質を発現させるコンストラクトを作成した。今後このコンストラクトをGFAP:GFF Tg fishの受精卵に導入して一過性に発現させ、Gal4-UAS systemが機能するかを確認した上で、UAS Tg fishの作成を行なう計画である。

成体脳での神経新生は、失われた神経機能を回復させる神経再生医療の面で注目されている。しかし、神経幹細胞・神経前駆細胞の増殖・分化を制御する分子機構は不明な点が多い。本研究では、哺乳類に比較して広範囲で神経新生が行われている生物モデルであるゼブラフィッシュ成魚を用いて、神経幹細胞の増殖・分化に係わる分子機構の解明を目的とした。我々は眼の視神経の投射先である視蓋における神経新生について研究を行なってきた。マウスなど哺乳類の研究で神経新生への関与が示唆されているシグナル伝達経路が、ゼブラフィッシュ成魚視蓋においてどのように神経幹細胞の増殖・分化に関与するかを、シグナル伝達の阻害剤や遺伝子発現制御により明らかにすることを目指した。　今回の解析では、特にNotchシグナルとWntシグナルに着目して研究を進めた。Notchシグナルの活性化にはリガンドであるNotchのgamma-セクレターゼによる切断が必要であることから、Notchシグナルの阻害剤として用いられる。実験では、gamma-セクレターゼ阻害剤LY450139を飼育水中に加え、BrdU取り込みで細胞増殖を評価することにより、ゼブラフィッシュ成魚視蓋での神経幹細胞増殖を増加させることが明らかとなった。また、Wntシグナルの阻害剤であるIWR1を投与することにより、Wntシグナルの関与を検討したところ、視蓋における細胞増殖は抑制された。逆に、Wntシグナル活性化状態を模倣するGSK3抑制剤の投与により、細胞増殖は増加傾向であった。従って、阻害剤などの薬剤を用いた実験結果より、神経幹細胞増殖・神経前駆細胞に対して、Notchシグナルは抑制的に、Wntシグナルは促進的に働いていることが示唆された。　Wntシグナルに関しては、薬剤投与による実験から得られた結果をさらに検討するため、ヒートショックによりWntシグナルが抑制されるトランスジェニックゼブラフィッシュを用いてさらに検討を行なっている。

脳機能の発現には正しく神経回路が形成される必要があり、その過程で軸索ガイダンスという機構が重要である。細胞外に分泌される軸索ガイダンス分子とそれを感受するシステムの組み合わせがあり、Sema3Aというガイダンス分子を感受し、それに反応するために必要な分子として同定されたものがCRMPである。我々はCdk5の基質としてCRMP2を同定し、そのSer522をCdk5がリン酸化することを示した。本研究では、CRMPの機能をそのリン酸化による調節を含めて、マウスとゼブラフィッシュをモデル生物として用いて個体レベルで明らかにすることを目的として研究を進めてきた。CRMPはCRMP1-5の５つの遺伝子でコードされたホモロジーの高いファミリータンパク質であり、それがヘテロテトラマーを形成して機能している。遺伝子改変マウスはCRMP1,CRMP4のKOマウスとSer522をAlaに１アミノ酸置換したCRMP2のみを有するCRMP2KIマウスを用いて研究を進めた。CRMP1KO;CRMP2KIの大脳皮質の神経細胞は特にbasal dendriteの投射領域が変化する異常が明らかとなり（Yamashitaら2012）、CRMP4KOマウスでは先に報告されたSema3AKOマウスと同様に、海馬CA1の錐体神経細胞のapical dendriteの分岐の異常がそれぞれ観察された（Niisatoら2012）。また、ゼブラフィッシュの胚に遺伝子のノックダウンが可能なCRMPのAMOを導入してその効果を調べたところ、脊髄内感覚神経細胞（RB細胞）の位置の異常がCRMP2, CRMP4AMOで観察され、同様の異常がCdk5AMOで起こることが判った。さらに、Cdk5AMOの異常がCRMP2,4のリン酸化により改善することから、CRMP2,4のリン酸化がRB細胞の位置の決定に重要であることが示唆された（Tanakaら投稿中）。以上より、神経系の発達、特に樹状突起の形成や細胞の位置決定にCRMPのリン酸化が必要であることが、個体レベルで明らかになった。

サイクリン依存性キナーゼ５(Cyclin-dependent kinase 5, Cdk5)は、脳形成過程での役割に加え、様々な脳機能への関与が知れらているが、その活性上昇が神経変性過程で報告されている。しかし、活性上昇が神経細胞死の原因であるとする報告があるものの、結果にすぎないとする反論があり、結論が出ていない。神経細胞中のCdk5活性を生きたままモニタリング出来れば、細胞死に先立って活性上昇があるのか、細胞死後に活性が上がるのかを検討できる。本研究では、Fluorescence Resonant Energy Transfer (FRET)を用いた生細胞でのCdk5活性のモニタリング法を開発して、神経細胞内での活性測定を試みることを目的とした。　FRETによるCdk5活性のモニタリング法は、1. Cdk5活性と活性型Cdk5の局在－Cdk5及びp35キメラタンパク質のヘテロ二量体形成による解析、２. Cdk5の基質タンパク質にCdk5のリン酸化依存的に起きるタンパク質相互作用を利用したものを用いた。１のCdk5/p35ヘテロ二量体形成に伴うFRETを培養神経細胞で観察した。グルタミン酸添加により一過性の活性上昇が知られているが、FRETの上昇が観察され、Cdk5/p35のヘテロ二量体形成の増加が推定された。２の基質リン酸化依存的なタンパク室結合を、FRETシグナルとして培養ＣＯＳ細胞を用いた系で観察することに成功した。これらの結果を踏まえて今後神経細胞にCdk5活性を上昇させる刺激を加えた時、細胞死を誘導した時などのFRETの変化を検討する予定である。本研究によるCdk5活性のFRET法を用いたモニタリング系をさらに発展させることで、神経細胞死におけるCdk5活性の意義、即ち原因であるか結果であるかを明らかにすることが可能であると考えた。

我々は、Cdk5がL-typeカルシウムチャネルのリン酸化を介してインシュリン分泌を負に制御している事を報告し、Cdk5阻害剤の糖尿病治療薬への応用を示唆した（WeiらNat. Med 11, 1104, 2005）。2008年度の特定課題研究Ａではbeta細胞特異的なCdk5抑制モデルマウスを作成し解析した。予想に反し、beta細胞特異的なCdk5抑制マウスは生後にbeta細胞の減少を示し、６週齢で糖尿病状態を示した。この事は、Cdk5がbeta細胞の生存に不可欠である事を示唆しているが、Cdk5活性の低下が、生存そのものに必須なのか、増殖・分化に必須なのか、不明である。Cdk5阻害剤の糖尿病治療薬としての応用を考えた場合には、Cdk5のbeta細胞への作用は、明確にする必要があり、以下の実験を行なった。1) beta細胞特異的なCdk5抑制モデルマウスのbata細胞の細胞死について、TUNEL染色で検討。2) beta細胞特異的なCdk5抑制モデルマウスのbeta細胞の増殖について、BrdUのラベルやKi67などの増殖細胞のマーカーで検討。3) beta細胞特異的なCdk5抑制モデルマウスとアポトーシスシグナル構成因子のBax欠損マウスを交配し、beta細胞の数を週齢ごとに、Tg;Bax+/+、 Tg;Bax-/-、 nonTg;Bax-/-の３群で比較。＜結果と考察＞1) beta細胞特異的なCdk5抑制モデルマウスにおいて、beta細胞が減少する原因を検討するため、TUNEL染色を行なったが、Tgマウスにおいて、明確なTUNEL陽性細胞数の上昇はなかった。2) 同様に、増殖細胞数に明らかな変化はなかった3) Tg;Bax-/- vs Tg;Bax+/+間で明確なbeta細胞が減少に差はなかった。すなわち、明確なrescueは得られなかった。Tg作成より、５－７世代目の解析となり、Tgの発現レベルが低下していた。このため、Tg単独でのbeta細胞の減少程度が軽度となり、細胞死、細胞増殖低下などの評価が困難となった。このため、1)-3)で行なった検討で、明確な結果が得られなかった。Tgは世代が進むにつれてTg由来の遺伝子産物の発現レベルが低下するという問題点があり、今回の解析でも、明確な結論が得られなかった大きな要因となった。今後検討を進めるために、再度Tgを作成する方向も検討することとした。

目的神経特異的サイクリン依存性キナーゼ５（Cdk5）は、中枢神経系の形成過程や、機能発現に寄与するセリン・スレオニンキナーゼであり、我々は、Cdk5欠損マウスや活性化サブユニットp35欠損マウスの解析を通じて、研究を行ってきた。、Cdk5欠損マウスは胎生致死であり、p35欠損マウスは脳の形成に軽度の異常がある。我々はこれまでにp35欠損マウスにおける記憶・学習の異常を報告したが、異常が脳組織構築の異常によるものか、機能喪失によるものかは結論を得ていない。胎生致死などの解決するため、近年の技術進歩により、時間・空間的に遺伝子の機能喪失を起こさせるコンディショナルKOマウスの技術が、Cre-loxP系を応用して確立されている。我々は、本研究において、p35遺伝子のコンディショナルマウスを作製する事を目的として、p35遺伝子座に２ヶ所loxPを有するマウス（p35-floxマウス）の作成を行った。結果まず、p35遺伝子を含むBACクローンを入手し、ターゲティングコンストラクトを作成し、ES細胞に導入して、相同組換えを起こしたクローンを得た。これをブラストシストへ導入し、キメラマウスを得る事が出来た。このキメラ♂を野生型B6♀マウスと交配して、F１ヘテロマウスを作製する事に成功している。今後は、frtにフランクされたネオマイシン耐性遺伝子を除くために、FLP Tgマウスと交配した後に、ヘテロのp35-loxPマウス同志を交配し、p35-loxPのホモマウスを得たのち、creマウスと交配する事により、コンディショナルKOマウスを得る予定である。結語今回の研究により、p35遺伝子座loxPサイトが導入されたマウスを作製する事に成功し、今後のp35コンディショナルKOマウス作成の基盤が確立できた。

我々は、Cdk5がL-typeカルシウムチャネルのリン酸化を介してインシュリン分泌を負に制御している事を報告し、Cdk5阻害剤の糖尿病治療薬への応用を示唆した（WeiらNat. Med 11, 1104, 2005）。Cdk5阻害によるインシュリン分泌の亢進作用は、高血糖時に顕著となるため、糖尿病薬の副作用である低血糖が起こさず、従来の治療薬とは作用点の異なる薬剤となる可能性がある。2005年の報告の際には、Cdk5活性が低下したマウスモデルとしてp35欠損マウスを用いた。p35はCdk5の活性化サブユニットであり、Cdk5はp35とのヘテロダイマーで活性型となる。p35欠損はCdk5の活性低下を来すが、全身性に遺伝子欠損の影響が出るため、Cdk5活性低下によるインシュリン分泌亢進がベータ細胞での直接的効果である事を示すに至らなかった。この問題をクリアするため、より適切なモデルマウスを作製する必要がある。　本研究では、insulin-promoter下にCdk5DNを発現するトランスジェニック(Tg)マウスラインを確立し、その解析を行った。ラインを確立した４系統のうち２系統でTg由来のCdk5DNタンパク質の膵臓ベータ細胞特異的な発現が確認された。Cdk5DNの発現により、インシュリンの分泌が亢進する事が期待されたが、Tgマウスにおいては、Cdk5DNの発現レベル依存的に、ベータ細胞が減少し、６週齢で糖尿病状態を示した。ベータ細胞の減少が観察される時期を検討した結果、生後１０日目には既にラ氏島の縮小とベータ細胞数の減少が認められる事が明らかとなった。今後は、このベータ細胞の減少が、増殖の低下によるものか、アポトーシスなどの細胞死の増加によるものか、検討を行う予定である。今回の研究により、Cdk5活性の低下はベータ細胞の生存にも関与する事が初めて明らかとなった