Cyclin-dependent kinase 5 (Cdk5) /p35 is involved in many developmental processes of the central nervous system. Cdk5/p35 is also implicated in synaptic plasticity, learning and memory. Several lines of conditional Cdk5 knockout mice (KO) have been generated and have shown different outcomes for learning and memory. Here, we present our analysis of p35 conditional KO mice (p35cKO) in hippocampal pyramidal neurons or forebrain GABAergic neurons using electrophysiological and behavioral methods. In the fear conditioning task, CamKII-p35cKO mice showed impaired memory retention. Furthermore, NMDAR-dependent long-term depression (LTD) induction by low-frequency stimuli in hippocampal slices from CamkII-p35cKO mice was impaired compared to that in control mice. In contrast, Dlx-p35cKO mice showed no abnormalities in behavioral tasks and electrophysiological analysis in their hippocampal slices. These results indicated that Cdk5/p35 in excitatory neurons is important for the hippocampal synaptic plasticity and associative memory retention.

The mammalian cerebral cortex is characterized by a 6-layer structure, and proper neuronal migration is critical for its formation. Cyclin-dependent kinase 5 (Cdk5) has been shown to be a critical kinase for neuronal migration. Several Cdk5 substrates have been suggested to be involved in ordered neuronal migration. However, in vivo loss-of-function studies on the function of Cdk5 phosphorylation substrates in neuronal migration in the developing cerebral cortex have not been reported. In this study, we demonstrated that Cdk5-mediated phosphorylation of collapsing mediator protein (CRMP) 2 is critical for neuronal migration in the developing cerebral cortex with redundant functions of CRMP1 and CRMP4. The cerebral cortices of triple-mutant CRMP1 knock-out (KO); CRMP2 knock-in (KI)/KI; and CRMP4 KO mice showed disturbed positioning of layers II-V neurons in the cerebral cortex. Further experiments using bromodeoxyuridine birthdate-labeling and in utero electroporation implicated radial migration defects in cortical neurons. Ectopic neurons were detected around the CA1 region and dentate gyrus in CRMP1 KO; CRMP2 KI/KI; and CRMP4 KO mice. These results suggest the importance of CRMP2 phosphorylation by Cdk5 and redundancy of CRMP1 and CRMP4 in proper neuronal migration in the developing cerebral cortex and hippocampus.

Since 1995, more than 100 transgenic (Tg) mouse models of Alzheimer's disease (AD) have been generated in which mutant amyloid precursor protein (APP) or APP/presenilin 1 (PS1) cDNA is overexpressed ( 1st generation models ). Although many of these models successfully recapitulate major pathological hallmarks of the disease such as amyloid β peptide (Aβ) deposition and neuroinflammation, they have suffered from artificial phenotypes in the form of overproduced or mislocalized APP/PS1 and their functional fragments, as well as calpastatin deficiency-induced early lethality, calpain activation, neuronal cell death without tau pathology, endoplasmic reticulum stresses, and inflammasome involvement. Such artifacts bring two important uncertainties into play, these being (1) why the artifacts arise, and (2) how they affect the interpretation of experimental results. In addition, destruction of endogenous gene loci in some Tg lines by transgenes has been reported. To overcome these concerns, single App knock-in mouse models harboring the Swedish and Beyreuther/Iberian mutations with or without the Arctic mutation (AppNL-G-F and AppNL-F mice) were developed ( 2nd generation models ). While these models are interesting given that they exhibit Aβ pathology, neuroinflammation, and cognitive impairment in an age-dependent manner, the model with the Artic mutation, which exhibits an extensive pathology as early as 6 months of age, is not suitable for investigating Aβ metabolism and clearance because the Aβ in this model is resistant to proteolytic degradation and is therefore prone to aggregation. Moreover, it cannot be used for preclinical immunotherapy studies owing to the discrete affinity it shows for anti-Aβ antibodies. The weakness of the latter model (without the Arctic mutation) is that the pathology may require up to 18 months before it becomes sufficiently apparent for experimental investigation. Nevertheless, this model was successfully applied to modulating Aβ pathology by genome editing, to revealing the differential roles of neprilysin and insulin-degrading enzyme in Aβ metabolism, and to identifying somatostatin receptor subtypes involved in Aβ degradation by neprilysin. In addition to discussing these issues, we also provide here a technical guide for the application of App knock-in mice to AD research. Subsequently, a new double knock-in line carrying the AppNL-F and Psen1 P117L/WT mutations was generated, the pathogenic effect of which was found to be synergistic. A characteristic of this 3rd generation model is that it exhibits more cored plaque pathology and neuroinflammation than the AppNL-G-F line, and thus is more suitable for preclinical studies of disease-modifying medications targeting Aβ. Furthermore, a derivative AppG-F line devoid of Swedish mutations which can be utilized for preclinical studies of β-secretase modifier(s) was recently created. In addition, we introduce a new model of cerebral amyloid angiopathy that may be useful for analyzing amyloid-related imaging abnormalities that can be caused by anti-Aβ immunotherapy. Use of the App knock-in mice also led to identification of the α-endosulfine-K ATP channel pathway as components of the somatostatin-evoked physiological mechanisms that reduce Aβ deposition via the activation of neprilysin. Such advances have provided new insights for the prevention and treatment of preclinical AD. Because tau pathology plays an essential role in AD pathogenesis, knock-in mice with human tau wherein the entire murine Mapt gene has been humanized were generated. Using these mice, the carboxy-terminal PDZ ligand of neuronal nitric oxide synthase (CAPON) was discovered as a mediator linking tau pathology to neurodegeneration and showed that tau humanization promoted pathological tau propagation. Finally, we describe and discuss the current status of mutant human tau knock-in mice and a non-human primate model of AD that we have successfully created.

We previously developed single App knock-in mouse models of Alzheimer's disease (AD) harboring the Swedish and Beyreuther/Iberian mutations with or without the Arctic mutation (AppNL-G-F and AppNL-F mice, respectively). These models showed Aβ pathology, neuroinflammation, and cognitive impairment in an age-dependent manner. The former model exhibits extensive pathology as early as 6 months, but is unsuitable for investigating Aβ metabolism and clearance because the Arctic mutation renders Aβ resistant to proteolytic degradation and prone to aggregation. In particular, it is inapplicable to preclinical immunotherapy studies due to its discrete affinity for anti-Aβ antibodies. The latter model may take as long as 18 months for the pathology to become prominent, which leaves an unfulfilled need for an Alzheimer's disease animal model that is both swift to show pathology and useful for antibody therapy. We thus utilized mutant Psen1 knock-in mice into which a pathogenic mutation (P117L) had been introduced to generate a new model that exhibits early deposition of wild-type human Aβ by crossbreeding the AppNL-F line with the Psen1P117L/WT line. We show that the effects of the pathogenic mutations in the App and Psen1 genes are additive or synergistic. This new 3rd generation mouse model showed more cored plaque pathology and neuroinflammation than AppNL-G-F mice, and will help accelerate the development of disease-modifying therapies to treat preclinical AD.

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

Impairment of episodic memory, a class of memory for spatiotemporal context of an event, is an early symptom of Alzheimer's disease. Both spatial and temporal information are encoded and represented in the hippocampal neurons, but how these representations are impaired under amyloid β (Aβ) pathology remains elusive. We performed chronic imaging of the hippocampus in awake male amyloid precursor protein (App) knock-in mice behaving in a virtual reality environment to simultaneously monitor spatiotemporal representations and the progression of Aβ depositions. We found that temporal representation is preserved, whereas spatial representation is significantly impaired in the App knock-in mice. This is because of the overall reduction of active place cells, but not time cells, and compensatory hyperactivation of remaining place cells near Aβ aggregates. These results indicate the differential impact of Aβ aggregates on two major modalities of episodic memory, suggesting different mechanisms for forming and maintaining these two representations in the hippocampus.

Parkinson's disease (PD) is a chronic neurodegenerative disorder characterized by impaired motor symptoms induced by the degeneration of dopaminergic neurons of the substantia nigra pars compacta (SNc). Many factors are speculated to operate in the mechanism of PD, including oxidative stress, mitochondrial dysfunction, abnormal protein handling, and PD induced apoptosis. Besides, researchers have recently shown that inflammatory secretions may engage neighboring cells such as astrocytes, which then induce autocrine and paracrine responses that amplify the inflammation, leading to neurodegeneration. In the present study, we analyzed the neuroprotective and anti-inflammatory effects of collapsin response mediator protein 4 (CRMP4) deletion in 6-hydroxydopamine (6-OHDA)-injected male mice, as well as its effects on motor impairments. Our findings indicated that the deletion of CRMP4 could maintain the TH-positive fibers in the striatum and the TH-positive cells in SNc, attenuate the inflammatory responses, and improve motor coordination and rotational behavior. Furthermore, based on our findings at the early time points, we hypothesized that primary differences between the Crmp4+/+ and Crmp4-/- mice may occur in microglia instead of neurons. Although further work should be carried out to clarify the specific role of CRMP4 in the pathogenesis of PD, our findings suggest that it could be a possible target for the treatment of PD.

Proper migration and positioning of Purkinje cells are important for formation of the developing cerebellum. Although several cyclin-dependent kinase 5 (Cdk5) substrates are known to be critical for ordered neuronal migration, there are no reports of mutant mouse-based, in vivo studies on the function of Cdk5-phosphorylation substrates in migration of Purkinje cells. We focused on the analysis of collapsin response mediator protein 2 (CRMP2), one of the Cdk5 substrates, because a previous study reported migration defects of cortical neurons with shRNA-mediated knockdown of CRMP2. However, CRMP2 KI/KI mice, in which Cdk5-phosphorylation is inhibited, showed little defects in Purkinje cell migration and positioning. We hypothesized compensatory redundant functions of the other CRMPs, and analyzed the migration and positioning of Purkinje cells in the cerebellum in every combination of CRMP1 knockout (KO), CRMP2 KI/KI, and CRMP4 KO mice. Severe disturbance of migration and positioning of Purkinje cells were observed in the triple mutant mice. We also found motor coordination defects in the triple CRMPs mutant mice. These results suggest the importance of both, phosphorylation of CRMP2 by Cdk5 and the redundant functions of CRMP1 and CRMP4 in proper migration and positioning of Purkinje cells in developing cerebellum.

Parkinson's disease (PD) is characterized by progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc). Levodopa (L-Dopa), the current main treatment for PD, reduces PD symptoms by partially replacing dopamine, but it does not slow neurodegeneration. Recent studies have evidenced that neuroinflammatory processes contribute to the degeneration of dopaminergic neurons in the SNc under cytopathic conditions, while other lines of inquiry have implicated phosphorylation of collapsin response mediator protein 2 (CRMP2) as a causal factor in axonal retraction after neural injury. We recently reported on the therapeutic effect of lanthionine ketimine ester (LKE) which associates with CRMP2 following axonal injury in the spinal cord. In the present study, we report that LKE protects SNc dopaminergic neurons after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) challenge, a common model for PD, and reduces the number of activated microglia proximal to the damaged SNc. The results also show that MPTP-induced motor impairment was suppressed in LKE treatment. Furthermore, the results show that LKE inhibits the elevation of CRMP2 phosphorylation in dopaminergic neurons in the SNc after MPTP injection. These data suggest that modification of CRMP2 phosphorylation and suppression of microglial activation with LKE administration may represent a novel strategy for slowing progress of pathological processes in PD.

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

Axonal degeneration occurs in patients with various neurological diseases and traumatic nerve injuries, and Wallerian degeneration is a phenomenon in the prototypical axonal degradation that is observed after injury. Collapsin response mediator protein 2 (CRMP2) is phosphorylated by glycogen synthase kinase 3β (GSK3β), and it is involved in Wallerian degeneration after optic nerve injury. We previously developed a CRMP2 knock-in (CRMP2 KI) mouse line, in which CRMP2 phosphorylation by GSK3β is inhibited; however, Wallerian degeneration in CRMP2 KI mice has not yet been examined. In this study, we examined whether Wallerian degeneration of the optic nerve is suppressed in CRMP2 KI mice. Using one eye removal model, we compared Wallerian degeneration of the optic nerve based on histological and biochemical analyses. Our experimental results indicated that the genetic inhibition of CRMP2 phosphorylation delays Wallerian degeneration after optic nerve injury.

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurological disease characterized by the selective degeneration of motor neurons leading to paralysis and immobility. Missense mutations in the gene coding for the Cu2+/Zn2+ superoxide dismutase 1 (SOD1) accounts for 15-20% of familial ALS, and mice overexpressing ALS-linked SOD1 mutants have been frequently used as an animal model for ALS. Degeneration of motor neurons in ALS progresses in a manner called "dying back", in which the degeneration of synapses and axons precedes the loss of cell bodies. Phosphorylation of collapsin response mediator protein 2 (CRMP2) is implicated in the progression of neuronal/axonal degeneration of different etiologies. To evaluate the role of CRMP2 phosphorylation in ALS pathogenesis, we utilized CRMP2 S522A knock-in (CRMP2ki/ki) mice, in which the serine residue 522 was homozygously replaced with alanine and thereby making CRMP2 no longer phosphorylatable by CDK5 or GSK3B. We found that the CRMP2ki/ki/SOD1G93A mice showed delay in the progression of the motor phenotype compared to their SOD1G93-Tg littermates. Histological analysis revealed that the CRMP2ki/ki/SOD1G93A mice retained more intact axons and NMJs than their SOD1G93A-Tg littermates. These results suggest that the phosphorylation of CRMP2 may contribute to the axonal degeneration of motor neurons in ALS.

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

Multisensory integration (MSI) is a fundamental emergent property of the mammalian brain. During MSI, perceptual information encoded in patterned activity is processed in multimodal association cortex. The systems-level neuronal dynamics that coordinate MSI, however, are unknown. Here, we demonstrate intrinsic hub-like network activity in the association cortex that regulates MSI. We engineered calcium reporter mouse lines based on the fluorescence resonance energy transfer sensor yellow cameleon (YC2.60) expressed in excitatory or inhibitory neurons. In medial and parietal association cortex, we observed spontaneous slow waves that self-organized into hubs defined by long-range excitatory and local inhibitory circuits. Unlike directional source/sink-like flows in sensory areas, medial/parietal excitatory and inhibitory hubs had net-zero balanced inputs. Remarkably, multisensory stimulation triggered rapid phase-locking mainly of excitatory hub activity persisting for seconds after the stimulus offset. Therefore, association cortex tends to form balanced excitatory networks that configure slow-wave phase-locking for MSI. Video Abstract [Figure presented] Kuroki et al. performed cell-type-specific, wide-field FRET-based calcium imaging to visualize cortical network activity induced by multisensory inputs. They observed phase-locking of cortical slow waves in excitatory neuronal hubs in association cortical areas that may underlie multisensory integration.

p35 is an activating co-factor of Cyclin-dependent kinase 5 (Cdk5), a protein whose dysfunction has been implicated in a wide-range of neurological disorders including cognitive impairment and disease. Inducible deletion of the p35 gene in adult mice results in profound deficits in hippocampal-dependent spatial learning and synaptic physiology, however the impact of the loss of p35 function on hippocampal in vivo physiology and spatial coding remains unknown. Here, we recorded CA1 pyramidal cell activity in freely behaving p35 cKO and control mice and found that place cells in the mutant mice have elevated firing rates and impaired spatial coding, accompanied by changes in the temporal organization of spiking both during exploration and rest. These data shed light on the role of p35 in maintaining cellular and network excitability and provide a physiological correlate of the spatial learning deficits in these mice.

Endoplasmic reticulum (ER) stress is believed to play an important role in the etiology of Alzheimer's disease (AD). The accumulation of misfolded proteins and perturbation of intracellular calcium homeostasis are thought to underlie the induction of ER stress, resulting in neuronal dysfunction and cell death. Several reports have shown an increased ER stress response in amyloid precursor protein (APP) and presenilin1 (PS1) double-transgenic (Tg) AD mouse models. However, whether the ER stress observed in these mouse models is actually caused byADpathology remains unclear. APP and PS1 contain one and nine transmembrane domains, respectively, for which it has been postulated that overexpressed membrane proteins can become wedged in a misfolded configuration in ER membranes, thereby inducing nonspecific ER stress. Here, we used an App-knockin (KI) AD mouse model that accumulates amyloid-β (Aβ) peptide without overexpressing APP to investigate whether the ER stress response is heightened because ofAβ pathology. Thorough examinations indicated that no ER stress responses arose in App-KI or single APP-Tg mice. These results suggest thatPS1overexpression or mutation induced a nonspecific ER stress response that was independent of Aβ pathology in the double-Tg mice. Moreover, we observed no ER stress in a mouse model of tauopathy (P301S-Tau-Tg mice) at various ages, suggesting that ER stress is also not essential in tau pathology-induced neurodegeneration. We conclude that the role of ER stress in AD pathogenesis needs to be carefully addressed in future studies.

© 2018 Elsevier B.V. Social recognition memory is crucial for survival across species, underlying the need to correctly identify conspecifics, mates and potential enemies. In humans the hippocampus is engaged in social and episodic memory, however the circuit mechanisms of social memory in rodent models has only recently come under scrutiny. Work in mice has established that the dorsal CA2 and ventral CA1 regions play critical roles, however a more comprehensive comparative analyses of the circuits and mechanisms required has not been reported. Here we employ conditional genetics to examine the differential contributions of the hippocampal subfields to social memory. We find that the deletion of NMDA receptor subunit 1 gene (NR1), which abolishes NMDA receptor synaptic plasticity, in CA3 pyramidal cells led to deficits in social memory; however, mice lacking the same gene in DG granule cells performed indistinguishable from controls. Further, we use conditional pharmacogenetic inhibition to demonstrate that activity in ventral, but not dorsal, CA3 is necessary for the encoding of a social memory. These findings demonstrated CA3 pyramidal cell plasticity and transmission contribute to the encoding of social stimuli and help further identify the distinct circuits underlying the role of the hippocampus in social memory.

Cyclin-dependent kinase 5 (Cdk5) is recognized as a unique member among other Cdks due to its versatile roles in many biochemical processes in the nervous system. The proper development of neuronal dendrites is required for the formation of complex neural networks providing the physiological basis of various neuronal functions. We previously reported that sparse dendrites were observed on cultured Cdk5-null Purkinje cells and Purkinje cells in Wnt1(cre)-mediated Cdk5 conditional knockout (KO) mice. In the present study, we generated L7(cre)-mediated p35; p39 double KO (L7(cre)-p35(f/f); p39(-/-)) mice whose Cdk5 activity was eliminated specifically in Purkinje cells of the developing cerebellum. Consequently, these mice exhibited defective Purkinje cell migration, motor coordination deficiency and a Purkinje dendritic abnormality similar to what we have observed before, suggesting that dendritic growth of Purkinje cells was cell-autonomous in vivo. We found that mixed and overlay cultures of WT cerebellar cells rescued the dendritic deficits in Cdk5-null Purkinje cells, however, indicating that Purkinje cell dendritic development was also supported by non-cell-autonomous factors. We then again rescued these abnormalities in vitro by applying exogenous brain-derived neurotrophic factor (BDNF). Based on the results from culture experiments, we attempted to rescue the developmental defects of Purkinje cells in L7(cre)-p35(f/f); p39(-/-) mice by using a TrkB agonist. We observed partial rescue of morphological defects of dendritic structures of Purkinje cells. These results suggest that Cdk5 activity is required for Purkinje cell dendritic growth in cell-autonomous and non-cell-autonomous manners. (c) 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1175-1187, 2017

Adult neurogenesis occurs more commonly in teleosts, represented by zebrafish, than in mammals. Zebrafish is therefore considered a suitable model to study adult neurogenesis, for which the regulatory molecular mechanisms remain little known. Our previous study revealed that neuroepithelial-like neural stem cells (NSCs) are located at the edge of the dorsomedial region. We also showed that Notch signaling inhibits NSC proliferation in this region. In the present study, we reported the expression of Wnt and Shh signaling components in this region of the optic tectum. Moreover, inhibitors of Wnt and Shh signaling suppressed NSC proliferation, suggesting that these pathways promote NSC proliferation. Shh is particularly required for maintaining Sox2-positive NSCs. Our experimental data also indicate the involvement of these signaling pathways in neural differentiation from NSCs. (c) 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1206-1220, 2017

Rett syndrome is a severe neurodevelopmental disorder. It is caused by a mutation in methyl-CpG binding protein 2 (MecP2), a transcriptional regulator that recruits protein complexes involved in histone modification and chromatin remodeling. However, the role of Mecp2 in Rett syndrome remains unclear. In this study, we investigated the function of Mecp2 in neuronal development using zebrafish embryos. Mecp2 expression was detected ubiquitously in the central nervous system and muscles at 28 h postfertilization (hpf). We injected an antisense morpholino oligonucleotide (AMO) to induce Mecp2 knockdown phenotype. In mecp2 morphants (embryos with Mecp2 knockdown by AMO) at 28 and 72 hpf, we found an increase in abnormal axonal branches of caudal primary motor neurons and a decrease in motor activity. In mecp2 morphants at 24 hpf, we observed an increase in the expression of an mecp2 downstream candidate gene, brain derived neurotrophic factor (bdnf). In mecp2 morphants at 72 hpf, the presynaptic area stained by an anti-SV2 antibody was increased at the neuromuscular junction (NMJ). Interestingly, the size of SV2-positive presynaptic area at the NMJ was also increased following bdnf mRNA injection, while it was normalized in a double knockdown of mecp2 and bdnf. These results imply that Mecp2 is an important functional regulator of bdnf gene expression during neural circuit formation in zebrafish embryo. (c) 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1101-1113, 2017

Neurodegeneration 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.

© 2017 the authors. Leukocytecommonantigen-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 growthconecollapseresponseofprimarycultured dorsal rootganglionneuronsfromMusmusculusembryos.Invivo,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 layerVneurons. This phenotype was observed in both male and female mutants. The double-heterozygous mutants, Ptpδ -/- ; Sema3a -/- , also showed a similar phenotype, indicating the geneticinteraction.InPtpδ -/- brains, Fynand Srckinaseswerehyper phosphorylatedat theirC-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.

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.

Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase, and its activity is dependent upon an association with a neuron-specific activating subunit. It was previously reported that Cdk5-/- mice exhibit perinatal lethality and defective neuronal positioning. In this study, they focused on the analysis of neuronal positioning of GABAergic neurons in the forebrain. Defective formation of the ventral striatum, nucleus accumbens, and olfactory tubercles was found in Cdk5-/- embryos. To further study this abnormal development, we generated and analyzed Dlx5/6-Cre p35 conditional KO (cKO); p39-/- mice in which forebrain GABAergic neurons have lost their Cdk5 kinase activity. Defective formation of the nucleus accumbens and olfactory tubercles as well as neuronal loss in the striatum of Dlx5/6-Cre p35cKO; p39-/- mice was found. Elevated levels of phosphorylated JNK were observed in neonatal striatal samples from Dlx5/6-Cre p35cKO; p39-/- mice, suggestive of neuronal death. These results indicate that Cdk5 is required for the formation of the ventral striatum in a cell-autonomous manner, and loss of the kinase activity of Cdk5 causes GABAergic neuronal death in the developing mouse forebrain. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 419-437, 2017.

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.

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.

© 2016 Elsevier Inc. All rights reserved.Alzheimer's disease (AD) is one of the best known neurodegenerative diseases; it causes dementia and its pathological features include accumulation of amyloid β (Aβ) 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 Aβ 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-Aβ 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 γ, 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-Aβ accumulation stage. These results suggest a correlation between CRMP2 phosphorylation and AD pathophysiology, and indicate the effectiveness of pioglitazone treatment at the pre-Aβ accumulation stage in AD model mice.

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: 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: 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.

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.

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.

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.

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.

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.

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.

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: 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.

: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 onths 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.

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.

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.

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.

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.

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.

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.

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. (c) 2012 Elsevier Inc. All rights reserved.

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. Copyright © American Society for Investigative Pathology.

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.

Collapsin response mediator protein 2 (CRMP2) is an intracellular protein that mediates signaling of Semaphorin3A (Sema3A), a repulsive axon guidance molecule. Fyn, a Src-type tyrosine kinase, is involved in the Sema3A signaling. However, the relationship between CRMP2 and Fyn in this signaling pathway is still unknown. In our research, we demonstrated that Fyn phosphorylated CRMP2 at Tyr(32) residues in HEK293T cells. Immunohistochemical analysis using a phospho-specific antibody at Tyr(32) of CRMP showed that Tyr(32)-phosphorylated CRMP was abundant in the nervous system, including dorsal root ganglion neurons, the molecular and Purkinje cell layer of adult cerebellum, and hippocampal fimbria. Overexpression of a nonphosphorylated mutant (Tyr(32) to Phe(32)) of CRMP2 in dorsal root ganglion neurons interfered with Sema3A-induced growth cone collapse response. These results suggest that Fyn-dependent phosphorylation of CRMP2 at Tyr(32) is involved in Sema3A signaling.

The dysfunction of proteasomes and mitochondria has been implicated in the pathogenesis of Parkinson disease. However, the mechanism by which this dysfunction causes neuronal cell death is unknown. We studied the role of cyclin-dependent kinase 5 (Cdk5)-p35 in the neuronal cell death induced by 1-methyl-4-phenylpyrinidinium ion (MPP(+)), which has been used as an in vitro model of Parkinson disease. When cultured neurons were treated with 100 mu M MPP(+), p35 was degraded by proteasomes at 3 h, much earlier than the neurons underwent cell death at 12-24 h. The degradation of p35 was accompanied by the down-regulation of Cdk5 activity. We looked for the primary target of MPP(+) that triggered the proteasome-mediated degradation of p35. MPP(+) treatment for 3 h induced the fragmentation of the mitochondria, reduced complex I activity of the respiratory chain without affecting ATP levels, and impaired the mitochondrial import system. The dysfunction of the mitochondrial import system is suggested to up-regulate proteasome activity, leading to the ubiquitin-independent degradation of p35. The overexpression of p35 attenuated MPP(+)-induced neuronal cell death. In contrast, depletion of p35 with short hairpin RNA not only induced cell death but also sensitized to MPP(+) treatment. These results indicate that a brief MPP(+) treatment triggers the delayed neuronal cell death by the down-regulation of Cdk5 activity via mitochondrial dysfunction-induced up-regulation of proteasome activity. We propose a role for Cdk5-p35 as a survival factor in countering MPP(+)-induced neuronal cell death.

Huntington's disease (HD) is a polyglutamine [ poly(Q)] disease with an expanded poly(Q) stretch in the N terminus of the huntingtin protein (htt). A major pathological feature of HD neurons is inclusion bodies, detergent-insoluble aggregates composed of poly(Q)expanded mutant htt (mhtt). Misfolding of mhtt is thought to confer a toxic property via formation of aggregates. Although toxic molecular species are still debated, it is important to clarify the aggregation mechanism to understand the pathogenesis of mhtt. Weshow Cdk5/p35 suppresses the formation of mhtt inclusion bodies in cell lines and primary neurons. Although we expressed the N-terminal exon 1 fragment of htt lacking phosphorylation sites for Cdk5 in COS-7 cells, the kinase activity of Cdk5 was required for the suppression. Furthermore, Cdk5/p35 suppressed inclusion formation of atrophin-1, another poly(Q) protein, raising the possibility that Cdk5/p35 generally suppresses inclusion formation of poly(Q) proteins. Microtubules (MTs) were a downstream component of Cdk5/p35 in the suppression of inclusion formation; Cdk5/p35 disrupted MTs, which were required for the formation of inclusions. Moreover, stabilization of MTs by Taxol induced inclusions even with overexpression of Cdk5/p35. The formation of inclusions was also regulated by manipulating the Cdk5/p35 activity in primary rat or mouse cortical neuron cultures. These results indicate that Cdk5-dependent regulation of MT organization is involved in the development of aggregate formation and subsequent pathogenesis of poly(Q) diseases. This Cdk5 inhibition of htt aggregates is a novel mechanism different from htt phosphorylation and interaction with Cdk5 reported previously.

The rat granular retrosplenial cortex (GRS) is a simplified cortex, with distinct stratification and, in the uppermost layers, distinct modularity. Thalamic and cortical inputs are segregated by layers and in layer 1 colocalize, respectively, with apical dendritic bundles originating from neurons in layers 2 or 5. To further investigate this organization, we turned to reelin-deficient reeler mouse and Shaking rat Kawasaki. We found that the disrupted lamination, evident in Nissl stains in these rodents, is in fact a patch-matrix mosaic of segregated afferents and dendrites. Patches consist of thalamocortical connections, visualized by vesicular glutamate transporter 2 (VGluT2) or AChE. The surrounding matrix consists of corticocortical terminations, visualized by VGluT1 or zinc. Dendrites concentrate in the matrix or patches, depending on whether they are OCAM positive (matrix) or negative (patches). In wild-type rodents and, presumably, mutants, OCAM(+) structures originate from layer 5 neurons. By double labeling for dendrites (filled by Lucifer yellow in fixed slice) and OCAM immunofluorescence, we ascertained 2 populations in reeler: dendritic branches either preferred (putative layer 5 neurons) or avoided (putative supra-granular neurons) the OCAM 1 matrix. We conclude that input-target relationships are largely preserved in the mutant GRS and that dendrite-dendrite interactions involving OCAM influence the formation of the mosaic configuration.

Collapsin response mediator protein 1(CRMP1) is one of the CRMP family members that mediates signal transduction of axonal guidance and neuronal migration. We show here evidence that CRMP1 is involved in semaphorin3A (Sema3A)-induced spine development in the cerebral cortex. In the cultured cortical neurons from crmp1(+/-) mice, Sema3A increased the density of clusters of synapsin I and postsynaptic density-95, but this increase was markedly attenuated in crmp1(-/-) mice. This attenuation was also seen in cyclin-dependent kinase 5 (cdk5)(-/-) neurons. Furthermore, the introduction of wild-type CRMP1 but not CRMP1-T509A/S522A, (Thr 509 and Ser 522 were replaced by Ala), a mutant that cannot be phosphorylated by Cdk5, into crmp1(-/-) neurons rescued the defect in Sema3A responsiveness. The Golgi-impregnation method showed that the crmp1(-/-) layer V cortical neurons showed a lower density of synaptic bouton-like structures and that this phenotype had genetic interaction with sema3A. These findings suggest that Sema3A-induced spine development is regulated by phosphorylation of CRMP1 by Cdk5.

At the lateral wall of the lateral ventricles in the adult rodent brain, neuroblasts form an extensive network of elongated cell aggregates called chains in the subventricular zone and migrate toward the olfactory bulb. The molecular mechanisms regulating this migration of neuroblasts are essentially unknown. Here, we report a novel role for cyclin-dependent kinase 5 (Cdk5), a neuronal protein kinase, in this process. Using in vitro and in vivo conditional knock-out experiments, we found that Cdk5 deletion impaired the chain formation, speed, directionality, and leading process extension of the neuroblasts in a cell-autonomous manner. These findings suggest that Cdk5 plays an important role in neuroblast migration in the postnatal subventricular zone.

The amygdaloid complex consists of diverse nuclei that belong to distinct functional systems, yet many issues about its development are poorly understood. Here, we identify a stream of migrating cells that form specific amygdaloid nuclei in mice. In utero electroporation showed that this caudal amygdaloid stream ( CAS) originated in a unique domain at the caudal telencephalic pole that is contiguous with the dorsal pallium, which was previously thought to generate only neocortical cells. The CAS and the neocortex share mechanisms for specification ( transcription factors Tbr1, Lhx2 and Emx1/ 2) and migration ( reelin and Cdk5). Reelin, a critical cue for migration in the neocortex, and Cdk5, which is specifically required for migration along radial glia in the neocortex, were both selectively required for the normal migration of the CAS, but not for that of other amygdaloid nuclei. This is first evidence of a dorsal pallial contribution to the amygdala, demonstrating a developmental and mechanistic link between the amygdala and the neocortex.

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.

Collapsin response mediator protein 1 ( CRMP1) is one of the CRMP family members that mediates signal transduction of axon guidance molecules. Here, we show evidence that CRMP1 is involved in Reelin ( Reln) signaling to regulate neuronal migration in the cerebral cortex. In crmp1(-/-) mice, radial migration of cortical neurons was retarded. This phenotype was not observed in the sema3A(-/-) and crmp1(+/+); sema3A(+/+) cortices. However, CRMP1 was colocalized with disabled- 1 ( Dab1), an adaptor protein in Reln signaling. In the Reln(rl/rl) cortex, CRMP1 and Dab1 were expressed at a higher level, yet tyrosine phosphorylated at a lower level. Loss of crmp1 in a dab1 heterozygous background led to the disruption of hippocampal lamination, a Reeler- like phenotype. In addition to axon guidance, CRMP1 regulates neuronal migration by mediating Reln signaling.

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.

Tight glycemic control in individuals with diabetes mellitus is essential to prevent or delay its complications(1). Present treatments to reduce hyperglycemia mainly target the ATP-sensitive K+ (K-ATP) channel of pancreatic beta cells to increase insulin secretion. These current approaches are often associated with the side effect of hypoglycemia. Here we show that inhibition of the activity of cyclin-dependent kinase 5 (Cdk5) enhanced insulin secretion under conditions of stimulation by high glucose but not low glucose in MIN6 cells and pancreatic islets. The role of Cdk5 in regulation of insulin secretion was confirmed in pancreatic beta cells deficient in p35, an activator of Cdk5. p35-knockout mice also showed enhanced insulin secretion in response to a glucose challenge. Cdk5 kinase inhibition enhanced the inward whole-cell Ca2+ channel current and increased Ca2+ influx across the L-type voltage-dependent Ca2+ channel ( L-VDCC) upon stimulation with high glucose in beta cells, but had no effect on Ca2+ influx without glucose stimulation. The inhibitory regulation by Cdk5 on the L-VDCC was attributed to the phosphorylation of loop II-III of the alpha(1C) subunit of L-VDCC at Ser783, which prevented the binding to SNARE proteins and subsequently resulted in a decrease of the activity of L-VDCC. These results suggest that Cdk5/p35 may be a drug target for the regulation of glucose-stimulated insulin secretion.

Cdk5 (cyclin-dependent kinase 5) activity is dependent upon association with one of two neuron-specific activators, p35 or p39. Genetic deletion of Cdk5 causes perinatal lethality with severe defects in corticogenesis and neuronal positioning. p35(-/-) mice are viable with milder histological abnormalities. Although substantial evidence implicates Cdk5 in synaptic plasticity, its role in learning and memory has not been evaluated using mutant mouse models. We report here that p35(-/-) mice have deficiencies in spatial learning and memory. Close examination of hippocampal circuitry revealed subtle histological defects in CA1 pyramidal cells. Furthermore, p35(-/-) mice exhibit impaired long-term depression and depotentiation of long-term potentiation in the Schaeffer collateral CA1 pathway. Moreover, the Cdk5-dependent phosphorylation state of protein phosphatase inhibitor-1 was increased in 4-week-old mice due to increased levels of p39, which co-localized with inhibitor-1 and Cdk5 in the cytoplasm. These results demonstrate that p35-dependent Cdk5 activity is important to learning and synaptic plasticity. Deletion of p35 may shift the substrate specificity of Cdk5 due to compensatory expression of p39.

Although the roles of cyclin-dependent kinase 5 (Cdk5) in neurodevelopment and neurodegeneration have been studied extensively, regulation of Cdk5 activity has remained largely unexplored. We report here that glutamate, acting via NMDA or kainate receptors, can induce a transient Ca2+/calmodulin-dependent activation of Cdk5 that results in enhanced autophosphorylation and proteasome-dependent degradation of a Cdk5 activator p35, and thus ultimately down-regulation of Cdk5 activity. The relevance of this regulation to synaptic plasticity was examined in hippocampal slices using theta burst stimulation. p35(-/-) mice exhibited a lower threshold for induction of long-term potentiation. Thus excitatory glutamatergic neurotransmission regulates Cdk5 activity through p35 degradation, and this pathway may contribute to plasticity.

Cocaine, a drug of abuse, increases synaptic dopamine levels in the striatum by blocking dopamine reuptake at axon terminals. Cyclin-dependent kinase 5 (Cdk5) and its activator p35, proteins involved in phosphorylation of substrates in postmitotic neurons, have been found to be up-regulated after chronic exposure to cocaine. To further examine the effects of Cdk5 and p35 induction on striatal dopamine signaling, we generated two independent transgenic mouse lines in which Cdk5 or p35 was overexpressed specifically in neurons. We report here that increased Cdk5 activity, as a result of p35 but not of Cdk5 overexpression, leads to attenuation of cocaine-mediated dopamine signaling. Increased Cdk5-mediated phosphorylation of dopamine and cAMP-regulated phosphoprotein, molecular mass 32 kDa (DARPP-32) at Thr-75, was accompanied by decreased phosphorylation of DARPP-32 at Thr-34. Increased Cdk5-mediated phosphorylation of extracellular signal-regulated kinase kinase 1 at Thr-286 was accompanied by decreased activation of extracellular signal-regulated kinase 1/2. These effects contributed to attenuation of cocaine-induced phosphorylation of cAMP response element-binding protein as well as a lesser induction of c-fos in the striatum. These results support the idea that Cdk5 activity is involved in altered gene expression after chronic exposure to cocaine and hence impacts the long-lasting changes in neuronal function underlying cocaine addiction.

Collapsin response mediating protein-2 (CRMP2) has been identified as an intracellular protein mediating Semaphorin3A (Sema3A), a repulsive guidance molecule. In this study, we demonstrate that cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3beta (GSK3beta) plays a critical role in Sema3A signalling. In In vitro kinase assay, Cdk5 phosphorylated CRMP2 at Ser522, while GSK3beta did not induce any phosphorylation of CRMP2. Phosphorylation by GSK3beta was exclusively observed in Cdk5-phosphorylated CRMP2, but barely in CRMP2T509A. These results indicate that Cdk5 primarily phosphorylates CRMP2 at Ser522 and GSK3beta secondarily phosphorylates at Thr509. The dual-phosphorylated CRMP2, but not non-phosphorylated or single-phosphorylated CRMP2, is recognized with the antibody 3F4, which is highly reactive with the neurofibrillary tangles of Alzheimer's disease. 3F4 recognized the CRMP2 in the wild-type but not cdk5(-/-) mouse embryonic brain lysates. The phosphorylation of CRMP2 at Ser522 caused reduction of its affinity to tubulin. In dorsal root ganglion neurones, Sema3A stimulation enhanced the levels of the phosphorylated form of CRMP2 detected by 3F4. Over-expression of CRMP2 mutant substituting either Ser522 or Thr509 to Ala attenuates Sema3A-induced growth cone collapse response. These results suggest that the sequential phosphorylation of CRMP is an important process of Sema3A signalling and the same mechanism may have some relevance to the pathological aggregation of the microtubule-associated proteins.

An impressive body of evidence has been accumulated indicating that local protein synthesis is implicated in navigation of neurite extension induced by guidance cues, such as semaphorin3A (Sema3A). We found previously that a Src type tyrosine kinase Fyn and cyclin-dependent kinase 5 (Cdk5) mediate Sema3A-signaling. We also showed that Sema3A elicits axonal transport through neuropilin-1, a receptor for Sema3A, located at the growth cones. Here, we investigate the relationship between Sema3A-induced local signaling, protein synthesis, and axonal transport. Lavendustin A, a tyrosine kinase inhibitor, and olomoucine, a cyclin-dependent kinase inhibitor, suppressed Sema3A-induced facilitation of anterograde and retrograde axonal transport in dorsal root ganglion (DRG) neuron with and without the cell body. Sema3A-induced facilitation of axonal transport was attenuated in DRG neurons of fyn- (fyn(-/-)) and a Cdk5 activator, p35 (p35(-/-))-deficient mice when compared with those of wild-type or heterozygous mice. Inhibition of protein synthesis suppressed Sema3A-induced facilitation of axonal transport in the DRG neuron with and without the cell body. Sema3A enhanced the level of immunoreactivity of phosphorylated eukaryotic translation initiation factor 4E (eIF-4E) within 5 min in growth cones in a time course similar to that of the facilitated axonal transport. This enhanced signal for phospho-eIF4E was blocked by lavendustin A or olomoucine and was not detected in the fyn(-/-) and p35(-/-) neurons. These results provide evidence for a mutual regulatory mechanism between local protein synthesis and axonal transport.

Cyclin-dependent kinase 5 (Cdk5) is essential for the proper development of the CNS, as is evident from the perinatal lethality of conventional Cdk5 knockout (Cdk5-/-) mice. Cdk5 is also implicated in numerous complex functions of the adult CNS such as synaptic transmission, synaptic plasticity, and neuronal signaling. To elucidate the molecular roles of Cdk5 in the adult CNS, we have abrogated neuronal expression of Cdk5 in perinatal mice by using a cre-loxP system. The Cdk5-loxP flanked mice were crossed with the cre-transgenic mice in which the cre expression is driven by the murine neurofilament-heavy chain promoter, resulting in generation of viable Cdk5 conditional knockout mice with the restricted deletion of the Cdk5 gene in specific neurons beginning around embryonic day 16.5. Twenty-five percent of the Cdk5 conditional knockout mice carrying the heterozygous cre allele had neuronal migration defects confined to brain areas where neuronal migration continues through the perinatal period. These results indicate that abrogation of Cdk5 expression in mature neurons results in a viable mouse model that offers further opportunities to investigate the molecular roles of Cdk5 in the adult CNS.

It has been thought that clathrin-mediated endocytosis is regulated by phosphorylation and dephosphorylation of many endocytic proteins, including amphiphysin I and dynamin I. Here, we show that Cdk5/p35-dependent cophosphorylation of amphiphysin I and dynamin I plays a critical role in such processes. Cdk5 inhibitors enhanced the electric stimulation-induced endocytosis in hippocampal neurons, and the endocytosis was also enhanced in the neurons of p35-deficient mice. Cdk5 phosphorylated the proline-rich domain of both amphiphysin I and dynamin I in vitro and in vivo. Cdk5-dependent phosphorylation of amphiphysin I inhibited the association with P-adaptin. Furthermore, the phosphorylation of dynamin I blocked its binding to amphiphysin I. The phosphorylation of each protein reduced the copolymerization into a ring formation in a cell-free system. Moreover, the phosphorylation of both proteins completely disrupted the copolymerization into a ring formation. Finally, phosphorylation of both proteins was undetectable in p35-deficient mice.

Disabled-1 (Dab I) is a cytosolic adaptor protein that plays critical roles in cortical development. However, a detailed mechanism of action has not yet been clearly defined. Through yeast two-hybrid screening, we observed that mouse Siah-1A, an E3 ubiquitin ligase containing a RING finger motif, interacts with Dab I. Co-immunoprecipitation experiments and in vitro binding experiments both indicated direct interaction between Siah and Dab I. Steady-state expression of Siah was enhanced by the presence of Dab I or lactacystin, a representative proteasomal inhibitor. Auto-ubiquitination of Siah was inhibited by the presence of Dabl, suggesting inhibition of Siah activity and subsequent increase of Siah expression by Dabl. Both Dab1-induced increase of steady-state expression of Deleted in cololectal cancer (DCC), one of the well-known substrates of Siah, and its inhibition by SiahDeltaR suggest that Dabl increases expression of DCC through inhibiting the activity of endogenous Siah. Our results suggest that Dabl inhibits the activity of Siah. (C) 2003 Elsevier Science (USA). All rights reserved.

Neuronal positioning is important for higher brain function because it is the architectural basis of the formation of precise synaptic circuits. Analysis of neurological mutant mice has led to dramatic progress in the identification and characterization of molecules important for neuronal positioning in the developing mammalian brain. Among these molecules, identification of signal pathways mediated by Reelin and Cdk5 kinase has provided a conceptual framework for exploring the molecular mechanisms underlying proper neuronal positioning in the developing mammalian brain. Recent evidence has implicated synergism between Reelin signaling and Cdk5 in contributing to the proper positioning of selective neuronal populations.

Dendrite development in neurons is one of the bases for the formation of a complex neuronal network in the nervous system, and involvement of the Rho family GTPases, including Rac1, Cdc42, and RhoA, in dendrite formation has been demonstrated. One of the effectors of Rac1 and Cdc42, p21-activated kinase 1 (Pak1), is abundant in the brain; however, the function of Pak1 in neurons remains unknown. In order to clarify the roles of Pak1 in neurons, we introduced mutant Pak1 into immature neurons by a novel gene transfer technique using in utero electroporation. Introduction of constitutive active (CA)-Pak1 led to increase the number of dendrites, whereas introduction of dominant negative (DN)-Pak1 caused a reduction, and coexpression of CA-Rac1 and DN-Pak1 also resulted in a reduction in the number of dendrites. These results suggest that Pak1 regulates dendrite initiation as a downstream effector of Rac1.

Semaphorin-3A (Sema3A), a member of class 3 semaphorins, regulates axon and dendrite guidance in the nervous system. How Sema3A and its receptors plexin-As and neuropilins regulate neuronal guidance is unknown. We observed that in fyn- and cdk5-deficient mice, Sema3A-induced growth cone collapse responses were attenuated compared to their heterologous controls. Cdk5 is associated with plexin-A2 through the active state of Fyn. Sema3A promotes Cdk5 activity through phosphorylation of Tyr15, a phosphorylation site with Fyn. A Cdk5 mutant (Tyr15 to Ala) shows a dominant-negative effect on the Sema3A-induced collapse response. The sema3A gene shows strong interaction with fyn for apical dendrite guidance in the cerebral cortex. We propose a signal transduction pathway in which Fyn and Cdk5 mediate neuronal guidance regulated by Sema3A.

Cyclin-dependent kinase (Cdk) 5 is a unique member of the Cdk family, because Cdk5 kinase activity is detected only in the nervous tissue. Two neuron-specific activating subunits of Cdk5, p35 and p39, have been identified. Overlapping expression pattern of these isoforms in the embryonic mouse brain and the significant residual Cdk5 kinase activity in brain homogenate of the p35-/- mice indicate the redundant functions of the Cdk5 activators in vivo. Severe neuronal migration defects in p35-/-Cdk5+/- mice further support the idea that the redundant expression of the Cdk5 activators may cause a milder phenotype in p35-/- mice compared with Cdk5-/- mice. Mutant mice lacking either Cdk5 or p35 exhibit certain similarities with Reelin/Dab1-mutant mice in the disorganization of cortical laminar structure in the brain. To elucidate the relationship between Cdk5/p35 and Reelin/Dab1 signaling, we generated mouse lines that have combined defects of these genes. The addition of heterozygosity of either Dab1 or Reelin mutation to p35-/- causes the extensive migration defects of cortical neurons in the cerebellum. In the double-null mice of p35 and either Dab1 or Reelin, additional migration defects occur in the Purkinje cells in the cerebellum and in the pyramidal neurons in the hippocampus. These additional defects in neuronal migration in mice lacking both Cdk5/p35 and Reelin/Dab1 indicate that Cdk5/p35 may contribute synergistically to the positioning of the cortical neurons in the developing mouse brain.