Rab21 regulates caveolin-1-mediated endocytic trafficking to promote immature neurite pruning.

Transmembrane proteins are internalized by clathrin- and caveolin-dependent endocytosis. Both pathways converge on early endosomes and are thought to share the small GTPase Rab5 as common regulator. In contrast to this notion, we show here that the clathrin- and caveolin-mediated endocytic pathways are differentially regulated. Rab5 and Rab21 localize to distinct populations of early endosomes in cortical neurons and preferentially regulate clathrin- and caveolin-mediated pathways, respectively, suggesting heterogeneity in the early endosomes, rather than a converging point. Suppression of Rab21, but not Rab5, results in decreased plasma membrane localization and total protein levels of caveolin-1, which perturbs immature neurite pruning of cortical neurons, an in vivo-specific step of neuronal maturation. Taken together, our data indicate that clathrin- and caveolin-mediated endocytic pathways run in parallel in early endosomes, which show different molecular regulation and physiological function.

[Ca2+]i fluctuation mediated by T-type Ca2+ channel is required for the differentiation of cortical neural progenitor cells.

The fluctuation of intracellular calcium concentration ([Ca2+]i) is known to be involved in various processes in the development of central nervous system, such as the proliferation of neural progenitor cells (NPCs), migration of intermediate progenitor cells (IPCs) from the ventricular zone (VZ) to the subventricular zone (SVZ), and migration of immature neurons from the SVZ to cortical plate. However, the roles of [Ca2+]i fluctuation in NPC development, especially in the differentiation of the self-renewing NPCs into neuron-generating NPCs and immature neurons have not been elucidated. Using calcium imaging of acute cortical slices and cells isolated from mouse embryonic cortex, we examined temporal changes in the pattern of [Ca2+]i fluctuations in VZ cells from E12 to E16. We observed intracellular Ca2+ levels in Pax6-positive self-renewing NPCs decreased with their neural differentiation. In E11, Pax6-positive NPCs and Tuj1-positive immature neurons exhibited characteristic [Ca2+]i fluctuations; few Pax6-positive NPCs exhibited [Ca2+]i transient, but many Tuj1-positive immature neurons did, suggesting that the change in pattern of [Ca2+]i fluctuation correlate to their differentiation. The [Ca2+]i fluctuation during NPCs development was mostly mediated by the T-type calcium channel and blockage of T-type calcium channel in neurosphere cultures increased the number of spheres and inhibited neuronal differentiation. Consistent with this finding, knockdown of Cav3.1 by RNAi in vivo maintained Pax6-positive cells as self-renewing NPCs, and simultaneously suppressing their neuronal differentiation of NPCs into Tbr1-positive immature neurons. These results reveal that [Ca2+]i fluctuation mediated by Cav3.1 is required for the neural differentiation of Pax6-positive self-renewing NPCs.

[Pharmacological approach to cerebral cortical development].

In the developing mammalian cerebral cortex, newly generated neurons migrate toward the pial surface to form a mammalian-specific six-layered cerebral cortex. Genetic studies of human neurological diseases have suggested the involvement of several molecules in cortical neuronal migration. In vivo electroporation is another powerful tool for understanding the molecular mechanisms of neuronal migration. By using these techniques, however, it is difficult to understand molecular basis of time-dependent changes of neuronal morphologies. Here, we introduce a pharmacological approach to cerebral cortical development. Major advantages of the pharmacological approach include the transient suppression of molecules of interest and analyzing time-dependent changes of neuronal morphologies. It also allows us to search molecules regulating neuronal migration with comparative ease. We propose the complementarity between the pharmacological approach and genetics or in vivo electroporation experiments.

Caveolin-1 Promotes Early Neuronal Maturation via Caveolae-Independent Trafficking of N-Cadherin and L1.

Axon specification is morphologically reproducible in vitro, whereas dendrite formation differs in vitro and in vivo. Cortical neurons initially develop immature neurites, but in vivo these are eliminated concurrently with the formation of a leading process, the future dendrite. However, the molecular mechanisms underlying these neuronal maturation events remain unclear. Here we show that caveolin-1, a major component of caveolae that are never observed in neurons, regulates in vivo-specific steps of neuronal maturation. Caveolin-1 is predominantly expressed in immature cortical neurons and regulates clathrin-independent endocytosis. In vivo knockdown of caveolin-1 disturbs immature neurite pruning, leading process elongation, and subsequent neuronal migration. Importantly, N-cadherin and L1, which are required for immature neurite formation, undergo caveolin-1-mediated endocytosis to eliminate immature neurites. Collectively, our findings indicate that caveolin-1 regulates N-cadherin and L1 trafficking independent of caveolae, which contributes to spatiotemporally restricted cellular events; immature neurite pruning and leading process elongation during early neuronal maturation.

Morphological and Molecular Basis of Cytoplasmic Dilation and Swelling in Cortical Migrating Neurons.

During corticogenesis, neuronal migration is an essential step for formation of a functional brain, and abnormal migration is known to cause various neurological disorders. Neuronal migration is not just a simple movement of the cell body, but a consequence of various morphological changes and coordinated subcellular events. Recent advances in in vivo and ex vivo cell biological approaches, such as in utero gene transfer, slice culture and ex vivo chemical inhibitor techniques, have revealed details of the morphological and molecular aspects of neuronal migration. Migrating neurons have been found to have a unique structure, dilation or swelling, at the proximal region of the leading process; this structure is not found in other migrating cell types. The formation of this structure is followed by nuclear deformation and forward movement, and coordination of this three-step sequential morphological change (the dilation/swelling formation, nuclear elongation and nuclear movement) is essential for proper neuronal migration and the construction of a functional brain structure. In this review, we will introduce the morphological features of this unique structure in migrating neurons and summarize what is known about the molecules regulating the dilation/swelling formation and nuclear deformation and movement.

Cdk5 and its substrates, Dcx and p27kip1, regulate cytoplasmic dilation formation and nuclear elongation in migrating neurons.

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.

SIL1, a causative cochaperone gene of Marinesco-Söjgren syndrome, plays an essential role in establishing the architecture of the developing cerebral cortex.

Marinesco-Sjögren syndrome (MSS) is a rare autosomal recessively inherited disorder with mental retardation (MR). Recently, mutations in the SIL1 gene, encoding a co-chaperone which regulates the chaperone HSPA5, were identified as a major cause of MSS. We here examined the pathophysiological significance of SIL1 mutations in abnormal corticogenesis of MSS. SIL1-silencing caused neuronal migration delay during corticogenesis ex vivo. While RNAi-resistant SIL1 rescued the defects, three MSS-causing SIL1 mutants tested did not. These mutants had lower affinities to HSPA5 in vitro, and SIL1-HSPA5 interaction as well as HSPA5 function was found to be crucial for neuronal migration ex vivo. Furthermore time-lapse imaging revealed morphological disorganization associated with abnormal migration of SIL1-deficient neurons. These results suggest that the mutations prevent SIL1 from interacting with and regulating HSPA5, leading to abnormal neuronal morphology and migration. Consistent with this, when SIL1 was silenced in cortical neurons in one hemisphere, axonal growth in the contralateral hemisphere was delayed. Taken together, abnormal neuronal migration and interhemispheric axon development may contribute to MR in MSS.

Biochemical and morphological characterization of MAGI-1 in neuronal tissue.

The membrane-associated guanylate kinase with inverted organization (MAGI) proteins consist of three members, MAGI-1, MAGI-2 (also known as S-SCAM), and MAGI-3. Although MAGI-2 has been analyzed and shown to interact with a variety of postsynaptic proteins, functional analyses and characterization of MAGI-1 in neuronal tissues have been rare. In this study, we prepared a specific antibody against MAGI-1, anti-MAGI-1, and carried out biochemical and morphological analyses of MAGI-1 in rat neuronal tissues. By Western blotting, a high level of MAGI-1 was detected in nervous tissues, especially in olfactory bulb. Biochemical fractionation clarified that MAGI-1 was relatively enriched in the synaptosomal vesicle and synaptic plasma membrane fractions, whereas MAGI-2 and MAGI-3 appeared to be in the synaptic plasma membrane and postsynaptic density fractions. Immunofluorescent analyses revealed diffuse distribution of MAGI-1 in the cell body and processes of primary cultured rat hippocampal neurons, whereas MAGI-2 and MAGI-3 were likely to be enriched at synapses. Immunohistochemical analyses demonstrated that MAGI-1 was expressed in Purkinje cells, in hypocampal neurons in CA1 region, in the glomerulus region of olfactory bulb, and at the dorsal root entry zone in embryonic rat spinal cord. These results suggest neuronal roles of MAGI-1 different from those of MAGI-2/3.

Application of in utero electroporation and live imaging in the analyses of neuronal migration during mouse brain development.

Correct neuronal migration is crucial for brain architecture and function. During cerebral cortex development (corticogenesis), excitatory neurons generated in the proliferative zone of the dorsal telencephalon (mainly ventricular zone) move through the intermediate zone and migrate past the neurons previously located in the cortical plate and come to rest just beneath the marginal zone. The in utero electroporation technique is a powerful method for rapid gain- and loss-of-function studies of neuronal development, especially neuronal migration. This method enabled us to introduce genes of interest into ventricular zone progenitor cells of mouse embryos and to observe resulting phenotypes such as proliferation, migration, and cell morphology at later stages. In this Award Lecture Review, we focus on the application of the in utero electroporation method to functional analyses of cytoskeleton-related protein septin. We then refer to, as an advanced technique, the in utero electroporation-based real-time imaging method for analyses of cell signaling regulating neuronal migration. The in utero electroporation method and its application would contribute to medical molecular morphology through identification and characterization of the signaling pathways disorganized in various neurological and psychiatric disorders.

Dissecting the factors involved in the locomotion mode of neuronal migration in the developing cerebral cortex.

Neuronal migration is essential for proper cortical layer formation and brain function, because migration defects result in neurological disorders such as mental retardation and epilepsy. Neuronal migration is divided into several contiguous steps: early phase (multipolar mode), locomotion mode, and terminal translocation mode. The locomotion mode covers most of the migration route and thereby is the main contributor to cortical layer formation. However, analysis of the molecular mechanisms regulating this mode is difficult due to the secondary effects of defects at the early phase of migration. In this study, we established an ex vivo chemical inhibitor screening, allowing us to directly analyze the locomotion mode of migration. Roscovitine and PP2, inhibitors for Cdk5 and Src family kinases, respectively, suppressed the locomotion mode of migration. In line with this, a small percentage of Cdk5- or Src family kinase (Fyn)-knockdown cells exhibited locomoting morphology but retarded migration, although the majority of cells were stalled at the early phase of migration. We also showed that rottlerin, widely used as a specific inhibitor for protein kinase Cdelta (PKCdelta), suppressed the locomotion mode. Unexpectedly, however, the dominant-negative form as well as RNA interference for PKCdelta hardly affected the locomotion, whereas they may disturb terminal translocation. In addition, we found JNK to be a potential downstream target of rottlerin. Taken together, our novel chemical inhibitor screening provides evidence that Cdk5 and Src family kinases regulate the locomotion mode of neuronal migration. It also uncovered roles for Fyn and PKCdelta in the early and final phases of migration, respectively.

The p21-activated kinase is required for neuronal migration in the cerebral cortex.

The normal formation and function of the mammalian cerebral cortex depend on the positioning of its neurones, which occurs in a highly organized, layer-specific manner. The correct morphology and movement of neurones rely on synchronized regulation of their actin filaments and microtubules. The p21-activated kinase (Pak1), a key cytoskeletal regulator, controls neuronal polarization, elaboration of axons and dendrites, and the formation of dendritic spines. However, its in vivo role in the developing nervous system is unclear. We have utilized in utero electroporation into mouse embryo cortices to reveal that both loss and gain of Pak1 function affect radial migration of projection neurones. Overexpression of hyperactivated Pak1 predominantly caused neurones to arrest in the intermediate zone (IZ) with apparently misoriented and disorganized leading projections. Loss of Pak1 disrupted the morphology of migrating neurones, which accumulated in the IZ and deep cortical layers. Unexpectedly, a significant number of neurones with reduced Pak1 expression aberrantly entered into the normally cell-sparse marginal zone, suggesting their inability to cease migrating that may be due to their impaired dissociation from radial glia. Our findings reveal the in vivo importance of temporal and spatial regulation of the Pak1 kinase during key stages of cortical development.

Localized activation of p21-activated kinase controls neuronal polarity and morphology.

In the developing forebrain, neuronal polarization is a stepwise and initially reversible process that underlies correct migration and axon specification. Many aspects of cytoskeletal changes that accompany polarization are currently molecularly undefined and thus poorly understood. Here we reveal that the p21-activated kinase (Pak1) is essential for the specification of an axon and dendrites. In hippocampal neurons, activation of Pak1 is spatially restricted to the immature axon despite its uniform presence in all neurites. Hyperactivation of Pak1 at the membrane of all neurites or loss of Pak1 expression disrupts both neuronal morphology and the distinction between an axon and dendrites. We reveal that Pak1 acts on polarity in a kinase-dependent manner, by affecting the F-actin and microtubule cytoskeleton at least in part through Rac1 and cofilin. Our data are the first to demonstrate the importance of localized Pak1 kinase activation for neuronal polarization and differentiation.

Ptf1a, a bHLH transcriptional gene, defines GABAergic neuronal fates in cerebellum.

The molecular machinery governing glutamatergic-GABAergic neuronal subtype specification is unclear. Here we describe a cerebellar mutant, cerebelless, which lacks the entire cerebellar cortex in adults. The primary defect of the mutant brains was a specific inhibition of GABAergic neuron production from the cerebellar ventricular zone (VZ), resulting in secondary and complete loss of external germinal layer, pontine, and olivary nuclei during development. We identified the responsible gene, Ptf1a, whose expression was lost in the cerebellar VZ but was maintained in the pancreas in cerebelless. Lineage tracing revealed that two types of neural precursors exist in the cerebellar VZ: Ptf1a-expressing and -nonexpressing precursors, which generate GABAergic and glutamatergic neurons, respectively. Introduction of Ptf1a into glutamatergic neuron precursors in the dorsal telencephalon generated GABAergic neurons with representative morphological and migratory features. Our results suggest that Ptf1a is involved in driving neural precursors to differentiate into GABAergic neurons in the cerebellum.

Involvement of a Rac activator,P-Rex1, in neurotrophin-derived signaling and neuronal migration.

Rho-family GTPases play key roles in regulating cytoskeletal reorganization, contributing to many aspects of nervous system development. Their activities are known to be regulated by guanine nucleotide exchange factors (GEFs), in response to various extracellular cues. P-Rex1, a GEF for Rac, has been mainly investigated in neutrophils, in which this molecule contributes to reactive oxygen species formation. However, its role in the nervous system is essentially unknown. Here we describe the expression profile and a physiological function of P-Rex1 in nervous system development. In situ hybridization revealed that P-Rex1 is dynamically expressed in a variety of cells in the developing mouse brain, including some cortical and DRG neurons. In migrating neurons in the intermediate zone, P-Rex1 protein was found to localize in the leading process and adjacent cytoplasmic region. When transfected in pheochromocytoma PC12 cells, P-Rex1 can be activated by NGF, causing an increase in GTP-bound Rac1 and cell motility. Deletion analyses suggested roles for distinct domains of this molecule. Experiments using a P-Rex1 mutant lacking the Dbl-homology domain, a dominant-negative-like form, and small interfering RNA showed that endogenous P-Rex1 was involved in cell migration of PC12 cells and primary cultured neurons from the embryonic day 14 cerebral cortices, induced by extracellular stimuli (NGF, BDNF, and epidermal growth factor). Furthermore, in utero electroporation of the mutant protein into the embryonic cerebral cortex perturbed radial neuronal migration. These findings suggest that P-Rex1, which is expressed in a variety of cell types, is activated by extracellular cues such as neurotrophins and contributes to neuronal migration in the developing nervous system.

MAP1B phosphorylation is differentially regulated by Cdk5/p35, Cdk5/p25, and JNK.

Mode I phosphorylated MAP1B is observed in developing and pathogenic brains. Although Cdk5 has been believed to phosphorylate MAP1B in the developing cerebral cortex, we show that a Cdk5 inhibitor does not suppress mode I phosphorylation of MAP1B in primary and slice cultures, while a JNK inhibitor does. Coincidently, an increase in phosphorylated MAP1B was not observed in COS7 cells when Cdk5 was cotransfected with p35, but this did occur with p25 which is specifically produced in pathogenic brains. Our primary culture studies showed an involvement of Cdk5 in regulating microtubule dynamics without affecting MAP1B phosphorylation status. The importance of regulating microtubule dynamics in neuronal migration was also demonstrated by in utero electroporation experiments. These findings suggest that mode I phosphorylation of MAP1B is facilitated by JNK but not Cdk5/p35 in the developing cerebral cortex and by Cdk5/p25 in pathogenic brains, contributing to various biological events.