論文

N-cadherin (NCad) is a classical cadherin that mediates cell–cell interactions in a Ca2+-dependent manner. NCad participates in various biological processes, from ontogenesis to higher brain functions, though the visualization of NCad interactions in living cells remains limited. Here, we present intensiometric NCad interaction indicators, named INCIDERs, that utilize dimerization-dependent fluorescent proteins. INCIDERs successfully visualize reversible NCad interactions across cells. Compared to FRET-based indicators, INCIDERs have a ~70-fold higher signal contrast, enabling clear identification of NCad interactions. In primary neuronal cells, NCad interactions are visualized between closely apposed processes. Furthermore, visualization of NCad interaction at cell adhesion sites in dense cell populations is achieved by two-photon microscopy. INCIDERs are useful tools in the spatiotemporal investigation of NCad interactions across cells; future research should evaluate the potential of INCIDERs in mapping complex three-dimensional architectures in multi-cellular systems.

Astrocytes are one of the most abundant cell types in the mammalian brain. They play essential roles in synapse formation, maturation, and elimination. However, how astrocytes migrate into the gray matter to accomplish these processes is poorly understood. Here, we show that, by combinational analyses of in vitro and in vivo time-lapse observations and lineage traces, astrocyte progenitors move rapidly and irregularly within the developing cortex, which we call erratic migration. Astrocyte progenitors also adopt blood vessel-guided migration. These highly motile progenitors are generated in the restricted prenatal stages and differentiate into protoplasmic astrocytes in the gray matter, whereas postnatally generated progenitors do not move extensively and differentiate into fibrous astrocytes in the white matter. We found Cxcr4/7, and integrin β1 regulate the blood vessel-guided migration, and their functional blocking disrupts their positioning. This study provides insight into astrocyte development and may contribute to understanding the pathogenesis caused by their defects.

Homer is a postsynaptic scaffold protein, which has long and short isoforms. The long form of Homer consists of an N-terminal target-binding domain and a C-terminal multimerization domain, linking multiple proteins within a complex. The short form of Homer only has the N-terminal domain and likely acts as a dominant negative regulator. Homer2a, one of the long form isoforms of the Homer family, expresses with a transient peak in the early postnatal stage of mouse cerebellar granule cells (CGCs); however, the functions of Homer2a in CGCs are not fully understood yet. In this study, we investigated the physiological roles of Homer2a in CGCs using recombinant adenovirus vectors. Overexpression of the Homer2a N-terminal domain construct, which was made structurally reminiscent with Homer1a, altered NMDAR1 localization, decreased NMDA currents, and promoted the survival of CGCs. These results suggest that the Homer2a N-terminal domain acts as a dominant negative protein to attenuate NMDAR-mediated excitotoxicity. Moreover, we identified a novel short form N-terminal domain-containing Homer2, named Homer2e, which was induced by apoptotic stimulation such as ischemic brain injury. Our study suggests that the long and short forms of Homer2 are involved in apoptosis of CGCs.

© 2019 Wiley Periodicals, Inc. During development of the mammalian cerebral neocortex, postmitotic excitatory neurons migrate toward the outermost region of the neocortex. We previously reported that this outermost region is composed of densely packed relatively immature neurons; we named this region, which is observed during the late stage of mouse neocortical development, the “primitive cortical zone (PCZ).” Here, we report that postmigratory immature neurons spend about 1–1.5 days in the PCZ. An electron microscopic analysis showed that the neurons in the PCZ tend to be in direct contact with each other, mostly in a radial direction, forming “primitive neuronal clusters” with a height of 3–7 cells and a width of 1–2 cells. A time-course analysis of fluorescently labeled neurons revealed that the neurons took their positions within the primitive clusters in an inside-out manner. The neurons initially participated in the superficial part of the clusters, gradually shifted their relative positions downward, and then left the clusters at the bottom of this structure. GABAergic inhibitory interneurons were also found within the primitive clusters in the developing mouse neocortex, suggesting that some clusters are composed of both excitatory neurons and inhibitory interneurons. Similar clusters were also observed in the outermost region of embryonic day (E) 78 cynomolgus monkey occipital cortex and 23 gestational week (GW) human neocortices. In the primate neocortices, including human, the presumptive primitive clusters seemed to expand in the radial direction more than that observed in mice, which might contribute to the functional integrity of the primate neocortex.

© 2019 the authors. The actin cytoskeleton is crucial for neuronal migration in the mammalian developing cerebral cortex. The adaptor protein Drebrin-like (Dbnl) plays important roles in reorganization of the actin cytoskeleton, dendrite formation, and endocytosis by interacting with F-actin, cobl, and dynamin. Although Dbnl is known to be expressed in the brain, the functions of this molecule during brain development are largely unknown. In this study, to examine the roles of Dbnl in the developing cerebral cortex, we conducted experiments using mice of both sexes with knockdown of Dbnl, effected by in utero electroporation, in the migrating neurons of the embryonic cortex. Time-lapse imaging of the Dbnl-knockdown neurons revealed that the presence of Dbnl is a prerequisite for appropriate formation of processes in the multipolar neurons in the multipolar cell accumulation zone or the deep part of the subventricular zone, and for neuronal polarization and entry into the cortical plate. We found that Dbnl knockdown decreased the amount of N-cadherin protein expressed on the plasma membrane of the cortical neurons. The defect in neuronal migration caused by Dbnl knockdown was rescued by moderate overexpression of N-cadherin and αN-catenin or by transfection of the phospho-mimic form(Y337E, Y347E), but not the phospho-resistant form(Y337F, Y347F), of Dbnl. These results suggest that Dbnl controls neuronal migration, neuronal multipolar morphology, and cell polarity in the developing cerebral cortex via regulating N-cadherin expression.

Mutations of PAFAH1B1 cause classical lissencephaly in humans. In addition, duplications and triplications of PAFAH1B1 are found in individuals with intellectual disability and other neurological disorders suggesting that proper brain development is highly sensitive to the PAFAH1B1 dosage. To examine the effect of PAFAH1B1 over-dosage in neural development, especially in migration of neurons and layer formation during cerebral cortical development, we overexpressed Pafah1b1 in migrating neurons in the mouse embryonic cortex using in utero electroporation. Enhanced expression of Pafah1b1 in radially-migrating neurons resulted in their over-migration into the marginal zone. Neurons that invaded the marginal zone were oriented abnormally. Layer distribution of Pafaha1b1-overexpressing neurons shifted more superficially than control neurons. Some of the Pafaha1b1-overexpressing future layer 4 neurons changed their positions to layers 2/3. Furthermore, they also changed their layer marker expression from layer 4 to layers 2/3. These results suggest that overexpression of Pafah1b1 affects the migration of neurons and disrupts layer formation in the developing cerebral cortex, and further support the idea that appropriate dosage of Pafah1b1 is crucial for the proper development of the brain.

Very-KIND/Kndc1/KIAA1768 (v-KIND) is a brain-specific Ras guanine nucleotide exchange factor carrying two sets of the kinase non-catalytic C-lobe domain (KIND), and is predominantly expressed in cerebellar granule cells. Here, we report the impact of v-KIND deficiency on dendritic and synaptic growth in cerebellar granule cells in v-KIND knockout (KO) mice. Furthermore, we evaluate motor function in these animals. The gross anatomy of the cerebellum, including the cerebellar lobules, layered cerebellar cortex and densely-packed granule cell layer, in KO mice appeared normal, and was similar to wild-type (WT) mice. However, KO mice displayed an overgrowth of cerebellar granule cell dendrites, compared with WT mice, resulting in an increased number of dendrites, dendritic branches and terminals. Immunoreactivity for vGluT2 (a marker for excitatory presynapses of mossy fiber terminals) was increased in the cerebellar glomeruli of KO mice, compared with WT mice. The postsynaptic density around the terminals of mossy fibers was also increased in KO mice. Although there were no significant differences in locomotor ability between KO and WT animals in their home cages or in the open field, young adult KO mice had an increased grip strength and a tendency to exhibit better motor performance in balance-related tests compared with WT animals. Taken together, our results suggest that v-KIND is required for compact dendritic growth and proper excitatory synaptic connections in cerebellar granule cells, which are necessary for normal motor coordination and balance.

Reelin is an essential glycoprotein for the establishment of the highly organized six-layered structure of neurons of the mammalian neocortex. Although the role of Reelin in the control of neuronal migration has been extensively studied at the molecular level, the mechanisms underlying Reelin-dependent neuronal layer organization are not yet fully understood. In this study, we directly showed that Reelin promotes adhesion among dissociated neocortical neurons in culture. The Reelin-mediated neuronal aggregation occurs in an N-cadherin-dependent manner, both in vivo and in vitro. Unexpectedly, however, in a rotation culture of dissociated neocortical cells that gradually reaggregated over time, we found that it was the neural progenitor cells [radial glial cells (RGCs)], rather than the neurons, that tended to form clusters in the presence of Reelin. Mathematical modeling suggested that this clustering of RGCs could be recapitulated if the Reelin-dependent promotion of neuronal adhesion were to occur only transiently. Thus, we directly measured the adhesive force between neurons and N-cadherin by atomic force microscopy, and found that Reelin indeed enhanced the adhesiveness of neurons to N-cadherin; this enhanced adhesiveness began to be observed at 30 min after Reelin stimulation, but declined by 3 h. These results suggest that Reelin transiently (and not persistently) promotes N-cadherin-mediated neuronal aggregation. When N-cadherin and stabilized β-catenin were overexpressed in the migrating neurons, the transfected neurons were abnormally distributed in the superficial region of the neocortex, suggesting that appropriate regulation of N-cadherin-mediated adhesion is important for correct positioning of the neurons during neocortical development.

During neural development, endosomal trafficking controls cell shape and motility through the polarized transport of membrane proteins related to cellcell and cellextracellular matrix interactions. ADP ribosylation factor 6 (Arf6) is a critical small GTPase that regulates membrane trafficking between the plasma membrane and endosomes. We herein demonstrated that the knockdown of endogenous Arf6 in mouse cerebral cortices led to impaired neuronal migration in the intermediate zone and cytoplasmic retention of N-cadherin and syntaxin12 in migrating neurons. Rescue experiments with separation-of-function Arf6 mutants identified Rab11 familyinteracting protein 3 (FIP3)/Arfophilin-1, a dual effector for Arf6 and Rab11, as a downstream effector of Arf6 in migrating neurons. The knockdown of FIP3 led to impaired neuronal migration in the intermediate zone and cytoplasmic retention of N-cadherin in migrating neurons, similar to that of Arf6, which could be rescued by the coexpression of wild-type FIP3 but not FIP3 mutants lacking the binding site for Arf6 or Rab11. These results suggest that Arf6 regulates cortical neuronal migration in the intermediate zone through the FIP3-dependent endosomal trafficking.

A fine structure of the hippocampus is required for proper functions, and disruption of this formation by neuronal migration defects during development may play a role in some psychiatric illnesses. During hippocampal development in rodents, pyramidal neurons in the Ammon's horn are mostly generated in the ventricular zone (VZ), spent as multipolar cells just above the VZ, and then migrate radially toward the pial surface, ultimately settling into the hippocampal plate. Although this process is similar to that of neocortical projection neurons, these are not identical. In addition to numerous histological studies, the development of novel techniques gives a clear picture of the cellular dynamics of hippocampal neurons, as well as neocortical neurons. In this article, we provide an overview of the cellular mechanisms of rodent hippocampal neuronal migration including those of dentate granule cells, especially focusing on the differences of migration modes between hippocampal neurons and neocortical neurons. The unique migration mode of hippocampal pyramidal neurons might enable clonally related cells in the Ammon's horn to distribute in a horizontal fashion.

The preoptic area (POa) of the rostral diencephalon supplies the neocortex and the amygdala with GABAergic neurons in the developing mouse brain. However, the molecular mechanisms that determine the pathway and destinations of POa-derived neurons have not yet been identified. Here we show that Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII)-induced expression of Neuropilin-2 (Nrp2) and its down-regulation control the destination of POa-derived GABAergic neurons. Initially, a majority of the POa-derived migrating neurons express COUP-TFII and form a caudal migratory stream toward the caudal subpallium. When a subpopulation of cells steers toward the neocortex, they exhibit decreased expression of COUP-TFII and Nrp2. The present findings show that suppression of COUP-TFII/Nrp2 changed the destination of the cells into the neocortex, whereas overexpression of COUP-TFII/Nrp2 caused cells to end up in the medial part of the amygdala. Taken together, these results reveal that COUP-TFII/Nrp2 is a molecular switch determining the pathway and destination of migrating GABAergic neurons born in the POa.

The cAMP and cAMP-dependent protein kinase A (PKA) signaling cascade is a ubiquitous pathway acting downstream of multiple neuromodulators. We found that the phosphorylation of phosphodiesterase-4 (PDE4) by cyclin-dependent protein kinase 5 (Cdk5) facilitated cAMP degradation and homeostasis of cAMP/PKA signaling. In mice, loss of Cdk5 throughout the forebrain elevated cAMP levels and increased PKA activity in striatal neurons, and altered behavioral responses to acute or chronic stressors. Ventral striatum-or D1 dopamine receptor-specific conditional knockout of Cdk5, or ventral striatum infusion of a small interfering peptide that selectively targeted the regulation of PDE4 by Cdk5, produced analogous effects on stress-induced behavioral responses. Together, our results demonstrate that altering cAMP signaling in medium spiny neurons of the ventral striatum can effectively modulate stress-induced behavioral states. We propose that targeting the Cdk5 regulation of PDE4 could be a new therapeutic approach for clinical conditions associated with stress, such as depression.

Cytoplasmic aggregation of fused in sarcoma (FUS) is detected in brain regions affected by amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), which compose the disease spectrum, FUS proteinopathy. To understand the pathomechanism of ALS-FTD-associated FUS, we examined the behavior and cellular properties of an ALS mouse model overexpressing FUS with nuclear localization signal deletion. Mutant FUS transgenic mice showed hyperactivity, social interactional deficits, and impaired fear memory retrieval, all of which are compatible with FTD phenotypes. Histological analyses showed decreased dendritic spine and synaptic density in the frontal cortex before neuronal loss. Examination of cultured cells confirmed that mutant but not wild-type FUS was associated with decreased dendritic growth, mRNA levels, and protein synthesis in dendrites. These data suggest that cytoplasmic FUS aggregates impair dendritic mRNA trafficking and translation, in turn leading to dendritic homeostasis disruption and the development of FTD phenotypes.

The kinase noncatalytic C-lobe domain (KIND) is a putative protein-protein interaction module. Four KIND-containing proteins, Spir-2 (actin-nuclear factor), PTPN13 (protein tyrosine phosphatase), FRMPD2 (scaffold protein) and very-KIND (v-KIND) (brain-specific Ras guanine nucleotide exchange factor), have been identified to date. Uniquely, v-KIND has two KINDs (i.e. KIND1 and KIND2), whereas the other three proteins have only one. The functional role of KIND, however, remains unclear. We previously demonstrated that v-KIND interacts with the high-molecular weight microtubule-associated protein 2 (MAP2), a dendritic microtubule-associated protein, leading to negative regulation of neuronal dendrite growth. In the present study, we analyzed the structure-function relationships of the v-KIND-MAP2 interaction by generating a series of mutant constructs. The interaction with endogenous MAP2 in mouse cerebellar granule cells was specific to v-KIND KIND2, but not KIND1, and was not observed for the KINDs from other KIND-containing proteins. The binding core modules critical for the v-KIND-MAP2 interaction were defined within 32 residues of the mouse v-KIND KIND2 and 43 residues of the mouse MAP2 central domain. Three Leu residues at amino acid positions 461, 474 and 477 in the MAP2-binding core module of KIND2 contributed to the interaction. The MAP2-binding core module itself promoted dendrite branching as a dominant-negative regulator of v-KIND in hippocampal neurons. The results reported in the present study demonstrate the structural and functional determinant underlying the v-KIND-MAP2 interaction that controls dendrite arborization patterns. Structured digital abstract to by with by,) with by with by with by The brain-specific Ras guanine exchange factor (RasGEF) very-kinase noncatalytic C-lobe domain (KIND) regulates neuronal dendrite growth by interacting with the high-molecular weight microtubule-associated protein 2 (MAP2). We describe the structural and functional determinants of the protein-protein interaction between very-KIND and MAP2. We suggest that the MAP2-binding core module of very-KIND is involved in negative regulation of dendrite branching in hippocampal neurons and cerebellar granule cells

Background: Phospholipase D (PLD) catalyzes conversion of phosphatidylcholine into choline and phosphatidic acid, leading to a variety of intracellular signal transduction events. Two classical PLDs, PLD1 and PLD2, contain phosphatidylinositide-binding PX and PH domains and two conserved His-x-Lys-(x)4-Asp (HKD) motifs, which are critical for PLD activity. PLD4 officially belongs to the PLD family, because it possesses two HKD motifs. However, it lacks PX and PH domains and has a putative transmembrane domain instead. Nevertheless, little is known regarding expression, structure, and function of PLD4. Methodology/Principal Findings: PLD4 was analyzed in terms of expression, structure, and function. Expression was analyzed in developing mouse brains and non-neuronal tissues using microarray, in situ hybridization, immunohistochem-istry, and immunocytochemistry. Structure was evaluated using bioinformatics analysis of protein domains, biochemical analyses of transmembrane property, and enzymatic deglycosylation. PLD activity was examined by choline release and transphosphatidylation assays. Results demonstrated low to modest, but characteristic, PLD4 mRNA expression in a subset of cells preferentially localized around white matter regions, including the corpus callosum and cerebellar white matter, during the first postnatal week. These PLD4 mRNA-expressing cells were identified as Iba1-positive microglia. In nonneuronal tissues, PLD4 mRNA expression was widespread, but predominantly distributed in the spleen. Intense PLD4 expression was detected around the marginal zone of the splenic red pulp, and splenic PLD4 protein recovered from subcellular membrane fractions was highly N-glycosylated. PLD4 was heterologously expressed in cell lines and localized in the endoplasmic reticulum and Golgi apparatus. Moreover, heterologously expressed PLD4 proteins did not exhibit PLD enzymatic activity. Conclusions/Significance: Results showed that PLD4 is a non-PLD, HKD motif-carrying, transmembrane glycoprotein localized in the endoplasmic reticulum and Golgi apparatus. The spatiotemporally restricted expression patterns suggested that PLD4 might play a role in common function(s) among microglia during early postnatal brain development and splenic marginal zone cells.

Background Attention-deficit/hyperactivity disorder (ADHD) may result from delayed establishment of corticolimbic circuitry or perturbed dopamine (DA) neurotransmission. Despite the widespread use of stimulants to treat ADHD, little is known regarding their long-term effects on neurotransmitter levels and metabolism. Cyclin-dependent kinase 5 (Cdk5) regulates DA signaling through control of synthesis, postsynaptic responses, and vesicle release. Mice lacking the Cdk5-activating cofactor p35 are deficient in cortical lamination, suggesting altered motor/reward circuitry. Methods We employed mice lacking p35 to study the effect of altered circuitry in vivo. Positron emission tomography measured glucose metabolism in the cerebral cortex using 2-deoxy-2-[ 18F] fluoro-d-glucose as the radiotracer. Retrograde dye tracing and tyrosine hydroxylase immunostains assessed the effect of p35 knockout on the medial prefrontal cortex (PFC), especially in relation to mesolimbic circuit formation. We defined the influence of Cdk5/p35 activity on catecholaminergic neurotransmission and motor activity via examination of locomotor responses to psychostimulants, monoamine neurotransmitter levels, and DA signal transduction. Results Here, we report that mice deficient in p35 display increased glucose uptake in the cerebral cortex, basal hyperactivity, and paradoxical decreased locomotion in response to chronic injection of cocaine or methylphenidate. Knockout mice also exhibited an increased susceptibility to changes in PFC neurotransmitter content after chronic methylphenidate exposure and altered basal DAergic activity in acute striatal and PFC slices. Conclusions Our findings suggest that dysregulation of Cdk5/p35 activity during development may contribute to ADHD pathology, as indicated by the behavioral phenotype, improperly established mesolimbic circuitry, and aberrations in striatal and PFC catecholaminergic signaling in p35 knockout mice.

The cerebellar Purkinje cell monolayer is organized into heterogeneous Purkinje cell compartments that have different molecular compositions. Here we describe a transgenic mouse line, 1NM13, that shows heterogeneous transgene expression in parasagittal Purkinje cell arrays. The transgene consists of a nuclear localization signal (nls) fused to the b-galactosidase (lacZ) composite gene driven by the type 1 inositol 1,4,5-trisphosphate receptor (IP 3R1) gene promoter. IP3R1-nls-lacZ transgene expression was detected at a single Purkinje cell level over the surface of a whole-mount X-gal-stained cerebellum because of nuclear accumulation of the nls-lacZ activity. Developing cerebella of 1NM13 mice showed stripe-like X-gal staining patterns of parasagittal Purkinje cell subsets. The X-gal stripe pattern was likely determined by an intrinsic property as early as E15 and showed increasing complexity with cerebellar development. The X-gal stripe pattern was reminiscent of, but not identical to, the stripe pattern of zebrin II immunoreactivity. We designated the symmetrical X-gal-positive (transgene-positive, Tg+) Purkinje cell stripes about the midline as vermal Tg1+, Tg2(a, b)+ and Tg3(a, b)+ stripes and hemispheric Tg4(a, b)+, Tg5(a, b)+, Tg6(a, b, c) +, and Tg7(a, b)+ stripes, where a, b, and c indicate substripes. We also assigned three parafloccular substripes Tg8(a, b, c) +. The boundaries of X-gal stripes at P5 were consistent with raphes in the Purkinje cell layer through which granule cells migrate, suggesting a possible association of the X-gal stripes with raphe formation. Our results indicate that 1NM13 is a good mouse model with a reproducible and clear marker for the compartmentalization of Purkinje cell arrays.

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase that has been implicated in learning, synaptic plasticity, neurotransmission, and numerous neurological disorders. We previously showed that conditional loss of Cdk5 in adult mice enhanced hippocampal learning and plasticity via modulation of calpain-mediated N-methyl-D-aspartic acid receptor (NMDAR) degradation. In the present study, we characterize the enhanced synaptic plasticity and examine the effects of long-term Cdk5 loss on hippocampal excitability in adult mice. Field excitatory post-synaptic potentials (fEPSPs) from the Schaffer collateral CA1 subregion of the hippocampus (SC/CA1) reveal that loss of Cdk5 altered theta burst topography and enhanced post-tetanic potentiation. Since Cdk5 governs NMDAR NR2B subunit levels, we investigated the effects of long-term Cdk5 knockout on hippocampal neuronal excitability by measuring NMDAR-mediated fEPSP magnitudes and population-spike thresholds. Long-term loss of Cdk5 led to increased Mg2+-sensitive potentials and a lower threshold for epileptiform activity and seizures. Biochemical analyses were performed to better understand the role of Cdk5 in seizures. Induced-seizures in wild-type animals led to elevated amounts of p25, the Cdk5-activating cofactor. Long-term, but not acute, loss of Cdk5 led to decreased p25 levels, suggesting that Cdk5/p25 may be activated as a homeostatic mechanism to attenuate epileptiform activity. These findings indicate that Cdk5 regulates synaptic plasticity, controls neuronal and behavioral stimulus-induced excitability and may be a novel pharmacological target for cognitive and anticonvulsant therapies.

Motor learning and neuro-adaptations to drugs of abuse rely upon neuronal signaling in the striatum. Cyclin-dependent kinase 5 (Cdk5) regulates striatal dopamine neurotransmission and behavioral responses to cocaine. Although the role for Cdk5 in neurodegeneration in the cortex and hippocampus and in hippocampal-dependent learning has been demonstrated, its dysregulation in the striatum has not been examined. Here we show that strong activation of striatal NMDA receptors produced p25, the truncated form of the Cdk5 co-activator p35. Furthermore, inducible overexpression of p25 in the striatum prevented locomotor sensitization to cocaine and attenuated motor coordination and learning. This corresponded with reduced dendritic spine density, increased neuro-inflammation, altered dopamine signaling, and shifted Cdk5 specificity with regard to physiological and aberrant substrates, but no apparent loss of striatal neurons. Thus, dysregulation of Cdk5 dramatically affects striatal-dependent brain function and may be relevant to non-neurodegenerative disorders involving dopamine neurotransmission.