Different exercises can modulate the differentiation/maturation of neural stem/progenitor cells after photochemically induced focal cerebral infarction.

INTRODUCTION: Exercise therapies during rehabilitation significantly promote recovery from various deficits after cerebral infarction, which is mediated by neuronal plasticity with distinct inputs. Although adult neurogenesis can also be modulated by neuronal activity before synaptogenesis, how distinct exercises contribute to the neurological reorganization of the injured cerebral cortex remains unclear. In the present study, we aimed to elucidate the effects of different exercise therapies on motor recovery and neuronal reorganization after photochemically induced focal cerebral infarction. METHODS: Here, we examined the effects of three different exercises-(a) forced lower-intensity and (b) higher-intensity treadmill exercises, and (c) voluntary exercise with wheel running-on motor recovery and adult neurogenesis in a rat model of focal cerebral infarction. Photochemically induced thrombosis (PIT) was used to generate focal infarction in rats that was mostly confined to their motor cortices. RESULTS: Beam walking tests showed that recovery after PIT-induced cortical infarction differed in acute and chronic stages and was influenced by the type of exercise. Furthermore, forced low-intensity training had more positive effects on functional recovery than other exercises or control. To evaluate the production of newly generated cells including de novo neurogenesis, we performed lineage analysis with BrdU labeling and immunofluorescence experiments. Lower-intensity treadmill exercise increased the number of BrdU/NeuN colabeled cells, but not total BrdU-retaining or BrdU/Sox2-colabeled cells, in the peri-infarct region of the ipsilateral cortex. In contrast, high-intensity treadmill or voluntary exercises had the opposite effects. CONCLUSIONS: These results suggest that neuronal maturation can be differently modulated by distinct exercises and that low-intensity treadmill exercise could result in more potent generation of mature neurons. This also suggests the possibility that the generation of neural stem/progenitor cells and differentiation might be modulated by rehabilitation-mediated neural plasticity.

G-protein-coupled receptor 40 agonist GW9508 potentiates glucose-stimulated insulin secretion through activation of protein kinase Cα and ε in INS-1 cells.

OBJECTIVE: The mechanism by which G-protein-coupled receptor 40 (GPR40) signaling amplifies glucose-stimulated insulin secretion through activation of protein kinase C (PKC) is unknown. We examined whether a GPR40 agonist, GW9508, could stimulate conventional and novel isoforms of PKC at two glucose concentrations (3 mM and 20 mM) in INS-1D cells. METHODS: Using epifluorescence microscopy, we monitored relative changes in the cytosolic fluorescence intensity of Fura2 as a marker of change in intracellular Ca2+ ([Ca2+]i) and relative increases in green fluorescent protein (GFP)-tagged myristoylated alanine-rich C kinase substrate (MARCKS-GFP) as a marker of PKC activation in response to GW9508 at 3 mM and 20 mM glucose. To assess the activation of the two PKC isoforms, relative increases in membrane fluorescence intensity of PKCα-GFP and PKCε-GFP were measured by total internal reflection fluorescence microscopy. Specific inhibitors of each PKC isotype were constructed and synthesized as peptide fusions with the third α-helix of the homeodomain of Antennapedia. RESULTS: At 3 mM glucose, GW9508 induced sustained MARCKS-GFP translocation to the cytosol, irrespective of changes in [Ca2+]i. At 20 mM glucose, GW9508 induced sustained MARCKS-GFP translocation but also transient translocation that followed sharp increases in [Ca2+]i. Although PKCα translocation was rarely observed, PKCε translocation to the plasma membrane was sustained by GW9508 at 3 mM glucose. At 20 mM glucose, GW9508 induced transient translocation of PKCα and sustained translocation as well as transient translocation of PKCε. While the inhibitors (75 µM) of each PKC isotype reduced GW9508-potentiated, glucose-stimulated insulin secretion in INS-1D cells, the PKCε inhibitor had a more potent effect. CONCLUSION: GW9508 activated PKCε but not PKCα at a substimulatory concentration of glucose. Both PKC isotypes were activated at a stimulatory concentration of glucose and contributed to glucose-stimulated insulin secretion in insulin-producing cells.

Ion channels, guidance molecules, intracellular signaling and transcription factors regulating nervous and vascular system development.

Our sophisticated thoughts and behaviors are based on the miraculous development of our complex nervous network system, in which many different types of proteins and signaling cascades are regulated in a temporally and spatially ordered manner. Here we review our recent attempts to grasp the principles of nervous system development in terms of general cellular phenomena and molecules, such as volume-regulated anion channels, intracellular Ca(2+) and cyclic nucleotide signaling, the Npas4 transcription factor and the FLRT family of axon guidance molecules. We also present an example illustrating that the same FLRT family may regulate the development of vascular networks as well. The aim of this review is to open up new vistas for understanding the intricacy of nervous and vascular system development.

Activity-dependent endogenous taurine release facilitates excitatory neurotransmission in the neocortical marginal zone of neonatal rats.

In the developing cerebral cortex, the marginal zone (MZ), consisting of early-generated neurons such as Cajal-Retzius cells, plays an important role in cell migration and lamination. There is accumulating evidence of widespread excitatory neurotransmission mediated by γ-aminobutyric acid (GABA) in the MZ. Cajal-Retzius cells express not only GABAA receptors but also α2/ß subunits of glycine receptors, and exhibit glycine receptor-mediated depolarization due to high [Cl(-)]i. However, the physiological roles of glycine receptors and their endogenous agonists during neurotransmission in the MZ are yet to be elucidated. To address this question, we performed optical imaging from the MZ using the voltage-sensitive dye JPW1114 on tangential neocortical slices of neonatal rats. A single electrical stimulus evoked an action-potential-dependent optical signal that spread radially over the MZ. The amplitude of the signal was not affected by glutamate receptor blockers, but was suppressed by either GABAA or glycine receptor antagonists. Combined application of both antagonists nearly abolished the signal. Inhibition of Na(+), K(+)-2Cl(-) cotransporter by 20 µM bumetanide reduced the signal, indicating that this transporter contributes to excitation. Analysis of the interstitial fluid obtained by microdialysis from tangential neocortical slices with high-performance liquid chromatography revealed that GABA and taurine, but not glycine or glutamate, were released in the MZ in response to the electrical stimulation. The ambient release of taurine was reduced by the addition of a voltage-sensitive Na(+) channel blocker. Immunohistochemistry and immunoelectron microscopy indicated that taurine was stored both in Cajal-Retzius and non-Cajal-Retzius cells in the MZ, but was not localized in presynaptic structures. Our results suggest that activity-dependent non-synaptic release of endogenous taurine facilitates excitatory neurotransmission through activation of glycine receptors in the MZ.

Accumulation of GABAergic neurons, causing a focal ambient GABA gradient, and downregulation of KCC2 are induced during microgyrus formation in a mouse model of polymicrogyria.

Although focal cortical malformations are considered neuronal migration disorders, their formation mechanisms remain unknown. We addressed how the γ-aminobutyric acid (GABA)ergic system affects the GABAergic and glutamatergic neuronal migration underlying such malformations. A focal freeze-lesion (FFL) of the postnatal day zero (P0) glutamic acid decarboxylase-green fluorescent protein knock-in mouse neocortex produced a 3- or 4-layered microgyrus at P7. GABAergic interneurons accumulated around the necrosis including the superficial region during microgyrus formation at P4, whereas E17.5-born, Cux1-positive pyramidal neurons outlined the GABAergic neurons and were absent from the superficial layer, forming cell-dense areas in layer 2 of the P7 microgyrus. GABA imaging showed that an extracellular GABA level temporally increased in the GABAergic neuron-positive area, including the necrotic center, at P4. The expression of the Cl(-) transporter KCC2 was downregulated in the microgyrus-forming GABAergic and E17.5-born glutamatergic neurons at P4; these cells may need a high intracellular Cl(-) concentration to induce depolarizing GABA effects. Bicuculline decreased the frequency of spontaneous Ca(2+) oscillations in these microgyrus-forming cells. Thus, neonatal FFL causes specific neuronal accumulation, preceded by an increase in ambient GABA during microgyrus formation. This GABA increase induces GABAA receptor-mediated Ca(2+) oscillation in KCC2-downregulated microgyrus-forming cells, as seen in migrating cells during early neocortical development.

De Novo mutations in GNAO1, encoding a Gαo subunit of heterotrimeric G proteins, cause epileptic encephalopathy.

Heterotrimeric G proteins, composed of α, ß, and γ subunits, can transduce a variety of signals from seven-transmembrane-type receptors to intracellular effectors. By whole-exome sequencing and subsequent mutation screening, we identified de novo heterozygous mutations in GNAO1, which encodes a Gαo subunit of heterotrimeric G proteins, in four individuals with epileptic encephalopathy. Two of the affected individuals also showed involuntary movements. Somatic mosaicism (approximately 35% to 50% of cells, distributed across multiple cell types, harbored the mutation) was shown in one individual. By mapping the mutation onto three-dimensional models of the Gα subunit in three different complexed states, we found that the three mutants (c.521A>G [p.Asp174Gly], c.836T>A [p.Ile279Asn], and c.572_592del [p.Thr191_Phe197del]) are predicted to destabilize the Gα subunit fold. A fourth mutant (c.607G>A), in which the Gly203 residue located within the highly conserved switch II region is substituted to Arg, is predicted to impair GTP binding and/or activation of downstream effectors, although the p.Gly203Arg substitution might not interfere with Gα binding to G-protein-coupled receptors. Transient-expression experiments suggested that localization to the plasma membrane was variably impaired in the three putatively destabilized mutants. Electrophysiological analysis showed that Gαo-mediated inhibition of calcium currents by norepinephrine tended to be lower in three of the four Gαo mutants. These data suggest that aberrant Gαo signaling can cause multiple neurodevelopmental phenotypes, including epileptic encephalopathy and involuntary movements.

Taurine inhibits K+-Cl- cotransporter KCC2 to regulate embryonic Cl- homeostasis via with-no-lysine (WNK) protein kinase signaling pathway.

GABA inhibits mature neurons and conversely excites immature neurons due to lower K(+)-Cl(-) cotransporter 2 (KCC2) expression. We observed that ectopically expressed KCC2 in embryonic cerebral cortices was not active; however, KCC2 functioned in newborns. In vitro studies revealed that taurine increased KCC2 inactivation in a phosphorylation-dependent manner. When Thr-906 and Thr-1007 residues in KCC2 were substituted with Ala (KCC2T906A/T1007A), KCC2 activity was facilitated, and the inhibitory effect of taurine was not observed. Exogenous taurine activated the with-no-lysine protein kinase 1 (WNK1) and downstream STE20/SPS1-related proline/alanine-rich kinase (SPAK)/oxidative stress response 1 (OSR1), and overexpression of active WNK1 resulted in KCC2 inhibition in the absence of taurine. Phosphorylation of SPAK was consistently higher in embryonic brains compared with that of neonatal brains and down-regulated by a taurine transporter inhibitor in vivo. Furthermore, cerebral radial migration was perturbed by a taurine-insensitive form of KCC2, KCC2T906A/T1007A, which may be regulated by WNK-SPAK/OSR1 signaling. Thus, taurine and WNK-SPAK/OSR1 signaling may contribute to embryonic neuronal Cl(-) homeostasis, which is required for normal brain development.

Guidepost neurons for the lateral olfactory tract: expression of metabotropic glutamate receptor 1 and innervation by glutamatergic olfactory bulb axons.

The guidepost neurons for the lateral olfactory tract, which are called lot cells, are the earliest-generated neurons in the neocortex. They migrate tangentially and ventrally further down this tract, and provide scaffolding for the olfactory bulb axons projecting into this pathway. The molecular profiles of the lot cells are largely uncharacterized. We found that lot cells specifically express metabotropic glutamate receptor subtype-1 at a very early stage of development. This receptor is functionally competent and responds to a metabotropic glutamate receptor agonist with a transient increase in the intracellular calcium ion concentration. When the glutamatergic olfactory bulb axons were electrically stimulated, lot cells responded to the stimulation with a calcium increase mainly via ionotropic glutamate receptors, suggesting potential neurotransmission between the axons and lot cells during early development. Together with the finding that lot cells themselves are glutamatergic excitatory neurons, our results provide another notable example of precocious interactions between the projecting axons and their intermediate targets.

Dominant-negative mutations in alpha-II spectrin cause West syndrome with severe cerebral hypomyelination, spastic quadriplegia, and developmental delay.

A de novo 9q33.3-q34.11 microdeletion involving STXBP1 has been found in one of four individuals (group A) with early-onset West syndrome, severe hypomyelination, poor visual attention, and developmental delay. Although haploinsufficiency of STXBP1 was involved in early infantile epileptic encephalopathy in a previous different cohort study (group B), no mutations of STXBP1 were found in two of the remaining three subjects of group A (one was unavailable). We assumed that another gene within the deletion might contribute to the phenotype of group A. SPTAN1 encoding alpha-II spectrin, which is essential for proper myelination in zebrafish, turned out to be deleted. In two subjects, an in-frame 3 bp deletion and a 6 bp duplication in SPTAN1 were found at the initial nucleation site of the alpha/beta spectrin heterodimer. SPTAN1 was further screened in six unrelated individuals with WS and hypomyelination, but no mutations were found. Recombinant mutant (mut) and wild-type (WT) alpha-II spectrin could assemble heterodimers with beta-II spectrin, but alpha-II (mut)/beta-II spectrin heterodimers were thermolabile compared with the alpha-II (WT)/beta-II heterodimers. Transient expression in mouse cortical neurons revealed aggregation of alpha-II (mut)/beta-II and alpha-II (mut)/beta-III spectrin heterodimers, which was also observed in lymphoblastoid cells from two subjects with in-frame mutations. Clustering of ankyrinG and voltage-gated sodium channels at axon initial segment (AIS) was disturbed in relation to the aggregates, together with an elevated action potential threshold. These findings suggest that pathological aggregation of alpha/beta spectrin heterodimers and abnormal AIS integrity resulting from SPTAN1 mutations were involved in pathogenesis of infantile epilepsy.

Possible contribution of 2-aminoethoxydiphenyl-borate-sensitive Ca2+ mobilization to adrenocorticotropin-induced glucocorticoid synthesis in rat adrenocortical cells.

Cytoplasmic calcium ([Ca(2+)](i)) provided through voltage-dependent Ca(2+) channels (VDCC) plays an important role in adrenocorticotropin (ACTH)-induced steroidogenesis in adrenocortical cells. To identify alternative mechanisms for [Ca(2+)](i) supply, we investigated the 2-aminoethoxydiphenyl borate (2APB)-sensitive pathway as one of the possible signaling pathways involved in [Ca(2+)](i) supply for ACTH-induced steroidogenesis. In monolayers of cultured rat adrenal fasciculate and reticularis cells, ACTH at 10(-11) M stimulated corticosterone synthesis without increasing intracellular cAMP, and corticosterone synthesis was decreased by 10 microM 2APB by 51.8% (6.71 +/- 0.97 vs. 3.23 +/- 0.05 ng/mL/4 hours; p<0.05). Furthermore, 2APB significantly decreased the 10(-11) M ACTH-stimulated [Ca(2+)](i). ACTH increased the intracellular inositol-1,4,5-trisphosphate (IP3) content with a peak at 10(-13) M ACTH, which illustrates the possibility that ACTH activates IP3/diacylglycerol- dependent protein kinase C signal transduction. However, the difference in ACTH concentrations between that responsible for the IP3 increase and steroidogenesis without elevated cAMP, suggest a hypothesis that IP3 is not required for steroidogenesis, but does involve an unknown messenger, which stimulates the release of Ca(2+) from the ER or the subsequent store-operated Ca(2+) entry (SOCE). The pregnenolone concentration in the culture medium was increased by ACTH, which was significantly suppressed by 2APB, showing that the 2APB-sensitive Ca(2+) supply affects cholesterol transport into the mitochondrial membrane via steroidogenic acute regulatory protein. Therefore, the SOCE may contribute to ACTH-induced steroidogenesis in the mitochondrial region. In conclusion, the [Ca(2+)](i) used for steroidogenesis may be derived from a 2APB-sensitive pathway and via VDCCs, particularly at physiological concentrations of ACTH. We suggest that ACTH receptors activate steroidogenesis via inositol triphosphate, or an unknown downstream messenger, which could be inhibited by 2APB.

Autonomous turning of cerebellar granule cells in vitro by intrinsic programs.

External guidance cues play a role in controlling neuronal cell turning in the developing brain, but little is known about whether intrinsic programs are also involved in controlling the turning. In this study, we examined whether granule cells undergo autonomous changes in the direction of migration in the microexplant cultures of the early postnatal mouse cerebellum. We found that granule cells exhibit spontaneous and periodical turning without cell-cell contact and in the absence of external guidance cues. The frequency of turning was increased by stimulating the Ca(2+) influx and the internal Ca(2+) release, or inhibiting the cAMP signaling pathway, while the frequency was reduced by inhibiting the Ca(2+) influx. Granule cell turning in vitro was classified into four distinct modes, which were characterized by the morphological changes in the leading process and the trailing process, such as bifurcating, turning, withdrawing, and changing the polarity. The occurrence of the 1st and 2nd modes of turning was differentially affected by altering the Ca(2+) and cAMP signaling pathways. Collectively, the results demonstrate that intrinsic programs regulate the autonomous turning of cerebellar granule cells in vitro. Furthermore, the results suggest that extrinsic signals play a role as essential modulators of intrinsic programs.

Crucial roles of Robo proteins in midline crossing of cerebellofugal axons and lack of their up-regulation after midline crossing.

BACKGROUND: Robo1, Robo2 and Rig-1 (Robo3), members of the Robo protein family, are candidate receptors for the chemorepellents Slit and are known to play a crucial role in commissural axon guidance in the spinal cord. However, their roles at other axial levels remain unknown. Here we examine expression of Robo proteins by cerebellofugal (CF) commissural axons in the rostral hindbrain and investigate their roles in CF axon pathfinding by analysing Robo knockout mice. RESULTS: We analysed the expression of Robo proteins by CF axons originating from deep cerebellar neurons in rodent embryos, focusing on developmental stages of their midline crossing and post-crossing navigation. At the stage of CF axon midline crossing, mRNAs of Robo1 and Robo2 are expressed in the nuclear transitory zone of the cerebellum, where the primordium of the deep cerebellar nuclei are located, supporting the notion that CF axons express Robo1 and Robo2. Indeed, immunohistochemical analysis of CF axons labelled by electroporation to deep cerebellar nuclei neurons indicates that Robo1 protein, and possibly also Robo2 protein, is expressed by CF axons crossing the midline. However, weak or no expression of these proteins is found on the longitudinal portion of CF axons. In Robo1/2 double knockout mice, many CF axons reach the midline but fail to exit it. We find that CF axons express Rig-1 (Robo3) before they reach the midline but not after the longitudinal turn. Consistent with this in vivo observation, axons elicited from a cerebellar explant in co-culture with a floor plate explant express Rig-1. In Rig-1 deficient mouse embryos, CF axons appear to project ipsilaterally without reaching the midline. CONCLUSION: These results indicate that Robo1, Robo2 or both are required for midline exit of CF axons. In contrast, Rig-1 is required for their approach to the midline. However, post-crossing up-regulation of these proteins, which plays an important role in spinal commissural axon guidance, does not appear to be required for the longitudinal navigation of CF axons after midline crossing. Our results illustrate that although common mechanisms operate for midline crossing at different axial levels, significant variation exists in post-crossing navigation.

Induction of amyloid beta accumulation by ER calcium disruption and resultant upregulation of angiogenic factors in ARPE19 cells.

PURPOSE: To investigate the intracellular mechanisms that induce amyloid beta (Abeta) accumulation and angiogenesis in the human retinal pigment epithelial cell line ARPE19. METHODS: The authors used two endoplasmic reticulum (ER) stress-inducing reagents, thapsigargin (TG), which inhibits the sarcoplasmic/endoplasmic calcium (Ca)2+-ATPase, and tunicamycin (TM), which inhibits N-linked glycosylation. The expression pattern of Abeta-precursor protein (APP) splice variants was investigated by reverse transcription (RT)-PCR. Cellular expressions of both a series of Abeta metabolism-related factors and angiogenic factors were evaluated by real-time RT-PCR and Western blot (VEGF). Expression of caspase-4 was examined by real-time RT-PCR and Western blot to evaluate the effect of the ER stressor. Intracellular Ca elevation by TG was evaluated by Ca2+ imaging experiments. Dimethyl sulfoxide and staurosporine were used as a nonreagent control and as an apoptosis-inducing reagent through mitochondria not ER, respectively. RESULTS: TG-treated ARPE19 cells increased the mRNA expression of Abeta production-inducing APP splice variants and reduced that of neprilysin, a catabolic enzyme for Abeta. TG-treated ARPE19 cells produced increases in VEGF, TNF-alpha, TACE mRNA, and VEGF protein expressions and a decrease in PEDF mRNA expression. TG-treated ARPE19 cells induced the expression of active more than TM-treated casepase-4. The intracellular Ca concentration was elevated in only TG-treated ARPE19 cells. CONCLUSIONS: TG-treated ARPE19 cells showed both Abeta accumulation-inducible and angiogenic factor mRNA expression patterns. This study suggests the possibility that ER stress through ER calcium disruption may induce the expression not only of Abeta deposit-promoting factors but also angiogenic factors in the retinal pigment epithelium.

De novo mutations in the gene encoding STXBP1 (MUNC18-1) cause early infantile epileptic encephalopathy.

Early infantile epileptic encephalopathy with suppression-burst (EIEE), also known as Ohtahara syndrome, is one of the most severe and earliest forms of epilepsy. Using array-based comparative genomic hybridization, we found a de novo 2.0-Mb microdeletion at 9q33.3-q34.11 in a girl with EIEE. Mutation analysis of candidate genes mapped to the deletion revealed that four unrelated individuals with EIEE had heterozygous missense mutations in the gene encoding syntaxin binding protein 1 (STXBP1). STXBP1 (also known as MUNC18-1) is an evolutionally conserved neuronal Sec1/Munc-18 (SM) protein that is essential in synaptic vesicle release in several species. Circular dichroism melting experiments revealed that a mutant form of the protein was significantly thermolabile compared to wild type. Furthermore, binding of the mutant protein to syntaxin was impaired. These findings suggest that haploinsufficiency of STXBP1 causes EIEE.

Cerebellar granule cell migration and the effects of alcohol.

In the developing brain the majority of neurons migrate from their birthplace to their final destination. This active movement is essential for the formation of cortical layers and nuclei. The impairment of migration does not affect the viability of neurons but often results in abnormal differentiation. The proper migration of neurons requires the orchestrated activities of multiple cellular and molecular events, such as pathway selection, the activation of specific receptors and channels, and the assembly and disassembly of cytoskeletal components. The migration of neurons is very vulnerable to exposure to environmental toxins, such as alcohol. In this article, we will focus on recent developments in the migration of cerebellar granule cells. First, we will describe when, where and how granule cells migrate through different cortical layers to reach their final destination. Second, we will present how internal programs control the sequential changes in granule cell migration. Third, we will review the roles of external guidance cues and transmembrane signals in granule cell migration. Finally, we will reveal mechanisms by which alcohol exposure impairs granule cell migration.

How does alcohol impair neuronal migration?

Maternal alcohol consumption during pregnancy can cause serious birth defects, of which fetal alcohol syndrome (FAS) is the most devastating. Recognized by characteristic craniofacial abnormalities and growth deficiency, this condition produces severe alcohol-induced damage in the developing brain. FAS children experience ataxia; deficits in intellectual functioning; and difficulties in learning, memory, problem solving, and attention. Multiple aspects of central nervous system development can be affected by alcohol exposure, but the most striking abnormalities are neuronal and glial migration. Little is known about cellular mechanisms by which alcohol affects the migration of immature neurons. Recently, it has been found that Ca(2+) signaling and cyclic nucleotide signaling are the central targets of the action of alcohol in neuronal cell migration. Most importantly, the aberrant migration of immature neurons caused by alcohol exposure is significantly ameliorated by controlling the activity of these second-messenger pathways. In this Mini-Review, we first describe how alcohol exposure impairs the migration of cerebellar granule cells and then discuss the signaling mechanisms involved.

Reversal of neuronal migration in a mouse model of fetal alcohol syndrome by controlling second-messenger signalings.

The brains of fetal alcohol syndrome patients exhibit impaired neuronal migration, but little is known about the mechanisms underlying this abnormality. Here we show that Ca2+ signaling and cyclic nucleotide signaling are the central targets of alcohol action in neuronal cell migration. Acute administration of ethanol reduced the frequency of transient Ca2+ elevations in migrating neurons and cGMP levels and increased cAMP levels. Experimental manipulations of these second-messenger pathways, through stimulating Ca2+ and cGMP signaling or inhibiting cAMP signaling, completely reversed the action of ethanol on neuronal migration in vitro as well as in vivo. Each second messenger has multiple but distinct downstream targets, including Ca2+/calmodulin-dependent protein kinase II, calcineurin, protein phosphatase 1, Rho GTPase, mitogen-activated protein kinase, and phosphoinositide 3-kinase. These results demonstrate that the aberrant migration of immature neurons in the fetal brain caused by maternal alcohol consumption may be corrected by controlling the activity of these second-messenger pathways.

Ca2+ transients control CNS neuronal migration.

In the developing CNS, postmitotic neurons exhibit dynamic changes in the mode, direction and rate of migration as they traverse different cortical layers, but the mechanisms underlying this process is largely unknown. Recent studies show that the changes in Ca2+ transient frequency play a central role in controlling the neuronal cell migration in a cortical layer-specific manner. In this article, we will first describe how granule cells migrate through different terrains of the developing cerebellar cortex. We will then present how such migration of granule cells is controlled by altering the Ca2+ transient frequency in their somata. Finally, we will discuss how the loss of Ca2+ transients triggers the completion of granule cell migration at their final destination.

Isolation of cystatin C via functional cloning of astrocyte differentiation factors.

We screened for factors upregulating glial fibrillary acidic protein (GFAP) promoter activity by functional cloning with an immature astrocyte cell line (HB108-10) harboring a GFAP-lacZ construct. One cDNA clone that repeatedly upregulated lacZ expression encoded cystatin C (CysC), a cysteine protease inhibitor. TGF-beta induced CysC and GFAP expression in AP-16 cells, an astrocyte progenitor-like cell line expressing GLAST (a glutamate transporter subtype specifically expressed in immature astrocytes). CysC gene expression started earlier than that of GFAP in the mouse forebrain. It started in the ventricular zone at a similar period as (or slightly after) GLAST expression, but before GFAP expression. Although previous data showed that CysC is involved in the maintenance of adult neural stem cells, our data indicate that it is involved in astrocyte differentiation during mouse brain development.

Completion of neuronal migration regulated by loss of Ca(2+) transients.

The migration of immature neurons constitutes one of the major processes by which the central nervous system takes shape. Completing the migration at the final destination requires the loss of cell body motility, but little is known about the signaling mechanisms underlying this process. Here, we show that a loss of transient Ca(2+) elevations triggers the completion of cerebellar granule cell migration. Simultaneous observation of the intracellular Ca(2+) levels and cell movement in cerebellar slices of the early postnatal mice revealed that granule cells exhibit distinct frequencies of the transient Ca(2+) elevations as they migrate in different cortical layers, and complete the migration only after the loss of Ca(2+) elevations. The reduction of the Ca(2+) elevation frequency by decreasing Ca(2+) influx, or by inhibiting the activity of phospholipase C, PKC, or Ca(2+)/calmodulin, halted the granule cell movement prematurely. In contrast, increasing the Ca(2+) elevation frequency by increasing Ca(2+) release from internal stores, or by elevating intracellular cAMP levels, significantly delayed the completion of granule cell migration. The timing of the loss of Ca(2+) elevations was intrinsically set in the granule cells and influenced by external cues. These results suggest that Ca(2+) signaling, dictated by multiple signaling systems, functions as a mediator for completing the migration of immature neurons.