Differential Spatiotemporal Expression of Type I and Type II Cadherins Associated With the Segmentation of the Central Nervous System and Formation of Brain Nuclei in the Developing Mouse.

Type I and type II classical cadherins comprise a family of cell adhesion molecules that regulate cell sorting and tissue separation by forming specific homo and heterophilic bonds. Factors that affect cadherin-mediated cell-cell adhesion include cadherin binding affinity and expression level. This study examines the expression pattern of type I cadherins (Cdh1, Cdh2, Cdh3, and Cdh4), type II cadherins (Cdh6, Cdh7, Cdh8, Cdh9, Cdh10, Cdh11, Cdh12, Cdh18, Cdh20, and Cdh24), and the atypical cadherin 13 (Cdh13) during distinct morphogenetic events in the developing mouse central nervous system from embryonic day 11.5 to postnatal day 56. Cadherin mRNA expression levels obtained from in situ hybridization experiments carried out at the Allen Institute for Brain Science (https://alleninstitute.org/) were retrieved from the Allen Developing Mouse Brain Atlas. Cdh2 is the most abundantly expressed type I cadherin throughout development, while Cdh1, Cdh3, and Cdh4 are expressed at low levels. Type II cadherins show a dynamic pattern of expression that varies between neuroanatomical structures and developmental ages. Atypical Cdh13 expression pattern correlates with Cdh2 in abundancy and localization. Analyses of cadherin-mediated relative adhesion estimated from their expression level and binding affinity show substantial differences in adhesive properties between regions of the neural tube associated with the segmentation along the anterior-posterior axis. Differences in relative adhesion were also observed between brain nuclei in the developing subpallium (basal ganglia), suggesting that differential cell adhesion contributes to the segregation of neuronal pools. In the adult cerebral cortex, type II cadherins Cdh6, Cdh8, Cdh10, and Cdh12 are abundant in intermediate layers, while Cdh11 shows a gradated expression from the deeper layer 6 to the superficial layer 1, and Cdh9, Cdh18, and Cdh24 are more abundant in the deeper layers. Person's correlation analyses of cadherins mRNA expression patterns between areas and layers of the cerebral cortex and the nuclei of the subpallium show significant correlations between certain cortical areas and the basal ganglia. The study shows that differential cadherin expression and cadherin-mediated adhesion are associated with a wide range of morphogenetic events in the developing central nervous system including the organization of neurons into layers, the segregation of neurons into nuclei, and the formation of neuronal circuits.

Autism throughout genetics: Perusal of the implication of ion channels.

BACKGROUND: Autism spectrum disorder (ASD) comprises a group of neurodevelopmental psychiatric disorders characterized by deficits in social interactions, interpersonal communication, repetitive and stereotyped behaviors and may be associated with intellectual disabilities. The description of ASD as a synaptopathology highlights the importance of the synapse and the implication of ion channels in the etiology of these disorders. METHODS: A narrative and critical review of the relevant papers from 1982 to 2017 known by the authors was conducted. RESULTS: Genome-wide linkages, association studies, and genetic analyses of patients with ASD have led to the identification of several candidate genes and mutations linked to ASD. Many of the candidate genes encode for proteins involved in neuronal development and regulation of synaptic function including ion channels and actors implicated in synapse formation. The involvement of ion channels in ASD is of great interest as they represent attractive therapeutic targets. In agreement with this view, recent findings have shown that drugs modulating ion channel function are effective for the treatment of certain types of patients with ASD. CONCLUSION: This review describes the genetic aspects of ASD with a focus on genes encoding ion channels and highlights the therapeutic implications of ion channels in the treatment of ASD.

Down-regulation of the Wnt/ß-catenin signaling pathway by Cacnb4.

The ß4 isoform of the ß-subunits of voltage-gated calcium channel regulates cell proliferation and cell cycle progression. Herein we show that coexpression of the ß4-subunit with actors of the canonical Wnt/ß-catenin signaling pathway in a hepatoma cell line inhibits Wnt-responsive gene transcription and decreases cell division, in agreement with the role of the Wnt pathway in cell proliferation. ß4-subunit-mediated inhibition of Wnt signaling is observed in the presence of LiCl, an inhibitor of glycogen synthase kinase (GSK3) that promotes ß-catenin translocation to the nucleus. Expression of ß4-subunit mutants that lost the ability to translocate to the nucleus has no effect on Wnt signaling, suggesting that ß4-subunit inhibition of Wnt signaling occurs downstream from GSK3 and requires targeting of ß4-subunit to the nucleus. ß4-subunit coimmunoprecipitates with the TCF4 transcription factor and overexpression of TCF4 reverses the effect of ß4-subunit on the Wnt pathway. We thus propose that the interaction of nuclear ß4-subunit with TCF4 prevents ß-catenin binding to TCF4 and leads to the inhibition of the Wnt-responsive gene transcription. Thereby, our results show that ß4-subunit is a TCF4 repressor and therefore appears as an interesting candidate for the regulation of this pathway in neurons where ß4-subunit is specifically expressed.

The ß4 subunit of the voltage-gated calcium channel (Cacnb4) regulates the rate of cell proliferation in Chinese Hamster Ovary cells.

The ß subunits of Voltage-Gated Calcium Channel (VGCC) are cytosolic proteins that interact with the VGCC pore -forming subunit and participate in the trafficking of the channel to the cell membrane and in ion influx regulation. ß subunits also exert functions independently of their binding to VGCC by translocation to the cell nucleus including the control of gene expression. Mutations of the neuronal Cacnb4 (ß4) subunit are linked to human neuropsychiatric disorders including epilepsy and intellectual disabilities. It is believed that the pathogenic phenotype induced by these mutations is associated with channel-independent functions of the ß4 subunit. In this report, we investigated the role of ß4 subunit in cell proliferation and cell cycle progression and examined whether these functions could be altered by a pathogenic mutation. To this end, stably transfected Chinese Hamster Ovary (CHO-K1) cells expressing either rat full-length ß4 or the rat C-terminally truncated epileptic mutant variant ß1-481 were generated. The subcellular localization of both proteins differed significantly. Full-length ß4 localizes almost exclusively in the cell nucleus and concentrates into the nucleolar compartment, while the C-terminal-truncated ß1-481 subunit was less concentrated within the nucleus and absent from the nucleoli. Cell proliferation was found to be reduced by the expression of ß4, while it was unaffected by the epileptic mutant. Also, full-length ß4 interfered with cell cycle progression by presumably preventing cells from entering the S-phase via a mechanism that partially involves endogenous B56δ, a regulatory subunit of the phosphatase 2A (PP2A) that binds ß4 but not ß1-481. Analysis of ß4 subcellular distribution during the cell cycle revealed that the protein is highly expressed in the nucleus at the G1/S transition phase and that it is translocated out of the nucleus during chromatin condensation and cell division. These results suggest that nuclear accumulation of ß4 at the G1/S transition phase affects the progression into S-phase resulting in a decrease in the rate of cell proliferation. Regulation of the cell cycle exit is a critical step in determining the number of neuronal precursors and neuronal differentiation suggesting that mutations of the ß4 subunit could affect neural development and formation of the mature central nervous system.

Protein partners of the calcium channel ß subunit highlight new cellular functions.

Calcium plays a key role in cell signalling by its intervention in a wide range of physiological processes. Its entry into cells occurs mainly via voltage-gated calcium channels (VGCC), which are found not only in the plasma membrane of excitable cells but also in cells insensitive to electrical signals. VGCC are composed of different subunits, α1, ß, α2δ and γ, among which the cytosolic ß subunit (Cavß) controls the trafficking of the channel to the plasma membrane, its regulation and its gating properties. For many years, these were the main functions associated with Cavß. However, a growing number of proteins have been found to interact with Cavß, emphasizing the multifunctional role of this versatile protein. Interestingly, some of the newly assigned functions of Cavß are independent of its role in the regulation of VGCC, and thus further increase its functional roles. Based on the identity of Cavß protein partners, this review emphasizes the diverse cellular functions of Cavß and summarizes both past findings as well as recent progress in the understanding of VGCC.

Regulation of neuronal high-voltage activated Ca(V)2 Ca(2+) channels by the small GTPase RhoA.

High-Voltage-Activated (HVA) Ca(2+) channels are known regulators of synapse formation and transmission and play fundamental roles in neuronal pathophysiology. Small GTPases of Rho and RGK families, via their action on both cytoskeleton and Ca(2+) channels are key molecules for these processes. While the effects of RGK GTPases on neuronal HVA Ca(2+) channels have been widely studied, the effects of RhoA on the HVA channels remains however elusive. Using heterologous expression in Xenopus laevis oocytes, we show that RhoA activity reduces Ba(2+) currents through CaV2.1, CaV2.2 and CaV2.3 Ca(2+) channels independently of CaVß subunit. This inhibition occurs independently of RGKs activity and without modification of biophysical properties and global level of expression of the channel subunit. Instead, we observed a marked decrease in the number of active channels at the plasma membrane. Pharmacological and expression studies suggest that channel expression at the plasma membrane is impaired via a ROCK-sensitive pathway. Expression of constitutively active RhoA in primary culture of spinal motoneurons also drastically reduced HVA Ca(2+) current amplitude. Altogether our data revealed that HVA Ca(2+) channels regulation by RhoA might govern synaptic transmission during development and potentially contribute to pathophysiological processes when axon regeneration and growth cone kinetics are impaired.

Cell surface localization of α3ß4 nicotinic acetylcholine receptors is regulated by N-cadherin homotypic binding and actomyosin contractility.

Neuronal nicotinic acetylcholine receptors (nAChRs) are widely expressed throughout the central and peripheral nervous system and are localized at synaptic and extrasynaptic sites of the cell membrane. However, the mechanisms regulating the localization of nicotinic receptors in distinct domains of the cell membrane are not well understood. N-cadherin is a cell adhesion molecule that mediates homotypic binding between apposed cell membranes and regulates the actin cytoskeleton through protein interactions with the cytoplasmic domain. At synaptic contacts, N-cadherin is commonly localized adjacent to the active zone and the postsynaptic density, suggesting that N-cadherin contributes to the assembly of the synaptic complex. To examine whether N-cadherin homotypic binding regulates the cell surface localization of nicotinic receptors, this study used heterologous expression of N-cadherin and α3ß4 nAChR subunits C-terminally fused to a myc-tag epitope in Chinese hamster ovary cells. Expression levels of α3ß4 nAChRs at cell-cell contacts and at contact-free cell membrane were analyzed by confocal microscopy. α3ß4 nAChRs were found distributed over the entire surface of contacting cells lacking N-cadherin. In contrast, N-cadherin-mediated cell-cell contacts were devoid of α3ß4 nAChRs. Cell-cell contacts mediated by N-cadherin-deleted proteins lacking the ß-catenin binding region or the entire cytoplasmic domain showed control levels of α3ß4 nAChRs expression. Inhibition of actin polymerization with latrunculin A and cytochalasin D did not affect α3ß4 nAChRs localization within N-cadherin-mediated cell-cell contacts. However, treatment with the Rho associated kinase inhibitor Y27632 resulted in a significant increase in α3ß4 nAChR levels within N-cadherin-mediated cell-cell contacts. Analysis of α3ß4 nAChRs localization in polarized Caco-2 cells showed specific expression on the apical cell membrane and colocalization with apical F-actin and the actin nucleator Arp3. These results indicate that actomyosin contractility downstream of N-cadherin homotypic binding regulates the cell surface localization of α3ß4 nAChRs presumably through interactions with a particular pool of F-actin.

N-cadherin induces partial differentiation of cholinergic presynaptic terminals in heterologous cultures of brainstem neurons and CHO cells.

N-cadherin is a calcium-sensitive cell adhesion molecule commonly expressed at synaptic junctions and contributes to formation and maturation of synaptic contacts. This study used heterologous cell cultures of brainstem cholinergic neurons and transfected Chinese Hamster Ovary (CHO) cells to examine whether N-cadherin is sufficient to induce differentiation of cholinergic presynaptic terminals. Brainstem nuclei isolated from transgenic mice expressing enhanced green fluorescent protein (EGFP) under the control of choline acetyltransferase (ChAT) transcriptional regulatory elements (ChAT(BAC)EGFP) were cultured as tissue explants for 5 days and cocultured with transfected CHO cells for an additional 2 days. Immunostaining for synaptic vesicle proteins SV2 and synapsin I revealed a ~3-fold increase in the area of SV2 immunolabeling over N-cadherin expressing CHO cells, and this effect was enhanced by coexpression of p120-catenin. Synapsin I immunolabeling per axon length was also increased on N-cadherin expressing CHO cells but required coexpression of p120-catenin. To determine whether N-cadherin induces formation of neurotransmitter release sites, whole-cell voltage-clamp recordings of CHO cells expressing α3 and ß4 nicotinic acetylcholine receptor (nAChR) subunits in contact with cholinergic axons were used to monitor excitatory postsynaptic potentials (EPSPs) and miniature EPSPs (mEPSPs). EPSPs and mEPSPs were not detected in both, control and in N-cadherin expressing CHO cells in the absence or presence of tetrodotoxin (TTX). These results indicate that expression of N-cadherin in non-neuronal cells is sufficient to initiate differentiation of presynaptic cholinergic terminals by inducing accumulation of synaptic vesicles; however, development of readily detectable mature cholinergic release sites and/or clustering of postsynaptic nAChR may require expression of additional synaptogenic proteins.

Maternal immune activation by poly I:C induces expression of cytokines IL-1ß and IL-13, chemokine MCP-1 and colony stimulating factor VEGF in fetal mouse brain.

BACKGROUND: Maternal viral infection during pregnancy is associated with an increase in the incidence of psychiatric disorders with presumed neurodevelopmental origin, including autism spectrum disorders and schizophrenia. The enhanced risk for developing mental illness appears to be caused by deleterious effects of innate immune response-associated factors on the development of the central nervous system, which predispose the offspring to pathological behaviors in adolescence and adulthood. To identify the immune response-associated soluble factors that may affect central nervous system development, we examined the effect of innate immune response activation by polyriboinosinic-polyribocytidylic acid (poly(I:C)), a synthetic analogue of viral double-stranded RNA, on the expression levels of pro- and anti-inflammatory cytokines, chemokines and colony stimulating factors in fetal and postnatal mouse brain 6 h and 24 h after treatment. METHODS: C57BL/6J pregnant mice (gestational day 16) or newborn mice (postnatal day 4) received a single intraperitoneal injection of the synthetic analogue of viral double-stranded RNA poly(I:C) (20 mg/kg). Thirty-two immune response-associated soluble factors, including pro- and anti-inflammatory cytokines, chemokines and colony stimulating factors, were assayed 6 h and 24 h after poly(I:C) injection using multiplexed bead-based immunoassay (Milliplex Map) and processed in a Luminex 100 IS instrument. RESULTS: Maternal exposure to poly(I:C) at gestational day 16 induced a significant increase in cytokines interleukin (IL)-1ß, IL-7 and IL-13; chemokines monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein (MIP)-1α, interferon gamma-induced protein (IP)-10 and monokine induced by IFN-gamma (MIG); and in the colony stimulating factor vascular endothelial growth factor (VEGF) in the fetal brain. IL-1ß showed the highest concentration levels in fetal brains and was the only cytokine significantly up-regulated 24 h after maternal poly(I:C) injection, suggesting that IL-1ß may have a deleterious impact on central nervous system development. In contrast, poly(I:C) treatment of postnatal day 4 pups induced a pronounced rise in chemokines and colony stimulating factors in their brains instead of the pro-inflammatory cytokine IL-1ß. CONCLUSIONS: This study identified a significant increase in the concentration levels of the cytokines IL-1ß and IL-13, the chemokine MCP-1 and the colony stimulating factor VEGF in the developing central nervous system during activation of an innate immune response, suggesting that these factors are mediators of the noxious effects of maternal immune activation on central nervous system development, with potential long-lasting effects on animal behavior.

In vivo labeling of zebrafish motor neurons using an mnx1 enhancer and Gal4/UAS.

The zebrafish spinal cord primary motor neurons are commonly used as an experimental model to study the molecular mechanisms that regulate axonal pathfinding and neuromuscular junction formation, and for the modeling of human neurodegenerative disorders. This study characterized a 125-bp mnx1 enhancer to direct gene expression in spinal cord motor neurons. A promoter containing three copies of the 125-bp mnx1 enhancer was generated in a Tol2 vector and used to drive enhanced green fluorescent protein (EGFP) expression either directly or in combination with the Gal4/UAS transcriptional activation system. Both methods induced protein expression for up to 5 days after fertilization, allowing the observation of the dendritic tree and axonal arborization of single motor neurons within a somitic segment in fixed and live animals. The use of the 125-bp mnx1 promoter for transient transgenic expression or for the generation of stable transgenic fish lines will facilitate the study of motor neuron development and neurodegenerative processes.

N-cadherin regulates primary motor axon growth and branching during zebrafish embryonic development.

N-cadherin is a classical type I cadherin that contributes to the formation of neural circuits by regulating growth cone migration and the formation of synaptic contacts. This study analyzed the role of N-cadherin in primary motor axons growth during development of the zebrafish (Danio rerio) embryo. After exiting the spinal cord, primary motor axons migrate ventrally through a common pathway and form the first neuromuscular junction with the muscle pioneer cells located at the horizontal myoseptum, which serves as a choice point for cell-type-specific pathway selection. Analysis of N-cadherin mutants (cdh2(hi3644Tg) ) and embryos injected with N-cadherin antisense morpholinos showed primary motor axons extending aberrant axonal branches at the choice point in ∼40% of the somitic hemisegments and an ∼150% increase in the number of branches per axon length within the ventral myotome. Analysis of individual axons trajectories showed that the caudal (CaP) and rostral (RoP) motor neurons axons formed aberrant branches at the choice point that abnormally extended in the rostrocaudal axis and ventrally to the horizontal myoseptum. Expression of a dominant-interfering N-cadherin cytoplasmic domain in primary motor neurons caused some axons to stall abnormally at the horizontal myoseptum and to impair their migration into the ventral myotome. However, in N-cadherin-depleted embryos, the majority of primary motor axons innervated their appropriate myotomal territories, indicating that N-cadherin regulates motor axon growth and branching without severely affecting the mechanisms that control axonal target selection.

Identification of gene transcripts expressed by postsynaptic neurons during synapse formation encoding cell surface proteins with presumptive synaptogenic activity.

Synapse formation is a well-programmed developmental process involving a variety of cell-cell interactions carried out by distinct groups of molecules. Various molecules that contribute to the assembly of synaptic contacts have been characterized; however, the repertoire of identified proteins expressed by postsynaptic neurons capable of inducing presynaptic differentiation is quite limited. To identify gene transcripts encoding cell surface proteins expressed by postsynaptic cells with molecular features suggestive of synaptogenic activity, this study carried out a genome-wide expression analysis in the chick ciliary ganglion during the different phases of synapse formation. It was found that from the 21,493 gene-probes detected throughout development, 302 protein-coding transcripts were upregulated during the initiation of synapse formation. Analysis of this pool of transcripts showed that 51 of them encoded cell surface proteins (27 membrane-bound and 24 secreted) with protein-protein interacting domains. This includes twelve cell adhesion molecules, six ligand-receptors, six proteins with ligand-like domains, three membrane bound enzymes, eight components of the extracellular matrix, three neuropeptides, three cytokines and growth factors, five extracellular modulators of cell signaling, and five unrelated secreted proteins. Furthermore, the role of synaptic transmission during the initiation of synapse formation was evaluated by assessing the effect of synaptic activity blockade with d-tubocurarine on the expression levels of the pool of 51 transcripts encoding cell surface proteins. Treatment with d-tubocurarine reduced the expression levels of 22% of the selected genes, while the expression levels of 78% of the genes was not affected or was enhanced.

N-cadherin modulates voltage activated calcium influx via RhoA, p120-catenin, and myosin-actin interaction.

N-cadherin is a transmembrane adhesion receptor that contributes to neuronal development and synapse formation through homophilic interactions that provide structural-adhesive support to contacts between cell membranes. In addition, N-cadherin homotypic binding may initiate cell signaling that regulates neuronal physiology. In this study, we investigated signaling capabilities of N-cadherin that control voltage activated calcium influx. Using whole-cell voltage clamp recording of isolated inward calcium currents in freshly isolated chick ciliary ganglion neurons we show that the juxtamembrane region of N-cadherin cytoplasmic domain regulates high-threshold voltage activated calcium currents by interacting with p120-catenin and activating RhoA. This regulatory mechanism requires myosin interaction with actin. Furthermore, N-cadherin homophilic binding enhanced voltage activated calcium current amplitude in dissociated neurons that have already developed mature synaptic contacts in vivo. The increase in calcium current amplitude was not affected by brefeldin A suggesting that the effect is caused via direct channel modulation and not by increasing channel expression. In contrast, homotypic N-cadherin interaction failed to regulate calcium influx in freshly isolated immature neurons. However, RhoA inhibitors enhanced calcium current amplitude in these immature neurons, suggesting that the inhibitory effect of RhoA on calcium entry is regulated during neuronal development and synapse maturation. These results indicate that N-cadherin modulates voltage activated calcium entry by a mechanism that involves RhoA activity and its downstream effects on the cytoskeleton, and suggest that N-cadherin provides support for synaptic maturation and sustained synaptic activity by facilitating voltage activated calcium influx.

N-cadherin signaling in synapse formation and neuronal physiology.

Neural cadherin (N-cadherin) is an adhesion receptor that is localized in abundance at neuronto- neuron synapses. N-cadherin contains an extracellular domain that binds to other cadherins on juxtaposed cell membranes, a single-pass transmembrane region, and a cytoplasmic tail that interacts with various proteins, including catenins, kinases, phosphatases, and presenilin 1. N-cadherin contributes to the structural and functional organization of the synaptic complex by ensuring the adhesion between synaptic membranes and organizing the underlying actin cytoskeleton. Additionally, recent findings have shown that N-cadherin may participate in synaptic physiology by regulating calcium influx through voltage-activated calcium currents. The diverse activities of N-cadherin stem from its ability to operate as both an adhesion molecule that links cytoskeletons across cell membranes and a ligand-activated homophilic receptor capable of initiating intracellular signaling. An important mechanism of cadherin signaling is the regulation of small Rho guanosine triphosphatase activity that affects cytoskeleton dynamics and calcium influx. Because both the regulation of cadherin adhesive activity and cadherin-mediated signaling are affected by the binding of molecules to the intracellular domain, changes in the composition of the N-cadherin complex are central to the regulation of cadherin-mediated functions. This article focuses on the roles that N-cadherin might play at the level of the synapse through its effect on adhesion and signaling in the proximity of the synaptic junction.

Assembly of the N-cadherin complex during synapse formation involves uncoupling of p120-catenin and association with presenilin 1.

N-cadherin is an adhesion receptor that participates in both interaction between immature pre- and postsynaptic neurons and in the stabilization and function of matured neuron-neuron synapses. To better understand how the N-cadherin complex contributes to synapse formation, we examined its distribution and composition during synapse formation in the chick ciliary neurons. It was found that at early phases of synaptogenesis, N-cadherin is distributed in small clusters on the cell surface and primarily associates with p120-catenin and beta-catenin. In contrast, as synaptic contacts matured, larger N-cadherin clusters were found localized adjacent to the active zone and associated with PS1 and gamma-catenin, while p120- and beta-catenin were dispersed among other cell regions, including axons. As it is known that PS1 binds gamma-catenin and that uncoupled p120-catenin can alter the cytoskeleton via its effect on Rho GTPases, these changes in the molecular composition of the N-cadherin complex (represented by the uncoupling of p120-catenin and association with PS1) may correspond to distinct functional states of the complex involved in synaptic maturation.

Adherens junctions in myelinating Schwann cells stabilize Schmidt-Lanterman incisures via recruitment of p120 catenin to E-cadherin.

Schwann cell myelin contains highly compacted layers of membrane as well as noncompacted regions with a visible cytoplasm. One of these cytoplasmic compartments is the Schmidt-Lanterman incisure, which spirals through the compacted layers and is believed to help sustain the growth and function of compact myelin. Incisures contain adherens junctions (AJs), the key components of which are E-cadherin, its cytoplasmic partners called catenins, and F-actin. To explore in vivo the role of cadherin and catenins in incisures, E-cadherin mutant proteins that completely replace endogenous cadherin have been delivered to the cells using adenovirus. When the introduced cadherin lacked its extracellular domain, association of p120 catenin (p120ctn) with the cadherin did not occur, and incisures disappeared. Remarkably, the additional replacement of two phosphorylatable tyrosines by phenylalanine in the cytoplasmic tail of the mutant cadherin restored both p120ctn binding and incisure architecture, indicating that p120ctn recruitment is critical for incisures maintenance and might be regulated by phosphorylations. In addition, the ability of the p120ctn/cadherin complex to support incisures was blocked by mutation of the Rho GTPase regulatory region of the p120ctn, and downregulation of Rac1 activity at the junction reversed this inhibition. Because Rho GTPases regulate the state of the actin filaments, these findings suggest that one role of p120ctn in incisures is to organize the cytoskeleton at the AJ. Finally, developmental studies of Schwann cells demonstrated that p120ctn recruitment from the cytoplasm to the AJ occurs before the appearance of Rac1 GTPase and F-actin at the junction.

Assembly of the N-cadherin complex during synapse formation involves uncoupling of p120-catenin and association with presenilin 1.

N-cadherin is an adhesion receptor that participates in both interaction between immature pre- and postsynaptic neurons and in the stabilization and function of matured neuron-neuron synapses. To better understand how the N-cadherin complex contributes to synapse formation, we examined its distribution and composition during synapse formation in the chick ciliary neurons. It was found that at early phases of synaptogenesis, N-cadherin is distributed in small clusters on the cell surface and primarily associates with p120-catenin and 3-catenin. In contrast, as synaptic contacts matured, larger N-cadherin clusters were found localized adjacent to the active zone and associated with PSI and y-catenin, while p120- and 3-catenin were dispersed among other cell regions, including axons. As it is known that PSI binds y-catenin and that uncoupled p120-catenin can alter the cytoskeleton via its effect on Rho GTPases, these changes in the molecular composition of the N-cadherin complex (represented by the uncoupling of p120-catenin and association with PS1) may correspond to distinct functional states of the complex involved in synaptic maturation.

N-cadherin juxtamembrane domain modulates voltage-gated Ca2+ current via RhoA GTPase and Rho-associated kinase.

The juxtamembrane domain (JMD) of N-cadherin cytoplasmic tail is an important regulatory region of the clustering and adhesion activities of the protein. In addition, the JMD binds a diversity of proteins capable of modifying intracellular processes including cytoskeletal rearrangement mediated by Rho GTPases. These GTPases also function as regulators of voltage-activated calcium channels, which in turn modulate neuronal excitability. The present study was designed to determine whether there is a direct functional link, via Rho GTPase, between the N-cadherin JMD and these voltage-activated channels. It was found that the infusion of the soluble JMD into chick ciliary neurons causes a substantial decrease in the amplitude of the high-threshold voltage-activated (HVA) calcium current. The activation time is increased while the inactivation process is reduced, suggesting that the decreased current amplitude reflects a reduction in the number of channels available to open. This effect was reversed by inhibition of RhoA or its downstream effector, Rho-associated kinase (ROCK). Because ROCK determines the active state of myosin, these results suggest that the modulation of HVA by the JMD could be mediated by changes in the status of the actin-myosin cytoskeleton.

Derivation of midbrain dopamine neurons from human embryonic stem cells.

Human embryonic stem (hES) cells are defined by their extensive self-renewal capacity and their potential to differentiate into any cell type of the human body. The challenge in using hES cells for developmental biology and regenerative medicine has been to direct the wide differentiation potential toward the derivation of a specific cell fate. Within the nervous system, hES cells have been shown to differentiate in vitro into neural progenitor cells, neurons, and astrocytes. However, to our knowledge, the selective derivation of any given neuron subtype has not yet been demonstrated. Here, we describe conditions to direct hES cells into neurons of midbrain dopaminergic identity. Neuroectodermal differentiation was triggered on stromal feeder cells followed by regional specification by means of the sequential application of defined patterning molecules that direct in vivo midbrain development. Progression toward a midbrain dopamine (DA) neuron fate was monitored by the sequential expression of key transcription factors, including Pax2, Pax5, and engrailed-1 (En1), measurements of DA release, the presence of tetrodotoxin-sensitive action potentials, and the electron-microscopic visualization of tyrosinehydroxylase-positive synaptic terminals. High-yield DA neuron derivation was confirmed from three independent hES and two monkey embryonic stem cell lines. The availability of unlimited numbers of midbrain DA neurons is a first step toward exploring the potential of hES cells in preclinical models of Parkinson's disease. This experimental system also provides a powerful tool to probe the molecular mechanisms that control the development and function of human midbrain DA neurons.

Neural subtype specification of fertilization and nuclear transfer embryonic stem cells and application in parkinsonian mice.

Existing protocols for the neural differentiation of mouse embryonic stem (ES) cells require extended in vitro culture, yield variable differentiation results or are limited to the generation of selected neural subtypes. Here we provide a set of coculture conditions that allows rapid and efficient derivation of most central nervous system phenotypes. The fate of both fertilization- and nuclear transfer-derived ES (ntES) cells was directed selectively into neural stem cells, astrocytes, oligodendrocytes or neurons. Specific differentiation into gamma-aminobutyric acid (GABA), dopamine, serotonin or motor neurons was achieved by defining conditions to induce forebrain, midbrain, hindbrain and spinal cord identity. Neuronal function of ES cell-derived dopaminergic neurons was shown in vitro by electron microscopy, measurement of neurotransmitter release and intracellular recording. Furthermore, transplantation of ES and ntES cell-derived dopaminergic neurons corrected the phenotype of a mouse model of Parkinson disease, demonstrating an in vivo application of therapeutic cloning in neural disease.