Balance between dopamine and adenosine signals regulates the PKA/Rap1 pathway in striatal medium spiny neurons.

Medium spiny neurons (MSNs) expressing dopamine D1 receptor (D1R) or D2 receptor (D2R) are major components of the striatum. Stimulation of D1R activates protein kinase A (PKA) through Golf to increase neuronal activity, while D2R stimulation inhibits PKA through Gi. Adenosine A2A receptor (A2AR) coupled to Golf is highly expressed in D2R-MSNs within the striatum. However, how dopamine and adenosine co-operatively regulate PKA activity remains largely unknown. Here, we measured Rap1gap serine 563 phosphorylation to monitor PKA activity and examined dopamine and adenosine signals in MSNs. We found that a D1R agonist increased Rap1gap phosphorylation in striatal slices and in D1R-MSNs in vivo. A2AR agonist CGS21680 increased Rap1gap phosphorylation, and pretreatment with the D2R agonist quinpirole blocked this effect in striatal slices. D2R antagonist eticlopride increased Rap1gap phosphorylation in D2R-MSNs in vivo, and the effect of eticlopride was blocked by the pretreatment with the A2AR antagonist SCH58261. These results suggest that adenosine positively regulates PKA in D2R-MSNs through A2AR, while this effect is blocked by basal dopamine in vivo. Incorporating computational model analysis, we propose that the shift from D1R-MSNs to D2R-MSNs or vice versa appears to depend predominantly on a change in dopamine concentration.

Dual role for DOCK7 in tangential migration of interneuron precursors in the postnatal forebrain.

Throughout life, stem cells in the ventricular-subventricular zone generate neuroblasts that migrate via the rostral migratory stream (RMS) to the olfactory bulb, where they differentiate into local interneurons. Although progress has been made toward identifying extracellular factors that guide the migration of these cells, little is known about the intracellular mechanisms that govern the dynamic reshaping of the neuroblasts' morphology required for their migration along the RMS. In this study, we identify DOCK7, a member of the DOCK180-family, as a molecule essential for tangential neuroblast migration in the postnatal mouse forebrain. DOCK7 regulates the migration of these cells by controlling both leading process (LP) extension and somal translocation via distinct pathways. It controls LP stability/growth via a Rac-dependent pathway, likely by modulating microtubule networks while also regulating F-actin remodeling at the cell rear to promote somal translocation via a previously unrecognized myosin phosphatase-RhoA-interacting protein-dependent pathway. The coordinated action of both pathways is required to ensure efficient neuroblast migration along the RMS.

Discovery of long-range inhibitory signaling to ensure single axon formation.

A long-standing question in neurodevelopment is how neurons develop a single axon and multiple dendrites from common immature neurites. Long-range inhibitory signaling from the growing axon is hypothesized to prevent outgrowth of other immature neurites and to differentiate them into dendrites, but the existence and nature of this inhibitory signaling remains unknown. Here, we demonstrate that axonal growth triggered by neurotrophin-3 remotely inhibits neurite outgrowth through long-range Ca2+ waves, which are delivered from the growing axon to the cell body. These Ca2+ waves increase RhoA activity in the cell body through calcium/calmodulin-dependent protein kinase I. Optogenetic control of Rho-kinase combined with computational modeling reveals that active Rho-kinase diffuses to growing other immature neurites and inhibits their outgrowth. Mechanistically, calmodulin-dependent protein kinase I phosphorylates a RhoA-specific GEF, GEF-H1, whose phosphorylation enhances its GEF activity. Thus, our results reveal that long-range inhibitory signaling mediated by Ca2+ wave is responsible for neuronal polarization.Emerging evidence suggests that gut microbiota influences immune function in the brain and may play a role in neurological diseases. Here, the authors offer in vivo evidence from a Drosophila model that supports a role for gut microbiota in modulating the progression of Alzheimer's disease.

Phospholipid localization implies microglial morphology and function via Cdc42 in vitro.

Under a quiescent state, microglia exhibit a ramified shape, rather than the amoeboid-like morphology following injury or inflammation. The manipulation of microglial morphology in vitro has not been very successful, which has impeded the progress of microglial studies. We demonstrate that lysophosphatidylserine (LysoPS), a kind of lysophospholipids, rapidly and substantially alters the morphology of primary cultured microglia to an in vivo-like ramified shape in a receptor independent manner. This mechanism is mediated by Cdc42 activity. LysoPS is incorporated into the plasma membrane and converted to phosphatidylserine (PS) via the Lands' cycle. The accumulated PS on the membrane recruits Cdc42. Both Cdc42 and PS colocalize predominantly in primary and secondary processes, but not in peripheral branches or tips of microglia. Along with the morphological changes LysoPS suppresses inflammatory cytokine production and NF-kB activity. The present study provides a tool to manipulate a microglial phenotype from an amoeboid to a fully ramified in vitro, which certainly contributes to studies exploring microglial physiology and pathology.

Survival of corticostriatal neurons by Rho/Rho-kinase signaling pathway.

Developing cortical neurons undergo a number of sequential developmental events including neuronal survival/apoptosis, and the molecular mechanism underlying each characteristic process has been studied in detail. However, the survival pathway of cortical neurons at mature stages remains largely uninvestigated. We herein focused on mature corticostriatal neurons because of their important roles in various higher brain functions and the spectrum of neurological and neuropsychiatric disorders. The small GTPase Rho is known to control diverse and essential cellular functions through some effector molecules, including Rho-kinase, during neural development. In the present study, we investigated the role of Rho signaling through Rho-kinase in the survival of corticostriatal neurons. We performed the conditional expression of Clostridium botulinum C3 ADP-ribosyltransferase (C3 transferase) or dominant-negative form for Rho-kinase (Rho-K DN), a well-known inhibitor of Rho or Rho-kinase, respectively, in corticostriatal neurons using a dual viral vector approach combining a neuron-specific retrograde gene transfer lentiviral vector and an adeno-associated virus vector. C3 transferase markedly decreased the number of corticostriatal neurons, which was attributed to caspase-3-dependent enhanced apoptosis. In addition, Rho-K DN produced phenotypic defects similar to those caused by C3 transferase. These results indicate that the Rho/Rho-kinase signaling pathway plays a crucial role in the survival of corticostriatal neurons.

Focused Proteomics Revealed a Novel Rho-kinase Signaling Pathway in the Heart.

Protein phosphorylation plays an important role in the physiological regulation of cardiac function. Myocardial contraction and pathogenesis of cardiac diseases have been reported to be associated with adaptive or maladaptive protein phosphorylation; however, phosphorylation signaling in the heart is not fully elucidated. We recently developed a novel kinase-interacting substrate screening (KISS) method for exhaustive screening of protein kinase substrates, using mass spectrometry and affinity chromatography. First, we examined protein phosphorylation by extracellular signal-regulated kinase (ERK) and protein kinase A (PKA), which has been relatively well studied in cardiomyocytes. The KISS method showed that ERK and PKA mediated the phosphorylation of known cardiac-substrates of each kinase such as Rps6ka1 and cTnI, respectively. Using this method, we found about 330 proteins as Rho-kinase-mediated substrates, whose substrate in cardiomyocytes is unknown. Among them, CARP/Ankrd1, a muscle ankyrin repeat protein, was confirmed as a novel Rho-kinase-mediated substrate. We also found that non-phosphorylatable form of CARP repressed cardiac hypertrophy-related gene Myosin light chain-2v (MLC-2v) promoter activity, and decreased cell size of heart derived H9c2 myoblasts more efficiently than wild type-CARP. Thus, focused proteomics enable us to reveal a novel signaling pathway in the heart.

Phosphoproteomics of the Dopamine Pathway Enables Discovery of Rap1 Activation as a Reward Signal In Vivo.

Dopamine (DA) type 1 receptor (D1R) signaling in the striatum presumably regulates neuronal excitability and reward-related behaviors through PKA. However, whether and how D1Rs and PKA regulate neuronal excitability and behavior remain largely unknown. Here, we developed a phosphoproteomic analysis method to identify known and novel PKA substrates downstream of the D1R and obtained more than 100 candidate substrates, including Rap1 GEF (Rasgrp2). We found that PKA phosphorylation of Rasgrp2 activated its guanine nucleotide-exchange activity on Rap1. Cocaine exposure activated Rap1 in the nucleus accumbens in mice. The expression of constitutively active PKA or Rap1 in accumbal D1R-expressing medium spiny neurons (D1R-MSNs) enhanced neuronal firing rates and behavioral responses to cocaine exposure through MAPK. Knockout of Rap1 in the accumbal D1R-MSNs was sufficient to decrease these phenotypes. These findings demonstrate a novel DA-PKA-Rap1-MAPK intracellular signaling mechanism in D1R-MSNs that increases neuronal excitability to enhance reward-related behaviors.

Radial Glial Cell-Neuron Interaction Directs Axon Formation at the Opposite Side of the Neuron from the Contact Site.

How extracellular cues direct axon-dendrite polarization in mouse developing neurons is not fully understood. Here, we report that the radial glial cell (RGC)-cortical neuron interaction directs axon formation at the opposite side of the neuron from the contact site. N-cadherin accumulates at the contact site between the RGC and cortical neuron. Inhibition of the N-cadherin-mediated adhesion decreases this oriented axon formation in vitro, and disrupts the axon-dendrite polarization in vivo. Furthermore, the RGC-neuron interaction induces the polarized distribution of active RhoA at the contacting neurite and active Rac1 at the opposite neurite. Inhibition of Rho-Rho-kinase signaling in a neuron impairs the oriented axon formation in vitro, and prevents axon-dendrite polarization in vivo. Collectively, these results suggest that the N-cadherin-mediated radial glia-neuron interaction determines the contacting neurite as the leading process for radial glia-guided neuronal migration and directs axon formation to the opposite side acting through the Rho family GTPases.

Extracellular and Intracellular Signaling for Neuronal Polarity.

Neurons are one of the highly polarized cells in the body. One of the fundamental issues in neuroscience is how neurons establish their polarity; therefore, this issue fascinates many scientists. Cultured neurons are useful tools for analyzing the mechanisms of neuronal polarization, and indeed, most of the molecules important in their polarization were identified using culture systems. However, we now know that the process of neuronal polarization in vivo differs in some respects from that in cultured neurons. One of the major differences is their surrounding microenvironment; neurons in vivo can be influenced by extrinsic factors from the microenvironment. Therefore, a major question remains: How are neurons polarized in vivo? Here, we begin by reviewing the process of neuronal polarization in culture conditions and in vivo. We also survey the molecular mechanisms underlying neuronal polarization. Finally, we introduce the theoretical basis of neuronal polarization and the possible involvement of neuronal polarity in disease and traumatic brain injury.

Phosphoproteomic Analysis Using the WW and FHA Domains as Biological Filters.

Protein phosphorylation plays a key role in regulating nearly all intracellular biological events. However, poorly developed phospho-specific antibodies and low phosphoprotein abundance make it difficult to study phosphoproteins. Cellular protein phosphorylation data have been obtained using phosphoproteomic approaches, but the detection of low-abundance or fast-cycling phosphorylation sites remains a challenge. Enrichment of phosphoproteins together with phosphopeptides may greatly enhance the spectrum of low-abundance but biologically important phosphoproteins. Previously, we used 14-3-3ζ to selectively enrich for HeLa cell lysate phosphoproteins. However, because 14-3-3 does not isolate phosphoproteins lacking the 14-3-3-binding motif, we looked for other domains that could complementarily enrich for phosphoproteins. We here assessed and characterized the phosphoprotein binding domains Pin1-WW, CHEK2-FHA, and DLG1-GK. Using a strategy based on affinity chromatography, phosphoproteins were collected from the lysates of HeLa cells treated with phosphatase inhibitor or cAMP activator. We identified different subsets of phosphoproteins associated with WW or FHA after calyculin A, okadaic acid, or forskolin treatment. Our Kinase-Oriented Substrate Screening (KiOSS) method, which used phosphoprotein-binding domains, showed that WW and FHA are applicable and useful for the identification of novel phospho-substrates for kinases and can therefore be used as biological filters for comprehensive phosphoproteome analysis.

In vivo screening for substrates of protein kinase A using a combination of proteomic approaches and pharmacological modulation of kinase activity.

Protein kinase A (PKA) is a serine/threonine kinase whose activity depends on the levels of cyclic AMP (cAMP). PKA plays essential roles in numerous cell types such as myocytes and neurons. Numerous substrate screens have been attempted to clarify the entire scope of the PKA signaling cascade, but it is still underway. Here, we performed a comprehensive screen that consisted of immunoprecipitation and mass spectrometry, with a focus on the identification of PKA substrates. The lysate of HeLa cells treated with Forskolin (FSK)/3-isobutyl methyl xanthine (IBMX) and/or H-89 was subjected to immunoprecipitation using anti-phospho-PKA substrate antibody. The identity of the phosophoproteins and phosphorylation sites in the precipitants was determined using liquid chromatography tandem mass spectrometry (LC/MS/MS). We obtained 112 proteins as candidate substrates and 65 candidate sites overall. Among the candidate substrates, Rho-kinase/ROCK2 was confirmed to be a novel substrate of PKA both in vitro and in vivo. In addition to Rho-kinase, we found more than a hundred of novel candidate substrates of PKA using this screen, and these discoveries provide us with new insights into PKA signaling.

Neuronal polarization in vivo: Growing in a complex environment.

Neurons are one of the most polarized cell types in the body. During the past three decades, many researchers have attempted to understand the mechanisms of neuronal polarization using cultured neurons. Although these studies have succeeded in discovering the various signal molecules that regulate neuronal polarization, one major question remains unanswered: how do neurons polarize in vivo?

Pioneering axons regulate neuronal polarization in the developing cerebral cortex.

The polarization of neurons, which mainly includes the differentiation of axons and dendrites, is regulated by cell-autonomous and non-cell-autonomous factors. In the developing central nervous system, neuronal development occurs in a heterogeneous environment that also comprises extracellular matrices, radial glial cells, and neurons. Although many cell-autonomous factors that affect neuronal polarization have been identified, the microenvironmental cues involved in neuronal polarization remain largely unknown. Here, we show that neuronal polarization occurs in a microenvironment in the lower intermediate zone, where the cell adhesion molecule transient axonal glycoprotein-1 (TAG-1) is expressed in cortical efferent axons. The immature neurites of multipolar cells closely contact TAG-1-positive axons and generate axons. Inhibition of TAG-1-mediated cell-to-cell interaction or its downstream kinase Lyn impairs neuronal polarization. These results show that the TAG-1-mediated cell-to-cell interaction between the unpolarized multipolar cells and the pioneering axons regulates the polarization of multipolar cells partly through Lyn kinase and Rac1.

ERK2-mediated phosphorylation of Par3 regulates neuronal polarization.

Axon formation is one of the most important events in neuronal polarization and is regulated by signaling molecules involved in cytoskeletal rearrangement and protein transport. We previously found that Partition-defective 3 (Par3) is associated with KIF3A (kinesin-2) and is transported into the nascent axon in a KIF3A-dependent fashion. Par3 interacts with the Rac-specific guanine nucleotide-exchange factors (GEFs) Tiam1/2, which activate Rac1, and participates in axon formation in cultured hippocampal neurons. However, the regulatory mechanism of the Par3-KIF3A interaction is poorly understood, and the role of Par3 in neuronal polarization in vivo remains elusive. Here, we found that extracellular signal-regulated kinase 2 (ERK2) directly interacts with Par3, that ERK2 phosphorylates Par3 at Ser-1116, and that the phosphorylated Par3 accumulates at the axonal tips in a manner dependent upon ERK2 activity. The phosphorylation of Par3 by ERK2 inhibited the interaction of Par3 with KIF3A but not with the other Par3 partners, including Par6 and aPKC. The phosphomimic mutant of Par3 (Par3-S1116D) showed less binding activity with the KIF3s and slower transport in the axons. The knockdown of Par3 by RNA interference impaired neuronal polarization, which was rescued with RNAi-resistant Par3, but not with the phosphomimic Par3 mutant, in cultured rat hippocampal neurons and mouse cortical projection neurons in vivo. These results suggest that ERK2 phosphorylates Par3 and inhibits its binding with KIF3A, thereby controlling Par3 transport and neuronal polarity.

Latent process genes for cell differentiation are common decoders of neurite extension length.

A latent process involving signal transduction and gene expression is needed as a preparation step for cellular function. We previously found that nerve growth factor (NGF)-induced cell differentiation has a latent process, which is dependent on ERK activity and gene expression and required for subsequent neurite extension. A latent process can be considered as a preparation step that decodes extracellular stimulus information into cellular functions; however, molecular mechanisms of this process remain unknown. We identified Metrnl, Dclk1 and Serpinb1a as genes that are induced during the latent process (LP) with distinct temporal expression profiles and are required for subsequent neurite extension in PC12 cells. The LP genes showed distinct dependency on the duration of ERK activity, and they were also induced during the latent process of PACAP- and forskolin-induced cell differentiation. Regardless of neurotrophic factors, expression levels of the LP genes during the latent process (0-12 hours), but not phosphorylation levels of ERK, always correlated with subsequent neurite extension length (12-24 hours). Overexpression of all LP genes together, but not of each gene separately, enhanced NGF-induced neurite extension. The LP gene products showed distinct spatial localization. Thus, the LP genes appear to be the common decoders for neurite extension length regardless of neurotrophic factors, and they might function in distinct temporal and spatial manners during the latent process. Our findings provide molecular insight into the physiological meaning of the latent process as the preparation step for decoding information for future phenotypic change.

Local application of neurotrophins specifies axons through inositol 1,4,5-trisphosphate, calcium, and Ca2+/calmodulin-dependent protein kinases.

Neurons are highly polarized cells that have structurally distinct processes-the axons and dendrites-that differentiate from common immature neurites. In cultured hippocampal neurons, one of these immature neurites stochastically initiates rapid extension and becomes an axon, whereas the others become dendrites. Various extracellular and intracellular signals contribute to axon specification; however, the specific intracellular pathways whereby particular extracellular stimuli lead to axon specification remain to be delineated. Here, we found that the neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) were required for axon specification in an autocrine or a paracrine fashion. Using local application with a micropipette to selectively stimulate individual neurites, we found that stimulation of a selected neurite by BDNF or NT-3 induced neurite outgrowth and subsequent axon formation. NT-3 induced a rapid increase in calcium ions (Ca(2+)) in an inositol 1,4,5-trisphosphate (IP(3))-dependent fashion as well as local activation of the Ca(2+) effector Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) in the growth cone. Inhibition of neurotrophin receptors or CaMKK attenuated NT-3-induced axon specification in cultured neurons and axon formation in cortical neurons in vivo. These results identify a role for IP(3)-Ca(2+)-CaMKK signaling in axon specification.

Flexible search for single-axon morphology during neuronal spontaneous polarization.

Polarization, a disruption of symmetry in cellular morphology, occurs spontaneously, even in symmetrical extracellular conditions. This process is regulated by intracellular chemical reactions and the active transport of proteins and it is accompanied by cellular morphological changes. To elucidate the general principles underlying polarization, we focused on developing neurons. Neuronal polarity is stably established; a neuron initially has several neurites of similar length, but only one elongates and is selected to develop into an axon. Polarization is flexibly controlled; when multiple neurites are selected, the selection is eventually reduced to yield a single axon. What is the system by which morphological information is decoded differently based on the presence of a single or multiple axons? How are stability and flexibility achieved? To answer these questions, we constructed a biophysical model with the active transport of proteins that regulate neurite growth. Our mathematical analysis and computer simulation revealed that, as neurites elongate, transported factors accumulate in the growth cone but are degraded during retrograde diffusion to the soma. Such a system effectively works as local activation-global inhibition mechanism, resulting in both stability and flexibility. Our model shows good accordance with a number of experimental observations.

The role of selective transport in neuronal polarization.

Neurons are functionally and morphologically polarized and possess two distinct types of neurites: axons and dendrites. Key molecules for axon formation are transported along microtubules and accumulated at the distal end of the nascent axons. In this review, we summarize recent advances in the understanding of the mechanisms involved in selective transport in neurons. In addition, we focus on motor proteins, cargo, cargo adaptors, and the loading and unloading of cargo.

CRMP-2 directly binds to cytoplasmic dynein and interferes with its activity.

The active transport of proteins and organelles is critical for cellular organization and function in eukaryotic cells. A substantial portion of long-distance transport depends on the opposite polarity of the kinesin and dynein family molecular motors to move cargo along microtubules. It is increasingly clear that many cargo molecules are moved bi-directionally by both sets of motors; however, the regulatory mechanism that determines the directionality of transport remains unclear. We previously reported that collapsin response mediator protein-2 (CRMP-2) played key roles in axon elongation and neuronal polarization. CRMP-2 was also found to associate with the anterograde motor protein Kinesin-1 and was transported with other cargoes toward the axon terminal. In this study, we investigated the association of CRMP-2 with a retrograde motor protein, cytoplasmic dynein. Immunoprecipitation assays showed that CRMP-2 interacted with cytoplasmic dynein heavy chain. Dynein heavy chain directly bound to the N-terminus of CRMP-2, which is the distinct side of CRMP-2's kinesin light chain-binding region. Furthermore, over-expression of the dynein-binding fragments of CRMP-2 prevented dynein-driven microtubule transport in COS-7 cells. Given that CRMP-2 is a key regulator of axon elongation, this interference with cytoplasmic dynein function by CRMP-2 might have an important role in axon formation, and neuronal development.

Anterograde transport of TrkB in axons is mediated by direct interaction with Slp1 and Rab27.

The neurotrophin receptors TrkA, TrkB, and TrkC are localized at the surface of the axon terminus and transmit key signals from brain-derived neurotrophic factor (BDNF) for diverse effects on neuronal survival, differentiation, and axon formation. Trk receptors are sorted into axons via the anterograde transport of vesicles and are then inserted into axonal plasma membranes. However, the transport mechanism remains largely unknown. Here, we show that the Slp1/Rab27B/CRMP-2 complex directly links TrkB to Kinesin-1, and that this association is required for the anterograde transport of TrkB-containing vesicles. The cytoplasmic tail of TrkB binds to Slp1 in a Rab27B-dependent manner, and CRMP-2 connects Slp1 to Kinesin-1. Knockdown of these molecules by siRNA reduces the anterograde transport and membrane targeting of TrkB, thereby inhibiting BDNF-induced ERK1/2 phosphorylation in axons. Our data reveal a molecular mechanism for the selective anterograde transport of TrkB in axons and show how the transport is coupled to BDNF signaling.