Synaptic proteomics decode novel molecular landscape in the brain.

Synapses play a pivotal role in forming neural circuits, with critical implications for brain functions such as learning, memory, and emotions. Several advances in synaptic research have demonstrated the diversity of synaptic structure and function, which can form thousands of connections depending on the neuronal cell types. Moreover, synapses not only interconnect neurons but also establish connections with glial cells such as astrocytes, which play a key role in the architecture and function of neuronal circuits in the brain. Emerging evidence suggests that dysfunction of synaptic proteins contributes to a variety of neurological and psychiatric disorders. Therefore, it is crucial to determine the molecular networks within synapses in various neuronal cell types to gain a deeper understanding of how the nervous system regulates brain function. Recent advances in synaptic proteome approaches, such as fluorescence-activated synaptosome sorting (FASS) and proximity labeling, have allowed for a detailed and spatial analysis of many cell-type-specific synaptic molecules in vivo. In this brief review, we highlight these novel spatial proteomic approaches and discuss the regulation of synaptic formation and function in the brain. This knowledge of molecular networks provides new insight into the understanding of many neurological and psychiatric disorders.

Rho-Rho-Kinase Regulates Ras-ERK Signaling Through SynGAP1 for Dendritic Spine Morphology.

The structural plasticity of dendritic spines plays a critical role in NMDA-induced long-term potentiation (LTP) in the brain. The small GTPases RhoA and Ras are considered key regulators of spine morphology and enlargement. However, the regulatory interaction between RhoA and Ras underlying NMDA-induced spine enlargement is largely unknown. In this study, we found that Rho-kinase/ROCK, an effector of RhoA, phosphorylated SynGAP1 (a synaptic Ras-GTPase activating protein) at Ser842 and increased its interaction with 14-3-3ζ, thereby activating Ras-ERK signaling in a reconstitution system in HeLa cells. We also found that the stimulation of NMDA receptor by glycine treatment for LTP induction stimulated SynGAP1 phosphorylation, Ras-ERK activation, spine enlargement and SynGAP1 delocalization from the spines in striatal neurons, and these effects were prevented by Rho-kinase inhibition. Rho-kinase-mediated phosphorylation of SynGAP1 appeared to increase its dissociation from PSD95, a postsynaptic scaffolding protein located at postsynaptic density, by forming a complex with 14-3-3ζ. These results suggest that Rho-kinase phosphorylates SynGAP1 at Ser842, thereby activating the Ras-ERK pathway for NMDA-induced morphological changes in dendritic spines.

Lemur tail kinase 1 (LMTK1) regulates the endosomal localization of ß-secretase BACE1.

Lemur tail kinase 1 (LMTK1), previously called apoptosis-associated tyrosine kinase (AATYK), is an endosomal Ser/Thr kinase. We recently reported that LMTK1 regulates axon outgrowth, dendrite arborization and spine formation via Rab11-mediated vesicle transport. Rab11, a small GTPase regulating recycling endosome trafficking, is shown to be associated with late-onset Alzheimer's disease (LOAD). In fact, genome-wide association studies identified many proteins regulating vesicle transport as risk factors for LOAD. Furthermore, LMTK1 has been reported to be a risk factor for frontotemporal dementia. Then, we hypothesized that LMTK1 contributes to AD development through vesicle transport and examined the effect of LMTK1 on the cellular localization of AD-related proteins, amyloid precursor protein (APP) and ß-site APP cleaving enzyme 1 (BACE1). The ß-cleavage of APP by BACE1 is the initial and rate-limiting step in Aß generation. We found that LMTK1 accumulated BACE1, but not APP, to the perinuclear endosomal compartment, whereas the kinase-negative(kn) mutant of LMTK1A did not. The ß-C-terminal fragment was prone to increase under overexpression of LMTK1A kn. Moreover, the expression level of LMTK1A was reduced in AD brains. These results suggest the possibility that LMTK1 is involved in AD development through the regulation of the proper endosomal localization of BACE1.

Tripartite synaptomics: Cell-surface proximity labeling in vivo.

The astrocyte is a central glial cell and plays a critical role in the architecture and activity of neuronal circuits and brain functions through forming a tripartite synapse with neurons. Emerging evidence suggests that dysfunction of tripartite synaptic connections contributes to a variety of psychiatric and neurodevelopmental disorders. Furthermore, recent advancements with transcriptome profiling, cell biological and physiological approaches have provided new insights into the molecular mechanisms into how astrocytes control synaptogenesis in the brain. In addition to these findings, we have recently developed in vivo cell-surface proximity-dependent biotinylation (BioID) approaches, TurboID-surface and Split-TurboID, to comprehensively understand the molecular composition between astrocytes and neuronal synapses. These proteomic approaches have discovered a novel molecular framework for understanding the tripartite synaptic cleft that arbitrates neuronal circuit formation and function. Here, this short review highlights novel in vivo cell-surface BioID approaches and recent advances in this rapidly evolving field, emphasizing how astrocytes regulate excitatory and inhibitory synapse formation in vitro and in vivo.

Chemico-genetic discovery of astrocytic control of inhibition in vivo.

Perisynaptic astrocytic processes are an integral part of central nervous system synapses1,2; however, the molecular mechanisms that govern astrocyte-synapse adhesions and how astrocyte contacts control synapse formation and function are largely unknown. Here we use an in vivo chemico-genetic approach that applies a cell-surface fragment complementation strategy, Split-TurboID, and identify a proteome that is enriched at astrocyte-neuron junctions in vivo, which includes neuronal cell adhesion molecule (NRCAM). We find that NRCAM is expressed in cortical astrocytes, localizes to perisynaptic contacts and is required to restrict neuropil infiltration by astrocytic processes. Furthermore, we show that astrocytic NRCAM interacts transcellularly with neuronal NRCAM coupled to gephyrin at inhibitory postsynapses. Depletion of astrocytic NRCAM reduces numbers of inhibitory synapses without altering glutamatergic synaptic density. Moreover, loss of astrocytic NRCAM markedly decreases inhibitory synaptic function, with minor effects on excitation. Thus, our results present a proteomic framework for how astrocytes interface with neurons and reveal how astrocytes control GABAergic synapse formation and function.

ARHGAP10, which encodes Rho GTPase-activating protein 10, is a novel gene for schizophrenia risk.

Schizophrenia (SCZ) is known to be a heritable disorder; however, its multifactorial nature has significantly hampered attempts to establish its pathogenesis. Therefore, in this study, we performed genome-wide copy-number variation (CNV) analysis of 2940 patients with SCZ and 2402 control subjects and identified a statistically significant association between SCZ and exonic CNVs in the ARHGAP10 gene. ARHGAP10 encodes a member of the RhoGAP superfamily of proteins that is involved in small GTPase signaling. This signaling pathway is one of the SCZ-associated pathways and may contribute to neural development and function. However, the ARHGAP10 gene is often confused with ARHGAP21, thus, the significance of ARHGAP10 in the molecular pathology of SCZ, including the expression profile of the ARHGAP10 protein, remains poorly understood. To address this issue, we focused on one patient identified to have both an exonic deletion and a missense variant (p.S490P) in ARHGAP10. The missense variant was found to be located in the RhoGAP domain and was determined to be relevant to the association between ARHGAP10 and the active form of RhoA. We evaluated ARHGAP10 protein expression in the brains of reporter mice and generated a mouse model to mimic the patient case. The model exhibited abnormal emotional behaviors, along with reduced spine density in the medial prefrontal cortex (mPFC). In addition, primary cultured neurons prepared from the mouse model brain exhibited immature neurites in vitro. Furthermore, we established induced pluripotent stem cells (iPSCs) from this patient, and differentiated them into tyrosine hydroxylase (TH)-positive neurons in order to analyze their morphological phenotypes. TH-positive neurons differentiated from the patient-derived iPSCs exhibited severe defects in both neurite length and branch number; these defects were restored by the addition of the Rho-kinase inhibitor, Y-27632. Collectively, our findings suggest that rare ARHGAP10 variants may be genetically and biologically associated with SCZ and indicate that Rho signaling represents a promising drug discovery target for SCZ treatment.

Phosphorylation of Npas4 by MAPK Regulates Reward-Related Gene Expression and Behaviors.

Dopamine (DA) activates mitogen-activated protein kinase (MAPK) via protein kinase A (PKA)/Rap1 in medium spiny neurons (MSNs) expressing the dopamine D1 receptor (D1R) in the nucleus accumbens (NAc), thereby regulating reward-related behavior. However, how MAPK regulates reward-related learning and memory through gene expression is poorly understood. Here, to identify the relevant transcriptional factors, we perform proteomic analysis using affinity beads coated with cyclic AMP response element binding protein (CREB)-binding protein (CBP), a transcriptional coactivator involved in reward-related behavior. We identify more than 400 CBP-interacting proteins, including Neuronal Per Arnt Sim domain protein 4 (Npas4). We find that MAPK phosphorylates Npas4 downstream of PKA, increasing the Npas4-CBP interaction and the transcriptional activity of Npas4 at the brain-derived neurotrophic factor (BDNF) promoter. The deletion of Npas4 in D1R-expressing MSNs impairs cocaine-induced place preference, which is rescued by Npas4-wild-type (WT), but not by a phospho-deficient Npas4 mutant. These observations suggest that MAPK phosphorylates Npas4 in D1R-MSNs and increases transcriptional activity to enhance reward-related learning and memory.

The LMTK1-TBC1D9B-Rab11A Cascade Regulates Dendritic Spine Formation via Endosome Trafficking.

Dendritic spines are postsynaptic protrusions at excitatory synapses that are critical for proper neuronal synaptic transmission. While lipid and protein membrane components are necessary for spine formation, it is largely unknown how they are recruited to developing spines. Endosomal trafficking is one mechanism that may influence this development. We recently reported that Lemur kinase 1A (LMTK1A), a membrane-bound Ser/Thr kinase, regulates trafficking of endosomes in neurons. LMTK1 has been shown to be a p35 Cdk5 activator-binding protein and a substrate for Cdk5-p35; however, its neuronal function has not been sufficiently studied. Here, we investigate the role of LMTK1 in spine formation. Depletion of LMTK1 increases spine formation, maturation, and density in primary cultured neurons and in mouse brain of either sex. Additionally, expression of kinase-negative LMTK1 stimulates spine formation in primary neurons and in vivo LMTK1 controls spine formation through Rab11, a regulator of recycling endosome trafficking. We identify TBC1D9B, a Rab11A GTPase-activating protein (Rab11A GAP), as a LMTK1 binding protein, and find that TBC1D9B mediates LMTK1 activity on Rab11A. TBC1D9B inactivates Rab11A under the control of LMTK1A. Further, by analyzing the effect of decreased TBC1D9B expression in primary neurons, we demonstrate that TBC1D9B indeed regulates spine formation. This is the first demonstration of the biological function of TBC1D9B. Together, with the regulation of LMTK1 by Cdk5-p35, we propose the Cdk5-LMTK1-TBC1D9B-Rab11A cascade as a novel signaling mechanism regulating endosomal transport for synapse formation and function.SIGNIFICANCE STATEMENT Dendritic spines are postsynaptic specializations essential for synaptic transmission. However, it is not known how critical membrane components are recruited to spines for their formation. Endosomal trafficking is one such mechanism that may mediate this process. Here we investigate regulators of endosomal trafficking and their contribution to spine formation. We identify two novel factors, LMTK1 and TBC1D9B, which regulate spine formation upstream of Rab11A, a small GTPase. LMTK1 is a membrane bound Ser/Thr kinase regulated by Cdk5-p35, and TBC1D9B is a recently identified Rab11 GAP. LMTK1 controls the GAP activity of TBC1D9B on Rab11A, and TBC1D9B mediates the LMTK1 activity on Rab11A. We propose the Cdk5-LMTK1-TBC1D9B-Rab11A cascade as a novel mechanism controlling spine formation and function.

Neuronal Polarity: Positive and Negative Feedback Signals.

Establishment and maintenance of neuronal polarity are critical for neuronal development and function. One of the fundamental questions in neurodevelopment is how neurons generate only one axon and several dendrites from multiple minor neurites. Over the past few decades, molecular and cell biological approaches have unveiled a large number of signaling networks regulating neuronal polarity in cultured hippocampal neurons and the developing cortex. Emerging evidence reveals that positive and negative feedback signals play a crucial role in axon and dendrite specification. Positive feedback signals are continuously activated in one of minor neurites and result in axon specification and elongation, whereas negative feedback signals are propagated from a nascent axon terminal to all minor neurites and inhibit the formation of multiple axon, thereby leading to dendrite specification, and maintaining neuronal polarity. This current insight provides a holistic picture of the signaling mechanisms underlying neuronal polarization during neuronal development. Here, our review highlights recent advancements in this fascinating field, with a focus on the positive, and negative feedback signals as key regulatory mechanisms underlying neuronal polarization.

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.

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.

Neuronal polarization.

Neurons are highly polarized cells with structurally and functionally distinct processes called axons and dendrites. This polarization underlies the directional flow of information in the central nervous system, so the establishment and maintenance of neuronal polarization is crucial for correct development and function. Great progress in our understanding of how neurons establish their polarity has been made through the use of cultured hippocampal neurons, while recent technological advances have enabled in vivo analysis of axon specification and elongation. This short review and accompanying poster highlight recent advances in this fascinating field, with an emphasis on the signaling mechanisms underlying axon and dendrite specification in vitro and in vivo.

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.

Preferential targeting of p39-activated Cdk5 to Rac1-induced lamellipodia.

Cdk5 is a member of the cyclin-dependent kinase (Cdk) family that plays a role in various neuronal activities including brain development, synaptic regulation, and neurodegeneration. Cdk5 requires the neuronal specific activators, p35 and p39 for subcellular compartmentalization. However, it is not known how active Cdk5 is recruited to F-actin cytoskeleton, which is a Cdk5 target. Here we found p35 and p39 localized to F-actin rich regions of the plasma membrane and investigated the underlying targeting mechanism in vitro by expressing them with Rho family GTPases in Neuro2A cells. Both p35 and p39 accumulated at the cell peripheral lamellipodia and perinuclear regions, where active Rac1 is localized. Interestingly, p35 and p39 displayed different localization patterns as p35 was found more at the perinuclear region and p39 was found more in peripheral lamellipodia. We then confirmed this distinct localization in primary hippocampal neurons. We also determined that the localization of p39 to lamellipodia requires myristoylation and Lys clusters within the N-terminal p10 region. Additionally, we found that p39-Cdk5, but not p35-Cdk5 suppressed lamellipodia formation by reducing Rac1 activity. These results suggest that p39-Cdk5 has a dominant role in Rac1-dependent lamellipodial activity.

LMTK1 regulates dendritic formation by regulating movement of Rab11A-positive endosomes.

Neurons extend two types of neurites-axons and dendrites-that differ in structure and function. Although it is well understood that the cytoskeleton plays a pivotal role in neurite differentiation and extension, the mechanisms by which membrane components are supplied to growing axons or dendrites is largely unknown. We previously reported that the membrane supply to axons is regulated by lemur kinase 1 (LMTK1) through Rab11A-positive endosomes. Here we investigate the role of LMTK1 in dendrite formation. Down-regulation of LMTK1 increases dendrite growth and branching of cerebral cortical neurons in vitro and in vivo. LMTK1 knockout significantly enhances the prevalence, velocity, and run length of anterograde movement of Rab11A-positive endosomes to levels similar to those expressing constitutively active Rab11A-Q70L. Rab11A-positive endosome dynamics also increases in the cell body and growth cone of LMTK1-deficient neurons. Moreover, a nonphosphorylatable LMTK1 mutant (Ser34Ala, a Cdk5 phosphorylation site) dramatically promotes dendrite growth. Thus LMTK1 negatively controls dendritic formation by regulating Rab11A-positive endosomal trafficking in a Cdk5-dependent manner, indicating the Cdk5-LMTK1-Rab11A pathway as a regulatory mechanism of dendrite development as well as axon outgrowth.

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.