Tomoki Nishioka

Behavioral alterations associated with targeted disruption of exons 2 and 3 of the Disc1 gene in the mouse

Disrupted-In-Schizophrenia 1 (DISC1) is a promising candidate gene for susceptibility to psychiatric disorders, including schizophrenia. DISC1 appears to be involved in neurogenesis, neuronal migration, axon/dendrite formation and synapse formation; during these processes, DISC1 acts as a scaffold protein by interacting with various partners. However, the lack of Disc1 knockout mice and a well-characterized antibody to DISC1 has made it difficult to determine the exact role of DISC1 in vivo. In this study, we generated mice lacking exons 2 and 3 of the Disc1 gene and prepared specific antibodies to the N- and C-termini of DISC1. The Disc1 mutant mice are viable and fertile, and no gross phenotypes, such as disorganization of the brains cytoarchitecture, were observed. Western blot analysis revealed that the DISC1-specific antibodies recognize a protein with an apparent molecular mass of ~100 kDa in brain extracts from wild-type mice but not in brain extracts from DISC1 mutant mice. Immunochemical studies demonstrated that DISC1 is mainly localized to the vicinity of the Golgi apparatus in hippocampal neurons and astrocytes. A deficiency of full-length Disc1 induced a threshold shift in the induction of long-term potentiation in the dentate gyrus. The Disc1 mutant mice displayed abnormal emotional behavior as assessed by the elevated plus-maze and cliff-avoidance tests, thereby suggesting that a deficiency of full-length DISC1 may result in lower anxiety and/or higher impulsivity. Based on these results, we suggest that full-length Disc1-deficient mice and DISC1-specific antibodies are powerful tools for dissecting the pathophysiological functions of DISC1.

Rho-kinase Contributes to Sustained RhoA Activation through Phosphorylation of p190A RhoGAP

RhoA is transiently activated by specific extracellular signals such as endothelin-1 (ET-1) in vascular smooth muscle cells. RhoGAP negatively regulates RhoA activity: thus, RhoA becomes the GDP-bound inactive form afterward. Sustained activation of RhoA is induced with high doses of the extracellular signals and is implicated in certain diseases such as vasospasms. However, it remains largely unknown how prolonged activation of RhoA is induced. Here we show that Rho-kinase, an effector of RhoA, phosphorylated p190A RhoGAP at Ser1150 and attenuated p190A RhoGAP activity in COS7 cells. Binding of Rnd to p190A RhoGAP is thought to enhance its activation. Phosphorylation of p190A RhoGAP by Rho-kinase impaired Rnd binding. Stimulation of vascular smooth muscle cells with a high dose of ET-1 provoked sustained RhoA activation and p190A RhoGAP phosphorylation, both of which were prohibited by a Rho-kinase inhibitor. The phosphomimic mutation of p190A RhoGAP weakened Rnd binding and RhoGAP activities. Taken together, these results suggest that ET-1 induces Rho-kinase activation and subsequent phosphorylation of p190A RhoGAP, leading to prolonged RhoA activation.

Cytoskeletal Regulation by AUTS2 in Neuronal Migration and Neuritogenesis

Mutations in the Autism susceptibility candidate 2 gene (AUTS2), whose protein is believed to act in neuronal cell nuclei, have been associated with multiple psychiatric illnesses, including autism spectrum disorders, intellectual disability, and schizophrenia. Here we show that cytoplasmic AUTS2 is involved in the regulation of the cytoskeleton and neural development. Immunohistochemistry and fractionation studies show that AUTS2 localizes not only in nuclei, but also in the cytoplasm, including in the growth cones in the developing brain. AUTS2 activates Rac1 to induce lamellipodia but downregulates Cdc42 to suppress filopodia. Our loss-of-function and rescue experiments show that a cytoplasmic AUTS2-Rac1 pathway is involved in cortical neuronal migration and neuritogenesis in the developing brain. These findings suggest that cytoplasmic AUTS2 acts as a regulator of Rho family GTPases to contribute to brain development and give insight into the pathology of human psychiatric disorders with AUTS2 mutations.

The inositol 5-phosphatase SHIP2 is an effector of RhoA and is involved in cell polarity and migration

Polarization in motile cells requires the coordination of several key signaling molecules, including RhoA small GTPases and phosphoinositides. It is found that SHIP2 interacts with RhoA in a GTP-dependent manner and this interaction is required for proper localization of PI(3,4,5)P3 and regulation of cell polarization and migration.

A proteomic approach for comprehensively screening substrates of protein kinases such as Rho-kinase.

Background Protein kinases are major components of signal transduction pathways in multiple cellular processes. Kinases directly interact with and phosphorylate downstream substrates, thus modulating their functions. Despite the importance of identifying substrates in order to more fully understand the signaling network of respective kinases, efficient methods to search for substrates remain poorly explored. Methodology/Principal Findings We combined mass spectrometry and affinity column chromatography of the catalytic domain of protein kinases to screen potential substrates. Using the active catalytic fragment of Rho-kinase/ROCK/ROK as the model bait, we obtained about 300 interacting proteins from the rat brain cytosol fraction, which included the proteins previously reported as Rho-kinase substrates. Several novel interacting proteins, including doublecortin, were phosphorylated by Rho-kinase both in vitro and in vivo. Conclusions/Significance This method would enable identification of novel specific substrates for kinases such as Rho-kinase with high sensitivity.

Kinase-interacting substrate screening is a novel method to identify kinase substrates

A novel method called kinase-interacting substrate screening based on affinity beads coated with the kinase of interest identifies phosphorylation sites for Rho-kinase and others, which reveals that Rho-kinase substrate Scrib plays a crucial role in the regulation of subcellular contractility by assembling with Rho-kinase and Shroom2.

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.

Identification of focal adhesion kinase (FAK) and phosphatidylinositol 3‐kinase (PI3‐kinase) as Par3 partners by proteomic analysis

Partition defective 3 (Par3) is involved in a variety of polarity events including establishment of apico‐basal polarity of epithelial cell, axon/dendrite specification of neurons and directional migration of cells with front‐rear polarity. Par3 is thought to regulate cell polarity as a scaffold protein by interacting with various partner proteins such as Par6, aPKC, Tiam1/2 and Numb. However, the mode of actions of Par3 in polarized migration remains largely unknown. To explore Par3 functions, we screened Par3‐interacting proteins by combining Par3 affinity column chromatography and shotgun analysis using liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). We obtained about two hundred Par3‐interacting proteins from the rat brain cytosol fraction. Among them, we focused on FAK and PI3‐kinase, as both of them participate in directional cell migration. FAK associated with the PDZ domain and the coiled‐coil region of Par3 and p110 of PI3‐kinase associated with the coiled‐coil region of Par3. Par3 was partially colocalized with FAK in spreading cells. Depletion of Par3 by RNA interference inhibited adhesion‐induced activation of FAK and PI3‐kinase, and RNA interference‐resistant Par3 restored the inhibitory effects. In addition, Par3 was required for the adhesion‐induced cell spreading as well as for directional cell migration toward collagen. These results suggest that Par3 directly interacts with FAK and PI3‐kinase, enhancing their activities for polarized cell migration.

Role of hemoglobin and transferrin in multi-wall carbon nanotube-induced mesothelial injury and carcinogenesis

Multi‐wall carbon nanotubes (MWCNT) are a form of flexible fibrous nanomaterial with high electrical and thermal conductivity. However, 50‐nm MWCNT in diameter causes malignant mesothelioma (MM) in rodents and, thus, the International Agency of Research on Cancer has designated them as a possible human carcinogen. Little is known about the molecular mechanism through which MWCNT causes MM. To elucidate the carcinogenic mechanisms of MWCNT in mesothelial cells, we used a variety of lysates to comprehensively identify proteins specifically adsorbed on pristine MWCNT of different diameters (50 nm, NT50; 100 nm, NT100; 150 nm, NT150; and 15 nm/tangled, NTtngl) using mass spectrometry. We identified >400 proteins, which included hemoglobin, histone, transferrin and various proteins associated with oxidative stress, among which we selected hemoglobin and transferrin for coating MWCNT to further evaluate cytotoxicity, wound healing, intracellular catalytic ferrous iron and oxidative stress in rat peritoneal mesothelial cells (RPMC). Cytotoxicity to RPMC was observed with pristine NT50 but not with NTtngl. Coating NT50 with hemoglobin or transferrin significantly aggravated cytotoxicity to RPMC, with an increase in cellular catalytic ferrous iron and DNA damage also observed. Knockdown of transferrin receptor with ferristatin II decreased not only NT50 uptake but also cellular catalytic ferrous iron. Our results suggest that adsorption of hemoglobin and transferrin on the surface of NT50 play a role in causing mesothelial iron overload, contributing to oxidative damage and possibly subsequent carcinogenesis in mesothelial cells. Uptake of NT50 at least partially depends on transferrin receptor 1. Modifications of NT50 surface may decrease this human risk.

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.