The Autism-Related Protein PX-RICS Mediates GABAergic Synaptic Plasticity in Hippocampal Neurons and Emotional Learning in Mice

GABAergic dysfunction underlies many neurodevelopmental and psychiatric disorders. GABAergic synapses exhibit several forms of plasticity at both pre- and postsynaptic levels. NMDA receptor (NMDAR)–dependent inhibitory long-term potentiation (iLTP) at GABAergic postsynapses requires an increase in surface GABAARs through promoted exocytosis; however, the regulatory mechanisms and the neuropathological significance remain unclear. Here we report that the autism-related protein PX-RICS is involved in GABAAR transport driven during NMDAR–dependent GABAergic iLTP. Chemically induced iLTP elicited a rapid increase in surface GABAARs in wild-type mouse hippocampal neurons, but not in PX-RICS/RICS–deficient neurons. This increase in surface GABAARs required the PX-RICS/GABARAP/14–3-3 complex, as revealed by gene knockdown and rescue studies. iLTP induced CaMKII–dependent phosphorylation of PX-RICS to promote PX-RICS–14-3-3 assembly. Notably, PX-RICS/RICS–deficient mice showed impaired amygdala–dependent fear learning, which was ameliorated by potentiating GABAergic activity with clonazepam. Our results suggest that PX-RICS–mediated GABAAR trafficking is a key target for GABAergic plasticity and its dysfunction leads to atypical emotional processing underlying autism. Keywords: Autism, Inhibitory synaptic plasticity, Emotion, GABAA receptor, Trafficking, Amygdal

Phosphoproteome of Human Glioblastoma Initiating Cells Reveals Novel Signaling Regulators Encoded by the Transcriptome

<div><h3>Background</h3><p>Glioblastoma is one of the most aggressive tumors with poor prognosis. Although various studies have been performed so far, there are not effective treatments for patients with glioblastoma.</p> <h3>Methodology/Principal Findings</h3><p>In order to systematically elucidate the aberrant signaling machinery activated in this malignant brain tumor, we investigated phosphoproteome dynamics of glioblastoma initiating cells using high-resolution nanoflow LC-MS/MS system in combination with SILAC technology. Through phosphopeptide enrichment by titanium dioxide beads, a total of 6,073 phosphopeptides from 2,282 phosphorylated proteins were identified based on the two peptide fragmentation methodologies of collision induced dissociation and higher-energy C-trap dissociation. The SILAC-based quantification described 516 up-regulated and 275 down-regulated phosphorylation sites upon epidermal growth factor stimulation, including the comprehensive status of the phosphorylation sites on stem cell markers such as nestin. Very intriguingly, our in-depth phosphoproteome analysis led to identification of novel phosphorylated molecules encoded by the undefined sequence regions of the human transcripts, one of which was regulated upon external stimulation in human glioblastoma initiating cells.</p> <h3>Conclusions/Significance</h3><p>Our result unveils an expanded diversity of the regulatory phosphoproteome defined by the human transcriptome.</p> </div

In vivo phosphorylation sites of nestin protein identified from human glioblastoma stem cells with multiple amino acid sequence alignment of Homo sapiens (NP_006608.1), Mus musculus (NP_057910.3) and Bos Taurus (NP_001193520.1).

<p>The numbers indicate the positions of the phosphorylated amino acid residues on the human sequence. The red vertical lines indicate novel phosphorylation sites, whereas the black ones represent known phosphorylation sites. The sequence alignment was performed using CLUSTALW ver. 1.83 (<a href="http://clustalw.ddbj.nig.ac.jp/top-j.html" target="_blank">http://clustalw.ddbj.nig.ac.jp/top-j.html</a>).</p

Functional classification of the glioblastoma phosphoproteome based on the Gene Ontology information in the Human Protein Reference Database (HPRD) (

<p><a href="http://www.hprd.org/" target="_blank">http://www.hprd.org/</a><b>).</b> The phosphorylated proteins identified in our analysis are classified by (A) molecular function, (B) biological process and (C) cellular component, respectively.</p

List of the novel phosphorylated peptides extracted from NCBI Refseq human RNA database.

a<p>Evaluation of the phosphorylated sites was conductd on the basis of the threshold of above 75% phosphoRS site probabilities, which were calculated by Proteome Discover (ver. 1.3; Thermo Fisher Scientific). The phosphorylated amino acid residues were indicated by pS, pT and pY, respectively.</p

List of the phosphorylated molecules involved in representative glioblastoma-related signaling pathways classified by DAVID^a.

a<p>http://david.abcc.ncifcrf.gov/home.jsp.</p

Schematic procedure for comprehensive identification and quantification of EGF-induced phosphoproteome based on SILAC technology.

<p>Two populations of glioblastoma initiating cells were grown in media supplemented with normal (L-¹²C₆-lysine) and stable isotopes (L-¹³C₆-lysine), respectively. Each cell population is unstimulated or stimulated with 20 ng/ml EGF for 15 min, lysed, and combined in equal ratio. Phosphopeptides enriched through TiO₂ columns are subjected to mass spectrometric analysis.</p

PX-RICS mediates ER-to-Golgi transport of the N-cadherin/β-catenin complex

Cadherins mediate Ca2+-dependent cell–cell adhesion. Efficient export of cadherins from the endoplasmic reticulum (ER) is known to require complex formation with β-catenin. However, the molecular mechanisms underlying this requirement remain elusive. Here we show that PX-RICS, a β-catenin-interacting GTPase-activating protein (GAP) for Cdc42, mediates ER-to-Golgi transport of the N-cadherin/β-catenin complex. Knockdown of PX-RICS expression induced the accumulation of the N-cadherin/β-catenin complex in the ER and ER exit site, resulting in a decrease in cell–cell adhesion. PX-RICS was also required for ER-to-Golgi transport of the fibroblast growth factor-receptor 4 (FGFR4) associated with N-cadherin. PX-RICS-mediated ER-to-Golgi transport was dependent on its interaction with β-catenin, phosphatidylinositol-4-phosphate (PI4P), Cdc42, and its novel binding partner γ-aminobutyric acid type A receptor-associated protein (GABARAP). These results suggest that PX-RICS ensures the efficient entry of the N-cadherin/β-catenin complex into the secretory pathway, and thereby regulates the amount of N-cadherin available for cell adhesion and FGFR4-mediated signaling

Ubiquitin ligase RNF167 regulates AMPA receptor-mediated synaptic transmission

AMPA receptors (AMPARs) mediate the majority of fast excitatory neurotransmission, and their density at postsynaptic sites determines synaptic strength. Ubiquitination is a posttranslational modification that dynamically regulates the synaptic expression of many proteins. However, very few of the ubiquitinating enzymes implicated in the process have been identified. In a screen to identify transmembrane RING domain-containing E3 ubiquitin ligases that regulate surface expression of AMPARs, we identified RNF167. Predominantly lysosomal, a subpopulation of RNF167 is located on the surface of cultured neurons. Using a RING mutant RNF167 or a specific shRNA to eliminate endogenous RNF167, we demonstrate that AMPAR surface expression increases in hippocampal neurons with disrupted RNF167 activity and that RNF167 is involved in activity-dependent ubiquitination of AMPARs. In addition, RNF167 regulates synaptic AMPAR currents, whereas synaptic NMDAR currents are unaffected. Therefore, our study identifies RNF167 as a selective regulator of AMPAR-mediated neurotransmission and expands our understanding of how ubiquitination dynamically regulates excitatory synapses