GPI Glycan Remodeling by PGAP5 Regulates Transport of GPI-Anchored Proteins from the ER to the Golgi

SummaryMany eukaryotic proteins are attached to the cell surface via glycosylphosphatidylinositol (GPI) anchors. How GPI-anchored proteins (GPI-APs) are trafficked from the endoplasmic reticulum (ER) to the cell surface is poorly understood, but the GPI moiety has been postulated to function as a signal for sorting and transport. Here, we established mutant cells that were selectively defective in transport of GPI-APs from the ER to the Golgi. We identified a responsible gene, designated PGAP5 (post-GPI-attachment to proteins 5). PGAP5 belongs to a dimetal-containing phosphoesterase family and catalyzed the remodeling of the glycan moiety on GPI-APs. PGAP5 catalytic activity is a prerequisite for the efficient exit of GPI-APs from the ER. Our data demonstrate that GPI glycan acts as an ER-exit signal and suggest that glycan remodeling mediated by PGAP5 regulates GPI-AP transport in the early secretory pathway

Trans-synaptic interaction of GluRdelta2 and Neurexin through Cbln1 mediates synapse formation in the cerebellum

SummaryElucidation of molecular mechanisms that regulate synapse formation is required for the understanding of neural wiring, higher brain functions, and mental disorders. Despite the wealth of in vitro information, fundamental questions about how glutamatergic synapses are formed in the mammalian brain remain unanswered. Glutamate receptor (GluR) δ2 is essential for cerebellar synapse formation in vivo. Here, we show that the N-terminal domain (NTD) of GluRδ2 interacts with presynaptic neurexins (NRXNs) through cerebellin 1 precursor protein (Cbln1). The synaptogenic activity of GluRδ2 is abolished in cerebellar primary cultures from Cbln1 knockout mice and is restored by recombinant Cbln1. Knockdown of NRXNs in cerebellar granule cells also hinders the synaptogenic activity of GluRδ2. Both the NTD of GluRδ2 and the extracellular domain of NRXN1β suppressed the synaptogenic activity of Cbln1 in cerebellar primary cultures and in vivo. These results suggest that GluRδ2 mediates cerebellar synapse formation by interacting with presynaptic NRXNs through Cbln1

Bamboo Shoot and Artemisia capillaris Extract Mixture Ameliorates Dextran Sodium Sulfate-Induced Colitis

Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract and is characterized by recurrent chronic inflammation and mucosal damage of the gastrointestinal tract. Recent studies have demonstrated that bamboo shoot (BS) and Artemisia capillaris (AC) extracts enhance anti-inflammatory effects in various disease models. However, it is uncertain whether there is a synergistic protective effect of BS and AC in dextran sodium sulfate (DSS)-induced colitis. In the current study, we tested the combined effects of BS and AC extracts (BA) on colitis using in vivo and in vitro models. Compared with control mice, oral administration of DSS exacerbated colon length and increased the disease activity index (DAI) and histological damage. In DSS-induced colitis, treatment with BA significantly alleviated DSS-induced symptoms such as colon shortening, DAI, histological damage, and colonic pro-inflammatory marker expression compared to single extracts (BS or AC) treatment. Furthermore, we found BA treatment attenuated the ROS generation, F-actin formation, and RhoA activity compared with the single extract (BS or AC) treatment in DSS-treated cell lines. Collectively, these findings suggest that BA treatment has a positive synergistic protective effect on colonic inflammation compared with single extracts, it may be a highly effective complementary natural extract mixture for the prevention or treatment of IBD

Analysis of IL1RAPL1-binding protein candidates (band #4).

<p><b>A, C, E, G,</b> Coimmunoprecipitation of FLAG-IL1RAPL1 with myc-PLCβ1 (<b>A</b>) and myc-SNIP (<b>E</b>) and that of YFP-IL1RAPL1 with FLAG-Snap91 (<b>C</b>) and FLAG-Mcf2l (<b>G</b>) in HEK 293T cells. Immunoprecipitation with anti-FLAG antibody followed by western blotting with anti-Myc antibody (<b>A</b>, <b>E</b>) and that with anti-GFP antibody followed by western blotting with anti-FLAG antibody (<b>C</b>, <b>G</b>) are shown. <b>B, D, F, H,</b> Colocalization of IL1RAPL1 (middle, green) and myc-PLCβ1 (<b>B</b>), myc-Snap91 (<b>D</b>), myc-SNIP (<b>F</b>) and myc-Mcf2l (<b>H</b>) (left, red) in HEK 293T cells. Merged images are shown (right). Scale bar, 10 μm.</p

IL1RAPL1 regulates AMPA receptor newly insertion to surface in cortical neurons.

<p><b>A,</b> pHluorin fluorescence of pH-GluA in neurons. pHluorin signals are invisible in Golgi and endosome (in low pH) and weakly visible in the endoplasmic reticulum (ER, pH ∼7.0). Bright punctate signals of fluorescence increase when pH-GluA is inserted to surface and the pHluorin tag is exposed to the extracellular space (pH 7.4). <b>B,</b> Representative real time visualization of typical pH-GluA1 insertion events. Signal position around a neuron (y-axis, 83 μm) and time (x-axis, 5 min). Each ‘comet-like’ event is indicated by a white arrowhead. The sudden rising and disappear in fluorescence represents individual surface expression of pH-GluA1. <b>C, E–G,</b> Effects of IL1RAPL1 overexpression on the insertion frequency of pH-GluA1 (<i>n</i> = 9, <i>n</i> = 7) (<b>C</b>), pH-GluA2/GluA3 (<i>n</i> = 10, respectively) (<b>E</b>), pH-GluA2 (<i>n</i> = 10, respectively) (<b>F</b>) and pH-GluA3 (<i>n</i> = 10, respectively) (<b>G</b>). <b>D,</b> Longer observation of IL1RAPL1 effects on the pH-GluA1 insertion frequency (<i>n</i> = 4, respectively). Signals existing on surface over 1 min were calculated. Student <i>t</i>-test. *, <i>p</i><0.05; **, <i>p</i><0.01. Error bars represent s.e.m.</p

Identification of IL1RAPL1-ICD-binding proteins by affinity chromatography.

<p><b>A,</b> Negative staining of IL1RAPL1-binding proteins from the brain extracts, resolved by SDS-PAGE. Affinity chromatography of brain extracts was performed with maltose binding protein (MBP) conjugated with or without the cytoplasmic domain of IL1RAPL1. Protein bands specific to or thicker on the “MBP-IL1RAPL1 (+ brain extract)” lane (arrowheads 1–5) were excised and analyzed by LC-MS/MS. After subtraction of proteins detected in the control “MBP (+ brain extract)” lane, 9 candidate interactors were identified. <b>B,</b> List of identified proteins from each gel band. Numbers of identified peptides for each protein and scores of Mascot searches are shown.</p

IL1RAPL1 Associated with Mental Retardation and Autism Regulates the Formation and Stabilization of Glutamatergic Synapses of Cortical Neurons through RhoA Signaling Pathway

<div><p>Interleukin-1 receptor accessory protein-like 1 (IL1RAPL1) is associated with X-linked mental retardation and autism spectrum disorder. We found that IL1RAPL1 regulates synapse formation of cortical neurons. To investigate how IL1RAPL1 controls synapse formation, we here screened IL1RAPL1-interacting proteins by affinity chromatography and mass spectroscopy. IL1RAPL1 interacted with Mcf2-like (Mcf2l), a Rho guanine nucleotide exchange factor, through the cytoplasmic Toll/IL-1 receptor domain. Knockdown of endogenous Mcf2l and treatment with an inhibitor of Rho-associated protein kinase (ROCK), the downstream kinase of RhoA, suppressed IL1RAPL1-induced excitatory synapse formation of cortical neurons. Furthermore, we found that the expression of IL1RAPL1 affected the turnover of AMPA receptor subunits. Insertion of GluA1-containing AMPA receptors to the cell surface was decreased, whereas that of AMPA receptors composed of GluA2/3 was enhanced. Mcf2l knockdown and ROCK inhibitor treatment diminished the IL1RAPL1-induced changes of AMPA receptor subunit insertions. Our results suggest that Mcf2l-RhoA-ROCK signaling pathway mediates IL1RAPL1-dependent formation and stabilization of glutamatergic synapses of cortical neurons.</p></div

Suppression of 6-Hydroxydopamine-Induced Oxidative Stress by Hyperoside Via Activation of Nrf2/HO-1 Signaling in Dopaminergic Neurons

In our ongoing research to discover natural products with neuroprotective effects, hyperoside (quercetin 3-O-galactoside) was isolated from Acer tegmentosum, which has been used in Korean traditional medicine to treat liver-related disorders. Here, we demonstrated that hyperoside protects cultured dopaminergic neurons from death via reactive oxygen species (ROS)-dependent mechanisms, although other relevant mechanisms of hyperoside activity remain largely uncharacterized. For the first time, we investigated the neuroprotective effects of hyperoside on 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in neurons, and the possible underlying mechanisms. Hyperoside significantly ameliorated the loss of neuronal cell viability, lactate dehydrogenase release, excessive ROS accumulation and mitochondrial membrane potential dysfunction associated with 6-OHDA-induced neurotoxicity. Furthermore, hyperoside treatment activated the nuclear erythroid 2-related factor 2 (Nrf2), an upstream molecule of heme oxygenase-1 (HO-1). Hyperoside also induced the expression of HO-1, an antioxidant response gene. Remarkably, we found that the neuroprotective effects of hyperoside were weakened by an Nrf2 small interfering RNA, which blocked the ability of hyperoside to inhibit neuronal death, indicating the vital role of HO-1. Overall, we show that hyperoside, via the induction of Nrf2-dependent HO-1 activation, suppresses neuronal death caused by 6-OHDA-induced oxidative stress. Moreover, Nrf2-dependent HO-1 signaling activation represents a potential preventive and therapeutic target in Parkinson&prime;s disease management

Pull down experiments of each IL1RAPL1 intracellular domain with interacting proteins.

<p><b>A,</b> Schematic structures (top) and SDS-PAGE followed by Coomassie Brilliant Blue staining (bottom) of purified MBP (1) and MBP-fusion proteins with the whole cytoplasmic region (2), the TIR domain (3) and the CT domain (4) of IL1RAPL1. <b>B,</b> Cell lysates from HEK 293T cells transfected with FLAG-Mcf2l, myc-PLCβ1, myc-SNIP, FLAG-Rasal1 or FLAG-PKCε were incubated with amylose resins coupled to MBP (1) or MBP-fusion protein with the whole cytoplasmic region (2), the TIR domain (3) or the CT domain (4) of IL1RAPL1. Precipitates were analyzed by SDS-PAGE followed by immunoblotting with anti-FLAG or anti-Myc antibody.</p