Abnormal expression of ER quality control and ER associated degradation proteins in the dorsolateral prefrontal cortex in schizophrenia

Abstract

Abnormalities in posttranslational protein modifications (PTMs) that regulate protein targeting, trafficking, synthesis, and function have been implicated in the pathophysiology of schizophrenia. The endoplasmic reticulum (ER) contains specialized machinery that facilitate protein synthesis, ER entry and exit, quality control, and post-translational processing, steps required for protein maturation. Dysregulation of these systems could represent potential mechanisms for abnormalities of neurotransmitter associated proteins in schizophrenia. We hypothesized that expression of ER processing pathways is dysregulated in schizophrenia. We characterized protein and complex expression of essential components from protein folding, ER quality control (ERQC), and ER associated degradation (ERAD) processes in the dorsolateral prefrontal cortex of 12 matched pairs of elderly schizophrenia and comparison subjects. We found increased expression of proteins associated with recognizing and modifying misfolded proteins, including UDP-glucose/glycoprotein glucosyltransferase 2 (UGGT2), ER degradation enhancing alpha-mannosidase like protein 2 (EDEM2), and synoviolin (SYVN1)/HRD1. As SYVN1/HRD1 is a component of the ubiquitin ligase HRD1-SEL1L complex that facilitates ERAD, we immunoprecipitated SEL1L and measured expression of other proteins in this complex. In schizophrenia, SYVN1/HRD1 and OS-9, ERAD promoters, have increased association with SEL1L, while XTP3-B, which can prevent ERAD of substrates, has decreased association. Abnormal expression of proteins associated with ERQC and ERAD suggests dysregulation in ER localized protein processing pathways in schizophrenia. Interestingly, the deficits we found are not in the protein processing machinery itself, but in proteins that recognize and target incompletely or misfolded proteins. These changes may reflect potential mechanisms of abnormal neurotransmitter associated protein expression previously observed in schizophrenia.

Introduction

While the pathophysiology of schizophrenia is not well-understood, many hypotheses focus on abnormalities of neurotransmission in this illness. The dopamine, serotonin, glutamate, and GABA neurotransmitter systems have all been implicated in schizophrenia (Baou et al., 2016; Gonzalez-Burgos and Lewis, 2012; Howes and Kapur, 2009; Nakazawa et al., 2012), and the plurality of abnormalities of neurotransmitter associated proteins suggests dysfunction of cellular processes common to the regulation of these systems. Multiple abnormalities in expression of receptors and transporters associated with glutamate and GABA signaling, including abnormal N-glycosylation, receptor localization, and expression of endoplasmic reticulum (ER) - retention signals suggest dysfunctional processing of neurotransmitter associated proteins in the ER (Bauer et al., 2010; Kristiansen et al., 2010; Mueller et al., 2014; Mueller et al., 2015; Tucholski et al., 2013; Tucholski et al., 2013).

The ER is the first compartment in the intracellular secretory pathway, through which the majority of secreted and intramembrane proteins pass (Ellgaard and Helenius, 2003; Vembar and Brodsky, 2008). Protein synthesis, folding, and post-translational processing occur within the ER and are essential for proper protein maturation (Ellgaard and Helenius, 2003; Vembar and Brodsky, 2008). As the initial stage in protein synthesis and processing, the ER contains a highly regulated system for recognizing unfolded and misfolded proteins (Ellgaard and Helenius, 2003; Vembar and Brodsky, 2008). Folding of nascent polypeptide chains in the ER is monitored and regulated by chaperone proteins and folding enzymes, which facilitate the folding and assembly of newly synthesized proteins (Ellgaard and Helenius, 2003; Vembar and Brodsky, 2008). Many newly synthesized proteins are glycosylated, and transfer of N-glycans occurs in a single enzymatic step. The two outermost glucose residues of the N-glycans are rapidly and sequentially removed by mannosyl-oligosaccharide glucosidase (MOGS, also called glucosidase I) and glucosidase II (Araki and Nagata, 2012; Grinna and Robbins, 1979). Glucosidase II is composed of a catalytic subunit, neutral alpha glucosidase AB (GANAB), and a regulatory subunit, glucosidase 2 subunit beta (PRKCSH) (Varki, 2009). The resulting N-glycan is recognized by the ER lectin-like chaperones calnexin (CNX) and/or calreticulin (CRT), which promote proper folding by protecting glycoproteins from aggregation or premature export from the ER(Araki and Nagata, 2012; Rutkevich and Williams, 2011; Williams, 2006). UDP-glucose/glycoprotein glucosyltransferase (UGGT) senses the folding state of glycoproteins released by CNX/CRT and targets correctly folded and assembled proteins for export to the Golgi (Ellgaard and Helenius, 2003; Vembar and Brodsky, 2008). If proteins are not correctly folded and assembled, UGGT reglucosylates the N-glycan to be reengaged by CNX/CRT (D'Alessio et al., 2010; Solda et al., 2007). Unfolded and terminally misfolded proteins within the CNX/CRT cycle are recognized, modified, and extracted from the ER for degradation by the ubiquitin (UB) proteasome system (UPS) (Ellgaard and Helenius, 2003), by the process of ER associated degradation (ERAD). ERAD glycoprotein substrates are recognized and modified by the lectins EDEM1/2/3, followed by association and modification by substrate recognition complexes including chaperones (GRP78, GRP94), lectins (OS.9, XTP3-B), and reductases (Christianson et al., 2011; Cormier et al., 2009; Maattanen et al., 2010; Ninagawa et al., 2014; Satoh et al., 2010; Vembar and Brodsky, 2008). OS-9 and XTP3-B directly bind with SEL1L, an adaptor protein that is essential for maintaining interaction with the E3 ubiquitin-protein ligase synoviolin (SYVN1; the human homolog of the E3 ligase HRD1 expressed in mice and yeast) (Christianson et al., 2011; Christianson et al., 2008). Substrates targeted to this complex are ubiquitinated by SYVN1/HRD1, a process that facilitates substrate recognition by the proteasome for degradation (MacGurn et al., 2012). The SYVN1/HRD1 UB ligase complex interacts with substrate extraction machinery (Christianson et al., 2008; St Pierre and Nabi, 2012; Vembar and Brodsky, 2008), including the transmembrane proteins derlin-1 and derlin-2 (DERL1/2), which eject substrates from the ER by forming a pore in the membrane (St Pierre and Nabi, 2012; Vembar and Brodsky, 2008). VCP-interacting membrane protein (VIMP) interacts with both DERLs and acts as a recruitment factor for cytosolic VCP-containing complexes (Christianson et al., 2011; Lee et al., 2015). VCP is an AAA-ATPase that is essential for substrate extraction from the ER into the cytosol in an ATP-dependent manner, where they are then degraded by the proteasome (Christianson et al., 2011; Zhong et al., 2004).

ER protein processing, ERQC, and ERAD are particularly important in neurons, and multiple reports suggest that Alzheimer's disease (AD) (Hoozemans et al., 2009; Hoozemans et al., 2005), Parkinson's disease (PD) (Hoozemans et al., 2007; Ryu et al., 2002), amyotrophic lateral sclerosis (ALS) (Atkin et al., 2008), Huntington's disease (HD) (Carnemolla et al., 2009; Duennwald and Lindquist, 2008), and Creutzfeldt Jacob disease (CJD) (Hetz et al., 2003) are all associated with ER stress and associated dysregulation. ER dysfunction has also been implicated in mood disorders and schizophrenia (Bown et al., 2000; Gold et al., 2013; Hunsberger et al., 2011; Nevell et al., 2014; So et al., 2007). To further characterize potential ER-associated abnormalities, we hypothesized that in schizophrenia there is dysregulation of ER protein processing pathways and proteins associated with protein folding, ERQC, and ERAD. This study sought to measure protein and complex expression of essential components of these processes in schizophrenia brain.