Neuropsychopharmacology (2013) 38, 1910–1920 & 2013 American College of Neuropsychopharmacology. All rights reserved 0893-133X/13 www.neuropsychopharmacology.org Dysfunction of the Ubiquitin Proteasome and Ubiquitin-Like Systems in Schizophrenia ,1 1 2 1 Marı´a D Rubio* , Krista Wood , Vahram Haroutunian and James H Meador-Woodruff 1 Department of Psychiatry and Behavioral Neurobiology, University of Alabama at Birmingham, Birmingham, AL, USA; 2 Department of Psychiatry, Mount Sinai School of Medicine, New York, NY, USA Protein expression abnormalities have been implicated in the pathophysiology of schizophrenia, but the underlying cause of these changes is not known. We sought to investigate ubiquitin and ubiquitin-like (UBL) systems (SUMOylation, NEDD8ylation, and Ufmylation) as putative mechanisms underlying protein expression abnormalities seen in schizophrenia. For this, we performed western blot analysis of total ubiquitination, free ubiquitin, K48- and K63-linked ubiquitination, and E1 activases, E2 conjugases, and E3 ligases involved in ubiquitination and UBL post-translational modifications in postmortem brain tissue samples from persons with schizophrenia (n ¼ 13) and comparison subjects (n ¼ 13). We studied the superior temporal gyrus (STG) of subjects from the Mount Sinai Medical Center brain collection that were matched for age, tissue pH, and sex. We found an overall reduction of protein ubiquitination, free ubiquitin, K48-linked ubiquitination, and increased K63 polyubiquitination in schizophrenia. Ubiquitin E1 activase UBA (ubiquitin activating enzyme)-6 and E3 ligase Nedd (neural precursor cell-expressed developmentally downregulated)-4 were decreased in this illness, as were E3 ligases involved in Ufmylation (UFL1) and SUMOylation (protein inhibitor of activated STAT 3, PIAS3). NEDD8ylation was also dysregulated in schizophrenia, with decreased levels of the E1 activase UBA3 and the E3 ligase Rnf7. This study of ubiquitin and UBL systems in schizophrenia found abnormalities of ubiquitination, Ufmylation, SUMOylation, and NEDD8ylation in the STG in this disorder. These results suggest a novel approach to the understanding of schizophrenia pathophysiology, where a disruption in homeostatic adaptation of the cell underlies discreet changes seen at the protein level in this illness. Neuropsychopharmacology (2013) 38, 1910–1920; doi:10.1038/npp.2013.84; published online 8 May 2013 Keywords: E3 ligase; E1 activase; polyubiquitination; SUMOylation; Ufmylation; Nedd8ylation INTRODUCTION The ubiquitin proteasome system (UPS), a protein degradation system, has been identified on the basis of Schizophrenia, a debilitating illness affecting 1% of the genetic reports as a canonical pathway associated with population, has a complex pathophysiology that is still not neuropsychiatric disorders, including Alzheimer’s (Lam completely understood. Until recently, efforts have been et al, 2000), Parkinson’s (Shimura et al, 2001), psychosis, made to unveil the neuropathology of this illness at the and bipolar disorder (Bousman et al, 2010a, b; Middleton molecular level. Numerous studies have found decreased et al, 2002). In addition to targeting proteins for degrada- expression of multiple proteins in schizophrenia, including tion, the UPS is involved in vesicle trafficking, trafficking of but not limited to dysbindin (Talbot et al, 2004; Weickert proteins from the endoplasmic reticulum to the plasma et al, 2008), cdc42 (Rubio et al, 2012), kalirin (Rubio et al, membrane, recycling of receptors at the cell membrane 2012), Akt1 (Zheng et al, 2012), Reelin and GAD67 (Guidotti (Haas and Broadie, 2008), and sorting of Golgi and plasma et al, 2000), Rap2 (Funk et al, 2012), JNK1 (Funk et al, membrane proteins into the vacuolar degradation pathway 2012), JNK2, (Funk et al, 2012), and PSD-95 (Funk (Scheuring et al, 2012). In addition, clathrin-coated vesicle et al, 2012). In addition, vesicle-sorting defects (Larimore trafficking interacting proteins have been found disrupted et al, 2011) and decreased expression of proteins involved in in schizophrenia. This is relevant as the clathrin inter- forward trafficking of glutamate receptors have also been actome regulates trafficking and targeting of ubiquitinated reported (Hammond et al, 2011). proteins (Schubert et al, 2012). Owing to the overlapping nature of the UPS function with abnormalities found in schizophrenia, the proteasome system emerged as a *Correspondence: Dr MD Rubio, Department of Psychiatry and putative candidate underlying protein changes in this Behavioral Neurobiology, University of Alabama at Birmingham, 1719 6th Avenue South, CIRC 590C, Birmingham, AL 35294-0021, illness. As such, abnormalities of the UPS have been USA, Tel: +1 205 996 6229, Fax: +1 205 975 4879, E-mail: repeatedly reported in mRNA expression studies performed [email protected] in blood cells (Bousman et al, 2010a), hippocampus Received 7 March 2013; revised 1 April 2013; accepted 2 April 2013; (Altar et al, 2005), prefrontal cortex, and temporal cortex accepted article preview online 9 April 2013 (Aston et al, 2004; Vawter et al, 2001) of patients suffering Ubiquitin and ubiquitin-like systems in schizophrenia MD Rubio et al 1911 from schizophrenia. In this study, we sought to determine similar 3D structures, with a ubiquitin fold and a C-terminal whether post-translational mechanisms regulating protein glycine that allows them to bind to lysine residues of degradation could be driving the abnormal protein substrates in a UBL fashion. Among them, NEDD8 (neural expression pattern seen in schizophrenia. Because UPS precursor cell-expressed developmentally downregulated-8) and ubiquitin-like (UBL) systems regulate protein expres- regulates ubiquitin E3 ligases and leads to proteasome sion, trafficking, and sorting, we hypothesized that degradation. Ufm1 (ubiquitin-fold modifier-1) is involved abnormalities in these pathways are likely to underlie the in the prevention of ER stress-induced apoptosis (Lemaire numerous protein expression disruptions that have been et al, 2011); and SUMO (small ubiquitin-like modifier) is described in schizophrenia. implicated in modulation of the activity of voltage-gated Protein ubiquitination is a three step process (Figure 1). K þ channels (Plant et al, 2011), repair of DNA single-strand Initially, ubiquitin is activated by E1 activases in an ATP- breaks in neurons (Hudson et al, 2012; Mao et al, 2000), dependent process, resulting in a thiol ester intermediate, axonal mRNA trafficking (Kadare et al, 2003), and E1-S-ubiquitin. This high-energy compound is then trans- neurodevelopment. NEDD8ylation dysfunction has been ferred to a target substrate by an E2 conjugase, also forming described in several degenerative disorders including a high-energy intermediate, E2-S-ubiquitin. The substrate is Parkinson’s disease (Choo et al, 2012) and Alzheimer’s bound to specific E3 ligases that catalyze the final process disease (Kee et al, 2012). Similarly, abnormalities of by covalently binding ubiquitin to an internal lysine SUMOylation have been implicated in neurodegenerative residue. During this step, the third and last high-energy disorders including Parkinson’s disease, Alzheimer’s dis- thiol ester intermediate, ubiquitin-S-E3, is formed ease, amyotrophic lateral sclerosis, spinocerebellar ataxia, (Varshavsky, 2012). By adding activated ubiquitin residues and Huntington’s disease (Krumova and Weishaupt, 2012). to the internal lysine, a polyubiquitin chain is synthesized. To assess the involvement of the ubiquitin and UBL The lysine residue at which polyubiquitination takes place systems in schizophrenia, we performed a detailed study determines the fate of the target protein. When polyubi- including measurement of total lysine 48 and 63 protein quitination occurs at lysine 48 (K48), the protein is likely to ubiquitination, polyubiquitination, and a series of mole- be recognized by the 26S proteasome and degraded; cules involved in activation, conjugation, and ligation polyubiquitin linkage at lysine 63 (K63) is known to have of ubiquitin in the left superior temporal gyrus (STG) in non-proteolytic functions associated with protein–protein schizophrenia. The left STG participates in the development interactions, cellular signaling, and endocytosis (Varshavsky, of auditory hallucinations (Barta et al, 1990; Nenadic et al, 2012). To date, there are at least 35 E2s and over 600 E3s that 2010; Silbersweig et al, 1995) and the volume of this have been described in mammals, resulting in a system with cortical area is decreased in subjects with schizophrenia a high degree of substrate specificity. (Sun et al, 2009). In addition, the STG has been associated In addition to ubiquitin, several other proteins termed with thought process abnormalites seen in this illness UBL modifiers have recently been described. The UBLs (Rajarethinam et al, 2000). Several reports have found share little sequence homology with ubiquitin but have abnormal expression of ubiquitination-related transcripts (Aston et al, 2004; Vawter et al, 2001), and the density of GABAA receptors is increased (Deng and Huang, 2006) in the STG in schizophrenia. We found decreased protein ubiquitination, accompanied by decreased ubiquitin, and UBL activases and ligases, suggesting the possibility that disruption of these systems may underlie protein expression abnormalities found in postmortem studies in schizophrenia. METHODS AND MATERIALS Subjects, Tissue Acquisition and Preparation Samples from the STG were obtained for