Blood and brain protein levels of ubiquitin-conjugating enzyme E2K (UBE2K) are elevated in individuals with schizophrenia

https://doi.org/10.1016/j.jpsychires.2019.03.005Get rights and content

Abstract

A number of recent studies have suggested the ubiquitin proteasome system (UPS) in schizophrenia is dysfunctional. The purpose of this study was to investigate UBE2K, a ubiquitin-conjugating (E2) enzyme within the UPS that has been associated with psychosis symptom severity, in the blood and brain of individuals with schizophrenia. Whole blood and erythrocytes from 128 (71 treatment-resistant schizophrenia, 57 healthy controls) individuals as well as frozen dorsolateral prefrontal cortex (DLPFC) and orbitofrontal cortex (OFC) post-mortem samples from 74 (37 schizophrenia, 37 controls) individuals were obtained. UBE2K gene expression was assayed in whole blood and DLPFC samples, whereas protein levels were assayed in erythrocytes and OFC samples. Elevated levels of UBE2K mRNA were observed in whole blood of individuals with schizophrenia (p = 0.03) but not in the DLPFC, while protein levels were raised in erythrocytes and the OFC (p < 0.001 and p = 0.002 respectively). Findings were not better explained by age, smoking, clozapine plasma levels or duration of illness. Although blood and brain samples were derived from independent samples, our findings suggest peripheral protein levels of UBE2K may serve as a surrogate of brain levels and further supports the notion of UPS dysfunction in schizophrenia. Future studies to determine the pathophysiological effects of elevated UBE2K protein levels in the brain of those with schizophrenia are warranted.

Introduction

The ubiquitin proteasome system (UPS) is present in all cells, where it plays a key role in the proper function of several biological processes (e.g. neurotransmitter synthesis and receptor recycling, cytokine production and activation) commonly associated with schizophrenia (Lecker et al., 2006; Lehman, 2009; Petroski, 2008). As such, perturbation of this essential system (e.g., changes in ubiquitination) may have numerous downstream biological consequences that could increase the risk for schizophrenia. Genomic, transcriptomic, and proteomic studies have suggested a dysregulation of the UPS among individuals with schizophrenia in blood and post-mortem human brain tissue (Altar et al., 2005; Arion et al., 2015; Bousman et al., 2010a, 2010b; Horton et al., 1993; Liu et al., 2017; Middleton et al., 2002; Nishimura et al., 2000; Rubio et al., 2013; Scott et al., 2016; Vawter et al., 2001, 2002). Interestingly, recent findings by our group showed an increase in the levels of ubiquitinated proteins in the blood and brain of individuals with chronic schizophrenia compared to those with recent-onset of the illness and healthy controls (Bousman et al., 2019). However, the mechanism by which this accumulation of ubiquitinated proteins occurs remains to be elucidated.

One potential mechanism for elevated ubiquitinated proteins in schizophrenia is an increase in ubiquitination activity. Ubiquitination of a target protein involves the activation, transfer, and attachment of ubiquitin to target proteins via ubiquitin-activating (E1), -conjugating (E2), and -ligase (E3) enzymes, respectively. Previous post-mortem brain and peripheral blood studies have implicated dysregulation of several of these ubiquitin enzymes in schizophrenia (Middleton et al., 2002; Rubio et al., 2013; Vawter et al., 2002) and bipolar disorder (Ryan et al., 2006). Among these ubiquitination enzymes, the ubiquitin-conjugating enzyme known as UBE2K (HIP2) is of particular interest. Expression of UBE2K's transcript has been shown to be dysregulated in the brain of individuals with a psychiatric illness (Ryan et al., 2006) and in the blood transcript expression was associated with positive symptom severity in schizophrenia (Bousman et al., 2010b). Furthermore, UBE2K is one of the few E2 enzymes capable of conjugating activated ubiquitin at lysine-48 residues, the canonical pathway for proteasomal degradation, and there is evidence that UBE2K is able to synthesise polyubiquitin chains in the absence of E3 ubiquitin ligases (Chen and Pickart, 1990; Haldeman et al., 1997; van Nocker and Vierstra, 1993; Wilson et al., 2009; Yao and Cohen, 2000) as well as inhibit proteasome function (Song et al., 2003). Collectively this evidence suggests that an increase in UBE2K expression could result in elevated levels of ubiquitinated proteins such as we recently observed among individuals with schizophrenia (Bousman et al., 2019). In fact, UBE2K's role in ubiquitinated protein accumulation and aggregation has already been implicated in several other brain disorders including Alzheimer's disease, Parkinson's disease, and Huntington's disease (de Pril et al., 2007; Hegde and Upadhya, 2007; Jia et al., 2012) but whether this is also true in schizophrenia is unknown. Thus, utilizing blood and brain samples from those with and without schizophrenia, we tested the hypothesis that UBE2K gene and protein expression would be increased in the blood and brain of those with schizophrenia relative to healthy controls. The rationale for investigating erythrocytes is based on the fact that the UPS is the sole system for protein degradation in erythrocytes due to the loss of all other organelles including autophagosomes during their maturation (Yousefi and Simon, 2009). In addition, we tested a secondary hypothesis that UBE2K's peripheral gene and/or protein expression would be positively correlated with symptom severity profiles in individuals with schizophrenia to determine if the correlation we previously observed between UBE2K gene expression and positive symptom severity (Bousman et al., 2010b) could be replicated.

Section snippets

Participants

Seventy-one individuals with schizophrenia were recruited from multiple clinical services and the community in Melbourne, Australia, with an additional 57 unrelated healthy controls matched for age, sex, and socioeconomic status recruited from the general community. All individuals with schizophrenia also met recent criteria for “treatment resistance” (Howes et al., 2016), having exhibited poor functioning and residual symptoms despite adequate trials of at least two antipsychotics before

UBE2K mRNA expression

UBE2K mRNA levels were found to be significantly elevated in peripheral blood of schizophrenia cases compared with healthy controls (padj = 0.020; pB-H = 0.03) (Fig. 1). No significant change in UBE2K mRNA levels was observed in the DLPFC in those with schizophrenia, although a trend-level decrease was demonstrated (padj = 0.05). Importantly, gene expression in schizophrenia cases for both cohorts was not correlated with clozapine plasma levels, chlorpromazine equivalent antipsychotic exposure,

Discussion

We found an elevation of UBE2K mRNA levels in whole blood of individuals with schizophrenia but not in the DLPFC. Whereas, UBE2K protein levels were raised in both erythrocytes and the OFC in schizophrenia. Our results, in part, support our primary hypothesis that UBE2K mRNA and protein levels would be increased in the blood and brain of those with schizophrenia and suggests that UBE2K-mediated ubiquitination may be one of several mechanisms contributing to the accumulation of ubiquitinated

Contributors

Authors CP, IE, AB, and CAB designed the study and wrote protocol. Authors HM and MSM managed the literature searches, conducted the experiments and analyses and wrote the first draft of the manuscript. Authors SL, DK, and SM helped in the experiments and analysis of data. All authors contributed to and have approved the final manuscript.

Conflicts of interest

Dr. Bush is a shareholder in Prana Biotechnology Ltd, Mesoblast Ltd, Grunbiotics Pty/Ltd, Cogstate Ltd, and a payed consultant for and receives profit share remuneration from Collaborative Medicinal Development Pty/Ltd. All other authors reported no competing interests. Dr. Cynthia Shannon Weickert is on an advisory board for Lundbeck Australia Pty Ltd. and in collaboration with Astellas Pharma Inc., Japan.

Acknowledgements

The authors acknowledge the financial support of the CRC for Mental Health. The Cooperative Research Centre (CRC) programme is an Australian Government Initiative. The authors also wish to acknowledge the CRC Scientific Advisory Committee, in addition to the contributions of study participants, clinicians at recruitment services, staff at the Murdoch Children's Research Institute, staff at the Australian Imaging, Biomarkers and Lifestyle Flagship Study of Aging, and research staff at the

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