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The ubiquitin proteasome system and schizophrenia
Review
The ubiquitin proteasome system and schizophrenia Sandra Luza*, Carlos M Opazo*, Chad A Bousman*, Christos Pantelis†, Ashley I Bush†, Ian P Everall†
The ubiquitin-proteasome system is a master regulator of neural development and the maintenance of brain structure and function. It influences neurogenesis, synaptogenesis, and neurotransmission by determining the localisation, interaction, and turnover of scaffolding, presynaptic, and postsynaptic proteins. Moreover, ubiquitin-proteasome system signalling transduces epigenetic changes in neurons independently of protein degradation and, as such, dysfunction of components and substrates of this system has been linked to a broad range of brain conditions. Although links between ubiquitin-proteasome system dysfunction and neurodegenerative disorders have been known for some time, only recently have similar links emerged for neurodevelopmental disorders, such as schizophrenia. Here, we review the components of the ubiquitin-proteasome system that are reported to be dysregulated in schizophrenia, and discuss specific molecular changes to these components that might, in part, explain the complex causes of this mental disorder.
Introduction Schizophrenia is characterised by a complex array of positive, negative, and cognitive symptoms that are associated with changes at the molecular level.1 Schizo phrenia has been linked to abnormalities in dopaminergic, glutamatergic, GABAergic (ie, γ-aminobutyric-acid), and serotoninergic neurotransmission pathways,1,2 as well as signalling pathways (eg, the Wnt/β-catenin pathway) that are crucial for brain growth and maturation.3 Common consequences of these extra cellular and intracellular pathway abnormalities in schizophrenia are changes in the levels of specific proteins, suggesting impaired proteostasis. Increased protein insolubility has been reported in autopsy brain samples from individuals with schizophrenia, which is a signature of proteostasis dysfunction.4 Proteostasis requires the control and harmonisation of protein synthesis, protein folding and conformational main tenance, protein—protein interac tion, protein trafficking, and protein degradation. The ubiquitin-proteasome system (UPS) is central to proteostasis5 and, as presented in this Review, multiple proteins that have been shown to be altered in schizo phrenia are regulated by this crucial system. As such, it could be argued that understanding the dysregulation of the UPS in schizophrenia would report novel molecular aspects for the basis of this mental disorder. Before addressing this argument, we will briefly describe the components and main features of the UPS. We will then summarise and discuss the evidence that implicates the involvement of specific components of the UPS in schizophrenia, before concluding with potential clinical implications of this evidence. Of note, other pathways involved in proteo stasis (eg, autophagy and unfolded protein response) could also be linked to the dysregulation of proteins in schizophrenia, but are beyond the scope of this Review and have been reviewed elsewhere.6,7 A flowchart summarising the article selection process is provided in figure 1.
The UPS The UPS is the principal pathway for the degradation of cytosolic, nuclear proteins, and transmembrane proteins8 (figure 2). This process involves the sequential conjugation
of one, or several, ubiquitin moieties to proteins (a process known as ubiquitination), which is catalysed by an enzymatic cascade composed of ubiquitin activases (E1), ubiquitin conjugases (E2), and ubiquitin ligases (E3).9 Multiple cycles of ubiquitination lead to the formation of a polyubiquitin chain on the protein substrate, which can function as a signal for degradation by three proteases (ie, caspase, trypsin, and chymotrypsin) that are part of the proteasome. Once the proteins are recognised by the 26S proteasome, the ubiquitin moiety is removed from the protein substrate by the action of deubiquitinating enzymes (DUBs) associated with the 26S proteasome, and thus ubiquitin is recycled.9,10 Then, the protein substrate binds the proteasome complex through the 19S regulatory particle, which governs their entry into the 20S catalytic chamber. The proteasome unfolds substrates and threads the polypeptide chains through the inner channel, where they are cleaved into short peptides by the catalytically active subunits caspase-like, trypsin-like, and chymotrypsinlike.9 Key challenges • Discerning whether the increased levels of ubiquitinated proteins observed in autopsy and peripheral tissue from indivduals with schizophrenia are primarily monoubiquitinated or polyubiquitinated • Assessing the effect that antipsychotic medications have on ubiquitin-proteasome system (UPS) function using in vivo human study designs • Surveying the distribution of UPS abnormalities across the brain and in various cell types to determine the specificity of UPS dysfunction • Evaluating the utility of peripheral UPS function as a predictor of clinical trajectories in individuals with schizophrenia • Determining the identity of proteins that are highly ubiquitinated in schizophrenia • Evaluating the therapeutic effect of E3-ligase inhibitors in animal models of schizophrenia • Investigating whether animal models of schizophrenia display changes in UPS components
www.thelancet.com/psychiatry Published online February 12, 2020 https://doi.org/10.1016/S2215-0366(19)30520-6
Lancet Psychiatry 2020 Published Online February 12, 2020 https://doi.org/10.1016/ S2215-0366(19)30520-6 *Contributed equally †These authors share senior authorship Melbourne Neuropsychiatry Centre, Department of Psychiatry (S Luza PhD, C M Opazo PhD, C A Bousman PhD, Prof C Pantelis MD, Prof I P Everall DSc), Melbourne Dementia Research Centre, Florey Institute of Neuroscience and Mental Health (S Luza, C M Opazo, Prof C Pantelis, Prof A I Bush PhD, Prof I P Everall), and Centre for Neural Engineering, Department of Electrical and Electronic Engineering (Prof C Pantelis, Prof I P Everall), The University of Melbourne & Melbourne Health, Parkville, VIC, Australia; The Cooperative Research Centre for Mental Health, Carlton South, VIC, Australia (C A Bousman, Prof C Pantelis, Prof I P Everall); Hotchkiss Brain Institute, Cumming School of Medicine (C A Bousman), Departments of Medical Genetics, Psychiatry, and Physiology & Pharmacology (C A Bousman), University of Calgary, Calgary, AB, Canada (C A Bousman); Alberta Children’s Hospital Research Institute, Calgary, AB, Canada; NorthWestern Mental Health, Melbourne, VIC, Australia (Prof C Pantelis); and Institute of Psychiatry, Psychology and Neuroscience, King’s College London, London, UK (Prof I P Everall) Correspondence to: Prof Ashley I Bush, Melbourne Dementia Research Centre, Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, VIC 3052, Australia [email protected]
1
Review
120 records identified through PubMed database searching
29 additional records identified through other sources
information together shows that UPS is defective in schizophrenia.
Ubiquitin and polyubiquitinated proteins 24 duplicate records removed
125 records screened 1 record excluded 1 no full text available 124 full-text articles assessed for eligibility 70 full-text articles excluded 3 in non-English language 15 non-human studies 52 no canonical ubiquitin-proteasome system component related to schizophrenia reported 54 studies included in the review
Figure 1: PRISMA flow chart detailing the article selection process
Beyond proteasomal degradation, the ubiquitination cascade enzymes and deubiquitinases can regulate protein function or signalling in many cellular processes by adding single moieties of ubiquitin or removing multiple moieties of ubiquitin to or from proteins, which regulates protein—protein interactions and, thus, protein endo cytosis, protein localisation, and protein expression.8 For example, monoubiquitination is a key trafficking mechanism for various cell surface proteins and is also involved in histone and transcription factor regulation, which are crucial to biological processes that are implicated in brain development (eg, monoubi quitination for the internalisation of G protein-coupled receptors).11,12 Changes in histone monoubiquitination have been linked to neurodevelopmental disorders,13 but its association to schizophrenia is unexplored. Thus, understanding the UPS in the context of schizophrenia has the potential to assist in reconciling or unifying the molecular bases that might explain, in part, the heterogeneity of pathways associated with this mental disorder. This notion is based on previous work from our group and other groups that have found pathway-level dysregulation of the UPS in schizophrenia using genomic,14,15 proteomic,16 and transcriptomic17 approaches. Yet, pathway-level findings alone have limited utility as they do not point to specific components of the pathway that are amenable to intervention. As such, we reviewed the literature to identify specific components of the UPS canonical pathway that are dysregulated at the genetic (table 1), transcriptomic (table 2), and proteomic (table 3) levels, as well as protein substrates that could serve as novel therapeutic targets in schizophrenia. Although some studies show conflicting data that require further clarification, overall all the 2
Ubiquitin is a 76 amino-acid peptide that has seven lysine residues and a N-terminus that serve as points of ubiquitination, that is, binding of additional ubiquitin moieties once a ubiquitin unit is conjugated to a protein substrate. As ubiquitin conjugation might occur at any of its seven lysine residues, a ubiquitin chain can grow into many different topologies. The best characterised chains are Lys48 (K48) and Lys63 (K63). Lys48 chains are involved in ubiquitin-dependent proteolysis by the 26S proteasome, and Lys63 chains are generally involved in other cellular processes (eg, DNA repair and signal transduction). The role of other ubiquitin chains (Lys6, Lys11, Lys27, Lys29, and Lys33), including mixed and branched chains, are less well understood.5 The measurement of ubiquitin immunoreactivity is widely used to evaluate changes of the UPS in tissues. Nishimura and colleagues18 assessed the ubiquitin immunoreactivity in autopsy hippocampal tissue from individuals with schizophrenia and were the first to show a higher immunoreactivity against ubiquitin relative to control tissue.18 Since this seminal observation, three studies have looked for ubiquitin or polyubiquitinated proteins in autopsy brain samples from individuals with schizophrenia.19–21 An mRNA analysis of laser-captured hippocampal neurons in multiple schizophrenia cohorts reported a decrease in the expression of ubiquitin,19 which might correspond to an independent phenomenon not related to changes in ubiquitin immunoreactivity or part of a homoeostatic response to the increased levels of ubiquitin immunoreactivity observed by Nishimura and colleagues.18 Protein analysis in autopsy superior temporal gyrus samples from elderly individuals with schizophrenia showed an overall decrease in free ubiquitin and Lys48-linked ubiquitin species, but an increase in Lys63-linked ubiquitin species.20 In agreement with this report, we showed in an independent cohort of individuals with late-onset schizophrenia that polyubiquitinated protein levels were increased in the orbitofrontal cortex and erythrocytes compared with healthy indvividuals.21 These latter two studies both suggest that polyubiquitinated protein levels might be affected in schizophrenia, but it remains to be determined which proteins are affected by these abnormalities. Collectively, studies of ubiquitin and polyubiquitinated proteins indicate that schizophrenia might result from a proteostatic imbalance in brain cells (as indicated by ubiquitin dysregulation and the accumulation of ubiquitinated proteins)—but evidence that this is the one underlying cause of the disorder is not available yet. A 2019 study found increased brain protein insolubility in a subset of individuals who have schizophrenia,4 but whether these abnormalities emerge before, early on, or as the illness progresses is not clear. In studies of erythrocytes from individuals with recent-onset
www.thelancet.com/psychiatry Published online February 12, 2020 https://doi.org/10.1016/S2215-0366(19)30520-6
Review
AMP ATP
Ub
Ub
Ub
Ub activation
Ub
E1
Ub
Ub Ub Ub
Free ubiquitin
Ub
Deubiquitination
E1 Ub Ub
Ub Ub
Ub Ub
Peptides 19S Proteasome
Ub
E2
Ub conjugation
E2
Degradation
E3
Ub
20S
26S
19S
E3
Ub Monoubiquitination
Target protein
Polyubiquitination Ub
Target protein Ub
Lys-63
Ub Ub
Ub Ub
Dubs
Lys-48 Ub
Ub
Ub Ub
Ub Ub
Modification of protein function, localisation, or binding
Ub Target protein
Ub
UB
P
Target protein Ub Ub
Ub Ub
Mediation of cell signalling Activation of endosomal pathway Activation of autophagy/lyosomal pathway
Figure 2: The ubiquitin proteasome system (UPS) The UPS in humans consists of two activating enzymes (E1s), approximately 40 conjugating enzymes (E2s) and hundreds of ligase enzymes (E3s) that result in the activation and conjugation of the 76 amino-acid ubiquitin onto the lysine residues of targeted proteins.78 Single or multiple ubiquitin molecules are added to target proteins, resulting in monoubiquitination or polyubiquitination, respectively. Monoubiquitination can result in modification of the target protein’s function, localisation, or binding, whereas polyubiquitination might target the protein for degradation by the 26S proteasome or result in activation of the autophagosome—lysosomal or endosomal pathways or mediation of cell signalling.78,79 Black arrows represent the intracellular destiny of ubiquitinated proteins. DUBs=deubiquitinating enzymes. Lys=lysine. Ub=ubiquitin.UBP=ubiquitin binding protein.
schizophrenia, we observed no changes in levels of ubiqui tinated proteins, suggesting that increased amounts of ubiquitinated proteins are found later in the disease course.21 Thus, the accumulation of polyubi quitinated proteins could represent an effect rather than a cause of the disease. Further studies that use brain tissue are needed to determine whether the accumulation of polyubiquitinated proteins precede onset of schizophrenia.
E1 activating enzymes The activation of ubiquitin by E1 enzymes is an essential step that triggers subsequent downstream processes in the UPS pathway. The human genome encodes two canonical E1 enzymes: ubiquitin activating enzyme 1 (UBA1) and ubiquitin activating enzyme 6 (UBA6). Ubiquitin is activated in an ATP-dependent process by E1 through the formation of a thioester bond between its C-terminal Gly76 residue and an active-site Cys of the E1. From samples derived from autopsies, superior temporal gyrus tissue shows a decrease in the protein levels of UBA6, with
no changes in UBA1 levels shown in schizophrenia compared with matched healthy controls.20 This result is in line with known links between UBA6 and neuronal development, spine architecture, and mouse behaviour,22 as well as the notion that a ubiquitination defect might precede changes found downstream of multiple molecular pathways in schizophrenia.20 The possible link between loss of UBA6 and loss of grey matter in the superior temporal gyrus in schizophrenia might be associated with the progressive grey matter reduction of the superior temporal gyrus that antecedes the first expression of florid psychosis in schizophrenia. Further studies are required to investigate a link between changes in UBA6 and loss of grey matter in superior temporal gyrus and psychosis. 23
E2 conjugating enzymes After its activation by E1, ubiquitin is transferred to a ubiquitin-conjugating enzyme (E2) via a transthiolation reaction. As of 2020, approximately 40 E2s had been described in humans. All E2s interact with an E1 enzyme
www.thelancet.com/psychiatry Published online February 12, 2020 https://doi.org/10.1016/S2215-0366(19)30520-6
3
Review
Sample size N
Risk of SZ
SZ
Reference
Controls
UBE2D1 rs11006122
536
268
268
No
Andrews and Fernandez-Enright (2014)25
rs1905455
536
268
268
No
Andrews and Fernandez-Enright (2014)25
1439
814
625
Yes (C-allele)
Chen et al (2008)46
rs1859427
536
268
268
No
Andrews and Fernandez-Enright (2014)25
rs6861170
1439
814
625
Yes (T-allele)
Chen et al (2008)46
rs6861170
536
268
268
No
Andrews and Fernandez-Enright (2014)25
FBXL21 rs1859427
dbSNP=Single Nucleotide Polymorphism Database identification number. rs=reference SNP cluster. SZ=individuals with schizophrenia.
Table 1: Ubiquitin-proteasome system genetic studies in schizophrenia by dbSNP ID (rs)
and one or more E3s. E2 enzymes determine the specificity of polyubiquitin chain-linkage types (eg, Lys48 and Lys63) and might act alone or in conjunction with other E2s to catalyse polyubiquitin chain formation.24 As of 2020, four canonical E2 conjugating enzymes (UBE2D1, UBE2K, UBE2M, and UBE2N) have been implicated in schizophrenia. In autopsy hippocampal tissue, Altar and colleagues19 found a decrease in the amount of the UBE2D1 transcript in schizophrenia. However, analyses of UBE2D1 genetic variants25 and UBE2D1 protein levels in the dorsolateral prefrontal cortex26 did not report changes. In addition, a decrease of UBE2N27 gene expression in the dorsolateral prefrontal cortex and a decrease of UBE2M16 protein expression in the hippocampus of individuals who have schizophrenia compared with matched healthy controls has been reported. Finally, UBE2K mRNA levels in lymphocytes have been shown to correlate (r=0·74; p<0·0005) with positive symptoms in schizophrenia.28 However, no changes in the level of UBE2K protein were reported in the superior temporal gyrus in a cohort of older (aged between 59 and 97 years) individuals who have schizophrenia,20 which could suggest that UBE2K expression is tissue specific or age dependent. Considering these results, a future study that explores these four canonical E2 enzymes at the gene, mRNA, and protein level in the same brain regions is required to elucidate the status of these enzymes in schizophrenia.
E3 ligase enzymes E3 ligases are the most studied UPS component in schizophrenia. The UPS includes more than 600 E3s that interact with E2s in unique configurations to dictate substrate specificity. Depending on the types of ubiquitination domains, they are subclassified as homo logous to the E6AP carboxyl terminus (HECT), really interesting new gene (RING), U-box E3, and PHD finger ligases. As of 2020, two HECT (UBE3B and UBE3A), three RING (SIAH2, Parkin, and MDM2), and three U-box E3 enzymes (FBXW7, FBXL21, and FBXO45) have been implicated in schizophrenia. 4
UBE3B expression is downregulated in pyramidal neurons of the prefrontal cortex in adolescents who have schizophrenia compared with controls.30 Moreover, the number of cells that express UBE3B, as well as the level of UBE3B protein, increases in normal adolescent brains, but is significantly decreased in adolescents with schizo phrenia.30,31 These findings suggest that UBE3B could contribute to the pathophysiology of schizophrenia by altering synaptic refinement on prefrontal cortex circuitry during adolescence.30 UBE3A, the paralogue of UBE3B, is also linked to schizophrenia, but in the opposite way. The UBE3A gene maps to chromosome 15q11-q13, a region prone to duplication that has been linked to increased transcription32 and risk for neuropsychiatric phenotypes, including schizophrenia.33,34 In addition, mutations and deletions in UBE3A play a role in other neurodevelopmental disorders (eg, Angelman syndrome).35 Therefore, investi gations of the UPS in these disorders might be informative to schizophrenia, and vice versa. UBE3A is an abundant protein in cytosolic and nuclear compartments and has diverse functions in cellular and transcriptional events in neurodevelopment.36 More studies are required to elucidate the role of UBE3A in illnesses of early and late neurodevelopment, like schizophrenia.37 Parkin, a RING E3 ligase, whose function is associated with energy metabolism and oxidative stress,38 is increased in autopsy prefrontal cortex samples from individuals with schizophrenia, which could be related to stress.39 By contrast, protein levels of the RING E3 enzyme, MDM2, are significantly reduced in the dorsolateral prefrontal cortex in people with schizophrenia compared with controls.26 Importantly for disorders like schizophrenia, MDM2 directly regulates synaptic activity by ubiquitinating PSD-95, which controls the localisation of AMPA receptors.40 PSD-95 and AMPA receptors have also been shown to be decreased in individuals with schizophrenia.41 More controversial is the role of the RING E3 family member SIAH2 in schizophrenia, which has been shown to be upregulated by hypoxia and oestrogen signalling42 and linked to the post-translational regulatory network that controls neuronal polarity during cerebellar granule neuron differentiation.43 Although peripheral mRNA levels of SIAH2 were positively correlated (r=0·73, p=0·0005) with positive symptoms in individuals with schizophrenia,28 the level of SIAH2 protein in autopsy superior temporal gyrus in schizophrenia was not changed.20 U-box E3 ligases can be a single polypeptide or a subunit of a protein complex, such as the Skip1-Cullin1-F-box (SCF) E3 complex. F-box proteins are the key recognition subunits of SCF, in the multimeric E3 ubiquitin ligase complexes. Human F-box proteins are classified into three categories according to specific substrate recognition domains: FBXW (comprising ten members), FBXL (comprising 22 members), and FBXO (comprising 37 members).44 mRNA expression of FBXW7, which regulates the stability of disrupted in schizophrenia 1 (DISC1), has been shown to be significantly lower in
www.thelancet.com/psychiatry Published online February 12, 2020 https://doi.org/10.1016/S2215-0366(19)30520-6
Review
Platform
Tissue
Sample size
Gene expression References relative to Controls
Healthy Individuals controls with schizophrenia
Category
Type
N
Brain
HIP
46
22 (48%)
24 (52%)
Decreased
Altar et al (2005)19
Ubiquitin encoding UBB
qTR-PCR
Ubiquitin activases (E1) No studies Ubiquitin conjugases (E2) UBE2N
Microarray
Brain
DLPFC
30
15 (50%)
15 (50%)
Decreased
Vawter et al (2002)27
UBE2D1
qTR-PCR
Brain
HIP
46
22 (48%)
24 (52%)
Decreased
Altar et al (2005)19
UBE2K
qTR-PCR
Brain
DLPFC
74
37 (50%)
37 (50%)
No change
Meiklejohn et al (2019)29
UBE2K
qTR-PCR
Blood
Whole blood
128
71 (56%)
57 (45%)
Increased
Meiklejohn et al (2019)29
UBE3A
qTR-PCR
Cells
Fibroblasts
6
3 (50%)
3 (50%)
Increased
Noor et al (2015)32
UBE3B
Microarray
Brain
DLPFC
30
15 (50%)
15 (50%)
Decreased
Kohlbrenner et al (2018)30
UBE3B
qTR-PCR
Brain
DLPFC
30
15 (50%)
15 (50%)
Decreased
Kohlbrenner et al (2018)30
FBXW7
Microarray
Brain
DLPFC
60
36 (60%)
24 (40%)
Decreased
Arion et al (2015)45
FBXW7
qTR-PCR
Brain
DLPFC
60
36 (60%)
24 (40%)
Decreased
Arion et al (2015)45
UCHL1
Microarray
Brain
PFC
20
10 (50%)
10 (50%)
Decreased
Middleton et al (2002)60
UCHL1
Microarray
Brain
PFC
6
3 (50%)
3 (50%)
Decreased
Vawter et al (2001)57
UCHL1
qTR-PCR
Brain
HIP
46
22 (48%)
24 (52%)
Decreased
Altar et al (2005)19
UCHL5
Microarray
Brain
DLPFC
60
36 (60%)
24 (40%)
Decreased
Arion et al (2015)45
UCHL5
qTR-PCR
Brain
DLPFC
60
36 (60%)
24 (40%)
Decreased
Arion et al (2015)45
USP14
Microarray
Brain
PFC
6
3 (50%)
3 (50%)
Decreased
Vawter et al (2001)57
USP9
Microarray
Brain
PFC
20
10 (50%)
10 (50%)
Decreased
Middleton et al (2002)60
qTR-PCR
Brain
HIP
46
22 (48%)
24 (52%)
Decreased
Altar et al (2005)19
Sub- unit RPn8
qTR-PCR
Brain
HIP
46
22 (48%)
24 (52%)
Decreased
Altar et al (2005)19
Sub-unit RPn9
qTR-PCR
Brain
HIP
46
22 (48%)
24 (52%)
Decreased
Altar et al (2005)19
PSMA1
Microarray
Brain
PFC
6
3 (50%)
3 (50%)
Decreased
Vawter et al (2001)57
PSMA1
qTR-PCR
Brain
HIP
46
22 (48%)
24 (52%)
Decreased
Altar et al (2005)19
PSMB4
Microarray
Brain
DLPFC
60
36 (60%)
24 (40%)
Decreased
Arion et al (2015)45
PSMB4
qTR-PCR
Brain
DLPFC
60
36 (60%)
24 (40%)
Decreased
Arion et al (2015)45
PSMB6
qTR-PCR
Brain
HIP
46
22 (48%)
24 (52%)
Decreased
Altar et al (2005)19
Ubiquitin ligases (E3)
Deubiquitination
Proteasome Sub-unit RPt6
DLPFC=Dorsolateral Prefrontal Cortex. HIP=hippocampus. PFC=prefrontal cortex
Table 2: Ubiquitin-proteasome system transcriptomic studies in people with schizophrenia
pyramidal neurons in the dorsolateral prefrontal cortex of individuals with schizophrenia compared with healthy controls.45 Within the FBXL category, genetic variation in and around FBXL21 was associated with schizophrenia in a case-control study of Irish families,46 but the association was not found in DNA samples from a replication cohort of white individuals obtained from the Australian Schizophrenia Research Bank.25 FBXL21 has been implicated in the regulation and stabilisation of the circadian clock proteins,47 such as CRY1 and PER2, which have been shown to have a loss of rhythmic expression in cultured fibroblasts from individuals with chronic schizophrenia compared with control individuals.48 Levels of FBXL21 protein were significantly reduced in autopsy
dorsolateral prefrontal cortex tissue from individuals with schizo phrenia compared with matched controls.26 Of the FBXO family, FBXO45 is required for normal synaptogenesis, axon navigation, and neuronal migration in the development of central and peripheral neurons.49 FBXO45 is included in the 3q29 microdeletion region that had been implicated as conferring a significant risk for schizophrenia.50 A study from Wang and colleagues51 reported a rare FBXO45 R108C mutation in an individual with schizophrenia that resulted in decreased mRNA expression. Bioinformatics analysis had predicted that the R108C mutation could have a damaging effect on the function of the FBXO45 protein, but empirical studies to confirm this are required.
www.thelancet.com/psychiatry Published online February 12, 2020 https://doi.org/10.1016/S2215-0366(19)30520-6
5
Review
Platform
Tissue
Sample size
Category
Type
N
SZ
Healthy controls
Analyte expression relative to controls
References
Nishimura et al (2000)18
Ubiquitin Ubiquitin
ICC
Brain
HIP
37
15
22
Increased
Ubiquitin
WB
Brain
STG
26
13
13
Decreased
Rubio et al (2013)20
Ubiquitin
WB
Brain
DLPFC
76
38
38
No change
Bousman et al (2019)21
Ubiquitin
WB
Blood
Erythrocytes
181
88
93
No change
Bousman et al (2019)21
Polyubiquitin K63-Linked
WB
Brain
STG
26
13
13
Increased
Rubio et al (2013)20
K48-Linked
WB
Brain
STG
26
13
13
Decreased
Rubio et al (2013)20
Total Poly-Ub
WB
Brain
DLPFC
Total Poly-Ub
WB
Blood
Erythrocytes
76
38
38
Increased
Bousman et al (2019)21
181
88
93
Increased
Bousman et al (2019)21
Ubiquitin activases (E1) UBA 1
WB
Brain
STG
26
13
13
No change
Rubio et al (2013)20
UBA 6
WB
Brain
STG
26
13
13
Decreased
Rubio et al (2013)20
Ubiquitin conjugases (E2) UBE2D1
WB
Brain
DLPFC
60
30
30
No change
Andrews et al (2017)26
UBE2K
WB
Brain
STG
26
13
13
No change
Rubio et al (2013)20
UBE2K
WB
Brain
DLPFC
74
37
37
Increased
Meiklejohn et al (2019)29
UBE2K
WB
Blood
Erythrocytes
128
71
57
Increased
Meiklejohn et al (2019)29
UBE2M
Mass spec
Brain
HIP
57
30
27
Decreased
Schubert et al (2015)16
UBE3B
IHC
Brain
DLPFC
30
15
15
Decreased
Kohlbrenner et al (2018)30
FBXL21
WB
Brain
DLPFC
60
30
30
Decreased
Andrews et al (2017)26
MDM2
WB
Brain
DLPFC
60
30
30
Decreased
Andrews et al (2017)26
Parkin
WB
Brain
DLPFC
26
13
13
Increased
Pandya et al (2014)39
UCHL1
WB
Brain
DLPFC
60
30
30
No change
Andrews et al (2017)26
UCHL1
Mass spectrometry
Brain
HIP
57
30
27
Decreased
Schubert et al (2015)16
UCHL1
Protein array
Brain
DLPFC
37
17
20
Decreased
Novikova et al (2006)58
UCHL1
ELISA
Blood
Serum
84
44
40
Decreased
Demirel et al (2017)57
Sub-unit Rpt1
WB
Brain
STG
28
14
14
Decreased
Scott et al (2016)65
Sub-unit Rpt3
WB
Brain
STG
28
14
14
Decreased
Scott et al (2016)65
Sub-unit Rpt6
WB
Brain
STG
28
14
14
Decreased
Scott et al (2016)65
Ubiquitin ligase (E3)
Deubiquitination
Proteasome
DLPFC=dorsolateral prefrontal cortex. ELISA=enzyme-linked immunosorbent assay. HIP=hippocampus. ICC=immunocytochemistry. IHC=immunohistochemistry. PFC=prefrontal cortex. STG=superior temporal gyrus. WB=Western blot.
Table 3: Ubiquitin-proteasome system (UPS) proteomic studies in people with schizophrenia.
DUBs The process of ubiquitination can be reversed by DUBs. Approximately 100 DUBs are encoded in the human genome. By reversing ubiquitination, DUBs affect the function, localisation, and stability of proteins. There are five DUB families classified on the basis of the homology of their catalytic domains: Ubiquitin C-terminal hydrolases (UCH, comprising four members), ubiquitin-specific proteases (USP, comprising 56 members), ovarian tumour proteases (OUT, comprising 16 members), Machadojosephin domain proteases (MJD, comprising 4 members) and Jab1/Mov34/Mpr1 Pad1 N-terminal (JAMM, comprising 11 members). The main function of DUBs is 6
maintaining an adequate pool of free ubiquitin for immediate conjugation, but DUBs can also act to edit or remodel ubiquitin chains on substrates and inhibit conjugation by binding to E2 enzymes and interfering with ubiquitin transfer to E3 enzymes.52 Among the DUBs, deubiquitinase ubiquitin carboxylterminal esterase L1 (UCHL1) has most often been associated with schizophrenia. UCHL1, a small protein that is highly specific to the human brain, adds or removes ubiquitin from proteins that are destined for degradation.53 By controlling the ubiquitin pool, UCHL1 plays a crucial role in the removal of excessive proteins, oxidised proteins, and misfolded proteins in the neuronal
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cyto sol during both normal and neuropathological conditions.54 UCHL1 is highly regulated at the mRNA level and protein level by a range of intracellular factors and post-translational modifications that are affected by cellular stress.54–56 In schizophrenia, the gene expression and protein levels of UCHL1 are decreased in the brain (dorsolateral prefrontal cortex [DLPFC], located in the hippocampus) and serum.16,26,54,57–59 Demirel and colleagues showed that the decrease in UCHL1 serum levels was correlated with the Positive and Negative Syndrome Scale (PANSS) total and PANSS general psychopathology scores in individuals with schizophrenia.59 Further studies are necessary to determine whether UCHL1 has a potential prognostic or therapeutic value specifically for schizophrenia. Decreases in the expression of UCHL5, USP9, and USP14 in the DLPFC from individuals with schizophrenia have been reported by different groups.45,57,60 Both UCHL5 (a nuclear protein) and USP14 (a cytosolic protein) are reversibly associated with the 19S regulatory cap of the proteasome. The binding of UCHL5 and USP14 to the 19S cap normally results in increased enzymatic activity for both DUBs, allowing substrate access to the active site and degradation by the proteasome.52 Finally, a negative association was observed between USP2 gene expression and positive symptom severity in individuals with schizophrenia.28 A decrease in the expression or protein levels of any of the DUBs might result in an accumulation of ubiquitinated proteins,20,21 however, most of the 100 DUBs are poorly characterised and require further investigation in schizophrenia.
The 26S proteasome The 26S proteasome is a large multi-subunit ATP-depen dent protease complex (appendix). It comprises a 20S core particle, capped on one or both sides by a 19S regulatory particle. The base of the regulatory particle is composed of six AAA-ATPases (Rpt1–Rpt6) and four non-ATPases (Rpn1, Rpn2, Rpn10, and Rpn13) and the lid has nine subunits (referred to as Rpn3, Rpn5–Rpn9, Rpn11, Rpn12, and Rpn15). The proteasome can recognise its substrates through several ubiquitin receptors, such as Rpn1, Rpn10, and Rpn13. The regulatory particle binds and unfolds the polyubiquitinated protein substrate and feeds the unfolded polypeptide chain into the chamber of the 20S particle. The regulatory particle also deubi quitinates the poly ubiquitinated substrates to recycle ubiquitin before the substrate passes through the proteasome α subunits (proteasome α subunits 1–7 compose the α-ring) within the 20S particle, where it is cleaved into small peptides by the catalytically active caspase-like, trypsin-like, and chymotrypsin-like β subunits.5 Studies in several brain regions have shown a decrease in the cluster of genes that encode various proteasome subunits, including lid subunits Rpn8 and Rpn9 in individuals with schizophrenia compared with healthy controls.19 Decreased expression of the genes for α-ring
E1 enzyme
UBA6
UBE38
UBE2M
UBE2K
FBL21
MDM2
E2 enzymes
Parkin
UCHL1
Rpt1
Rpt3
E3 enzymes
Deubiquitinase
Rpt6
Proteasome ATPase subunits
Figure 3: Ubiquitin proteasome system (UPS) enzymes that are altered in people with schizophrenia The diagram indicates UPS enzymes that are downregulated (dashed blue circles) or upregulated (dashed red circles) in people with schizophrenia. Black circles denote healthy control conditions.
subunit 1 (PSMA1)19,57 and β-ring subunits 4 and 6 (PSMB4 and PSMB6)19,45 has also been reported in this mental disorder. These deficiencies in proteasome gene expression occurred concomitantly with decreases in expression of synaptic plasticity genes,19 which might be associated with the excessive complement activity that has been proposed as the link to the reduced numbers of synapses in the brain of individuals with schizophrenia.61,62 This observed downregulation of proteasome-related genes appears to be unique to schizophrenia, as brain samples from patients with major depression and bipolar mood disorder did not show similar patterns of dysregulation.63 In 2019, it was shown that some of the proteasome activities are altered in subcellular fractions in the superior temporal gyrus from autopsies of individuals with schizophrenia,64 which is line with the decrease in protein expression of 19S Rpt base subunits that are important regulators of proteasome activity.65 However, we found that total proteasome activity was not changed in autopsy orbitofrontal cortex tissue from individuals with schizo phrenia compared with healthy controls;21 a discrepancy that might be explained by differences in the brain regions or methods used to measure proteasome activity between these studies. The most consistently reported changes are for deubiquitinases and proteasome subunits (tables 2, 3), which are nearly all downregulated in individuals with schizophrenia (figure 3). The studies on other components of the UPS are more heterogenous, but show signs of protein deficiency in individuals with schizophrenia that might implicate changes in ubiquitin conjugation to specific substrates. This observation suggests that components of the UPS are deficient in
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See Online for appendix
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Search strategy and selection criteria PubMed was searched without any filter up to May 31, 2019, using the search terms “ubiquitin”, “proteasome”, and “schizophrenia”. Bibliographies of all research articles were hand searched for additional references not identified in the primary searches. Studies were retained if the following inclusion criteria were met: (1) included data derived from humans with schizophrenia; (2) one or more of the components of the canonical UPS was reported; and (3) the article was published in a peer-reviewed, English-language journal and the full text was available. Review articles, commentaries, books, book chapters, editorial pieces, or any published material not deemed original research were excluded. A flowchart summarising the article selection process is provided in Figure 1. In total, 54 full-text articles met inclusion criteria and were reviewed.
schizophrenia, which might explain the loss of proteostasis of presynaptic and postsynaptic proteins that are found to be altered in this disorder and discussed in the section titled schizophrenia-related proteins that are substrates of the UPS below. Therefore, dysfunction of the UPS might be central to explaining the multiple changes observed in schizophrenia at the synaptic level, which might constitute the molecular basis of this disorder, an idea first proposed by Feinberg.66 Importantly, however, genome-wide asso ciation studies have not identified genetic variants in components of the UPS meeting genome-wide signifi cance for schizophrenia, suggesting that the molecular basis of dysregulation of the UPS observed in studies reside at the epigenomic or post-translational levels.
Schizophrenia-related proteins that are substrates of the UPS Using our search strategy and selection criteria, we identified from the published literature several proteins that are known substrates of the UPS that have been associated with schizophrenia and have functions related to neurodevelopment or synaptic regulation. These proteins include sulfo transferase 4A1,67 glutamate 68 carboxypeptidase II, DISC1,69–73 dysbindin,17,74 SH3, and multiple ankyrin repeat domains 3 (SHANK3).75,76 Although this list is undoubtedly an underestimation of schizophrenia-relevant proteins undergoing abberant ubiquitination and proteasome degradation, they serve as a starting point for further research. In fact, as previously noted, the UPS as a whole seems to be altered in schizo phrenia (figure 3), which might be part of a homoeostatic response to reduce protein ubiquitination and proteasome degradation of these important proteins and others yet to be identified that are necessary for normal synaptic function. Further studies are required to systematically identify and characterise schizophrenia-relevant proteins that aberrantly interact with the UPS. 8
Effect of antipsychotics on the UPS Antipsychotic exposure is known to induce peripheral and central changes in gene and protein expression and would presumably also influence the UPS. However, studies suggest that the expression of UPS-related genes in autopsy prefrontal and hippocampal tissue is not correlated with cumulative lifetime antipsychotic exposure.19,60 Moreover, no associations have been reported between chlorpromazine equivalent dose and polyubiquitinated protein levels, ubiquitination activity, or proteasome activity in erythrocytes or orbitofrontal cortex brain samples from individuals with schizophrenia.20,21 Further more, there is no association between UBE2K gene expression or protein levels and chlorpromazine equivalent antipsychotic exposure.29 Although these results suggest UPS gene and protein expression are unlikely to be significantly affected by antipsychotic exposure, no study to date has been specifically designed to test this association and all published studies have relied on imprecise measures of antipsychotic clinical exposure. Studies on rodents have shown that the administration of anti psychotics does not change the levels of components of the UPS.20,64,65,77 Future in vitro or animal studies are needed to support or refute these initial findings.
Conclusions Studies of autopsy brain tissue done three decades ago provided the first direct evidence for the association between schizophrenia and the UPS. Since then, several studies have supported the notion that the UPS is dysfunctional in schizophrenia. However, it is still unclear whether this abnormality is a cause or consequence of this disease. We reported that accumulation of ubiqutinated proteins is detected at the later stages of schizophrenia. Further histological studies are required to determine whether schizophrenia is a ubiquinopathy. Moreover, both peripheral and central dysregulation of the UPS have been identified in samples from individuals with schizophrenia, implicating the UPS as a potential site of clinically relevant biomarkers that might assist in the early identification, prognosis, and treatment of this mental disorder. This Review shows evidence that the UPS is altered at different biological levels across several stages of the illness. As such, we hypothesise that the downregulation of enzymes of the UPS occurs upstream of the disease manifestations and is related to changes in neuronal development, synaptic consolidation, and function. We believe that these biochemical changes in the UPS might not show detectable clinical manifestations. Other changes within the UPS are likely to be a homoeostatic response and, therefore, represent a secondary event that involves the upregulation of UPS enzymes. This response might explain the decrease of specific proteins that have been associated with early onset (eg, DISC1 and SHANK3) and more advanced states (eg, SULT41 and dysbindin) of schizophrenia. Looking forward, a major challenge will be the
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identification, refinement, and classification of these two groups of enzymes, which could uncover biomarkers for schizophrenia and inform development of new therapies for early and later stages of this illness. If our working hypothesis is correct, specific inhibitors for E3-ligases might be novel therapeutic candidates for the treatment of schizophrenia. Considering how the clinical complexity of schizophrenia parallels the myriad of alterations detected in the UPS in people with this disorder, longitudinal studies that use the full spectrum of socalled omic approaches to interrogate the UPS in schizo phrenia, from pre-psychosis onset to chronic illness, are warranted. Contributors SL was responsible for searching the literature for articles included in this Review. SL, CMO, and CAB prepared the figures and tables. SL, CMO, CAB, CP, AIB, and IPE contributed to the design, writing, preparation, and discussion of the manuscript. All authors read and approved the final manuscript. Declaration of interests AIB is a shareholder in Prana Biotechnology, Mesoblast, Grunbiotics Pty, Cogstate, and a paid consultant for and receives profit share remuneration from Collaborative Medicinal Development. These conflicts of interest do not overlap with the topic of this Review. In the past 5 years, CP served on an advisory board for Lundbeck Australia. He has received honoraria for talks presented at educational meetings that were organised by JanssenCilag, AstraZeneca, Shire, and Lundbeck, and has received an unrestricted grant from Servier Laboratories Australia. All other authors declare no competing interests. Acknowledgments The authors acknowledge the financial support of the The Cooperative Research Centre (CRC) for Mental Health. The CRC programme is an Australian Government initiative. CAB was supported by the Alberta Children’s Hospital Research Inisititue and Cumming School of Medicine, University of Calgary (Canada). CP was supported by the National Health and Medical Research Council (NHMRC) senior principal research fellowship, a grant from the Lundbeck Foundation, and a NHMRC programme grant. AIB was supported by a NHMRC senior principal research fellowship. None of the funding sources played any role in the writing of this Review or in the decision to submit the manuscript for publication. References 1 Horváth S, Mirnics K. Schizophrenia as a disorder of molecular pathways. Biol Psychiatry 2015; 77: 22–28. 2 Lang UE, Puls I, Muller DJ, Strutz-Seebohm N, Gallinat J. Molecular mechanisms of schizophrenia. Cell Physiol Biochem 2007; 20: 687–702. 3 Peng Y, Xu Y, Cui D. Wnt signaling pathway in schizophrenia. CNS Neurol Disord Drug Targets 2014; 13: 755–64. 4 Nucifora LG, MacDonald ML, Lee BJ, et al. Increased protein insolubility in brains from a subset of patients with schizophrenia. Am J Psychiatry 2019; 176: 730–43. 5 Yu H, Matouschek A. Recognition of client proteins by the proteasome. Annu Rev Biophys 2017; 46: 149–73. 6 Bar-Yosef T, Damri O, Agam G. Dual role of autophagy in diseases of the central nervous system. Front Cell Neurosci 2019; 13: 196. 7 Kim P, Scott MR, Meador-Woodruff JH. Dysregulation of the unfolded protein response (UPR) in the dorsolateral prefrontal cortex in elderly patients with schizophrenia. Mol Psychiatry 2019; published online Oct 2. DOI.org/10.1038/s41380-019-0537-7. 8 Dantuma NP, Bott LC. The ubiquitin-proteasome system in neurodegenerative diseases: precipitating factor, yet part of the solution. Front Mol Neurosci 2014; 7: 70. 9 Hershko A, Ciechanover A. The ubiquitin system. Annu Rev Biochem 1998; 67: 425–79. 10 Kleiger G, Mayor T. Perilous journey: a tour of the ubiquitinproteasome system. Trends Cell Biol 2014; 24: 352–59.
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33 Stewart LR, Hall AL, Kang SH, Shaw CA, Beaudet AL. High frequency of known copy number abnormalities and maternal duplication 15q11-q13 in patients with combined schizophrenia and epilepsy. BMC Med Genet 2011; 12: 154. 34 Boot E, Kant SG, Otter M, Cohen D, Nabanizadeh A, Baas RW. Overexpression of chromosome 15q11-q13 gene products: a risk factor for schizophrenia and associated psychoses? Am J Psychiatry 2012; 169: 96–97, author reply 97. 35 Malzac P, Webber H, Moncla A, et al. Mutation analysis of UBE3A in Angelman syndrome patients. Am J Hum Genet 1998; 62: 1353–60. 36 LaSalle JM, Reiter LT, Chamberlain SJ. Epigenetic regulation of UBE3A and roles in human neurodevelopmental disorders. Epigenomics 2015; 7: 1213–28. 37 Pantelis C, Yücel M, Wood SJ, et al. Structural brain imaging evidence for multiple pathological processes at different stages of brain development in schizophrenia. Schizophr Bull 2005; 31: 672–96. 38 Seirafi M, Kozlov G, Gehring K. Parkin structure and function. FEBS J 2015; 282: 2076–88. 39 Pandya CD, Crider A, Pillai A. Glucocorticoid regulates parkin expression in mouse frontal cortex: implications in schizophrenia. Curr Neuropharmacol 2014; 12: 100–07. 40 Colledge M, Snyder EM, Crozier RA, et al. Ubiquitination regulates PSD-95 degradation and AMPA receptor surface expression. Neuron 2003; 40: 595–607. 41 Toro C, Deakin JF. NMDA receptor subunit NRI and postsynaptic protein PSD-95 in hippocampus and orbitofrontal cortex in schizophrenia and mood disorder. Schizophr Res 2005; 80: 323–30. 42 Qi J, Nakayama K, Gaitonde S, et al. The ubiquitin ligase Siah2 regulates tumorigenesis and metastasis by HIF-dependent and -independent pathways. Proc Natl Acad Sci USA 2008; 105: 16713–18. 43 Ong T, Solecki DJ. Seven in absentia E3 ubiquitin ligases: central regulators of neural cell fate and neuronal polarity. Front Cell Neurosci 2017; 11: 322. 44 Shen J, Spruck C. F-box proteins in epigenetic regulation of cancer. Oncotarget 2017; 8: 110650–55. 45 Arion D, Corradi JP, Tang S, et al. Distinctive transcriptome alterations of prefrontal pyramidal neurons in schizophrenia and schizoaffective disorder. Mol Psychiatry 2015; 20: 1397–405. 46 Chen X, Wang X, Sun C, et al. FBXL21 association with schizophrenia in Irish family and case-control samples. Am J Med Genet B Neuropsychiatr Genet 2008; 147B: 1231–37. 47 Hirano A, Yumimoto K, Tsunematsu R, et al. FBXL21 regulates oscillation of the circadian clock through ubiquitination and stabilization of cryptochromes. Cell 2013; 152: 1106–18. 48 Johansson AS, Owe-Larsson B, Hetta J, Lundkvist GB. Altered circadian clock gene expression in patients with schizophrenia. Schizophr Res 2016; 174: 17–23. 49 Saiga T, Fukuda T, Matsumoto M, et al. Fbxo45 forms a novel ubiquitin ligase complex and is required for neuronal development. Mol Cell Biol 2009; 29: 3529–43. 50 International Schizophrenia Consortium. Rare chromosomal deletions and duplications increase risk of schizophrenia. Nature 2008; 455: 237–41. 51 Wang C, Koide T, Kimura H, et al. Novel rare variants in F-box protein 45 (FBXO45) in schizophrenia. Schizophr Res 2014; 157: 149–56. 52 Coyne ES, Wing SS. The business of deubiquitination—location, location, location. F1000 Res 2016; 5: 5. 53 Liu Y, Fallon L, Lashuel HA, Liu Z, Lansbury PT Jr. The UCH-L1 gene encodes two opposing enzymatic activities that affect alpha-synuclein degradation and Parkinson’s disease susceptibility. Cell 2002; 111: 209–18. 54 Wang KK, Yang Z, Sarkis G, Torres I, Raghavan V. Ubiquitin C-terminal hydrolase-L1 (UCH-L1) as a therapeutic and diagnostic target in neurodegeneration, neurotrauma and neuro-injuries. Expert Opin Ther Targets 2017; 21: 627–38. 55 Gu Y, Ding X, Huang J, et al. The deubiquitinating enzyme UCHL1 negatively regulates the immunosuppressive capacity and survival of multipotent mesenchymal stromal cells. Cell Death Dis 2018; 9: 459. 56 Nakashima R, Goto Y, Koyasu S, et al. UCHL1-HIF-1 axis-mediated antioxidant property of cancer cells as a therapeutic target for radiosensitization. Sci Rep 2017; 7: 6879. 57 Vawter MP, Barrett T, Cheadle C, et al. Application of cDNA microarrays to examine gene expression differences in schizophrenia. Brain Res Bull 2001; 55: 641–50. 10
58 Novikova SI, He F, Cutrufello NJ, Lidow MS. Identification of protein biomarkers for schizophrenia and bipolar disorder in the postmortem prefrontal cortex using SELDI-TOF-MS ProteinChip profiling combined with MALDI-TOF-PSD-MS analysis. Neurobiol Dis 2006; 23: 61–76. 59 Demirel OF, Cetin İ, Turan Ş, Sağlam T, Yıldız N, Duran A. Decreased expression of α-synuclein, Nogo-A and UCH-L1 in patients with schizophrenia: a preliminary serum study. Psychiatry Investig 2017; 14: 344–49. 60 Middleton FA, Mirnics K, Pierri JN, Lewis DA, Levitt P. Gene expression profiling reveals alterations of specific metabolic pathways in schizophrenia. J Neurosci 2002; 22: 2718–29. 61 Sekar A, Bialas AR, de Rivera H, et al. Schizophrenia risk from complex variation of complement component 4. Nature 2016; 530: 177–83. 62 Laskaris L, Chana G, Weickert CS, et al. Increased C3 and C4 proteins in Serum of FEP and UHR patients: implications for inflammatory subtyping in Scz. Biol Psychiatry 2017; 81: S27–28. 63 Chu CI, Liu CY, Sun CT, Lin J. The effect of animal-assisted activity on inpatients with schizophrenia. J Psychosoc Nurs Ment Health Serv 2009; 47: 42–48. 64 Scott MR, Meador-Woodruff JH. Intracellular compartment-specific proteasome dysfunction in postmortem cortex in schizophrenia subjects. Mol Psychiatry 2019; published online Jan 25. DOI:10.1038/s41380-019-0359-7. 65 Scott MR, Rubio MD, Haroutunian V, Meador-Woodruff JH. Protein expression of proteasome subunits in elderly patients with schizophrenia. Neuropsychopharmacology 2016; 41: 896–905. 66 Feinberg I. Schizophrenia: caused by a fault in programmed synaptic elimination during adolescence? J Psychiatr Res 1982–1983; 17: 319–34. 67 Sidharthan NP, Butcher NJ, Mitchell DJ, Minchin RF. Expression of the orphan cytosolic sulfotransferase SULT4A1 and its major splice variant in human tissues and cells: dimerization, degradation and polyubiquitination. PLoS One 2014; 9: e101520. 68 Choi JY, Kim JH, Jo SA. Acetylation regulates the stability of glutamate carboxypeptidase II protein in human astrocytes. Biochem Biophys Res Commun 2014; 450: 372–77. 69 Watanabe Y, Khodosevich K, Monyer H. Dendrite development regulated by the schizophrenia-associated gene FEZ1 involves the ubiquitin proteasome system. Cell Rep 2014; 7: 552–64. 70 Lee SA, Kim SM, Suh BK, et al. Disrupted-in-schizophrenia 1 (DISC1) regulates dysbindin function by enhancing its stability. J Biol Chem 2015; 290: 7087–96. 71 Barodia SK, Park SK, Ishizuka K, Sawa A, Kamiya A. Half-life of DISC1 protein and its pathological significance under hypoxia stress. Neurosci Res 2015; 97: 1–6. 72 Tankou S, Ishii K, Elliott C, et al. SUMOylation of DISC1: a potential role in neural progenitor proliferation in the developing cortex. Mol Neuropsychiatry 2016; 2: 20–27. 73 Yalla K, Elliott C, Day JP, et al. FBXW7 regulates DISC1 stability via the ubiquitin-proteosome system. Mol Psychiatry 2018; 23: 1278–86. 74 Fu C, Chen D, Chen R, Hu Q, Wang G. The schizophrenia-related protein Dysbindin-1A is degraded and facilitates NF-Kappa B activity in the nucleus. PLoS One 2015; 10: e0132639. 75 Kerrisk Campbell M, Sheng M. USP8 deubiquitinates SHANK3 to control synapse density and SHANK3 activity-dependent protein levels. J Neurosci 2018; 38: 5289–301. 76 Liu X, Zhao B, Sun L, et al. Orthogonal ubiquitin transfer identifies ubiquitination substrates under differential control by the two ubiquitin activating enzymes. Nat Commun 2017; 8: 14286. 77 Iwata S, Morioka H, Iwabuchi M, et al. Administration of haloperidol with biperiden reduces mRNAs related to the ubiquitin-proteasome system in mice. Synapse 2005; 56: 175–84. 78 Chaugule VK, Walden H. Specificity and disease in the ubiquitin system. Biochem Soc Trans 2016; 44: 212–27. 79 Cohen-Kaplan V, Livneh I, Avni N, et al. p62- and ubiquitin-dependent stress-induced autophagy of the mammalian 26S proteasome. Proc Natl Acad Sci USA 2016; 113: e7490–99. © 2020 Elsevier Ltd. All rights reserved
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The ubiquitin proteasome system and schizophrenia Sandra Luza*, Carlos M Opazo*, Chad A Bousman*, Christos Pantelis†, Ashley I Bush†, Ian P Everall†
The ubiquitin-proteasome system is a master regulator of neural development and the maintenance of brain structure and function. It influences neurogenesis, synaptogenesis, and neurotransmission by determining the localisation, interaction, and turnover of scaffolding, presynaptic, and postsynaptic proteins. Moreover, ubiquitin-proteasome system signalling transduces epigenetic changes in neurons independently of protein degradation and, as such, dysfunction of components and substrates of this system has been linked to a broad range of brain conditions. Although links between ubiquitin-proteasome system dysfunction and neurodegenerative disorders have been known for some time, only recently have similar links emerged for neurodevelopmental disorders, such as schizophrenia. Here, we review the components of the ubiquitin-proteasome system that are reported to be dysregulated in schizophrenia, and discuss specific molecular changes to these components that might, in part, explain the complex causes of this mental disorder.
Introduction Schizophrenia is characterised by a complex array of positive, negative, and cognitive symptoms that are associated with changes at the molecular level.1 Schizo phrenia has been linked to abnormalities in dopaminergic, glutamatergic, GABAergic (ie, γ-aminobutyric-acid), and serotoninergic neurotransmission pathways,1,2 as well as signalling pathways (eg, the Wnt/β-catenin pathway) that are crucial for brain growth and maturation.3 Common consequences of these extra cellular and intracellular pathway abnormalities in schizophrenia are changes in the levels of specific proteins, suggesting impaired proteostasis. Increased protein insolubility has been reported in autopsy brain samples from individuals with schizophrenia, which is a signature of proteostasis dysfunction.4 Proteostasis requires the control and harmonisation of protein synthesis, protein folding and conformational main tenance, protein—protein interac tion, protein trafficking, and protein degradation. The ubiquitin-proteasome system (UPS) is central to proteostasis5 and, as presented in this Review, multiple proteins that have been shown to be altered in schizo phrenia are regulated by this crucial system. As such, it could be argued that understanding the dysregulation of the UPS in schizophrenia would report novel molecular aspects for the basis of this mental disorder. Before addressing this argument, we will briefly describe the components and main features of the UPS. We will then summarise and discuss the evidence that implicates the involvement of specific components of the UPS in schizophrenia, before concluding with potential clinical implications of this evidence. Of note, other pathways involved in proteo stasis (eg, autophagy and unfolded protein response) could also be linked to the dysregulation of proteins in schizophrenia, but are beyond the scope of this Review and have been reviewed elsewhere.6,7 A flowchart summarising the article selection process is provided in figure 1.
The UPS The UPS is the principal pathway for the degradation of cytosolic, nuclear proteins, and transmembrane proteins8 (figure 2). This process involves the sequential conjugation
of one, or several, ubiquitin moieties to proteins (a process known as ubiquitination), which is catalysed by an enzymatic cascade composed of ubiquitin activases (E1), ubiquitin conjugases (E2), and ubiquitin ligases (E3).9 Multiple cycles of ubiquitination lead to the formation of a polyubiquitin chain on the protein substrate, which can function as a signal for degradation by three proteases (ie, caspase, trypsin, and chymotrypsin) that are part of the proteasome. Once the proteins are recognised by the 26S proteasome, the ubiquitin moiety is removed from the protein substrate by the action of deubiquitinating enzymes (DUBs) associated with the 26S proteasome, and thus ubiquitin is recycled.9,10 Then, the protein substrate binds the proteasome complex through the 19S regulatory particle, which governs their entry into the 20S catalytic chamber. The proteasome unfolds substrates and threads the polypeptide chains through the inner channel, where they are cleaved into short peptides by the catalytically active subunits caspase-like, trypsin-like, and chymotrypsinlike.9 Key challenges • Discerning whether the increased levels of ubiquitinated proteins observed in autopsy and peripheral tissue from indivduals with schizophrenia are primarily monoubiquitinated or polyubiquitinated • Assessing the effect that antipsychotic medications have on ubiquitin-proteasome system (UPS) function using in vivo human study designs • Surveying the distribution of UPS abnormalities across the brain and in various cell types to determine the specificity of UPS dysfunction • Evaluating the utility of peripheral UPS function as a predictor of clinical trajectories in individuals with schizophrenia • Determining the identity of proteins that are highly ubiquitinated in schizophrenia • Evaluating the therapeutic effect of E3-ligase inhibitors in animal models of schizophrenia • Investigating whether animal models of schizophrenia display changes in UPS components
www.thelancet.com/psychiatry Published online February 12, 2020 https://doi.org/10.1016/S2215-0366(19)30520-6
Lancet Psychiatry 2020 Published Online February 12, 2020 https://doi.org/10.1016/ S2215-0366(19)30520-6 *Contributed equally †These authors share senior authorship Melbourne Neuropsychiatry Centre, Department of Psychiatry (S Luza PhD, C M Opazo PhD, C A Bousman PhD, Prof C Pantelis MD, Prof I P Everall DSc), Melbourne Dementia Research Centre, Florey Institute of Neuroscience and Mental Health (S Luza, C M Opazo, Prof C Pantelis, Prof A I Bush PhD, Prof I P Everall), and Centre for Neural Engineering, Department of Electrical and Electronic Engineering (Prof C Pantelis, Prof I P Everall), The University of Melbourne & Melbourne Health, Parkville, VIC, Australia; The Cooperative Research Centre for Mental Health, Carlton South, VIC, Australia (C A Bousman, Prof C Pantelis, Prof I P Everall); Hotchkiss Brain Institute, Cumming School of Medicine (C A Bousman), Departments of Medical Genetics, Psychiatry, and Physiology & Pharmacology (C A Bousman), University of Calgary, Calgary, AB, Canada (C A Bousman); Alberta Children’s Hospital Research Institute, Calgary, AB, Canada; NorthWestern Mental Health, Melbourne, VIC, Australia (Prof C Pantelis); and Institute of Psychiatry, Psychology and Neuroscience, King’s College London, London, UK (Prof I P Everall) Correspondence to: Prof Ashley I Bush, Melbourne Dementia Research Centre, Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, VIC 3052, Australia [email protected]
1
Review
120 records identified through PubMed database searching
29 additional records identified through other sources
information together shows that UPS is defective in schizophrenia.
Ubiquitin and polyubiquitinated proteins 24 duplicate records removed
125 records screened 1 record excluded 1 no full text available 124 full-text articles assessed for eligibility 70 full-text articles excluded 3 in non-English language 15 non-human studies 52 no canonical ubiquitin-proteasome system component related to schizophrenia reported 54 studies included in the review
Figure 1: PRISMA flow chart detailing the article selection process
Beyond proteasomal degradation, the ubiquitination cascade enzymes and deubiquitinases can regulate protein function or signalling in many cellular processes by adding single moieties of ubiquitin or removing multiple moieties of ubiquitin to or from proteins, which regulates protein—protein interactions and, thus, protein endo cytosis, protein localisation, and protein expression.8 For example, monoubiquitination is a key trafficking mechanism for various cell surface proteins and is also involved in histone and transcription factor regulation, which are crucial to biological processes that are implicated in brain development (eg, monoubi quitination for the internalisation of G protein-coupled receptors).11,12 Changes in histone monoubiquitination have been linked to neurodevelopmental disorders,13 but its association to schizophrenia is unexplored. Thus, understanding the UPS in the context of schizophrenia has the potential to assist in reconciling or unifying the molecular bases that might explain, in part, the heterogeneity of pathways associated with this mental disorder. This notion is based on previous work from our group and other groups that have found pathway-level dysregulation of the UPS in schizophrenia using genomic,14,15 proteomic,16 and transcriptomic17 approaches. Yet, pathway-level findings alone have limited utility as they do not point to specific components of the pathway that are amenable to intervention. As such, we reviewed the literature to identify specific components of the UPS canonical pathway that are dysregulated at the genetic (table 1), transcriptomic (table 2), and proteomic (table 3) levels, as well as protein substrates that could serve as novel therapeutic targets in schizophrenia. Although some studies show conflicting data that require further clarification, overall all the 2
Ubiquitin is a 76 amino-acid peptide that has seven lysine residues and a N-terminus that serve as points of ubiquitination, that is, binding of additional ubiquitin moieties once a ubiquitin unit is conjugated to a protein substrate. As ubiquitin conjugation might occur at any of its seven lysine residues, a ubiquitin chain can grow into many different topologies. The best characterised chains are Lys48 (K48) and Lys63 (K63). Lys48 chains are involved in ubiquitin-dependent proteolysis by the 26S proteasome, and Lys63 chains are generally involved in other cellular processes (eg, DNA repair and signal transduction). The role of other ubiquitin chains (Lys6, Lys11, Lys27, Lys29, and Lys33), including mixed and branched chains, are less well understood.5 The measurement of ubiquitin immunoreactivity is widely used to evaluate changes of the UPS in tissues. Nishimura and colleagues18 assessed the ubiquitin immunoreactivity in autopsy hippocampal tissue from individuals with schizophrenia and were the first to show a higher immunoreactivity against ubiquitin relative to control tissue.18 Since this seminal observation, three studies have looked for ubiquitin or polyubiquitinated proteins in autopsy brain samples from individuals with schizophrenia.19–21 An mRNA analysis of laser-captured hippocampal neurons in multiple schizophrenia cohorts reported a decrease in the expression of ubiquitin,19 which might correspond to an independent phenomenon not related to changes in ubiquitin immunoreactivity or part of a homoeostatic response to the increased levels of ubiquitin immunoreactivity observed by Nishimura and colleagues.18 Protein analysis in autopsy superior temporal gyrus samples from elderly individuals with schizophrenia showed an overall decrease in free ubiquitin and Lys48-linked ubiquitin species, but an increase in Lys63-linked ubiquitin species.20 In agreement with this report, we showed in an independent cohort of individuals with late-onset schizophrenia that polyubiquitinated protein levels were increased in the orbitofrontal cortex and erythrocytes compared with healthy indvividuals.21 These latter two studies both suggest that polyubiquitinated protein levels might be affected in schizophrenia, but it remains to be determined which proteins are affected by these abnormalities. Collectively, studies of ubiquitin and polyubiquitinated proteins indicate that schizophrenia might result from a proteostatic imbalance in brain cells (as indicated by ubiquitin dysregulation and the accumulation of ubiquitinated proteins)—but evidence that this is the one underlying cause of the disorder is not available yet. A 2019 study found increased brain protein insolubility in a subset of individuals who have schizophrenia,4 but whether these abnormalities emerge before, early on, or as the illness progresses is not clear. In studies of erythrocytes from individuals with recent-onset
www.thelancet.com/psychiatry Published online February 12, 2020 https://doi.org/10.1016/S2215-0366(19)30520-6
Review
AMP ATP
Ub
Ub
Ub
Ub activation
Ub
E1
Ub
Ub Ub Ub
Free ubiquitin
Ub
Deubiquitination
E1 Ub Ub
Ub Ub
Ub Ub
Peptides 19S Proteasome
Ub
E2
Ub conjugation
E2
Degradation
E3
Ub
20S
26S
19S
E3
Ub Monoubiquitination
Target protein
Polyubiquitination Ub
Target protein Ub
Lys-63
Ub Ub
Ub Ub
Dubs
Lys-48 Ub
Ub
Ub Ub
Ub Ub
Modification of protein function, localisation, or binding
Ub Target protein
Ub
UB
P
Target protein Ub Ub
Ub Ub
Mediation of cell signalling Activation of endosomal pathway Activation of autophagy/lyosomal pathway
Figure 2: The ubiquitin proteasome system (UPS) The UPS in humans consists of two activating enzymes (E1s), approximately 40 conjugating enzymes (E2s) and hundreds of ligase enzymes (E3s) that result in the activation and conjugation of the 76 amino-acid ubiquitin onto the lysine residues of targeted proteins.78 Single or multiple ubiquitin molecules are added to target proteins, resulting in monoubiquitination or polyubiquitination, respectively. Monoubiquitination can result in modification of the target protein’s function, localisation, or binding, whereas polyubiquitination might target the protein for degradation by the 26S proteasome or result in activation of the autophagosome—lysosomal or endosomal pathways or mediation of cell signalling.78,79 Black arrows represent the intracellular destiny of ubiquitinated proteins. DUBs=deubiquitinating enzymes. Lys=lysine. Ub=ubiquitin.UBP=ubiquitin binding protein.
schizophrenia, we observed no changes in levels of ubiqui tinated proteins, suggesting that increased amounts of ubiquitinated proteins are found later in the disease course.21 Thus, the accumulation of polyubi quitinated proteins could represent an effect rather than a cause of the disease. Further studies that use brain tissue are needed to determine whether the accumulation of polyubiquitinated proteins precede onset of schizophrenia.
E1 activating enzymes The activation of ubiquitin by E1 enzymes is an essential step that triggers subsequent downstream processes in the UPS pathway. The human genome encodes two canonical E1 enzymes: ubiquitin activating enzyme 1 (UBA1) and ubiquitin activating enzyme 6 (UBA6). Ubiquitin is activated in an ATP-dependent process by E1 through the formation of a thioester bond between its C-terminal Gly76 residue and an active-site Cys of the E1. From samples derived from autopsies, superior temporal gyrus tissue shows a decrease in the protein levels of UBA6, with
no changes in UBA1 levels shown in schizophrenia compared with matched healthy controls.20 This result is in line with known links between UBA6 and neuronal development, spine architecture, and mouse behaviour,22 as well as the notion that a ubiquitination defect might precede changes found downstream of multiple molecular pathways in schizophrenia.20 The possible link between loss of UBA6 and loss of grey matter in the superior temporal gyrus in schizophrenia might be associated with the progressive grey matter reduction of the superior temporal gyrus that antecedes the first expression of florid psychosis in schizophrenia. Further studies are required to investigate a link between changes in UBA6 and loss of grey matter in superior temporal gyrus and psychosis. 23
E2 conjugating enzymes After its activation by E1, ubiquitin is transferred to a ubiquitin-conjugating enzyme (E2) via a transthiolation reaction. As of 2020, approximately 40 E2s had been described in humans. All E2s interact with an E1 enzyme
www.thelancet.com/psychiatry Published online February 12, 2020 https://doi.org/10.1016/S2215-0366(19)30520-6
3
Review
Sample size N
Risk of SZ
SZ
Reference
Controls
UBE2D1 rs11006122
536
268
268
No
Andrews and Fernandez-Enright (2014)25
rs1905455
536
268
268
No
Andrews and Fernandez-Enright (2014)25
1439
814
625
Yes (C-allele)
Chen et al (2008)46
rs1859427
536
268
268
No
Andrews and Fernandez-Enright (2014)25
rs6861170
1439
814
625
Yes (T-allele)
Chen et al (2008)46
rs6861170
536
268
268
No
Andrews and Fernandez-Enright (2014)25
FBXL21 rs1859427
dbSNP=Single Nucleotide Polymorphism Database identification number. rs=reference SNP cluster. SZ=individuals with schizophrenia.
Table 1: Ubiquitin-proteasome system genetic studies in schizophrenia by dbSNP ID (rs)
and one or more E3s. E2 enzymes determine the specificity of polyubiquitin chain-linkage types (eg, Lys48 and Lys63) and might act alone or in conjunction with other E2s to catalyse polyubiquitin chain formation.24 As of 2020, four canonical E2 conjugating enzymes (UBE2D1, UBE2K, UBE2M, and UBE2N) have been implicated in schizophrenia. In autopsy hippocampal tissue, Altar and colleagues19 found a decrease in the amount of the UBE2D1 transcript in schizophrenia. However, analyses of UBE2D1 genetic variants25 and UBE2D1 protein levels in the dorsolateral prefrontal cortex26 did not report changes. In addition, a decrease of UBE2N27 gene expression in the dorsolateral prefrontal cortex and a decrease of UBE2M16 protein expression in the hippocampus of individuals who have schizophrenia compared with matched healthy controls has been reported. Finally, UBE2K mRNA levels in lymphocytes have been shown to correlate (r=0·74; p<0·0005) with positive symptoms in schizophrenia.28 However, no changes in the level of UBE2K protein were reported in the superior temporal gyrus in a cohort of older (aged between 59 and 97 years) individuals who have schizophrenia,20 which could suggest that UBE2K expression is tissue specific or age dependent. Considering these results, a future study that explores these four canonical E2 enzymes at the gene, mRNA, and protein level in the same brain regions is required to elucidate the status of these enzymes in schizophrenia.
E3 ligase enzymes E3 ligases are the most studied UPS component in schizophrenia. The UPS includes more than 600 E3s that interact with E2s in unique configurations to dictate substrate specificity. Depending on the types of ubiquitination domains, they are subclassified as homo logous to the E6AP carboxyl terminus (HECT), really interesting new gene (RING), U-box E3, and PHD finger ligases. As of 2020, two HECT (UBE3B and UBE3A), three RING (SIAH2, Parkin, and MDM2), and three U-box E3 enzymes (FBXW7, FBXL21, and FBXO45) have been implicated in schizophrenia. 4
UBE3B expression is downregulated in pyramidal neurons of the prefrontal cortex in adolescents who have schizophrenia compared with controls.30 Moreover, the number of cells that express UBE3B, as well as the level of UBE3B protein, increases in normal adolescent brains, but is significantly decreased in adolescents with schizo phrenia.30,31 These findings suggest that UBE3B could contribute to the pathophysiology of schizophrenia by altering synaptic refinement on prefrontal cortex circuitry during adolescence.30 UBE3A, the paralogue of UBE3B, is also linked to schizophrenia, but in the opposite way. The UBE3A gene maps to chromosome 15q11-q13, a region prone to duplication that has been linked to increased transcription32 and risk for neuropsychiatric phenotypes, including schizophrenia.33,34 In addition, mutations and deletions in UBE3A play a role in other neurodevelopmental disorders (eg, Angelman syndrome).35 Therefore, investi gations of the UPS in these disorders might be informative to schizophrenia, and vice versa. UBE3A is an abundant protein in cytosolic and nuclear compartments and has diverse functions in cellular and transcriptional events in neurodevelopment.36 More studies are required to elucidate the role of UBE3A in illnesses of early and late neurodevelopment, like schizophrenia.37 Parkin, a RING E3 ligase, whose function is associated with energy metabolism and oxidative stress,38 is increased in autopsy prefrontal cortex samples from individuals with schizophrenia, which could be related to stress.39 By contrast, protein levels of the RING E3 enzyme, MDM2, are significantly reduced in the dorsolateral prefrontal cortex in people with schizophrenia compared with controls.26 Importantly for disorders like schizophrenia, MDM2 directly regulates synaptic activity by ubiquitinating PSD-95, which controls the localisation of AMPA receptors.40 PSD-95 and AMPA receptors have also been shown to be decreased in individuals with schizophrenia.41 More controversial is the role of the RING E3 family member SIAH2 in schizophrenia, which has been shown to be upregulated by hypoxia and oestrogen signalling42 and linked to the post-translational regulatory network that controls neuronal polarity during cerebellar granule neuron differentiation.43 Although peripheral mRNA levels of SIAH2 were positively correlated (r=0·73, p=0·0005) with positive symptoms in individuals with schizophrenia,28 the level of SIAH2 protein in autopsy superior temporal gyrus in schizophrenia was not changed.20 U-box E3 ligases can be a single polypeptide or a subunit of a protein complex, such as the Skip1-Cullin1-F-box (SCF) E3 complex. F-box proteins are the key recognition subunits of SCF, in the multimeric E3 ubiquitin ligase complexes. Human F-box proteins are classified into three categories according to specific substrate recognition domains: FBXW (comprising ten members), FBXL (comprising 22 members), and FBXO (comprising 37 members).44 mRNA expression of FBXW7, which regulates the stability of disrupted in schizophrenia 1 (DISC1), has been shown to be significantly lower in
www.thelancet.com/psychiatry Published online February 12, 2020 https://doi.org/10.1016/S2215-0366(19)30520-6
Review
Platform
Tissue
Sample size
Gene expression References relative to Controls
Healthy Individuals controls with schizophrenia
Category
Type
N
Brain
HIP
46
22 (48%)
24 (52%)
Decreased
Altar et al (2005)19
Ubiquitin encoding UBB
qTR-PCR
Ubiquitin activases (E1) No studies Ubiquitin conjugases (E2) UBE2N
Microarray
Brain
DLPFC
30
15 (50%)
15 (50%)
Decreased
Vawter et al (2002)27
UBE2D1
qTR-PCR
Brain
HIP
46
22 (48%)
24 (52%)
Decreased
Altar et al (2005)19
UBE2K
qTR-PCR
Brain
DLPFC
74
37 (50%)
37 (50%)
No change
Meiklejohn et al (2019)29
UBE2K
qTR-PCR
Blood
Whole blood
128
71 (56%)
57 (45%)
Increased
Meiklejohn et al (2019)29
UBE3A
qTR-PCR
Cells
Fibroblasts
6
3 (50%)
3 (50%)
Increased
Noor et al (2015)32
UBE3B
Microarray
Brain
DLPFC
30
15 (50%)
15 (50%)
Decreased
Kohlbrenner et al (2018)30
UBE3B
qTR-PCR
Brain
DLPFC
30
15 (50%)
15 (50%)
Decreased
Kohlbrenner et al (2018)30
FBXW7
Microarray
Brain
DLPFC
60
36 (60%)
24 (40%)
Decreased
Arion et al (2015)45
FBXW7
qTR-PCR
Brain
DLPFC
60
36 (60%)
24 (40%)
Decreased
Arion et al (2015)45
UCHL1
Microarray
Brain
PFC
20
10 (50%)
10 (50%)
Decreased
Middleton et al (2002)60
UCHL1
Microarray
Brain
PFC
6
3 (50%)
3 (50%)
Decreased
Vawter et al (2001)57
UCHL1
qTR-PCR
Brain
HIP
46
22 (48%)
24 (52%)
Decreased
Altar et al (2005)19
UCHL5
Microarray
Brain
DLPFC
60
36 (60%)
24 (40%)
Decreased
Arion et al (2015)45
UCHL5
qTR-PCR
Brain
DLPFC
60
36 (60%)
24 (40%)
Decreased
Arion et al (2015)45
USP14
Microarray
Brain
PFC
6
3 (50%)
3 (50%)
Decreased
Vawter et al (2001)57
USP9
Microarray
Brain
PFC
20
10 (50%)
10 (50%)
Decreased
Middleton et al (2002)60
qTR-PCR
Brain
HIP
46
22 (48%)
24 (52%)
Decreased
Altar et al (2005)19
Sub- unit RPn8
qTR-PCR
Brain
HIP
46
22 (48%)
24 (52%)
Decreased
Altar et al (2005)19
Sub-unit RPn9
qTR-PCR
Brain
HIP
46
22 (48%)
24 (52%)
Decreased
Altar et al (2005)19
PSMA1
Microarray
Brain
PFC
6
3 (50%)
3 (50%)
Decreased
Vawter et al (2001)57
PSMA1
qTR-PCR
Brain
HIP
46
22 (48%)
24 (52%)
Decreased
Altar et al (2005)19
PSMB4
Microarray
Brain
DLPFC
60
36 (60%)
24 (40%)
Decreased
Arion et al (2015)45
PSMB4
qTR-PCR
Brain
DLPFC
60
36 (60%)
24 (40%)
Decreased
Arion et al (2015)45
PSMB6
qTR-PCR
Brain
HIP
46
22 (48%)
24 (52%)
Decreased
Altar et al (2005)19
Ubiquitin ligases (E3)
Deubiquitination
Proteasome Sub-unit RPt6
DLPFC=Dorsolateral Prefrontal Cortex. HIP=hippocampus. PFC=prefrontal cortex
Table 2: Ubiquitin-proteasome system transcriptomic studies in people with schizophrenia
pyramidal neurons in the dorsolateral prefrontal cortex of individuals with schizophrenia compared with healthy controls.45 Within the FBXL category, genetic variation in and around FBXL21 was associated with schizophrenia in a case-control study of Irish families,46 but the association was not found in DNA samples from a replication cohort of white individuals obtained from the Australian Schizophrenia Research Bank.25 FBXL21 has been implicated in the regulation and stabilisation of the circadian clock proteins,47 such as CRY1 and PER2, which have been shown to have a loss of rhythmic expression in cultured fibroblasts from individuals with chronic schizophrenia compared with control individuals.48 Levels of FBXL21 protein were significantly reduced in autopsy
dorsolateral prefrontal cortex tissue from individuals with schizo phrenia compared with matched controls.26 Of the FBXO family, FBXO45 is required for normal synaptogenesis, axon navigation, and neuronal migration in the development of central and peripheral neurons.49 FBXO45 is included in the 3q29 microdeletion region that had been implicated as conferring a significant risk for schizophrenia.50 A study from Wang and colleagues51 reported a rare FBXO45 R108C mutation in an individual with schizophrenia that resulted in decreased mRNA expression. Bioinformatics analysis had predicted that the R108C mutation could have a damaging effect on the function of the FBXO45 protein, but empirical studies to confirm this are required.
www.thelancet.com/psychiatry Published online February 12, 2020 https://doi.org/10.1016/S2215-0366(19)30520-6
5
Review
Platform
Tissue
Sample size
Category
Type
N
SZ
Healthy controls
Analyte expression relative to controls
References
Nishimura et al (2000)18
Ubiquitin Ubiquitin
ICC
Brain
HIP
37
15
22
Increased
Ubiquitin
WB
Brain
STG
26
13
13
Decreased
Rubio et al (2013)20
Ubiquitin
WB
Brain
DLPFC
76
38
38
No change
Bousman et al (2019)21
Ubiquitin
WB
Blood
Erythrocytes
181
88
93
No change
Bousman et al (2019)21
Polyubiquitin K63-Linked
WB
Brain
STG
26
13
13
Increased
Rubio et al (2013)20
K48-Linked
WB
Brain
STG
26
13
13
Decreased
Rubio et al (2013)20
Total Poly-Ub
WB
Brain
DLPFC
Total Poly-Ub
WB
Blood
Erythrocytes
76
38
38
Increased
Bousman et al (2019)21
181
88
93
Increased
Bousman et al (2019)21
Ubiquitin activases (E1) UBA 1
WB
Brain
STG
26
13
13
No change
Rubio et al (2013)20
UBA 6
WB
Brain
STG
26
13
13
Decreased
Rubio et al (2013)20
Ubiquitin conjugases (E2) UBE2D1
WB
Brain
DLPFC
60
30
30
No change
Andrews et al (2017)26
UBE2K
WB
Brain
STG
26
13
13
No change
Rubio et al (2013)20
UBE2K
WB
Brain
DLPFC
74
37
37
Increased
Meiklejohn et al (2019)29
UBE2K
WB
Blood
Erythrocytes
128
71
57
Increased
Meiklejohn et al (2019)29
UBE2M
Mass spec
Brain
HIP
57
30
27
Decreased
Schubert et al (2015)16
UBE3B
IHC
Brain
DLPFC
30
15
15
Decreased
Kohlbrenner et al (2018)30
FBXL21
WB
Brain
DLPFC
60
30
30
Decreased
Andrews et al (2017)26
MDM2
WB
Brain
DLPFC
60
30
30
Decreased
Andrews et al (2017)26
Parkin
WB
Brain
DLPFC
26
13
13
Increased
Pandya et al (2014)39
UCHL1
WB
Brain
DLPFC
60
30
30
No change
Andrews et al (2017)26
UCHL1
Mass spectrometry
Brain
HIP
57
30
27
Decreased
Schubert et al (2015)16
UCHL1
Protein array
Brain
DLPFC
37
17
20
Decreased
Novikova et al (2006)58
UCHL1
ELISA
Blood
Serum
84
44
40
Decreased
Demirel et al (2017)57
Sub-unit Rpt1
WB
Brain
STG
28
14
14
Decreased
Scott et al (2016)65
Sub-unit Rpt3
WB
Brain
STG
28
14
14
Decreased
Scott et al (2016)65
Sub-unit Rpt6
WB
Brain
STG
28
14
14
Decreased
Scott et al (2016)65
Ubiquitin ligase (E3)
Deubiquitination
Proteasome
DLPFC=dorsolateral prefrontal cortex. ELISA=enzyme-linked immunosorbent assay. HIP=hippocampus. ICC=immunocytochemistry. IHC=immunohistochemistry. PFC=prefrontal cortex. STG=superior temporal gyrus. WB=Western blot.
Table 3: Ubiquitin-proteasome system (UPS) proteomic studies in people with schizophrenia.
DUBs The process of ubiquitination can be reversed by DUBs. Approximately 100 DUBs are encoded in the human genome. By reversing ubiquitination, DUBs affect the function, localisation, and stability of proteins. There are five DUB families classified on the basis of the homology of their catalytic domains: Ubiquitin C-terminal hydrolases (UCH, comprising four members), ubiquitin-specific proteases (USP, comprising 56 members), ovarian tumour proteases (OUT, comprising 16 members), Machadojosephin domain proteases (MJD, comprising 4 members) and Jab1/Mov34/Mpr1 Pad1 N-terminal (JAMM, comprising 11 members). The main function of DUBs is 6
maintaining an adequate pool of free ubiquitin for immediate conjugation, but DUBs can also act to edit or remodel ubiquitin chains on substrates and inhibit conjugation by binding to E2 enzymes and interfering with ubiquitin transfer to E3 enzymes.52 Among the DUBs, deubiquitinase ubiquitin carboxylterminal esterase L1 (UCHL1) has most often been associated with schizophrenia. UCHL1, a small protein that is highly specific to the human brain, adds or removes ubiquitin from proteins that are destined for degradation.53 By controlling the ubiquitin pool, UCHL1 plays a crucial role in the removal of excessive proteins, oxidised proteins, and misfolded proteins in the neuronal
www.thelancet.com/psychiatry Published online February 12, 2020 https://doi.org/10.1016/S2215-0366(19)30520-6
Review
cyto sol during both normal and neuropathological conditions.54 UCHL1 is highly regulated at the mRNA level and protein level by a range of intracellular factors and post-translational modifications that are affected by cellular stress.54–56 In schizophrenia, the gene expression and protein levels of UCHL1 are decreased in the brain (dorsolateral prefrontal cortex [DLPFC], located in the hippocampus) and serum.16,26,54,57–59 Demirel and colleagues showed that the decrease in UCHL1 serum levels was correlated with the Positive and Negative Syndrome Scale (PANSS) total and PANSS general psychopathology scores in individuals with schizophrenia.59 Further studies are necessary to determine whether UCHL1 has a potential prognostic or therapeutic value specifically for schizophrenia. Decreases in the expression of UCHL5, USP9, and USP14 in the DLPFC from individuals with schizophrenia have been reported by different groups.45,57,60 Both UCHL5 (a nuclear protein) and USP14 (a cytosolic protein) are reversibly associated with the 19S regulatory cap of the proteasome. The binding of UCHL5 and USP14 to the 19S cap normally results in increased enzymatic activity for both DUBs, allowing substrate access to the active site and degradation by the proteasome.52 Finally, a negative association was observed between USP2 gene expression and positive symptom severity in individuals with schizophrenia.28 A decrease in the expression or protein levels of any of the DUBs might result in an accumulation of ubiquitinated proteins,20,21 however, most of the 100 DUBs are poorly characterised and require further investigation in schizophrenia.
The 26S proteasome The 26S proteasome is a large multi-subunit ATP-depen dent protease complex (appendix). It comprises a 20S core particle, capped on one or both sides by a 19S regulatory particle. The base of the regulatory particle is composed of six AAA-ATPases (Rpt1–Rpt6) and four non-ATPases (Rpn1, Rpn2, Rpn10, and Rpn13) and the lid has nine subunits (referred to as Rpn3, Rpn5–Rpn9, Rpn11, Rpn12, and Rpn15). The proteasome can recognise its substrates through several ubiquitin receptors, such as Rpn1, Rpn10, and Rpn13. The regulatory particle binds and unfolds the polyubiquitinated protein substrate and feeds the unfolded polypeptide chain into the chamber of the 20S particle. The regulatory particle also deubi quitinates the poly ubiquitinated substrates to recycle ubiquitin before the substrate passes through the proteasome α subunits (proteasome α subunits 1–7 compose the α-ring) within the 20S particle, where it is cleaved into small peptides by the catalytically active caspase-like, trypsin-like, and chymotrypsin-like β subunits.5 Studies in several brain regions have shown a decrease in the cluster of genes that encode various proteasome subunits, including lid subunits Rpn8 and Rpn9 in individuals with schizophrenia compared with healthy controls.19 Decreased expression of the genes for α-ring
E1 enzyme
UBA6
UBE38
UBE2M
UBE2K
FBL21
MDM2
E2 enzymes
Parkin
UCHL1
Rpt1
Rpt3
E3 enzymes
Deubiquitinase
Rpt6
Proteasome ATPase subunits
Figure 3: Ubiquitin proteasome system (UPS) enzymes that are altered in people with schizophrenia The diagram indicates UPS enzymes that are downregulated (dashed blue circles) or upregulated (dashed red circles) in people with schizophrenia. Black circles denote healthy control conditions.
subunit 1 (PSMA1)19,57 and β-ring subunits 4 and 6 (PSMB4 and PSMB6)19,45 has also been reported in this mental disorder. These deficiencies in proteasome gene expression occurred concomitantly with decreases in expression of synaptic plasticity genes,19 which might be associated with the excessive complement activity that has been proposed as the link to the reduced numbers of synapses in the brain of individuals with schizophrenia.61,62 This observed downregulation of proteasome-related genes appears to be unique to schizophrenia, as brain samples from patients with major depression and bipolar mood disorder did not show similar patterns of dysregulation.63 In 2019, it was shown that some of the proteasome activities are altered in subcellular fractions in the superior temporal gyrus from autopsies of individuals with schizophrenia,64 which is line with the decrease in protein expression of 19S Rpt base subunits that are important regulators of proteasome activity.65 However, we found that total proteasome activity was not changed in autopsy orbitofrontal cortex tissue from individuals with schizo phrenia compared with healthy controls;21 a discrepancy that might be explained by differences in the brain regions or methods used to measure proteasome activity between these studies. The most consistently reported changes are for deubiquitinases and proteasome subunits (tables 2, 3), which are nearly all downregulated in individuals with schizophrenia (figure 3). The studies on other components of the UPS are more heterogenous, but show signs of protein deficiency in individuals with schizophrenia that might implicate changes in ubiquitin conjugation to specific substrates. This observation suggests that components of the UPS are deficient in
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Search strategy and selection criteria PubMed was searched without any filter up to May 31, 2019, using the search terms “ubiquitin”, “proteasome”, and “schizophrenia”. Bibliographies of all research articles were hand searched for additional references not identified in the primary searches. Studies were retained if the following inclusion criteria were met: (1) included data derived from humans with schizophrenia; (2) one or more of the components of the canonical UPS was reported; and (3) the article was published in a peer-reviewed, English-language journal and the full text was available. Review articles, commentaries, books, book chapters, editorial pieces, or any published material not deemed original research were excluded. A flowchart summarising the article selection process is provided in Figure 1. In total, 54 full-text articles met inclusion criteria and were reviewed.
schizophrenia, which might explain the loss of proteostasis of presynaptic and postsynaptic proteins that are found to be altered in this disorder and discussed in the section titled schizophrenia-related proteins that are substrates of the UPS below. Therefore, dysfunction of the UPS might be central to explaining the multiple changes observed in schizophrenia at the synaptic level, which might constitute the molecular basis of this disorder, an idea first proposed by Feinberg.66 Importantly, however, genome-wide asso ciation studies have not identified genetic variants in components of the UPS meeting genome-wide signifi cance for schizophrenia, suggesting that the molecular basis of dysregulation of the UPS observed in studies reside at the epigenomic or post-translational levels.
Schizophrenia-related proteins that are substrates of the UPS Using our search strategy and selection criteria, we identified from the published literature several proteins that are known substrates of the UPS that have been associated with schizophrenia and have functions related to neurodevelopment or synaptic regulation. These proteins include sulfo transferase 4A1,67 glutamate 68 carboxypeptidase II, DISC1,69–73 dysbindin,17,74 SH3, and multiple ankyrin repeat domains 3 (SHANK3).75,76 Although this list is undoubtedly an underestimation of schizophrenia-relevant proteins undergoing abberant ubiquitination and proteasome degradation, they serve as a starting point for further research. In fact, as previously noted, the UPS as a whole seems to be altered in schizo phrenia (figure 3), which might be part of a homoeostatic response to reduce protein ubiquitination and proteasome degradation of these important proteins and others yet to be identified that are necessary for normal synaptic function. Further studies are required to systematically identify and characterise schizophrenia-relevant proteins that aberrantly interact with the UPS. 8
Effect of antipsychotics on the UPS Antipsychotic exposure is known to induce peripheral and central changes in gene and protein expression and would presumably also influence the UPS. However, studies suggest that the expression of UPS-related genes in autopsy prefrontal and hippocampal tissue is not correlated with cumulative lifetime antipsychotic exposure.19,60 Moreover, no associations have been reported between chlorpromazine equivalent dose and polyubiquitinated protein levels, ubiquitination activity, or proteasome activity in erythrocytes or orbitofrontal cortex brain samples from individuals with schizophrenia.20,21 Further more, there is no association between UBE2K gene expression or protein levels and chlorpromazine equivalent antipsychotic exposure.29 Although these results suggest UPS gene and protein expression are unlikely to be significantly affected by antipsychotic exposure, no study to date has been specifically designed to test this association and all published studies have relied on imprecise measures of antipsychotic clinical exposure. Studies on rodents have shown that the administration of anti psychotics does not change the levels of components of the UPS.20,64,65,77 Future in vitro or animal studies are needed to support or refute these initial findings.
Conclusions Studies of autopsy brain tissue done three decades ago provided the first direct evidence for the association between schizophrenia and the UPS. Since then, several studies have supported the notion that the UPS is dysfunctional in schizophrenia. However, it is still unclear whether this abnormality is a cause or consequence of this disease. We reported that accumulation of ubiqutinated proteins is detected at the later stages of schizophrenia. Further histological studies are required to determine whether schizophrenia is a ubiquinopathy. Moreover, both peripheral and central dysregulation of the UPS have been identified in samples from individuals with schizophrenia, implicating the UPS as a potential site of clinically relevant biomarkers that might assist in the early identification, prognosis, and treatment of this mental disorder. This Review shows evidence that the UPS is altered at different biological levels across several stages of the illness. As such, we hypothesise that the downregulation of enzymes of the UPS occurs upstream of the disease manifestations and is related to changes in neuronal development, synaptic consolidation, and function. We believe that these biochemical changes in the UPS might not show detectable clinical manifestations. Other changes within the UPS are likely to be a homoeostatic response and, therefore, represent a secondary event that involves the upregulation of UPS enzymes. This response might explain the decrease of specific proteins that have been associated with early onset (eg, DISC1 and SHANK3) and more advanced states (eg, SULT41 and dysbindin) of schizophrenia. Looking forward, a major challenge will be the
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Review
identification, refinement, and classification of these two groups of enzymes, which could uncover biomarkers for schizophrenia and inform development of new therapies for early and later stages of this illness. If our working hypothesis is correct, specific inhibitors for E3-ligases might be novel therapeutic candidates for the treatment of schizophrenia. Considering how the clinical complexity of schizophrenia parallels the myriad of alterations detected in the UPS in people with this disorder, longitudinal studies that use the full spectrum of socalled omic approaches to interrogate the UPS in schizo phrenia, from pre-psychosis onset to chronic illness, are warranted. Contributors SL was responsible for searching the literature for articles included in this Review. SL, CMO, and CAB prepared the figures and tables. SL, CMO, CAB, CP, AIB, and IPE contributed to the design, writing, preparation, and discussion of the manuscript. All authors read and approved the final manuscript. Declaration of interests AIB is a shareholder in Prana Biotechnology, Mesoblast, Grunbiotics Pty, Cogstate, and a paid consultant for and receives profit share remuneration from Collaborative Medicinal Development. These conflicts of interest do not overlap with the topic of this Review. In the past 5 years, CP served on an advisory board for Lundbeck Australia. He has received honoraria for talks presented at educational meetings that were organised by JanssenCilag, AstraZeneca, Shire, and Lundbeck, and has received an unrestricted grant from Servier Laboratories Australia. All other authors declare no competing interests. Acknowledgments The authors acknowledge the financial support of the The Cooperative Research Centre (CRC) for Mental Health. The CRC programme is an Australian Government initiative. CAB was supported by the Alberta Children’s Hospital Research Inisititue and Cumming School of Medicine, University of Calgary (Canada). CP was supported by the National Health and Medical Research Council (NHMRC) senior principal research fellowship, a grant from the Lundbeck Foundation, and a NHMRC programme grant. AIB was supported by a NHMRC senior principal research fellowship. None of the funding sources played any role in the writing of this Review or in the decision to submit the manuscript for publication. References 1 Horváth S, Mirnics K. Schizophrenia as a disorder of molecular pathways. Biol Psychiatry 2015; 77: 22–28. 2 Lang UE, Puls I, Muller DJ, Strutz-Seebohm N, Gallinat J. Molecular mechanisms of schizophrenia. Cell Physiol Biochem 2007; 20: 687–702. 3 Peng Y, Xu Y, Cui D. Wnt signaling pathway in schizophrenia. CNS Neurol Disord Drug Targets 2014; 13: 755–64. 4 Nucifora LG, MacDonald ML, Lee BJ, et al. Increased protein insolubility in brains from a subset of patients with schizophrenia. Am J Psychiatry 2019; 176: 730–43. 5 Yu H, Matouschek A. Recognition of client proteins by the proteasome. Annu Rev Biophys 2017; 46: 149–73. 6 Bar-Yosef T, Damri O, Agam G. Dual role of autophagy in diseases of the central nervous system. Front Cell Neurosci 2019; 13: 196. 7 Kim P, Scott MR, Meador-Woodruff JH. Dysregulation of the unfolded protein response (UPR) in the dorsolateral prefrontal cortex in elderly patients with schizophrenia. Mol Psychiatry 2019; published online Oct 2. DOI.org/10.1038/s41380-019-0537-7. 8 Dantuma NP, Bott LC. The ubiquitin-proteasome system in neurodegenerative diseases: precipitating factor, yet part of the solution. Front Mol Neurosci 2014; 7: 70. 9 Hershko A, Ciechanover A. The ubiquitin system. Annu Rev Biochem 1998; 67: 425–79. 10 Kleiger G, Mayor T. Perilous journey: a tour of the ubiquitinproteasome system. Trends Cell Biol 2014; 24: 352–59.
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