In this study, we identified 53 proteins that were differentially expressed between a pair of monozygotic twins discordant for bipolar disorder; 34 were up-regulated and 19 were down-regulated. The differentially expressed proteins included those previously implicated in psychiatric disorders, such as ALDOC, ENO1, and PRDX2 
. Differences for ALDOC, ANXA4, PGAM1, PSME1, RPLP0, TPI1, and WARS between twins were regarded as robust because they were identified in three of four experiments with high scores.
To evaluate whether identified proteins might be biomarkers for bipolar disorder, we performed a case–control study for several proteins by Western blot analysis using available antibodies. An increased level of PGAM1 was observed in samples from patients with bipolar disorder. PGAM1 is an enzyme of the glycolytic pathway that catalyzes the conversion of 3-phosphoglycerate to 2-phosphoglycerate 
. This enzyme also promotes glycolysis and ATP production via the TCA cycle and the electron transport system. Although previous studies using postmortem brains of patients with bipolar disorder and schizophrenia suggested altered protein expression of glycolysis enzymes, including PGAM1 
, the results were controversial. The differentially expressed proteins between bipolar disorder and healthy control including PGAM1, might be a clue to understand the biological basis of bipolar disorder.
To examine whether the 53 identified proteins were related to each other and constituted a global molecular network, pathway, or category, we applied IPA to our data. The results showed that the networks having a high score belonged to cell death, energy production, and glucose metabolism categories. The cell death category included the following proteins: NPM1, P4HB, LGALS3, CASP3, PDIA3, ATP5A1, GAPDH, ANXA4, HSPA5, RPLP0, UCHL1, STMN1, ENO1, ANXA5, MZB1, PSMB1, ALDOA, VDAC1, LDHA, HSPB1, and PRDX2 (). These results are consistent with previous studies. Benes et al
showed increased expression of pro-apoptotic gene transcripts in postmortem brains of bipolar disorder patients. Furthermore, Herbeth et al
indicated altered cell death and inflammation-related proteins in peripheral blood mononuclear cells and serum from patients with euthymic bipolar disorder. Brain imaging studies demonstrated reductions in the mean gray matter volume of brains from patients with bipolar disorder 
. Previous studies reported a decreased density of nonpyramidal neurons in layer II of the anterior cingulate and a lower number of glial cells in layer III with bipolar disorder 
. Meta-analyses of volumetric magnetic resonance imaging studies showed reduced volume of gray matter in the anterior cingulate and bilateral insula 
. Neuropathological studies of bipolar disorder showed decreases of each brain field and neuronal cells. Because mood stabilizers and antidepressants, which are used for treatment of bipolar disorder, have neuroprotective actions 
, it has been suggested that cells derived from patients with bipolar disorder are more vulnerable to factors related to cell death than those from controls. Patients with unipolar or bipolar depression exhibit decreased brain-derived neurotrophic factor levels 
. Moreover, mood stabilizers have neuroprotective effects by increasing bcl-2 levels 
. These findings suggest cellular vulnerability has a role in the pathology of bipolar disorder. Dysregulation of the apoptotic process found in the monozygotic twins discordant for bipolar disorder might be relevant to this hypothesis.
We examined the relationship of the identified proteins with canonical pathways and found that the proteins were related to the glycolysis pathway. The proteins included PKM2, ALDH2, ENO1, PGAM1, GAPDH, ALDOA, LDHA, and ALDOC. Glycolysis, or anaerobic respiration, is a fundamental metabolic process that produces energy for all cells. In order to maintain its functions, the brain needs an enormous amount of energy compared with other tissues. ALDOC is a brain-specific glycolysis enzyme that catalyzes the reversible aldol cleavage of fructose-1,6-biphosphate and fructose-1-phosphate to dihydroxyacetone phosphate and either glyceraldehyde-3-phosphate or glyceraldehyde 
. In the present study, we found a decrease of the ALDOC protein level in the affected twin. However, previous reports showed that protein expression level of ALDOC was increased in the frontal cortex of patients, including those with mood disorder 
. This discrepancy might reflect differences between tissues. Moreover, we found differential expression of many essential enzymes of glycolysis such as TPI1, ALDOA, and PGAM1. A previous report using positron emission tomography showed that familial bipolar depressive patients had decreased blood flow in the cerebrum and a decreased rate of glucose metabolism in the ventral anterior cingulate cortex 
. As indicated by an alteration in energy metabolism, compromised metabolic function has been reported in bipolar disorder 
. In these studies, alteration of mitochondrial proteins was reported. Mitochondria are involved in processes including the TCA cycle, glycolysis and gluconeogenesis, lipogenesis, and malate-asparate shuttle 
. Thus, changes in these proteins may lead to major alterations in the energy pathways, thus affecting ATP production. Recently, many reports have suggested that mitochondrial dysfunction is involved in bipolar disorder and other psychiatric disorders 
. Mitochondria are also involved in other essential processes such as apoptosis, oxidative stress, and calcium regulation 
. Thus, a decrease in energy production due to mitochondrial dysfunction in the brains of patients with bipolar disorder may be compensated for by an increase in energy production by glycolysis. It is possible that mitochondrial dysfunction affects neuronal cell death. Further study is needed to know whether these alterations in glycolysis-related proteins are a cause or consequence of the disease process.
This is the first study to our knowledge to apply proteomics for the analysis of monozygotic twins discordant for bipolar disorder, and it has major limitations. First of all, we analyzed only a single pair of monozygotic twins. Thus, results cannot be applied to bipolar disorder in general. Another limitation is the tissue examined; that is, lymphoblastoid cells. Although brain samples may be optimal to identify molecules directly related to bipolar disorder, brain samples of twins are difficult to access. In addition, accessible tissues such as body fluid and peripheral cells such as serum, plasma, cerebrospinal fluids, saliva, urine, and peripheral blood cells should be used for biomarkers. In this study, we used lymphoblastoid cells and avoided a possible effect of medication by culturing the cells in drug-free media. However, a possibility that the effect of medication at the collection of blood last even after culturing the cells in drug-free media for a month cannot be totally ruled out. The other major limitation is the small number of case–control samples.
In summary, we performed a proteomic analysis of lymphoblastoid cells in a pair of monozygotic twins discordant for bipolar disorder. The identified proteins were mainly categorized as those involved in cell death and glycolysis. In a case–control study, protein expression of PGAM1, which is related to glycolysis, was significantly higher in patients than in healthy controls. The present findings suggest future new targets that may be relevant to the pathology of bipolar disorder. The present results need to be tested in a larger, independent sample set to reach a valid conclusion.