Marked alteration of phosphoinositide signaling‐associated molecules in postmortem prefrontal cortex with bipolar disorder

Abstract

Aim

The etiology of bipolar disorder (BD) remains unknown; however, lipid abnormalities in BD have received increasing attention in recent years. In this study, we examined the expression levels of enzyme proteins associated with the metabolic pathway of phosphoinositides (PIs) and their downstream effectors, protein kinase B (Akt1) and glycogen synthase kinase 3β (GSK3β), which have been assumed to be the targets of mood stabilizers such as lithium, in the postmortem brains of patients with BD.

Methods

The protein expression levels of phosphatidylinositol 4‐phosphate 5‐kinase type‐1 gamma (PIP5K1C), phosphatidylinositol 4‐kinase alpha (PIK4CA), phosphatase and tensin homolog deleted from chromosome 10 (PTEN), Akt1, and GSK3β were measured using enzyme‐linked immunosorbent assays and multiplex fluorescent bead‐based immunoassays in the prefrontal cortex (PFC). Specifically, PTEN, Akt1, GSK3β, and PIP5K1C were measured in seven BD patients and 48 controls. Additionally, PIK4CA was analyzed in 10 cases and 34 controls.

Results

PTEN expression levels were markedly decreased in the PFCs of patients with BD, whereas those of Akt and GSK3β were prominently elevated. Moreover, patients medicated with lithium exhibited higher Akt1 expression levels and lower PTEN expression levels in comparison with the untreated group.

Conclusion

Our results suggest that the expression levels of Akt1/GSK3β and its upstream regulator PTEN are considerably altered.

INTRODUCTION

Bipolar disorder (BD) is a chronic psychiatric disorder characterized by recurrent manic or hypomanic episodes and depressive state, with an estimated lifetime prevalence of approximately 1% according to epidemiological studies.¹ BD has a significant socioeconomic impact as it impairs social life due to manic state-induced abnormal behavior, prolonged work absence due to depression, recent evidence of accelerated age-related cognitive decline,² and a high suicide rate.³ The precise etiology of BD remains unknown; however, several factors are potentially involved, including biological differences and genetics. From a first-degree relative, such as a sibling, parent, or twin, and from adoption studies, the heritability of BD is estimated at approximately 70%–80%.^4,5 Several recent genome-wide association studies (GWASs) in Europe have identified numerous susceptibility loci for BD.^5,6 As the cohort's genetic background changes or sample size increases, new risk loci may be discovered. For example, in 2018, a GWAS of a Japanese sample of approximately 3000 BD cases and 60 000 controls identified novel BD risk loci in the gene region encoding the fatty acid desaturase,⁷ which is strongly associated with blood lipid traits^8,9 and n-3/n-6 polyunsaturated fatty acids.¹⁰ Efforts to comprehensively analyze related genes, as represented by GWASs, are important in elucidating the pathogenesis of BD; however, they have not made any clinical impact, such as drug discovery. Moreover, the current treatment options for BD are limited, and long-term prognosis, including social functioning, remains to be undesirable and unacceptable for patients and their families or psychiatrists. Therefore, elucidating BD's biological pathology and developing therapeutic strategies based on this understanding are imperative.

Since inositol monophosphatase (IMPase) and glycogen synthase kinase 3β (GSK3β) are the most promising targets of lithium (Li), the most effective first-choice treatment for BD, both the inositol and GSK3β hypotheses are currently the leading molecular pathological hypotheses for BD.¹¹ The fact that valproate, another agent for BD, also inhibits inositol biosynthesis and GSK3β activity further supports these hypotheses.¹² IMPase catalyzes the dephosphorylation of inositol phosphate (IP) to inositol^13,14 (Figure 1).

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Inositol is used to produce phosphatidylinositol (PI), which is further phosphorylated at positions 3, 4, and 5 of the inositol group to produce seven types of phosphoinositides (PIs; collective term for PI and its phosphorylated derivatives).¹⁵ Phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2) is degraded by phospholipase C (PLC) into diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), which act as secondary messengers, and phosphatidylinositol 3,4,5-trisphosphate (PI[3,4,5]P3) activates Akt in the plasma membrane.¹⁴ Since Akt is a major GSK3β regulator, two important Li targets, IMPase and GSK3β, are linked via the metabolic pathway of PIs. Altogether, PIs are essential for normal brain function; thus, it is plausible that their fluctuations are related to the pathophysiology of BD.^16–18

Collectively, the overall picture of the metabolic pathway of PIs is complicated, and its involvement in the pathophysiology of BD remains unknown. In addition, few postmortem brain studies have measured the PIs kinase/phosphatase in BD. Thus, the current study measured the expression of proteins involved in phosphorylation/dephosphorylation of PIs, such as phosphatidylinositol 4-phosphate 5-kinase type-1 gamma (PIP5K1C), phosphatidylinositol 4-kinase alpha (PIK4CA), and phosphatase and tensin homolog deleted from chromosome 10 (PTEN), and subsequent signal transduction pathways (Akt1 and GSK3β) in the postmortem prefrontal cortex (PFC) of patients with BD.

MATERIALS AND METHODS

Human postmortem brain tissue

Postmortem brain tissue samples from individuals with BD and control subjects were obtained from three different sources: the Fukushima Brain Bank at the Department of Neuropsychiatry, Fukushima Medical University; the Brain Research Institute, Niigata University; and Choju Medical Institute, Fukushimura Hospital as described previously.^19,20 The acquisition of postmortem human brain tissue for this study was approved by the Ethics Committee of Fukushima Medical University, Niigata University, and Fukushimura Hospital, and it complied with the principles of the Declaration of Helsinki and its later amendments. All procedures were performed after obtaining written consent from close relatives. The analysis was divided into two phases: the first half for Akt, GSK3β, PTEN, and PIP5K1C included seven BD cases and 48 controls, and the second half for PIK4CA included 10 cases and 34 controls. Detailed demographic and brain tissue information for the patients with BD is summarized in Table 1. Patients with BD fulfilled the diagnostic criteria established by the American Psychiatric Association (Diagnostic and Statistical Manual of Mental Disorders). No statistical differences in sex, age, or postmortem interval (PMI) were noted between participants and controls. Patient history of Li prescription was also provided (Table 1).

TABLE 1 Demographic information of patients with bipolar disorder and matched controls.

	Akt/GSK3β/PTEN/PIP5K1C	PIK4CA
Cont	BD	p-Value	Cont	BD	p-Value
Number of Samples	48	7	—	34	10	—
Sex
F	21	4		15	4
M	27	3	Fisher exact p-value = 0.689	19	6	Fisher exact p-value = 1.00
Age (year)	75.0 ± 15.9(SD)	67.5 ± 23.6(SD)	Welch's t-test p-value = 0.436	63.5 ± 15.7 (SD)	59.0 ± 24.5(SD)	Welch's t-test p-value = 0.595
PMI (h)	12.1 ± 16.17 (SD)	14.7 ± 15.2 (SD)	Welch's t-test p-value = 0.678	8.7 ± 11.0 (SD)	21.4 ± 24.5 (SD)	Welch's t-test p-value = 0.144
	DOI (year)	History of Li treatment	Akt/GSK3β/PTEN/PIP5K1C	PIK4CA
1	56	No	✓	✓
2	9	Yes	✓	✓
3	n.d.	Yes	✓	✓
4	12	Yes	✓	✓
5	9	Yes	✓	✓
6	34	Yes	✓	✓
7	29	No	✓	✓
8	15	Yes		✓
9	7	No		✓
10	19	No		✓

Protein expression analysis using the enzyme-linked immunosorbent assay (ELISA) and multiplex assay

Protein expression analysis of PIP5K1C, PTEN, Akt1, and GSK3β

The expression levels of PIP5K1C, PTEN, Akt1, and GSK3β proteins in postmortem brains were determined using ELISA and multiplex fluorescent bead-based immunoassay, as described previously.²⁰ In brief, we collected small samples of gray matter (approximately 100 mg each) from Brodmann area (BA) 10 within the prefrontal cortex (PFC) of frozen brains. These samples were placed in a 2% sodium dodecyl sulfate (SDS) solution and underwent three cycles of freezing and thawing and sonicated. Then, the samples were diluted in phosphate-buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 1.76 mM KH₂PO₄) to ensure that the SDS concentration did not exceed 0.2%. After centrifugation (10 000 g for 3 min at 4°C), total protein concentration in the supernatant was measured using the Bradford method (Bradford protein assay kit, Bio-Rad Laboratories, Hercules, CA, USA) with bovine serum albumin as the standard. The expression of proteins was determined by using a commercial ELISA kit (MBS282297, MyBioSource, San Diego, CA, USA) for PIP5K1C and multiplex fluorescent bead-based immunoassay kits (MAPmateTM 46-678MAG for PTEN, 46-675MAG for Akt/PKB, and 46-689MAG for GSK3β; Merck Millipore, Tokyo, Japan). The expression levels of each protein were normalized against the total protein concentration. For more information about the raw data, see Appendix S1.

Protein expression analysis of PIK4CA

Pieces (about 100 mg) of gray matter from the BA10 within the PFC were obtained from the frozen brain. The tissues were suspended in N-PER™ Neuronal Protein Extraction Reagent (Thermo Fisher Scientific, USA) and sonicated and diluted with PBS as described.²¹ The expression of PIK4CA was determined by using a commercial ELISA kit (SEG843Hu, Cloud Clone Corp., Houston, TX, USA). For more information about the raw data, see Appendix S1.

Statistical analysis

Demographic variables (sex, age, and PMI) were compared between groups using Fisher's exact test and Welch's t-test. To compare protein expression levels, the normality and homogeneity of data variances were verified using the Shapiro–Wilk and Brown–Forsythe tests, respectively, followed by the most suitable analytical method among the four statistical methods (i.e., Student t-test, Welch's t-test, Mann–Whitney U-test, and Brunner–Munzel test) indicated in figure legends. In particular, the Brunner–Munzel test was used when both normality and homogeneity of data variances were absent. For these tests, statistical significance was set at p < 0.05. Statistical analysis of the effect of Li medication on protein expression levels was performed using the Brunner–Munzel test because of the small sample size of the Li-treated group. SigmaPlot 14.0 (Systat Software Inc., San Jose, CA, USA) and R (version 4.3.1) were used for analysis.

RESULTS

Expression of phospholipid signaling-associated molecules between BD and controls

Akt1 and GSK3β levels in the PFC were significantly higher in patients with BD than in controls (Akt1: BD/controls = 8.85, p < 0.001, GSK3β: BD/controls = 3.84, p = 0.046, respectively; Figure 2A,B). However, PTEN levels in the PFC were significantly lower in these patients (BD/controls = 0.47, p = 0.019, Figure 2C). PIP5K1C and PIK4CA levels did not differ significantly between the two groups (Figure 2D,E).

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Effects of Li medication on phospholipid signaling-associated protein expression levels

The Akt1 protein expression levels in the Li-treated group were higher than those in the non-treated group (control and Li-naive patient group) [Li (+)/(−) = 8.60, p < 0.001, Figure 3A]. In contrast, the PTEN protein expression level of the Li-treated group was lower than that of the non-treated group [Li (+)/(−) = 0.128, p < 0.001, Figure 3C].

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DISCUSSION

In this study, we examined the expression levels of enzyme proteins associated with the metabolic pathway of PIs, including those of their downstream effectors, Akt1 and GSK3β, which have been considered targets of mood stabilizers, such as Li, in the postmortem brains of patients with BD. We initially revealed that PTEN expression levels were markedly decreased in the PFCs of patients with BD and also found that Akt1 and GSK3β expression levels were prominently increased in the PFCs of patients with BD. Moreover, Li-treated patients exhibited higher Akt1 expression levels and lower PTEN expression levels compared to the control and Li-naive patient group.

Our study is the first to analyze the expression levels of enzyme proteins associated with the metabolic pathway of PIs, including PIP5K1C, PIK4CA, and PTEN, in the postmortem PFCs of patients with BD. We demonstrated decreased expression levels of PTEN, which dephosphorylates PI(3,4,5)P3 to produce PI(4,5)P2 (Figure 1). PI(4,5)P2 is known to play a vital role in membrane trafficking and regulating the exocytosis of synaptic and dense-core vesicles with the plasma membrane, namely, glutamate and dopamine release,²² which have been well-established as crucial etiological neurotransmitters of BD. Moreover, PI(4,5)P2 is hydrolyzed by PLC, which is stimulated by multiple neurotransmitters and neuromodulators, to produce the secondary messengers IP3 and DAG^15,23 (Figure 1). Interestingly, Li affects the synthesis of PI(4,5)P2 and subsequent generation of IP3 and DAG.²⁴ In addition, the impairment of the PLC-mediated pathway is known to be associated with BD.^25–27 Several GWASs have identified PLC signaling as a pathway contributing to BD risk,²⁸ and genome-wide linkage analysis studies have also identified the gene encoding phospholipase Cγ1 (PLCG1) as a susceptibility locus for BD.²⁹ Patients with BD possessing a dinucleotide repeat within the PLCG1 genomic region have been found to respond well to Li medication.²⁷ In addition, manic-like behavior has been associated with forebrain-selective deletion of PLCG1 in mice,³⁰ PLCβ1 (PLCB1) gene deletion in a female patient with BD,³¹ and decreased PLC expression in the platelets of patients with BD.³² Overall, the alteration of the PTEN-PI(4,5)P2-PLC-mediated pathway is potentially involved in the etiology of BD and appears to be a promising target for novel therapeutic agents.

As previously outlined, the PI-to-GSK3β pathway, including the metabolic pathway of PIs, is probably key to pathology of BD, and this supposition has also been based on point of action of Li (Figure 1). Indeed, several postmortem brain studies examining GSK3β and Akt1 expression in BD have been performed. A postmortem brain study indicated that the protein and mRNA expression levels of GSK3β decreased in the dorsolateral prefrontal (DLPFC) and temporal cortex of BD³³; nonetheless, another study revealed increased GSK3β protein expression levels in the anterior prefrontal cortex (BA10) of BD.³⁴ Moreover, several studies have not demonstrated any changes in GSK3β protein expression levels in the prefrontal cortex (BA8 and 9),³⁵ DLPFC (BA9 and 46),³⁶ or BA10.³⁷ Additionally, postmortem Akt-expression results have remained inconsistent across the relatively few studies that have reported this phenomenon. One study revealed a reduction in phosphorylated Akt1 in the PFCs of male patients with BD without psychosis compared with that of male controls³⁸; however, another study highlighted that the Akt1 protein level did not differ between patients with BD and controls.³⁹ In contrast, in the current study, the expression levels of both GSK3β and Akt1 increased in the PFCs of patients with BD. Despite the differences in the direction of change, GSK3β and Akt1 expression certainly appeared to exhibit alterations in the brains of patients with BD. Since PI(3,4,5)P3, which is a fully phosphorylated PI, activates Akt by recruiting it to the plasma membrane, lower PTEN levels possibly affect Akt/GSK3β signaling, and dysfunction of this pathway may be integral to the mechanism underlying BD's pathology.

Contrary to the inositol hypothesis of BD, which focuses on the inhibition of IMPase as the action of Li, the two enzymes of the metabolic pathway of PIs, PIK4CA and PI5KA, showed no significant differences in their expression levels between patients and control subjects. This result might suggest that the final stage of metabolic pathway of PIs is more important than its initial stage in the development of BD. However, there are many more proteins that make up this pathway, and these need to be tested as well. Analysis of local concentrations of IMP, inositol, PI, and PIs may also be needed.

Repeatedly, the molecules examined in this study have been assumed to be targets of mood stabilizers, such as Li. Thus, the postmortem brain expression levels of these molecules in the present study potentially reflect the effects of mood stabilizers taken internally before death. Several studies have reported a decrease in PTEN protein levels and an increase in GSK3β protein levels in mouse astrocyte primary culture cells after 2 weeks of 0.5/1 mM Li treatment⁴⁰ as well as an increase in Akt1 protein and mRNA expression levels after 24–48 h of 1 mM Li treatment.⁴¹ However, these studies were characterized by different Li treatment concentrations, durations, and cell types, and their results vary and remain controversial. Therefore, we compared the expression levels of these proteins between patients who had received Li treatment and those who did not receive Li treatment and the control group and found that the expression levels of Akt1 were higher and those of PTEN were lower in the Li-treated patients. The results of the comparison between BD cases and controls might have been influenced to some extent by the effect of Li administration. We assume that the mechanisms behind this finding may be Li-induced microRNA expression and epigenetic changes. Several microRNAs are known to be altered by Li treatment, among which mirR-144 is widely expressed in the brain and is upregulated by Li, and its target is considered to be the PTEN pathway.⁴² In addition, mood stabilizers, including Li, have been suggested to potentially alter the DNA methylation status⁴³ in many regions, which were also altered in the postmortem brains of patients with BD in this study, suggesting that Li may affect the expression of several genes involved in BD's pathology by altering their DNA methylation.

Our study on brain tissues has certain limitations that require careful consideration. First, the sample size of the BD in this study is particularly small, making it impossible to compare patients with and without Li treatment. Second, confounding variables like age, sex, duration of illness, and PMI might have influenced protein expression in this study, even though there were no statistically significant differences in sex, age, or PMI between BD and control groups, (Table 1). Specifically, the limited sample size of the BD makes the results of the analysis vulnerable to sample heterogeneity. Therefore, the current study's findings must be confirmed using a larger postmortem brain resource.

In conclusion, our results suggest that the expression levels of Akt1, GSK3b, and its upstream regulator PTEN are considerably altered. This pathway has been extensively studied in previous studies focusing on the pathogenesis of BD. This study will advance our understanding of the molecular pathways underlying BD and contribute to the elucidation of molecular targets for Li therapy. However, further large-scale studies are needed to validate these results and account for various confounding factors.

AUTHOR CONTRIBUTIONS

M. H., Y. K., and J. M. designed and Y.K. supervised the study. M. H., Y. K., R. S., A. N., J. M., H. A., Y. H., H. H., A. K., H. T., and H. Y. contributed to the accumulation, quality control, and provision of human resources. M. H. and J. M. carried out the experiment. M. H. analyzed the data. All authors have read and approved the final version of the manuscript.

ACKNOWLEDGMENTS

We thank Ms. H. Onuma for her contribution in coordinating for donations. We deeply appreciate all brain donors and their families for the time and effort they devoted to the consent process and interviews.

FUNDING INFORMATION

This work was supported in part by the Strategic Research Program for Brain Sciences of the Japan Agency for Medical Research and Development under grant numbers JP22dm0207074 (Y. K.), JP22wm0425019 (H. Y.), and JP22wm0425019 (A. K.); the Grant-in-Aid for Scientific Research on Innovative Areas from the Ministry of Education, Culture, Sports, Science, and Technology of Japan under grant numbers JP21H00180 (Y. K.) and JP21K07524 (M. H.); and the Collaborative Research Project of the Brain Research Institute, Niigata University, under grant number 22002 (Y. K.).

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are attached as Appendix S1.

ETHICS STATEMENT

Approval of the Research Protocol by an Institutional Reviewer Board: The acquisition of postmortem human brain tissue for this study was approved by the Ethics Committee of Fukushima Medical University, Niigata University, and Fukushimura Hospital, and it complied with the principles of the Declaration of Helsinki and its later amendments.

Informed Consent: All procedures were performed after obtaining written consent from close relatives.

Registry and the Registration No. of the Study/Trial: N/A.

Animal Studies: N/A.

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