Lithium response in bipolar disorder correlates with improved cell viability of patient derived cell lines

Abstract

Lithium is an effective, well-established treatment for bipolar disorder (BD). However, the mechanisms of its action, and reasons for variations in clinical response, are unclear. We used neural precursor cells (NPCs) and lymphoblastoid cell lines (LCLs), from BD patients characterized for clinical response to lithium (using the "Alda scale" and "NIMH Retrospective Life chart method"), to interrogate cellular phenotypes related to both disease and clinical lithium response. NPCs from two biologically related BD patients who differed in their clinical response to lithium were compared with healthy controls. RNA-Seq and analysis, mitochondrial membrane potential (MMP), cell viability, and cell proliferation parameters were assessed, with and without in vitro lithium. These parameters were also examined in LCLs from 25 BD patients (16 lithium responders and 9 non-responders), and 12 controls. MMP was lower in both NPCs and LCLs from BD; but it was reversed with in vitro lithium only in LCLs, and this was unrelated to clinical lithium response. The higher cell proliferation observed in BD was unaffected by in vitro lithium. Cell death was greater in BD. However, LCLs from clinical lithium responders could be rescued by addition of in vitro lithium. In vitro lithium also enhanced BCL2 and GSK3B expression in these cells. Our findings indicate cellular phenotypes related to the disease (MMP, cell proliferation) in both NPCs and LCLs; and those related to clinical lithium response (cell viability, BCL2/GSK3B expression) in LCLs.

Figure 1

Family A pedigree with clinical details of B1 (lithium non-responder) and B2 (lithium responder).

Figure 2

Experiments using NPCs. (A) Representative immunocytochemistry of NPCs showing Nestin+ cells in all the four cell lines used for experiments. (B) Comparison of high MMP population percentage (MTDR) in the three groups at baseline and after in-vitro treatment with lithium (1 mM) or valproate (0.7 mM) for 7 days by flow cytometry (N = 5). (C) Representative immunocytochemistry of NPCs showing Nestin+ cells with mitochondria localized using TOMM22, and selection of regions of interest for calculation of mitochondrial area fraction. (D) Comparison of mitochondrial area (N = 3). (E) Comparison of dead cell population percentage in the three groups at baseline and after in-vitro treatment by flow cytometry (N = 5). (F) Representative immunocytochemistry of NPCs showing EdU+ cells indicating cells in S phase of cell cycle. (G) Comparison of EdU+ cells in the three groups at baseline and after in-vitro treatment (N = 3). All data are shown as mean ± s.e.m.; Experiments were performed in at least 3 independent experiments (N) for each cell line; For group comparisons, initially appropriate ANOVA test was applied and if significant, independent multiple comparison test were performed. Significance level: *p ≤ 0.05, **p ≤ 0.01, ^***p ≤ 0.001, ****p ≤ 0.0001. Abbreviations: NPCs, neural precursor cells, MTDR, mitotracker deep red; MMP, mitochondrial membrane potential, EdU, 5-ethynyl-2′-deoxyuridine.

Figure 3

Experiments using LCLs. (A) Comparison of high MMP population percentage in the three groups at baseline and after in-vitro treatment with lithium (1 mM) or valproate (0.7 mM) for 7 days. (B) Comparison of relative quantification values of mitochondrial DNA- Cyt B, and (C) ND1, from qPCR normalized to PK (single copy nuclear DNA) at baseline and after treatment with lithium. (D) Comparison of G2/M population percentage in the three groups at baseline and after in-vitro treatment. (E) Comparison of dead cell population percentage in the three groups at baseline and after in-vitro treatment. (F) Relative gene expression of GSK3B across groups - comparison for RQ expression values of GSK3B gene from qPCR normalized to GAPDH (endogenous control). (G) Relative gene expression of BCL2 across groups - comparison for RQ expression values of BCL2 gene from qPCR normalized to GAPDH (endogenous control). All data are shown as mean ± s.e.m.; Experiments were performed in 3 independent experiments for each cell line; N, number of subject LCLs in each group. For group comparisons, initially appropriate ANOVA test was applied and if significant, independent multiple comparison test were performed. Significance level: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Abbreviations: LCLs, lymphoblastoid cell lines, MTDR, mitotracker deep red; MMP, mitochondrial membrane potential, PI, propidium iodide.