The Bcl-2 gene polymorphism rs956572AA increases inositol 1,4,5-trisphosphate receptor-mediated endoplasmic reticulum calcium release in subjects with bipolar disorder

Abstract

Background: Bipolar disorder (BPD) is characterized by altered intracellular calcium (Ca(2+)) homeostasis. Underlying mechanisms involve dysfunctions in endoplasmic reticulum (ER) and mitochondrial Ca(2+) handling, potentially mediated by B-cell lymphoma 2 (Bcl-2), a key protein that regulates Ca(2+) signaling by interacting directly with these organelles, and which has been implicated in the pathophysiology of BPD. Here, we examined the effects of the Bcl-2 gene single nucleotide polymorphism (SNP) rs956572 on intracellular Ca(2+) dynamics in patients with BPD.

Methods: Live cell fluorescence imaging and electron probe microanalysis were used to measure intracellular and intra-organelle free and total calcium in lymphoblasts from 18 subjects with BPD carrying the AA, AG, or GG variants of the rs956572 SNP. Analyses were carried out under basal conditions and in the presence of agents that affect Ca(2+) dynamics.

Results: Compared with GG homozygotes, variant AA-which expresses significantly reduced Bcl-2 messenger RNA and protein-exhibited elevated basal cytosolic Ca(2+) and larger increases in inositol 1,4,5-trisphosphate receptor-mediated cytosolic Ca(2+) elevations, the latter in parallel with enhanced depletion of the ER Ca(2+) pool. The aberrant behavior of AA cells was reversed by chronic lithium treatment and mimicked in variant GG by a Bcl-2 inhibitor. In contrast, no differences between SNP variants were found in ER or mitochondrial total Ca(2+) content or in basal store-operated Ca(2+) entry.

Conclusions: These results demonstrate that, in patients with BPD, abnormal Bcl-2 gene expression in the AA variant contributes to dysfunctional Ca(2+) homeostasis through a specific ER inositol 1,4,5-trisphosphate receptor-dependent mechanism.

Copyright © 2011 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1

Basal cytosolic calcium in B-cell lymphoma 2 (Bcl-2) single nucleotide polymorphism (SNP) variants. (A, B) Basal cytosolic free calcium (Ca2+) concentrations measured with fura-2 in B-lymphoblastoid cell lines (BLCL) from individual BPD patients (A) and in individual BLCL cells (B) from 18 patients of three genotypes incubated in extracellular medium containing 1.0 mmol/L Ca2+. Both patient/patient and cell/cell analysis indicated that [Ca2+]i in variant AA was significantly higher than in variant GG (p <.01). (C, D) Plot of the inverse correlations between individual patient-averaged cytosolic Ca2+ concentrations and Bcl-2 messenger RNA expression (C) (r = .60, p <.01) and protein expression (D) (r =.49, p <.05) as a function of genotype. By both measures, reduced Bcl-2 expression in variant AA was associated with elevated basal [Ca2+]i. All statistical tests were one-way analysis of variance with post hoc Bonferroni correction. a.u., arbitrary unit.

Figure 2

Cytosolic calcium dynamics in Bcl-2 SNP variants. (A) Representative cytosolic Ca2+ traces (fura-2) from BLCL of the three variants after stimulation with thapsigargin (Tg) (1 μmol/L) in Ca2+-free medium containing 200 μmol/L ethylene glycol bis-(β-aminoethyl ether)-N,N′-tetraacetic acid (lower traces) or medium containing 1 mmol/L extracellular Ca2+ (upper traces). Tg induced significant [Ca2+]i elevations only in the presence of extracellular Ca2+. (B) Average (per-patient) [Ca2+]i elevations in BLCL of the three different genotypes after Tg incubation in Ca2+-containing medium did not significantly differ. (C, D) Typical Ca2+ traces (C) and average [Ca2+]i elevations (D) in cells from the three Bcl-2 SNP variants after exposure to the InsP3R agonist adenophostin A (AdA) (75 nmol/L) in a medium containing 1 mmol/L extracellular Ca2+ revealed a graded, genotype-dependent response. The [Ca2+]i elevations in variant AA were significantly higher than in variant GG (p < .05). Other abbreviations as in Figure 1.

Figure 3

Basal endoplasmic reticulum (ER) and mitochondrial Ca2+ stores are not altered in cells expressing the Bcl-2 SNP variant AA. (A, B) Typical Ca2+ traces (A) and per-patient averages (B) of low-affinity fura-2FF fluorescence emission (ratio 340/380 nm) from AA and GG BLCL permeabilized with digitonin (10 μg/mL) in an intracellular buffer (ICB) supplemented with 500 μmol/L adenosine triphosphate. Fluorescence gradually increased to a steady-state maximum that reflects the level of luminal ER Ca2+(23). Basal ER free Ca2+ was similar in Bcl-2 SNP variants AA and GG. (C) Total ER and mitochondrial matrix calcium concentrations, as measured by electron probe microanalysis, were similar in variants AA and GG, although the former was tenfold higher. (D, E) Electron micrographs of BLCL perinuclear regions. Left panel is a conventionally fixed and embedded specimen; right panel is a rapidly frozen, cryosectioned, and freeze-dried preparation as used for electron probe microanalysis. Both panels illustrate typical organelle abundance and distribution, including ER (arrows) and mitochondria (arrowheads). Bar = .5 μm for both panels. ECB, extracellular buffer; other abbreviations as in Figure 1.

Figure 4

Inositol 1,4,5-trisphosphate (InsP3)-induced ER Ca2+ release is enhanced in Bcl-2 SNP variant AA. (A, B) Inositol 1,4,5-trisphosphate receptor (InsP3R)–dependent reduction in ER Ca2+ directly measured with fura-2FF in permeabilized cells. Traces in panel A are a continuation of Figure 3A, with the InsP3 agonist AdA (75 nmol/L) added after a steady-state fluorescence ratio was reached; the sharp decline in fluorescence after AdA application corresponds to the decrease of ER luminal free Ca2+. Per-patient averages of residual ER Ca2+ after AdA (B) showed significantly greater Ca2+ release in the variant AA compared with GG (p < .05). (C) Representative Western blots and quantitative estimation of InsP3R total protein show no differences between AA and GG cells. (D) Similarly, mitochondrial Ca2+ uptake, measured with the Ca2+-selective dye Rhod-2, detected no differences among the three variants after AdA application (p = .77). Other abbreviations as in Figures 1 and 3.

Figure 5

Lithium reduces InsP3R-dependent ER Ca2+ release in Bcl-2 SNP variant AA. (A) Representative Western blots (top) and per-patient averages (duplicate analysis, n = 6 each) showed a significant increase in Bcl-2 expression in lithium-treated (1 mmol/L for 7 days) AA BLCL relative to untreated cells (**p < .01). (B, C) Representative cytosolic Ca2+ traces (fura-2) and per-patient averages showed reduced AdA-stimulated (75 nmol/L) [Ca2+]i elevations in lithium-treated lymphoblasts (**p <.01). (D, E) In an experiment analogous to Figure 4A, typical traces of fura-2FF fluorescence emission (340/380 nm ratio, panel D) and per-patient averages (E) from digitonin-permeabilized, ER loaded cells showed reduced AdA-induced ER Ca2+ release in lithium-treated AA lymphoblasts (*p < .05). Abbreviations as in Figures 1 and 3.

Figure 6

The Bcl-2 inhibition elevates InsP3R-dependent cytosolic Ca2+ in the SNP variant GG. (A) One-hour pre-incubation with the Bcl-2 inhibitor BH3-I significantly increased fura-2-measured basal [Ca2 +]i in BLCL from the variant GG (*p < .05 relative to control). (B, C) Representative cytosolic Ca2+ traces (B) and per-patient averages (C) show enhanced AdA-stimulated (75 nmol/L) [Ca2+]i elevation in GG lymphoblasts after Bcl-2 inhibition (*p < .05). Abbreviations as in Figures 1 and 3.