Chronic LiCl pretreatment suppresses thrombin-stimulated intracellular calcium mobilization through TRPC3 in astroglioma cells.

OBJECTIVES: Transient receptor potential canonical type 3 (TRPC3) channels are activated in B lymphoblast cell lines from patients with bipolar disorder (BD), and its expression is reduced by chronic lithium treatment, implicating TRPC3 in the intracellular calcium (Ca2+ ) dyshomeostasis of BD. Thrombin, via a protease-activated receptor, moderates Ca2+ signaling and TRPC3 in astrocytes, and also cell proliferation. We examined whether lithium pretreatment attenuates thrombin-stimulated TRPC3 expression and function in astrocytes, and levels of the calcium-binding peptide, S100B, which is expressed mainly in these cells. METHODS: Human astroglioma, U-87MG, cells were pretreated with 1 mmol L-1 LiCl for 1 day (acute), 3 days (subacute), and 7 days (chronic). To examine the role of TRPC3, genetically stable knockdown TRPC3 cells (TRPC3Low cells) were constructed using U-87MG cells. Thrombin (2.0 U/mL)-stimulated Ca2+ mobilization was measured by ratiometric fluorimetry. Changes in TRPC3 and S100B expression levels were determined by quantitative reverse transcription-polymerase chain reaction and immunoblotting, respectively. Cell proliferation was also measured using the WST-8 assay. RESULTS: In this cell model, thrombin-stimulated Ca2+ mobilization, and both TRPC3 and S100B expression were suppressed by chronic LiCl pretreatment and the knockdown of TRPC3. Additionally, cell proliferation was attenuated in TRPC3Low cells, compared with the negative control vector-transfected cell. CONCLUSIONS: The reduced Ca2+ mobilization and S100B expression levels following chronic LiCl pretreatment and in TRPC3Low cells support the notion that TRPC3 modulates S100B expression and is the target of the LiCl effect. Downregulation of TRPC3 may be an important mechanism by which lithium ameliorates pathophysiological intracellular Ca2+ disturbances as observed in BD, accounting, in part, for its mood-stabilizing effects.

Bcl-2 SNP rs956572 associates with disrupted intracellular calcium homeostasis in bipolar I disorder.

OBJECTIVES: Disrupted intracellular calcium (Ca(2+) ) homeostasis (ICH) related to mitochondrial and/or endoplasmic reticulum (ER) dysfunction has been implicated in bipolar disorder (BD). The anti-apoptotic protein B-cell CLL/lymphoma 2 (Bcl-2), encoded in a putative BD susceptibility locus, modulates ER-Ca(2+) dynamics. Recently, an intronic single-nucleotide polymorphism (SNP) in the Bcl-2 gene, rs956572, was suggested as a functionally active SNP that influences its messenger RNA (mRNA) and protein level as well as human gray matter volume. We sought to evaluate the impact of this variant on ICH in BD. METHODS: Basal intracellular Ca(2+) concentrations ([Ca(2+) ](B) ) and rs956572 genotypes were determined in B lymphoblast cell lines (BLCLs) from bipolar I disorder (BD-I) (n=150), bipolar II disorder (BD-II) (n=65), and major depressive disorder (n=30) patients, and from healthy subjects (n=70). Bcl-2 mRNA and protein levels were determined by quantitative reverse transcriptase polymerase chain reaction and immunoblotting, respectively. Functional interactions of rs956572 with ICH were assessed by thapsigargin- and lysophosphatidic acid (LPA)-stimulated Ca(2+) responses. RESULTS: Although rs956572 variation was not significantly associated with BD, BD-I, or BD-II, BLCL [Ca(2+) ](B) was significantly higher in BD-I G/G patients compared with other genotypes and with healthy subjects. Bcl-2 mRNA and protein levels were lowest in BD-I G/G patients. Compared with A carriers, BD-I patients with G/G variants showed a modest enhancing effect on thapsigargin- and LPA-stimulated Ca(2+) responses. CONCLUSIONS: These findings support the notion that genetic variation in Bcl-2 affecting its expression impacts ICH in BD. Moreover, we show here for the first time that this interactive effect is diagnostically specific to BD-I.

CACNA1C SNP rs1006737 associates with bipolar I disorder independent of the Bcl-2 SNP rs956572 variant and its associated effect on intracellular calcium homeostasis.

OBJECTIVES: Intracellular calcium (Ca(2+)) dyshomeostasis (ICDH) has been implicated in bipolar disorder (BD) pathophysiology. We previously showed that SNP rs956572 in the B-cell CLL/lymphoma 2 (Bcl-2) gene associates with elevated B lymphoblast (BLCL) intracellular Ca(2+) concentrations ([Ca(2+)]B) differentially in BD-I. Genome-wide association studies strongly support the association between BD and the SNP rs1006737, located within the L-type voltage-dependent Ca(2+) channel α1C subunit gene (CACNA1C). Here we investigated whether this CACNA1C variant also associates with ICDH and interacts with SNP rs956572 on [Ca(2+)]B in BD-I. METHODS: CACNA1C SNP rs1006737 was genotyped in 150 BD-I, 65 BD-II, 30 major depressive disorder patients, and 70 healthy subjects with available BLCL [Ca(2+)]B and Bcl-2 SNP rs956572 genotype measures. RESULTS: SNP rs1006737 was significantly associated with BD-I. The [Ca(2+)]B was significantly higher in BD-I rs1006737 A compared with healthy A allele carriers and also in healthy GG compared with A allele carriers. There was no significant interaction between SNP rs1006737 and SNP rs956572 on [Ca(2+)]B. CONCLUSIONS: Our study further supports the association of SNP rs1006737 with BD-I and suggests that CACNA1C SNP rs1006737 and Bcl-2 SNP rs956572, or specific causal variants in LD with these proxies, act independently to increase risk and ICDH in BD-I.

Further support for association of the mitochondrial complex I subunit gene NDUFV2 with bipolar disorder.

BACKGROUND: The nuclear-encoded mitochondrial complex I subunit gene, NDUFV2, has been implicated in the pathogenesis of bipolar disorder (BD) in Japanese by virtue of association of variants in its promoter with BD and decreased NDUFV2 messenger ribonucleic acid (mRNA) levels in B lymphoblasts (BLCL) in BD patients compared to controls. We sought to determine if these same changes occur in non-Japanese populations and, if so, their relationship to altered basal intracellular Ca(2+) ([Ca(2+)](B)) in BLCL from BD patients. METHODS: Bipolar disorder patients and healthy subjects included 298 subjects of European Caucasian descent. The 5'-nuclease allelic discrimination TaqMan assay was used to detect selected single nucleotide polymorphisms (SNPs) in promoter, introns and 3'UTR regions, spanning the NDUFV2 gene. NDUFV2 mRNA levels and [Ca(2+)](B) in BLCLs were determined. RESULTS: The A allele of the NDUFV2 SNP rs1156044 was significantly associated (Bonferroni-corrected) with BD (p = 0.013) but differed in allele (rs1156044 G allele) from that previously reported as associated with BD. There was a trend for elevated BLCL [Ca(2+)](B) associated with SNP rs977581 in BD patients, but NDUFV2 mRNA levels in BLCLs did not differ between patients and controls, nor represented genotypes. CONCLUSIONS: While genetic variants of NDUFV2 may increase risk for BD, the role of its altered expression and the link to intracellular Ca(2+) abnormalities in BD remains equivocal.