Calcium channel genes associated with bipolar disorder modulate lithium's amplification of circadian rhythms

Abstract

Bipolar disorder (BD) is associated with mood episodes and low amplitude circadian rhythms. Previously, we demonstrated that fibroblasts grown from BD patients show weaker amplification of circadian rhythms by lithium compared to control cells. Since calcium signals impact upon the circadian clock, and L-type calcium channels (LTCC) have emerged as genetic risk factors for BD, we examined whether loss of function in LTCCs accounts for the attenuated response to lithium in BD cells. We used fluorescent dyes to measure Ca2+ changes in BD and control fibroblasts after lithium treatment, and bioluminescent reporters to measure Per2∷luc rhythms in fibroblasts from BD patients, human controls, and mice while pharmacologically or genetically manipulating calcium channels. Longitudinal expression of LTCC genes (CACNA1C, CACNA1D and CACNB3) was then measured over 12-24 hr in BD and control cells. Our results indicate that independently of LTCCs, lithium stimulated intracellular Ca2+ less effectively in BD vs. control fibroblasts. In longitudinal studies, pharmacological inhibition of LTCCs or knockdown of CACNA1A, CACNA1C, CACNA1D and CACNB3 altered circadian rhythm amplitude. Diltiazem and knockdown of CACNA1C or CACNA1D eliminated lithium's ability to amplify rhythms. Knockdown of CACNA1A or CACNB3 altered baseline rhythms, but did not affect rhythm amplification by lithium. In human fibroblasts, CACNA1C genotype predicted the amplitude response to lithium, and the expression profiles of CACNA1C, CACNA1D and CACNB3 were altered in BD vs. controls. We conclude that in cells from BD patients, calcium signaling is abnormal, and that LTCCs underlie the failure of lithium to amplify circadian rhythms.

Disinhibition of the extracellular-signal-regulated kinase restores the amplification of circadian rhythms by lithium in cells from bipolar disorder patients

“…While ERK1/2 could be directly affected in BD, several other genes functioning upstream of ERK1/2 have been implicated in BD [e.g. NTRK2/3 , ADCY2 , CACNA1C (McCarthy et al, 2015;. Given the polygenic nature of BD, and its phenotypic heterogeneity, it is possible that pathogenic variation could be located in one or more of these interconnected genes ( Figure S3).…”

“…Skin biopsies were obtained from BD (type I) patients who consented to research while hospitalized or participating in a clinical trial. Additional details regarding the sample have been reported previously (McCarthy et al, 2015;McCarthy et al, 2013). Use of human subjects was conducted in accordance with all pertinent regulations and approved by the VASDHS IRB.…”

“…For siRNA transfection experiments in human fibroblasts, a modified Per2::luc lentiviral reporter containing blasticidin resistance gene was used to stably express the Per2::luc reporter, allowing selection and expansion of cell lines that expressed the reporter (Liu et al, 2007). NIH3T3 cells were stably transfected with the Per2::luc reporter gene (NIH3T3 P2L ) as described previously (McCarthy et al, 2015; (Table S1) and sample rhythm traces ( Figure S1) are provided. Protein solutions were made from frozen cell pellets using ice cold lysis buffer specially designed for assessment phospho-proteins (R&D Systems).…”

“…The mood stabilizing actions of lithium have been attributed to GSK3B inhibition (Klein and Melton, 1996), and genetic variants in CACNA1C, an L-type calcium channel gene have been strongly associated with disease risk for BD . We have shown more recently that genetic variants in GSK3B (McCarthy et al, 2013) and CACNA1C (McCarthy et al, 2015) predict rhythm amplification by lithium in BD cell lines, suggesting overlap across BD susceptibility genes, regulators of circadian rhythms, and targets of lithium. However, how these molecular links are affected in BD remain incompletely described.…”

Entrainment of Circadian Rhythms to Temperature Reveals Amplitude Deficits in Fibroblasts from Patients with Bipolar Disorder and Possible Links to Calcium Channels

“…The attenuation of amplitude in modulation by lithium in BD cells has been linked to LTCC, reduced calcium signaling and genetic variation in CACNA1C and downstream deficits affecting the extracellular signal-regulated kinase (ERK) signal transduction pathway [12, 13]. In the course of this work, it was discovered that ­ CACNA1C is expressed rhythmically in fibroblasts and that, in BD patients harboring the CACNA1C risk allele rs4765913 (a common T/A SNP), the rhythmic expression of CACNA1C is greatly reduced [13]. …”