Genetic and clinical factors predict lithium's effects on PER2 gene expression rhythms in cells from bipolar disorder patients

Abstract

Bipolar disorder (BD) is associated with abnormal circadian rhythms. In treatment responsive BD patients, lithium (Li) stabilizes mood and reduces suicide risk. Li also affects circadian rhythms and expression of 'clock genes' that control them. However, the extent to which BD, Li and the circadian clock share common biological mechanisms is unknown, and there have been few direct measurements of clock gene function in samples from BD patients. Hence, the role of clock genes in BD and Li treatment remains unclear. Skin fibroblasts from BD patients (N=19) or healthy controls (N=19) were transduced with Per2::luc, a rhythmically expressed, bioluminescent circadian clock reporter gene, and rhythms were measured for 5 consecutive days. Rhythm amplitude and period were compared between BD cases and controls with and without Li. Baseline period was longer in BD cases than in controls. Li 1 mM increased amplitude in controls by 36%, but failed to do so in BD cases. Li 10 mM lengthened period in both BD cases and controls. Analysis of clock gene variants revealed that PER3 and RORA genotype predicted period lengthening by Li, whereas GSK3β genotype predicted rhythm effects of Li, specifically among BD cases. Analysis of BD cases by clinical history revealed that cells from past suicide attempters were more likely to show period lengthening with Li 1 mM. Finally, Li enhanced the resynchronization of damped rhythms, suggesting a mechanism by which Li could act therapeutically in BD. Our work suggests that the circadian clock's response to Li may be relevant to molecular pathology of BD.

Figure 1

Fibroblasts from BD patients have an attenuated response to Li. Vehicle, Li 1 mM or Li 10 mM was included in the growth medium for 48 h prior to, and continuously during 5 days of recording. Rhythmic Per2::luc expression was measured as counts per second (cps) with baseline subtraction. (a) Amplitude increased in cells from control (N=19, P<0.01), but not BD (N=19, P=0.32) subjects after Li 1 mM (error bars indicate 95% confidence intervals). (b) There was a non-significant trend toward greater period lengthening in controls compared with BD cells after Li 10 mM (error bars indicate 95% confidence intervals). Representative vehicle-treated (black) and Li-treated (red) rhythm traces are shown for representative control and BD cell lines at 1 mM (c, d) and 10 mM (e, f).

Figure 2

Rhythm modulation by Li is associated with clock gene variants. (a) Period lengthening after Li 1 mM for RORA rs12912233 homozygous common (T/T), heterozygous (C/T) and homozygous variant (C/C) genotypes [N=5/11/3 (controls), N=7/9/3 (BD), P<0.05]. (b) Period lengthening after Li 10 mM for PER3 rs228729 homozygous common (T/T), heterozygous (T/C) and homozygous variant (C/C) genotypes [N=6/12/1 (controls), N=11/6/2 (BD), P<0.001]. In both cases (a,b), genotype effects were significant regardless of diagnosis so BD cases and controls have been combined. (c) Amplitude increase after Li 1 mM among GSK3β −50T/C homozygous common (T/T) and rare variant carriers (T/C and C/C). Two-way ANOVA revealed a significant genotype × diagnosis interaction (P<0.05), but no significant difference between genotypes or diagnoses alone. (d) Period lengthening after Li 10 mM. Two-way ANOVA revealed effect of BD (P<0.05), and significant genotype × diagnosis interaction (P<0.001). For homozygous common/heterozygous/homozygous variant genotypes, N=8/11/0 (controls) and N=9/9/1 (BD), respectively. Error bars indicate s.e.m.

Figure 3

Suicide in BD is associated with period lengthening and amplitude increase after Li 1 mM. When measured categorically, BD patients with a history of suicide were more sensitive to (a) period lengthening, but not (b) amplitude increase after treatment with Li 1 mM. P-values indicate the results of 1-way ANOVA with N=6 PSA positive/13 PSA negative/19 controls. Error bars indicate s.e.m. When measured quantitatively, suicide history is significantly correlated with both (c) period lengthening and (d) amplitude increase after Li 1 mM.

Figure 4

Li improves rhythm resynchronization. Cellular rhythms (N=10 each BD and control) were measured without treatment until damped (dashed line, day 1–5 in panel a). In order to restore rhythms, medium change with Li 10 mM (red) or vehicle (black) was performed on day 5.5 (arrow in panel a). Omitting the signal artifact associated with media change, rhythms were then measured another 2 days (solid line in panel a). After media change, Li-treated cells had (b) significantly higher rhythm amplitudes (P<0.05), and (c) lower periods (P<0.001) compared with vehicle-treated cells. Following media change, period in Li-treated cells was in the circadian range (23.1±0.5 h (Ave±s.e.m.)); whereas period in the vehicle-treated cells was not (31.1±1.9 h (Ave±s.e.m.)).