Cell death in mouse neurons and NPCs. To establish models of neuronal cell death and lithium neuroprotection, we tested the effects of STS on NPCs and IMHNs. After confirming NPC identity using markers of neuronal lineage (Figure S1), microscopic examination of control NPCs indicated that STS reduced cell count and that lithium generally attenuated STS-induced cell loss (Fig. 1A). We then conducted additional biochemical experiments to quantify cell loss and neuroprotection. In IMHN, STS treatment for 20 h caused quantifiable cell death that was significantly attenuated by co-treatment with lithium in a concentration-dependent manner (Fig. 1B). We next used STS to induce caspase activation and apoptosis in NPCs from BD patient and controls. After a brief 6 h exposure to STS, caspase activation in NPCs (N = 3 control, 2 Li-R, 3 Li-NR), increased by approximately five-fold and viability decreased by approximately 20% (Fig. 1C-D). After a longer 20 h STS exposure, caspase activity increased further by approximately ten-fold, and viability decreased further to approximately 50% compared to control levels (Fig. 1C-D). At both times, co-treatment of NPCs with lithium at a therapeutically relevant concentration (1 mM) was neuroprotective. At 6 h after STS, despite having relatively little effect on caspase activity, lithium increased viability, with 90% of NPCs remaining viable (Fig. 1E-F). At 20 h, lithium significantly mitigated this cell loss and improved viability by an average of approximately 5% compared to vehicle co-treatment (Fig. 1E-F). For both caspase activity and viability, there were main effects of lithium treatment and time, but there were no significant group differences between control and BD, or Li-R/Li-NR (Fig. 1C-F). To further characterize neuronal mechanisms underlying apoptosis in BD and control cells, we examined BCL2 expression, a putative mediator of lithium neuroprotection [37]. Consistent with past reports, BCL2 expression was decreased by STS and this effect was uniformly reversed by lithium. There were no significant group differences between Control, Li-R and Li-NR NPCs (Fig. 1G). We conclude from these results that both IMHN and human iPSC-derived NPC recapitulate key aspects of lithium’s neuroprotective effects in vitro using the STS apoptosis model.
Clock gene knockdown and viability. To determine the contributions of individual clock genes to apoptosis, we next knocked down expression of BMAL1, PER1 and REV-ERBα in IMHN prior to STS. For each gene, knockdown caused specific changes in the cell death responses to STS. After PER1 knockdown in IMHN, viability was modestly decreased compared to controls at baseline, but knockdown did not affect the response to STS (Fig. 2A). In contrast, knockdown of BMAL1 and REV-ERBα both significantly reduced viability in vehicle- and STS-treated IMHN (Fig. 2B-C). Knockdown of both BMAL1 and REV-ERBα independently reduced viability, and following STS, knockdown additively increased cell death (Fig. 2B, 2D). Following REV-ERBα knockdown, there was an additional significant siRNA x STS interaction, whereby REV-ERBα knockdown modestly attenuated the amount of cell death caused by STS: -17% viability for REV-ERBα vs. -25% for sham (Fig. 2C, 2D). Having established these findings in IMHNs, we next conducted similar experiments in control and BD patient NPCs, including Li-R and Li-NR. Compared to control siRNA, PER1 knockdown increased cell death, in both vehicle- and STS-treated NPCs. However, the amount of additional cell death differed significantly by group (Fig. 2E). Viability decreased the most in NPCs from Li-NR (-12.9%) compared to Li-R (-7.4%) and controls (-6.2%). Following BMAL1 knockdown (Fig. 2F), viability was decreased − 16.1% in controls. In Li-NR, viability was significantly lower (-31.9%), while in Li-R NPCs, BMAL1 knockdown increased viability (+ 21.2%). REV-ERBα knockdown caused a modest increase in viability in control NPCs after STS (+ 7.6%) but as in IMHN, caused a nominal decrease in viability in the two BD groups (-1.2% to -2.0%). The overall effect of REV-ERBα knockdown was significantly different between control and BD samples, but not between Li-R and Li-NR (Fig. 2G). These results reveal differences in the role of clock genes regulating cell death in BD patient NPCs that associate with lithium response.
Pharmacological modulators of the circadian clock. Given the indications that there may be distinct contributions to cell survival from clock genes in NPC from BD patients, we next used pharmacological modulators of the circadian clock to further investigate these interactions. Both in IMHN and NPC, we first tested lithium, a drug that has effects on REV-ERBα [26]. In cellular rhythm assays, lithium increased amplitude in IMHN, similar to its effects on rhythms in other cells [13] (Figure S1A). In NPCs, lithium increased amplitude in control, but not Li-R or Li-NR samples (Figure S1B-D). We next tested four drugs that target the REV-ERB and ROR nuclear receptors. Compared to NPC, IMHN are better suited to studies using multiple drug conditions in parallel and were the focus of these experiments. The REV-ERB agonist GSK4112 also increased rhythm amplitude, whereas the REV-ERB antagonist SR8278 decreased amplitude. The ROR agonist SR1078 decreased amplitude, whereas the ROR inverse-agonist SR1001 had no effect on rhythms (Fig. 3). Since GSK4112 and lithium both increased circadian rhythm amplitude, we hypothesized that these shared amplitude-increasing effects may be relevant for neuroprotection and focused on GSK4112 in subsequent studies.
Neuroprotective properties of a REV-ERB agonist. Knockdown of REV-ERBα altered cell death responses in IMHN and NPC. Moreover, the REV-ERB agonist GSK4112 and lithium both increase amplitude, perhaps indicating neuroprotection may be related to the strength of circadian rhythms. Therefore, we hypothesized that a REV-ERB agonist may be neuroprotective. To test this, we conducted viability experiments with GSK4112 both in IMHN and NPCs. Compared STS exposure alone, co-treatment with GSK4112 and STS increased IMHN viability in a concentration dependent manner that was statistically significant at 10 µM (Fig. 4). In NPCs from controls, GSK4112 (10 µM) had a similar protective effect, significantly increasing viability after STS exposure by 10%, similar to the effects observed in IMHN. However, in NPCs from either BD group (Li-R and Li-NR), GSK4112 had no effect on viability, leading to a statistically significant group difference between control and BD in the protective benefit from the drug (Fig. 4, p < 0.005).
Mechanisms of REV-ERB agonist neuroprotection in NPCs. The differences in neuroprotective effects of GSK4112 in control and BD NPC were evaluated further to identify potential underlying mechanisms. In NPCs from control and BD (Li-R and Li-NR), we compared the effects of STS and GSK4112 (alone and in combination) on the expression of nine genes selected for their involvement in cell death and neuroprotection (Fig. 5). Most of the genes selected (7/9) showed significant differences in expression after drug treatments, indicating that the drug interventions effectively engaged the intended target genes. Following drug treatment, BAD, BCL2, BMAL1, BRCA1, CASP1, CHEK2 and IL6 showed significantly altered expression (all p < 0.05 in 2-way ANOVA, main effect of drug). An overlapping, but distinct set of genes (7/9) showed significant group differences and differed among control, Li-R and/or Li-NR samples: BAD, BMAL1, BRCA1, CASP1, CHEK2, IL6 and TP53. ATM expression did not differ by group (trend p = 0.05) or drug alone, but revealed a significant drug x group interaction. Based on the findings that GSK4112 increased viability after STS treatment in control, but not in BD samples, we looked for gene expression patterns that correlated with these viability findings. Following treatment with STS, IL6 expression was massively upregulated in NPCs from controls, but significantly less so in BD cells (Control 2210% vs 188% Li-R and 766% in Li-NR). Following combined treatment with STS + GSK4112, BAD expression directly correlated with the viability results, with significantly increased levels in control NPC, but not in either BD group (Control 313%, Li-R 124% and Li-NR 60%). Other gene expression patterns inversely correlated with that viability effects using the same drugs. BMAL1, BRCA1, CHEK2 and TP53 were all downregulated by combination treatment with STS + GSK4112 in controls, but upregulated by STS + GSK4112 in BD NPCs (Li-R and Li-NR, Fig. 5). Interestingly, other effects on gene expression of combined treatment of STS + GSK4112 did not correlate well with viability following STS + GSK4112, but showed significant differences between Li-R and Li-NR, and distinguished the BD sub-groups: CASP1, BAD, BMAL1 and TP53.