Circadian rhythms in bipolar disorder patient-derived neurons predict lithium response

Himanshu K. Mishra, Noelle M. Ying, Angelica Luis, Heather Wei, Metta Nguyen, Timothy Nakhla, Sara Vandenburgh, Martin Alda, Wade H. Berrettini, Kristen J. Brennand, Joseph R. Calabrese, William H. Coryell, Mark A. Frye, Fred H. Gage, Elliot S. Gershon, Melvin G. McInnis, Caroline M. Nievergelt, John I. Nurnberger, Paul D. Shilling, Ketil J. Oedegaard, Peter P. Zandi, The Pharmacogenomics of Bipolar Disorder Study, John R. Kelsoe, David K Welsh, Michael J. McCarthy

Preprint posted on 15 December 2020

Broken clocks and mood swings: The link between circadian clock changes and Lithium treatment in Bipolar disorder patients.

Selected by Sejal Davla

Categories: cell biology, neuroscience


Sleep and circadian rhythm disruptions are highly prevalent in patients with neuropsychiatric disorders. Bipolar disorder (BD) is one of the major neuropsychiatric illnesses characterized by cognitive dysfunction, mood changes and sleep/activity pattern shifts. Insomnia and poor sleep quality are some of the most widespread symptoms in BD and these sleep disturbances are often good predictors of mood swings. Hence establishing the stability of sleep-wake and circadian cycles is a key therapeutic strategy in BD patients1.

Lithium is a wonder drug that everyone knows about as a mood stabilizer in BD patients. Lithium is shown to be a definitive neuroprotective agent in BD with beneficial effects in the prevention of severe mood changes, psychosis and suicide2. While Lithium continues to be the main pharmacotherapy drug for the treatment of BD, 30-40% of patients fail to respond to the treatment. BD is a complex, polygenic disorder where patients display heterogeneous genotypic and phenotypic disparities. Lithium responsiveness, therefore, can be an alternate strategy to reduce heterogeneity in clinical studies.

Lithium treatment altered phase and period of circadian activity patterns in many animal models and fibroblast cells harvested from BD patients. Besides, chronotype is a predictor of Lithium response where patients with ‘high morningness’ show higher Li responsiveness3. While animal models are informative to chart out behavioral and pathological hallmarks of Li responsiveness, human cellular models are irreplaceable in understanding biological mechanisms of Lithium response in BD patients.

Patient-derived induced pluripotent stem cells (iPSCs) provide an excellent tool to characterize the etiology of any human disease. A recent preprint from Mishra et al investigates the cellular and molecular changes in circadian rhythmicity from patient-derived iPSCs from Lithium-responsive and non-responsive BD patients.

About the preprint

Circadian rhythm is a ubiquitous, cell-autonomous process that displays an oscillation of around 24-hours. Circadian rhythms are generated and regulated by circadian clock genes whose mRNA and protein products oscillate in a periodic manner. The clock genes are expressed in all tissues but are required specifically in central clock neurons that communicate with each other to generate a synchronous circadian output. How does the clock function in the neurons of BD patients had not been investigated before. The authors in this manuscript use live imaging and gene expression analyses to identify the relationship between Lithium response and circadian rhythm disruption in BD patients.

The authors tested a cohort of BD patients and chose the most and least lithium responsive people to harvest skin cells for the study. Bioluminescence reporters are crucial in the studies of circadian molecular oscillators. The authors used luciferase activity of core clock gene PER2 in iPSC-derived neuronal precursor cells (NPCs) and glutamatergic neurons to compare and contrast the hallmarks of circadian rhythmicity in healthy vs lithium-responsive and non-responsive BD patients.

Time series analysis

Using live imaging time series analysis, Mishra et al unraveled profound circadian rhythm disruptions in BD cells, particularly in cells from Lithium non-responders. Control and Lithium-responsive NPCs and neurons displayed robust, high amplitude period rhythms whereas cells from lithium non-responders showed weak, low-amplitude rhythms and faster rhythm dampening. The only identifiable difference between control and Lithium responder cells was observed in NPCs where period length was significantly shorter in BD NPCs compared to controls.

Another crucial difference in rhythmicity was identified in healthy control cell lines where differentiated mature neurons were more rhythmic compared to NPCs. Meanwhile, the overall proportion and rhythmicity of neurons remained similar to NPCs in BD cell lines. The authors further analyzed cells that were highly rhythmic to identify phase differences. Control NPCs and neurons showed clustered phase compared to BD cells with dispersed phase. When the authors treated these cells with Lithium, they further identified a concentration-dependent effect on period lengthening in control and Lithium responder cells. As expected, Lithium treatment had no effect on Lithium non-responder cells.

Gene expression changes

The authors also analyzed the levels of seven core clock genes that provided the contribution of the molecular clock network in the circadian disruption phenotypes observed in the luciferase assay. Among the clock genes, BD cells showed a significant increase in PER2 mRNA and protein levels. The overexpression of PER2 which is a negative regulator of the clock might explain the weak rhythmicity phenotype in BD cells. Among the BD cohort, the Lithium responder neurons had higher CRY1 and BMAL1 mRNA expression compared to the Lithium non-responder neurons. This particular observation might pave the way in developing new targets for drug therapies to correct circadian rhythmicity defects in BD patients.

Circadian Entrainment

The ability to synchronize circadian activity at different temperatures is an important feature of circadian systems. When the authors used a temperature ramp entrainment protocol where cells experienced different temperatures every 12 hours, all groups displayed an increase in the amplitude of the rhythm and a more synchronized and lasting rhythm. This result is valuable for the Lithium non-responsive BD cohort in the management of circadian disruptions in using temperature entrainment protocols.

Why I chose this preprint

Lithium is an old drug with promising beneficial effects not just in BD but also in depression, psychosis and a number of neurodegenerative disorders. The molecular and cellular targets of Lithium’s action are numerous. Work from McCarthy and colleagues directly demonstrates circadian disruptions in patient-derived neurons. I chose to highlight this preprint because it undoubtedly provides an important framework for future large-scale studies in iPSC cell lines to characterize circadian disruptions in BD patients with varying genetic backgrounds. Further, the use of the circadian entrainment technique to improve cellular rhythmicity and stabilize sleep disturbances in Lithium non-responsive BD patients might lead to a game-changing therapeutic strategy.

Questions to the authors

  1. Were the Lithium responder BD patients on Lithium therapy when their skin cells were harvested for iPSC? This clarification will be helpful to interpret the nature of Lithium effect on circadian rhythms (short-term vs long-term).
  2. What percentage of Lithium non-responder neurons showed increased amplitude under temperature ramp conditions?
  3. Did the temperature entrainment alter PER2 levels in Lithium non-responsive neurons?
  4. Are there any genetic forms of BD that show higher Lithium non-responsiveness?


  1. Steardo L Jr, de Filippis R, Carbone EA, Segura-Garcia C, Verkhratsky A, De Fazio P. Sleep Disturbance in Bipolar Disorder: Neuroglia and Circadian Rhythms. Front Psychiatry. 2019;10:501. Published 2019 Jul 18. doi:10.3389/fpsyt.2019.00501
  2. Machado-Vieira R, Manji HK, Zarate CA Jr. The role of lithium in the treatment of bipolar disorder: convergent evidence for neurotrophic effects as a unifying hypothesis. Bipolar Disord. 2009;11 Suppl 2(Suppl 2):92-109. doi:10.1111/j.1399-5618.2009.00714.x
  3. McCarthy, M.J., Wei, H., Nievergelt, C.M. et al. Chronotype and cellular circadian rhythms predict the clinical response to lithium maintenance treatment in patients with bipolar disorder. Neuropsychopharmacol 44, 620–628 (2019).



Posted on: 19 January 2021


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Author's response

Michael McCarthy shared

We are really excited that the method we developed to study rhythms in human clinical samples using fibroblasts translated so well into the iPSC-neuron model. It will add nicely to the experimental tools available to researchers to study other neuronal models of psychiatric disorders. The demonstration of rhythm deficits in bipolar disorder neurons shows that the dimension of time is important to consider in future studies.

Questions to the authors

  1.  Were the Lithium responder BD patients on Lithium therapy when their skin cells were harvested for iPSC? This clarification will be helpful to interpret the nature of Lithium effect on circadian rhythms (short-term vs long-term).
    Yes- all of the patients, both responders and non-responders were on lithium when they gave us the biopsy.  However, we don’t think the exposure to lithium at the time of the biopsy plays a major role in our findings. The cells were cultured and passaged several times before the experiments were performed, as well as re-programmed into iPSC, then differentiated into neurons. After all of that, the residual amount of lithium in the cell cultures would be expected to be vanishingly low, and too low for a pharmacological effect of lithium. We can’t rule out that lithium exposure caused some other kind of long-lasting changes to the cells (i.e. epigenetic modifications), but we are confident we washed out any transient drug effects. We expect that the effects we saw relate more to stable genetic differences in the cells that impact circadian rhythms and/or bipolar disorder.
  2. What percentage of Lithium non-responder neurons showed increased amplitude under temperature ramp conditions?  The sample size is too small to say with any certainty. This is an area we hope to study more in the future.
  3. Did the temperature entrainment alter PER2 levels in Lithium non-responsive neurons? Previous work in mice has shown that entrainment to light causes upregulation of another clock gene, PER1. A similar mechanism may be present in the temperature cycle experiments we conducted in neurons, but we did not measure gene expression or protein for any of the clock genes under these conditions.
  4. Are there any genetic forms of BD that show higher Lithium non-responsiveness? Genome-wide association studies have shown genetic differences between lithium responders and non-responders (see Hou et al Lancet 2016, Amare et al JAMA Psych 2017, Amare et al Mol Psych 2020). There are some clinical features that may predict a good response as well including strong family history, euphoric manias, and a symptom course of mania followed by depression.


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