Differential responses to lithium in hyperexcitable neurons from patients with bipolar disorder

  • An Erratum to this article was published on 25 November 2015

Abstract

Bipolar disorder is a complex neuropsychiatric disorder that is characterized by intermittent episodes of mania and depression; without treatment, 15% of patients commit suicide1. Hence, it has been ranked by the World Health Organization as a top disorder of morbidity and lost productivity2. Previous neuropathological studies have revealed a series of alterations in the brains of patients with bipolar disorder or animal models3, such as reduced glial cell number in the prefrontal cortex of patients4, upregulated activities of the protein kinase A and C pathways5,6,7 and changes in neurotransmission8,9,10,11. However, the roles and causation of these changes in bipolar disorder have been too complex to exactly determine the pathology of the disease. Furthermore, although some patients show remarkable improvement with lithium treatment for yet unknown reasons, others are refractory to lithium treatment. Therefore, developing an accurate and powerful biological model for bipolar disorder has been a challenge. The introduction of induced pluripotent stem-cell (iPSC) technology has provided a new approach. Here we have developed an iPSC model for human bipolar disorder and investigated the cellular phenotypes of hippocampal dentate gyrus-like neurons derived from iPSCs of patients with bipolar disorder. Guided by RNA sequencing expression profiling, we have detected mitochondrial abnormalities in young neurons from patients with bipolar disorder by using mitochondrial assays; in addition, using both patch-clamp recording and somatic Ca2+ imaging, we have observed hyperactive action-potential firing. This hyperexcitability phenotype of young neurons in bipolar disorder was selectively reversed by lithium treatment only in neurons derived from patients who also responded to lithium treatment. Therefore, hyperexcitability is one early endophenotype of bipolar disorder, and our model of iPSCs in this disease might be useful in developing new therapies and drugs aimed at its clinical treatment.

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Figure 1: Hippocampal DG granule cell-like neurons derived from patients with BD show gene expression and mitochondrial abnormalities.
Figure 2: Hippocampal neurons derived from patients with BD show hyperexcitability.
Figure 3: Li rescues hyperexcitability in hippocampal neurons derived from iPSCs of patients with BD.
Figure 4: Somatic Ca2+ imaging analysis reveals hyperactivity in the neural network formed by the BD iPSC-derived neurons.

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Acknowledgements

We thank the patients who participated in this study. We thank M. Ku for help in the RNA-seq analysis, and L. McHenry, D. Lisuk and C. Bardy for help with the somatic Ca2+ imaging experiments. We thank L. Moore, E. Mejia, B. Miller, R. Wright, T. Berggren and S. Lu for technical assistance. We also thank C. O’Connor for help on flow cytometry. This work was supported by the National Natural Science Foundation of China (grant numbers 31471020, 31161120358, 31123004), the National Basic Research Program of China (2015CB910603, 2011CB510106), the Open Project of Key Laboratory of Genomic and Precision Medicine, Chinese Academy of Sciences, by the Engmann Foundation, the JPB Foundation, the Helmsley Trust, the Mather’s Foundation, the Glenn Foundation for Aging Research, by National Institute of Health grant MH106056 (K.J.B.), New York Stem Cell Foundation – Robertson Award (K.J.B.), and by grants/contracts to J.R.K. from the National Institute of Mental Health (U01 MH92758) supporting the Pharmacogenomics of Bipolar Disorder Study and from the Department of Veterans Affairs (5I01CX000363). K.J.B. is a New York Stem Cell Foundation - Robertson Investigator. J.Y. is an Investigator of the Young Thousand Talents Program of China.

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J.M., Q.W.W., K.E.D., L.B., K.J.B., T.J.E. and J.Y. conducted the iPSC reprogramming and differentiation experiments. Q.W.W., B.Y. and J.Y. conducted the electrophysiological recording experiments. J.M., Q.W.W., Y.Z., S.S. and J.Y. conducted immunostaining experiments. D.X.Y., J.M., Q.W.W., B.Y. S.T.S. and J.Y. conducted Ca2+ imaging experiments. Y.K., Q.W.W. and J.Y. performed mitochondrial assays. J.M., S.P., B.Y., J.Z., Y.Z., S.M. and J.Y. conducted RNA-seq and qRT-PCR analysis. J.R.K., J.I.N., J.R.C., K.J.O., M.J.M., P.P.Z., M.A., C.M.N. and the Pharmacogenomics of Bipolar Disorder Study designed and conducted the clinical trial and provided samples from patients. J.Y. designed the experiments with F.H.G. and wrote the manuscript with J.M. and Q.W.W.

Corresponding authors

Correspondence to John R. Kelsoe or Fred H. Gage or Jun Yao.

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The authors declare no competing financial interests.

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Lists of participants and their affiliations appear in the Supplementary Information.

Extended data figures and tables

Extended Data Figure 1 Generation of iPSCs from patients with BD and healthy people.

a, Human fibroblasts generated from punch biopsy. b, The iPSC colonies appeared after fibroblasts were reprogrammed using the Sendai virus. c, Purified iPSC colonies were cultured in Matrigel-coated plate. d, Immunostaining of iPSCs with DAPI and pluripotency markers Nanog and TRA-1-60. e, RT–PCR results showing that the introduced Sendai virus genes were cleared from the generated iPSCs. f, RT–PCR results showing that the generated iPSCs expressed human pluripotency markers NANOG, LIN28, OCT4, TDGF and cMYC. g, Representative karyotyping image of generated iPSCs showing normal chromosomal structure. hj, Bar graphs of quantitative RT–PCR showing that the iPSCs can randomly differentiate into cells expressing the markers for endoderm, mesoderm and ectoderm. Data are representative for a total of 20 iPS cell lines from 10 patients (2 clones per patient). Scale bar, 50 μm. Bars, mean ± s.e.m.

Extended Data Figure 2 Lentiviral transduction of Prox1::eGFP efficiently labels Prox1-positive DG granule cell-like neurons.

a, Sample immunostaining images showing the expression of Prox1 and Prox1::eGFP in the normal and BD neurons. Scale bar, 100 μm. b, Bar graph showing that, both in the normal and in BD groups, more than 90% of Prox1::eGFP-positive neurons express nuclear Prox1 protein. Normal, 92.1 ± 2.4%, n = 4 lines; BD, 93.3 ± 1.2%, n = 12 lines. c, Bar graph showing that, both in the normal and in BD groups, approximately 90% of Prox1-positive DG-like neurons express Prox1::eGFP. Bars, mean ± s.e.m.

Extended Data Figure 3 Bar graphs summarizing the similarity between different cell lines of the same subject and comparison of low and high passage cells.

a, b, Bar graph comparing the MMP (n = 20 lines) (a) and mitochondria size (n = 68 images from 20 lines) (b) of different cell lines of one subject. cf, Electrophysiological recording experiments, including peak Na+ currents (n = 92 neurons from 20 lines) (c), AP threshold (94 neurons from 20 lines) (d), total evoked AP number (n = 97 neurons from 20 lines) (e) and maximal AP amplitude (n = 96 neurons from 20 lines) (f). g, Bar graph comparing the frequency of Ca2+ transient events. Black bar, cell line/clone 1; grey bar, cell line/clone 2 (178 videos from 20 lines). h, Bar graph showing the normalized peak Na+ current in normal (NM) and BD neurons derived from <P5 and >P9 cell lines (P5: normal, n = 40 neurons from 8 lines; BD, n = 52 from 12 lines. P9: normal, n = 11 from 2 lines; BD, n = 23 from 5 lines). Student’s t-test, *P < 0.05. Bars, mean ± s.e.m.

Extended Data Figure 4 K+ currents in the BD neurons.

a, Average peak values of K+ currents in the BD and normal neurons. b, Normalized average K+ currents at different membrane potentials (normal, n = 35 neurons from 7 lines; BD, n = 41 from 10 lines). Student’s t-test. Bars, mean ± s.e.m.

Extended Data Figure 5 Prox1:eGFP expression in the BD LR and NR neurons and AP amplitude and threshold of BD neurons.

a, Sample immunostaining images showing the expression of Prox1::eGFP in the BD LR and NR neurons. Scale bar, 50 μm. b, Quantitative analysis revealed a similar percentage of Prox1::eGFP-positive DG-like neurons in the LR and NR groups (n = 32 images from 4 lines). c, d, Bar graphs showing the Li-induced effects in the maximal amplitude of evoked APs (LR without Li treatment, n = 27 neurons from 5 lines; with Li treatment, n = 18 from 5 lines) (c) and mean amplitude of spontaneous APs (LR without Li, n = 11 neurons from 3 lines; with Li, n = 10 from 3 lines) (d) of the LR neurons. e, Bar graph showing that the threshold of AP firing was not changed by Li (LR without Li, n = 11 neurons from 3 lines; with Li, n = 10 from 3 lines). Bars, mean ± s.e.m.

Extended Data Figure 6 AP firing in the BD NR neurons treated with lamotrigine (LTG).

a, Representative traces of APs evoked during 300 ms stepwise depolarization periods in the normal and NR neurons with and without 100 μm lamotrigine treatment. b, c, Bar graphs summarizing the effects of lamotrigine on the total number (b) and maximal amplitude (c) of evoked APs in the normal and BD NR neurons (normal: without lamotrigine, n = 7 neurons; with lamotrigine, n = 8. BD NR: without lamotrigine,n = 5; with lamotrigine, n = 6). Student’s t-test, *P < 0.05; **P < 0.001. Bars, mean ± s.e.m.

Extended Data Figure 7 Effect of Li on the normal neurons.

a, Representative traces of Na+/K+ currents in the normal neurons treated with Li at different concentrations. b, Representative traces of APs evoked during 300 ms stepwise depolarization periods in the normal neurons treated with Li at different concentrations. c, d, Bar graphs summarizing the effects of different concentrations of Li on the total number (c) and maximal amplitude (d) of evoked APs in the normal neurons (n = 4 neurons). Student’s t-test. *P < 0.05. Bars, mean ± s.e.m.

Extended Data Figure 8 Reversal of hyperexcitability in old BD neurons.

a, b, Sample traces (a) and scatter graph (b) showing that the 8-week-old BD neurons exhibited weaker Na+ currents than the normal neurons (normal, n = 28 neurons from 4 lines; BD, n = 37 from 6 lines). c, d, Sample traces (c) and scatter graph (d) showing that the 8-week-old BD neurons exhibited a lower frequency of Ca2+ transient events than the normal neurons (n = 30 videos from 10 patients). e, Scatter graphs showing the MMP of 6- and 8-week-old BD and normal neurons (normal, n = 3 lines; BD, n = 3 lines). Student’s t-test, *P < 0.05; **P < 0.001. Bars, mean ± s.e.m.

Extended Data Figure 9 The Prox1::eGFP-positive BD cells have similar expression of differentially expressed genes to the whole differentiation culture.

a, Sorting of cells strongly expressing Prox1::eGFP using flow cytometry. b, Bar graph showing that Prox1::eGFP expression is enriched in the selected cells. c, Enrichment of differentially expressed genes in the Prox1 + DG-like neurons (c) and non-DG cells (d) (n = 6 patients). Bars, mean ± s.e.m.

Extended Data Figure 10 Representative icons of the subjects in the figures.

a, Representative icons of the patients with BD and healthy people used in the experiments shown in the figures. Identical symbols indicate the same subject.

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Supplementary Information

This file contains Supplementary Tables 1-7 and full RT-PCR gels for Extended Data Figure 1. (PDF 1430 kb)

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Mertens, J., Wang, Q., Kim, Y. et al. Differential responses to lithium in hyperexcitable neurons from patients with bipolar disorder. Nature 527, 95–99 (2015). https://doi.org/10.1038/nature15526

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