Significance One-third of bipolar disorder (BPD) patients are lithium-responsive (LiR) for unknown reasons. Were lithium’s target to be identified, then BPD’s pathogenesis might be unraveled. We identified and mapped the “lithium-response pathway,” which governs the phosphorylation of CRMP2, a cytoskeleton regulator, particularly for dendritic spines: hence, a neural network modulator. Although “toggling” between inactive (phosphorylated) and active (nonphosphorylated) CRMP2 is physiologic, the “set-point” in LiR BPD is abnormal. Lithium (and other pathway-modulators) normalize that set-point. Hence, BPD is a disorder not of a gene but of the posttranslational regulation of a developmentally critical molecule. Such knowledge should enable better mechanistically based treatments and bioassays. Instructively, lithium was our “molecular can-opener” for “prying” intracellularly to reveal otherwise inscrutable pathophysiology in this complex polygenic disorder.

Abstract The molecular pathogenesis of bipolar disorder (BPD) is poorly understood. Using human-induced pluripotent stem cells (hiPSCs) to unravel such mechanisms in polygenic diseases is generally challenging. However, hiPSCs from BPD patients responsive to lithium offered unique opportunities to discern lithium's target and hence gain molecular insight into BPD. By profiling the proteomics of BDP–hiPSC-derived neurons, we found that lithium alters the phosphorylation state of collapsin response mediator protein-2 (CRMP2). Active nonphosphorylated CRMP2, which binds cytoskeleton, is present throughout the neuron; inactive phosphorylated CRMP2, which dissociates from cytoskeleton, exits dendritic spines. CRMP2 elimination yields aberrant dendritogenesis with diminished spine density and lost lithium responsiveness (LiR). The “set-point” for the ratio of pCRMP2:CRMP2 is elevated uniquely in hiPSC-derived neurons from LiR BPD patients, but not with other psychiatric (including lithium-nonresponsive BPD) and neurological disorders. Lithium (and other pathway modulators) lowers pCRMP2, increasing spine area and density. Human BPD brains show similarly elevated ratios and diminished spine densities; lithium therapy normalizes the ratios and spines. Consistent with such “spine-opathies,” human LiR BPD neurons with abnormal ratios evince abnormally steep slopes for calcium flux; lithium normalizes both. Behaviorally, transgenic mice that reproduce lithium's postulated site-of-action in dephosphorylating CRMP2 emulate LiR in BPD. These data suggest that the “lithium response pathway” in BPD governs CRMP2's phosphorylation, which regulates cytoskeletal organization, particularly in spines, modulating neural networks. Aberrations in the posttranslational regulation of this developmentally critical molecule may underlie LiR BPD pathogenesis. Instructively, examining the proteomic profile in hiPSCs of a functional agent—even one whose mechanism-of-action is unknown—might reveal otherwise inscrutable intracellular pathogenic pathways.

Although human induced pluripotent stem cells (hiPSCs) have proven valuable for studying the molecular pathology of monogenic diseases, one of the technique’s greatest challenges has been to offer similar insights into the molecular pathogenesis of polygenic, multifactorial disorders for which the underlying pathophysiology is unknown. The struggle has been to go beyond phenotypic description to discerning underlying molecular mechanisms. Neuropsychiatric illnesses are a prototype for such complex conditions (1⇓–3). They are difficult to model not only because of the likelihood of polygenic influences, but also because of the subjectivity with which these diseases must often be diagnosed, the empirical fashion with which drugs are prescribed, and the heterogeneity of patient response. Of such maladies, bipolar disorder (BPD) type 1, a chronic illness of episodic mania with intervening periods of depression for which the interplay between genetic and environmental factors is poorly understood, is unique in that, for unclear reasons, ∼35% of patients respond to monotherapy with lithium salts (4⇓⇓–7); indeed, lithium responsiveness (LiR) is often regarded as pathognomic of BPD. However, despite our knowledge of lithium’s ubiquitous multisystemic influences and pleiotropic actions (4), the molecular mechanism underlying this drug responsiveness specifically in BPD, as well as BPD’s molecular pathogenesis, are poorly understood. The former, however, could lend insight into the latter. For example, although lithium may suppress hyperexcitability of a subset of neurons in culture (2) [many mechanisms have been proffered (4)], clinical trials have shown that drugs that simply suppress neuronal activity, such as calcium channel blockers, are ineffective in BPD (7). Furthermore, of the one-third of patients who are LiR, many become noncompliant because of frequent adverse side effects (e.g., weight gain, hypothyroidism, tremor, kidney dysfunction, dermatologic reactions, teratogenicity). Such pleiotropic effects underscore our ignorance with regard to lithium’s action specifically for BPD. Additionally, the safety index of lithium is narrow (5, 8). In view of its prevalence (the sixth leading cause of disability worldwide), suboptimal treatment options, and absence of biomarkers for onset and progression, neuropsychiatric disorders in general—and BPD in particular—represent a pressing unmet medical need (1 in 250 sufferers die from complications of BPD). Two obstacles to developing safer, more effective mood stabilizers have been a lack of known clinically relevant molecular drug targets and of drug-screening assays that are rooted in the molecular pathogenesis and pathophysiology of the disorder. Although heritability of BPD is ∼80%, few disease-specific gene associations have been identified with sufficient consistency and statistical significance to guide further studies (9, 10); multiple loci are more likely to contribute to LiR than any single reliable genetic marker, making it challenging for hiPSC disease-modeling technology. The approach presented here might help address these challenges.

Because most of lithium’s actions have been linked to posttranslational regulation rather than to transcription (4), we elected to start with an unbiased differential proteomic approach. Thus, whereas lithium’s action as a modifier of kinase signaling has been described for numerous substrates (4), precisely how phosphorylation plays a role, and what the substrate of that phosphorylation might be that is relevant to BPD, are not understood. Here we describe inroads into probing, mapping, and understanding the regulation of the molecular “lithium-response pathway” in BPD initially using proteomic profiling (by two independent techniques) of patient-derived hiPSCs to identify putative lithium targets, followed by bioinformatic pathway analyses to determine the hierarchy and convergence of these candidates. We validated our conclusions in: (i) biochemical analyses comparing hiPSC-derived neurons from LiR, lithium-nonresponsive (LiNR), and unaffected individuals (as well as those with other psychiatric and neurological conditions); (ii) assays of neuronal function; (iii) neurocytological and behavioral analyses of transgenic mice in which the pathway’s putative central node is eliminated or lithium’s putative site-of-action is reproduced; and (iv) biochemical and histological assessment of primary human patient brain specimens. Extrapolating from the mediators of LiR to conclusions regarding the molecular underpinnings of BPD, our data implicate not a gene defect per se, but rather aberrant posttranslational modification of a developmentally critical molecule: an abnormally high phosphoregulatory set-point for the central cytoskeletal modulator Collapsin Response Mediator Protein-2 (CRMP2) (11⇓⇓⇓⇓–16) which, by determining CRMP2’s active state, in turn influences dendritic form and function and hence, presumably, neural network development and activity.

Discussion In summary, through a combination of unbiased, differential proteomic and bioinformatic pathway analyses of hiPSC-derived NPCs and neurons from LiR BPD patients (and control patients with other disorders, including LiNR BPD and other psychiatric and neurological conditions), followed by node-by-node mapping, animal modeling, functional validation in vitro and in vivo, and corroboration in human BPD postmortem brains, our results suggest that the molecular lithium-response pathway in BPD acts via CRMP2 to alter neuronal cytoskeletal dynamics, most particularly dendrite and dendritic spine formation, and presumably function: hence, neural network development and activity. By “mapping” the upstream and downstream interactors of CRMP2, we observed that lithium does not impact its direct upstream activator, collapsin (SEMA3A), but does regulate GSK3β and AKT kinases, the arrestin–PP2A complex, and hence, the phospho-sites (e.g., T514, S522) that govern CRMP2’s central role in cytoskeleton regulation. The phosphorylation state of CRMP2 (influenced by both GSK3β-dependent and -independent pathways) determines its association with cytoskeletal elements: nonphosphorylated active CRMP2 binds them, phosphorylated inactive CRMP2 dissociates from them. Our observations in hiPSCs and then in human postmortem brain specimens suggests that the inactive CRMP2-p-T514:active CRMP2 ratio set-point is uniquely elevated in LiR BPD patients. Lithium lowers this ratio to a level observed in unaffected patients. Nullifying CRMP2 function entirely by KO elicits dendrite and spine pathology. It also eliminates lithium’s increase of spine density. Abrogating phosphorylation of CRMP2 at lithium’s postulated site of action, hence emulating lithium’s proposed action, also reproduces lithium’s therapeutic action in accepted behavioral models of LiR BPD. Data from primary BPD patient brains further confirm the predicted link between abnormally elevated CRMP2-p-T514 and dendritic spine abnormalities, as well as evidence that lithium treatment of patients acts to normalize both CRMP2 ratios and dendritic spine density and length. We emphasize that these actions of lithium in BPD do not rule out potential roles for that cation’s multiple other actions (4⇓⇓–7), which may function additively or synergistically in this condition. We now simply identify a promising regulatable molecular pathway upon which to focus etiologically and pharmacologically. Elevated baseline CRMP2-p-T514 might be associated with LiR as a clinical classification of BPD (potentially a biomarker). (Prospective studies in large cohorts of living patients will inevitably be required to help define what the critical clinical threshold for a CRMP2-p-T514:CRMP2 ratio should be.) Our data cannot yet determine whether the CRMP2-p-T514:CRMP2 set-point is chronically high in LiR BPD patients or rather that the response of LiR BPD patients to stimuli that increase CRMP2-p-T514 is more pronounced, prolonged, or of earlier onset than in unaffected patients, or a combination of these. Nevertheless, these results may provide impetus for biomarker assay development, for example measuring CRMP2-p-T514:CRMP2 ratios in reprogrammed patient-derived cells (including those obtained from the peripheral blood, like some of ours) as a diagnostic aid to predict drug responsiveness. A qualitative, not just quantitative, distinction between LiR and LiNR BPD based on an abnormally high set-point for an otherwise physiologic posttranslational modification of a cytoskeletal regulator (uniquely in LiR BPD) invites speculation that LiNR BPD is actually a separate disease that “pheno-copies” BPD but is unrelated pathophysiologically to the lithium-response pathway. (See SI Appendix, Figs. S9 and S12 for further discussion of this possibility based on biochemical and functional data, respectively.) Although CRMP2 may play a role in other neuropsychiatric diseases—for example, total CRMP2 levels may be abnormal in postmortem brains of schizophrenics—aberrantly elevated inactive:active CRMP2 ratios with excessively high CRMP2-p-T514 seems specific to LiR BPD. The lithium-response pathway impinging on CRMP2 likely has additional modes of input that go beyond those illustrated in Fig. 1B. For example, brain-derived neurotrophic factor (BDNF) also reduced CRMP2-p-T514 in a manner similar to lithium (SI Appendix, Fig. S13A), suggesting that systems downstream of tyrosine receptor kinase B (TRKB, the BDNF receptor) might be integrated into the pathway (SI Appendix, Fig. S13B). Moreover, CRMP2 has been associated with tauopathies via its interactions with microtubule-associated protein tau and presenillin 1. Indeed, CRMP2 interacts with amyloid precursor protein, and has been noted to be a component of neurofibrillary tangles in Alzheimer’s disease (41). Hence, not only might this axis be implicated in diseases other than BPD (cytoskeletal dynamics coming to be recognized as central to a growing number of neuropathological processes), but also drugs that lower CRMP2-p-T514 may potentially be more widely therapeutic (particularly in disorders characterized by deposition of cytoskeletal elements such as tau either as a cause or a biomarker). If our premise is correct that identifying a therapeutic pathway also implicates that pathway as central to that disease’s pathogenesis, then certain broader ideas warrant mentioning. First, whereas BPD has heritable features and developmental underpinnings, it would appear to be a disorder not of a defective gene per se but rather of dysregulated posttranslational modulation of a normally produced gene product that, although developmentally critical, plays a physiologic role in all individuals throughout life. Second, attributing pathophysiology to aberrant cytoskeletal dynamics (dendritic disorganization representing one consequence) would appear to implicate not merely defective neurons but also dysregulated interneuronal networks. Whether these observations are particular to LiR BPD or are applicable more widely to other neuropsychiatric disorders warrants study. With regard to disease modeling in general, this study suggests a strategy for merging hiPSC technology with proteomics to discern underlying pathophysiological mechanisms in complex, polygenic, multifactorial diseases in which causative genes, cells, proteins, and pathways are not well-understood. If there exists an agent that is known to be functionally impactful even if its molecular mechanism-of-action is uncertain (like lithium in BPD), such an agent may allow an investigator to probe otherwise inscrutable intracellular signaling by identifying its target and then reconstructing the regulatory molecular routes upstream and downstream of that node with an eye toward mapping underlying pathogenic pathways and identifying more specific drug targets for the development of safer, cheaper, or more effective pharmacotherapeutics. In this way, hiPSCs may be used in the most challenging diseases not only to reflect phenomenology and a phenotype, but also to identify underlying molecular mechanisms.

Materials and Methods Human fibroblasts or lymphoblasts from multiple well-sourced patients (SI Appendix, Fig. S1) were reprogrammed to hiPSCs via nonintegrating episomal-mediated (42), lentivirus-mediated (1), or retrovirus-mediated (19) gene transfer, characterized (43), and differentiated to NPCs and cortical interneurons, as per our routine and as previously described (17⇓⇓–20). Protein isolation, 2D-DIGE and SILAC, Western blotting, and coimmunoprecipitation were performed as described previously (17, 28). Immunofluorescence was quantified by pixel number captured via image analysis software using unbiased stereology. All MS data are publicly accessible; for SILAC data, the mass spectra may be downloaded from MassIVE (massive.ucsd.edu) using accession no. MSV000080975; the data are directly accessible via ftp://massive.ucsd.edu (MSV000080975). Creation and analysis of CRMP2 knockout and knockin mice used standard transgenic techniques. Analysis of primary cultures of rodent hippocampal neurons also followed standard techniques (44). Ca i 2+ transients and flux were measured via kinetic imaging analysis (34, 35). Animal modeling of BPD behavior (to determine the response to lithium-response pathway manipulation) was as described previously (37⇓–39). All animal use was conducted in accordance with the NIH guidelines and approved by the Yokohama City University Institutional Animal Care and Use Committee. Human postmortem material was obtained from the University of Pittsburgh and from the McLean Hospital. Patient samples were obtained following informed consent; all patient identification was removed and the material was processed according to IRB approvals including from Nova Scotia Health Authority Research Ethics Board, National Institutes of Mental Health, and the Sanford-Burnham Prebys Medical Discovery Institute. Dendrite and dendritic spine morphology were assessed as per routine procedures with Golgi stains and image-assisted quantification (44, 45). Bioinformatic analysis was performed as per the Sullivan Lab Evidence Project, ProtKIN, and IPA. See SI Appendix, Supplemental Methods for details.

Acknowledgments We thank M. Niepel, N. Moerke, C. Shamu, and P. Sorger for kinase perturbagen screens; J. Wong and V. Chen for help with Ca i 2+ imaging; S. Ghose and the Harvard Brain Tissue Resource Center for access to postmortem brains. This study was supported by Grant RC2MH090011 (to E.Y.S.); NIH’s Library of Integrated Network-based Cellular Signatures Program (E.Y.S.); the Viterbi Foundation Neuroscience Initiative (E.Y.S.); Stanley Medical Research Institute Grants R21MH093958, R33MH087896, and R01MH095088 (to S.J.H.); the Tau Consortium (S.J.H.); NIH Grant R01MH087823 (to S.H.); California Institute of Regenerative Medicine training grants (to B.T.D.T., L.D., and C.D.); a University of California, San Diego T32 training grant in psychiatry (to B.T.D.T.); the California Bipolar Foundation; the International Bipolar Foundation; and Creation of Innovation Centers for Advanced Interdisciplinary Research Areas Program in the Project for Developing Innovation Systems from the Ministry of Education, Science, Sports and Culture in Japan (Grant 42890001) (Y.G.). Dedicated to the memory of Dr. Jeffrey Nye and his contributions to neuropsychopharmacology.

Footnotes Author contributions: B.T.D.T., A.M.C., H. Makihara, F.N., G.J.G., L.M.B., J.S.N., H. Manji, J.H.P., C.A.M., H.S.A., M.A., D.-M.M.C., Y.L., Y.D.T., R.L.S., S.H., S.J.H., Y.G., and E.Y.S. designed research; B.T.D.T., A.M.C., A.M.W., B.C., W.-n.Z., J. Lalonde, H.N., G.K., M.S., C.D.P., N.Y., M.W., Y.I., S.D.S., R.W.L., M.B., D.W., J.H., L.D., C.D., R.C.B.B., M.J.M., N.D.U., P.M., S.A.C., G.A.R., L.M., J. Li, G.J.G., and J.T.C. performed research; B.T.D.T., A.M.C., A.M.W., M.B., D.W., J.H., J. Li, N.V., G.C., M.A., Y.L., T.O., and K.M. contributed new reagents/analytic tools; B.T.D.T., A.M.C., C.D.P., M.B., M.J.M., R.L.S., S.H., S.J.H., Y.G., and E.Y.S. analyzed data; B.T.D.T., R.L.S., and E.Y.S. wrote the paper; and B.T.D.T., A.M.W., B.C., M.B., S.J.H., Y.G., and E.Y.S. generated figures.

Reviewers: M.P., Sahlgrenska Academy at University of Gothenburg; and D.E.R., Yale University School of Medicine.

Conflict of interest statement: R.C.B.B., L.M.B., G.C., J.S.N., H.M., and J.H.P. are employees of private companies. Their role in the study was solely as researchers with no financial or proprietary involvement.

Data deposition: All MS data are publicly accessible; for SILAC data, the mass spectra may be downloaded from MassIVE, massive.ucsd.edu (accession no. MSV000080975); the data are directly accessible via ftp://massive.ucsd.edu/MSV000080975.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1700111114/-/DCSupplemental.