Discovery of suppressors of CRMP2 phosphorylation reveals compounds that mimic the behavioral effects of lithium on amphetamine-induced hyperlocomotion

Abstract

The effective treatment of bipolar disorder (BD) represents a significant unmet medical need. Although lithium remains a mainstay of treatment for BD, limited knowledge regarding how it modulates affective behavior has proven an obstacle to discovering more effective mood stabilizers with fewer adverse side effects. One potential mechanism of action of lithium is through inhibition of the serine/threonine protein kinase GSK3β, however, relevant substrates whose change in phosphorylation may mediate downstream changes in neuroplasticity remain poorly understood. Here, we used human induced pluripotent stem cell (hiPSC)-derived neuronal cells and stable isotope labeling by amino acids in cell culture (SILAC) along with quantitative mass spectrometry to identify global changes in the phosphoproteome upon inhibition of GSK3α/β with the highly selective, ATP-competitive inhibitor CHIR-99021. Comparison of phosphorylation changes to those induced by therapeutically relevant doses of lithium treatment led to the identification of collapsin response mediator protein 2 (CRMP2) as being highly sensitive to both treatments as well as an extended panel of structurally distinct GSK3α/β inhibitors. On this basis, a high-content image-based assay in hiPSC-derived neurons was developed to screen diverse compounds, including FDA-approved drugs, for their ability to mimic lithium's suppression of CRMP2 phosphorylation without directly inhibiting GSK3β kinase activity. Systemic administration of a subset of these CRMP2-phosphorylation suppressors were found to mimic lithium's attenuation of amphetamine-induced hyperlocomotion in mice. Taken together, these studies not only provide insights into the neural substrates regulated by lithium, but also provide novel human neuronal assays for supporting the development of mechanism-based therapeutics for BD and related neuropsychiatric disorders.

Clinical evidence that a dysregulated master neural network modulator may aid in diagnosing schizophrenia

“…These observations in brain suggested to us that, if elevated phosphorylated CRMP2 (and an elevated p-CRMP2:CRMP2 ratio) could be a BPD-specific biomarker [as we previously reported ( 14 , 20 )], then an elevated nonphosphorylated CRMP2 (and a diminished ratio) might serve as a biomarker for SCZ. We sought to support that speculation prospectively in not only a larger and better stratified patient population (particularly with regard to age), but also by going to a clinically accessible cell type in living patients, PBMCs.…”

“…With an excess of active (nonphosphorylated) CRMP2-in contrast to the equilibrium between active and inactive (phosphorylated) CRMP2 that normally exists-neural network function, particularly at dendritic spines, might be expected to be aberrant (14). Notably, the lower p-CRMP2:CRMP2 ratio in SCZ contrasted dramatically with the higher ratio previously reported by us in patients with LiR BPD (14,20), enhancing the potential utility of this diagnostic aid.…”

“…In the brain, these changes are associated with abnormalities in dendritogenesis. In contrast to other psychiatric disorders, including those in which CRMP2 has been implicated [e.g., LiR BPD ( 14 , 20 )], the higher active CRMP2 level with a constant inactive p-CRMP2 level makes for a unique lowering of the p-CRMP2:CRMP2 ratio below that of unaffected age-matched control subjects. This disruption in the normal equilibrium between active and inactive CRMP2 might plausibly lead to neural network imbalances.…”

“…These alterations of phosphorylation status affect regulation of cytoskeletal dynamics. “Toggling” between inactive (phosphorylated) and active (nonphosphorylated) CRMP2 is physiologic ( 14 , 20 ). However, an abnormal balance between active and inactive CRMP2—too much of one or the other—could plausibly lead to aberrations in synaptogenesis, synapse maturation, and synaptic transmission ( 11 , 28 , 32 ).…”

“…Phenotypic analysis of crmp1 and crmp2 gene-deficient mice ( crmp −/− and crmp2 −/− , respectively) revealed that both of these gene-deficient animals show behavioral abnormalities that model neuropsychiatric symptoms ( 15 – 17 ). We recently implicated aberrant CRMP2 posttranslational regulation as central to the pathogenesis of lithium-responsive (LiR) bipolar disorder (BPD) ( 14 , 20 ). Specifically, the “set-point” for the ratio of phosphorylated (inactive)-to-nonphosphorylated (active) CRMP2 was abnormally high in LiR BPD human brains and patient-specific human induced pluripotent stem cell (hiPSC)-derived neurons; lithium, a CRMP2 pathway modulator, normalized that set-point by reducing the levels of phosphorylated CRMP2 (pCRMP2) and, concomitantly, dendritic structural pathology and neuronal hyperexcitability.…”