A large-scale chemical screen for regulators of the arginase 1 promoter identifies the soy isoflavone daidzeinas a clinically approved small molecule that can promote neuronal protection or regeneration via a cAMP-independent pathway

Abstract

An ideal therapeutic for stroke or spinal cord injury should promote survival and regeneration in the CNS. Arginase 1 (Arg1) has been shown to protect motor neurons from trophic factor deprivation and allow sensory neurons to overcome neurite outgrowth inhibition by myelin proteins. To identify small molecules that capture Arg1's protective and regenerative properties, we screened a hippocampal cell line stably expressing the proximal promoter region of the arginase 1 gene fused to a reporter gene against a library of compounds containing clinically approved drugs. This screen identified daidzein as a transcriptional inducer of Arg1. Both CNS and PNS neurons primed in vitro with daidzein overcame neurite outgrowth inhibition from myelin-associated glycoprotein, which was mirrored by acutely dissociated and cultured sensory neurons primed in vivo by intrathecal or subcutaneous daidzein infusion. Further, daidzein was effective in promoting axonal regeneration in vivo in an optic nerve crush model when given intraocularly without lens damage, or most importantly, when given subcutaneously after injury. Mechanistically, daidzein requires transcription and induction of Arg1 activity for its ability to overcome myelin inhibition. In contrast to canonical Arg1 activators, daidzein increases Arg1 without increasing CREB phosphorylation, suggesting its effects are cAMP-independent. Accordingly, it may circumvent known CNS side effects of some cAMP modulators. Indeed, daidzein appears to be safe as it has been widely consumed in soy products, crosses the blood-brain barrier, and is effective without pretreatment, making it an ideal candidate for development as a therapeutic for spinal cord injury or stroke.

Figure 1.

Biological screen for inducers of Arg1. A drug library containing 2000 FDA-approved drugs, natural products, and other biologically active small molecules was screened for inducers of Arg1 using a luciferase reporter driven by 4.8 kb of the Arg1 promoter in HT22 cells. A, Schematic representation of the screening procedure. B, Representative plot of data from one 96 well plate. Molecules that induced Arg1 reporter activity by at least twofold were selected as hits. C, Daidzein concentration—response curve (μm) for activity at the Arg1 promoter. D, Protein lysates from cells treated with indentified compounds were immunoblotted for Arg1 protein levels as a secondary screen. Not all compounds identified in the reporter screen increased Arg1 protein levels. The lanes are: S, recombinant Arg1 protein; 12, 3,7-dimethyloxyflavone; 13, daidzein; 20, genistein; 31, methoxyvone; and 32, ginkgetin. E, Of the 10 compounds identified and confirmed in the secondary screen, many were flavanoids including daidzein, methoxyvone, and derrustone.

Figure 2.

Daidzein overcomes MAG inhibition through inducing Arg1 activity. A, In a blind screen of 12 compounds (3 true hits and 9 false positives), only CGNs primed with methoxyvone and daidzein overcame MAG-mediated inhibition of neurite outgrowth. Daidzein was further characterized due to its superior efficacy. B, Arg1 protein levels are increased by daidzein (20 μm) to the same extent as db-cAMP (2 mm). Arginase activity is required for daidzein-mediated axonal outgrowth on MAG-CHO cells (C–G). CGNs grown on control CHO cells incubated with NOHA (C) exhibit normal neurite outgrowth, whereas CGNs plated on MAG-CHO cells (D) fail to extend neurites. E, Priming CGNs with daidzein (20 μm) for 24 h overcomes MAG inhibition, which was reversed by the inclusion of nor-NOHA (10 μm), an inhibitor of arginase activity. G–I, Neurite outgrowth was quantitated in neurons primed with daidzein and then plated on control CHO cells (closed bars), or in neurons grown on MAG-CHO cells (diagonal bars). P0 CGNs (G), P5–P6 DRG neurons (H), or E18 cortical neurons (I) treated with daidzein were resistant to inhibition by MAG. These data were repeated in at least three independent experiments, *p < 0.001, one-way ANOVA with Tukey's post hoc test.

Figure 3.

Daidzein prevents growth cone collapse and repulsion from MAG-coated microparticles. Adult DRGs were grown for 12 h in the presence or absence of the RNA polymerase II inhibitor DRB and then treated with vehicle (DMSO) or increasing concentrations of daidzein for an additional 12 h. A, A representative micrograph of a terminal axon and growth cone, cultured in the absence of DRB, stimulated with a MAG-Fc-coated microparticle. B, In the absence of DRB, pretreatment with 40 μm daidzein prevents the axon from responding to the MAG stimulus and the axon continues to grow along its trajectory, growing past the microparticle within 120 min. C, A representative micrograph of a terminal axon and growth cone cultured in the presence of 80 μm DRB, stimulated with a MAG-Fc-coated microparticle. D, Pretreatment with DRB blocks the daidzein effect and the axon turns away from the MAG stimulus. E, Quantification of axon turning/retraction in response to microparticles loaded with MAG-Fc. Pretreatment with daidzein at 30 or 40 μm attenuates the MAG response. This attenuation is blocked by addition of the transcriptional inhibitor DRB. These data were repeated in three independent experiments, with at least 20 axons imaged per experiment, *p < 0.001, two-way ANOVA followed by Bonferroni's post hoc test.

Figure 4.

Daidzein primes in DRG neurons when administered in vivo and crosses the blood–brain barrier. Daidzein was administered as indicated, after which DRG neurons were plated on control or MAG-CHO cells for 24 h, and assayed for neurite outgrowth by βIII-tubulin fluorescence immunolabeling. A–C, To determine whether in vivo administration of daidzein primes DRG neurons to overcome MAG inhibition, we infused P28–P30 rats with daidzein intrathecally using osmotic minipumps for 24 h before removing DRG neurons for culture on CHO cells. A, Neurons from vehicle-infused animals on control CHO cells; B, neurons from vehicle-infused animals on MAG-CHO cells; C, neurons from animals infused with 40 μm daidzein on MAG-CHO cells. D–F, To test whether daidzein crossed the blood–brain barrier and was effective in priming DRG neurons when given peripherally, daidzein was administered to P28–P30 rats through subcutaneously implanted osmotic minipumps for 24 h before removal of DRG neurons. D, Neurons from vehicle-treated animas on control CHO cells; E, neurons from vehicle-treated animals on MAG-CHO cells; F, neurons from animals treated with 8 mm daidzein on MAG-CHO cells. G, H, Analysis of neurite lengths from intrathecal infusion (G) and subcutaneous administration (H) show that daidzein primes DRG neurons to overcome MAG inhibition when given in vivo (black bars, neurons plated on control CHO cells; diagonal bars, neurons plated on MAG-CHO cells). The efficacy of subcutaneous administration suggests that daidzein crosses the blood–brain barrier. Data from in vivo priming experiment represent two animals per condition from two independent experiments, *p < 0.01, one-way ANOVA with Tukey's post hoc test.

Figure 5.

Daidzein promotes optic nerve regeneration in vivo following an optic nerve crush injury. The optic nerves of P28–P30 rats were crushed approximate 2 mm behind the eye and allowed to recover for 2 weeks. Daidzein was administered intraocularly immediately following the injury, taking care to not damage the lens, or subcutaneously for the 2-week recovery period. Optic nerves were removed, sectioned, and immunolabeled for GAP43-positive axons (arrows). A, GAP43-labeled injured optic nerve from an animal that received intraocular vehicle. B, An injured optic nerve from an animal that received intraocular daidzein. Note the increase in GAP43-positive axons (arrows) growing beyond the lesion site (asterisk) in daidzein-treated animals. C, The distance of regeneration beyond the injury site of the three longest axons was measured in each section from animals treated subcutaneously with vehicle or daidzein. Importantly, these data show that daidzein is effective in an in vivo injury model when given peripherally and after the injury. Data represent mean ± SEM for 5 animals per group, *p < 0.05, one-way ANOVA with Tukey's post hoc test.

Figure 6.

Daidzein does not increase cAMP levels or induce CREB phosphorylation but requires estrogen receptor binding to activate the Arg1 promoter. A, Cortical neurons were treated with daidzein or db-cAMP (5 mm) for 24 h and then assayed for intracellular cAMP levels. In contrast to db-cAMP, daidzein did not change intracellular cAMP levels. B, Protein lysates from cortical neuron cultures treated with db-cAMP (5 mm) or daidzein (20 μm) were immunoblotted to determine CREB phosphorylation at Ser-133. A time course shows that db-cAMP maximally induced pCREB levels by 2 h, which gradually declined over 24 h. CREB phosphorylation was inhibited by H89 (10 μm), a PKA inhibitor, indicating that CREB phosphorylation was PKA dependent. In contrast, daidzein did not change pCREB levels at any time point. C, HT22 Arg1 reporter cells were treated with daidzein (20 μm) and fulvestrant, an estrogen receptor antagonist. Basal and daidzein-induced Arg1 promoter activity were inhibited by fulvestrant. D, E, 17β-Estradiol alone (D) is insufficient to activate the Arg1 promoter at concentrations that activate the ERE in HT22 cells (E). Experiments were repeated at least three times with identical results. *p < 0.001, two-way ANOVA with Bonferroni's post hoc test.

Figure 7.

Daidzein protects neurons in two models of oxidative injury. Protein lysates from spinal cord motor neurons treated with daidzein (0–20 μm) for 24 h were immunoblotted to determine Arg1 protein levels. A, Representative Western blot of Arg1 levels in motor neurons following daidzein treatment. B, Motor neurons were plated in the presence (closed bars) or absence (diagonal bars) of BDNF. Omission of BDNF causes peroxynitrite-dependent motor neuron apoptosis, which was blocked by daidzein (20 μm). C, Cortical neurons were treated with (diagonal bars) or without (closed bar) HCA, a compound that depletes intraneuronal glutathione and increases oxidative stress. Neurons treated with HCA undergo apoptosis, which is also blocked by daidzein (20 μm). Data represent mean ± SEM of 4–6 independent experiments, *p < 0.05 one-way ANOVA with Newman–Keuls post hoc test.