Enhanced Neuronal Survival and Neurite Outgrowth Triggered by Novel Small Organic Compounds Mimicking the LewisX Glycan

Abstract

Glycosylation fine-tunes signal transduction of adhesion molecules during neural development and supports synaptic plasticity and repair after injury in the adult nervous system. One abundantly expressed neural glycan is LewisX (LeX). Although it is known that its expression starts at the formation of the neural tube during the second embryonic week in the mouse and peaks during the first postnatal week, its functional relevance is only rudimentarily understood. To gain better insights into the functions of this glycan, we identified small organic compounds that mimic structurally and functionally this glycan glycosidically linked to several neural adhesion molecules. Mimetic compounds were identified by competitive enzyme-linked immunosorbent assay (ELISA) using the LeX-specific monoclonal antibodies L5 and SSEA-1 for screening a library of small organic molecules. In this assay, antibody binding to substrate-coated LeX glycomimetic peptide is measured in the presence of compounds, allowing identification of molecules that inhibit antibody binding and thereby mimic LeX. Gossypol, orlistat, ursolic acid, folic acid, and tosufloxacin inhibited antibody binding in a concentration-dependent manner. With the aim to functionally characterize the molecular consequences of the compounds' actions, we here present evidence that, at nM concentrations, the mimetic compounds enhance neurite outgrowth and promote neuronal survival of cultured mouse cerebellar granule cells via, notably, distinct signal transduction pathways. These findings raise hopes that these LeX mimetics will be powerful tools for further studying the functions of LeX and its effects in acute and chronic nervous system disease models. It is worth mentioning in this context that the LeX compounds investigated in the present study have been clinically approved for different therapies.

Keywords: Glycomimetic peptide; LewisX mimetics; Neurite outgrowth; Neuronal survival; Signal transduction; Small organic compounds.

Fig. 1

Concentration-dependent binding of the LeX glycomimetic peptide and the LeX mimetic compounds gossypol, folic acid, orlistat, to sufloxacin, and ursolic acid to the LeX-specific monoclonal antibody SSEA-1. a The graph shows a binding curve of the SSEA-1 antibody in different concentrations (5–120 μM) to the substrate-coated linear LeX glycomimetic peptide. The binding curve is fitted to a logarithmic regression function. b LeX-specific antibody SSEA-1 was preincubated with different concentrations (1–80 μM) of gossypol (red), folic acid (blue), orlistat (orange), tosufloxacin tosylate (purple), ursolic acid (green), or the control compound idarubicin (black) and added to wells coated with the LeX glycomimetic peptide. The signal of LeX-specific SSEA-1 antibody alone without any competing chemical compound was set on 100%. Asterisks signify differences in competition between all LeX mimetic compounds and the negative control compound idarubicin as calculated by one-way analysis of variance (ANOVA) followed by Fisher’s PLSD test (F =9.468, p = 0.0001; PLSD *p<0.05). a, b Mean values are shown from eight wells in four independent experiments. c Chemical structure of the LeX epitope and the LeX mimetic compounds gossypol, folic acid, orlistat, tosufloxacin tosylate, and ursolic acid

Fig. 2

Gossypol, but not orlistat, ursolic acid, folic acid, and tosufloxacin, is toxic to cultured cerebellar granule cells at 50 μM. Representative images of cerebellar granule cells treated only with vehicle DMSO (control), or treated with 50 μM LeX mimetic compounds and stained with calcein-AM (green) and propidium iodide (red). Bar diagram shows percentage of viable cells (mean + SEM, 36 images captured from nine wells carried out in three independent experiments) treated with different concentrations of the mimetics or treated only with vehicle DMSO (control). Asterisk signifies differences between the vehicle control group and the LeX mimetic compound-treated group as determined by one-way ANOVA followed by Fisher’s PLSD test (F = 140.61, p < 0.0001; PLSD *p < 0.05). Scale bar, 20 μm

Fig. 3

LeX mimetics enhance neuronal survival of stressed cerebellar granule cells. Bar diagram shows the percentage of viable cerebellar granule cells (mean + SEM, 60 images captured from 15 wells carried out in five independent experiments) treated with different concentrations of the linear LeX glycomimetic peptide or with different concentrations of the LeX mimetic compounds and subsequently stressed with 20 μM H₂O₂. The hash sign indicates significant differences between the unstressed group (control, − H₂O₂) and stressed group (control, + H₂O₂). Asterisks signify differences between the stressed group (control, + H₂O₂) and compound-treated groups as determined by oneway ANOVA with Fisher’s PLSD test (F = 1.61, p <0.0329; PLSD *p <0.05)

Fig. 4

LeX mimetics enhance neurite outgrowth of cerebellar granule cells. Representative images of primary cultured cerebellar granule cells treated only with vehicle DMSO (control), or treated with the LeX mimetic compounds. Bar diagram shows average longest neurite lengths (300 neurons from six wells out of three independent experiments, mean + SEM) exposed to different concentrations of the linear LeX glycomimetic peptide (LeX) or the LeX mimetic compounds. Asterisks signify differences versus vehicle control (control) as determined by one-way ANOVA with Fisher’s PLSD test (F =27.744, p < 0.0001; PLSD *p < 0.05). Scale bar, 20 μm

Fig. 5

LeX mimetics trigger cell signaling that is required for neurite outgrowth and neuronal survival. a, b Cerebellar granule cells were pretreated with different inhibitors of signal transducing molecules. Shown are inhibitors (KT 5720 (PKA), HBDDE (PKC), TBCA (CKII), Erk inhibitor III (Erk), PP121 (Src), 1-naphthyl PP1 (Fyn), bpV(HOpic) (PTEN)) versus cells not treated with inhibitor (control, black bars). After exposure to the inhibitors, cells were treated with the linear LeX glycomimetic peptide (LeX) (80 μM) or with LeX mimetic compounds (1 nM). a Bar diagram shows the average percentage ofviable cells (mean + SEM, 48 images captured from 12 wells carried out in four independent experiments) that were stressed with H₂O₂. b Bar diagram shows average longest neurite lengths (300 neurons from six wells out of three independent experiments, mean + SEM). Asterisks signify differences between cells only treated with the mimetics (control, black bars) and cells pretreated with the inhibitors as determined by oneway ANOVA with Fisher’s PLSD test (a F =3.542, p < 0.0001; b F = 9.505, p <0.0001; PLSD *p <0.05)