Fig 2: Reduced phosphorylation of CRMP2 at Thr514 upon treatment with CHIR-99021 and lithium.a CRMP2 was identified with reduced phosphorylation at Thr514 as previously reported in Tobe et al.15. A bar graph of CRMP2T514 phosphopeptide ratios in CHIR-99021-treated over control is shown. The UniProt gene symbol for CRMP2 is DPYSL2. b Schematic of key phosphorylation events on CRMP2 involving CDK5 and GSK3ß. CDK5 phosphorylates CRMP2 at Ser522, providing a primed substrate for subsequent phosphorylation by GSK3 at Ser518, Thr514, and Thr509. Phosphorylation of CRMP2 at Thr514 has been shown to be GSK3ß-specific. The phosphorylation events are essential for diverse CRMP2 cellular functions. c Western blot analysis validated that p-CRMP2T514 levels were decreased after 6-h treatment of NPCs with CHIR-99021 in a dose-dependent manner. Total CRMP2 protein was not affected by the treatments. d Lithium treatment in NPCs for 6 h effectively reduced p-CRMP2T514 levels in a dose-dependent manner. e CHIR-99021 and f lithium also exhibited dose-dependent reduction of p-CRMP2T514 on the 4-week differentiated neuronal cultures. Total CRMP2 protein was not affected by the treatments.

Fig 3: High-content screen for CRMP2-phosphorylation modulators.a Z' factor = 0.6 was calculated with 10 µM CHIR-99021 used as positive control in the high-content screen. b High-content screen of 240 compounds for p-CRMP2T514 modulators (n = 3 replicates). GSK3 inhibitors are most effective modulators down-regulating p-CRMP2T514 levels to below 80%, indicated by blue bracket. Smaller effect is detected for a group of potential hits (green bracket). c Graph of the data for GSK3 inhibitors from the screen. d Re-test of a group of novel modulators that are lysine deacetylase inhibitors. Crebinostat, a novel HDAC inhibitor demonstrated to enhance fear conditioning86, was included though it was not present in the library. All compounds are retested at 10 µM except crebinostat at 1 µm. Lithium’s effect at 1 mM was set to 100%. e Re-test of other novel modulators (10 µM), some of which are involved in regulation of neurotransmission. In vitro GSK3ß enzymatic assay showing no direct GSK3ß inhibitory activity detected for the group of compounds. Unpaired t-test: *0.01 = p < 0.05, **0.001 = p < 0.01, ***0.0001 = p < 0.001, ****p < 0.0001. f Chemical structures of novel p-CRMP2T514 modulators that are non-lysine deacetylase inhibitors. g Chemical–protein interaction network presented by STITCH reveals connectivity of novel p-CRMP2T514 modulators to lysine deacetylation and neurotransmission.

Fig 4: Expression levels of TLR4 and phosphorylation of CRMP2 in the cortex via western blotting. (A) Western blotting was performed to reveal the expression levels of TLR4 and p-CRMP2 in each group. (B) Quantitative analysis of TLR4 expression relative to ß-actin from western blotting as presented in (A). (C) Quantitative analysis of p-CRMP2 expression relative to ß-actin from western blotting a presented in (A). Data are expressed as the mean ± standard deviation (n=5). ??P<0.01 vs. the normal group; #P<0.05 vs. the MCAO group; *P<0.05 vs. the LPS group; $P<0.05 vs. the LPS+neutral group. LPS, lipopolysaccharide; p-CRMP2, phosphorylated collapsin response mediator protein 2; MCAO, middle cerebral artery occlusion and reperfusion group; Normal, rats that did not undergo MCAO surgery; LPS+neutral, rats treated with LPS and TLR-neutralizing antibody prior to MCAO; Saline, rats administered an intracerebroventricular injection of saline prior to MCAO; Neutral, rats treated with TLR-neutralizing antibody only prior to MCAO; Sham, rats that underwent surgery without MCAO; TLR4, Toll-like receptor 4.

Fig 5: Nogo-66 inhibits neurite outgrowth and induces Aß production by regulating ROCK2 protein expression and CRMP2 phosphorylation. (A, C and E) Cortical neurons were treated with various concentrations of Nogo-66. Western blot analysis results showing the protein expression levels of ROCK2, p-CRMP2 and CRMP2. Equal protein loading was confirmed using GAPDH. Data are presented as the mean OD values ± SD of triplicate samples. **P<0.01, ***P<0.001 vs. control. (B, D and F) N2a cells were treated with various concentrations of Nogo-66. Western blot analysis results showing the protein expression levels of ROCK2, p-CRMP2 and CRMP2. Equal protein loading was confirmed using GAPDH. Data are presented as the mean OD values ± SD of triplicate samples. *P<0.05, ***P<0.001 vs. control. Aß, ß-amyloid protein; N2a, Neuro2a; ROCK2, Rho-associated coiled-coil containing kinase 2; CRMP2, collapsin response mediator protein-2; p-, phosphorylated; OD, optical density.