Fig 1: Targeting CRMP2 prevents activity-dependent increase in neurite outgrowth. (A) Timeline of experimental procedures. (B–E) Representative tracings of cortical neurons expressing EGFP and incubated for 96 h in vehicle, 25 mM KCl, 200 µM (S)-LCM, or 25 mM KCl + 200 µM (S)-LCM. (F) Total outgrowth of cortical neurons exposed to 25 mM KCl in the presence or absence of 200 µM (S)-LCM. (G) In naïve neurons, KCl exposure increased outgrowth by ~40% compared to vehicle. Co-application of (S)-LCM blunted the KCl-induced increase to ~10% (*p < 0.05 compared to control; Student's t-test) (n = 110–379 cells, across 8 separate culture wells) (scale bar = 50 µm).
Fig 2: Interaction of CRMP2 with brain mitochondria. (A–C) Immunocytochemistry staining with anti-CRMP2 antibody (A, red) and visualization of mitochondria with mitochondrially targeted enhanced yellow fluorescent protein (mito-eYFP) (B, green) illustrates co-localization of CRMP2 and mitochondria (yellow, C) in cultured striatal neuron from FVB/NJ mouse. (D,E), CRMP2 binds to synaptic brain mitochondria isolated from FVB/NJ mice. Mitochondria were purified either on discontinuous 26/40% Percoll gradient (D) or on continuous 30% Percoll gradient (E) as described in the Materials and Methods. H: homogenate; C: cytosolic fraction; Mtc: mitochondrial fraction. MEK1/2, calnexin, ß-tubulin, Histone H3, and COX IV are cytosolic, ER, microtubule, nuclear, and mitochondrial markers, respectively. Representative western blots from 3 independent experiments are shown.
Fig 3: Alterations of CRMP2 expression and phosphorylation levels in L5 DRG neuron subpopulations. Bar graphs showing the comparison between contralateral and ipsilateral side of a spared nerve injury of total CRMP2 or CRMP2 p522 signal depending of NF200, NaV1.8 of IB4 neuronal subpopulations. a total CRMP2 level in NF200 neurons (contra, n = 1257 individual neurons, ipsi n = 1187 individual neurons). b CRMP2 p522 level in NF200 neurons (contra, n = 1040 individual neurons, ipsi n = 899 individual neurons). c total CRMP2 level in NaV1.8 neurons (contra, n = 1369 individual neurons, ipsi n = 1528 individual neurons). d CRMP2 p522 level in NaV1.8 neurons (contra, n = 1408 individual neurons, ipsi n = 885 individual neurons). e total CRMP2 level in IB4 neurons (contra, n = 772 individual neurons, ipsi n = 1251 individual neurons). f CRMP2 p522 level in IB4 neurons (contra, n = 902 individual neurons, ipsi n = 906 individual neurons). *p < 0.05; Student’s t-test compared to the contralateral side
Fig 4: CRMP2 expression and phosphorylation is detected in L5 DRG neuron subpopulations. Micrographs of a 10 µm section of adult L5 DRG from animals having received a spared nerve injury and immunostained with antibodies against CRMP2, CRMP2 p522, NF200, NaV1.8 or for the lectin IB4 as indicated. Micrographs are representative of N = 4 animals with 8 independent slices per animal per staining combination; ipsi = ipsilateral (injured side) and contra = contralateral (non-injured side)
Fig 5: Increased CRMP2 and Drp1 phosphorylation in human striatal neurons derived from induced pluripotent stem cells (iPSCs) from fibroblasts of HD patients compared to striatal neurons from unaffected individuals (control). (S)-lacosamide ((S)-LCM) prevented CRMP2 hyperphosphorylation but failed to diminish Drp1 phosphorylation in HD neurons. In these experiments, human neurons from three control cell lines (hFib2-iPS5, TiPS5, and miPS-2) and three HD cell lines (HD-iPS4, CS04iHD-46n10, and CS03iHD-53n2) were used. With neurons from miPS-2 cell line (control) and CS03iHD-53n2 cell line (HD), immunoblotting experiments were performed twice. Scale bar = 50 µm (A,C,E), representative immunocytochemistry images of MAP2 expression in control, HD, and HD neurons, treated with 10 µM of (S)-LCM for the last 7 days prior to imaging, respectively. (B,D,F), representative immunocytochemistry images of DARPP32 (the striatal marker) expression in control, HD, and HD neurons, treated with 10 µM of (S)-LCM for the last 7 days prior to imaging, respectively. (G), representative Western blots of lysates prepared with human neurons from HD patients and unaffected individuals. Where indicated, neurons were treated with 10 µM of (S)-LCM for the last 7 days prior to analysis. GAPDH and ß-actin are loading controls. (H–L), densitometry data. Here and in other Figure legends, the colored circles indicate data from individual measurements. (H,I), pCRMP2 normalized by total CRMP2. Data are mean ± SD, N = 4, ** p < 0.01 comparing HD neurons with and without treatment with (S)-LCM, *** p < 0.001 comparing control and HD neurons. (J), pDrp1 normalized by total Drp1. Data are mean ± SD, N = 4, *** p < 0.001 comparing control with HD and HD + (S)-LCM neurons. (K,L), total CRMP2 normalized by GAPDH and total Drp1 normalized by ß-Actin, respectively, N = 4.
Supplier Page from MilliporeSigma for Anti-CRMP2 antibody produced in rabbit