Journal: Cells

Article Title: Involvement of CRMP2 in Regulation of Mitochondrial Morphology and Motility in Huntington’s Disease

doi: 10.3390/cells10113172

Figure Lengend Snippet: CRMP2 hyperphosphorylation in postmortem brain tissues of HD patients compared to postmortem brain samples of unaffected individuals. ( A ), Western blots with pCRMP2 antibodies for Thr 509/514 and Ser 522, total CRMP2, and GAPDH (loading control). Molecular weight markers (kilodalton, kDa) are indicated in the left margin of the blots. ( B – G ), densitometry data. All samples were immunoblotted three times ( B , D , F ), and then individual data were averaged across the different blots ( C , E , G ). Here, and in other figures, the data from individual experiments are shown as the colored circles. In ( B , D , F ), pCRMP2 are normalized by total CRMP2 and total CRMP2 are normalized by GAPDH in 9 individual samples from 4 control and 5 HD human brain samples, respectively. Here, and in other figures, the phosphorylation sites are indicated in the panels. Data are mean ± SD, N = 3 technical replicates (3 separate immunoblotting experiments). In ( C , E ), averaged pCRMP2 are normalized by total CRMP2 from individual samples pooled together for statistical analysis. Data are mean ± SD. N = 4–5, data are pooled together from separate immunoblotting experiments with 4 control and 5 HD human brain samples shown in ( B , D ), *** p < 0.001 comparing brain tissue from HD patients (HD) and unaffected individuals (control). In G , total CRMP2 normalized by GAPDH from experiments shown in ( F ). Data are mean ± SD. N = 4–5 separate immunoblotting experiments with 4 control and 5 HD human brain samples.

Article Snippet: After blocking, blots were incubated with either rabbit anti-CRMP2 (Sigma, St. Louis, MO, USA, Cat # C2993, 1:1000), sheep anti-CRMP2 pThr 509/514 (KinaSource, Dundee, UK, Cat # PB-043, 1:1500), rabbit anti-CRMP2 pSer522 (ECM Biosciences, Cat # CP2191, 1:1500), mouse anti-GAPDH (Abcam, Cambridge, MA, USA, Cat # ab9484, 1:2000), rabbit anti-Drp1 pSer 616 (Cell Signaling, Danvers, MA, USA, Cat# 3455, 1:1000), mouse anti-β-actin (ThermoFisher Scientific, Carlsbad, CA, USA, Cat# MA5-15739, 1:1000), or rabbit anti-Miro 2 (Proteintech, Rosemont, IL, USA, Cat # 11237-1-AP, 1:1000) antibodies.

Techniques: Western Blot, Molecular Weight

Journal: Cells

Article Title: Involvement of CRMP2 in Regulation of Mitochondrial Morphology and Motility in Huntington’s Disease

doi: 10.3390/cells10113172

Figure Lengend Snippet: 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 β-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 β-Actin, respectively, N = 4.

Article Snippet: After blocking, blots were incubated with either rabbit anti-CRMP2 (Sigma, St. Louis, MO, USA, Cat # C2993, 1:1000), sheep anti-CRMP2 pThr 509/514 (KinaSource, Dundee, UK, Cat # PB-043, 1:1500), rabbit anti-CRMP2 pSer522 (ECM Biosciences, Cat # CP2191, 1:1500), mouse anti-GAPDH (Abcam, Cambridge, MA, USA, Cat # ab9484, 1:2000), rabbit anti-Drp1 pSer 616 (Cell Signaling, Danvers, MA, USA, Cat# 3455, 1:1000), mouse anti-β-actin (ThermoFisher Scientific, Carlsbad, CA, USA, Cat# MA5-15739, 1:1000), or rabbit anti-Miro 2 (Proteintech, Rosemont, IL, USA, Cat # 11237-1-AP, 1:1000) antibodies.

Techniques: Derivative Assay, Western Blot, Immunocytochemistry, Expressing, Imaging, Marker

Journal: Cells

Article Title: Involvement of CRMP2 in Regulation of Mitochondrial Morphology and Motility in Huntington’s Disease

doi: 10.3390/cells10113172

Figure Lengend Snippet: Increased CRMP2 ( A ) and Drp1 ( B ) phosphorylation in cultured striatal neurons from YAC128 mice compared to striatal neurons from FVB/NJ mice. (S)-lacosamide ((S)-LCM) prevented CRMP2 hyperphosphorylation but failed to diminish Drp1 phosphorylation in YAC128 neurons. YAC128 neurons were treated with 10 µM of (S)-LCM for the last 7 days prior to analysis. GAPDH and β-actin are loading controls. In ( A ) and ( B ), representative Western blots are shown. In ( C – I ), densitometry data are shown. ( C – G ), pCRMP2 normalized by total CRMP2 ( C – F ) and total CRMP2 normalized by GAPDH ( G ), respectively. In ( H , I ), pDrp1 normalized by total Drp1 and total Drp1 normalized by β-Actin, respectively. Data are mean ± SD, N = 5 separate experiments. ( C ), ** p < 0.01, comparing FVB/NJ neurons and YAC128 neurons; comparing YAC128 neurons with and without (S)-LCM treatment. ( D ), * p < 0.05 comparing FVB/NJ and YAC128 neurons, ** p < 0.01, comparing YAC128 neurons with and without (S)-LCM treatment. ( E ), * p < 0.05 comparing FVB/NJ and YAC128 neurons, ** p < 0.01, comparing FVB/NJ neurons with YAC128 neurons treated with (S)-LCM. ( F , H ), *** p < 0.01, comparing FVB/NJ neurons and YAC128 neurons with and without (S)-LCM treatment.

Article Snippet: After blocking, blots were incubated with either rabbit anti-CRMP2 (Sigma, St. Louis, MO, USA, Cat # C2993, 1:1000), sheep anti-CRMP2 pThr 509/514 (KinaSource, Dundee, UK, Cat # PB-043, 1:1500), rabbit anti-CRMP2 pSer522 (ECM Biosciences, Cat # CP2191, 1:1500), mouse anti-GAPDH (Abcam, Cambridge, MA, USA, Cat # ab9484, 1:2000), rabbit anti-Drp1 pSer 616 (Cell Signaling, Danvers, MA, USA, Cat# 3455, 1:1000), mouse anti-β-actin (ThermoFisher Scientific, Carlsbad, CA, USA, Cat# MA5-15739, 1:1000), or rabbit anti-Miro 2 (Proteintech, Rosemont, IL, USA, Cat # 11237-1-AP, 1:1000) antibodies.

Techniques: Cell Culture, Western Blot