G . Western blotting and quantitative analysis of MBP protein levels from homogenates of organotypic cerebellar slices at 6 dpl cultured in absence or in presence of 100 μΜ D - Asp Data information : The values represent the means ± S . E . M . Level of significance was determined by usin G , one - way ANOVA P = 0 . 003 followed by Bonferroni post hoc test , * P < 0 . 05 versus control , ˄ P < 0 . 05 versus LPC ( n =

O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-EXP_ASSAY O O O O B-TISSUE O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O

G. Western blotting and quantitative analysis of MBP protein levels from homogenates of organotypic cerebellar slices at 6 dpl cultured in absence or in presence of 100 μΜ D-Asp Data information: The values represent the means ± S.E.M. Level of significance was determined by usin G, one-way ANOVA P=0.003 followed by Bonferroni post hoc test, *P<0.05 versus control, ˄P< 0.05 versus LPC (n=3


H . Scatter plot histogram analysis of the remyelination index in cerebellar explants at 6dpl after LPC exposure cultured in absence or in presence of 100 μΜ D - Asp . Data were normalized to vehicle control Data information : The values represent the means ± S . E . M . Level of significance was determined by using ) ; H , one - way ANOVA P < 0 . 001 followed by Bonferroni post hoc test , * P < 0 . 05 versus controls , ˄ P < 0 . 05 versus LPC ( n = 4 animals , 3 - 4 slices per group )

O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O B-TISSUE O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-ORGANISM O O O O O O O O

H. Scatter plot histogram analysis of the remyelination index in cerebellar explants at 6dpl after LPC exposure cultured in absence or in presence of 100 μΜ D-Asp. Data were normalized to vehicle control Data information: The values represent the means ± S.E.M. Level of significance was determined by using ); H, one-way ANOVA P<0.001 followed by Bonferroni post hoc test, *P<0.05 versus controls, ˄P< 0.05 versus LPC (n=4 animals, 3-4 slices per group)


I . Maximum intensity projection of z - stack confocal images displaying MBP ( red ) and NF200 ( green ) immunoreactivities in control cerebellar organotypic slices ( b - d ) , and in cerebellar slices at 10 dpl in absence ( f - h ) or in presence of 100 μM D - Asp ( j - l ) . Panels d , h , and l display colocalized points ( white ) . Panels a , e , and i show representative low magnification images of 7 DIV cerebellar slices cultured cultures in absence ( a ) or in presence of 100 μM D - Asp ( e ) . Scale bars in a , e , i : 200 μm ; in b - d , f - h , and j - l : 50

O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O B-TISSUE O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-TISSUE O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I. Maximum intensity projection of z-stack confocal images displaying MBP (red) and NF200 (green) immunoreactivities in control cerebellar organotypic slices (b-d), and in cerebellar slices at 10 dpl in absence (f-h) or in presence of 100 μM D-Asp (j-l). Panels d, h, and l display colocalized points (white). Panels a, e, and i show representative low magnification images of 7 DIV cerebellar slices cultured cultures in absence (a) or in presence of 100 μM D-Asp (e). Scale bars in a, e ,i: 200 μm; in b-d, f-h,and j-l: 50 μm


J . Scatter plot histogram analysis of the remyelination index in cerebellar explants at 10 dpl cultured in absence or in presence of 100 μΜ D - Asp . Data were normalized to vehicle control Data information : The values represent the means ± S . E . M . Level of significance was determined by usin J , one - way ANOVA P < 0 . 001 followed by Bonferroni post hoc test , * P < 0 . 05 versus controls , ˄ P < 0 . 05 versus LPC ( n = 4 animals , 3 - 4 slices per group )

O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O B-TISSUE O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-ORGANISM O O O O O O O O

J. Scatter plot histogram analysis of the remyelination index in cerebellar explants at 10 dpl cultured in absence or in presence of 100 μΜ D-Asp. Data were normalized to vehicle control Data information: The values represent the means ± S.E.M. Level of significance was determined by usin J, one-way ANOVA P<0.001 followed by Bonferroni post hoc test, *P<0.05 versus controls, ˄P< 0.05 versus LPC (n=4 animals, 3-4 slices per group)


A . RT - PCR of CNPase mRNA expression in oligodendrocyte MO3 . 13 progenitors under control conditions and following 200 μM D - Asp exposure for 3 days , in absence or in presence of 10 μM MK - 801 ( left panel ) , or 30 nM YM - 244769 or 100 nM BED ( right panel ) . Graphs show quantification of ratio of CNPase to L19 Data information : The values represent the means ± S . E . M from 3 independent experimental sessions . Level of significance was determined by using : in A , left , one - way ANOVA P = 0 . 009 followed by Tukey ' s post hoc test , * P < 0 . 05 versus control , ˄ P < 0 . 05 versus D - Asp ( n = 3 ) ; A , right , one - way ANOVA P = 0 . 0001 followed by Tukey ' s post hoc test , * P < 0 . 05 versus control , ˄ P < 0 . 05 versus D - Asp ( n = 3

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O

A. RT-PCR of CNPase mRNA expression in oligodendrocyte MO3.13 progenitors under control conditions and following 200 μM D-Asp exposure for 3 days, in absence or in presence of 10 μM MK-801 (left panel), or 30 nM YM-244769 or 100 nM BED (right panel). Graphs show quantification of ratio of CNPase to L19 Data information: The values represent the means ± S.E.M from 3 independent experimental sessions. Level of significance was determined by using: in A, left, one-way ANOVA P=0.009 followed by Tukey's post hoc test, *P< 0.05 versus control, ˄P< 0.05 versus D-Asp (n=3); A, right, one-way ANOVA P=0.0001 followed by Tukey's post hoc test, *P< 0.05 versus control, ˄P< 0.05 versus D-Asp (n=3


B . RT - PCR of MBP mRNA expression in MO3 . 13 cells under control conditions and following D - Asp exposure for 3 days , in absence or in presence of 10 μΜ MK - 801 ( left panel ) , or 30 nM YM - 244769 or 100 nM BED ( right panel ) Graphs show quantification of ratio of MBP to L19 Data information : The values represent the means ± S . E . M from 3 independent experimental sessions . Level of significance was determined by usin B , left , one - way ANOVA P = 0 . 0001 followed by Tukey ' s post hoc test , * P < 0 . 05 versus control , ˄ P < 0 . 05 versus D - Asp ( n = 3 ) ; B , right , one - way ANOVA P = 0 . 0004 followed by Tukey ' s post hoc test , * P < 0 . 05 versus control , ˄ P < 0 . 05 versus D - Asp ( n =

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O

B. RT-PCR of MBP mRNA expression in MO3.13 cells under control conditions and following D-Asp exposure for 3 days, in absence or in presence of 10 μΜ MK-801 (left panel), or 30 nM YM-244769 or 100 nM BED (right panel) Graphs show quantification of ratio of MBP to L19 Data information: The values represent the means ± S.E.M from 3 independent experimental sessions. Level of significance was determined by usin B, left, one-way ANOVA P=0.0001 followed by Tukey's post hoc test, *P< 0.05 versus control, ˄P< 0.05 versus D-Asp (n=3); B, right, one-way ANOVA P=0.0004 followed by Tukey's post hoc test, *P< 0.05 versus control, ˄P< 0.05 versus D-Asp (n=3


C . ( a - j ) ; Confocal images displaying the expression of NG2 and MAG proteins in rat primary OPC cultured in presence of D - Asp for 3 days , in absence ( a - c ) or in presence of 10 μM MK - 801 ( d - f ) , or 30 nM YM - 244769 ( g - i ) or 100 nM BED ( j - l ) C . ( m - n ) ; Quantitative analysis of NG2 + and MAG + cells in rat primary OPC cultured in presence of D - Asp for 3 days , in absence or in presence of 10 μM MK - 801 , 30 nM YM - 244769 or 100 nM BED Data information : The values represent the means ± S . E . M from 3 independent experimental sessions . Level of significance was determined by using C , m and C , n , one - way ANOVA P < 0 . 0001 followed by Bonferroni post hoc test , * P < 0 . 05 versus control , ˄ P < 0 . 05 versus D - Asp ( n = 3 )

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O

C. (a-j); Confocal images displaying the expression of NG2 and MAG proteins in rat primary OPC cultured in presence of D-Asp for 3 days, in absence (a-c) or in presence of 10 μM MK-801 (d-f), or 30 nM YM-244769 (g-i) or 100 nM BED (j-l) C. (m-n); Quantitative analysis of NG2+ and MAG+ cells in rat primary OPC cultured in presence of D-Asp for 3 days, in absence or in presence of 10 μM MK-801, 30 nM YM-244769 or 100 nM BED Data information: The values represent the means ± S.E.M from 3 independent experimental sessions. Level of significance was determined by using C, m and C, n, one-way ANOVA P<0.0001 followed by Bonferroni post hoc test, *P< 0.05 versus control, ˄P< 0.05 versus D-Asp (n=3)


D . RT - PCR of NCX3 mRNA expression under control conditions and following 10 - 200 μM D - Asp exposure or 100 nM PMA for 3 days Data information : The values represent the means ± S . E . M from 3 independent experimental sessions . Level of significance was determined by using : i D , one - way ANOVA P = 0 . 0001 followed by Tukey ' s post hoc test , * P < 0 . 05 versus control , ˄ P < 0 . 05 versus D - Asp ( n = 3 )

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O

D. RT-PCR of NCX3 mRNA expression under control conditions and following 10-200 μM D-Asp exposure or 100 nM PMA for 3 days Data information: The values represent the means ± S.E.M from 3 independent experimental sessions. Level of significance was determined by using: i D, one-way ANOVA P=0.0001 followed by Tukey's post hoc test, *P< 0.05 versus control, ˄P< 0.05 versus D-Asp (n=3)


E . RT - PCR of NCX3 mRNA expression following 200 μM D - Asp exposure , in absence or in presence of 10 μM MK - 801 Data information : The values represent the means ± S . E . M from 3 independent experimental sessions . Level of significance was determined by using E , one - way ANOVA P = 0 . 0083 followed by Tukey ' s post hoc test , * P < 0 . 05 versus control , ˄ P < 0 . 05 versus D - Asp ( n = 3 ) , n . s , not

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O

E. RT-PCR of NCX3 mRNA expression following 200 μM D-Asp exposure, in absence or in presence of 10 μM MK-801 Data information: The values represent the means ± S.E.M from 3 independent experimental sessions. Level of significance was determined by using E, one-way ANOVA P=0.0083 followed by Tukey's post hoc test, *P<0.05 versus control, ˄P< 0.05 versus D-Asp (n=3), n.s, not significant


F . RT - PCR of NCX1 mRNA expression following 200 μM D - Asp exposure , in absence or in presence of 10 μM MK - 801 . Graphs show quantification of ratio of NCX1 and NCX3 to L19

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD

F. RT-PCR of NCX1 mRNA expression following 200 μM D-Asp exposure, in absence or in presence of 10 μM MK-801. Graphs show quantification of ratio of NCX1 and NCX3 to L19


left panels . Superimposed single - cell traces representative of the effect of 100 μM D - Asp on [ Ca2 + ] i detected in MO3 . 13 cells ( A in absence or in presence of 10 μM MK - 801 or 150 μM APV right panels . Quantification of the oscillation index in MO3 . 13 cells ( A in absence or in presence of 10 μM MK - 801 or 150 μM APV Quantification of the initial [ Ca2 + ] i increase elicited by D - Asp measured as ∆ % of peak versus basal values in absence or in presence of 10 μM MK - 801 or 150 μM AP in M03 . 13 cells ( a MK - 801 and APV were preincubated 10 minutes before registration Data information : The values represent the mean ± S . E . M from 3 independent experimental sessions . Level of significance was determined by using : in A and a , one way - ANOVA P < 0 . 001 followed by Bonferroni post hoc test , * P < 0 . 05 versus control ( basal value ) , ˄ P < 0 . 05 versus D - Asp . Data are reported as mean of 23 - 30 cells in each group , n = 3 biological replicate

O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O

left panels. Superimposed single-cell traces representative of the effect of 100 μM D-Asp on [Ca2+]i detected in MO3.13 cells (A in absence or in presence of 10 μM MK-801 or 150 μM APV right panels. Quantification of the oscillation index in MO3.13 cells (A in absence or in presence of 10 μM MK-801 or 150 μM APV Quantification of the initial [Ca2+]i increase elicited by D-Asp measured as ∆% of peak versus basal values in absence or in presence of 10 μM MK-801 or 150 μM AP in M03.13 cells (a MK-801 and APV were preincubated 10 minutes before registration Data information: The values represent the mean ± S.E.M from 3 independent experimental sessions. Level of significance was determined by using: in A and a, one way-ANOVA P<0.001 followed by Bonferroni post hoc test, *P<0.05 versus control (basal value), ˄P<0.05 versus D-Asp. Data are reported as mean of 23-30 cells in each group, n=3 biological replicate


left panels . Superimposed single - cell traces representative of the effect of 100 μM D - Asp on [ Ca2 + ] i detected i primary OP ( B ) in absence or in presence of 10 μM MK - 801 or 150 μM APV right panels . Quantification of the oscillation index i primary OPC ( B ) in absence or in presence of 10 μM MK - 801 or 150 μM APV Quantification of the initial [ Ca2 + ] i increase elicited by D - Asp measured as ∆ % of peak versus basal values in absence or in presence of 10 μM MK - 801 or 150 μM APV - i primary OPC ( b ) . MK - 801 and APV were preincubated 10 minutes before registration Data information : The values represent the mean ± S . E . M from 3 independent experimental sessions . Level of significance was determined by using B and b , one way - ANOVA , P < 0 . 001 followed by Bonferroni post hoc test , * P < 0 . 05 versus control ( basal value ) , ˄ P < 0 . 05 versus D - Asp . Data are reported as mean of 20 - 23 cells in each group , n = 3 biological

O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-CELL_TYPE I-CELL_TYPE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

left panels. Superimposed single-cell traces representative of the effect of 100 μM D-Asp on [Ca2+]i detected i primary OP (B) in absence or in presence of 10 μM MK-801 or 150 μM APV right panels. Quantification of the oscillation index i primary OPC (B) in absence or in presence of 10 μM MK-801 or 150 μM APV Quantification of the initial [Ca2+]i increase elicited by D-Asp measured as ∆% of peak versus basal values in absence or in presence of 10 μM MK-801 or 150 μM APV -i primary OPC (b). MK-801 and APV were preincubated 10 minutes before registration Data information: The values represent the mean ± S.E.M from 3 independent experimental sessions. Level of significance was determined by using B and b, one way-ANOVA, P<0.001 followed by Bonferroni post hoc test, *P<0.05 versus control (basal value), ˄P< 0.05 versus D-Asp. Data are reported as mean of 20-23 cells in each group, n=3 biological replicate


left panels ; Superimposed single - cell traces representative of the effect of 100 μM D - Asp on [ Ca2 + ] i detected in MO3 . 13 cells ( C in absence or in presence of 30 nM YM - 244769 or 100 nM BED right panels ; Quantification of the oscillation index in MO3 . 13 cells ( C in absence or in presence of 30 nM YM - 244769 or 100 nM BED d , Quantification of the initial [ Ca2 + ] i increase elicited by D - Asp measured as ∆ % of peak versus basal values in absence or in presence of 30 nM YM - 244769 or 100 nM BED in M03 . 13 cells ( c YM - 244769 or BED were preincubated 10 minutes before registration Data information : The values represent the mean ± S . E . M from 3 independent experimental sessions . Level of significance was determined by using C and c , one way - ANOVA P < 0 . 001 followed by Bonferroni post hoc test , * p < 0 . 05 versus control ( basal value ) , ˄ P < 0 . 05 versus D - Asp . Data are reported as mean of 19 - 30 cells in each group , n = 3 biological

O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

left panels; Superimposed single-cell traces representative of the effect of 100 μM D-Asp on [Ca2+]i detected in MO3.13 cells (C in absence or in presence of 30 nM YM-244769 or 100 nM BED right panels; Quantification of the oscillation index in MO3.13 cells (C in absence or in presence of 30 nM YM-244769 or 100 nM BED d, Quantification of the initial [Ca2+]i increase elicited by D-Asp measured as ∆% of peak versus basal values in absence or in presence of 30 nM YM-244769 or 100 nM BED in M03.13 cells (c YM-244769 or BED were preincubated 10 minutes before registration Data information: The values represent the mean ± S.E.M from 3 independent experimental sessions. Level of significance was determined by using C and c, one way-ANOVA P<0.001 followed by Bonferroni post hoc test, *p< 0.05 versus control (basal value), ˄ P< 0.05 versus D-Asp. Data are reported as mean of 19-30 cells in each group, n=3 biological replicate


left panels ; Superimposed single - cell traces representative of the effect of 100 μM D - Asp on [ Ca2 + ] i detected i primary OPC ( D ) in absence or in presence of 30 nM YM - 244769 or 100 nM BED D , right panels ; Quantification of the oscillation index i primary OPC ( D ) in absence or in presence of 30 nM YM - 244769 or 100 nM BED Quantification of the initial [ Ca2 + ] i increase elicited by D - Asp measured as ∆ % of peak versus basal values in absence or in presence of 30 nM YM - 244769 or 10 nM BED - i primary OPC ( d ) . YM - 244769 or BED were preincubated 10 minutes before registration Data information : The values represent the mean ± S . E . M from 3 independent experimental sessions . Level of significance was determined by usin D and d , one way - ANOVA P < 0 . 001 followed by Bonferroni post hoc test , * P < 0 . 05 versus control ( basal value ) , ˄ P < 0 . 05 versus D - Asp . Data are reported as mean of 18 - 20 cells in each group , n = 3 biological

O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-CELL_TYPE I-CELL_TYPE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

left panels; Superimposed single-cell traces representative of the effect of 100 μM D-Asp on [Ca2+]i detected i primary OPC (D) in absence or in presence of 30 nM YM-244769 or 100 nM BED D, right panels; Quantification of the oscillation index i primary OPC (D) in absence or in presence of 30 nM YM-244769 or 100 nM BED Quantification of the initial [Ca2+]i increase elicited by D-Asp measured as ∆% of peak versus basal values in absence or in presence of 30 nM YM-244769 or 10 nM BED -i primary OPC (d). YM-244769 or BED were preincubated 10 minutes before registration Data information: The values represent the mean ± S.E.M from 3 independent experimental sessions. Level of significance was determined by usin D and d, one way-ANOVA P<0.001 followed by Bonferroni post hoc test, *P< 0.05 versus control (basal value), ˄P< 0.05 versus D-Asp. Data are reported as mean of 18-20 cells in each group, n=3 biological replicate


E . left panel ; Superimposed single - cell traces representative of the effect of 100 μM D - Asp on [ Ca2 + ] i detected in MO3 . 13 cells in presence of siCtl or sincx3 silencing . E , right panel ; Quantification of the oscillation index in MO3 . 13 cells in absence or in presence of sincx3 . e , Quantification of the initial [ Ca2 + ] i increase elicited by D - Asp and measured as ∆ % of peak versus basal values in absence or in presence of sincx3 Data information : The values represent the mean ± S . E . M from 3 independent experimental sessions . Level of significance was determined by usin E and e , one way - ANOVA P < 0 . 001 followed by Bonferroni post hoc test , * P < 0 . 05 versus sictl , ˄ P < 0 . 05 versus D - Asp + sictl . Data are reported as mean of 25 - 30 cells in each group , n = 3 biological

O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O

E. left panel; Superimposed single-cell traces representative of the effect of 100 μM D-Asp on [Ca2+]i detected in MO3.13 cells in presence of siCtl or sincx3 silencing. E, right panel; Quantification of the oscillation index in MO3.13 cells in absence or in presence of sincx3. e, Quantification of the initial [Ca2+]i increase elicited by D-Asp and measured as ∆% of peak versus basal values in absence or in presence of sincx3 Data information: The values represent the mean ± S.E.M from 3 independent experimental sessions. Level of significance was determined by usin E and e, one way-ANOVA P<0.001 followed by Bonferroni post hoc test, *P< 0.05 versus sictl, ˄P< 0.05 versus D-Asp + sictl. Data are reported as mean of 25-30 cells in each group, n=3 biological replicate


F . left panel . Superimposed single - cell traces representative of the effect of 100 μM D - Asp on [ Ca2 + ] i detected in primary OPC obtained from wild - type ncx3 + / + , heterozygous ncx3 + / - , and knock - out ncx3 - / - mice . F . right panel . Quantification of the oscillation index elicited by D - Asp in primary mouse OPC obtained from ncx3 + / + , ncx3 + / - , and ncx3 - / - mice . f , Quantification of the initial [ Ca2 + ] i increase measured as ∆ % of peak versus basal values Data information : The values represent the mean ± S . E . M from 3 independent experimental sessions . Level of significance was determined by using F and f , one way - ANOVA P < 0 . 001 and P = 0 . 003 , respectively , followed by Bonferroni post hoc test , * P < 0 . 05 versus basal value , ˄ P < 0 . 05 versus ncx3 + / + and ncx3 + / - . Data are reported as mean of 10 - 19 cells in each group , n = 3 biological

O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-CELL_TYPE I-CELL_TYPE O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-ORGANISM B-CELL_TYPE O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

F. left panel. Superimposed single-cell traces representative of the effect of 100 μM D-Asp on [Ca2+]i detected in primary OPC obtained from wild-type ncx3+/+, heterozygous ncx3 +/-, and knock-out ncx3-/- mice. F. right panel. Quantification of the oscillation index elicited by D-Asp in primary mouse OPC obtained from ncx3+/+, ncx3 +/-, and ncx3-/- mice. f, Quantification of the initial [Ca2+]i increase measured as ∆% of peak versus basal values Data information: The values represent the mean ± S.E.M from 3 independent experimental sessions. Level of significance was determined by using F and f, one way-ANOVA P<0.001 and P=0.003, respectively, followed by Bonferroni post hoc test, *P< 0.05 versus basal value, ˄P< 0.05 versus ncx3+/+ and ncx3+/-. Data are reported as mean of 10-19 cells in each group, n=3 biological replicates


A . Representative inward current traces elicited by 10μM AMPA in human oligodendrocyte MO3 . 13 progenitor cells in the absence or in the presence of 10μM DNQX . The concentration - dependent curve of AMPA ( 1 - 100 μM ) on inward currents is showed at the bottom of panel A Data information : The values represent the mean ± S . E . M from 3 independent experimental sessions . Level of significance was determined by using : in A , one - way ANOVA P < 0 . 001 followed by Bonferroni post hoc test , * P < 0 . 05 versus 1μM AMPA , n = 3 biological replicate

O O O O O O O O O B-SMALL_MOLECULE O B-ORGANISM B-CELL_TYPE B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O

A. Representative inward current traces elicited by 10μM AMPA in human oligodendrocyte MO3.13 progenitor cells in the absence or in the presence of 10μM DNQX. The concentration-dependent curve of AMPA (1-100 μM) on inward currents is showed at the bottom of panel A Data information: The values represent the mean ± S.E.M from 3 independent experimental sessions. Level of significance was determined by using: in A, one-way ANOVA P<0.001 followed by Bonferroni post hoc test, *P< 0.05 versus 1μM AMPA, n=3 biological replicate


B . Representative inward current traces elicited by 100μM D - Asp application in MO3 . 13 cells in the absence or in the presence of 10μM DNQX

O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O B-SMALL_MOLECULE

B. Representative inward current traces elicited by 100μM D-Asp application in MO3.13 cells in the absence or in the presence of 10μM DNQX


C . Representative traces of the inward current ( upper panel ) and the corresponding quantification ( bottom panel ) observed in human oligodendrocyte MO3 . 13 progenitors after : ( I ) the first application of 10μM AMPA alone ; ( II ) the second application of 10μM AMPA alone ; ( III ) the third application of 10μM AMPA in the presence of 10μM DNQX ; ( IV ) the fourth application of 10μM AMPA alone . Data are expressed as percentage of control Data information : The values represent the mean ± S . E . M from 3 independent experimental sessions . Level of significance was determined by usin i one - way ANOVA P < 0 . 001 followed by Bonferroni post hoc test , * P < 0 . 05 versus versus AMPA ( I ) . Data are reported as mean of at least 25 cells in each group , n = 3 biological

O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM B-CELL_TYPE B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O

C. Representative traces of the inward current (upper panel) and the corresponding quantification (bottom panel) observed in human oligodendrocyte MO3.13 progenitors after: (I) the first application of 10μM AMPA alone; (II) the second application of 10μM AMPA alone; (III) the third application of 10μM AMPA in the presence of 10μM DNQX; (IV) the fourth application of 10μM AMPA alone. Data are expressed as percentage of control Data information: The values represent the mean ± S.E.M from 3 independent experimental sessions. Level of significance was determined by usin i one-way ANOVA P<0.001 followed by Bonferroni post hoc test, *P< 0.05 versus versus AMPA(I). Data are reported as mean of at least 25 cells in each group, n=3 biological replicate


D . Representative traces of the inward current ( upper panel ) and the corresponding quantification ( bottom panel ) observed in human oligodendrocyte MO3 . 13 progenitors after : ( I ) the first application of 100μM D - Asp alone ; ( II ) the second application of 100μM D - Asp alone ; ( III ) the third application of 100μM D - Asp in presence of 10μM DNQX ; ( IV ) the fourth application of 100μM D - Asp alone . Data are expressed as percentage of control Data information : The values represent the mean ± S . E . M from 3 independent experimental sessions . Level of significance was determined by using : i one - way ANOVA P < 0 . 001 followed by Bonferroni post hoc test , * P < 0 . 05 versus D - Asp ( I ) . Data are reported as mean of at least 25 cells for each group , n = 3 biological

O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM B-CELL_TYPE B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O

D. Representative traces of the inward current (upper panel) and the corresponding quantification (bottom panel) observed in human oligodendrocyte MO3.13 progenitors after: (I) the first application of 100μM D-Asp alone; (II) the second application of 100μM D-Asp alone; (III) the third application of 100μM D-Asp in presence of 10μM DNQX; (IV) the fourth application of 100μM D-Asp alone. Data are expressed as percentage of control Data information: The values represent the mean ± S.E.M from 3 independent experimental sessions. Level of significance was determined by using: i one-way ANOVA P<0.001 followed by Bonferroni post hoc test, *P< 0.05 versus D-Asp (I). Data are reported as mean of at least 25 cells for each group, n=3 biological replicates


E . Representative inward current traces elicited by 10μM AMPA in rat primary OPC in the absence or in the presence of 10μM DNQX

O O O O O O O O O B-SMALL_MOLECULE O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O O O O O O O B-SMALL_MOLECULE

E. Representative inward current traces elicited by 10μM AMPA in rat primary OPC in the absence or in the presence of 10μM DNQX


F , Representative inward current traces elicited by 100μM D - Asp in rat primary OPC in the absence or in presence of 10μM DNQX or 10μM DNQX + 20μM PDC

O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE

F, Representative inward current traces elicited by 100μM D-Asp in rat primary OPC in the absence or in presence of 10μM DNQX or 10μM DNQX + 20μM PDC


G . Representative traces of the inward current ( upper panel ) and the corresponding quantification ( lower panel ) observed in rat primary OPC after : ( I ) the first application of 10μM AMPA alone ; ( II ) the second application of 10μM AMPA alone ; ( III ) the third application of 10μM AMPA in the presence of 10μM DNQX ; ( IV ) the fourth application of 10μM AMPA alone . Data are expressed as percentage of control Data information : The values represent the mean ± S . E . M from 3 independent experimental sessions . Level of significance was determined by using : i G , one - way ANOVA P < 0 . 001 followed by Bonferroni post hoc test , * P < 0 . 05 versus versus AMPA ( I ) . Data are reported as mean of at least 25 cells in each group , n = 3 biological

O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O

G. Representative traces of the inward current (upper panel) and the corresponding quantification (lower panel) observed in rat primary OPC after: (I) the first application of 10μM AMPA alone; (II) the second application of 10μM AMPA alone; (III) the third application of 10μM AMPA in the presence of 10μM DNQX; (IV) the fourth application of 10μM AMPA alone. Data are expressed as percentage of control Data information: The values represent the mean ± S.E.M from 3 independent experimental sessions. Level of significance was determined by using: i G, one-way ANOVA P<0.001 followed by Bonferroni post hoc test, *P< 0.05 versus versus AMPA(I). Data are reported as mean of at least 25 cells in each group, n=3 biological replicate


H , Representative traces of the inward current ( upper panel ) and the corresponding quantification ( lower panel ) observed in rat primary OPC after : ( I ) the first application of 100μM D - Asp alone ; ( II ) the second application of 100μM D - Asp alone ; ( III ) the third application of 100μM D - Asp in presence of 10μM DNQX ; ( IV ) the fourth application of 100μM D - Asp in presence of 10μM DNQX + 10μM MK801 ; ( V ) the fifth application of 100μM D - Asp in presence of 10μM DNQX + 10μM MK - 801 + 20μM PDC ; ( VI ) the sixth application of 100μM D - Asp alone . Data are expressed as percentage of control Data information : The values represent the mean ± S . E . M from 3 independent experimental sessions . Level of significance was determined by using : i H , one - way ANOVA P < 0 . 001 followed by Bonferroni post hoc test , * P < 0 . 05 versus D - Asp ( I ) . Data are reported as mean of at least 25 cells for each group , n = 3 biological

O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O

H, Representative traces of the inward current (upper panel) and the corresponding quantification (lower panel) observed in rat primary OPC after: (I) the first application of 100μM D-Asp alone; (II) the second application of 100μM D-Asp alone; (III) the third application of 100μM D-Asp in presence of 10μM DNQX; (IV) the fourth application of 100μM D-Asp in presence of 10μM DNQX + 10μM MK801; (V) the fifth application of 100μM D-Asp in presence of 10μM DNQX + 10μM MK-801+ 20μM PDC; (VI) the sixth application of 100μM D-Asp alone. Data are expressed as percentage of control Data information: The values represent the mean ± S.E.M from 3 independent experimental sessions. Level of significance was determined by using: i H, one-way ANOVA P<0.001 followed by Bonferroni post hoc test, *P< 0.05 versus D-Asp (I). Data are reported as mean of at least 25 cells for each group, n=3 biological replicates


B - C . Beam crossing latency during daily training ( left panel , averaged across 3 trials per day ) and average latency to cross the beam over 3 consecutive days ( right panel ) , recorded in control ( open circles ) , cuprizone - ( filled black circles ) and in cuprizone + D - Asp - treated mice ( filled red triangles ) during 3 weeks ( B ) , and 5 weeks ( C ) of cuprizone feeding The values represent the means ± S . E . M ( n = 8 mice for each group ) Level of significance was determined by using left panels ( daily training ) , for each day ( d ) one - way ANOVA P = 0 . 0013 ( B , 1d ) , P = 0 . 00 followed by Bonferroni post hoc test , # P < 0 . 05 versus cpz at day 1 , § P < 0 . 05 versus cpz at day 2 , * P < 0 . 05 versus cpz at day 3 right panels ( average ) : one - way ANOVA P = 0 . 0001 ( B ) , P < 0 . 0001 ( C ) , followed by Bonferroni post hoc test * P < 0 . 05 versus control ; ^ P < 0 . 05 versus cpz or vehicle ( veh )

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O

B-C. Beam crossing latency during daily training (left panel, averaged across 3 trials per day) and average latency to cross the beam over 3 consecutive days (right panel), recorded in control (open circles), cuprizone- (filled black circles) and in cuprizone + D-Asp-treated mice (filled red triangles) during 3 weeks (B), and 5 weeks (C) of cuprizone feeding The values represent the means ± S.E.M (n=8 mice for each group) Level of significance was determined by using left panels (daily training), for each day (d) one-way ANOVA P=0.0013 (B, 1d), P=0.00 followed by Bonferroni post hoc test, #P<0.05 versus cpz at day 1, §P<0.05 versus cpz at day 2, *P<0.05 versus cpz at day 3 right panels (average): one-way ANOVA P=0.0001 (B), P<0.0001 (C ), followed by Bonferroni post hoc test *P<0.05 versus control; ^P<0.05 versus cpz or vehicle (veh)


D . Beam crossing latency during daily training ( left panel , averaged across 3 trials per day ) and average latency to cross the beam over 3 consecutive days ( middle and right panels ) recorded after 2 . 5 weeks of remyelination in control ( open circles ) , vehicle - ( filled black circles ) and in D - Asp - treated mice . D - Asp ( I ) ( filled grey triangles ) refers to the group of mice which received D - Asp only for 2 . 5 weeks after cuprizone withdrawal ; D - Asp ( II ) ( filled red triangles ) refers to the group of mice which received D - Asp during the last week of cuprizone feeding and for 2 . 5 additional weeks after cuprizone withdrawal The values represent the means ± S . E . M ( n = 8 mice for each group ) Level of significance was determined by using left panels ( daily training ) , for each day ( d ) one - way ANOV P = 0 . 0027 ( D , 3d ) , P = 0 . 0025 ( D , 2d followed by Bonferroni post hoc test , # P < 0 . 05 versus cpz at day 1 , § P < 0 . 05 versus cpz at day 2 , * P < 0 . 05 versus cpz at day 3 right panels ( average ) : one - way ANOV P < 0 . 0001 ( D , for both middle and right panels followed by Bonferroni post hoc test * P < 0 . 05 versus control ; ^ P < 0 . 05 versus cpz or vehicle ( veh )

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-ORGANISM O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-ORGANISM O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-ORGANISM O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O

D. Beam crossing latency during daily training (left panel, averaged across 3 trials per day) and average latency to cross the beam over 3 consecutive days (middle and right panels) recorded after 2.5 weeks of remyelination in control (open circles), vehicle- (filled black circles) and in D-Asp- treated mice. D-Asp (I) (filled grey triangles) refers to the group of mice which received D-Asp only for 2.5 weeks after cuprizone withdrawal; D-Asp (II) (filled red triangles) refers to the group of mice which received D-Asp during the last week of cuprizone feeding and for 2.5 additional weeks after cuprizone withdrawal The values represent the means ± S.E.M (n=8 mice for each group) Level of significance was determined by using left panels (daily training), for each day (d) one-way ANOV P=0.0027 (D, 3d), P=0.0025 (D, 2d followed by Bonferroni post hoc test, #P<0.05 versus cpz at day 1, §P<0.05 versus cpz at day 2, *P<0.05 versus cpz at day 3 right panels (average): one-way ANOV P<0.0001 (D, for both middle and right panels followed by Bonferroni post hoc test *P<0.05 versus control; ^P<0.05 versus cpz or vehicle (veh)


E - F . Latency to fall during daily training in accelerating rotarod test ( left panel , averaged across 3 trials per day ) and average over 3 consecutive days ( right panel ) recorded in control ( open circles ) , cuprizone - ( filled black circles ) and D - Asp - treated ( filled red triangles ) mice at 3 weeks ( E ) , and 5 weeks ( F ) of cuprizone feeding The values represent the means ± S . E . M ( n = 8 mice for each group ) Level of significance was determined by usin left panels ( daily training ) , for each day ( d ) one - way ANOV P < 0 . 0001 ( E , 3d ) , P = 0 . 0082 ( F , 3d followed by Bonferroni post hoc test , # P < 0 . 05 versus cpz at day 1 , § P < 0 . 05 versus cpz at day 2 , * P < 0 . 05 versus cpz at day 3 right panels ( average ) : one - way ANOV ) , P = 0 . 0002 ( E ) , P < 0 . 0001 ( F ) followed by Bonferroni post hoc test * P < 0 . 05 versus control ; ^ P < 0 . 05 versus cpz or vehicle ( veh )

O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-ORGANISM O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O

E-F. Latency to fall during daily training in accelerating rotarod test (left panel, averaged across 3 trials per day) and average over 3 consecutive days (right panel) recorded in control (open circles), cuprizone- (filled black circles) and D-Asp- treated (filled red triangles) mice at 3 weeks (E), and 5 weeks (F) of cuprizone feeding The values represent the means ± S.E.M (n=8 mice for each group) Level of significance was determined by usin left panels (daily training), for each day (d) one-way ANOV P<0.0001 (E, 3d), P=0.0082 (F, 3d followed by Bonferroni post hoc test, #P<0.05 versus cpz at day 1, §P<0.05 versus cpz at day 2, *P<0.05 versus cpz at day 3 right panels (average): one-way ANOV ), P=0.0002 (E), P<0.0001 (F) followed by Bonferroni post hoc test *P<0.05 versus control; ^P<0.05 versus cpz or vehicle (veh)


G . Latency to fall during daily training in accelerating rotarod test ( left panel , averaged across 3 trials per day ) and average over 3 consecutive days ( right panel ) recorded after 2 . 5 weeks of remyelination ( REM ) in control , vehicle - and in D - Asp - treated mice . D - Asp ( II ) refers to the group of mice which received D - Asp during the last week of cuprizone feeding and 2 . 5 additional weeks after cuprizone withdrawal The values represent the means ± S . E . M ( n = 8 mice for each group ) Level of significance was determined by using left panels ( daily training ) , for each day ( d ) one - way ANOV P < 0 . 0001 ( G , 1d ) , P = 0 . 0044 ( G , 2d ) , P = 0 . 0038 ( G , 3d ) , followed by Bonferroni post hoc test , # P < 0 . 05 versus cpz at day 1 , § P < 0 . 05 versus cpz at day 2 , * P < 0 . 05 versus cpz at day 3 right panels ( average ) : one - way ANOV P < 0 . 0001 ( G ) , followed by Bonferroni post hoc test * P < 0 . 05 versus control ; ^ P < 0 . 05 versus cpz or vehicle ( veh )

O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-ORGANISM O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-ORGANISM O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O

G. Latency to fall during daily training in accelerating rotarod test (left panel, averaged across 3 trials per day) and average over 3 consecutive days (right panel) recorded after 2.5 weeks of remyelination (REM) in control, vehicle- and in D-Asp- treated mice. D-Asp (II) refers to the group of mice which received D-Asp during the last week of cuprizone feeding and 2.5 additional weeks after cuprizone withdrawal The values represent the means ± S.E.M (n=8 mice for each group) Level of significance was determined by using left panels (daily training), for each day (d) one-way ANOV P<0.0001 (G, 1d), P=0.0044 (G, 2d), P=0.0038 (G, 3d), followed by Bonferroni post hoc test, #P<0.05 versus cpz at day 1, §P<0.05 versus cpz at day 2, *P<0.05 versus cpz at day 3 right panels (average): one-way ANOV P<0.0001 (G), followed by Bonferroni post hoc test *P<0.05 versus control; ^P<0.05 versus cpz or vehicle (veh)


A . Representative confocal double immunofluorescence images displaying MBP ( red ) and NF200 ( green ) distribution and their coexpression ( white ) in the middle corpus callosum of septostriatal sections of control mice ( a - c ) , and of mice fed with cuprizone for 5 weeks in the absence ( d - f ) or in the presence of D - Asp ( g - i ) . Scale bars in a - i : 20 μm . ( j ) , Densitometric analysis of MBP immunofluorescence signal and ( k ) quantification of MBP - NF200 colocalized points in the middle corpus callosum of septostriatal sections from control mice , and from mice fed with cuprizone for 5 weeks in the absence or in the presence of D - Asp Data information The values represent the means ± S . E . M . Level of significance was determined by using in : A , j , one - way ANOVA P < 0 . 0001 followed by Bonferroni post hoc test , * P < 0 . 05 versus control , ^ P < 0 . 05 versus cpz ( n = 4 mice for each group ) ; A , k , one - way ANOVA P = 0 . 0162 followed by Newman - Keuls post hoc test , * P < 0 . 05 versus control , ^ P < 0 . 05 versus cpz ( n = 4 mice for each group

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O B-TISSUE I-TISSUE O O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O B-TISSUE I-TISSUE O O O O O B-ORGANISM O O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-ORGANISM O O O

A. Representative confocal double immunofluorescence images displaying MBP (red) and NF200 (green) distribution and their coexpression (white) in the middle corpus callosum of septostriatal sections of control mice (a-c), and of mice fed with cuprizone for 5 weeks in the absence (d-f) or in the presence of D-Asp (g-i). Scale bars in a-i: 20 μm. (j), Densitometric analysis of MBP immunofluorescence signal and (k) quantification of MBP-NF200 colocalized points in the middle corpus callosum of septostriatal sections from control mice, and from mice fed with cuprizone for 5 weeks in the absence or in the presence of D-Asp Data information The values represent the means ± S.E.M. Level of significance was determined by using in: A, j, one-way ANOVA P<0.0001 followed by Bonferroni post hoc test, *P<0.05 versus control, ^P<0.05 versus cpz (n=4 mice for each group); A, k, one-way ANOVA P=0.0162 followed by Newman-Keuls post hoc test, *P<0.05 versus control, ^P<0.05 versus cpz (n=4 mice for each group


B . Western blot and densitometric analysis of MBP levels in corpus callosum lysates from control and cuprizone treated - mice for 5 weeks in the absence or in the presence of D - Asp , respectively . Data were normalized on the basis of α - tubulin and expressed as percentage of controls Data information The values represent the means ± S . E . M . Level of significance was determined by using i B , one - way ANOVA P < 0 . 0001 followed by Bonferroni post hoc test , * P < 0 . 05 versus control , ^ P < 0 . 05 versus cpz ( n = 3 mice for each

O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O B-TISSUE I-TISSUE O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-ORGANISM O O

B. Western blot and densitometric analysis of MBP levels in corpus callosum lysates from control and cuprizone treated-mice for 5 weeks in the absence or in the presence of D-Asp, respectively. Data were normalized on the basis of α-tubulin and expressed as percentage of controls Data information The values represent the means ± S.E.M. Level of significance was determined by using i B, one-way ANOVA P<0.0001 followed by Bonferroni post hoc test, *P<0.05 versus control, ^P<0.05 versus cpz (n=3 mice for each group


C . Representative confocal double immunofluorescence images displaying GFAP ( green ) and Iba1 ( red ) distribution in the middle corpus callosum of septostriatal sections of control mice ( a - c ) , and of mice fed with cuprizone for 5 weeks in the absence ( d - f ) or in the presence of D - Asp ( g - i ) . Scale bars in a - i : 50 μm . ( j - k ) Quantification of GFAP + and Iba1 + cells in the middle corpus callosum of septostriatal sections of control mice and of mice fed with cuprizone for 5 weeks in the absence or in the presence of D - Asp . Data were normalized to the total cell number ( Hoechst signal ) and expressed as percentage of controls Data information The values represent the means ± S . E . M . Level of significance was determined by using i C , j - k , one - way ANOVA P < 0 . 0001 followed by Bonferroni post hoc test , * P < 0 . 05 versus control , ^ P < 0 . 05 versus cpz ( n = 4 mice for each

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-TISSUE I-TISSUE O O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O B-TISSUE I-TISSUE O O O O O B-ORGANISM O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-ORGANISM O O

C. Representative confocal double immunofluorescence images displaying GFAP (green) and Iba1 (red) distribution in the middle corpus callosum of septostriatal sections of control mice (a-c), and of mice fed with cuprizone for 5 weeks in the absence (d-f) or in the presence of D-Asp (g-i). Scale bars in a-i: 50 μm. (j-k) Quantification of GFAP+ and Iba1+ cells in the middle corpus callosum of septostriatal sections of control mice and of mice fed with cuprizone for 5 weeks in the absence or in the presence of D-Asp. Data were normalized to the total cell number (Hoechst signal) and expressed as percentage of controls Data information The values represent the means ± S.E.M. Level of significance was determined by using i C, j-k, one-way ANOVA P<0.0001 followed by Bonferroni post hoc test, *P<0.05 versus control, ^P<0.05 versus cpz (n=4 mice for each group


D . Western blot of GFAP and Iba1 protein levels in corpus callosum lysates obtained from control or cuprizone treated - mice for 5 weeks in the absence or in the presence of D - Asp , respectively Data information The values represent the means ± S . E . M . Level of significance was determined by using i D middle panel , one - way ANOVA P = 0 . 0146 followed by Bonferroni post hoc test , * P < 0 . 05 versus control , ^ P < 0 . 05 versus cpz ( n = 3 mice for each group ) ; D right panel , one - way ANOVA P = 0 . 0026 followed by Bonferroni post hoc test , * P < 0 . 05 versus control , ^ P < 0 . 05 versus cpz ( n = 3 mice for each group )

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O B-TISSUE I-TISSUE O O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-ORGANISM O O O O

D. Western blot of GFAP and Iba1 protein levels in corpus callosum lysates obtained from control or cuprizone treated-mice for 5 weeks in the absence or in the presence of D-Asp, respectively Data information The values represent the means ± S.E.M. Level of significance was determined by using i D middle panel, one-way ANOVA P=0.0146 followed by Bonferroni post hoc test, *P<0.05 versus control, ^P<0.05 versus cpz (n=3 mice for each group); D right panel, one-way ANOVA P= 0.0026 followed by Bonferroni post hoc test, *P<0.05 versus control, ^P<0.05 versus cpz (n=3 mice for each group)


A . Confocal double immunofluorescence images depicting Olig2 + ( a - e ) and Olig2 + - CC1 + ( f - j ) double labeled cells in the middle corpus callosum of control , cuprizone - treated mice at 5 weeks , in absence ( cpz ) or in presence of D - Asp ( cpz + D - Asp ) , and 2 . 5 weeks after cuprizone withdrawal , in absence ( vehicle ) or in presence of D - Asp ( D - Asp ) . ( k , left panel ) , Quantitative analysis of Olig2 + cells ( red ) and Olig2 + - CC1 + ( blue ) double labeled cells in the middle corpus callosum of control , cuprizone - treated mice , and cuprizone + D - Asp - treated mice at 5 weeks . ( k , right panel ) , Quantitative analysis of Olig2 + cells ( red ) and Olig2 + - CC1 + ( blue ) double labeled cells in the middle corpus callosum of control , vehicle , and D - Asp - treated mice 2 . 5 weeks after cuprizone withdrawal . Arrows point to Olig2 + - CC1 + cells . Arrowheads point to Olig2 + - CC1 + cells . Scale bar in a - j : 50 μm Data information : The values represent the means ± S . E . M . Level of significance was determined by using : in A , k left panel , one - way ANOVA P < 0 . 0001 followed by Newman - Keuls post hoc test , * P < 0 . 05 versus ctrl ; ^ P < 0 . 05 versus cpz ( n = 3 mice for each group ) . A , k right panel , one - way ANOVA P < 0 . 0001 followed by Newman - Keuls post hoc test , * P < 0 . 05 versus ctrl ; ^ P < 0 . 05 versus vehicle ( n = 3 mice for each group

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-TISSUE I-TISSUE O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O B-TISSUE I-TISSUE O O O B-SMALL_MOLECULE O O B-ORGANISM O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O

A. Confocal double immunofluorescence images depicting Olig2+ (a-e) and Olig2+-CC1+ (f-j) double labeled cells in the middle corpus callosum of control, cuprizone-treated mice at 5 weeks, in absence (cpz) or in presence of D-Asp (cpz + D-Asp), and 2.5 weeks after cuprizone withdrawal, in absence (vehicle) or in presence of D-Asp (D-Asp). (k, left panel), Quantitative analysis of Olig2+ cells (red) and Olig2+-CC1+ (blue) double labeled cells in the middle corpus callosum of control, cuprizone-treated mice, and cuprizone+D-Asp-treated mice at 5 weeks. (k, right panel), Quantitative analysis of Olig2+ cells (red) and Olig2+-CC1+ (blue) double labeled cells in the middle corpus callosum of control, vehicle, and D-Asp-treated mice 2.5 weeks after cuprizone withdrawal. Arrows point to Olig2+-CC1+ cells. Arrowheads point to Olig2+-CC1+ cells. Scale bar in a-j: 50 μm Data information: The values represent the means ± S.E.M. Level of significance was determined by using: in A, k left panel, one-way ANOVA P<0.0001 followed by Newman-Keuls post hoc test, *P<0.05 versus ctrl; ^P<0.05 versus cpz (n=3 mice for each group). A, k right panel, one-way ANOVA P<0.0001 followed by Newman-Keuls post hoc test, *P<0.05 versus ctrl; ^P<0.05 versus vehicle (n=3 mice for each group


B . Representative electron microscopy images of middle corpus callosum sections from control mice ( a ) , cuprizone treated - mice for 5 weeks in the absence ( b ) or in the presence of D - Asp ( c ) , and from mice 2 . 5 weeks after cuprizone withdrawal in the absence ( d , vehicle ) or in presence of D - Asp ( e ) . Scale bars in a - e : 1 μ

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE I-TISSUE O O O B-ORGANISM O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O

B. Representative electron microscopy images of middle corpus callosum sections from control mice (a), cuprizone treated-mice for 5 weeks in the absence (b) or in the presence of D-Asp (c), and from mice 2.5 weeks after cuprizone withdrawal in the absence (d, vehicle) or in presence of D-Asp (e). Scale bars in a-e: 1 μ


C . Percentage of myelinated axons in the middle corpus callosum of cuprizone ( blue ) and cuprizone + D - Asp - treated mice ( red ) for 5 weeks Data information : The values represent the means ± S . E . M . Level of significance was determined by using C , two - tailed Student ' s t test , * P < 0 . 05 versus cpz ( n = 4 mice for each

O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-TISSUE I-TISSUE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-ORGANISM O O

C. Percentage of myelinated axons in the middle corpus callosum of cuprizone (blue) and cuprizone + D-Asp-treated mice (red) for 5 weeks Data information: The values represent the means ± S.E.M. Level of significance was determined by using C, two-tailed Student's t test, *P<0.05 versus cpz (n=4 mice for each group


D . Scatter plot analysis of myelin thickness , expressed as individual G - ratios against axon diameters , after 2 . 5 weeks of cuprizone withdrawal in the middle corpus callosum of vehicle ( blue circle ) or D - Asp - treated mice ( red triangle ) . Linear regression was used to indicate the differences between the groups in myelin thickness across the range of axon diameters

O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O B-SUBCELLULAR O O O O O O O O B-SMALL_MOLECULE O O O O B-TISSUE I-TISSUE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O B-SUBCELLULAR O

D. Scatter plot analysis of myelin thickness, expressed as individual G-ratios against axon diameters, after 2.5 weeks of cuprizone withdrawal in the middle corpus callosum of vehicle (blue circle) or D-Asp-treated mice (red triangle). Linear regression was used to indicate the differences between the groups in myelin thickness across the range of axon diameters


E . Histogram analysis displaying the G - ratio values of myelinated fibers across the range of axon diameters . Data represent the mean S . E . M . for at least 100 axons from each group Data information : The values represent the means ± S . E . M . Level of significance was determined by usin E , for each axon diameter group , Student ' s t test , * P < 0 . 05 versus vehicle ( n = 4 mice for each

O O B-EXP_ASSAY O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O

E. Histogram analysis displaying the G-ratio values of myelinated fibers across the range of axon diameters. Data represent the mean S.E.M. for at least 100 axons from each group Data information: The values represent the means ± S.E.M. Level of significance was determined by usin E, for each axon diameter group, Student's t test, *P<0.05 versus vehicle (n=4 mice for each group


F . Percentage of myelinated axons after 2 . 5 weeks of cuprizone withdrawal in the middle corpus callosum of vehicle ( blue ) or D - Asp - treated mice ( red ) . Abbreviations : DEM , demyelination ; REM , remyelination Data information : The values represent the means ± S . E . M . Level of significance was determined by usin F , for each axon diameter group , Student ' s t test , * P < 0 . 05 versus vehicle ( n = 4 mice for each group )

O O O O O B-SUBCELLULAR O O O O O O B-SMALL_MOLECULE O O O O B-TISSUE I-TISSUE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O

F. Percentage of myelinated axons after 2.5 weeks of cuprizone withdrawal in the middle corpus callosum of vehicle (blue) or D-Asp- treated mice (red). Abbreviations: DEM, demyelination; REM, remyelination Data information: The values represent the means ± S.E.M. Level of significance was determined by usin F, for each axon diameter group, Student's t test, *P<0.05 versus vehicle (n=4 mice for each group)


C , D Two types of TNTs were detected in H1299 ( C ) and A549 ( D ) cells , Type 1 , containing F - actin with little or no detectable microtubules ( white box ) and Type2 , containing both microtubules and F - actin ( yellow box ) . Anti - β - tubulin antibody ( green ) and phalloidin ( red ) were used for fluorescent staining . Scale bar : 20 μm . Data information : nuclei were stained with Hoechst 33342 ( blue ) . Boxed areas are enlarged and shown in panels on the right .

O O O O O O B-SUBCELLULAR O O O B-CELL_LINE O O O O B-CELL_LINE O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O

C, D Two types of TNTs were detected in H1299 (C) and A549 (D) cells, Type 1, containing F-actin with little or no detectable microtubules (white box) and Type2, containing both microtubules and F-actin (yellow box). Anti-β-tubulin antibody (green) and phalloidin (red) were used for fluorescent staining. Scale bar: 20 μm. Data information: nuclei were stained with Hoechst 33342 (blue). Boxed areas are enlarged and shown in panels on the right.


C , D TNT formation ( marked by black triangles and white arrowheads ) was imaged in control and shM2 H1299 ( C ) and shM2 A549 ( D ) cells by confocal microscopy . Scale bar : 60 μm . Boxed areas are enlarged and shown at the bottom of the corresponding images . E , F Quantification of the percentage of cells forming TNTs in control and shM2 H1299 ( E ) and shM2 A549 ( F ) cells . 13 images per group ( E ) and 10 per group ( F ) were captured for quantification . Three independent experiments were conducted . G Data information : data were analyzed by one - way ANOVA with Bonferroni post - test and shown as mean ± SEM ; * * * P < 0 . 001 . Three independent experiments were

O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-CELL_LINE O O O O O B-CELL_LINE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-CELL_LINE O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, D TNT formation (marked by black triangles and white arrowheads) was imaged in control and shM2 H1299 (C) and shM2 A549 (D) cells by confocal microscopy. Scale bar: 60 μm. Boxed areas are enlarged and shown at the bottom of the corresponding images. E, F Quantification of the percentage of cells forming TNTs in control and shM2 H1299 (E) and shM2 A549 (F) cells. 13 images per group (E) and 10 per group (F) were captured for quantification. Three independent experiments were conducted. G Data information: data were analyzed by one-way ANOVA with Bonferroni post-test and shown as mean ±SEM; *** P < 0.001. Three independent experiments were performed


H , I H1299 cells stably expressing the Ctr or GFP - M2 were cultured in the presence of 20 μM 2 - DG for 24 h to induce TNT formation . TNTs were imaged by confocal microscopy ( A ) and the percentage of cells forming TNTs was quantified ( B ) . n = 21 , 20 and 19 images were captured from Ctr , GFP - M2 # 1 and GFP - M2 # 2 , respectively . Three independent experiments were performed . Scale bar : 60 μm . Data information : data were analyzed by one - way ANOVA with Bonferroni post - test and shown as mean ± SEM ; * * * P < 0 . 001 . Three independent experiments were

O O O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H,I H1299 cells stably expressing the Ctr or GFP-M2 were cultured in the presence of 20 μM 2-DG for 24 h to induce TNT formation. TNTs were imaged by confocal microscopy (A) and the percentage of cells forming TNTs was quantified (B). n=21, 20 and 19 images were captured from Ctr, GFP-M2#1 and GFP-M2#2, respectively. Three independent experiments were performed. Scale bar: 60 μm. Data information: data were analyzed by one-way ANOVA with Bonferroni post-test and shown as mean ±SEM; *** P < 0.001. Three independent experiments were performed


A Time - lapse microscopic images showing that H1299 shCtr or shM2 cells stably expressing Mito - GFP were connected by TNTs containing mitochondria . Cells were seeded in collagen - coated dishes and cultured for 24 h before time - lapse life cell images were acquired by confocal microscopy ( also see Movies EV1 and EV2 ) . Fluorescent images ( with bright field ) at different time points show the mitochondria moving along the TNT and entering in one of the two connected cells . Scale bar : 20 μm . The moving mitochondria are marked by the white arrows . Boxed areas ( shCtr in yellow dotted boxes ; shM2 in red dotted boxes ) are enlarged and shown in the middle panel . B Confocal microscopic images of control and shM2 H1299 cells following immunofluorescence staining with anti - Tom20 antibody ( green ) and staining using phalloidin ( red ) . The white arrows marked TNTs , whereas the white triangles marked mitochondria inside TNTs . Scale bar : 25 μm . C Data information : nuclei were stained with Hoechst 33342 ( blue ) .

O O O O O O O O B-CELL_LINE O O O O O O B-SUBCELLULAR O B-GENEPROD O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_LINE O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O

A Time-lapse microscopic images showing that H1299 shCtr or shM2 cells stably expressing Mito-GFP were connected by TNTs containing mitochondria. Cells were seeded in collagen-coated dishes and cultured for 24 h before time-lapse life cell images were acquired by confocal microscopy (also see Movies EV1 and EV2). Fluorescent images (with bright field) at different time points show the mitochondria moving along the TNT and entering in one of the two connected cells. Scale bar: 20 μm. The moving mitochondria are marked by the white arrows. Boxed areas (shCtr in yellow dotted boxes; shM2 in red dotted boxes) are enlarged and shown in the middle panel. B Confocal microscopic images of control and shM2 H1299 cells following immunofluorescence staining with anti-Tom20 antibody (green) and staining using phalloidin (red). The white arrows marked TNTs, whereas the white triangles marked mitochondria inside TNTs. Scale bar: 25 μm. C Data information: nuclei were stained with Hoechst 33342 (blue).


C Quantification of TNTs containing mitochondria in shCtr and shM2 H1299 cells . The percentage of TNTs containing mitochondria was determined . n = 168 , 187 and 192 for shCtr , shM2 # 1 and shM2 # 2 groups , respectively ; three independent experiments . Data information : data were analyzed using FlowJo analysis software . When co - cultured through the filter in transwells , only 0 . 01 % or

O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O B-CELL_LINE O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O

C Quantification of TNTs containing mitochondria in shCtr and shM2 H1299 cells. The percentage of TNTs containing mitochondria was determined. n=168, 187 and 192 for shCtr, shM2#1 and shM2#2 groups, respectively; three independent experiments. Data information: data were analyzed using FlowJo analysis software. When co-cultured through the filter in transwells, only 0.01% or f


E Representative flow cytometry plot of shCtr and shM2 cells following co - culture for 2 days . Experiments were conducted under three conditions : co - cultured in direct contact in normal glucose medium or in galactose medium or co - cultured through a filter .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O

E Representative flow cytometry plot of shCtr and shM2 cells following co-culture for 2 days. Experiments were conducted under three conditions: co-cultured in direct contact in normal glucose medium or in galactose medium or co-cultured through a filter.


F The percentage of mCherry - expressing recipient cells containing Mito - GFP was quantified . Twenty thousand events were acquired for each condition and data were analyzed using FlowJo analysis software . When co - cultured through the filter in transwells , only 0 . 01 % or fewer recipient cells contained Mito - GFP , and this group is not included in the graph to avoid an overcrowded figure . Three independent experiments were conducted . Data information : data were analyzed using FlowJo analysis software . When co - cultured through the filter in transwells , only 0 . 01 % or

O O O O B-GENEPROD O O O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O

F The percentage of mCherry-expressing recipient cells containing Mito-GFP was quantified. Twenty thousand events were acquired for each condition and data were analyzed using FlowJo analysis software. When co-cultured through the filter in transwells, only 0.01% or fewer recipient cells contained Mito-GFP, and this group is not included in the graph to avoid an overcrowded figure. Three independent experiments were conducted. Data information: data were analyzed using FlowJo analysis software. When co-cultured through the filter in transwells, only 0.01% or f


A Down - regulating MICAL2PV promotes mitochondrial infiltration into the cortical cytoskeleton . Control and shM2 H1299 cells were subjected to immunofluorescence staining with anti - Tom20 antibody ( green ) and staining using phalloidin ( red ) . Scale bar : 25 μm . Data information : nuclei were stained with Hoechst 33342 ( blue ) .

O O O O B-GENEPROD O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-CELL_LINE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O

A Down-regulating MICAL2PV promotes mitochondrial infiltration into the cortical cytoskeleton. Control and shM2 H1299 cells were subjected to immunofluorescence staining with anti-Tom20 antibody (green) and staining using phalloidin (red). Scale bar: 25 μm. Data information: nuclei were stained with Hoechst 33342 (blue).


C Down - regulating MICAL2PV enhances the mitochondrial localization of Miro1 , Miro2 and KIF5B . Mitochondria were purified from control and shM2 H1299 cells and subjected to Western blotting . The purity of the mitochondrial fraction was confirmed by detection of mitochondrial ATP5B and the absence of the cytoplasmic β - tubulin protein .

O O O O B-GENEPROD O O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O O O O O B-CELL_LINE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR B-GENEPROD O O O O O B-SUBCELLULAR B-GENEPROD I-GENEPROD I-GENEPROD O O

C Down-regulating MICAL2PV enhances the mitochondrial localization of Miro1, Miro2 and KIF5B. Mitochondria were purified from control and shM2 H1299 cells and subjected to Western blotting. The purity of the mitochondrial fraction was confirmed by detection of mitochondrial ATP5B and the absence of the cytoplasmic β-tubulin protein.


E MICAL2PV interacts with the C - terminal domain of Miro2 containing the transmembrane region . H1299 cells were co - transfected with plasmids encoding Flag - MICAL2PV and Myc - tagged Miro2 mutants as indicated . Following transfection , cells were lysed for immunoprecipitation using the anti - Flag antibody and Western blotting . Please note the Myc - Miro2 ( 410 - 618 ) protein band in lane 4 migrated as a faint band , slightly faster than the IgG light chain .

O B-GENEPROD B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O B-CELL_LINE O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O

E MICAL2PV interacts with the C-terminal domain of Miro2 containing the transmembrane region. H1299 cells were co-transfected with plasmids encoding Flag-MICAL2PV and Myc-tagged Miro2 mutants as indicated. Following transfection, cells were lysed for immunoprecipitation using the anti-Flag antibody and Western blotting. Please note the Myc-Miro2 (410-618) protein band in lane 4 migrated as a faint band, slightly faster than the IgG light chain.


F MICAL2PV inhibits the mitochondrial localization of Miro2 and mitochondrial infiltration . H1299 cells were co - transfected with Myc - tagged Miro2 and GFP - tagged MCAL2PV ( GFP - M2 ) or control vector . Following transfection , cells were fixed and stained with anti - Tom20 ( red ) and anti - Myc antibodies ( magenta ) . Confocal microscopic images were taken under the same objective and the same microscope settings . In cells co - expressing the GFP vector and Myc - Miro2 ( upper panels ) , most of Miro2 signals ( magenta ) are co - colocalized with mitochondria ( red Tom20 signals ) . However , a significant fraction of Miro2 signals is not colocalized with mitochondria in cells co - expressing GFP - M2 and Myc - Miro2 ( marked by the yellow arrowheads ) . Mitochondria in cells expressing GFP - M2 show the perinuclear distribution pattern , whereas mitochondria in cells transfected by Myc - Miro2 without GFP - M2 ( marked by the yellow stars ) exhibit an infiltration pattern into the cortical cytoplasm . Scale bar : 20 μm .

O B-GENEPROD O O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O B-SUBCELLULAR O O B-CELL_LINE O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-TISSUE B-SUBCELLULAR O O O O O O O

F MICAL2PV inhibits the mitochondrial localization of Miro2 and mitochondrial infiltration. H1299 cells were co-transfected with Myc-tagged Miro2 and GFP-tagged MCAL2PV (GFP-M2) or control vector. Following transfection, cells were fixed and stained with anti-Tom20 (red) and anti-Myc antibodies (magenta). Confocal microscopic images were taken under the same objective and the same microscope settings. In cells co-expressing the GFP vector and Myc-Miro2 (upper panels), most of Miro2 signals (magenta) are co-colocalized with mitochondria (red Tom20 signals). However, a significant fraction of Miro2 signals is not colocalized with mitochondria in cells co-expressing GFP-M2 and Myc-Miro2 (marked by the yellow arrowheads). Mitochondria in cells expressing GFP-M2 show the perinuclear distribution pattern, whereas mitochondria in cells transfected by Myc-Miro2 without GFP-M2 (marked by the yellow stars) exhibit an infiltration pattern into the cortical cytoplasm. Scale bar: 20 μm.


A Control and shM2 H1299 cells were transfected with control siRNA or siRNAs targeting Miro2 . Following transfection , cells were stained with anti - Tom20 antibody ( green ) and phalloidin ( red ) . Confocal microscopic images are shown . Scale bar : 20 μm . Data information : nuclei were stained with Hoechst 33342 ( blue ) .

O O O O B-CELL_LINE O O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O

A Control and shM2 H1299 cells were transfected with control siRNA or siRNAs targeting Miro2. Following transfection, cells were stained with anti-Tom20 antibody (green) and phalloidin (red). Confocal microscopic images are shown. Scale bar: 20 μm. Data information: nuclei were stained with Hoechst 33342 (blue).


F Down - regulating Miro2 inhibits mitochondrial transport in shM2 cells . Mito - GFP labelled control and shM2 H1299 cells were transfected with control siRNA or siRNAs targeting Miro2 . These cells were then co - cultured with mCherry labelled recipient cells and analyzed by flow cytometry The percentage of mCherry recipient cells containing Mito - GFP was quantified . Twenty thousand events were analyzed for each condition Data information : Data were analyzed using two - way ANOVA with Bonferroni post - test and presented as mean ± SEM ; * P < 0 . 05 , * * * P < 0 . 01 , * * * P < 0 . 001 , ns , not significant . Three independent experiments were conducted .

O O O O B-GENEPROD O B-SUBCELLULAR O O O O O B-SUBCELLULAR O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F Down-regulating Miro2 inhibits mitochondrial transport in shM2 cells. Mito-GFP labelled control and shM2 H1299 cells were transfected with control siRNA or siRNAs targeting Miro2. These cells were then co-cultured with mCherry labelled recipient cells and analyzed by flow cytometry The percentage of mCherry recipient cells containing Mito-GFP was quantified. Twenty thousand events were analyzed for each condition Data information: Data were analyzed using two-way ANOVA with Bonferroni post-test and presented as mean ± SEM; * P < 0.05, *** P < 0.01, *** P < 0.001, ns, not significant. Three independent experiments were conducted.


A Stable H1299 cells ( shCtr or shM2 ) were treated by 5 - FU ( 50 μM , 24 h ) and then cultured alone or co - cultured with non - drug treated vehicle - treated cells stably expressing mito - mCherry in the absence or presence of Cyto B ( 500 nM , 24 h ) . The percentage of dead cells in each group was quantified . Three independent experiments were conducted . Data information : data were analyzed using two - way ANOVA with Bonferroni post - test and shown as mean ± SEM ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns , not significant .

O O B-CELL_LINE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Stable H1299 cells (shCtr or shM2) were treated by 5-FU (50 μM, 24 h) and then cultured alone or co-cultured with non-drug treated vehicle-treated cells stably expressing mito-mCherry in the absence or presence of Cyto B (500 nM, 24 h). The percentage of dead cells in each group was quantified. Three independent experiments were conducted. Data information: data were analyzed using two-way ANOVA with Bonferroni post-test and shown as mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant.


D Confocal microscopic images of TNTs in each group , white arrowheads and yellow triangle in enlarged views ( zoom - in panels , zoom ) marked the TNTs formed between non - drug treated ( mCherry positive ) and drug - treated cells . Scale bar : 25 μm .

O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O B-GENEPROD O O O O O O O O O O O O O O

D Confocal microscopic images of TNTs in each group, white arrowheads and yellow triangle in enlarged views (zoom-in panels, zoom) marked the TNTs formed between non-drug treated (mCherry positive) and drug-treated cells. Scale bar: 25 μm.


D MICAL2PV mutants containing the MO domain are active in depolymerizing F - actin . Following transfection of plasmids expressing different HA - MICAL2PV mutants ( # 1 , # 2 , or # 4 ) into shM2 cells , confocal microscopy was performed following staining with phalloidin ( red ) and anti - HA antibody ( green ) . F - actin signals were reduced in cells expressing different mutants ( HA - positive green cells marked by dashed lines ) . Scale bar : 20 μm . Data information : nuclei were stained with Hoechst 33342 ( blue ) .

O B-GENEPROD O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O

D MICAL2PV mutants containing the MO domain are active in depolymerizing F-actin. Following transfection of plasmids expressing different HA-MICAL2PV mutants (#1, #2, or #4) into shM2 cells, confocal microscopy was performed following staining with phalloidin (red) and anti-HA antibody (green). F-actin signals were reduced in cells expressing different mutants (HA-positive green cells marked by dashed lines). Scale bar: 20 μm. Data information: nuclei were stained with Hoechst 33342 (blue).


G , H The MICAL2PV mutant containing an inactive MO domain does not inhibited TNT formation . Stable shM2 H1299 ( G ) or A549 ( H ) cells were transfected with plasmids expressing the MICAL2PV mutant in which the MO domain was inactivated by mutations in the FAD binding site ( MOmut : amino acid residues 93 - 98 GxGxxG mutated to WxWxxW ) or the control vector ( Ctr ) . Following transfection , Western blotting and quantification of TNT formation were carried out as in panel E . 10 images were captured for quantification in each group . Three independent experiments were performed . Data information : Data were analyzed using a one - way ANOVA with Bonferroni post - test and shown as mean ± SEM ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns , not significant .

O O O O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O O O O B-CELL_LINE O O O O B-CELL_LINE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G, H The MICAL2PV mutant containing an inactive MO domain does not inhibited TNT formation. Stable shM2 H1299 (G) or A549 (H) cells were transfected with plasmids expressing the MICAL2PV mutant in which the MO domain was inactivated by mutations in the FAD binding site (MOmut: amino acid residues 93-98 GxGxxG mutated to WxWxxW) or the control vector (Ctr). Following transfection, Western blotting and quantification of TNT formation were carried out as in panel E. 10 images were captured for quantification in each group. Three independent experiments were performed. Data information: Data were analyzed using a one-way ANOVA with Bonferroni post-test and shown as mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant.


( A ) HEK293 cells were transfected with DAPK ΔCaM or with a control vector ( pcDNA3 - luciferase , LUC ) , together with shRNAs targeting beclin 1 or HcRed , and with GFP - LC3 plasmid . After 72 h , cells were counted and lysates were prepared . ( A ) The percentage of cells with punctate GFP - LC3 fluorescence per total GFP - LC3 - positive cells was quantified . Data presented are the mean ± s . d . from a triplicate of 100 transfected cells . The asterisks denote a significance level of P = 0 . 001 .

O O O B-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) HEK293 cells were transfected with DAPK ΔCaM or with a control vector (pcDNA3-luciferase, LUC), together with shRNAs targeting beclin 1 or HcRed, and with GFP-LC3 plasmid. After 72 h, cells were counted and lysates were prepared. (A) The percentage of cells with punctate GFP-LC3 fluorescence per total GFP-LC3-positive cells was quantified. Data presented are the mean±s.d. from a triplicate of 100 transfected cells. The asterisks denote a significance level of P=0.001.


( B ) Western blot analysis was performed using the indicated antibodies .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O

(B) Western blot analysis was performed using the indicated antibodies.


( C ) Representative GFP - LC3 staining of cells transfected with the control shRNA ( HcRed ) together with pcDNA3 ‐ luciferase or DAPKΔCaM . CaM , calmodulin ; DAPK , death ‐ associated protein kinase ; GFP , green fluorescent protein ; HEK , human embryonic kidney ; shRNA , short hairpin RNA .

O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Representative GFP-LC3 staining of cells transfected with the control shRNA (HcRed) together with pcDNA3‐luciferase or DAPKΔCaM. CaM, calmodulin; DAPK, death‐associated protein kinase; GFP, green fluorescent protein; HEK, human embryonic kidney; shRNA, short hairpin RNA.