D Cell viability was determined using Cell Biolabs CytoSelect ™ Cell Transformation Assay using 2 - control , 2C3 , 9 - control and 9C2 cells in CSM - charcoal stripped media , E2 ( β - estradiol ; 10 - 10 M ) and 10 % fetal bovine serum . Data ( mean + SD ) were calculated using two - way ANOVA based on three independent biological replicates .

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D Cell viability was determined using Cell Biolabs CytoSelect™ Cell Transformation Assay using 2-control, 2C3, 9-control and 9C2 cells in CSM-charcoal stripped media, E2 (β-estradiol; 10-10 M) and 10% fetal bovine serum. Data (mean + SD) were calculated using two-way ANOVA based on three independent biological replicates.


E Impact of ESRP1 knockdown on in vivo tumor growth . Five million cells ( 2 - control , 2C3 , 9 - control and 9C2 ) were implanted into mammary fat pads of athymic mice ( five mice per group ) in the presence of supplemental estrogen . Tumors were measured weekly using calipers for external measurements . Tumor volume was calculated as L x W2 / 2 , where L is length and W is width ( note the different scale for tumor volumes ) . Data ( mean ± SD ) were calculated using two - way ANOVA ( n = 5 mice per group ) .

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E Impact of ESRP1 knockdown on in vivo tumor growth. Five million cells (2-control, 2C3, 9-control and 9C2) were implanted into mammary fat pads of athymic mice (five mice per group) in the presence of supplemental estrogen. Tumors were measured weekly using calipers for external measurements. Tumor volume was calculated as L x W2/2, where L is length and W is width (note the different scale for tumor volumes). Data (mean ± SD) were calculated using two-way ANOVA (n= 5 mice per group).


A Cell morphology using high - power magnification ( 40x ) of the cultured cell lines under a microscope in LCC2 model - 2 - control and 2C3 cells . Scale bar : 50 µm .

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A Cell morphology using high-power magnification (40x) of the cultured cell lines under a microscope in LCC2 model-2-control and 2C3 cells. Scale bar: 50 µm.


B Cell morphology using high - power magnification ( 40x ) of the cultured cell lines under a microscope in LCC9 model - 9 - control and 9C2 cells . Scale bar : 50 µm .

B Cell morphology using high-power magnification (40x) of the cultured cell lines under a microscope in LCC9 model-9-control and 9C2 cells. Scale bar: 50 µm.


C - D Protein expression of key EMT - TFs in ESRP1 knockdown cells ( 2C3 and 9C2 ) compared to endocrine resistant control cell lines ( 2 - control and 9 - control ) using Western blotting analysis .

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C-D Protein expression of key EMT-TFs in ESRP1 knockdown cells (2C3 and 9C2) compared to endocrine resistant control cell lines (2-control and 9-control) using Western blotting analysis.


E Protein expression of key EMT - TFs in ESRP1 knockdown cells ( T - 47D - kESRP1 ) and ESRP1 - overexpressing MCF - 7 cells compared to their control counterparts ( T - 47D - control and MCF - 7 - control , respectively ) .

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E Protein expression of key EMT-TFs in ESRP1 knockdown cells (T-47D-kESRP1) and ESRP1-overexpressing MCF-7 cells compared to their control counterparts (T-47D-control and MCF-7-control, respectively).


A Doughnut chart for RNA - seq analysis showing the differentially regulated alternative splicing events ( ASEs ) and their corresponding genes in ESRP1 knockdown cells compared to control cells ; SE : skipped exon , RI : retained intron , MXE : mutually exclusive exons , A5SS : alternative 5 ' splice site , and A3SS : alternative 3 ' splice site ) . The " percent spliced in " ( PSI or ψ ) value was estimated . Differences in inclusion levels ( ∆ ψ ) between samples and their significance were calculated ( P < 0 . 05 and IΔψI > 0 . 1 ) . Circle 1 : all ASEs ; Circle 2 : all genes corresponding to all ASEs ; Circle 3 : significant ASEs ; Circle 4 : significant genes corresponding to significantly altered ASEs ; Circle 5 : significant ASEs with ESRP1 motifs ; Circle 6 : significant genes corresponding to significantly altered ASEs with ESRP1 motifs . ESRP - binding motifs were obtained from Dittmar et al . 31 [ ] and analyzed based on the following assumption : the presence of the motif in the downstream region of an exon results in inclusion of the exon , while upstream or intraexonic location leads to exclusion of the exon .

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A Doughnut chart for RNA-seq analysis showing the differentially regulated alternative splicing events (ASEs) and their corresponding genes in ESRP1 knockdown cells compared to control cells; SE: skipped exon, RI: retained intron, MXE: mutually exclusive exons, A5SS: alternative 5' splice site, and A3SS: alternative 3' splice site). The "percent spliced in" (PSI or ψ) value was estimated. Differences in inclusion levels (∆ψ) between samples and their significance were calculated (P<0.05 and IΔψI>0.1). Circle 1: all ASEs; Circle 2: all genes corresponding to all ASEs; Circle 3: significant ASEs; Circle 4: significant genes corresponding to significantly altered ASEs; Circle 5: significant ASEs with ESRP1 motifs; Circle 6: significant genes corresponding to significantly altered ASEs with ESRP1 motifs. ESRP-binding motifs were obtained from Dittmar et al.31[] and analyzed based on the following assumption: the presence of the motif in the downstream region of an exon results in inclusion of the exon, while upstream or intraexonic location leads to exclusion of the exon.


B Radar plot showing that the EMT gene signature was not altered at the gene level in the ESRP1 knockdown model ( LCC2 set ) . C Radar plot showing that the EMT gene signature was altered at the ASEs level ( SE ) in the ESRP1 knockdown model ( LCC2 set ) .

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B Radar plot showing that the EMT gene signature was not altered at the gene level in the ESRP1 knockdown model (LCC2 set). C Radar plot showing that the EMT gene signature was altered at the ASEs level (SE) in the ESRP1 knockdown model (LCC2 set).


D Radar plot showing that the EMT gene signature was not altered at the gene level in the ESRP1 knockdown model ( LCC9 set ) . E Radar plots showing that the EMT gene signature was altered at the ASEs level ( SE ) in the ESRP1 knockdown model ( LCC9 set ) .

D Radar plot showing that the EMT gene signature was not altered at the gene level in the ESRP1 knockdown model (LCC9 set). E Radar plots showing that the EMT gene signature was altered at the ASEs level (SE) in the ESRP1 knockdown model (LCC9 set).


B TCGA Splicegraphs for the EMT signature genes in ESRP1high versus ESRP1low tumors . A splice graph of the gene ' s exons is shaded based on expression level and shows the selected splice event outlined in red .

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B TCGA Splicegraphs for the EMT signature genes in ESRP1high versus ESRP1low tumors. A splice graph of the gene's exons is shaded based on expression level and shows the selected splice event outlined in red.


A Volcano plots showing the distribution of significant gene expression changes using the HTA 2 . 0 platform .

A Volcano plots showing the distribution of significant gene expression changes using the HTA 2.0 platform.


C Validation of the protein levels of FASN , SCD1 and PHGDH protein levels in ESRP1 knockdown versus control resistant cell lines using Western blot analysis . GAPDH was used as the loading control . The data are representative of three individual biological replicates .

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C Validation of the protein levels of FASN, SCD1 and PHGDH protein levels in ESRP1 knockdown versus control resistant cell lines using Western blot analysis. GAPDH was used as the loading control. The data are representative of three individual biological replicates.


B . Diluting concentrations of 4D7 to bound full - length 30 amino acid peptide compared to scrambled , C - terminal and N - terminal 11 amino acid peptides as shown in A ( n = 3 biological replicates ) .

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B. Diluting concentrations of 4D7 to bound full-length 30 amino acid peptide compared to scrambled, C-terminal and N-terminal 11 amino acid peptides as shown in A (n=3 biological replicates).


F . Proliferation curves of factor - independent TF - 1 TpoR CALRdel61 cells cultured with 2 , 10 or 20 µg / mL 4D7 or 20 µg / mL of control IgG antibody ( n = 3 biological replicates ) . G Proliferation curves of factor - independent TF - 1 TpoR CALRdel52 cells cultured with 10 or 20 µg / mL 4D7 or 20 µg / mL of control IgG antibody ( n = 3 biological replicates ) . Data information : bars represent standard error of the mean summarizing 3 biological replicates performed in triplicate , with a one - way ANOVA with Bonferroni correction used to determine statistical significance .

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F. Proliferation curves of factor-independent TF-1 TpoR CALRdel61 cells cultured with 2, 10 or 20 µg/mL 4D7 or 20 µg/mL of control IgG antibody (n=3 biological replicates). G Proliferation curves of factor-independent TF-1 TpoR CALRdel52 cells cultured with 10 or 20 µg/mL 4D7 or 20 µg/mL of control IgG antibody (n=3 biological replicates). Data information: bars represent standard error of the mean summarizing 3 biological replicates performed in triplicate, with a one-way ANOVA with Bonferroni correction used to determine statistical significance.


B . Similar experiment using TPO - independent TF - 1 TpoR CALRdel61 cells . C . Similar experiment using TPO - independent TF - 1 TpoR CALRdel52 cells at 8 hours . D . Similar experiment using PBMNCs from CALRdel52 PMF primary cells at 8 hours . Additionally , cells were treated with 280 nM of ruxolitinib as a positive control .

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B. Similar experiment using TPO-independent TF-1 TpoR CALRdel61 cells. C. Similar experiment using TPO-independent TF-1 TpoR CALRdel52 cells at 8 hours. D. Similar experiment using PBMNCs from CALRdel52 PMF primary cells at 8 hours. Additionally, cells were treated with 280 nM of ruxolitinib as a positive control.


F . Western blot showing caspase 3 cleavage occurring within 24 hours of TPO withdrawal in TPO dependent TF - 1 TpoR cells . An increase in cleaved caspase 3 is observed after 48 hours of treatment with 20 µg / mL 4D7 in TF - 1 TpoR CALRdel61 and TF - 1 TpoR CALRdel52 cells .

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F. Western blot showing caspase 3 cleavage occurring within 24 hours of TPO withdrawal in TPO dependent TF-1 TpoR cells. An increase in cleaved caspase 3 is observed after 48 hours of treatment with 20 µg/mL 4D7 in TF-1 TpoR CALRdel61 and TF-1 TpoR CALRdel52 cells.


G . Western blot of TpoR immunoprecipitation under non - reducing conditions showing associated CALR 50 kDa monomers and 100 kDa dimers ( red arrowheads ) present only in TF - 1 TpoR CALRdel61 disrupted by 8 - hour treatment with 20 µg / mL 4D7 but not PBS or 20 µg / mL IgG . CALR monomers & dimers are detectable by polyclonal anti - wild type CALR or anti - mutant CALR monoclonal antibodies . Red arrowheads , detected mutant CALR protein ; brown arrowheads , detected wildtype CALR protein , asterisk , non - specific bands .

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G. Western blot of TpoR immunoprecipitation under non-reducing conditions showing associated CALR 50 kDa monomers and 100 kDa dimers (red arrowheads) present only in TF-1 TpoR CALRdel61 disrupted by 8-hour treatment with 20 µg/mL 4D7 but not PBS or 20 µg/mL IgG. CALR monomers & dimers are detectable by polyclonal anti-wild type CALR or anti-mutant CALR monoclonal antibodies. Red arrowheads, detected mutant CALR protein; brown arrowheads, detected wildtype CALR protein, asterisk, non-specific bands.


B . Graph showing the decreased fold change of CD41 + CD61 + megakaryocytes cultured in 4D7 compared to IgG . FACS - sorted CD34 + from myelofibrosis patients with CALR or JAK2 mutation were cultured over 12 days without TPO in the presence of SCF , IL - 6 and IL - 9 . Black columns show JAK2V617F mutation positive samples ( n = 4 technical replicates of samples from different patients ) . Data information : Unpaired students t - test used to determine statistical significance Bars represent standard deviations * , P = 0 . 05 - 0 . 01 , * * , P = 0 . 01 - 0 . 001 , * * * , P = 0 . 001 - 0 . 0001 , * * * * , P < 0 . 0001 , n . s , not significant .

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B. Graph showing the decreased fold change of CD41+CD61+ megakaryocytes cultured in 4D7 compared to IgG. FACS-sorted CD34+ from myelofibrosis patients with CALR or JAK2 mutation were cultured over 12 days without TPO in the presence of SCF, IL-6 and IL-9. Black columns show JAK2V617F mutation positive samples (n= 4 technical replicates of samples from different patients). Data information: Unpaired students t-test used to determine statistical significance Bars represent standard deviations *, P = 0.05 - 0.01, **, P = 0.01 - 0.001, ***, P = 0.001 - 0.0001, ****, P <0.0001, n.s, not significant.


C . Number of CD41 + megakaryocytes derived from isolated CD34 + progenitors from myelofibrosis patients . Number of CD41 / CD61 + cells counted on day 12 using trypan blue exclusion ( n = 1 technical replicate ) . D . Summary of fold change reduction of CD41 / CD61 + megakaryocytes by 4D7 in all tested CALR mutated patient samples compared to CALR wild type , normalised to IgG ( n = 11 samples from different patients , with 4 technical replicates per sample ) . Data information : Bars represent standard error of means Unpaired students t - test used to determine statistical significance * , P = 0 . 05 - 0 . 01 , * * , P = 0 . 01 - 0 . 001 , * * * , P = 0 . 001 - 0 . 0001 , * * * * , P < 0 . 0001 , n . s , not significant .

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C. Number of CD41+ megakaryocytes derived from isolated CD34+ progenitors from myelofibrosis patients. Number of CD41/CD61+ cells counted on day 12 using trypan blue exclusion (n=1 technical replicate). D. Summary of fold change reduction of CD41/CD61+ megakaryocytes by 4D7 in all tested CALR mutated patient samples compared to CALR wild type, normalised to IgG (n=11 samples from different patients, with 4 technical replicates per sample). Data information: Bars represent standard error of means Unpaired students t-test used to determine statistical significance *, P = 0.05 - 0.01, **, P = 0.01 - 0.001, ***, P = 0.001 - 0.0001, ****, P <0.0001, n.s, not significant.


E Number of CD41 + megakaryocyte colonies from patient samples after 4D7 treatment . CD34 + from patients with myelofibrosis with CALRdel52 or CALRins5 were plated on collagen - based matrix in presence of 20 µg / mL 4D7 or IgG control ( n = 3 biological replicates ) . Data information : Unpaired students t - test used to determine statistical significance Bars represent standard deviations * , P = 0 . 05 - 0 . 01 , * * , P = 0 . 01 - 0 . 001 , * * * , P = 0 . 001 - 0 . 0001 , * * * * , P < 0 . 0001 , n . s , not significant .

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E Number of CD41+ megakaryocyte colonies from patient samples after 4D7 treatment. CD34+ from patients with myelofibrosis with CALRdel52 or CALRins5 were plated on collagen-based matrix in presence of 20 µg/mL 4D7 or IgG control (n=3 biological replicates). Data information: Unpaired students t-test used to determine statistical significance Bars represent standard deviations *, P = 0.05 - 0.01, **, P = 0.01 - 0.001, ***, P = 0.001 - 0.0001, ****, P <0.0001, n.s, not significant.


F . Representative micrographs showing CD41 + colonies in pink and CD41 - colonies in blue after treatment with 4D7 or IgG at 100 X or 40X magnification . Scale bar indicates 100µm . G . Summary of fold change reduction of CD41 + megakaryocytes in CALR mutated samples cultured MegaCult treated with 4D7 compared to IgG ( n = 4 biological replicates ) . Data information : Bars represent standard error of means Unpaired students t - test used to determine statistical significance * , P = 0 . 05 - 0 . 01 , * * , P = 0 . 01 - 0 . 001 , * * * , P = 0 . 001 - 0 . 0001 , * * * * , P < 0 . 0001 , n . s , not significant .

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F. Representative micrographs showing CD41+ colonies in pink and CD41- colonies in blue after treatment with 4D7 or IgG at 100 X or 40X magnification. Scale bar indicates 100µm. G. Summary of fold change reduction of CD41+ megakaryocytes in CALR mutated samples cultured MegaCult treated with 4D7 compared to IgG (n= 4 biological replicates). Data information: Bars represent standard error of means Unpaired students t-test used to determine statistical significance *, P = 0.05 - 0.01, **, P = 0.01 - 0.001, ***, P = 0.001 - 0.0001, ****, P <0.0001, n.s, not significant.


H . Number of megakaryocyte colony forming units grouped according to colony cell number after 4D7 treatment in a mutant CALRins5 sample ( n = 3 biological replicates ) . Data information : Unpaired students t - test used to determine statistical significance Bars represent standard deviations * , P = 0 . 05 - 0 . 01 , * * , P = 0 . 01 - 0 . 001 , * * * , P = 0 . 001 - 0 . 0001 , * * * * , P < 0 . 0001 , n . s , not significant .

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H. Number of megakaryocyte colony forming units grouped according to colony cell number after 4D7 treatment in a mutant CALRins5 sample (n=3 biological replicates). Data information: Unpaired students t-test used to determine statistical significance Bars represent standard deviations *, P = 0.05 - 0.01, **, P = 0.01 - 0.001, ***, P = 0.001 - 0.0001, ****, P <0.0001, n.s, not significant.


B . Effect of 4D7 on TPO - dependent megakaryocyte differentiation of healthy CD34 + cells cultured with 20 µg / mL 4D7 or IgG control ( n = 2 biological replicates , with 2 technical replicates ) . Data information : Error bars indicate standard error of mean .

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B. Effect of 4D7 on TPO-dependent megakaryocyte differentiation of healthy CD34+ cells cultured with 20 µg/mL 4D7 or IgG control (n= 2 biological replicates, with 2 technical replicates). Data information: Error bars indicate standard error of mean.


C . Total numbers of hematopoietic colonies plated in MethoCult from healthy cord blood after treatment with 4D7 or IgG . CFU - GM , colony forming unit - granulocyte macrophage ; BFU - E , blast forming unit - erythroid ; CFU - GEMM , colony forming unit granulocyte , erythroid , monocyte , megakaryocyte ( n = 3 biological replicates ) . Data information : Error bars indicate standard error of mean .

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C. Total numbers of hematopoietic colonies plated in MethoCult from healthy cord blood after treatment with 4D7 or IgG. CFU-GM, colony forming unit- granulocyte macrophage; BFU-E, blast forming unit-erythroid; CFU-GEMM, colony forming unit granulocyte, erythroid, monocyte, megakaryocyte (n=3 biological replicates). Data information: Error bars indicate standard error of mean.


F . Western blot showing signalling in ruxolitinib - resistant TF - 1 TpoR CALRdel61 compared to ruxolitinib sensitive TF - 1 TpoR CALRdel61 cells after treatment with 100nM ruxolitinib for 16 hours or 30 minutes and blotted for phospho - STAT5 , total STAT5 , phospho - ERK , total ERK and actin as indicated . Ruxolitinib - sensitive cells were non - viable after 16 hours of treatment .

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F. Western blot showing signalling in ruxolitinib-resistant TF-1 TpoR CALRdel61 compared to ruxolitinib sensitive TF-1 TpoR CALRdel61 cells after treatment with 100nM ruxolitinib for 16 hours or 30 minutes and blotted for phospho-STAT5, total STAT5, phospho-ERK, total ERK and actin as indicated. Ruxolitinib-sensitive cells were non-viable after 16 hours of treatment.


G . Comparison of cell growth after DMSO , 20 µg / mL 4D7 , IgG or 100 nM ruxolitinib treatment over 4 days of ruxolitinib - resistant TF - 1 TpoR CALRdel61 . Cells counted using Trypan Blue exclusion ( n = 3 biological replicates with 3 technical replicates ) . H . Number of colonies observed from cells plated in MethoCult following 72 hours of treatment with DMSO , 20 µg / mL 4D7 , IgG or 100nM ruxolitinib performed in ruxolitinib - resistant TF - 1 TpoR CALRdel61 cells ( n = 3 biological replicates with 3 technical replicates ) . Data information : Error bars on represent standard deviation . All P values are unpaired Student ' s unpaired t - test .

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G. Comparison of cell growth after DMSO, 20 µg/mL 4D7, IgG or 100 nM ruxolitinib treatment over 4 days of ruxolitinib-resistant TF-1 TpoR CALRdel61. Cells counted using Trypan Blue exclusion (n=3 biological replicates with 3 technical replicates). H. Number of colonies observed from cells plated in MethoCult following 72 hours of treatment with DMSO, 20 µg/mL 4D7, IgG or 100nM ruxolitinib performed in ruxolitinib-resistant TF-1 TpoR CALRdel61 cells (n=3 biological replicates with 3 technical replicates). Data information: Error bars on represent standard deviation. All P values are unpaired Student's unpaired t-test.


B . Pharmacokinetic measurements of serum levels of 4D7 in mice after intraperitoneal injection at 0 , 1 , 24 , 48 , 72 and 110 hours since administration .

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B. Pharmacokinetic measurements of serum levels of 4D7 in mice after intraperitoneal injection at 0, 1, 24, 48, 72 and 110 hours since administration.


D . Kaplan - Meier survival curve of bone marrow engraftment model of TF - 1 TpoR CALRdel61 cells showing improved survival of mice treated with 4D7 compared with IgG control commencing 1 week after tail vein injection ( n = 5 mice per treatment ) . Data information : For all survival curves the log - rank Mantel - Cox test P value is shown .

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D. Kaplan-Meier survival curve of bone marrow engraftment model of TF-1 TpoR CALRdel61 cells showing improved survival of mice treated with 4D7 compared with IgG control commencing 1 week after tail vein injection (n=5 mice per treatment). Data information: For all survival curves the log-rank Mantel-Cox test P value is shown.


F . Kaplan - Meier survival curve of TF - 1 TpoR CALRdel61 chloroma mice treated with 4D7 or IgG until humane killing due to tumour diameter > 30 mm or ulceration . ( n = 3 mice per treatment ) G . Kaplan - Meier survival curve of NSG mice engrafted with ruxolitinib - resistant TF - 1 TpoR CALRdel61 treated with 12 . 5 mg / kg 4D7 or IgG twice weekly ( n = 5 and 6 mice for IgG and 4D7 respectively ) . Data information For all survival curves the log - rank Mantel - Cox test P value is shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Kaplan-Meier survival curve of TF-1 TpoR CALRdel61 chloroma mice treated with 4D7 or IgG until humane killing due to tumour diameter > 30 mm or ulceration. (n=3 mice per treatment) G. Kaplan-Meier survival curve of NSG mice engrafted with ruxolitinib-resistant TF-1 TpoR CALRdel61 treated with 12.5 mg/kg 4D7 or IgG twice weekly (n=5 and 6 mice for IgG and 4D7 respectively). Data information For all survival curves the log-rank Mantel-Cox test P value is shown.


A . RT - PCR of CNPase ( left ) and MBP ( right ) mRNAs expression in MO3 . 13 precursors under control conditions and following 10 - 200 μM D - Asp exposure for 3 days . Graphs show quantification of ratio of CNPase , and MBP to L19 Data information : The values represent the means ± S . E . M . Level of significance was determined by using : in A , left panel , one - way ANOVA P < 0 . 0001 followed by Tukey ' s post hoc test , * P < 0 . 05 versus control ( n = 3 ) ; A , right panel , one - way ANOVA P < 0 . 0001 followed by Tukey ' s post hoc test , * P < 0 . 05 versus control ( n = 3

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A. RT-PCR of CNPase (left) and MBP (right) mRNAs expression in MO3.13 precursors under control conditions and following 10-200 μM D-Asp exposure for 3 days. Graphs show quantification of ratio of CNPase, and MBP to L19 Data information: The values represent the means ± S.E.M. Level of significance was determined by using: in A, left panel, one-way ANOVA P<0.0001 followed by Tukey's post hoc test, *P< 0.05 versus control (n=3); A, right panel, one-way ANOVA P<0.0001 followed by Tukey's post hoc test, *P<0.05 versus control (n=3


B . Western blotting ( left ) and densitometric analysis ( right ) of MBP expression in absence or in presence of 10 - 200 μΜ D - Asp exposure for 5 days Data information : The values represent the means ± S . E . M . Level of significance was determined by using ) ; B , one - way ANOVA P = 0 . 0001 followed by Bonferroni post hoc test , * P < 0 . 05 versus control ( n =

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B. Western blotting (left) and densitometric analysis (right) of MBP expression in absence or in presence of 10-200 μΜ D-Asp exposure for 5 days Data information: The values represent the means ± S.E.M. Level of significance was determined by using ); B, one-way ANOVA P=0.0001 followed by Bonferroni post hoc test, *P<0.05 versus control (n=3


C . Cell growth analysis of human MO3 . 13 oligodendrocytes in absence or in presence of 200 μM D - Asp for 1 - 5 days . The density of M03 . 13 oligodendrocytes was daily recorded through trypan blue dye exclusion . Mean of daily measurements were recorded . The data of each experimental group were normalized to the density of cells plated at day 0 and expressed as percentage of ctrlday0 Data information : The values represent the means ± S . E . M . Level of significance was determined by using C , for each day , two - tailed Student ' s t test , * P < 0 . 05 versus control at 3 days ( n = 5 )

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C. Cell growth analysis of human MO3.13 oligodendrocytes in absence or in presence of 200 μM D-Asp for 1-5 days. The density of M03.13 oligodendrocytes was daily recorded through trypan blue dye exclusion. Mean of daily measurements were recorded. The data of each experimental group were normalized to the density of cells plated at day 0 and expressed as percentage of ctrlday0 Data information: The values represent the means ± S.E.M. Level of significance was determined by using C, for each day, two-tailed Student's t test, *P<0.05 versus control at 3 days (n=5)


D . FACS - based cell cycle distribution analysis after PI incorporation of MO3 . 13 oligodendrocytes in absence or in presence of 200 μM D - Asp for 3 days . Representative FACS plots of biological replicates are shown ( n = 5 independent experimental sessions )

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D. FACS-based cell cycle distribution analysis after PI incorporation of MO3.13 oligodendrocytes in absence or in presence of 200 μM D-Asp for 3 days. Representative FACS plots of biological replicates are shown (n=5 independent experimental sessions)


E . FACS - based cell cycle distribution analysis after PI incorporation of rat primary OPC in absence or in presence of 100 μM D - Asp for 1 day . Representative FACS plots of biological replicates are shown ( n = 3 independent experimental sessions )

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E. FACS-based cell cycle distribution analysis after PI incorporation of rat primary OPC in absence or in presence of 100 μM D-Asp for 1 day. Representative FACS plots of biological replicates are shown (n=3 independent experimental sessions)


F . Confocal microscopic images displaying the coexpression of MAG or MBP with Phalloidin - 594 ( a - b and c - d , respectively ) in rat primary OPC cultured in absence or in presence of 100 μM D - Asp for 4 days . ( e ) , Single representative MBP + + cell . Scale bars : 50 μm in a - d ; 10 μm in e . ( f - g ) ; Quantitative analysis of Alexa - 594 - phalloidin - positive oligodendrocytes ( f ) or MAG + and MBP + + oligodendrocytes ( g ) scored in control and D - Asp exposed cells . Alexa - 594 - phalloidin - positive oligodendrocytes were scored in 4 categories , according to their morphological complexity . In each category , data were normalized on the total number of oligodendrocytes Data information : The values represent the means ± S . E . M . Level of significance was determined by usin F , for each category two - tailed Student ' s t test , * P < 0 . 05 versus control ( n = 3 )

O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Confocal microscopic images displaying the coexpression of MAG or MBP with Phalloidin-594 (a-b and c-d, respectively) in rat primary OPC cultured in absence or in presence of 100 μM D-Asp for 4 days. (e), Single representative MBP++ cell. Scale bars: 50 μm in a-d; 10 μm in e. (f-g); Quantitative analysis of Alexa-594-phalloidin-positive oligodendrocytes (f) or MAG+ and MBP++ oligodendrocytes (g) scored in control and D-Asp exposed cells. Alexa-594-phalloidin-positive oligodendrocytes were scored in 4 categories, according to their morphological complexity. In each category, data were normalized on the total number of oligodendrocytes Data information: The values represent the means ± S.E.M. Level of significance was determined by usin F, for each category two-tailed Student's t test, *P<0.05 versus control (n=3)


B . Maximum intensity projection of z - stack confocal images displaying MBP ( red ) and NF200 ( green ) immunoreactivities in 7 DIV cerebellar organotypic slices cultured in absence ( b - d ) or in presence of 100 μM D - Asp ( f - h ) . Panels d and h display colocalized points ( white ) . Panels a and e 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 and e : 200 μm ; in b - d and f - h : 50 μm

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B. Maximum intensity projection of z-stack confocal images displaying MBP (red) and NF200 (green) immunoreactivities in 7 DIV cerebellar organotypic slices cultured in absence (b-d) or in presence of 100 μM D-Asp (f-h). Panels d and h display colocalized points (white). Panels a and e 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 and e: 200 μm; in b-d and f-h: 50 μm


C . Scatter plot histogram analysis of the myelination index in 7 DIV cerebellar slices 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 using : in C , two - tailed Student ' s t test , * P < 0 . 05 versus control ( n = 4 animals , 3 - 4 slices per group

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C. Scatter plot histogram analysis of the myelination index in 7 DIV cerebellar slices 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 using: in C, two-tailed Student's t test, *P<0.05 versus control (n=4 animals, 3-4 slices per group


D . Western blotting analysis of MBP protein levels from homogenates of organotypic cerebellar slices culture in absence or in presence of 100 μΜ D - Asp . Data were normalized on the basis of β - actin and expressed as percentage of controls Data information : The values represent the means ± S . E . M . Level of significance was determined by using D , two - tailed Student ' s t test , * P < 0 . 05 versus controls ( n =

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D. Western blotting analysis of MBP protein levels from homogenates of organotypic cerebellar slices culture in absence or in presence of 100 μΜ D-Asp. Data were normalized on the basis of β-actin and expressed as percentage of controls Data information: The values represent the means ± S.E.M. Level of significance was determined by using D, two-tailed Student's t test, *P<0.05 versus controls (n=3


F . 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 6 days after LPC exposure ( dpl ) in absence ( f - h ) or in presence 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

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F. 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 6 days after LPC exposure (dpl) in absence (f-h) or in presence 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


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 =

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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 )

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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

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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 )

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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

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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 =

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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 )

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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 O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O

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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR

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 O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O B-CONTROLLED_VAR

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 O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR

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 O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR 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 O O O O O O O O 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-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O 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 O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O 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 O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O 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 O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O 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)