Journal: Scientific Reports

Article Title: Ebselen analogues delay disease onset and its course in fALS by on-target SOD-1 engagement

doi: 10.1038/s41598-024-62903-5

Figure Lengend Snippet: Cell viability of human and mouse neuronal cells expressing wild-type SOD1 and ALS-associated mutants of A4V and G93A under treatments of ebselen-based compounds and ALS-approved drugs. ( A ) Chemical structures of approved ALS drugs, ebselen and derivatives. ( B ) MTS assay of mouse N2a neuroblastoma cells transfected with G93A SOD1. ( C ) human H4 neuroglioma cells transfected with A4V SOD1, and ( D , E ) ATP bioluminescent assay for single compound and in combination with approved drugs using human H4 neuroglioma cells transfected with wild-type, A4V and G93A SOD1. Cell viability levels are shown in bar chart with data points and error bars representing standard deviation of the mean from at least 6 separate measurements. Asterisks above bar charts indicate statistically significant improvements ( p < 0.05) against untreated control groups.

Article Snippet: The Venus-bimolecular fluorescence complementation (BiFC) plasmids containing wild-type SOD1 gene were generated by gene synthesis and cloning service of Genscript, USA.

Techniques: Expressing, MTS Assay, Transfection, ATP Bioluminescent Assay, Standard Deviation, Control

Journal: Scientific Reports

Article Title: Ebselen analogues delay disease onset and its course in fALS by on-target SOD-1 engagement

doi: 10.1038/s41598-024-62903-5

Figure Lengend Snippet: The BiFC assay reliably captures the differences between wild-type and mutant SOD1. ( A ) Schematic illustration of the DNA constructs of the human SOD1 fused to truncated Venus fluorescent protein segments using a larger N-terminal fragment of Venus (V N ), corresponding to amino acids 1–158, and a smaller C-terminal fragment (V C ), corresponding to amino acids 159–239. ( B ) Schematic presentation of BiFC assay. Dimerization of WT SOD1 fused to Venus fragments generates fluorescence from the fusion of V N and V C fragments. ( C ) In the A4V SOD1 mutant, Venus fluorescence complementation allows to detect amyloid-like filament formation (aggregates/inclusion) due to non-native (unstable) A4V SOD1 dimerization. Representative images showing the different negative and positive controls to demonstrate the ability of BiFC assay to differentiate between stable, unstable or no SOD1 dimerization. Reciprocal negative controls of SOD1 V C with empty V N (V N + SOD1 V C or V N SOD1 with empty V C (V C + V N SOD1) vectors showing no fluorescence due to lack of dimerization in the absence of complimentary SOD1. Venus is pVenus N1 empty plasmid that does not contain SOD1 and only expresses VFP; C6S, a positive control, is an internal non-specific mutation and interaction of V N C6S SOD1 and C6S SOD1 V C presents uniform fluorescence WT represents the interaction of V N WT SOD1 and WT SOD1 V C displayingeven fluorescence A4V, aSOD1 mutant and represents the interaction of V N A4V SOD1 and A4V SOD1 V C leading to the aggregates/inclusion formation (shown by white arrows), scale bar: 25 μm. Left to right panel for each control shows transmitted light (TL), DRAQ5 stained nuclei, SOD1 VFP (after complementation of SOD1 Vc or SOD1 V N ) and merged images respectively.

Article Snippet: The Venus-bimolecular fluorescence complementation (BiFC) plasmids containing wild-type SOD1 gene were generated by gene synthesis and cloning service of Genscript, USA.

Techniques: Bimolecular Fluorescence Complementation Assay, Mutagenesis, Construct, Fluorescence, Plasmid Preparation, Positive Control, Control, Staining

Journal: Scientific Reports

Article Title: Ebselen analogues delay disease onset and its course in fALS by on-target SOD-1 engagement

doi: 10.1038/s41598-024-62903-5

Figure Lengend Snippet: Ebselen and its derivatives reduce aggregating species in human cell model. ( A ) Effects of drug (MR6-26-2) doses 25, 50 and 100 µM on SOD1 dimerization following transfection and drug treatment of H4 cells with A4V (V N + V C ), scale bar: 25 µm. Left to right panel for each dose shows transmitted light (TL), DRAQ5 stained nuclei, SOD1 VFP (after complementation of SOD1 V C or SOD1 V N ) and merged images respectively. ( B ) Quantification of the number of inclusions per cell. 50–200 cells were counted per condition and per experiment and classified into three groups: blue, orange, and grey bars represent the percentage of cells without inclusions, with 5 or less inclusions and cells with more than 5 inclusions respectively. Data are expressed as mean ± SD of at least three independent experiments. One-way ANOVA, with Dunnet’s multiple comparison, was used for statistical analysis with significance level of * p < 0.05 ***p < 0.001 or ****p < 0.0001, which represents statistically different results between mutant SOD1 with and without compound treatment. ( C ) Representative images showing reduction in the aggregation propensity of H4 cells expressingA4V, G93A, H46R and D90A following treatment with most effective MR6-26-2 compound versus non-treated cells (scale bar: 50 µm). White arrows indicate the mutant SOD1 aggregation. The scaling of the images has been kept different to make the aggregates/inclusion bodies clearly visible in non-treated conditions, and to show the maximum possible number of cells within a single field as proof of drug efficacy in stable SOD1 dimerization and uniform fluorescence following treatment with MR6-26-2.

Article Snippet: The Venus-bimolecular fluorescence complementation (BiFC) plasmids containing wild-type SOD1 gene were generated by gene synthesis and cloning service of Genscript, USA.

Techniques: Transfection, Staining, Comparison, Mutagenesis, Fluorescence

Journal: Scientific Reports

Article Title: Ebselen analogues delay disease onset and its course in fALS by on-target SOD-1 engagement

doi: 10.1038/s41598-024-62903-5

Figure Lengend Snippet: Co-crystalised structures of G93A SOD1 with ebselen, MR6-8-2 and MR6-26-2. Electron density (2F o –F c ) maps of ( A ) ebselen, ( B ) MR6-8-2, and ( C ) MR6-26-2 are contoured at 1σ in green mesh. Ligands and waters are illustrated as yellow sticks and red sphere, respectively. Individual SOD1 monomers are coloured in dark and light blue surface. Overlaid ligand poses in co-crystalised G93A (yellow sticks) and A4V SOD1 (pink sticks) structures of ( D ) ebselen, ( E ) MR6-8-2, and ( F ) MR6-26-2. Structures of A4V SOD1 with corresponding compounds are obtained from PDBs: 6Z4G, 6Z4L and 6Z4M respectively. Binding modes of ( G MR6-8-2 and ( H ) MR6-26-2 are shown among amino acid residues (dark and light blue sticks) and water molecules in SOD1 dimer interface. Hydrogen bonds and distances between molecules are shown as green and black dashes, respectively. Numbers represent distances in Ångstroms.

Article Snippet: The Venus-bimolecular fluorescence complementation (BiFC) plasmids containing wild-type SOD1 gene were generated by gene synthesis and cloning service of Genscript, USA.

Techniques: Binding Assay

Journal: Scientific Reports

Article Title: Ebselen analogues delay disease onset and its course in fALS by on-target SOD-1 engagement

doi: 10.1038/s41598-024-62903-5

Figure Lengend Snippet: MR6-26-2 decreased misfolded SOD1 species and delayed the disease onset of SOD1 G93A mice. ( A , B ) Onset ( A ) and survival ( B ) curves of the control or MR6-26-2 treated female SOD1 G93A mice plotted over time (n = 20, each). The mean ages for onset or survival are shown with SD. ( C ) Changes in clasping score of the control or MR6-26-2 treated SOD1 G93A mice plotted over time (n = 20, each). The data were analyzed by two-way ANOVA following posthoc multiple comparisons with Šidák correction. ( D , E ) Decrease of insoluble SOD1 species in the spinal cords of MR6-26-2 treated SOD1 G93A mice at 120 days old. A representative immunoblotting image is shown ( D) , and the relative expressions of insoluble SOD1 per soluble SOD1 were quantified and plotted as mean ± SEM ( E ). ( F , G ) SOD1 oligomers produced by aberrant disulfide bonds (hSOD1 S-S oligomers) were decreased in the spinal cords of MR6-26-2 treated SOD1 G93A mice at 120 days old. A representative immunoblotting image indicates the hSOD1 S-S oligomers (a bold line) ( F ). Relative expressions of hSOD1 S-S oligomers were quantified and plotted as mean ± SEM ( G ). ( H – J ) Immunofluorescent analyses of lumbar spinal cord sections of early symptomatic SOD1 G93A mice showed the decrease of misfolded SOD1 species, detected using a C4F6 antibody that specifically recognizes misfolded SOD1, by the MR6-26-2 treatment. Motor neurons were identified using an anti-choline acetyltransferase (ChAT) antibody. Representative immunofluorescent image is shown ( H ), and the relative fluorescence intensity of C4F6 (I) or the number of ChAT-positive motor neurons (J) in the ventral horn averaged per mouse was quantified and plotted as mean ± SD. Each dot represents the section used for the quantification ( I , J ). Scale bar: 50 µm.

Article Snippet: The Venus-bimolecular fluorescence complementation (BiFC) plasmids containing wild-type SOD1 gene were generated by gene synthesis and cloning service of Genscript, USA.

Techniques: Control, Western Blot, Produced, Fluorescence

Journal: Scientific Reports

Article Title: Ebselen analogues delay disease onset and its course in fALS by on-target SOD-1 engagement

doi: 10.1038/s41598-024-62903-5

Figure Lengend Snippet: MR6-26-2 ameliorated a neuromuscular junction pathology of SOD1 G93A mice and improved proteostasis in vitro and in vivo . ( A , B ) The MR6-26-2 treatment reduced poly-ubiquitin- (poly-Ubi) and p62/SQSTM1-positive inclusions in the 120-days-old SOD1 G93A lumbar spinal cord. Representative immunofluorescent images are shown ( A ). The number of the inclusions per anterior horn (AH) quantified from three mice are plotted as mean ± SEM (n = 3) ( B ). Scale bar: 75 µm. ( C – E ) Immunoblot analyses of poly-Ubi and p62 expressions in insoluble fractions of spinal cords from the control or MR6-26 treated SOD1 G93A mice at 120 days old. Representative immunoblotting images are shown ( C ), and the quantification results of insoluble poly-Ubi (bold line in C ) and insoluble p62 (black rectangle) are expressed as mean ± SEM in ( D ) and ( E ), respectively. An asterisk indicates non-specific bands. ( F , G ) MR6-26-2 reduced denervation at neuromuscular junctions (NMJs) of 120-days-old SOD1 G93A mice. Representative immunofluorescent images of tibialis anterior muscles are shown ( F ). Bungarotoxin (BTX) and synaptophysin indicate postsynaptic acetylcholine receptors on muscles and motor nerve terminal ends, respectively. Innerved NMJ ratio was quantified as a co-localization ratio of BTX and synaptophysin and plotted as mean ± SD (n = 3) ( G ). ( H – L ) MR6-26-2 improved proteostasis in cultured Neuro2a cells. Representative immunoblotting images are shown ( H ), and the quantification results of insoluble SOD1 ratio against soluble SOD1 ( I ), insoluble poly-Ubi (indicated by a double line in H ) ( J ), and insoluble ( K ) or soluble ( L ) p62 are expressed as mean ± SEM (n = 3, each), respectively. Three independent experiments were performed, and the amount of each protein was quantified as relative to the DMSO-treated controls with SOD1 G93A expression. Scale bar: 10 µm.

Article Snippet: The Venus-bimolecular fluorescence complementation (BiFC) plasmids containing wild-type SOD1 gene were generated by gene synthesis and cloning service of Genscript, USA.

Techniques: In Vitro, In Vivo, Ubiquitin Proteomics, Western Blot, Control, Muscles, Cell Culture, Expressing

Journal: Scientific Reports

Article Title: Ebselen analogues delay disease onset and its course in fALS by on-target SOD-1 engagement

doi: 10.1038/s41598-024-62903-5

Figure Lengend Snippet: Cell viability of human and mouse neuronal cells expressing wild-type SOD1 and ALS-associated mutants of A4V and G93A under treatments of ebselen-based compounds and ALS-approved drugs. ( A ) Chemical structures of approved ALS drugs, ebselen and derivatives. ( B ) MTS assay of mouse N2a neuroblastoma cells transfected with G93A SOD1. ( C ) human H4 neuroglioma cells transfected with A4V SOD1, and ( D , E ) ATP bioluminescent assay for single compound and in combination with approved drugs using human H4 neuroglioma cells transfected with wild-type, A4V and G93A SOD1. Cell viability levels are shown in bar chart with data points and error bars representing standard deviation of the mean from at least 6 separate measurements. Asterisks above bar charts indicate statistically significant improvements ( p < 0.05) against untreated control groups.

Article Snippet: The Venus-BiFC plasmids of A4V SOD1 were generated by site-direct mutagenesis service (Genscript, USA) using the Venus-BiFC plasmids of wild-type SOD1 as starting templates.

Techniques: Expressing, MTS Assay, Transfection, ATP Bioluminescent Assay, Standard Deviation, Control

Journal: Scientific Reports

Article Title: Ebselen analogues delay disease onset and its course in fALS by on-target SOD-1 engagement

doi: 10.1038/s41598-024-62903-5

Figure Lengend Snippet: The BiFC assay reliably captures the differences between wild-type and mutant SOD1. ( A ) Schematic illustration of the DNA constructs of the human SOD1 fused to truncated Venus fluorescent protein segments using a larger N-terminal fragment of Venus (V N ), corresponding to amino acids 1–158, and a smaller C-terminal fragment (V C ), corresponding to amino acids 159–239. ( B ) Schematic presentation of BiFC assay. Dimerization of WT SOD1 fused to Venus fragments generates fluorescence from the fusion of V N and V C fragments. ( C ) In the A4V SOD1 mutant, Venus fluorescence complementation allows to detect amyloid-like filament formation (aggregates/inclusion) due to non-native (unstable) A4V SOD1 dimerization. Representative images showing the different negative and positive controls to demonstrate the ability of BiFC assay to differentiate between stable, unstable or no SOD1 dimerization. Reciprocal negative controls of SOD1 V C with empty V N (V N + SOD1 V C or V N SOD1 with empty V C (V C + V N SOD1) vectors showing no fluorescence due to lack of dimerization in the absence of complimentary SOD1. Venus is pVenus N1 empty plasmid that does not contain SOD1 and only expresses VFP; C6S, a positive control, is an internal non-specific mutation and interaction of V N C6S SOD1 and C6S SOD1 V C presents uniform fluorescence WT represents the interaction of V N WT SOD1 and WT SOD1 V C displayingeven fluorescence A4V, aSOD1 mutant and represents the interaction of V N A4V SOD1 and A4V SOD1 V C leading to the aggregates/inclusion formation (shown by white arrows), scale bar: 25 μm. Left to right panel for each control shows transmitted light (TL), DRAQ5 stained nuclei, SOD1 VFP (after complementation of SOD1 Vc or SOD1 V N ) and merged images respectively.

Article Snippet: The Venus-BiFC plasmids of A4V SOD1 were generated by site-direct mutagenesis service (Genscript, USA) using the Venus-BiFC plasmids of wild-type SOD1 as starting templates.

Techniques: Bimolecular Fluorescence Complementation Assay, Mutagenesis, Construct, Fluorescence, Plasmid Preparation, Positive Control, Control, Staining

Journal: Scientific Reports

Article Title: Ebselen analogues delay disease onset and its course in fALS by on-target SOD-1 engagement

doi: 10.1038/s41598-024-62903-5

Figure Lengend Snippet: Ebselen and its derivatives reduce aggregating species in human cell model. ( A ) Effects of drug (MR6-26-2) doses 25, 50 and 100 µM on SOD1 dimerization following transfection and drug treatment of H4 cells with A4V (V N + V C ), scale bar: 25 µm. Left to right panel for each dose shows transmitted light (TL), DRAQ5 stained nuclei, SOD1 VFP (after complementation of SOD1 V C or SOD1 V N ) and merged images respectively. ( B ) Quantification of the number of inclusions per cell. 50–200 cells were counted per condition and per experiment and classified into three groups: blue, orange, and grey bars represent the percentage of cells without inclusions, with 5 or less inclusions and cells with more than 5 inclusions respectively. Data are expressed as mean ± SD of at least three independent experiments. One-way ANOVA, with Dunnet’s multiple comparison, was used for statistical analysis with significance level of * p < 0.05 ***p < 0.001 or ****p < 0.0001, which represents statistically different results between mutant SOD1 with and without compound treatment. ( C ) Representative images showing reduction in the aggregation propensity of H4 cells expressingA4V, G93A, H46R and D90A following treatment with most effective MR6-26-2 compound versus non-treated cells (scale bar: 50 µm). White arrows indicate the mutant SOD1 aggregation. The scaling of the images has been kept different to make the aggregates/inclusion bodies clearly visible in non-treated conditions, and to show the maximum possible number of cells within a single field as proof of drug efficacy in stable SOD1 dimerization and uniform fluorescence following treatment with MR6-26-2.

Article Snippet: The Venus-BiFC plasmids of A4V SOD1 were generated by site-direct mutagenesis service (Genscript, USA) using the Venus-BiFC plasmids of wild-type SOD1 as starting templates.

Techniques: Transfection, Staining, Comparison, Mutagenesis, Fluorescence

Journal: Scientific Reports

Article Title: Ebselen analogues delay disease onset and its course in fALS by on-target SOD-1 engagement

doi: 10.1038/s41598-024-62903-5

Figure Lengend Snippet: Co-crystalised structures of G93A SOD1 with ebselen, MR6-8-2 and MR6-26-2. Electron density (2F o –F c ) maps of ( A ) ebselen, ( B ) MR6-8-2, and ( C ) MR6-26-2 are contoured at 1σ in green mesh. Ligands and waters are illustrated as yellow sticks and red sphere, respectively. Individual SOD1 monomers are coloured in dark and light blue surface. Overlaid ligand poses in co-crystalised G93A (yellow sticks) and A4V SOD1 (pink sticks) structures of ( D ) ebselen, ( E ) MR6-8-2, and ( F ) MR6-26-2. Structures of A4V SOD1 with corresponding compounds are obtained from PDBs: 6Z4G, 6Z4L and 6Z4M respectively. Binding modes of ( G MR6-8-2 and ( H ) MR6-26-2 are shown among amino acid residues (dark and light blue sticks) and water molecules in SOD1 dimer interface. Hydrogen bonds and distances between molecules are shown as green and black dashes, respectively. Numbers represent distances in Ångstroms.

Article Snippet: The Venus-BiFC plasmids of A4V SOD1 were generated by site-direct mutagenesis service (Genscript, USA) using the Venus-BiFC plasmids of wild-type SOD1 as starting templates.

Techniques: Binding Assay

Journal: Scientific Reports

Article Title: Ebselen analogues delay disease onset and its course in fALS by on-target SOD-1 engagement

doi: 10.1038/s41598-024-62903-5

Figure Lengend Snippet: MR6-26-2 decreased misfolded SOD1 species and delayed the disease onset of SOD1 G93A mice. ( A , B ) Onset ( A ) and survival ( B ) curves of the control or MR6-26-2 treated female SOD1 G93A mice plotted over time (n = 20, each). The mean ages for onset or survival are shown with SD. ( C ) Changes in clasping score of the control or MR6-26-2 treated SOD1 G93A mice plotted over time (n = 20, each). The data were analyzed by two-way ANOVA following posthoc multiple comparisons with Šidák correction. ( D , E ) Decrease of insoluble SOD1 species in the spinal cords of MR6-26-2 treated SOD1 G93A mice at 120 days old. A representative immunoblotting image is shown ( D) , and the relative expressions of insoluble SOD1 per soluble SOD1 were quantified and plotted as mean ± SEM ( E ). ( F , G ) SOD1 oligomers produced by aberrant disulfide bonds (hSOD1 S-S oligomers) were decreased in the spinal cords of MR6-26-2 treated SOD1 G93A mice at 120 days old. A representative immunoblotting image indicates the hSOD1 S-S oligomers (a bold line) ( F ). Relative expressions of hSOD1 S-S oligomers were quantified and plotted as mean ± SEM ( G ). ( H – J ) Immunofluorescent analyses of lumbar spinal cord sections of early symptomatic SOD1 G93A mice showed the decrease of misfolded SOD1 species, detected using a C4F6 antibody that specifically recognizes misfolded SOD1, by the MR6-26-2 treatment. Motor neurons were identified using an anti-choline acetyltransferase (ChAT) antibody. Representative immunofluorescent image is shown ( H ), and the relative fluorescence intensity of C4F6 (I) or the number of ChAT-positive motor neurons (J) in the ventral horn averaged per mouse was quantified and plotted as mean ± SD. Each dot represents the section used for the quantification ( I , J ). Scale bar: 50 µm.

Article Snippet: The Venus-BiFC plasmids of A4V SOD1 were generated by site-direct mutagenesis service (Genscript, USA) using the Venus-BiFC plasmids of wild-type SOD1 as starting templates.

Techniques: Control, Western Blot, Produced, Fluorescence

Journal: Scientific Reports

Article Title: Ebselen analogues delay disease onset and its course in fALS by on-target SOD-1 engagement

doi: 10.1038/s41598-024-62903-5

Figure Lengend Snippet: MR6-26-2 ameliorated a neuromuscular junction pathology of SOD1 G93A mice and improved proteostasis in vitro and in vivo . ( A , B ) The MR6-26-2 treatment reduced poly-ubiquitin- (poly-Ubi) and p62/SQSTM1-positive inclusions in the 120-days-old SOD1 G93A lumbar spinal cord. Representative immunofluorescent images are shown ( A ). The number of the inclusions per anterior horn (AH) quantified from three mice are plotted as mean ± SEM (n = 3) ( B ). Scale bar: 75 µm. ( C – E ) Immunoblot analyses of poly-Ubi and p62 expressions in insoluble fractions of spinal cords from the control or MR6-26 treated SOD1 G93A mice at 120 days old. Representative immunoblotting images are shown ( C ), and the quantification results of insoluble poly-Ubi (bold line in C ) and insoluble p62 (black rectangle) are expressed as mean ± SEM in ( D ) and ( E ), respectively. An asterisk indicates non-specific bands. ( F , G ) MR6-26-2 reduced denervation at neuromuscular junctions (NMJs) of 120-days-old SOD1 G93A mice. Representative immunofluorescent images of tibialis anterior muscles are shown ( F ). Bungarotoxin (BTX) and synaptophysin indicate postsynaptic acetylcholine receptors on muscles and motor nerve terminal ends, respectively. Innerved NMJ ratio was quantified as a co-localization ratio of BTX and synaptophysin and plotted as mean ± SD (n = 3) ( G ). ( H – L ) MR6-26-2 improved proteostasis in cultured Neuro2a cells. Representative immunoblotting images are shown ( H ), and the quantification results of insoluble SOD1 ratio against soluble SOD1 ( I ), insoluble poly-Ubi (indicated by a double line in H ) ( J ), and insoluble ( K ) or soluble ( L ) p62 are expressed as mean ± SEM (n = 3, each), respectively. Three independent experiments were performed, and the amount of each protein was quantified as relative to the DMSO-treated controls with SOD1 G93A expression. Scale bar: 10 µm.

Article Snippet: The Venus-BiFC plasmids of A4V SOD1 were generated by site-direct mutagenesis service (Genscript, USA) using the Venus-BiFC plasmids of wild-type SOD1 as starting templates.

Techniques: In Vitro, In Vivo, Ubiquitin Proteomics, Western Blot, Control, Muscles, Cell Culture, Expressing

Journal: Stem Cell Research & Therapy

Article Title: Oxidized SOD1 accelerates cellular senescence in neural stem cells

doi: 10.1186/s13287-024-03669-5

Figure Lengend Snippet: Cellular senescence of NSCs WT after repeated passages in culture. A. Sequences of CT4 and mCT4 peptides. B. Number of NSCs WT after each passage. CT4 at 5 μM once every other day increased the number of NSCs at every passage. C. CT4 doubled the size of neurospheres at P5. a), representative image, scale bar: 100 μm. b) quantification of the diameter of spheres. D. Levels of hSOD1 detected by western blot in NSCs WT treated with CT4 or mCT4. Full-length blots are presented in Additional file 1: Fig. 1D. a) gel image; b) densitometry. E. Levels of hSOD1 detected by ELISA in NSCs WT treated with CT4 or mCT4. F. Levels of misfolded hSOD1 detected by Western blot. Full-length blots are presented in Additional file 1: Fig. 1F. a) gel image; b) densitometry. G. Thiol-disulfide status of the hSOD1 in NSCs WT treated with CT4 or mCT4 at P3 and P5. The thiol content was normalized to total hSOD1. For C and F , Student’s t-test. Two-way ANOVA followed by Sidak’s multiple comparisons test was performed for D , E and G . * P < 0.05; ** P < 0.01. All analysis was done in triplicate from three independent cultures

Article Snippet: Transgenic mice carrying human G93A mutant SOD1 (B6.Cg-Tg(SOD1*G93A)1Gur/J; 004435) and wild-type human SOD1 (B6.Cg-Tg(SOD1)2Gur/J; 002298) were obtained from the Jackson Laboratory.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Stem Cell Research & Therapy

Article Title: Oxidized SOD1 accelerates cellular senescence in neural stem cells

doi: 10.1186/s13287-024-03669-5

Figure Lengend Snippet: NSCs undergo early entrance into replicative senescence in the presence of misfolded hSOD . A. Expression of the WT hSOD1 and the hSOD1 G93A mutation in NSCs results in diminished proliferation. Cell proliferation was visualized by immunostaining with Ki67 (Green) and neural stem cell marker SOX2 (Red) in P5 NSCs WT . a) fluorescence image, scale bar: 20 μm. b) quantification of Ki-67 positive cell count. One-way ANOVA followed by Bonferroni’s Multiple Comparison Post-Test. B. P5 NSCs WT were treated with mCT4 and CT4 for 24 h, and cell proliferation was detected by immunostaining with antibodies to Ki67 (Green) and to the neural stem cell marker SOX2 (Red). a) fluorescence image, scale bar: 50 μm. Quantification of b) Ki-67 and c) SOX2 + positive cell count. Student’s t-test. C. Western blot detection of P53 at different passages of NSCs WT and NSCs G93A . Full-length blots are presented in Additional file 1: Fig. 2C. a) gel image; b) densitometry. Two-way ANOVA followed by Sidak’s multiple comparisons test was performed. All in vitro analysis was done in triplicate from three independent cultures. D. Quantitative detection of hSOD1 thiol in the spinal cord of the WT hSOD1 mice at 135 days of age. Student’s t-test. E. a) Representative immunoblot and quantitative data of b) P16 and c) P53 expression in the spinal cord of the WT hSOD1 mice (n = 5 in control, and n = 6 in AAV-CT4), Student’s t-test. Full-length blots are presented in Additional file 1: Fig. 2E

Article Snippet: Transgenic mice carrying human G93A mutant SOD1 (B6.Cg-Tg(SOD1*G93A)1Gur/J; 004435) and wild-type human SOD1 (B6.Cg-Tg(SOD1)2Gur/J; 002298) were obtained from the Jackson Laboratory.

Techniques: Expressing, Mutagenesis, Immunostaining, Marker, Fluorescence, Cell Counting, Comparison, Western Blot, In Vitro

Journal: Stem Cell Research & Therapy

Article Title: Oxidized SOD1 accelerates cellular senescence in neural stem cells

doi: 10.1186/s13287-024-03669-5

Figure Lengend Snippet: EVs from senescent NSCs WT contain misfolded and oxidized hSOD1 . A. Level of misfolded hSOD1 in EVs isolated from P3 and P5 NSCs WT and NSCs G93A as detected by affinity binding assay with the GST-tagged CT4. Blots were probed with an anti-hSOD1 antibody. Input: total SOD1 in each lysate. Full-length blots/gels are presented in Additional file 1: Fig. 3A. B. EM analysis revealed the presence of EVs by negative staining. Scale bar: 100 nm. C. Thiol-disulfide status of the SOD1 in EVs WT and EVs G93A from indicated passages of NSCs. The thiol content was normalized to total hSOD1. Two-way ANOVA followed by Sidak’s multiple comparisons test was performed. D. SOD1 expression in EVs from P5 NSCs WT and NSCs G93A was analyzed by non-reducing SDS-PAGE with antibodies to hSOD1 and misfolded hSOD1 (A5C3). Full-length blots are presented in Additional file 1: Fig. 3D. The experiments were done in triplicate from three independent cultures.

Article Snippet: Transgenic mice carrying human G93A mutant SOD1 (B6.Cg-Tg(SOD1*G93A)1Gur/J; 004435) and wild-type human SOD1 (B6.Cg-Tg(SOD1)2Gur/J; 002298) were obtained from the Jackson Laboratory.

Techniques: Isolation, Affinity Binding Assay, Negative Staining, Expressing, SDS Page

Journal: Stem Cell Research & Therapy

Article Title: Oxidized SOD1 accelerates cellular senescence in neural stem cells

doi: 10.1186/s13287-024-03669-5

Figure Lengend Snippet: Exogenous EVs from senescent NSCs activate the p53 pathway . A. Treatment with P3 and P5 EVs WT diminished proliferation in the recipient P3 NSCs WT . Cell proliferation was visualized by a) immunostaining with Ki67 (Red) and Nestin (Green). Scale bar: 20 μm. b) quantification of Ki-67 positive cell count. Two-way ANOVA followed by Sidak’s multiple comparisons test was performed. B. and C. Expression of P16 and P53 detected by Western blot in P3 NSCs WT treated with P3 or P5 EVs WT for 24 h. Full-length gels are presented in Additional file 1: Figs. 4B and 4C. a) gel images; b) densitometry. One-way ANOVA followed by Bonferroni’s Multiple Comparison Post-Test was performed. D. a) Removal of misfolded SOD1 from P5 EVs WT restored the proliferation of NSCs. b) Incubation of GST-tagged CT4 and P5 EVs WT for 2 h resulted in a pulldown of a misfolded SOD1 band as detected by Western blot. After removing misfolded SOD1, P5 EVs WT lost its inhibition on the proliferation of P3 NSCs WT as determined by immunostaining with antibodies to Ki67 (Red) and Nestin (Green). Scale bar: 20 μm. c) For quantitative comparison of Ki-67 positive cells, a Student’s t-test was performed. The experiments were done in triplicate from three independent cultures

Article Snippet: Transgenic mice carrying human G93A mutant SOD1 (B6.Cg-Tg(SOD1*G93A)1Gur/J; 004435) and wild-type human SOD1 (B6.Cg-Tg(SOD1)2Gur/J; 002298) were obtained from the Jackson Laboratory.

Techniques: Immunostaining, Cell Counting, Expressing, Western Blot, Comparison, Incubation, Inhibition

Journal: Stem Cell Research & Therapy

Article Title: Oxidized SOD1 accelerates cellular senescence in neural stem cells

doi: 10.1186/s13287-024-03669-5

Figure Lengend Snippet: Exogenous EVs from senescent NSCs induce aging-like phenotypes . A. Internalization of exogenous EVs. Shown are representative immunofluorescence staining of misfolded hSOD1 (B8H10, Green) and hSOD1 (blue) after treatment with EVs WT and EVs G93A from P5 NSCs. EVs were labeled with Texas Red. Scale bar: 2 μm. B. Immunofluorescence staining of P3 NSCs WT with HMGB1 (Green) and Nestin (Red) after treatment with P5 EVs WT and EVs G93A . Senescent NSCs were detected by cytoplasmic translocation of HMGB1 and by expressing SA-β-gal. Scale bar: 10 μm . C. The translocation of HMGB1 from the nucleus to the cytosol in the P3 recipient NSCs WT . D. Quantification of SA-β-gal positive cells. For C and D, one-way ANOVA followed by Bonferroni’s Multiple Comparison Post-Test was performed. The experiments were conducted in triplicate from three independent cultures

Article Snippet: Transgenic mice carrying human G93A mutant SOD1 (B6.Cg-Tg(SOD1*G93A)1Gur/J; 004435) and wild-type human SOD1 (B6.Cg-Tg(SOD1)2Gur/J; 002298) were obtained from the Jackson Laboratory.

Techniques: Immunofluorescence, Staining, Labeling, Translocation Assay, Expressing, Comparison

Journal: Stem Cell Research & Therapy

Article Title: Oxidized SOD1 accelerates cellular senescence in neural stem cells

doi: 10.1186/s13287-024-03669-5

Figure Lengend Snippet: Blocking misfolded SOD1 reduces SOD1 aggregation and mitigates the neurotoxicity of EVs isolated from senescent NSCs. A. and B. Blocking misfolded SOD1 reduces SOD1 aggregation. P5 EVs WT were incubated for 2 h with or without the CT4 motif before adding it to P3 NSCs WT . After 24 h, protein samples were separated into detergent-insoluble and soluble fractions and analyzed by Western blot with an anti-hSOD1 antibody. Full-length blots/gels are presented in Additional file 1: Fig. 6A. Total protein generated by stain-free visualization was used to ensure equal loading. Two-way ANOVA followed by Sidak’s multiple comparisons test was performed. C. Neurotoxicity of P5 EVs to P3 NSCs WT . Cells were immunostained with antibodies to misfolded hSOD1 (B8H10, Red) and MAP2 (Green). Scale bar: 50 μm . D. Quantification of MAP2 positive neurons. E. Release of lactate dehydrogenase. For D and E, one-way ANOVA followed by Bonferroni’s Multiple Comparison Post-Test was performed. The experiments were done in triplicate from three independent cultures

Article Snippet: Transgenic mice carrying human G93A mutant SOD1 (B6.Cg-Tg(SOD1*G93A)1Gur/J; 004435) and wild-type human SOD1 (B6.Cg-Tg(SOD1)2Gur/J; 002298) were obtained from the Jackson Laboratory.

Techniques: Blocking Assay, Isolation, Incubation, Western Blot, Generated, Staining, Comparison