Journal: International Journal of Molecular Sciences

Article Title: A Metal-Free, Disulfide Oxidized Form of Superoxide Dismutase 1 as a Primary Misfolded Species with Prion-Like Properties in the Extracellular Environments Surrounding Motor Neuron-Like Cells

doi: 10.3390/ijms22084155

Figure Lengend Snippet: Generation of motor neuron models of ALS in which misfolded and aggregated hSOD1 are intracellularly deposited. NSC-34 cells were transiently transfected with expression vectors encoding human wild-type superoxide dismutase 1 (hSOD1 WT ) or the ALS-causing hSOD1 mutants (G93A, G37R, D90A, and G85R) tagged with green fluorescent protein (GFP), treated with all trans -retinoic acid, and cultured for 72 h. ( a ) Western blots of hSOD1 in the Nonidet P-40 (NP-40) soluble and insoluble factions from NSC-34 cells, β-tubulin was used as a loading control. No Vec., untransfected cells; ( b ) florescence microscopy of cells transfected with hSOD1 WT GFP or the ALS-linked hSOD1 mutant GFP. Arrows indicate protein aggregations containing hSOD1 GFP. Scale bar = 10 µm; ( c ) enzyme-linked immunosorbent assay signals of (black) EDI-positive and (gray) C4F6-positive misfolded SOD1 in the NP-40 soluble fraction. All data are shown as the mean ± SD, n = 3 in each transfection.

Article Snippet: For experiments on intracellular propagation of hSOD1 WT misfolding by the conditioned medium, wild-type SOD1 with untagged GFP was also cloned into pCMV3 from human cDNA, (HG11727-UT, Sino Biological, Beijing, China).

Techniques: Transfection, Expressing, Cell Culture, Western Blot, Microscopy, Mutagenesis, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: A Metal-Free, Disulfide Oxidized Form of Superoxide Dismutase 1 as a Primary Misfolded Species with Prion-Like Properties in the Extracellular Environments Surrounding Motor Neuron-Like Cells

doi: 10.3390/ijms22084155

Figure Lengend Snippet: Decrease in the total level of extracellular hSOD1 mutants, but not murine SOD1, in the conditioned medium. The NSC-34 cells were transiently transfected with human wild-type superoxide dismutase 1 (hSOD1 WT ) and the ALS-causing hSOD1 mutants (G93A, G37R, D90A, and G85R) fused with green fluorescent protein (GFP). After 72 h of transfection, the conditioned medium from the cells was collected; ( a ) Western blots of hSOD1-GFP and endogenous murine SOD1 in the medium, β-tubulin was used as a marker for intracellular non-secreted protein to confirm whether cell lysis or debris could be contaminated in the conditioned medium. The amount of protein loaded for sodium dodecyl sulfate polyacrylamide gel electrophoresis was validated by Ponceau S staining; ( b , c ) quantification of the absolute total protein level of extracellular ( b ) hSOD1 and ( c ) murine SOD1. Data are shown as the mean ± SD, n = 3 in each transfection. Statistical analysis was performed using one-way ANOVA followed by the Tukey–Kramer post hoc test. ** p < 0.01 vs. hSOD1 WT .

Article Snippet: For experiments on intracellular propagation of hSOD1 WT misfolding by the conditioned medium, wild-type SOD1 with untagged GFP was also cloned into pCMV3 from human cDNA, (HG11727-UT, Sino Biological, Beijing, China).

Techniques: Transfection, Western Blot, Marker, Lysis, Polyacrylamide Gel Electrophoresis, Staining

Journal: International Journal of Molecular Sciences

Article Title: A Metal-Free, Disulfide Oxidized Form of Superoxide Dismutase 1 as a Primary Misfolded Species with Prion-Like Properties in the Extracellular Environments Surrounding Motor Neuron-Like Cells

doi: 10.3390/ijms22084155

Figure Lengend Snippet: The thiol/disulfide redox balance of extracellular hSOD1 shifts toward oxidation because of increased oxidative stress in the conditioned medium. The conditioned medium of the NSC-34 cells expressing human wild-type superoxide dismutase 1 (hSOD1 WT )-green fluorescence protein (GFP), the ALS-linked hSOD1-GFP, or GFP alone was treated with 100 mM iodoacetamide to block artificial oxidation of the thiol group of proteins. ( a ) Non-reducing Western blots of disulfide oxidized (S-S) and reduced (-SH) forms of hSOD1-GFP in the medium. β-ME = beta-mercaptoethanol; ( b ) quantification of absolute levels of (black) hSOD1 S-S and (gray) hSOD1 SH in the medium; ( c ) Western blots of 4-hydroxynonenal (4-HNE)-conjugated proteins in the conditioned medium; ( d ) densitometrical calculation of relative levels of 4-HNE-conjugated proteins in the medium. The amount of protein loaded for sodium dodecyl sulfate polyacrylamide gel electrophoresis was validated by Ponceau S staining. Data are expressed as the mean ± SD. Statistical analysis was performed using one-way ANOVA followed by the Tukey–Kramer post hoc test, n = 3 for each transfection. ** p < 0.01 vs. no vector.

Article Snippet: For experiments on intracellular propagation of hSOD1 WT misfolding by the conditioned medium, wild-type SOD1 with untagged GFP was also cloned into pCMV3 from human cDNA, (HG11727-UT, Sino Biological, Beijing, China).

Techniques: Expressing, Fluorescence, Blocking Assay, Western Blot, Polyacrylamide Gel Electrophoresis, Staining, Transfection, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: A Metal-Free, Disulfide Oxidized Form of Superoxide Dismutase 1 as a Primary Misfolded Species with Prion-Like Properties in the Extracellular Environments Surrounding Motor Neuron-Like Cells

doi: 10.3390/ijms22084155

Figure Lengend Snippet: The population of extracellular Cu/Zn-deficient hSOD1 was increased in the conditioned medium. The conditioned medium from the NSC-34 cells transfected with human wild-type superoxide dismutase 1 (hSOD1 WT )-green fluorescence protein (GFP), the ALS-linked hSOD1-GFP, or GFP alone, was treated with 100 mM iodoacetamide to block artificial oxidation of the thiol group of proteins. Then, extracellular human and murine SOD1 enzymatic activity in the medium was measured by a SOD Assay Kit-WST. The level of active SOD1 in the medium was calculated based on a calibration curve that was generated from a Cu/Zn SOD1 standard. The extracellular level of active ( a ) hSOD1 and ( b ) endogenous murine SOD1 in the medium; ( c ) the medium was treated with 1 mM CuSO 4 , 1 mM ZnSO 4 , or both, for 24 h, and active hSOD1 levels in the medium were quantified using a SOD Assay Kit-WST. All data are expressed as the mean ± SD. Statistical analysis was performed using one-way ANOVA followed by the Tukey–Kramer post hoc test, n = 3 for each treatment or for each transfection. ** p < 0.01 vs. H 2 O-added medium that corresponds to the same transfection.

Article Snippet: For experiments on intracellular propagation of hSOD1 WT misfolding by the conditioned medium, wild-type SOD1 with untagged GFP was also cloned into pCMV3 from human cDNA, (HG11727-UT, Sino Biological, Beijing, China).

Techniques: Transfection, Fluorescence, Blocking Assay, Activity Assay, Generated

Journal: International Journal of Molecular Sciences

Article Title: A Metal-Free, Disulfide Oxidized Form of Superoxide Dismutase 1 as a Primary Misfolded Species with Prion-Like Properties in the Extracellular Environments Surrounding Motor Neuron-Like Cells

doi: 10.3390/ijms22084155

Figure Lengend Snippet: Extracellular misfolded human superoxide dismutase 1 (hSOD1) presents as a metal-free, disulfide oxidized form (apo-SOD1 S-S ). Enzyme-linked immunosorbent assay signals of ( a ) EDI-positive misfolded SOD1; ( b ) C4F6-positive misfolded SOD1; ( c ) total hSOD1, in the conditioned medium treated with or without both 1 mM CuSO 4 and 1 mM ZnSO 4 for 24 h. Data are expressed as the mean ± SD, n = 3 for each treatment per each transfection. Statistical analysis was performed using a two-tailed unpaired Student’s t test. ** p < 0.01 vs. Cu/Zn-untreated medium that corresponds to the same transfection. N.S., not significant; ( d ) the extracellular levels of different folding states of hSOD1 in the mediums; ( e ) extracellular misfolded SOD1 in the medium was detected by immunoprecipitation with C4F6 followed by non-reducing Western blotting to determine the thiol/disulfide redox balance of the misfolded species. β-ME = beta-mercaptoethanol.

Article Snippet: For experiments on intracellular propagation of hSOD1 WT misfolding by the conditioned medium, wild-type SOD1 with untagged GFP was also cloned into pCMV3 from human cDNA, (HG11727-UT, Sino Biological, Beijing, China).

Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Two Tailed Test, Immunoprecipitation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: A Metal-Free, Disulfide Oxidized Form of Superoxide Dismutase 1 as a Primary Misfolded Species with Prion-Like Properties in the Extracellular Environments Surrounding Motor Neuron-Like Cells

doi: 10.3390/ijms22084155

Figure Lengend Snippet: Extracellular misfolded apo-SOD1 S-S induces intracellular propagation of hSOD1 WT misfolding in recipient cells. A procedure to investigate intracellular prion-like propagation of human wild-type superoxide dismutase 1 (hSOD1 WT ) misfolding was performed using immunoprecipitation of the Nonidet P (NP-40) soluble fraction with C4F6 followed by Western blotting with antibodies to ( a , c ) SOD1 or (f) green fluorescence protein (GFP). The conditioned media from NSC-34 cells transfected with hSOD1-GFP containing misfolded apo-SOD1 S-S were used as a donor medium, whereas NSC-34 cells transfected with GFP-untagged hSOD1 WT were used as a recipient cell. ( a ) The recipient cells were exposed to conditioned medium pretreated with or without C4F6 to remove misfolded apo-SOD1 S-S ; ( b ) relative level of intracellular misfolded hSOD1 WT in the recipient cells exposed to medium pretreated with or without C4F6, ** p < 0.01 vs. C4F6-untreated cells that correspond to the same transfection, n = 3 for each treatment; ( c ) the recipient cells were pretreated with endocytosis inhibitors, 50 nM wortmannin, or 100 µM 5-( N -ethyl- N -isopropyl) amiloride (EIPA), for 1 h, and the cells were exposed to the medium for 24 h; ( d ) relative level of intracellular misfolded hSOD1 WT in endocytosis-impaired cells, ** p < 0.01 vs. DMSO-treaded cells that were exposed to the corresponding medium, n = 3 for each treatment; ( e ) the recipient cells expressing GFP-tagged hSOD1 WT were exposed to 0.5 µg/mL purified hSOD1 WT proteins with a disulfide bond (Cu/Zn SOD1 S-S , Zn SOD1 S-S , and apo-SOD1 S-S ) for 24 h, n = 3 for each treatment; ( f ) relative level of intracellular misfolded hSOD1 WT cells treated with the purified hSOD1 WT protein, ** p < 0.01 vs. HEPES-treated cells, N.S. = not significant. All data are expressed as the mean ± SD. Statistical analysis was performed using one-way ANOVA followed by the Tukey–Kramer post hoc test.

Article Snippet: For experiments on intracellular propagation of hSOD1 WT misfolding by the conditioned medium, wild-type SOD1 with untagged GFP was also cloned into pCMV3 from human cDNA, (HG11727-UT, Sino Biological, Beijing, China).

Techniques: Immunoprecipitation, Western Blot, Fluorescence, Transfection, Expressing, Purification

Journal: International Journal of Molecular Sciences

Article Title: A Metal-Free, Disulfide Oxidized Form of Superoxide Dismutase 1 as a Primary Misfolded Species with Prion-Like Properties in the Extracellular Environments Surrounding Motor Neuron-Like Cells

doi: 10.3390/ijms22084155

Figure Lengend Snippet: The conditioned medium containing extracellular misfolded metal free, disulfide oxidized superoxide dismutase 1 (apo-SOD1 S-S ) exerted cytotoxicity to motor neuron-like cells. (white) NSC-34 cells expressing green fluorescence protein (GFP) untagged human wild-type SOD1 (hSOD1 WT ) were exposed to conditioned medium from NSC-34 harboring hSOD1-GFP containing misfolded apo-SOD1 S-S for 24 h. Cell viability and cytotoxicity were assessed using ( a ) a Cell Counting Kit 8 assay and ( b ) a lactate dehydrogenase (LDH) release assay, respectively. (black) Extracellular misfolded apo-SOD1 S-S was removed from the conditioned medium by immunoprecipitation with C4F6. (gray) As a control, the medium was immunoprecipitated with normal mouse IgG. In the data set of cell viability, the results are expressed as the cell viability of recipient cells relative to that of untransfected cells, which were incubated with normal culture medium, Dulbecco’s modified Eagle’s medium and F-12 with GlutaMAX™ containing 1% ( v / v ) fetal bovine serum and 0.1 mM non-essential amino acids, instead of the conditioned medium. In the data set of cytotoxicity, the results are given as the amounts of released LDH to the medium relative to that of cells exposed to 100 μM H 2 O 2 for 24 h. All data are expressed as the mean ± SD, n = 3 for each treatment. Statistical analysis was performed using one-way ANOVA followed by the Tukey–Kramer post hoc test. ** p < 0.01.

Article Snippet: For experiments on intracellular propagation of hSOD1 WT misfolding by the conditioned medium, wild-type SOD1 with untagged GFP was also cloned into pCMV3 from human cDNA, (HG11727-UT, Sino Biological, Beijing, China).

Techniques: Expressing, Fluorescence, Cell Counting, Lactate Dehydrogenase Assay, Immunoprecipitation, Incubation, Modification

Journal: ACS chemical biology

Article Title: Amyotrophic Lateral Sclerosis-Associated Mutants of SOD1 Modulate miRNA Biogenesis through Aberrant Interactions with Exportin 5.

doi: 10.1021/acschembio.2c00591

Figure Lengend Snippet: Figure 1. Combination of APEX2 labeling with LC−MS/MS revealed XPO5 as a candidate interaction partner of SOD1G85R and SOD1G93A. (a) Schematic diagram showing the workflow for APEX labeling. “BP” and “AP” represent biotin phenol and affinity pull-down, respectively. (b) Western blot showing a similar level of expression for different SOD1-APEX2 variants in HEK293T cells. (c) Western blot indicated similar APEX labeling efficiencies for SOD1WT, SOD1G85R, and SOD1G93A. Cells were transfected with plasmids encoding APEX2-conjugated SOD1WT, SOD1G85R, and SOD1G93A for 24 h, followed by treatments with biotin phenol for 30 min and then with H2O2 for 1 min. “SA-HRP” represents streptavidin-horseradish peroxidase. (d) Venn diagrams comparing the proximity proteomes of SOD1G85R vs SOD1WT and SOD1G93A vs SOD1WT. In total, 1999 proteins were identified. (e) Volcano plots displaying fold changes for the proteins being pulled down with streptavidin beads. The p values were calculated by using paired, two-tailed Student’s t-test. The red and blue labels designate those with statistically significant fold changes (p < 0.05).

Article Snippet: Plasmids for wild-type (WT) SOD1 (plasmid #26397), SOD1G85R (plasmid #26400), and SOD1G93A (plasmid#26401) were obtained from Addgene.

Techniques: Labeling, Liquid Chromatography with Mass Spectroscopy, Western Blot, Expressing, Transfection, Two Tailed Test

Journal: ACS chemical biology

Article Title: Amyotrophic Lateral Sclerosis-Associated Mutants of SOD1 Modulate miRNA Biogenesis through Aberrant Interactions with Exportin 5.

doi: 10.1021/acschembio.2c00591

Figure Lengend Snippet: Figure 3. Ectopic expression of the G85R and G93A mutants of SOD1 perturbs the nucleocytoplasmic transport of pre-miRNAs and the maturation of miRNAs. (a) RT-qPCR results showing increases in the nuclear levels and decreases in the cytosol levels of pre-miR-let7a, pre-miR- 30a, and pre-miR-143 in cells expressing SOD1G93A and SOD1G95R compared to those expressing the WT protein. (b) RT-qPCR results depicting substantially increased levels of mature miR-30a, miR-let7b, miR-100, and miR-15a in cells with the ectopic expression of SOD1G85R and SOD1G93A

Article Snippet: Plasmids for wild-type (WT) SOD1 (plasmid #26397), SOD1G85R (plasmid #26400), and SOD1G93A (plasmid#26401) were obtained from Addgene.

Techniques: Expressing, Quantitative RT-PCR

Journal: Cellular and Molecular Life Sciences

Article Title: Cellular therapy to target neuroinflammation in amyotrophic lateral sclerosis

doi: 10.1007/s00018-013-1480-4

Figure Lengend Snippet: Recent advancements in astrocyte [glial-restricted precursors (GRP)] transplantation for ALS

Article Snippet: Wild-type mouse BMCs , Myelo-ablated SOD1 G93A mice , No benefit on disease progression , [ ] .

Techniques: Transplantation Assay

Journal: Cellular and Molecular Life Sciences

Article Title: Cellular therapy to target neuroinflammation in amyotrophic lateral sclerosis

doi: 10.1007/s00018-013-1480-4

Figure Lengend Snippet: Recent advancements in bone marrow-derived cell (BMC) transplantation for ALS

Article Snippet: Wild-type mouse BMCs , Myelo-ablated SOD1 G93A mice , No benefit on disease progression , [ ] .

Techniques: Transplantation Assay, Expressing, Irradiation