Cellular analysis of SOD1 protein-aggregation propensity and toxicity: a case of ALS with slow progression harboring homozygous SOD1-D92G mutation – Scientific Reports

Ethics statements and consent to participate

This study was approved by the Kyoto University Graduate School of Medicine Ethical Committee (G1178, R91). Written, informed consent was obtained from the participants prior to inclusion in the study. Samples from the participants were identified by numbers, not by names. All methods were performed in accordance with the relevant guidelines and regulations.

MRI data acquisition

MRI scans were performed using the 3 T Magnetom Prisma or Avanto system for the brain or muscles, respectively (Siemens, Erlangen, Germany). Data analysis was performed with Centricity PACS 4.0 (GE Healthcare, Chicago, IL, USA).

DNA sequencing

DNA was extracted from a blood sample, and previously reported primers were useda of SOD1 gene is not an ALS causing mutation. Amyotroph. Lateral Scler. Other Motor Neuron Disord. 6, 45–49 (2005).” href=”http://news.google.com/articles/s41598-022-16871-3#ref-CR28″ id=”ref-link-section-d329520438e1811″>28. The exons of SOD1 were amplified and Sanger sequencing was performed on 3730xl DNA Analyzer (Thermo Fisher Scientific, Waltham, MA, USA) with Thermo Fisher ScientificTerminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific). A novel homD92G mutation in the SOD1 gene was identified (Fig. 1).

Whole exome sequencing

For whole-exome sequencing, genomic DNA was extracted, and 1 μg was sheared and used for the construction of a paired-end sequencing library as described in the protocol provided by Illumina. Enrichment of exonic sequences was then performed for each library using the SureSelect Human All Exon V6 (Agilent Technologies Inc., Santa Clara, CA, USA) following the manufacturer’s instructions. Libraries for whole-exome sequencing were sequenced with a NovaSeq 6000 (Illumina Inc., San Diego, CA, USA).

In silico analysis

The effects of the newly detected SOD1-homD92G and previously reported mutations of FALS cases (SOD1-L84F, N86S, D90A, D92G, G93A, and L126S) were analyzed with Mutation Taster (http://www.mutationtaster.org), Sorting Intolerant from Tolerant (SIFT, https://sift.bii.a-star.edu.sg/), PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/), PROVEAN (http://provean.jcvi.org/index.php), and PANTHER (http://www.pantherdb.org/).

Plasmids and cell culture

KOD-Plus-Mutagenesis kits (TOYOBO, Osaka, Japan) were used to generate pSOD1-D92G-EGFP and pSOD1-G93A-EGFP plasmids. The mouse neuroblastoma Neuro2a cells were plated onto 24-well plastic plates (Greiner Bio-One, Kremsmünster, Austria) or glass-bottom dishes (Matsunami, Osaka, Japan), and maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Wako, Tokyo, Japan) with 5% fetal bovine serum (FBS) (Thermo Fisher Scientific). The Neuro2a cells were transfected with plasmid carrying pSOD1-(WT/D92G/G93A)-EGFP using lipofectamine polyethyleneimine “MAX” PEI (Polysciences, Warrington, UK) reagent according to the manufacturer’s protocol. After overnight incubation, the medium was changed with new growth medium. At 48 h after transfection, the cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS). After 10-min incubation with PBS/0.1% Tween, the samples were incubated overnight at 4 °C with primary antibodies against misfolded SOD1 (A5C3, 1:200, MEDIMABS, Montreal, Canada). The samples were subsequently incubated with donkey-derived secondary antibodies (Alexa Fluor 594, 1:1000, Thermo Fisher Scientific) for 1 h at room temperature, and they were then covered with antifade mounting medium, VECTASHIELD® with DAPI, (Vector Laboratories, Burlingame, CA, USA). High magnification images were acquired with an FV-1000 confocal laser scanning microscope (Olympus, Tokyo, Japan). SOD1-EGFP aggregates were observed with a BZ-X710 fluorescence microscope (KEYENCE, Osaka, Japan). A ‘SOD1-EGFP-aggregate’ was defined as a dense round formation more than 10 µm in diameter and counted in a double-blind manner. An ‘aggregate-positive cell’ was defined as a cell having an ‘EGFP-aggregate’. The ratio of the number of ‘aggregate-positive cells’ to that of whole EGFP-expressing cells was calculated.

Western-blotting (WB)

The Neuro2a cells were plated onto 6-well plastic plates (Greiner Bio-One). The Neuro2a cells were transfected with the plasmid carrying SOD1-EGFP (WT, D92G, or G93A) using LTX (Thermo Fisher Scientific) reagent according to the manufacturer’s protocol. After overnight incubation, the medium was changed with new growth medium. At 48 h after transfection, the cells in 6-well dishes were washed twice with PBS and then scraped in 300 µl of PBS. The cells were sonicated (Cosmo Bio, Bioruptor, Tokyo, Japan) for 5 min and shaken for 15 min at 4 °C, and then the lysates were centrifuged at 12,000g for 10 min at 4 °C. The resulting pellet was resolved in 20 µl of 2% SDS as the PBS-insoluble fraction, and the supernatant was designated the PBS-soluble fraction. These lysates were incubated in 2 × SDS sample buffer and boiled at 95 °C for 5 min. One-fourth of the insoluble fraction and 1/60 of the soluble fraction were processed for the detection of SOD1. These proteins (PBS-soluble 5 µl, PBS-insoluble 5 µl) were loaded onto SuperSep™ Ace 10–20% (Wako, Tokyo, Japan). The proteins were then transferred onto polyvinylidene difluoride membranes and blocked with 5% skim milk in Tris-buffered saline Tween-20 (TBST) for 30 min. The membranes were incubated with an anti-SOD1 primary antibody (Enzo Life Science abi-sod-100F, 1:1000) at 4 °C overnight. Next, the membranes were incubated for 1 h at room temperature with a horseradish peroxidase secondary antibody (Santa Cruz #sc-2005, 1:10,000), and the protein bands were visualized using ECL Western Blotting Substrate (Thermo Fisher Scientific). Chemiluminescent signals were detected using an Amersham Imager 600 imager (GE Healthcare, Chicago, IL, USA). To evaluate the protein levels, these bands were analyzed with ImageJ ver. 1.50i (https://imagej.nih.gov/ij/). Total protein was measured by Coomassie Brilliant Blue staining (CBB Stain One, Nacalai Tesque, Kyoto, Japan). The insoluble fraction ratio was calculated as I/S, where S is the band intensity of the soluble fraction and I of the insoluble fraction.

Cell viability assay

Neuro2a cells were plated onto 24-well plastic plates. Cell viability was evaluated with the LDH assay kit (Dojindo, Tokyo, Japan) 48 h after transfection. The LDH assay was performed according to the manufacturer’s protocol.

Generation of human iPSCs

Human iPSCs were generated from peripheral blood mononuclear cells (PBMCs) using episomal vectors (Sox2, Klf4, Oct3/4, L-Myc, Lin28, and p53-shRNA) as reported previously29 and cultured by a feeder-free culture system with StemFit (Ajinomoto, Tokyo, Japan). Karyotype analysis of iPSCs was conducted by LSI Medience (Tokyo, Japan). Established iPSCs were cultured under feeder-free conditions on iMatrix (Nippi, Tokyo, Japan)-coated plates with StemFit AK01 (Ajinomoto).

Generation of motor neurons

Motor neurons were generated from iPSCs as previously described13. Briefly, iPSCs carrying the tetracycline-inducible motor neuron differentiation cassette containing Lhx3, Ngn2, and Isl1 (LNI cassette) under control of the tetracycline operator were established. The iPSCs were dissociated to single cells using Accumax and plated onto Matrigel-coated 96-well plates with the Neuronal Medium containing DMEM/F12 (Thermo Fisher Scientific), N2 (Thermo Fisher Scientific) containing 1 μM retinoic acid (Sigma), 1 μM Smoothened Agonist (SAG), 10 ng/ml BDNF (R&D Systems, Minneapolis, MN, USA), 10 ng/ml GDNF (R&D Systems), and 10 ng/ml NT-3 (R&D Systems) with 1 μg/ml doxycycline (TAKARA, Kusatsu, Japan), and cultured for 7 days.

Motor neuron survival assay

The iPSCs derived motor neurons were cultured on iMatrix-coated 96-well plates (BD Bioscience, San Jose, CA) for 7 days and 14 days. The number of surviving motor neurons stained with the βIII-tubulin antibody was quantified by IN Cell Analyzer 6000 and IN Cell Developer toolbox software 1.9, and the ratio of surviving neurons on day 14 to those on day 7 was shown as a percent, as previously described13.

Enzyme-linked immunosorbent assay (ELISA)

For ELISA of misfolded SOD1, motor neurons on day 7 were harvested and dissolved in buffer containing 1% Triton-X, 0.5% deoxycholate, 50 mM Tris–HCl, 1 mM EDTA, 0.1% SDS, 150 mM NaCl, 0.1% sodium deoxycholate, protease inhibitor (Roche), and phosphatase inhibitor (Roche). Samples were centrifuged at 13,000×g for 15 min at 4 °C. Then, 96-well plates (Thermo Fisher Scientific) were coated with 3 μg/ml MS785 antibody in 0.05 M sodium carbonate buffer at 4 °C overnight. After washing and blocking with TBS-T containing 1% BSA, 200 μg protein/100 µl of samples were added, and incubation was carried out for 2 h at room temperature. Recombinant mutant SOD1 protein (G93A) was used to obtain a standard curve. For detection, the plates were incubated with 3 μg/ml anti-SOD1 antibody (ENZO), followed by sheep anti-rabbit IgG F(ab)’2 fragment linked to horseradish peroxidase (1:3000; GE Healthcare). After incubation with tetramethylbenzidine solution (BD Bioscience) at room temperature for 30 min, absorbance at 450 nm was measured by VersaMax (Molecular Device, Sunnyvale, CA, USA).

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