Cellular analysis of SOD1 protein aggregation propensity and toxicity: a case of slowly progressing ALS harboring a homozygous SOD1-D92G mutation

Statements of ethics and consent to participate

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

MRI data acquisition

MRIs were performed using the 3T Magnetom Prisma or Avanto system for brain or muscle, 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 used²⁸. The exons of SOD1 were amplified and Sanger sequencing was performed on a 3730xl DNA Analyzer (Thermo Fisher Scientific, Waltham, MA, USA) with the Thermo Fisher Scientific Terminator 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 construction of a paired sequencing library as described in the protocol provided by Illumina. Exon sequence enrichment 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 new detection 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/), PROVEN (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 the pSOD1-D92G-EGFP and pSOD1-G93A-EGFP plasmids. Neuro2a mouse neuroblastoma cells were plated on 24-well plastic plates (Greiner Bio-One, Kremsmünster, Austria) or glass-bottom dishes (Matsunami, Osaka, Japan) and maintained in Eagle’s modified medium. Dulbecco (DMEM) (Wako, Tokyo, Japan) with 5% fetal bovine serum (FBS) (Thermo Fisher Scientific). Neuro2a cells were transfected with the plasmid carrying pSOD1-(WT/D92G/G93A)-EGFP using lipofectamine polyethyleneimine “MAX” PEI reagent (Polysciences, Warrington, UK) according to the manufacturer’s protocol. After overnight incubation, the medium was replaced with new growth medium. 48 h after transfection, cells were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS). After 10 minutes of incubation with PBS/0.1% Tween, samples were incubated overnight at 4°C with primary antibodies against misfolded SOD1 (A5C3, 1:200, MEDIMABS, Montreal, Canada ). Samples were then incubated with donkey-derived secondary antibodies (Alexa Fluor 594, 1:1000, Thermo Fisher Scientific) for 1 h at room temperature, followed by overlaying with antifade mounting medium, VECTASHIELD^® with DAPI, (Vector Laboratories, Burlingame, CA, USA). High magnification images were acquired with a confocal laser scanning microscope FV-1000 (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 greater than 10 µm in diameter and counted double-blind. 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 cells expressing EGFP was calculated.

Western blot (WB)

Neuro2a cells were plated on 6-well plastic plates (Greiner Bio-One). Neuro2a cells were transfected with the plasmid carrying SOD1-EGFP (WT, D92GWhere G93A) using LTX reagent (Thermo Fisher Scientific) according to the manufacturer’s protocol. After overnight incubation, the medium was replaced with new growth medium. 48 h after transfection, cells in 6-well dishes were washed twice with PBS and then scraped in 300 μl of PBS. Cells were sonicated (Cosmo Bio, Bioruptor, Tokyo, Japan) for 5 min and shaken for 15 min at 4°C, then 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 referred to as the PBS-soluble fraction. These lysates were incubated in 2 × SDS sample buffer and boiled at 95°C for 5 min. A quarter of the insoluble fraction and 1/60 of the soluble fraction were processed for the detection of SOD1. These proteins (5 µl soluble in PBS, 5 µl insoluble in PBS) were loaded onto SuperSep™ Ace 10–20% (Wako, Tokyo, Japan). Proteins were then transferred to polyvinylidene difluoride membranes and blocked with 5% skim milk in Tris Tween-20 buffered saline (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. Then, membranes were incubated for 1 h at room temperature with horseradish peroxidase secondary antibody (Santa Cruz #sc-2005, 1:10,000) and protein bands were visualized using blot substrate Western ECL (ThermoFisher Scientific). Chemiluminescent signals were detected using an Amersham Imager 600 imager (GE Healthcare, Chicago, IL, USA). To assess 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 ratio of insoluble fraction 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 on 24-well plastic plates. Cell viability was assessed 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 (PBMC) using episomal vectors (Sox2, Klf4, Oct3/4, L-Myc, Lin28and p53-shRNA) as stated previously²⁹ and cultured by a no-feed culture system with StemFit (Ajinomoto, Tokyo, Japan). Karyotype analysis of iPSCs was performed by LSI Medience (Tokyo, Japan). Established iPSCs were cultured under loaderless conditions on iMatrix-coated plates (Nippi, Tokyo, Japan) with StemFit AK01 (Ajinomoto).

Generation of motor neurons

Motor neurons were generated from iPSCs as previously described¹³. Briefly, iPSCs carrying the tetracycline-induced motor neuron differentiation cassette containing Lhx3, Ngn2and Isl1 (LNI cassette) under the control of the tetracycline operator have been established. iPSCs were dissociated into single cells using Accumax and plated on 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 Smoothed 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 test

iPSC-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 software and IN Cell Developer toolbox 1.9, and the ratio between surviving neurons at day 14 and those at day 7 was indicated as a percentage. , like before. describe¹³.

Enzyme immunoassay (ELISA)

For the misfolded SOD1 ELISA, 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 MS785 antibody at 3 μg/ml 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 sample was added and incubation was performed for 2 h at room temperature. The recombinant mutant SOD1 protein (G93A) was used to obtain a standard curve. For detection, plates were incubated with 3 μg/ml anti-SOD1 antibody (ENZO), followed by horseradish peroxidase-linked sheep anti-rabbit IgG F(ab)’2 fragment ( 1:3000; GE Healthcare). After incubation with a solution of tetramethylbenzidine (BD Bioscience) at room temperature for 30 min, the absorbance at 450 nm was measured by VersaMax (Molecular Device, Sunnyvale, CA, USA).