Cell culture and plasmid
DAP (triple affinity-purification tag; a biotinylation sequence and FLAG tags, and N-terminal epitope tag; 6-histidine epitope tag) -TDP-43-inducible 293 T Rex cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, glucose, 4.5 g/l) supplemented with Tet System Approved FBS (Takara Bio USA, CA, USA) at 37 °C in a 5% CO2/95% air atmosphere19. The pSyn-SRE-Luc vector was kindly provided by Dr. Elena Cattaneo (Univ. of Milan), and pFLAG-N-SREBP2 (1–481) was generously donated by Dr. Yoshihiro Yoneda (Osaka Univ.). pcDNA3.1-Myc-SREBP2 (1–3423) and pcDNA3.1-TDP-43 (1–1253) were generated using the In-Fusion HD cloning Kit (Takara Bio USA).
Construction of doxycycline-inducible cell lines
To establish a cell line stably expressing DAP-TDP-43 upon doxycycline application, we used an Flp-In T-Rex Expression System (Thermo Fisher Scientific). Flp-In T-Rex 293 cells (293 T Rex) were transfected with pOG44 (Flp-recombinase expression vector) (Thermo Fisher Scientific) and DAP-TDP-43 pcDNA5/FRT (FLP Recombination Target) /TO (Tet-On) and cultured for 48 h in culture medium containing 200 μg/ml of hygromycin B (Thermo Fisher Scientific)19.
Gene expression analysis
One hundred ng of total RNA extracted from DAP-TDP-43 cells was processed by using the Ambion WT Expression Kit (Thermo Fisher Scientific) and WT Terminal Labeling and Controls Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The sample was then hybridized onto GeneChip Human Transcriptome Array 2.0 (Thermo Fisher Scientific). After washing and staining, the microarray was scanned by GeneChip Scanner 3000 7G (Thermo Fisher Scientific). The sample was re-hybridized onto a GeneChip Human Gene 1.0 ST Array (Thermo Fisher Scientific), and the microarray was washed, stained and scanned. Signal data were analyzed with Transcriptome Analysis Console software (Thermo Fisher Scientific) for the Human Transcriptome Array and Partek Genomic Suite software (Partek Inc., St. Louis, MO, USA). IPA analysis (QIAGEN, Germantown, MD, USA) and gene set analysis for genes found with a fold-change greater than 1.2 were performed using Benjamini Hochberg FDR (p < 0.05). The data were examined using Genespring GX software (Agilent Technologies, La Jolla, CA, USA) for the gene set analysis.
RNA sequence analysis (RNA-seq)
We extracted total RNA from DAP-TDP-43 cells using the RNeasy Plus Kit (QIAGEN, Hilden, Germany). After the depletion of ribosomal RNA by Ribo-Zero Gold (Illumina, San Diego, CA, USA), libraries were prepared using the Illumina TruSeq Stranded Total RNA Sample Prep Kit (Illumina). The libraries were sequenced in 100 cycle Single-Read mode of HiSeq2500 (Illumina). All sequence reads were extracted in FASTQ format using BCL2FASTQ Conversion Software 1.8.4 in the CASAVA 1.8.4 pipeline. The number of sequence reads is listed in Supplementary Information (SI): Table S1. The sequence reads were mapped to hg19 reference genes, downloaded on 10 December 2012, using Tophat v2.0.8b, and quantified by RPKMforGenes, downloaded on 19 October 2012. Gene Ontology analysis was performed with GOstats and GOdb v2.14.0 in R package 3.1.0. To estimate transcript variants, Partek Genomics Suite v6.6 with Gencode v19 reference annotation was used. Mapping was performed using TopHat (CCB at JHU, USA).
Quantitative reverse transcription PCR (qRT-PCR)
QRT-PCR was performed using SYBR green and analyzed with StepOne software v2.1. Primers used for the measurement of SREBP2, TDP-43, HMGCS1, HMGCR, SQLE, LDLR, DHCR24 and LXR mRNA in amplified cDNA from cells are listed in SI: Table S2.
Cells were harvested and lysed in buffer (50 mM Tris–HCl buffer, pH 7.6, 10 mM ethylenediaminetetraacetic acid, protease inhibitor cocktail (Roche, Basel, Switzerland)) and phosphatase inhibitor (Roche) containing 1% SDS. After sonication, each 100-μg sample of protein was subjected to SDS-PAGE (5–15% polyacrylamide gels, BIO CRAFT, Tokyo, Japan), with 2-ME, and separated proteins were transferred to PVDF. The membranes were incubated with primary antibodies, then by appropriate secondary antibodies, and they were finally visualized using ECL plus or ECL chemiluminescence (GE Healthcare, Chicago, IL, USA). The following primary antibodies were used in this assay: TDP-43 (Proteintech, #10782-1-AP, 1:1,000), β-actin (Sigma-Aldrich, #A5441, 1:5,000), SREBP2 (Cayman Chemical, #10007663, 1:200), LDL-R (Abcam, #ab30532, 1:100), SCAP (Protein tech, #12266-1-AP, 1:200), Insig-1 (Novus Biologicals, #NB110-55244, 1:500), S1P (Sigma-Aldrich, #HPA040702, 1:1,000) and S2P (Cell Signaling Technology, #2157S, 1:1,000).
Cells were fixed in 4% paraformaldehyde (pH 7.4) for 10 min at room temperature and rinsed with PBS. The cells were permeabilized in PBS containing 0.2% Triton X-100 for 10 min at room temperature, followed by rinsing with PBS. Nonspecific binding was blocked with Blocking One HIST (Nacalai, Kyoto, Japan) for 10 min at room temperature. Cells were incubated with primary antibodies overnight at 4 °C, and then labeled with appropriate fluorescent-tagged secondary antibodies. DAPI (Thermo Fisher Scientific) was used to label nuclei. Acquisition of fluorescence images and quantification were performed using In Cell Analyzer 6500 (GE Healthcare). The following primary antibodies were used in this assay: SREBP2 (Abcam, Emeryville, CA, 1:50).
Equipment and settings
The image acquisition tool LAS 4000 (GE Healthcare) was used for immunoblots, and images were processed on imageJ (https://imagej.nih.gov/ij/) software.
HEK293T cells were transiently transfected with SRE-luciferase reporter construct (pSynSRE, 1.0 μg) and pRL-SV40 (Promega, Madison, WI, USA) (0.2 μg) along with pcDNA3.1-CMV-TDP-43 (0, 0.5, 1.0, 2.0 μg) or pcDNA3.1 (2.0, 1.0, 0.5, 0 μg) using Lipofectamine LTX (Thermo Fisher Scientific). After 72 h, the cells were lysed for luciferase assay. The lysates were measured in triplicate using a Dual Luciferase Reporter Assay System (Promega) on Envision Multilabel Reader (PerkinElmer, Waltham, MA, USA). Relative activity was defined as the ratio of firefly luciferase activity to Renilla luciferase activity to normalize for transfection efficiency.
Prp-TDP43A315T mice (B6Cg-Tg (Prnp-TARDBP*A315T) 95Balo/J, 010700) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). As described previously20, their asymptomatic stage is between 1 and 2 months of age. The onset of gait disorder appeared at ∼ 3 months of age in transgenic mutant TDP-43 mice and increased little by little over the next few months. The animals became paralyzed at ∼ 5 months of age, corresponding to symptomatic stage (gender-independent average); they were unable to move their hindlimbs or right themselves when placed on their backs. For cholesterol measurements, at 3 months of age (pre-symptomatic stage) and 4–6 months of age (symptomatic stage) the mice were perfused with cold PBS and spinal lipids were extracted with chloroform/methanol (2:1) according to the Folch technique21 at Skylight Biotech (Akita, Japan). All surgical procedures were performed according to the rules established by the Ethics Committee of Kyoto University. All experiments were performed in accordance with ARRIVE guidelines and relevant regulations.
Human spinal fluid samples
For disease diagnosis, spinal fluid samples were obtained by lumbar puncture; initial pressure, cell count, total protein level, and glucose level were measured. The remaining samples were frozen at − 80 °C until further analysis. Spinal fluids were collected from both ALS patients (N = 20) and disease-control patients (N = 20). The entity providing the material and following the patients is Tokushima University Hospital. All experiments were performed in accordance with relevant guidelines and regulations.
For quantitative analysis of cholesterol, cells were homogenized in PBS and lipids were extracted by modified Bligh and Dyer extraction method21,22. Briefly, 100 μl of chloroform plus methanol (2:1) was added to 100 μl of PBS including homogenized cells after counting the cell number. After centrifugation, the chloroform layer was collected and dried with a centrifugal dryer. The dry matter was lysed in 100 μl of ethanol to measure free cholesterol using a Cholesterol Assay kit (Cayman Chemical Company, Ann Arbor, MI, USA). Fluorescence signals were read with an Envision Multilabel Reader (PerkinElmer). Total lipids including sterols in spinal fluids from patients were extracted by the same method, with the extracts being subjected to LC–MS/MS-based quantification (Agilent 6400). Briefly, 100 μl of chloroform plus methanol (2:1) was added to 10 μl of spinal fluids mixed with 90 μl of PBS. After centrifugation, the chloroform layer was collected and dried with a centrifugal dryer. The dry matter was then lysed in 100 μl of ethanol, filtered with a 0.2-μm centrifugal filter tube, and measured for cholesterol by the use of Agilent 6400.
All data are shown as mean ± s.e.m. or ± s.d. Two group-analyses were performed using unpaired two-tailed Student’s t-test or paired t-test. One- or two-way ANOVA was performed for each comparison, followed by Tukey’s post hoc tests for evaluation of pair-wise group differences. A p value < 0.05 was considered statistically significant. In a comparative analysis of the amount of cholesterol, an alternative hypothesis was applied, namely that sample data could differ between the two groups by 10% (α = 0.05, β = 0.2, power = 0.8 and effect size = 10% of the average), and the sample size was then determined. Analyses were performed by JMP (SAS Institute Inc., Cary, NC, USA) and Excel Tokei (Social Survey Research Information, Tokyo, Japan).
Ethics approval and consent to participate
This study was approved by the Institutional Review Boards of Kyoto University and Tokushima University (Approval number: 2572-6, reference year: 2022), and all surgical procedures for animal studies were performed according to the rules set forth by the Ethics Committee of Kyoto University (Approval number: 17-91-11, reference year: 2022). Written informed consent was received from the participants prior to inclusion in the study. Samples from the participants were identified by numbers, not by names.
Consent for publication
Written informed consent for publication was received from each participant.