Cell culture, plasmids, knockdown
DAP (triple affinity-purification tag; biotin and FLAG tags, and N-terminal epitope tag; 6×histidine) -TDP-43-inducible 293T Rex cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, glucose, 4.5 g/liter) supplemented with Tet System Approved FBS (Takara Bio USA, CA, USA) at 37°C in a 5% CO2/95% air atmosphere. The pSyn-SRE-Luc vector was kindly provided by Dr. Elena Cattaneo (Univ. of Milan), and pFLAG-N-SREBP2 (1-481) was generously given 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). For the knockdown of TDP-43 and mTOR, we transfected 50 nM and 200 nM mission siRNA (Sigma-Aldrich, St. Louis, MO, USA) into cultured cells using Lipofectamine LTX (Invitrogen, Carlsbad, CA, USA), respectively. The two kinds of siRNA were attempted in order to rule out off-target effects.
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 (Invitrogen). Flp-In T-Rex 293 cells (293T Rex) were transfected with pOG44 (Invitrogen) and DAP-TDP-43 pcDNA5/FRT/TO and cultured for 48 hours in culture medium containing 200 µg/ml of hygromycin B (Invitrogen)(19).
Gene expression analysis
One hundred ng of total RNA was processed by using the Ambion WT Expression Kit (Life Technologies, Carlsbad, CA, USA) and WT Terminal Labeling and Controls Kit (Affymetrix, Santa Clara, CA, USA) according to the manufacturers’ instructions. The sample was then hybridized onto GeneChip Human Transcriptome Array 2.0 (Affymetrix). After washing and staining, the microarray was scanned by GeneChip Scanner 3000 7G (Affymetrix). The sample was re-hybridized onto a GeneChip Human Gene 1.0 ST Array (Affymetrix), and the microarray was washed, stained and scanned. Signal data were analyzed by using Affymetrix Transcriptome Analysis Console software (Affymetrix) for the Human Transcriptome Array and Partek Genomic Suite software (Partek Inc., St. Louis, MO, USA). IPA analysis 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 analyzed by Genespring GX software (Agilent Technologies, La Jolla, CA, USA) for the gene set analysis.
RNA sequence analysis (RNA-seq)
We extracted total RNA using the RNeasy Plus Kit (QIAGEN, Hilden, Germany). After the depletion of ribosomal RNA by Ribo-Zero Gold (Illumina, San Diego, CA, USA), we prepared libraries using the Illumina TruSeq Stranded Total RNA Sample Prep Kit (Illumina). The libraries were sequenced in the 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 Additional file 1: Table S4. 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 by 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 Additional file 1: Table S5. In HEK293T or DAP-TDP-43 293T Rex cells for TDP-43 knockdown, control and TDP-43 siRNA (Sigma-Aldrich) were transfected (50 µM) using RNAiMAX (Invitrogen).
Induced pluripotent stem cells (iPSCs)
Human cDNAs for reprogramming factors were transduced in human dermal fibroblasts with retrovirus (Sox2, Klf4, Oct3/4 and/or c-Myc) or episomal vectors (Sox2, Klf4, Oct3/4, L-Myc, Lin28, shRNA for p53). We used a human control iPSC line (HPS0063) and a familial ALS iPSC line with TDP-43 mutation M337V (A3416). Several days after the transduction, the fibroblasts were harvested and replated on an SNL feeder layer. On the next day, the medium was changed to primate embryonic stem cell medium (Reprocell, Kanagawa, Japan) supplemented with 4 ng/ml bFGF (Wako Chemicals, Osaka, Japan). The medium was changed every other day. Thirty days after the transduction, iPSC colonies were gathered.
Induction of spinal motor neurons by quick embryoid body-like aggregates method (SFEBq)
iPSCs were dissociated to single cells and quickly reaggregated in low cell adhesion U-shaped 96-well plates (Lipidure Coat Plate A-U96, NOF Corporation, Tokyo, Japan). Aggregations were cultured in 5% DFK medium (5% KSR Medium (DFK5%, DMEM/Ham's F12 (Sigma-Aldrich), 5% KSR (Invitrogen), MEM-NEAA (Invitrogen), L-glutamine (Sigma-Aldrich), 2-mercaptoethanol (Wako)) with 2 µM dorsomorphin in a neural inductive stage (P1) for 12 days. After patterning using neurobasal medium supplemented with B27, 1 µM retinoic acid and 100–500 ng/ml Sonic Hedgehog, the aggregates were adhered to Matrigel (BD Biosciences, San Jose, CA, USA)-coated dishes on day 22. Adhesive embryoid bodies were cultured in neurobasal medium with 10 ng/ml BDNF, 10 ng/ml GDNF and 10 ng/ml NT-3 in P2 culture. They were then separated from the dish by Accutase (Sigma-Aldrich), dissociated into a small clump or single cells, and cultured at 500,000 cells/well on Matrigel-coated 24-well dishes on day 35 as the P3 maturation stage.
Immunoblots
Differentiated cells were treated with cytosine arabinoside (AraC) (Sigma-Aldrich). Three days after the removal of AraC, the cells were harvested and lysed in TS buffer (50 mM Tris-HCl buffer, pH 7.5, 0.15 M NaCl, 5 mM ethylenediaminetetraacetic acid, 5 mM ethylene glycol bis (β-aminoethyl ether)-N,N,N,N-tetraacetic acid, and protease inhibitor cocktail (Roche, Basel, Switzerland)] containing 1% Triton X-100 on ice for 10 minutes. After sonication, samples were centrifuged at 10,000 x g for 15 minutes at 4°C. The supernatant was saved as the soluble fraction, and the pellet was resuspended, sonicated in 2% SDS buffer and saved as the insoluble fraction. Each 10-µg sample of protein was subjected to SDS-PAGE (10–20% polyacrylamide gels, BIO CRAFT, Tokyo, Japan), with or without 2-ME, and separated proteins were transferred to PVDF. The membranes were incubated with primary antibodies, followed by appropriate secondary antibodies, and then visualized using ECL plus or ECL chemiluminescence (GE Healthcare, Chicago, IL, USA). The images were acquired on an LAS 4000 (GE Healthcare). The following primary antibodies were used in this assay: TDP-43 (Protein Tech, 1:1,000), β-actin (Sigma-Aldrich, 1:5,000), SREBP1 (Protein Tech, 1:500), SREBP2 (Cayman Chemical, 1:200), mTOR (Cell Signaling Technology, 1:1000), Raptor (Cell Signaling Technology, 1:1000), Lipin-1 (Cell Signaling Technology, 1:1000), p70S6K (Cell Signaling Technology, 1:1000), and p-p70S6K (Cell Signaling Technology, 1:1000).
Reporter assay
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 (Invitrogen). After 72 hours, the cells were lysed for the 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.
Mice
We purchased Prp-TDP43A315T mice (B6Cg-Tg (Prnp-TARDBP*A315T) 95Balo/J, 010700) from The Jackson Laboratory (location?). As described previously(21), the asymptomatic stage is between 1–2 months of age. The onset of gait disorder appeared at ∼3 months of age in transgenic mutant TDP-43 mice (symptomatic stage) and increased slightly over the next several months. The animals became paralyzed at ∼5 months of age, corresponding to the end stage (average including males and females); they were unable to move their hindlimbs or right themselves when placed on their backs. For cholesterol measurements, the mice were perfused with cold PBS. All surgical procedures were performed according to the rules set by the Ethics Committee of Kyoto University.
Human spinal fluid samples
For disease diagnosis, we obtained spinal fluid samples by lumbar puncture; initial pressure, cell count, total protein level, and glucose level were measured. The remaining samples were frozen at 80℃ until further investigation. We collected spinal fluids from ALS patients (N = 20) and control patients (N = 20).
Cholesterol measurement
Cells were homogenized in PBS, and the lipids were extracted by modified Bligh and Dyer extraction method for the quantitative analysis of cholesterol(22, 23). 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 lysed in 100 µl of ethanol and filtered with a 0.2-µm centrifugal filter tube, and measured for cholesterol by using Agilent 6400.
Statistics
All data are shown as mean ± s.e.m. or ± s.d. The comparison of two groups was analyzed using unpaired two-tailed Student’s t-test or paired t-test. One-way ANOVA was performed for each comparison followed by Tukey’s post hoc tests for the evaluation of pair-wise group differences. A P value < 0.05 was considered statistically significant. Analyses were performed by JMP (SAS Institute Inc., Cary, NC, USA) and Excel Tokei (Social Survey Research Information, Tokyo, Japan).