Postmortem brain samples
Postmortem human tissue samples used in this study were collected from the Brain Bank for Aging Research (BBAR) in the Tokyo Metropolitan Geriatric Hospital and Institute of Gerontology (TMGHIG), and the National Center of Neurology and Psychiatry (NCNP) Brain Bank. sALS cases (n = 7) were diagnosed based on El Escorial and Airlie House revised criteria with no familial history of the disease, and confirmed pathological inclusions of TDP-43 in brain lesions. Age- and sex-matched control cases (n = 7) had no neurological pathologies (Table 1). The pyramidal tract of the medulla oblongata was chosen for this study because this area is rich in axons of upper motor neurons, which are affected in ALS.
cDNA preparation for small RNA-Seq
Total RNA used for RNA-seq was extracted using Isogen-LS (Nippon Gene # 311–02501). RNA concentration was measured with a Qubit RNA Assay Kit in a Qubit 2.0 Fluorometer (Life Technologies, CA, USA). RNA integrity was evaluated with RNA Pico 6000 Assay Kit and a Bioanalyzer 2100 System (Agilent Technologies, CA, USA). The mean RIN for ALS samples was 7.9 (range 6.9–8.5). (See Supplementary Information for raw values.) 50 ng of each RNA sample were used to construct a cDNA library for each sample using a NEXTFLEX Combo-Seq mRNA/miRNA kit (Bio-Scientific), according to the manufacturer’s protocol (Nova-5139-01). cDNAs were sequenced on an Illumina HiSeq 3000 with 74-bp single reads (10M reads/sample).
RNA-Seq bioinformatic analysis
We excluded adapter sequences using the cutadapt tool V.1.9.2. We excluded reads shorter than 15 bases using trimmomatic version 0.36. Then, we conducted comprehensive small RNA-seq analysis using the exceRpt extra-cellular RNA processing toolkit.
Raw FASTQ files for the RNA-seq libraries have been submitted to the DNA Data Bank of Japan (DDBJ) with Temporary Submission ID: SSUB016807.
RNA extraction for reverse transcription-quantitative PCR (RT-qPCR) validation
Total RNA was extracted from medulla oblongata samples using a mirVana kit (Ambion), according to manufacturer instructions. Small RNA (sRNA) was isolated from total RNA using the mirVana kit (Ambion), according to the manufacturer’s instructions for sRNA isolation.
RT-qPCR for target piRNA
In brief, 3’-ends of 60 ng of sRNA from each sample were ligated to 5’ pre-adenylated 3’-adaptors (5′- rApp/CTGTAGGCACCATCAAT/3ddC-3′) using truncated T4RNA ligase 2 enzyme (NEB). 3’- adaptor-ligated sRNAs were reverse transcribed with ReverTraAce, reverse transcription polymerase (Toyobo) using an oligonucleotide complementary to the 3’-adaptor (IDT) in a MiniAmp plus Thermal Cycler (Applied Biosystems). RT-qPCR was performed using forward primers for each target piRNA and the oligonucleotide complementary to the 3’-adaptor was used as a reverse primer (Table 5). Data were collected in triplicate for each sample on an ABI 7900 Prism qPCR machine and normalized using U6RNA as an internal control. Relative gene-expression levels were calculated using the fold-change method.
RT-qPCR for coding genes
RT-qPCR was performed using ReverTraAce (Toyobo), in a MiniAmp plus Thermal Cycler (Applied Biosystems). The glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) was used as an internal control to normalize coding genes. All quantitative PCR was performed using SYBRGreen qPCR Master Mix (Applied Biosystems) on an ABI 7900 Prism qPCR machine. Primer sets used for RT-qPCR are listed in Table 5.
Standard curve method for RT-qPCR
cDNA generated by reverse transcription for seven controls and seven ALS samples were used for the fold-change calculation. Equal volumes of control cDNA were pooled as a control stock. The control stock was then diluted 10 times with nuclease-free water in a dilution series to produce a control standard curve. sALS cDNA samples were diluted 20 times and then plotted against the control standard curve for each gene in triplicate. GAPDH was used as an internal control for normalization.
△CT method for RT-qPCR
The △CT RT-qPCR method was used to calculate the difference in cycle numbers needed to amplify target genes after normalization using the internal control. Therefore, △CT denotes the CT value of a target gene substituted by the CT value of the internal control. Internal controls for piRNA and coding genes were U6RNA and GAPDH, respectively.
Tissue lysates and protein quantification
CellLytic MT Mammalian Tissue Lysis Reagent (C3228, Sigma-Aldrich), mixed with a protease inhibitor cocktail (1:100, EMD Millipore), was added to frozen tissue in 2-mL Lysing Matrix D tubes (P000912-LYSK0, Precellys) to be homogenized using a MINILYS personal homogenizer (Bretin Instruments). Homogenized tissue lysates were centrifuged and supernatants were used for protein concentration determinations with a Pierce TM BCA protein assay (23225, Thermo Fisher Scientific).
Immunoblotting for PIWI proteins
30 µg of protein from lysates were separated on 5–20% SDS–PAGE gels (E-T520L, ATTO) and transferred to 0.2-µm PVDF membranes (1620177, Bio-Rad). Membranes were blocked for 1 h in EZ blocking solution (AE-1475, ATTO) and then incubated with primary antibodies diluted in Can Get Signal Solution 1 (TOYOBO) overnight at 4°C. Primary antibodies used included PIWIL1 (1:1000; #701177, Novex), PIWIL2 (1:500; ab181340, Abcam), PIWIL3 (1:500; ab77088, Abcam), PIWIL4 (1:1000; ab111714, Abcam), and PIWIL4 (1:1000; PA-49710, Thermo Fisher Scientific).
After primary antibody incubation, membranes were washed with 0.1% T-TBS buffer three times (for 15, 10, 5 min each) before incubation with secondary antibody. Secondary antibodies were ECL anti-mouse antibody (1:10000; NA931V, GE Healthcare) or anti-rabbit antibody (1:20000; NA934V, GE Healthcare) diluted in Can get Signal solution2 (TOYOBO) or EZ block solution and supplemented with STREP-TACTIN (#1616380, Bio-Rad) for marker band detection. After secondary antibody incubation, membranes were washed with 0.1% T-TBS buffer 3 x 10 min before visualization.
Data analysis and statistics (Immunoblotting and RT-qPCR)
Statistics were performed using GraphPad Prism, version9 software. Two-way ANOVA was performed with Bonferroni’s multiple comparison test to compare two or more independent groups. Pairwise comparisons were made using the Mann-Whitney test for △CT with unpaired and nonparametric settings. Pairwise comparisons for fold change using the standard curve method were made using one-sample t-tests (one-tailed Wilcoxon test) with paired and nonparametric settings.
For immunoblot statistical analysis, two-way ANOVA was performed using Bonferroni’s multiple comparison test to compare two or more proteins. Pairwise comparisons were made using the Mann-Whitney test for PIWI proteins with unpaired and nonparametric, two-tailed p-value settings. p-values < 0.05 were considered significant (* indicates p < 0.05 and ** p < 0.01). All numbers in plots represent means ± SEM.
Clinical profiles of patients examined in the present study are shown in Supplementary Table 1. Samples from the lumbar cord (L5) were fixed in 10% buffered formalin. For immunohistochemistry (IHC), 6-µm sections were prepared. Deparaffinized sections were incubated 30 min with 0.3% hydrogen peroxide to quench endogenous peroxidase activity and then washed with PBS. The primary antibody was a mouse monoclonal antibody against PIWIL1 (1:500). Samples were autoclaved 15 min before incubation with antibody. Secondary antibody was goat anti-mouse immunoglobulin conjugated to peroxidase-labeled dextran polymer (Dako Envision+, Dako). Reaction products were visualized with 3,3'-diaminobenzidine tetrahydrochloride (ImmPACT DAB, Vector Laboratories), and hematoxylin was used to counterstain cell nuclei. For double IHC, two primary antibodies were combined, including antibodies against TDP-43 (1:1000, rabbit polyclonal, Proteintech) and PIWIL1 (1:500). Alexa Fluor® 488 goat anti-mouse IgG (H + L) antibody (A-11008, Thermo Fisher Scientific) and Alexa Fluor® 568 goat anti-rabbit IgG (H + L) antibody (A-11004, Thermo Fisher Scientific) were used as secondary antibodies. Sudan Black B treatment was performed to reduce autofluorescence from lipofustin. Images were obtained using an all-in-one fluorescence microscope (BZ-X710, Keyence).