Study cohort
All patients were diagnosed according to the El-Escorial criteria for definite and probable ALS. Patients were considered sporadic based upon a negative family history. Individuals with confounding conditions affecting the immune system were excluded. Age- and sex-matched healthy controls were chosen, as listed in Table s1.
Animal models
Tbk1fl/fl mice were generated by Taconic, and SOD1G93A transgenic mice (B6SJL-TgN [SOD1-G93A] 1Gur) were obtained from Jackson Laboratories. Tbk1fl/fl mice were crossed with LysM-cre mice to generate Tbk1fl/fl LysM-cre (termed Tbk1-LKO) and Tbk1fl/fl WT mice. Animals were bred and maintained under controlled conditions (12-h light/dark cycles, 60% ± 10% relative humidity, 22 ± 1°C). The copy number of the hSOD1 gene was evaluated by real-time PCR. Body weight was measured every 7 days, starting at approximately 60 days of age.
Animal experiments were carried out according to the laboratory animal management regulations promulgated by the Ministry of Science and Technology of the People’s Republic of China, which are in accordance with the NIH Guide for the Care and Use of Laboratory Animals.
PEI-mannose packaging
According to the protocol of In vivo-jetPEI®-Man (Polyplus transfection), the nucleic acids, including TBK1 plasmid and AAV-SaCas9-sgRNA targeting SOD1, and reagent were separately diluted in a 5% glucose solution and then mixed together, and after a 15 min incubation at room temperature (RT), the nucleic acid/in vivo-jetPEI®-Man complexes were injected into the animals via the tail vein.
Cell culture
The NSC-34 cell line was routinely maintained in DMEM (Invitrogen, CA, USA; cat. No: 21063-029) supplemented with 10% heat-inactivated FBS (Invitrogen; cat. No: 16000-044) and antibiotics (100 IU/mL penicillin and 100 µg/mL streptomycin).
THP-1 cells were routinely maintained in RPMI-1640 medium (Gibco, 8119144) supplemented with 10% heat-inactivated FBS (Invitrogen; cat. No: 16000-044) and antibiotics (100 IU/mL penicillin and 100 µg/mL streptomycin).
Human primary monocyte culture
After written informed consent was obtained, we collected peripheral blood from subjects. Human primary PBMCs were isolated with HISTOPAQUE-1077 (Sigma-Aldrich) according to the manufacturer’s protocols. Cells were maintained in RPMI-1640 medium (Gibco, 8119144) supplemented with 10% (vol/vol) FBS, 2 mM glutamine, penicillin (100 U/mL) and streptomycin (100 mg/mL). For monocyte differentiation into macrophages, M-CSF (20 ng/mL, Sino Biological, 11792-H08H) was added for 7 days.
Inhibitors and activators
Amlexanox (LKT Labs, A4944), Nik smi1 (MCE, HY-112433), BV6 (MCE, HY-16701 ), human TNF alpha (Sino Biological, 10602-HNAE), and mouse Tnf alpha (Sino Biological, 50349-MNAE) were used.
Immunohistochemistry
Following transcardiac perfusion with 4% paraformaldehyde, mouse tissues were removed and further fixed for 24 h in the same fixative, followed by paraffin embedding. The tissue was cut into 5 μm sections. The sections were permeabilized with 0.3% Triton X-100 and then washed three times in 0.01 M phosphate-buffered saline (PBS). After blocking with 3% H2O2 in methanol for 15 min and in 10% horse serum for 1 h at RT, the sections were incubated overnight at 4℃ with antibodies against IBA1 (1:500, Wako, 019-19741), Chat (1:500, Abcam, 178850), NeuN (1:500, Millipore, MAB377), CD11B (1:100, Proteintech, 20991-1-AP), Arginase1 (1:100, Proteintech, 16001-1-AP), or GFAP (1:200, Millipore, MAB360). The sections were subsequently incubated at RT with a biotin-conjugated secondary antibody (ZSGB-BIO, 1:200) for 2 h, followed by incubation with HRP-conjugated streptavidin (ZSGB-BIO, 1:200) for 1 h and 0.03% diaminobenzidine as a chromogen for 10 min. Slides were mounted and analyzed by light microscopy (Olympus BX51).
Muscle morphology
Ten-micron frozen cross-sections were stained with HE, and the stained sections were visualized using an Olympus microscope (BX53). Images were captured with a digital camera and analyzed using image analysis software (DP73). Approximately 300 fibers of the gastrocnemius were recorded, and the fiber area was calculated using ImageJ.
Confocal laser scanning microscopy
Spinal cord sections (obtained as described above) were washed three times in 0.3% Triton X-100/PBS and blocked in 10% horse serum for 30 min. Primary antibodies against P100 (1:100, Cell Signaling, 4882), Neu N (1:100, Millipore, MAB377), GFAP (1:200, Millipore, MAB360), phospho-Tbk1 (1:50, Cell Signaling, 5483), and IBA1 (1:200, Wako, 019-19741) were applied overnight at 4℃. After washing, the sections were incubated with fluorescent secondary antibodies for 1 h at RT. The slides were observed using confocal fluorescence microscopy (Olympus FV1000).
Western blotting
Protein expression in the cortex and cerebellum was quantified by western blotting. Total protein was extracted using a total protein extraction kit (Applygen Technologies Inc., P1250). Fifty micrograms of protein from each sample was separated on a 10% or 12% SDS-PAGE gel and transferred onto PVDF membranes. The membranes were incubated overnight at 4°C with primary antibodies, including those against β-actin (1:2000, Proteintech, 60008-1-Ig), CD11B (1:1000, Proteintech, 20991-1-AP), P100 (1:1000, Cell Signaling, 4882), Arginase1 (1:1000, Proteintech, 16001-1-AP), GAPDH (1:5000, Proteintech, 10494), or hSOD1 (1:10000, Abcam, ab51254). Following incubation with fluorescent secondary antibodies for 1 h at RT, an Odyssey Infrared Imaging System (LI-COR, Lincoln, NE) was used to detect the bands of interest.
Quantitative RT-PCR
Copy number: DNA was isolated from the mouse tail using an extraction kit (Generay Biotechnology, Shanghai, China) according to the manufacturer’s instructions. Quantitative PCR was performed using synthetic primers, SYBR Green (Generay Biotech Co., Ltd., GK8020) and an M3005P system (Agilent Technologies, Santa Clara, CA, USA). The following sequences were used in this assay: hSOD1, 5’-CATCAGCCCTAATCCATCTGA-3’ (forward) and 5’-CGCGACTAACAATCAAAGTGA-3’ (reverse); and mIL2, 5’-CTAGGCCACAGAATTGAAAGATCT-3’ (forward) and 5’-GTAGGTGGAAATTCTAGCATCATCC-3′ (reverse). The amplification conditions were as follows: 95°C for 10 min followed by 40 cycles of amplification at 95°C for 30 s, 60°C for 1 min, and 72°C for 30 s. The difference in CT (ΔCT) values was computed as the difference between the human SOD1 CT value and the mouse IL2 CT value (ΔCT=mIL2 CT-hSOD1 CT).
Total RNA was isolated from the tissues using an RNA rapid extraction kit (Generay Biotech Co., Ltd., GK3016) in accordance with the manufacturer’s protocol. cDNA was prepared using AMV reverse transcriptase and random primers (Promega, A3500). Targeted gene primers and SYBR Green (Generay Biotech Co., Ltd., GK8020) were used for quantitative PCR on an M3005P (Agilent Technologies, Santa Clara, USA). After incubating the samples at 95°C for 10 min, 40 cycles of amplification at 95°C for 10 s, 60°C for 20 s, and 72°C for 15 s were performed. GAPDH was used as a reference gene. The synthesized primers used here are listed in Table s2.
Flow cytometry
Osmotic lysis of red blood cells was performed using 45 mL of RBLB (red blood cell lysis buffer: 50 mM NH4Cl, 70 mM NaHCO3, 0.1% EDTA, ddH2O) for 5 mL of whole blood for 10 min at RT. Leukocytes were centrifuged for 5 min at 500×g at 4°C and washed twice in PBS. Leukocytes were resuspended in 400 μL of PBS and stained with antibodies specific for cell surface antigens (CD11B-FITC, 1:100, BD, 557396; Ly6c-APC, 1:100, eBioscience, 17-5932-82; CD206-PE, 1:100, eBioscience, 12-2061-82; F4/80-APC, 1:100, eBioscience, 17-4801; CD14-APC, 1:100, BD, 555399; and CD16-perCY-CY5.5, 1:100, BD, 560717) for 30 min on ice in the dark. After washing in PBS, the stained cells were fixed and permeabilized by adding 500 µL of Fixation/Permeabilization solution (Cytofix/CytopermTM Plus Fixation/Permeabilization Kit, BD, 554714) and incubated at RT in the dark for 20 min. Thoroughly resuspended fixed/permeabilized cells were incubated with p-TBK1-PE (1:100, Cell Signaling, 13498) at 4°C for 30 min in the dark. After washing cells twice with 1×BD Perm/WashTM buffer and resuspending in staining buffer, flow cytometric analysis was then performed.
Electron microscopy
Tissues were fixed in 4% glutaraldehyde and treated with 1% OsO4 in 0.1 M PBS. The samples were dehydrated through graded acetone solutions and embedded in EPON 812. Ultrathin sections (70 nm) were placed on a copper grid and stained with uranyl acetate and lead citrate. The samples were observed by transmission electron microscopy (TEM, JEM-1230).
Bielschowsky staining
Staining of nerve fibers was carried out using a Hito Bielschowsky OptimStain Kit (HTKNS1126). Briefly, the slides were incubated with Solution 1 for 15 min, followed by immersion in the Developer solution for 1-8 min until the tissues became golden-brown. After incubation with Solution 4, sections were rinsed with distilled water, dehydrated, cleared, and coverslipped.
Motor function test
A rotarod (Rota-Rod; Ugo Basile, Gemonio, Italy) was used to assess motor function in mice. With an arbitrary cut-off time of 180 s, three once-per-week trials were performed, and the longest time was recorded. The onset of disease was determined as a reduction in rotarod performance between weekly time points. The weight of the mice was measured every 7 days, starting at 60 days of age.
Accelerated rotarod testing. Mice were placed on the top of the revolving beam for 4 successive trials/day for 2 days, with 20-min intertrial intervals. The rod was accelerated gradually from 4 to 28 rpm over 2 min. Latencies before falling from the rod were recorded.
The footprints of the mice were recorded during continuous locomotion. The stride length was measured as the distance between footprints, and the average stride length was calculated from three stride lengths.
The weight of the mice was measured every 7 days, starting at 60 days of age.
Microbiota analysis
Total DNA was extracted from the feces of mice. PCRs targeting the V3-V4 regions of the 16S rDNA genes (probes used: 319F, 5’-ACTCCTACGGGAGGCAGCAG-3’ and 806R, 5’-GGACTACHVGGGTWTCTAAT-3) were performed, and sequencing reactions were performed using Illumina HiSeq sequencing technology for paired-end reads. Paired-end reads obtained were merged using FLASH v1.2.11 software, and reads with a length ≥ 400 bp were kept for the following analysis. The merged sequences were processed with QIIME v1.8.0, and the sequences were binned into operational taxonomic units (OTUs) based on 97% identity. Alpha diversity, richness, Venn diagrams, heat maps, principal coordinate analysis, and linear discriminant analysis were further processed with a bioinformatic pipeline tool, BMK Cloud online (Biomarker Technologies Corporation, Beijing, China).
Statistical analysis
Data were analyzed using SPSS 13.0 software. Lifespan was compared between groups of mice by the Kaplan–Meier method. Comparisons among multiple groups were performed using one-way ANOVA followed by Student–Newman–Keuls or Dunn’s T tests. All values are expressed as the mean ± S. E., and P < 0.05 was considered statistically significant.