All patients in this study were diagnosed with definite or probable ALS according to EI criteria  and were enrolled in the Department of Neurology, the First Affiliated Hospital of Nanchang University and the Peking University First Hospital from January 2010 to December 2018, respectively. Additionally，subjects initially suspected with myopathy but without any abnormal pathological changes in muscle biopsy were used to be controls. All clinical materials used in this study were obtained for diagnostic purposes with written informed consent. This study was approved by the Ethics Committee of the First Affiliated Hospital of Nanchang University and the Peking University First Hospital.
Muscle pathological examination
Muscle biopsies were performed on the affected limb in all patients. The muscle tissue was frozen and then cut at 8 μm sections. These sections were stained with standard histological and enzyme histochemical procedures as in our previous study .
Cell culture and transfection
C2C12 cells were cultured in DMEM (BI) supplemented with 10% fetal bovine serum (FBS) (BI), and100 unit/ml penicillin-streptomycin in a humidified incubator at 37℃ under 5% CO2/ 95% air. C2C12 cells were respectively transfected with the Ctr (empty vector) or FUS-K510Q mutant (full length of FUS-K510Q protein without any tag) plasmids. For oleic acid treatment, cells were expressing Ctr or FUS-K510Q for 24 hours, and then 0.25 mM oleic acid was added to continue the culture for 24 hours after treatment [13,14]. The adenovirus AdMax system (vigenebio) was used for transduction according to the manufacturer's instructions.
Fly strains and antibodies
Transgenic fly expressing the human K510Q-FUS was described previously . Mef2-Gal4 and Tubulin-Gal80ts (Tub-Gal80ts) were kindly provided by Dr. Tao Wang (National Institute of Biological Sciences). For flies under the Mef2-Gal4/Tub-Gal80ts-driver, parental flies were crossed and cultured at 18°C, young flies after eclosion were transferred to 28°C for 4 h every day to induce FUS-K510Q expression [18-20]. Other flies were all cultured at 25°C. All flies were raised in standard fly food, 50% relative humidity, and 12 h-12 h light-dark cycles as described previously .
Antibodies used in this study include polyclonal rabbit-antibodies against FUS, CPT1A, PLIN2, ATGL (ProteinTechGroup Inc), as well as a mouse monoclonal antibody anti-β-actin (ProteinTech Group Inc).
Fly motility assay
The motility of adult flies expressing control or FUS-K510Q mutant were determined by using the climbing assay with small modification from the previous study . Briefly, 35-day-old flies were collected in each group. Fly climbing ability was examined as the number of flies climbing above a 3-cm line in 20 seconds after they were tapped to the bottom of an empty vial. The experiment was repeated 6 times for each group.
C2C12 cells were rinsed with 1X PBS, fixed with 4% polyformaldehyde in 1×PBS after expression of GFP (Ctr) or K510Q FUS-EGFP for 48 h. The samples were mounted using prolong gold antifade mounting medium with DAPI (Invitrogen). Images were acquired at 60 × magnification by using a confocal microscopy (Nikon A1MP).
Oil Red O staining
Slides were fixed with 4% paraformaldehyde (PFA) solution for 20 min and then placed in ORO solution for 25 min, rinsed with distilled water for 5 min and counterstained with Mayer's hematoxylin (C0157S, Beyotime) for 5 min. The slides were then coverslipped with glycerin and observed by light microscopy (Nikon), and myocytes were photographed at 20x and 40x.Image analysis was performed with Image Pro Plus 6 software to calculate the lipid droplet ratio (the total area of red fat droplets accounted for the percentage of the entire x20 image area used). The lipid droplet area ratio was calculated and averaged by two investigators [22,23].
Measurement of mitochondrial membrane potential by TMRM
Mitochondrial membrane potential was measured in cells expressing control vector or K510Q mutant FUS using the tetramethylrhodamine methyl ester (TMRM) (Invitrogen) following a published protocol . Briefly, cells were rinsed in warm PBS and then stained using TMRM (40nM) for 20 minutes at 37°C. After washes 2 times, the images were immediately observed and photographed using an inverted research microscope ECLIPSE Ti2-E (Nikon). Fluorescent images were processed and quantified using Image J.
Mitochondrial ROS detection assay
Cells expressing control vector or K510Q mutant FUS for 48 h were rinsed in PBS and then stained with 10μM Dihydroethidium (DHE) for 30 min at 37°C. After washes, cells were fixed with 4% PFA for 10 min at room temperature. The images were immediately observed and photographed under an inverted research microscope ECLIPSE Ti2-E(Nikon), and images were processed and quantified using ImageJ.
Measurement of the total cellular ATP levels
The ATP level was detected using a rapid bioluminescent ATP assay Kit (S0026, Beyotime Corporation). The measurement was performed according to the manufacturer’s instructions. Briefly, C2C12 cells expressing control vector or K510Q mutant FUS were seeded in 96-well plates. Following removal of the culture media, cell lysis with reaction mixtures were transferred to another opaque 96-well plate to measure luminescence. Luminescent signal values were normalized by the protein amount in each group to determine the total cellular ATP levels.
Measurement of the triglyceride levels
The triglyceride level was detected using GPO-PAP assay Kit (A110-1-1, NanjingJiancheng Institute of Biological Engineering). The measurement was performed according to the manufacturer’s instructions.
Western blot analysis
Thoracic flight muscles from flies, as well as cultured cells were lysed in RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.25% sodium deoxycholate, 0.1% SDS, 1% NP-40, supplemented with complete protease inhibitor mixture; Roche). Lysates were analyzed by Western blot using the corresponding specific antibodies: anti-FUS (1:1000, Rabbit, Proteintech, # 11570-1-AP), anti-CPT1A (1:1000, Rabbit, Proteintech, #15184-1-AP), anti-ATGL (1:1000, Rabbit, Proteintech, #55190-1-AP) and anti-PLIN2 (1:1000, Rabbit, Proteintech, #15294-1-AP) The same antibodies are applied to both C2C12 cells and drosophila for Western blot.
Transmission electron microscopy
For EM study on C2C12 cells, cells were rinsed with PBS and then fixed in 2.5% glutaraldehyde overnight at 4°C. EM samples were prepared following protocols as described previously [16,24]. They were sectioned on a Leica EM UC6/FC6 Ultramicrotome. After sections were transferred to copper grids, counter staining was performed with uranyl acetate and lead acetate before EM imaging.
Statistical analysis was performed using the SPSS 24.0 statistical package (Chicago, IL, USA). A parametric test was applied if the data followed normal distribution. Otherwise, non-parametric tests were used. When a correlation between two variables was assessed, Pearson’s R correlation coefficient was calculated. When two groups were compared, parametric t or non-parametric Mann–Whitney tests were used. When more than two groups were compared, parametric ANOVA with indicated post hoc tests or non-parametric Kruskal–Wallis were used. The bar graphs represent mean ± SD. Significance is indicated by asterisks: N.S. P >0.05, *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001. Actual p-values of each test are indicated in the corresponding figure legend.