Cell culture
All neuronal culture techniques were performed under sterile conditions. Coverslips were coated with 1% Poly-D-lysine hydrobromide in PBS (Sigma) and incubated at 37°C overnight and washed with PBS prior to the seeding of rat primary cortical cells at 70,000 cells/ml in 500μl/well.
DNA Transfection
For each transfection, 500ng DNA was mixed with 1μl of Lipofectamine 2000 (Invitrogen) in 25μl of HEPES (Gibco) and DMEM (Gibco) solution per well. Prior to addition of DNA, coverslips were removed and placed in a 350μl of fresh media (without pen/strep). DNA mix was incubated for 1 hour at 37°C before adding in a drop wise manner to DIV6 neurons. 6-8 hours post transfection coverslips were replaced in the old media, before fixation 48 hours later.
Viral transduction
Virus was added to rat primary neurons at DIV14 to achieve an infection rate of 1 x 107 virus particles/ml. Following the day of transduction, 250μl of cortical media was removed and 300μl of fresh cortical media was added onto the cells before fixation occurred at DIV21.
Immunofluorescence
Primary neurons were fixed in 4% paraformaldehyde for 10 minutes and then ice cold methanol for 10 minutes before being probed with selected primary antibodies overnight at 4 °C. Antibodies were used as follows rabbit anti-synaptophysin I (1:300, Synaptic Systems), rabbit anti-FUS (1:300, Sigma), mouse anti-FUS (1:200, Proteintech), mouse anti-PSD95 (1:500, AbCam), mouse anti-HA (1:1000, CoVance), chicken anti-MAP2 (1:1000, AbCam), mouse anti-FUS (H6) (1:200, Santa Cruz), rabbit anti-SNPH (1:200, ProteinTech). Goat anti-rabbit, anti-mouse or anti-chicken Alexaflour 488/564/640 secondary antibodies (1:500, Invitrogen) were added the following day for two hours at room temperature in the dark. DAPI (4ʹ,6-diamidino-2-phenylindole (Sigma) counterstaining (1.25μg/ml) was added as a nuclear stain before cover slips were mounted.
Quantitative image analysis
Images were taken on a Leica TCS-SP5 laser scanning confocal microscope and imaged at x63 with a numerical aperture of 1.4 (with a digital zoom of 2.5). Images were taken at 10-14 Z-sacks with 0.5μm increment before being processed in Image J.
Colocalisation analysis
Images of selected neurites were extracted, and their length (100μm) was recorded. After splitting each channel, a Gaussian and median filter (with a radius of 10 pixels) was applied to the channel of interest, individually. The median image was subtracted from the Gaussian channel and a threshold was selected. Particles were then analysed from the channel before overlaying puncta onto the other channel of interest to allow measurement of colocalization which was determined by subtracting the area of the overlaid puncta from the under laid puncta (colocalisation was only counted if 50% and over). Analysis was performed on three separate dendrites per cell (N=9) from three individual experiments at DIV21. All statistical analysis to determine significance between groups was performed using GraphPad Prism 9 using a student’s T-Test.
Analysis of synaptic puncta and mitochondria
Maximum projection images were converted to 8-bit grayscale and individual channels were then used to select the threshold which was kept consistent to the control (eGFP) channel. Puncta and mitochondria were thresholded to be bigger than five pixels in size. Images were obtained from three independent experiments and three dendrites from five different cells were analysed for each repeat for each condition. All statistical analysis to determine significance between groups was performed using GraphPad Prism 9 using a One-Way ANOVA with post-hoc tukey’s multiple comparisons test.
Analysis of dendritic complexity
Maximum projection images of the MAP2 channel were then converted into an 8-bit gray scale image and dendrites were traced using an available plugin (Neuron J). After tracing, sholl analysis was performed to assess dendritic complexity from the soma at 10μm increments. Sholl analysis is a quantitative measure of the shape and/size of a dendritic tree. To measure dendritic complexity, concentric circles were drawn from the centre of the neuron and the number of times each dendritic branch intersects, branching is assessed (number of intersections divided by area against distance). Traces were analysed from 10 individual cells across three individual experiments per condition. All statistical analysis to determine significance between groups was performed using GraphPad Prism 9 using a Two-Way ANOVA with a post-hoc tukey’s multiple comparisons test which compared the sample effects within each row.
Quantitative image analysis of mitochondrial dynamic using kymographs
Cells were plated in an Ibidi 8 well plate at 50, 000 cell/ml. Following co-transfection with GFP-FUS constructs (250ng) and a Ds-Mitored plasmid (250ng) at DIV6, cells were incubated for 48hr before imaging on the Nikon Eclipse Ti Spinning disk confocal microscope at 63X. Images were taken every 30 seconds over 10 minutes. Channels were split using Image J and a line was drawn along a dendrite (100μm), to create a kymograph using an Image J plugin. After a kymograph had been created, individual particles were traced using neuron J plugin on Image J to calculate if a particle moved in a retrograde or anterograde direction. Stationary mitochondria were counted if there was no visible movement of that particle in the kymograph. Mitochondrial dynamics were performed on a single dendrite from eight individual cells across three individual experiments per condition. All statistical analysis to determine significance between groups was performed using GraphPad Prism 9 using a One-Way ANOVA with post-hoc tukey’s multiple comparisons test.
Proximity Ligation Assay
Proximity ligation assays (PLAs) were performed essentially as the manufacture instructions (Sigma-Aldrich). Briefly, neurons were fixed in 4% paraformaldehyde in PBS and probed with mouse anti-FUS (1:200, Proteintech) and anti-SNPH (1:200, Proteintech), and signals developed using a Duolink In Situ Orange kit (Sigma-Aldrich). Following PLAs, neurons were immunolabeled for chicken anti-MAP2 (1:1000, AbCam). Images were taken at x60 (oil) on a Nikon Ti-E Two Camera microscope. Images were analysed in ImageJ and positive puncta counted using the cell counting tool. Five somas for each image were analysed for the soma count and 3 different neurites for each of five cells per image were analysed with three biological replicates carried out.
Puromycin assay
Following transfection of DIV6 neurons as previously described, neurons were treated with 1mL/well of 1x ACSF (10x ACSF with H2O, Glucose 11mM and HEPES 5mM), MgCl2 1.25 mM and CaCl2 mM (pH 7.4) at DIV8. Following a 1-hour incubation, 5μL of puromycin (P8833, 10mg/mL, Sigma Aldrich) was added to each well. After 10 minutes, cells were fixed and immunostained with mouse anti-puromycin (1:1000, 3RH11, Kerafast) and chicken anti-MAP2 (1:1000, AbCam). Images were taken with a Nikon iSIM super resolution microscope at x100 (oil) objective. Puromycin puncta were thresholded to be bigger than five pixels in size and average intensity was calculated. Images were obtained from three independent experiments and five different somas and neurites were analysed for each repeat for each condition and values were normalised to threshold. All statistical analysis to determine significance between groups was performed using GraphPad Prism 9 using a One-Way ANOVA with post-hoc tukey’s multiple comparisons test.
Fish stock maintenance, husbandry and embryo collection
All Danio rerio lines were raised and maintained at 28°C on a 14hour light/ 10hour dark cycle in the Guy’s Campus Zebrafish facility, London. Embryos were collected and incubated in dishes filled with system water with methylene blue in a 28°C incubator until experimentation. Morphological staging was determined in accordance with (27). The animal experiments have been authorised by the KCL ethics Review Committee and under the HO license 70/7577.
Microinjection procedure
To deliver the plasmid into individual embryos, 1mm single capillary needles with filament (world precision instruments) were pulled on a model P-97 flaming/brown micropipette puller (Sutter instrument Co.). Once the micropipette was created, 2.8μl of plasmid was taken up and attached to manual micromanipulator apparatus. 0.5nl of plasmid solution (50ng/μl) was measured on a graticule (Pyser-SGI) and injected into a 1-cell stage embryo using the Picospritzer 111 microinjector (Parker instrumentation).
UAS: eGFP-FUS constructs
Homologous sticky-end restriction sites were used (PciI and NheI) to allow insertion of the UAS promoter. Initially, both the pN2 5UAS eGFP and the pC1 CMV eGFP-FUS were digested with PciI (NEB). Following digestion, each digested plasmid was purified and then digested with NheI (NEB). After each plasmid had been digested with both enzymes, the UAS insert and eGFP vector were gel extracted, and gel purified (Qiagen) before being ligated and transformed into competent cells (NEB). UAS: eGFP-FUSWT and eGFP-FUSR514G and eGFP-FUSΔNLS constructs were microinjected at 25ng/μl along with 25ng/μl of MNX1:Gal4 plasmid.
Immunofluorescence
2-day post fertilisation (Long pec) embryos were fixed and, if needed, stained overnight with primary antibody mouse anti-SV2 (1:100, DSHB) followed by overnight staining with alexa-555 conjugated anti-alpha Bungarotoxin (1:100, Invitrogen) and alexa-conjugated goat anti-mouse (1:1000, Invitrogen).
Morphological analysis of motor neurons
images were taken on a Nikon Eclipse C1 confocal microscope using a x40 water objective (N.A. 0.8). Images were based on eGFP expression and z-stacks taken at 1μm increments. Maximum projection images of the eGFP channel were converted into an 8-bit gray scale image and axons were traced using an available plugin on Image J (Neuron J). After tracing, sholl analysis was performed to assess dendritic complexity from the soma at 10μm increments. To measure axonal length and branch numbers, axonal branches that were 0.5μm and larger were included in the axonal branching analysis. Neurons were obtained from six independent experiments and six axons of each condition were chosen. All statistical analysis to determine significance between groups was performed using GraphPad Prism 9 using a One-Way ANOVA with post-hoc tukey’s multiple comparisons test, unless otherwise stated.
Quantification of synaptic density and colocalisation
Z-sacks images with 1 μm increment which were obtained using a x40 oil objective (N.A. 1.3) and obtained on the Zeiss Axio Imager Z2 LSM 800 Confocal. Maximum projection images were converted to 8-bit gray-scale in Image J and individual channels were then used to select the threshold which was kept consistent to the control (eGFP) channel. Following imaging, selected axons were extracted and their length was recorded (which allowed calculation of density). Colocalisation analysis was performed as for the rat primary neuron analysis. Analysis was performed on six separate axons from six biological replicates. All statistical analysis to determine significance between groups was performed using GraphPad Prism 9 using a One-Way ANOVA with post-hoc tukey’s multiple comparisons test.