Computational protocol: ALS-linked FUS exerts a gain of toxic function involving aberrant p38 MAPK activation

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Protocol publication

[…] Motor neurons were isolated from embryonic (E)12.5 mouse embryos and cultured as described. FLAGHA-FUS expression cassette vectors were generated from pFRT-TO-DEST-FLAGHA-FUS-WT, which was obtained from Addgene (#26373). ALS-linked mutations were introduced with the QuikChange II Mutagenesis kit (Stratagene; 200523) according to the manufacturer’s instructions. Cells were transfected at 2 days in vitro (DIV) with green fluorescent protein (GFP) and FLAGHA-tagged FUS constructs in a 1:2 ratio with NeuroMag (OZ Biosciences) as previously described. FLAGHA-FUS plasmids were generated from pFRT-TO-DEST-FLAGHA-FUS-WT obtained from Addgene (#26373). The QuikChange II Mutagenesis kit (Stratagene; 200523) was used to introduce the R521G and P525L mutations according to the manufacture’s instructions. Twenty µM MW069 or its inactive form (MW069_inactive) were added to the culture medium after transfection to inhibit p38 activity and maintained throughout the experiment for a total of ~18–24 hours. Cells were imaged at 3 days in vitro (DIV) using a Nikon TiE widefield microscope equipped with temperature- and CO2-controlled environmental chamber. Neurobasal medium was replaced with Hybernate E Low Fluorescence (BrainBits) 1 hour prior to imaging to reduce autofluorescence and light toxicity. GFP was used to identify transfected cells. Movies were acquired with a 20x lens at a rate of 1 frame every 10 minutes for 1 hour. The speed of axon outgrowth was measured using the ImageJ plugin MTrackJ, . Data from three independent, biological replicates were normalized to the condition with FUS WT in the presence of MW069_inactive. Statistical analysis (2-way ANOVA followed by Tukey’s multiple comparisons test) was performed using Graph Pad Prism 6 to establish statistical differences (p < 0.05) between conditions. To assess FLAGHA-FUS expression, cells were post-fixed in 4% paraformaldehyde for 15 minutes and stained with an anti-HA antibody (Cell Signaling - C29F4; 1:500) overnight at 4 °C. Secondary antibody (donkey anti-Rabbit conjugated with Alexa594; Jackson ImmunoResearch) was incubated at room temperature for 1 hour. […]

Pipeline specifications

Software tools ImageJ, MTrackJ
Databases Addgene
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus, Homo sapiens
Diseases Amyotrophic Lateral Sclerosis, Liposarcoma, Sarcoma, Neurodegenerative Diseases