Computational protocol: Mutant torsinA in the heterozygous DYT1 state compromises HSV propagation in infected neurons and fibroblasts

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Protocol publication

[…] Human DYT1 patient-derived and control fibroblasts were infected with HSV-VP26-RFP under different conditions. For the virus-induced cytopathic effect experiments, we used a range of M.O.I. Cells were viewed under the microscope at 12, 20, 24 and 44 h post-infection. For the plaque assay, we plated 100,000 cells/well in a 12-well plate. Two days later, when the cells reached confluency, we added HSV-VP26-RFP in serial dilutions, ranging from 10−5 to 1 plaque forming units (PFUs) for 6 h at 37 °C. Then we aspirated the virus-containing culture media and overlaid 0.4% agarose dissolved in media + serum. The plates were left at room temperature for 30 min to allow for solidification of the agarose. Forty-eight to 72 h later, we visualized the red plaques using epifluorescent microscopy. Finally, we counted the number and measured the area of red plaques in each well using ImageJ v1.43. [...] Timed pregnancies of DYT1 Het mice were monitored in-house and dissociated cortical neuronal cultures were prepared from individual E15 embryos, as described. Cells from each embryo were saved for genotyping using primers described. Mouse cortical neurons from WT, Het and KI embryos were plated on coverslips pre-coated with poly-D-lysine and laminin (Corning, New York, NY USA). The purity of neuronal cultures was determined by beta-III tubulin staining (data not shown).Neurons were infected in triplicate with HSV-VP26-RFP at the M.O.I. of 1. Cells were analyzed uninfected and 8 h and 15 h post-infection. Cells were fixed for 20 min with 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) and then stained for DAPI (1:10,000; Life Technologies) and WGA-488 (10 µg/mL; Invitrogen, Carlsbad, CA USA). Images were captured with a Zeiss LSM 710 inverted confocal microscope (Carl Zeiss AG, Germany) using a 63x oil objective (plan-apochromat SF25, DIC, NA = 1.4) and a z thickness of 1 μm. Eighteen images were taken for each embryo. Cytoplasm fluorescence was analyzed by ImageJ version 2.0.0-rc-54/1.51 h (Supplementary Fig. ). Briefly, images were acquired in three channels (red, blue, and green) and overlaid into composites. The images were analyzed using ImageJ v2.0.0 to quantify the red fluorescence signal, indicating the intensities of HSV-VP26-RFP, in the cytoplasm of cells from each genotype (WT, Het and KI). To select only nuclear areas in the images, the threshold tool was used in ImageJ for the DAPI channel, and then the red fluorescence intensity within the selected regions was measured using the Analyze Particles tool again with an optical thickness of 1 μm. This value was subtracted from the total red fluorescence intensity for the image to determine the fluorescence value for the cytoplasm. For nuclear fluorescence analysis, we segmented cells based on DAPI staining using Imaris 9.0.0 (Bitplane, Zurich, Switzerland) (Supplementary Fig. ). RFP fluorescence intensity was determined for all cells or only for infected cells. When analyzing all cells, no thresholding was done, i.e. infected and also non-infected cells (having background red fluorescence) were included in the analysis (data is presented in Fig. ). We also analyzed nuclear fluorescent intensity only in infected cells (Fig. ). In this case, cells that had a nuclear fluorescent intensity twice as high as the background level were considered ‘infected’. Finally, we analyzed the percentage of highly infected cells (defined as having 3 times above the background level) among all infected cells (Fig. ). Image analysis was performed in a blinded manner with the genotypes unknown to the person performing the analysis. […]

Pipeline specifications

Software tools ImageJ, Imaris
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus, Homo sapiens
Diseases Infection, Machado-Joseph Disease, Epstein-Barr Virus Infections, Genetic Diseases, Inborn
Chemicals Glutamic Acid