Fig. 8: Kapβ2_W460A:W730A reduces (GR)₁₀₀ aggregation and counteracts neuronal toxicity of poly(GR).

A Representative images of primary cortical neurons co-transfected with (GR)₁₀₀-mCherry and GFP or GFP-Kapβ2_W460A:W730A. GFP signal is shown in green, mCherry is shown in red, Tuj1 is shown in magenta, and nuclei are shown in blue. Scale bar = 10 μm. B Quantification of the number of (GR)₁₀₀ aggregates per neuron in primary cortical neurons co-transfected with (GR)₁₀₀-mCherry and GFP or GFP-Kapβ2_W460A:W730A. The number of neurons quantified (n) in each condition is indicated in the figure. Data shown are mean ± SEM. Unpaired Student’s t-tests were used to compare different conditions; ***p ≤ 0.001. C Quantification of the size of (GR)₁₀₀ aggregates formed in primary cortical neurons co-transfected with (GR)₁₀₀-mCherry and GFP or GFP-Kapβ2_W460A:W730A. The number of aggregates quantified (n) in each condition is indicated in the figure. Data shown are mean ± SEM. Unpaired Student’s t-tests were used to compare different conditions; ns: p > 0.05. D Quantification of the mean mCherry fluorescence intensity in each (GR)₁₀₀ aggregates formed in primary cortical neurons co-transfected with (GR)₁₀₀-mCherry and GFP or GFP-Kapβ2_W460A:W730A. The number of aggregates quantified (n) in each condition is indicated in the figure. Data shown are mean ± SEM. Unpaired Student’s t-tests were used to compare different conditions; ***p ≤ 0.001. E Analysis of percentage of survival neurons 5 days after co-transfected with (GR)₁₀₀-mCherry and GFP or GFP-Kapβ2_W460A:W730A. N = 3. Unpaired Student’s t-test was used to compare different conditions. F Viability assay of primary cortical neurons treated with indicated R-DPR alone or with Kapβ2 WT or Kapβ2_W460A:W730A. 5μM of DRAQ7, 2.5μM of the indicated R-DPRs and proteins were added into the culture media of treated cortical neurons. Cells positive for DRAQ7 fluorescence (observed in Cy5 channel) were counted 18 hours after treatment to assess the viability of the neurons. Data are represented as median ± S.E.M., n = 3, m (fields of view) = 5. Nested Student’s t-test was used to compare different conditions, ****p ≤ 0.0001. G The indicated R-DPRs (2.5μM) and Kapβ2 WT or Kapβ2_W460A:W730A (2.5 μM) were added into the culture media of treated primary cortical neurons. DIC images of the treated primary cortical neurons were taken 18 hours after treatment to assess the morphology of the neurons. Scale bar = 20 μm.