Fig. 8: The GFP tag alters the kinetic, ultrastructural properties, protein content and toxicity of Httex1 72Q inclusions in neurons. | Nature Communications

Fig. 8: The GFP tag alters the kinetic, ultrastructural properties, protein content and toxicity of Httex1 72Q inclusions in neurons.

From: Nuclear and cytoplasmic huntingtin inclusions exhibit distinct biochemical composition, interactome and ultrastructural properties

Fig. 8

a Httex1 72Q-GFP was directly visualized with GFP (green) at 3 (D3), 7 (D7), and 14 (D14) days after lentiviral transduction. The nucleus was counterstained with DAPI, and neurons were stained with the MAP2 antibody. Scale bar = 20 μm. b Image-based quantification of the number of transduced neurons bearing Httex1 72Q-GFP inclusions over time. c Morphological and subcellular localization classifications (see Fig. 5c) of the 72Q-GFP inclusions formed in transduced neurons. n = 219 cells were examined over 3 independent experiments. d TUNEL cell death assay in cortical neurons transduced with Httex1. b, c, d The graphs represent the mean ± SD of 3 independent experiments. b, d One-way ANOVA followed by an HSD post hoc test was performed. *P < 0.05, **P < 0.005, ***P < 0.001 for multiple comparisons. #P < 0.05 (72Q vs. 72Q-GFP). c Two-way repeated-measures ANOVA revealed no significant differences. e Representative EM images of Httex1 72Q-GFP inclusions formed in cortical neurons at D7 post-transduction. Green dashed lines represent Httex1 inclusions. Scale bars = 500 nm and 50 μm (inset). f-i Urea soluble proteins extracted from cortical neurons expressing Httex1 72Q or Httex1 16Q (f) or Httex1 72Q-GFP or GFP (h) at D7 post-transduction were analyzed using LC-MS/MS. n = 3 independent experiments. Identified proteins were plotted using volcano plots (f, Httex1 72Q vs. Httex1 16Q; h, Httex1 72Q-GFP vs. GFP). Dotted lines represent the false discovery rate <0.05, and an absolute log2 fold change threshold of significance of 1 was assigned. Upregulated proteins are represented in red, downregulated in blue and non-significant proteins in grey. g The online platform STRING underscores an association between the upregulated Httex1 72Q proteins and protein related to the degradation pathways (red circle). i Venn diagram comparison of proteins enriched in Httex1 72Q vs. Httex1 16Q (blue) to Httex1 72Q-GFP vs. Httex1 16Q (green). 24 proteins were unique to Httex1 72Q vs. Httex1 16Q (44.4%), 17 unique to Httex1 72Q-GFP vs. Httex1 16Q (31.5%) and 13 (24.1%) common to both co-aggregated proteins. Source data are provided as a Source Data file.

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