Article Figures & Data
Figures
- Figure 1.
The anti-Htt intrabodies reduce mHDx-1-induced toxicity and aggregation in cell culture. A , The epitopes in HDx-1 for the various intrabodies are depicted. B , To quantify mHtt toxicity, HEK293 cells were cotransfected with mHDx-1-GFP and intrabody at various intrabody/Htt ratios and incubated for 48 h. Cells were stained with EthD-2 to identify dead cell nuclei, fixed, and stained with DAPI to identify all cell nuclei. The number of dead cells was normalized to total cell number. All of the intrabodies reduce mHDx-1-induced cell death in a saturable, dose-dependent manner, with maximal effects at different intrabody/Htt ratios (1:1 for VL12.3, 2:1 for Happ1 and Happ3, and 4:1 for MW7). C , Aggregation was determined by counting GFP foci and normalizing to total cell number. *Different from VL12.3 at p < 0.05; **p < 0.01. The point labeled as 0 on the intrabody:Htt axis corresponds to the value for HDx-1 + CVL.
- Figure 2.
Protective effects of anti-Htt intrabodies against mHDx-1-induced neurodegeneration in corticostriatal brain slice explants. Corticostriatal brain slices were biolistically transfected with plasmid expression constructs encoding YFP, mHDx-1 (N1–66 with 73 Q), and the indicated intrabody. The number of healthy medium spiny neurons in the striatal region of each slice was scored visually 4–5 d after slice preparation and transfection. A , Slices were transfected with YFP + CVL; YFP + CVL + mHDx-1; YFP + mHDx-1 + VL12.3; or YFP + mHDx-1 + Happ1. *Different from YFP + CVL at p < 0.01. B , Slices were transfected with YFP + CVL; YFP + vector (Vec) + mHDx-1; YFP + CVL + mHDx-1; or YFP + mHDx-1 + MW7. *p < 0.01. The data in A and B are from independent experiments.
- Figure 3.
VL12.3 increases the level of nuclear HDx-1. ST14A cells were cotransfected with mHDx-1-GFP with 103Q and intrabody at optimal ratios. A , At 48 h after transfection, cells were fixed, stained for the appropriate intrabody and cell nuclei, and analyzed by confocal microscopy. B , Mean whole-cell fluorescence intensity (int.) and mean nuclear fluorescence intensity of HDx-1 were compared. Whereas MW7, Happ1, and Happ3 have no effect on HDx-1 localization, VL12.3 significantly increases nuclear HDx-1. *Different from HDx-1 at p < 0.01.
- Figure 4.
All of the anti-Htt intrabodies reduce insoluble HDx-1, whereas only the anti-PRR intrabodies also reduce soluble HDx-1. HEK293 cells were cotransfected with intrabody and mHDx-1 (76 kDa) or wtHDx-1 (40 kDa) at the optimal ratio for each intrabody. A , At 48 h after transfection, cells were lysed with detergent. The soluble protein fraction was recovered, and the insoluble fraction was treated with urea. Samples were then separated by SDS-PAGE and blotted for HDx-1. Nontransfected cells were used as a negative control (NEG). B , Quantification of bands shows that reduction of HDx-1 by PRR-binding intrabodies is significantly greater for the mutant form of Htt. Chemiluminescence densitometry was used to compare the levels of soluble mHDx-1 and wtHDx-1. Each band was normalized to the level of β-tubulin (54 kDa) in that sample. Bands for HDx-1 + intrabody (iAb) were then normalized to the level of soluble HDx-1 for that blot. n = 4 independent experiments, and values for each blot were used to compile a mean. *p < 0.01.
- Figure 5.
Anti-PRR intrabodies increase mHDx-1 turnover. A–C , ST14A cells were transfected with mHDx-1-SNAPtag 97Q ( A , B ) or wtHDx-1-SNAPtag 25Q ( C ) and intrabody at the optimal ratio for each intrabody. DAF green fluorescent SNAP substrate was added to cultures at 24 h after transfection. Some cultures were then fixed and stained with Toto-3 iodide nuclear marker, whereas others were incubated an additional 24 h to allow turnover of labeled HDx-1. Mean fluorescence intensity of cells at 24 h was compared with intensity at 48 h to determine the percentage of labeled HDx-1 remaining. VL12.3 has no effect on mHDx-1 or wtHDx-1 turnover, whereas MW7, Happ1, and Happ3 significantly increase the rate of mHDx-1 turnover. n = 4; *p < 0.01.
Additional Files
Supplemental Data
Files in this Data Supplement:
- supplemental material - Supplemental Material
- supplemental material - Supplemental Figure 1
- supplemental material - Supplemental Figure 2
