Figure 4.
Accumulation of the UPS reporter is also transient in neuronal primary cultures expressing N-mutHtt. A, Diagram of the transfected constructions. Open reading frames of exon 1 and ubiquitin (Ub) are shown in open boxes. An empty triangle indicates where the expanded polyQ tract (94 CAGs) is located. The head arrow indicates the place where the mutation G76V is placed in the reporter construction. CFP and YFP tags are indicated by cyan and yellow boxes, respectively. B, Histogram of average percentage of striatal neurons transfected with UbG76VYFP (YFP), N-wtHtt-CFP + UbG76VYFP (N-wtHtt+YFP), or N-mutHtt-CFP + UbG76VYFP (N-mutHtt+YFP), showing above threshold-yellow fluorescence 16 h after transfection. This histogram does not show error bars because significant differences between groups were evaluated using two proportion comparison test. C, Histogram showing the yellow fluorescence intensity in striatal neurons transfected with YFP, N-wtHtt+YFP, or N-mutHtt+YFP, 16 h after transfection (in arbitrary units of YFP fluorescence). Data are presented as the mean ± SEM. D, Striatal neuron 24 h after transfection with N-mutHtt-CFP + YFP with no degradation signal. E, Striatal neurons transfected with N-mutHtt-CFP + UbG76VYFP, 16 h or 24 h after transfection. Scale bar, 20 μm. F, Histogram showing average percentage of striatal neurons transfected with N-mutHtt-CFP + UbG76VYFP (N-mutHtt) (black bars) or N-wtHtt-CFP + UbG76VYFP (N-wtHtt) (white bars) in which yellow fluorescence does not change, decreases, or increases during the 8 h observation period. This histogram does not show error bars because significant differences between groups were evaluated using two-proportion comparison test. G, H, Cortical (G) and striatal (H) neurons transfected with N-mutHtt-CFP + UbG76VYFP in which yellow fluorescence decreases with time. 36% of the cortical transfected neurons and 30% of the striatal transfected neurons build visible IBs, whereas YFP fluorescence decreases with time as shown at 16, 18, 20, 22, and 24 h posttransfection. Scale bars, 15 μm. **p < 0.005, ***p < 0.001).