Fluc-EGFP reporter mice reveal differential alterations of neuronal proteostasis in aging and disease (2021)
Figure 3

Figure 3. Fluc-EGFP reporter reveals proteostasis impairment in tauopathy mice.

A, Cortical sections from rTg4510:Fluc-EGFP mice (right) and control littermates (left) stained for total human tau (HT7, magenta) and p-tau (AT8, yellow). Fluc-EGFP was detected by EGFP fluorescence (green), nuclei were labeled with DAPI (blue). Images on the right show higher magnification of the areas indicated by the boxes, with neuronal cell bodies outlined. Note the presence of EGFP foci (arrowheads) in the majority of neurons in rTg4510:Fluc-EGFP mice. Occasional foci of lower relative fluorescence intensity can be observed in control mice (arrow).

B, Quantification of fluorescence intensity of EGFP foci, normalized to cytoplasmic EGFP fluorescence in cells with and without tau expression and p-tau pathology. N=4 rTg4510:Fluc-EGFP mice, 3 WT:Fluc-EGFP mice, 3 CaMKIIα-tTA:Fluc-EGFP mice and 3 tetO-tauP301L:Fluc-EGFP mice. WT:Fluc-EGFP, CaMKIIα-tTA:Fluc-EGFP and tetO-tauP301L:Fluc-EGFP littermates did not differ from each other and were pooled together as controls. One-way ANOVA with Tukey's multiple comparisons test. ANOVA: ****p < 0.0001. Pairwise comparisons between groups are indicated on the graph.

C, Contour plot of fluorescence intensity distribution of EGFP foci in cells with and without tau expression and p-tau pathology. N = 174 tau- / p-tau- neurons; 178 tau+ / p-tau- neurons, and 69 tau+ / p-tau+ neurons.

D, Quantification of the fraction of cells with EGFP foci among the cells with and without tau expression and p-tau pathology. N=4 rTg4510:Fluc-EGFP mice, 3 WT:Fluc-EGFP mice, 3 CaMKIIα-tTA:Fluc-EGFP mice and 3 tetO-tauP301L:Fluc-EGFP mice. WT:Fluc-EGFP, CaMKIIα-tTA:Fluc-EGFP and tetO-tauP301L:Fluc-EGFP littermates did not differ from each other and were pooled together as controls. One-way ANOVA with Tukey's multiple comparisons test. ANOVA: ****p < 0.0001. Pairwise comparisons between groups are indicated on the graph.

E, F, Luciferase assay in rTg4510:Fluc-EGFP mice. E, Left, scheme of brain regions used for luciferase assay color-coded by their vulnerability to tau pathology. Right, specific luciferase activity measured in the indicated brain regions of 4-month-old and 16-month-old rTg4510:Fluc-EGFP mice normalized to control littermates. F, Fluc-EGFP protein quantity in the indicated brain regions of rTg4510:Fluc-EGFP mice measured by Western blot (for representative blots, see Fig. EV3B). Values are normalized to control littermates. N=6 rTg4510:Fluc-EGFP mice, 4 WT:Fluc-EGFP mice, 6 CaMKIIα-tTA:Fluc-EGFP mice and 1 tetO-tauP301L:Fluc-EGFP mouse at 4 months; 4-5 rTg4510:Fluc-EGFP mice, 5 WT:Fluc-EGFP mice, 3 CaMKIIα-tTA:Fluc-EGFP mice and 4 tetO-tauP301L:Fluc-EGFP mice at 16 months. WT:Fluc-EGFP, CaMKIIα-tTA:Fluc-EGFP and tetO-tauP301L:Fluc-EGFP littermates did not differ from each other and were pooled together as controls. Light-red and dark-red asterisks indicate comparisons to the corresponding littermate group (one-sample t-test), black asterisks indicate significant comparisons between corresponding brain regions of rTg4510:Fluc-EGFP mice at 4 vs. 16 months (two-tailed t-test).

Data information: Error bars represent SD. Significance: *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; n.s. - not significant. Scale bars in A, 10 µm.