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. Author manuscript; available in PMC: 2019 May 3.
Published in final edited form as: Cell Rep. 2019 Apr 23;27(4):1133–1150.e8. doi: 10.1016/j.celrep.2019.03.093

Figure 4. RNA Binding and Protein Stability Are Essential for TDP43-Dependent Neurode-generation.

Figure 4.

(A) Schematic of TDP43 hybrid constructs.

(B) TDP43(PUM2)-EGFP expression significantly increased the cumulative risk of death compared to EGFP (HR = 2.54, p < 2 × 10−16). Expression of the PUM2 RNA binding domain (RBD) marginally elevated the risk of death compared to EGFP (HR =1.19, p = 3.78 × 10−5). RNA binding-deficient versions of the PUM2 RBD (mPUM2) significantly reduced the risk of death compared to TDP43(PUM2)-EGFP (HR = 0.45, p < 2 × 10−16).

(C) Images of neurons overexpressing mApple (red fluorescent protein [RFP]) and EGFP-tagged TDP43(WT), TDP43(PUM2), or TDP43(mPUM2). Scale bar: 5 μm.

(D) Expression of TDP43(PP7) reduced the risk of death in comparison to TDP43(WT) (HR = 0.73, p < 2 × 10−16) but remained significantly toxic compared to EGFP (HR = 2.05, p < 2 × 10−16).

(E) Boxplot of TDP43-Dendra2 variant half-life, determined by OPL.

(F) Median TDP43-Dendra2 half-life decreased from 51.9 to 19.8 h with the addition of the destabilizing CL degron.

(G) Both TDP43(WT)-Dendra2 and TDP43(WT)-Dendra2-CL significantly elevated the risk of death compared to Dendra2 (HR = 1.82 and 1.18, p < 2 × 10−16 and p = 1.23 × 10−10, respectively). TDP43(WT)-Dendra2-CL was significantly less toxic compared to TDP43(WT)-Dendra2 (HR = 0.64, p < 2 × 10−16).

Data pooled from 3 independent replicates for (B) and (D) and 4 replicates for (E), (F), and (G). For (B) and (D)–(G), n, number of neurons; ***p < 2 × 10−16; Cox proportional hazards. In (E), ***p < 0.05, 1-way ANOVA with Dunnett’s test. In (F), ****p < 0.0001, unpaired t test. Plots in (E) and (F) show median (horizontal line), interquartile range (box), and maximum/minimum (vertical lines).