Figure 4

Upregulation of Hsc70.3 mitigates TDP-43-induced toxicity in the Drosophila eye. (A) Compared to the normal control, expression of human TDP-43 (ctrl) in the Drosophila eye disrupts the external eye (white hatched line, top panel) and internal retina (white double headed arrow, and white hatched line lower panel). (B) Expression of Hsc70.3 suppresses TDP-43-induced disruption of the external eye. Data is presented as Mean ± SD, one way ANOVA and a Fisher’s LSD test. ****P < 0.0001 and ns not significant. (C) Expression of Hsc70.3^WT suppresses TDP-43-induced reduction of retinal width (see double headed arrow, lower panel in A). Data is presented as Mean ± SD, one way ANOVA and a Fisher’s LSD test. ****P < 0.0001, *P < 0.05 and ns not significant. (D) Expression of Hsc70.3^WT, and Hsc70.3^K97S reduces TDP-43-induced vacuolization of the internal eye (white hatched line, lower panel in A.). Data is presented as Mean ± SD, one way ANOVA and a Fisher’s LSD test. ****P < 0.0001, *P < 0.05 and ns not significant. (E) Upregulation of Hsc70.3^WT or Hsc70.3^K97S had no effect of the total protein levels of TDP-43. Protein isolated from ~ 5 to 10 male heads immunoblotted for TDP-43 and Tubulin. Protein levels were quantified from 3 independent biological repeats. Data is presented as Mean ± SD, one-way ANOVA and Tukey’s test, ns not significant. (F) Upregulation of Hsc70.3^WT or Hsc70.3-^K97S had no effect of the total protein levels of β-galactosidase. Protein isolated from ~ 5 to 10 male heads immunoblotted for β-galactosidase and Tubulin. Protein levels were quantified from 3 independent biological repeats. Data is presented as Mean ± SD, one-way ANOVA and Tukey’s test, ns: not significant. (G) Expression of TDP-43 with gmr-GAL4 leads to an increase in Hsc70.3 mRNA levels compared to control (ctrl). Data is presented as Mean ± SD. An unpaired and two-tailed T test was used to determine significance. Genotypes are (A–E) normal is y, sc, v, sev/w¹¹¹⁸; +/+; gmr-GAL4 (YH3)/si.mCherry³⁵⁷⁸³, ctrl is y, sc, v, sev/w¹¹¹⁸; UAS-TDP-43/+; gmr-GAL4 (YH3)/ si.mCherry³⁵⁷⁸³, Hsc70.3^WT is w-; UAS-TDP-43/ UAS-Hsc70-3^WT; gmr-GAL4 (YH3)/+ and Hsc70.3^K97S is w-; UAS-TDP-43/UAS- Hsc70-3^K97S; gmr-GAL4 (YH3)/+. (F) ctrl is y, sc, v, sev/w¹¹¹⁸; UAS-LacZ/+; gmr-GAL4 (YH3)/si.mCherry³⁵⁷⁸³, Hsc70.3^WT is w−; UAS-LacZ/UAS-Hsc70-3^WT; gmr-GAL4 (YH3)/+ and Hsc70.3^K97S is w-; UAS-LacZ/UAS-Hsc70-3^K97S; gmr-GAL4 (YH3).