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. 2016 Jul 21;113(32):E4688–E4697. doi: 10.1073/pnas.1523597113

Fig. 6.

Fig. 6.

Knockdown of endogenous Usp8 in Drosophila protects against α-synuclein–induced toxicity. (A and B) Overexpression of α-synuclein caused a rough eye phenotype, which was more severe in flies expressing A53T mutant α-synuclein, as detected by SEM. This phenotype was rescued in double-transgenic flies with eye-specific Usp8 knockdown and either wild-type (A) or A53T mutant α-synuclein (B). *P < 0.05. Quantitative PCR did not show a reduction in α-synuclein mRNA levels between single- and double-transgenic lines to explain the phenotype. (C) Knockdown of AMSH did not rescue this phenotype, whereas knockdown of Vps28 made it worse. (D) Usp8 knockdown did not affect the expanded Ataxin 3 phenotype. (E) Representative immunoblot of fractionated lysate (C, cytosol; P, pellet) from A53T mutant α-synuclein flies and flies expressing A53T mutant α-synuclein with Usp8 knockdown (+Usp8 KD). Quantitative band densitometry showed that, relative to the actin loading control, protein levels of either wild-type or A53T mutant monomeric α-synuclein (indicated by an asterisk) were significantly reduced in flies coexpressing Usp8 RNAi (n = 4 biological replicates) in the absence of a reduction in mRNA levels, as shown in A and B. *P < 0.05; ****P < 0.0001. (F) Accelerated loss of climbing ability was seen in transgenic flies expressing human A53T mutant α-synuclein in dopaminergic neurons (ddc-GAL4 driver) with increasing age. The climbing ability of double-transgenic lines expressing A53T α-synuclein with Usp8 knockdown in dopaminergic cells was significantly improved (shown with asterisks) compared with A53T α-synuclein–expressing flies and was similar to the control genotype, ddc-GAL4/+ (*P = 0.0381 for day 10; **P = 0.01 for day 12; *P = 0.0159 for day 14; n = 50 flies per group). (G) Knockdown of JosD2 or AMSH in dopaminergic neurons slightly worsened the A53T mutant α-synuclein phenotype. (H) Expression of A53T mutant α-synuclein but not control constructs in dopaminergic neurons (ddc-GAL4 driver) led to loss of TH-immunoreactive neurons in the PPM1/2 cluster, which was prevented by concomitant Usp8 knockdown (***P < 0.001). NS, nonspecific band. Error bars correspond to standard error of the mean.