Drosophila histone deacetylase 6 protects dopaminergic neurons against {alpha}-synuclein toxicity by promoting inclusion formation

Abstract

Parkinson's disease (PD) is associated with progressive degeneration of dopaminergic (DA) neurons. We report for the first time that the Drosophila histone deacetylase 6 (dHDAC6) plays a critical role in the protection of DA neurons and the formation of alpha-synuclein inclusions by using a Drosophila PD model constructed by ectopic expression of human alpha-synuclein. Depletion of dHDAC6 significantly enhances the effects caused by ectopic expression of alpha-synuclein, namely, loss of DA neurons, retinal degeneration, and locomotor dysfunction. Expression of alpha-synuclein in the DA neurons leads to fewer inclusions in the brains of dHDAC6 mutant flies than in wild-type flies. Conversely, overexpression of dHDAC6 is able to suppress the alpha-synuclein-induced DA neuron loss and retinal degeneration and promote inclusion formation. Furthermore, mutation of dHDAC6 reinforces the accumulation of oligomers that are suggested to be a toxic form of alpha-synuclein. We propose that alpha-synuclein inclusion formation in the presence of dHDAC6 protects DA neurons from being damaged by oligomers, which may uncover a common mechanism for synucleinopathies.

Figure 1.

Characterization of the dHDAC6^KO mutant. (A) Genomic organization of the Drosophila dHDAC6 locus and the mutant allele of dHDAC6. Asterisk indicates the mutation of the ATG start codon. Filled bars are the coding regions; open bars are the untranslated regulatory regions. (B) Part of the dHDAC6^KO mutant genomic sequence that contains point mutation and frame shift at the dHDAC6 translation start codon. The boxed sequence is dHDAC6 start codon in the wild-type gene. (C) dHDAC6 protein is absent in the dHDAC6 mutant flies as shown by Western blot. Protein extracts from wild-type (WT) and dHDAC6 knockout (KO) flies were blotted with anti-HDAC6 and anti-actin antibodies. Actin was used as a loading control.

Figure 2.

dHDAC6^KO promotes α-synuclein–induced dopaminergic neuron loss. Confocal images of dopaminergic neurons in DM, PM, and DL₁ regions immunostained with anti-GFP antibody on 1-d-old (A), 10-d-old (B), and 20-d-old (C) fly brains, respectively, are shown, with arrows for PM and arrowheads for DM clusters and circles for DL₁ clusters. (D–F) Quantitative graphs are shown corresponding to confocal images of 1-d-old, 10-d-old, and 20-d-old fly brains, respectively. (G) Graphs showing total numbers of dopaminergic neurons in the DM, PM, and DL₁ clusters of different genotypes as indicated at 1 d, 10 d, 20 d. Data were analyzed by Student's t test and presented as mean ± SEM (n = 20∼40) with * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. Bar, 50 μm. Genotypes: control flies are w; UAS-mCD8::GFP/+; TH-GAL4/+. dHDAC6^KO flies are w, dHDAC6^KO; UAS-mCD8::GFP/+; TH-GAL4/+. α-syn flies are w; UAS-mCD8::GFP/+; TH-GAL4, UAS-α-synuclein/+. dHDAC6^KO; α-syn flies are w, dHDAC6^KO; UAS-mCD8::GFP/+; TH-GAL4, UAS-α-synuclein/+.

Figure 3.

Loss of dHDAC6 exacerbates retinal degeneration in α-synuclein–expressing eyes and overexpression of dHDAC6 counteracts the α-synuclein neurotoxicity. Semithin sections of retina showing inner retinal structures of 1-d-old (A and B, top), 20-d-old (A, bottom), and 30-d-old (B, bottom) flies. Arrows indicate outer ring disintegration of the ommatidia. Circles indicate ommatidia with loss of photoreceptor cells. (C) Quantification of disrupted ommatidia in control, dHDAC6-L^KO, α-syn and dHDAC6-L^KO;α-syn flies at 1 and 20 d after eclosion. (D) Quantification of disrupted ommatidia in control, α-syn, and α-syn;dHDAC6 flies at 1 and 30 d. (E) Quantification of ommatidia with photoreceptor cell loss in 20-d-old dHDAC6^KO;α-syn flies, 30-d-old α-syn flies, and α-syn;dHDAC6 flies. Note that the retinal pattern and seven photoreceptor cells were normal in GMR-GAL4 control and dHDAC6-L^KO flies. No discernible photoreceptor loss was detected in 1-d-old and 20-d-old α-syn flies. Data were analyzed by Student's t test and presented as mean ± SEM (n > 5) with *p < 0.05, **p < 0.01, and ***p < 0.001. Genotypes: control flies are w; GMR-GAL4/+. dHDAC6-L^KO flies are w, dHDAC6-L^KO. α-syn flies are w; GMR-GAL4/+; UAS-α-synuclein/+. dHDAC6-L^KO; α-syn flies are w, dHDAC6-L^KO; GMR-GAL4/+; UAS-α-synuclein/+. α-syn; dHDAC6 flies are w; GMR-GAL4/+; UAS-α-synuclein/UAS-dHDAC6.

Figure 4.

dHDAC6^KO enhances the climbing defects caused by α-synuclein overexpression. Bars show the percentage of the flies that climbed to reach 15 cm in 30 s at different ages with the genotypes as indicated. Data were analyzed by Student's t test and presented as mean ± SEM (n = 120) with *p < 0.05, **p < 0.01, and ***p < 0.001. Genotypes: control flies are w; TH-GAL4/+. dHDAC6-L^KO flies are w, dHDAC6-L^KO; TH-GAL4/+. α-syn flies are w; TH-GAL4/UAS-α-synuclein. dHDAC6^KO; α-syn flies are w, dHDAC6^KO; TH-GAL4/UAS-α-synuclein.

Figure 5.

The amount of LB-like inclusion decreases in dHDAC6^KO flies with α-synuclein overexpression. LB-like inclusions in the DM region of α-syn (A and C) and dHDAC6^KO; α-syn flies (B and D) at 10 d (A and B) and 20 d (C and D) were labeled by whole-mount immunostaining with anti-α-synuclein antibody. (E) Inclusions when UAS-dHDAC6 transgene is expressed in α-syn flies at 10 d old. Bar, 40 μm. (F and G) Quantification of the numbers of total inclusions (F) and large inclusions that are >1 μm (G) at 10 d old and 20 d old with the genotypes as indicated on the right. Data were analyzed by Student's t test and presented as mean ± SEM (n > 5) with *p < 0.05, **p < 0.01, and ***p < 0.001. Genotypes: flies in A and C are w; TH-GAL4/UAS-α-synuclein. Flies in B and D are w, dHDAC6^KO; TH-GAL4/UAS-α-synuclein. Fly in E is w; UAS-dHDAC6/+; TH-GAL4, UAS-α-synuclein/+.

Figure 6.

dHDAC6 positively regulates the accumulation of insoluble α-synuclein. (A) Immunoblots detecting α-synuclein from the fractions (see Materials and Methods for details of fractionation) of Tris-soluble supernatant (S) and Sarkosyl-insoluble/urea-soluble pellets (P). The flies were 1 d old, 10 d old, and 20 d old, respectively. (B) Western blot of α-synuclein from the heads of 1-d-old flies that express α-synuclein under the control of TH-GAL4 in the backgrounds of wild-type, dHDAC6^KO, and dHDAC6 overexpression, as indicated on the top. The membrane was first probed with α-synuclein antibody and then stripped before immunoblotted with anti-actin that serves as a loading control. Genotypes: α-syn flies are w; TH-GAL4/UAS-α-synuclein. dHDAC6^KO; α-syn flies are w, dHDAC6^KO; TH-GAL4/UAS-α-synuclein. α-syn; dHDAC6 flies are w; UAS-dHDAC6/+; TH-GAL4, UAS-α-synuclein/+.

Figure 7.

Oligomers accumulate in dHDAC6^KO fly brains with α-synuclein overexpression. Oligomers in the dorsal brain area of α-syn flies (A and C) and dHDAC6^KO; α-syn flies (B and D) at 10 (A and B) and 20 d (C and D) were visualized in red by oligomer-specific antibody A11. (E) α-syn flies at 20 d with dHDAC6 overexpression showing A11 signals. (F) Wild-type control flies at 20 d probed with A11. Bar, 20 μm. (G) Quantitative graphs showing the total A11-positive signals (oligomers) in the dorsal region of the brain as shown in A–F. Data were analyzed by Student's t test and presented as mean ± SEM (n > 5) with *p < 0.05, **p < 0.01, and ***p < 0.001. Genotypes: α-syn flies are w; TH-GAL4/UAS-α-synuclein. dHDAC6^KO; α-syn flies are w, dHDAC6^KO; TH-GAL4/UAS-α-synuclein. Control flies are TH-GAL4/+.

Figure 8.

dHDAC6 physically interacts with oligomeric α-synuclein in Drosophila. Protein extracts for immunoprecipitation were prepared from the brains of 20-d-old flies: w; TH-GAL4/UAS-α-synuclein (A, with α-synuclein expression) and w; TH-GAL4/+ (B, without α-synuclein expression). The SDS-10% PAGE of the coimmunoprecipitation samples was immunoblotted with anti-dHDAC6 (A, top; and B) or anti-α-synuclein antibody (A, bottom). Input: 20% of the extracts that were used for immunoprecipitation.