Mutations in String/CDC25 inhibit cell cycle re-entry and neurodegeneration in a Drosophila model of Ataxia telangiectasia

Abstract

Mutations in ATM (Ataxia telangiectasia mutated) result in Ataxia telangiectasia (A-T), a disorder characterized by progressive neurodegeneration. Despite advances in understanding how ATM signals cell cycle arrest, DNA repair, and apoptosis in response to DNA damage, it remains unclear why loss of ATM causes degeneration of post-mitotic neurons and why the neurological phenotype of ATM-null individuals varies in severity. To address these issues, we generated a Drosophila model of A-T. RNAi knockdown of ATM in the eye caused progressive degeneration of adult neurons in the absence of exogenously induced DNA damage. Heterozygous mutations in select genes modified the neurodegeneration phenotype, suggesting that genetic background underlies variable neurodegeneration in A-T. The neuroprotective activity of ATM may be negatively regulated by deacetylation since mutations in a protein deacetylase gene, RPD3, suppressed neurodegeneration, and a human homolog of RPD3, histone deacetylase 2, bound ATM and abrogated ATM activation in cell culture. Moreover, knockdown of ATM in post-mitotic neurons caused cell cycle re-entry, and heterozygous mutations in the cell cycle activator gene String/CDC25 inhibited cell cycle re-entry and neurodegeneration. Thus, we hypothesize that ATM performs a cell cycle checkpoint function to protect post-mitotic neurons from degeneration and that cell cycle re-entry causes neurodegeneration in A-T.

Figure 1.

ATM RNAi in pATM flies reduced the level of ATM mRNA and protein. (A) A schematic diagram of the pWIZ-ATM (pATM) transformation vector (Lee and Carthew 2003). (B) Quantitative PCR analysis of ATM mRNA in heat-shocked hsp70-GAL4/+; pATM^T4/+ (hs-ATMi) and hsp70-GAL4/+ (hs-GAL4) flies. Graphed is the level of ATM mRNA relative to actin 5C mRNA in hs-ATMi flies normalized to hs-GAL4 flies. Error bars represent the standard error of the mean of experiments performed in triplicate. (C, lanes 3,4) Western blot analysis of ATM in hsp70-GAL4/pATM^T4 and hsp70-GAL4/+ embryos. Extracts were probed with an α-ATM antibody and an α-SIN3 antibody as a loading control (Pile and Wassarman 2000). Note that lane 4 was overloaded relative to lane 3. (Lanes 1,2) RNAi-mediated knockdown of ATM in S2 cells by long dsRNA served as a marker for ATM.

Figure 2.

RNAi-mediated knockdown of ATM causes degeneration of photoreceptor neurons. Shown are representative TEM micrographs of single ommatidia (A–H) and high-magnification micrographs of single photoreceptor neurons from flies of the indicated genotypes (A′–H′). (A) Three-day-old Elav-GAL4/+ (Elav-GAL4). (B) Three-day-old Elav-GAL4/pATM^T4 (Elav-ATMi). (C) Three-day-old GMR-GAL4/+ (GMR-GAL4). (D) Three-day-old GMR-GAL4/+; pATM^T4/+ (GMR-ATMi). (E) Three-day-old hs-ATMi not heat-shocked. (F) Three-day-old hs-ATMi heat-shocked throughout development. (G) Fifteen-day-old hs-ATMi not heat-shocked. (H) Fifteen-day-old hs-ATMi heat-shocked days 3–15 of adulthood. (A′–H′) Red circles indicate the photoreceptor neuron that is shown in high magnification. Red arrows indicate defects in rhabdomere structure.

Figure 3.

ATM knockdown causes progressive degeneration of adult neurons. Zero-day-old to 4-d-old GMR-GAL4, GMR-ATMi, and GMR-GAL4/+; pATMT4/Stg^EY12388 (GMR-ATMi, Stg) adult flies were collected and aged for 9, 27, and 48 d. The number of morphologically wild-type photoreceptor neurons per ommatidium was determined by analyzing TEM images of sections at the R7 level in the center of each fly eye. Error bars represent standard error of the mean. P-values were calculated using two-way ANOVA and Bonferonni post-tests. (A) Flies of the same genotype were compared at each time point. (B) Flies of different genotypes were compared at each time point.

Figure 4.

Suppressors of the GMR-ATMi rough eye phenotype. Shown are representative SEM micrographs of 0- to 4-d-old control flies (A,B) and suppressors of GMR-ATMi (C–F). (A′–F′) High-magnification micrographs of the center of the eye are shown below. (A) GMR-GAL4. (B) GMR-ATMi. (C) GMR-P35/+; GMR-GAL4/+; pATM^T4/+ (GMR-P35; GMR-ATMi). (D) GMR-GAL4/RAD50^EP1; pATM^T4/+ (RAD50; GMR-ATMi). (E) GMR-GAL4/+; pATMT4/RPD3⁰⁴⁵⁵⁶ (GMR-ATMi, RPD3). (F) GMR-GAL4/+; pATMT4/Stg^EY12388 (GMR-ATMi, Stg).

Figure 5.

Enhancers of the GMR-ATMi rough eye phenotype. Shown are representative SEM micrographs of 0- to 4-d-old control flies (A,B) and enhancers of GMR-ATMi (C–E). (A′–E′) High-magnification micrographs of the center of the eye are shown below. Note the increased incidence of ommatidium fusions in the presence of enhancer mutations. (A) GMR-GAL4. (B) GMR-ATMi. (C) GMR-GAL4/+; pATM^T4/ MEKK4^EY02276 (GMR-ATMi, MEKK4). (D) GMR-GAL4/+; pATM^T4/PP2A-B′^A131 (GMR-ATMi, PP2A-B′). (E) GMR-GAL4/+; pATM^T4/Delta⁰⁵¹⁵¹ (GMR-ATMi, Delta).

Figure 6.

Genetic background modified the severity of the GMR-ATMi neurodegeneration phenotype. The number of morphologically wild-type photoreceptor neurons per ommatidium was determined by analyzing TEM images of sections at the R7 level in the center of eyes from 0- to 4-d-old flies of the indicated genotypes. More than 260 ommatidia were scored from at least four eyes of each genotype. RAD50^EP1, RPD3⁰⁴⁵⁵⁶, Stg^EY12388, or GMR-P35 alleles were used for this analysis. (A) Graphed is the average number of normal photoreceptors per ommatidium for each genotype. Error bars represent the standard error of the mean. P-values were calculated using one-way ANOVA and Dunnett’s multiple comparison post-tests where all genotypes were compared with GMR-ATMi flies. The statistical difference between genotypes is not attributable to the variability seen in flies of a given genotype. (B) Graphed is the percentage of total ommatidia with zero to seven normal photoreceptor neurons for each genotype.

Figure 7.

DNA replication occurred in neurons of GMR-ATMi flies. Eye discs from control GMR-GAL4 (A,A′,A″) or GMR-ATMi (B,B′,B″) third instar larvae were costained with α-Elav (red) and α-BrdU (green) antibodies. A′ and A″ and B′ and B″ are higher-magnification images of A and B, respectively. A″ and B″ are merged images of α-Elav and α-BrdU. Arrows indicate a neuron in which DNA replication has occurred. MF indicates the position of the MF.

Figure 8.

Cell cycle re-entry in Elav-ATMi flies is inhibited by mutation of Stg but not expression of P35. FACS analysis was carried out on eye imaginal discs dissected from control Elav-GAL4,UAS-GFP/+ (Elav-GFP, n = 9) (A); Elav-GAL4, UAS-GFP/+; Stg^EY12388/+ (Elav-GFP; Stg, n = 4) (B); Elav-GAL4, UAS-GFP/+; pATM^T4/+ (Elav-GFP; Elav-ATMi, n = 8) (C); Elav-GAL4, UAS-GFP/+; pATM^T4/Stg^EY12388 (Elav-GFP; Elav-ATMi, Stg, n = 6) (D); Elav-GAL4, UAS-GFP/GMR-P35 (Elav-GFP, GMR-P35, n = 2) (E); and Elav-GAL4, UAS-GFP/GMR-P35; pATM^T4/+ (Elav-GFP, GMR-P35; Elav-ATMi, n = 4) (F) third instar larvae. (G) Quantitation of cell cycle phases from multiple, independent samples. For each sample, 5000 live, single, GFP-positive events were analyzed. P-values were calculated using one-way ANOVA and Tukey’s multiple comparison post-tests.

Figure 9.

HDAC2 interacts with ATM and represses ATM activation. (A) Western blot analysis of lysates and immunoprecipitates from HEK 293T cells transfected with empty plasmid (vector) or plasmids that expressed Flag-epitope-tagged human HDAC1 (FLAG-H1) or HDAC2 (FLAG-H2). Cells were either exposed to 20 Gy of IR (+) or not exposed (−), and cell lysates were immunoprecipitated with an α-Flag antibody. Immunoprecipitates (IP) or total lysates (Input) were immunoblotted (WB) for ATM or the Flag tag. (B) Lysates prepared from HEK 293T cells were immunoprecipitated with a rabbit IgG antibody (C), an α-ATM antibody from Santa Cruz Biotechnology (A1), and α-ATM antibody from Genetex (A2), or an α-HDAC2 antibody (H2). Immunoprecipitates (IP) were immunoblotted (WB) for ATM or HDAC2. (C) HEK 293T cells were transfected with control scrambled (S) or HDAC2 (H2)-specific siRNAs. Cell lysates were immunoblotted (WB) for pS1981 on ATM (pS1981-ATM), total ATM (ATM) as a loading control, or HDAC2. The triangle indicates increasing amounts of siRNAs.