Figure 3. The Drosophila Eip74EF gene is a target of miR-34 in modulation of the aging process.
a. E74A mRNA is robustly expressed in the adult and unchanged between age-matched controls and miR-34 mutants. In control flies, E74A mRNA is significantly upregulated in 30d compared to 3d animals. RNA was from male heads. Mean ± s.e.m., n = 3 independent experiments; signal density of E74A mRNA normalized to 18S rRNA loading control (*=p < 0.01, one-way analysis of variance, with post test: Tukey's multiple comparison test). Genotypes: control: 5905. miR-34 −/−: miR-34 null-1 in 5905 homogenous genetic background.
b. E74A protein is deregulated in miR-34 mutants. Protein was from male heads. Mean ± s.e.m., n = 3 independent experiments; signal density of E74A protein normalized to tubulin loading control (*=p < 0.01, one-way analysis of variance, with post test: Tukey's multiple comparison). Genotypes as in a.
c. De-regulation of E74A protein is diminished in miR-34 rescue flies. Protein was from male heads. Mean ± s.e.m., n = 3 independent experiments; signal density normalized to tubulin loading control (*=p < 0.05, one-way analysis of variance, with post test: Tukey's multiple comparison test). Genotypes: control: 5905. miR-34 −/−: miR-34 null-1 in 5905 homogenous genetic background. miR-34 −/−; miR-34 (+): miR-34 genomic rescue in miR-34 null-1 in 5905 homogenous genetic background.
d. miR-34 mutants homozygous for the E74ABG01805 allele have lower levels of E74A protein. Protein was from male heads of 20d flies raised at 29°C. Mean ± s.e.m., n = 3 independent experiments; signal density normalized to tubulin loading control (*=p < 0.01, one-way analysis of variance, with post test: Tukey's multiple comparison test). Genotypes: control: 5905. miR-34−/−, E74ABG/+: E74ABG01805/+, miR-34 null-1 in 5905 homogenous genetic background. miR-34−/−, E74ABG/E74ABG: E74ABG01805/E74ABG01805, miR-34 null-1 in 5905 homogenous genetic background.
e–f. Reducing E74A protein levels in the adult mitigates age-related defects of miR-34 mutants.miR-34 mutants also homozygous for the E74ABG01805 show rescued lifespan (e) and brain morphology (f), compared to miR-34 mutants heterozygous for the E74ABG01805 (these flies have a lifespan that is the same as miR-34 mutants alone; see Supplemental Table 4). Flies raised at 29°C. Lifespan: p<0.0001 (log-rank test). Mean ± s.e., n>=150 male flies. Brain vacuoles: *=p < 0.01 (one-way analysis of variance, with post test: Tukey's multiple comparison test). Mean ± s.e.m.,, n=10 independent male animals. Genotypes as in d.
g. Upregulation of miR-34 reduces accumulation of pathogenic polyQ protein inclusions. Left panels, in the retina of flies expressing SCA3trQ78 alone, pathogenic polyQ protein is initially diffuse (1d, top), but gradually accumulates into nuclear inclusions (3d, bottom). Right panels, upregulation of miR-34 reduces inclusion formation. DAPI staining highlights nuclei. 3d controls show 53.75 ± 12.55 inclusions in a retinal section, vs 23.67 ± 7.57 with miR-34 upregulation; mean ± s.d., n≥3 cryosections from independent male animals; p<0.01 (t-test). Genotypes: SCA3trQ78 is w+; rh1-gal4, UAS-SCA3trQ78/+. SCA3trQ78; miR34 (+) is w+; rh1-gal4, SCA3trQ78/+; UAS-miR-34/+. Scale bar: 0.05mm.
h. Upregulation of miR-34 prevents neural degeneration. At 21d, male flies expressing SCA3trQ78 show a dramatic loss of photoreceptor neuronal integrity (middle panel), with an average of only 2.46 ±1.32 photoreceptors per ommatidium remaining by pseudopupil analysis. Flies with upregulated miR-34 (right panel) retain 6.90 ± 0.34 photoreceptors per ommatidium. Control (left panel) and upregulation of miR-34 alone (not shown) have normal photoreceptor numbers per ommatidium. Mean ± s.d., n= 619, 722 and 700 ommatidia, for SCA3trQ78, SCA3trQ78; miR-34 (+) and control, respectively; ***=p< 0.0001 (one-way analysis of variance, with Bonferroni’s multiple comparison test). Genotypes as in b; control: w+; rh1-gal4/+. Scale bar: 0.05mm.
i. Flies with upregulated miR-34 (color) have an extended median lifespan compared to control flies (black and grey curves for repeats 1 and 2, respectively) (log-rank test). Lifespan result for each genotype is indicated in median and maximal days. Mean ± s.e., n>=150 male flies per genotype, 25°C. Three independent miR-34 genomic transgenic lines (4, 8, 9) were analyzed. Genotypes: control: 5905, miR-34 (+): miR-34 genomic rescue in 5905 homogenous genetic background.