Drosophila ref(2)P is required for the parkin-mediated suppression of mitochondrial dysfunction in pink1 mutants

Abstract

Autophagy is a critical regulator of organellar homeostasis, particularly of mitochondria. Upon the loss of membrane potential, dysfunctional mitochondria are selectively removed by autophagy through recruitment of the E3 ligase Parkin by the PTEN-induced kinase 1 (PINK1) and subsequent ubiquitination of mitochondrial membrane proteins. Mammalian sequestrome-1 (p62/SQSTM1) is an autophagy adaptor, which has been proposed to shuttle ubiquitinated cargo for autophagic degradation downstream of Parkin. Here, we show that loss of ref(2)P, the Drosophila orthologue of mammalian P62, results in abnormalities, including mitochondrial defects and an accumulation of mitochondrial DNA with heteroplasmic mutations, correlated with locomotor defects. Furthermore, we show that expression of Ref(2)P is able to ameliorate the defects caused by loss of Pink1 and that this depends on the presence of functional Parkin. Finally, we show that both the PB1 and UBA domains of Ref(2)P are crucial for mitochondrial clustering. We conclude that Ref(2)P is a crucial downstream effector of a pathway involving Pink1 and Parkin and is responsible for the maintenance of a viable pool of cellular mitochondria by promoting their aggregation and autophagic clearance.

Figure 1

Characterisation of ref(2)P-mutant flies. (a) Structural domain organisation of mammalian p62, Drosophila Ref(2)P, and Ref(2)P^od2 and Ref(2)P^od3 mutants. p62 has a PB1 domain, a ZZ-type zinc-finger domain, a TRAF6-binding (TB) domain, an LC3-interacting region (LIR) and a UBA. Ref(2)P has in common with the mammalian p62, the PB1 domain, the ZZ-type finger domain and the UBA domain. Ref(2)P^od2 mutant protein lacks the PB1 domain, and Ref(2)P^od3 mutant protein lacks the UBA domain. (b–e) Schematic and phase contrast micrographs of mitochondrial morphogenesis in spermatids during the ‘onion stage'. Mutations in ref(2)P lead to morphological defects in the mitochondrial derivative (Nebenkern) of the onion-stage early spermatids. ref(2)P^od2 mutants present an atypical localisation of the nebenkern, surrounding the white nuclei (c). ref(2)P^od3 mutants exhibit an abnormally pale mitochondrial derivative (d). Scale bar, 50 μm (f–k) TEM images of single post-individualisation cysts containing 64 spermatids. (f and i) In the control (w¹¹¹⁸), each spermatid contains an axoneme (arrowhead) and mitochondrial derivative (arrow) within an individual plasma membrane. Both ref(2)P mutant cyst show individualisation defects. (g and j) In ref(2)P^od2 mutants, the mitochondrial derivative appears bigger and contains a large mass of electron-dense material (arrow); (h and k) the ref(2)P^od3 mutant cysts have an overall disorganised architecture, with a rather normal axoneme (arrowhead) but a large vacuolised mitochondrial derivative (arrow). Scale bar, 2 μm (f–h), 500 nm (i–k)

Figure 2

Mutations in ref(2)P result in the accumulation of mtDNA. (a) Regular mitochondrial distribution is disrupted in ref(2)P mutants and the number of mtDNA nucleoids (mt) is increased. PicoGreen also labels the dsDNA of muscle nuclei (nuc). (b) ref(2)P mutants flies showed an increase in mtDNA. The ratio of mtDNA to nDNA was measured using real-time quantitative PCR in flies with the indicated ages (mean±S.D., n=9 per genotype). Statistically significant values relative to the control (w¹¹¹⁸) are indicated by asterisks (one-way ANOVA with Dunnett's multiple comparison test). (c and d) Mutations in ref(2)P do not cause alterations in mitochondrial protein density. Protein density was determined in 26-day-old flies with the indicated genotypes by determining the concentration of Cyt c using an ELISA assay (d) and by measuring the activity of the mitochondrial matrix enzyme citrate synthase (c). Data are shown as the mean ±S.E.M (n=3 in each group). Statistical significance relative to the control (w¹¹¹⁸) is indicated (one-way ANOVA with Dunnett's multiple comparison test). (e) Analysis of the mtTFA levels in ref(2)P mutants. Lysates prepared from adult flies were subjected to western blot analysis with the indicated antibodies. (f) Normal respiration in ref(2)P mutant flies. Activity of the indicated complexes in coupled mitochondria was measured in 3-day-old flies using high-resolution respirometry. Data are shown as the mean±S.E.M. (n≥4 in each genotype). Statistical significance relative to the control (w¹¹¹⁸) is indicated (one-way ANOVA with Dunnett's multiple comparison test)

Figure 3

Detection of heteroplasmic mtDNA variants in Drosophila ref(2)P mutants. (a) Next-Generation Sequencing analysis of the Drosophila mitochondrial genome. Horizontal scale corresponds to the position of individual features in the Drosophila mitochondria genome. Vertical scale indicates the average read depth obtained for each individual base. Genes and features shown in red indicate areas where heteroplasmy was detected in individual genomes. The circle indicates the origin of replication for the mitochondrial genome (mt:ori) and is located to the left of the A+T-rich region of the mitochondrial genome. (b) Heteroplasmic variants in the mtDNA of adult flies. Frequencies of 1.6% or more of the variant allele when compared with the reference allele are shown

Figure 4

ref(2)P mutant flies have a decreased lifespan and motor impairment. (a–c) Fly viability was scored over the indicated periods, using a minimum of 80 flies per genotype. Statistical significance is indicated (log-rank, Mantel–Cox test). (d) Mutations in ref(2)P result in significant motor impairment. Flies with the indicated genotypes and ages were tested using a standard climbing assay (mean ±S.D., n≥100 flies for each genotype)). Statistical significance relative to the control (w¹¹¹⁸) is indicated (two-way ANOVA with Dunnett's multiple comparison test)

Figure 5

ref(2)P gain-of-function rescues pink1 mutant flies. (a) Analysis of Ref(2)P levels. Whole-fly lysates were analysed using western blotting with the indicated antibodies. (b) Reduced fertility in ref(2)P^od3 mutants is rescued by expression of ref(2)P. Single male of the indicated genotypes were mated with w¹¹¹⁸ virgin females for 24 h before the males were removed. Total number of vials with progenies per genotype were scored (n). (c) Thoracic defects of pink1 mutants. SEM micrographs of thoraces from flies with the indicated genotypes show that the collapsed-thorax phenotype of pink1 mutants. A control thorax (w¹¹¹⁸) is also shown. (d) Expression of ref(2)P rescued the thoracic indentations of pink1 mutants (n≥60 flies per genotype). Asterisk indicates statistical significance relative to pink1^B9, da>Ref(2)P (χ²). (e–g) ref(2)P expression rescues the mitochondrial abnormalities observed in the indirect flight muscles of the pink1 null flies. Tissues were analysed using TEM. Scale bar, 2 μm. (h) Expression of ref(2)P rescued the climbing defects of pink1 flies. Flies were tested using a standard climbing assay (mean ±S.D., n≥60 flies per genotype). Asterisks indicate statistically significant values relative to the control (daGal4/+) (one-way ANOVA with Dunnett's multiple comparison test). (i) Expression of ref(2)P restored mitochondrial Complex I and V protein levels in pink1 mutant flies. Whole-fly protein lysates were subjected to western blot analysis with the indicated antibodies. (j) Neuronal expression of ref(2)P rescued the loss of dopaminergic neurons in the PPL1 cluster of pink1 mutant flies (mean ±S.D., n≥16 flies per genotype). The asterisks indicate statistically significant values (one-way ANOVA with Bonferroni's multiple comparison test) relative to pink1B9. (k) Neuronal expression of ref(2)P rescued the thoracic defects of pink1 mutants (n≥76 flies per genotype). Asterisk(s) indicate statistical significance (Fisher's exact test)

Figure 6

Drosophila ref(2)P is required for mitochondrial aggregation and parkin-mediated suppression of the pink1 mutant phenotype. (a–d) The Ref(2)P PB1 and UBA domains are essential for mitochondrial aggregation in Drosophila. Mitochondria were visualised by confocal analysis of the indirect flight muscles in a mitochondria-targeted protein (mitoGFP) transgenic animals with the da-GAL4 driver. Arrows point to aggregated mitochondria. Purple: nuclei, green: mitoGFP. Scale bar, 20 μm. (e) ref(2)P mutations blocked the Parkin-mediated suppression of the thoracic indentations in pink1 flies. Thoracic indentations were scored in flies of each indicated genotype (n≥60 flies per genotype). Asterisks indicate the statistical significance relative to pink1^B9, da>Parkin (χ²). (f) ref(2)P mutations blocked the Parkin-mediated suppression of mitochondrial loss in pink1 mutant flies. Whole-fly lysates were analysed using western blotting with the indicated antibodies

Figure 7

Autophagy is required for parkin- and ref(2)P-mediated suppression of the pink1 mitochondrial phenotype. Autophagy is required for parkin-mediated (a) suppression of thoracic indentations (n≥60 flies per genotype), asterisk indicates statistical significance relative to pink1B9, da>Parkin (χ²); (b) recovery of mitochondrial loss, flies were analysed using western blotting using the indicated antibodies; and (c) rescuing of the motor impairment in pink1 mutants, flies with the indicated genotypes were tested using a standard climbing assay (mean ±S.D., n≥60 flies for each genotype), asterisks indicate statistically significant values relative to the control (daGal4/+) (one-way ANOVA with Dunnett's multiple comparison test). Autophagy is also essential for ref(2)P-mediated (d) suppression of thoracic indentations in pink1 flies, asterisk indicates statistical significance relative to pink1B9, da>Ref(2)P (χ²); and (e) climbing defects observed in pink1 mutants. Flies were tested using a standard climbing assay (mean±S.D., n≥60 flies per genotype); asterisks indicate statistically significant values relative to pink1B9 (one-way ANOVA with Dunnett's multiple comparison test). Contrary to the pink1 mutants, ref(2)P failed to suppress the thoracic indentations in parkin flies (d, green)