A Personalized Model of COQ2 Nephropathy Rescued by the Wild-Type COQ2 Allele or Dietary Coenzyme Q₁₀ Supplementation

Coq2 gene silencing induced abnormal slit diaphragm localization and collapsed lacunar channels with multiple slit diaphragms (A) TEM showing normal (control) nephrocyte ultrastructure with slit diaphragms and lacunar channels uniformly spaced along the circumference of the cell. Slit diaphragms localized exclusively at the mouth of the channel. In Coq2-IR nephrocytes, channels appeared narrower (collapsed) and interchannel spacing was interrupted and irregular. Slit diaphragms occurred not only at the channel mouth but also ectopically along the interior channel membranes. Scale bar, 200 nm. (B) Higher magnification TEM comparing normal control and Coq2-IR slit diaphragm and lacunar channel ultrastructure. Ectopic slit diaphragms arranged in ladder-like configuration at points of channel narrowing are indicated by arrows. Scale bar, 100 nm. (C) Quantitation of normally localized slit diaphragms in control versus Coq2-IR nephrocytes. Average number of slit diaphragms positioned at mouths of lacunar channels per 2000 nm of cell circumference (*P<0.05). (D) The average distance (in nm) between normally localized slit diaphragms in control versus Coq2-IR nephrocytes (*P<0.05). (E) Quantitation of ectopic slit diaphragms in control versus Coq2-IR nephrocytes. Average number of slit diaphragms positioned along interior channel membranes per 2000 nm of cell circumference (*P<0.05). (F) Quantitation of slit diaphragms (normally localized plus ectopic) in control versus Coq2-IR nephrocytes. Total number of slit diaphragms per 2000 nm of cell circumference (*P<0.05).

Coq2 gene silencing induced elevated numbers of autophagosomes and abnormal mitochondria in nephrocytes. (A) Upper panels: TEM showing normal mitochondria (m) in control nephrocyte of a wild-type fly. In Coq2-IR nephrocytes the mitochondria are more abundant and morphologically abnormal. Autophagosomes (ap) are in evidence. Scale bar, 300 nm. Lower panels: higher magnification electron micrographs comparing normal control and Coq2-IR nephrocyte mitochondria. In nephrocytes in which Coq2 expression was silenced the mitochondria exhibited shortened cristae and fewer inner mitochondrial membranes. Scale bar, 200 nm. (B) Quantitation of mitochondria in control versus Coq2-IR nephrocytes. Average number of mitochondria per 4 μm² area of cytoplasm (*P<0.05). (C) Percentage of mitochondria exhibiting abnormal morphology in control versus Coq2-IR nephrocytes (*P<0.05). (D) TEM showing mitochondria and autophagosomes in control nephrocyte of a wild-type fly. In Coq-IR nephrocytes, the number of autophagosomes was increased. Scale bar, 300 nm. (E) Quantitation of autophagosomes in control versus Coq2-IR nephrocytes. Average number of autophagosome per 4 μm² area of cytoplasm (*P<0.05).

Coq2 gene silencing induced autophagy and mitophagy. (A) Fluorescence micrographs showing nephrocytes of adult female flies 1-day postemergence, expressing a dual-labeled GFP-Atg8-mCherry autophagy marker protein specifically in nephrocytes (UAS-GFP-Atg8-mCherry construct driven by Dot-Gal4). Dotted lines indicate nephrocyte cell boundary. Atg8 is present in autophagosomes and subsequently accumulates in autolysosomes. Fluorescence from the compartmentalized marker protein is punctate in appearance. Both GFP and mCherry fluorescence can be detected in autophagosomes. In the acidic lysosome lumen, GFP fluorescence undergoes rapid quenching whereas mCherry retains fluorescence activity. Therefore only very early autolysosomes will transiently exhibit GFP fluorescence. Because Atg8 accumulates to high levels in autolysosomes, mCherry fluorescence is relatively stronger in this compartment. Upper panels show control nephrocytes (normal Coq2 gene expression) in which autophagy is essentially undetectable. Lower panels show nephrocytes in which Coq2 gene expression was silenced (Coq2-IR) in which a few autophagosomes were present (GFP, green; GFP-Atg8-mCherry, yellow) and autolysosomes were highly abundant (mCherry, red). (B) Fluorescence micrographs showing nephrocytes of adult female flies 1-day postemergence, expressing GFP-labeled mitochondria and Atg8-RFP marker protein in nephrocytes. Dotted line indicates nephrocyte cell boundary. Upper panels show control nephrocytes (normal Coq2 gene expression) with labeled mitochondria (Mito-GFP, green), essentially undetectable autophagy (Atg8-RFP, red), and no overlapping yellow fluorescence in merged images (Mito-GFP Atg8-RFP). Lower panels show nephrocytes in which Coq2 was silenced (Coq2-IR) with increased mitochondrial fluorescence (higher Mito-GFP fluorescence, green), fluorescence because of Atg8 in autophagosomes/autolysosomes (Atg8-RFP, red), and overlap of fluorescence indicating mitophagy (Mito-GFP Atg8-RFP, yellow). (C) Quantitative comparison of Mito-GFP fluorescence in control versus Coq2-IR nephrocytes, expressed relative to control. The increased fluorescence due to Coq2 silencing is shown relative to control. For each group, 30 nephrocytes (six nephrocytes from each of five flies) were examined (*P<0.05). (D) Quantitative comparison of Atg8-RFP fluorescence in control versus Coq2-IR nephrocytes, expressed relative to Control. For each group, 30 nephrocytes (six nephrocytes from each of five flies) were examined (*P<0.05). (E) Quantitative spatial comparison of Mito-GFP (green), Atg8-RFP (red), and Mito-GFP Atg8-RFP (yellow) fluorescence in control versus Coq2-IR nephrocytes.

Simultaneous silencing of Coq2 and autophagy genes induces synergistic effects. (A) Fluorescence micrographs showing nephrocytes of 1-day postemergence adult flies transgenic for Hand-GFP (GFP expressed under the control of a Hand gene enhancer to confirm pericardial nephrocyte cell identity).²³ ANF-RFP (red) is merged with GFP (green, predominantly in nucleus). Control flies carry the Dot-Gal4 driver but no RNAi construct. Gene-IR flies carry Dot-Gal4 driving RNAi transgenes silencing endogenous Coq2, Atg1, or Atg6 genes singly or in combination. (B) Quantitation of nephrocyte RFP levels, relative to control. For each genotype, 30 nephrocytes (six nephrocytes from each of five flies) were examined (* indicates significance compared with control, ^# indicates significance compared with Coq2-IR; P<0.05). (C) Oxidized DHE fluorescence (red) in nephrocytes of third instar larvae. Dashed lines indicate cell nuclei (determined from DAPI staining, not shown). (D) Quantitation of DHE fluorescence intensity expressed relative to control nephrocytes. For each genotype, 30 nephrocytes (six nephrocytes from each of five larvae) were examined (* indicates significance compared with control, ^# indicates significance compared with Coq2-IR; P<0.05).

CoQ₁₀ administration rescued nephrocyte functional and ultrastructural defects induced by Coq2 gene silencing. (A) Fluorescence micrographs showing nephrocytes of 1-day postemergence adult flies. Flies were reared from embryos on standard food supplemented with the indicated concentrations (1% or 5%) of CoQ₁₀ (Q₁₀). Left panels (MHC-ANF-RFP) show intracellular ANF-RFP fluorescence (red). Right panels (MHC-ANF-RFP Hand-GFP) show RFP (red) merged with GFP (green, mostly nuclear). A GFP transgene is expressed under the control of a Hand gene enhancer (Hand-GFP) to confirm pericardial nephrocyte cell identity.²³ All flies are transgenic for Hand-GFP. Control flies carry the Dot-Gal4 driver but no RNAi construct. Coq2 flies carry Dot-Gal4 driving RNAi transgene silencing Coq2 expression. (B) Quantitation of RFP levels in control versus Coq2-IR nephrocytes (expressed relative to control) with no Q₁₀, 1% Q₁₀, or 5% Q₁₀ dietary supplementation. For each genotype, 30 nephrocytes (six nephrocytes from each of five flies) were examined (*P<0.05). (C) TEM showing mislocalized slit diaphragms and irregularly spaced and collapsed lacunar channel ultrastructure induced by Coq2 silencing (upper panel) rescued by administration of 5% Q₁₀ (lower panel). Scale bar, 300 nm. (D) Quantitation of ectopic slit diaphragms in Coq2-IR versus Coq2-IR plus 5% Q₁₀ supplementation nephrocytes. Average number of slit diaphragms positioned along interior channel membranes per 2000 nm length of cell circumference (*P<0.05). (E) The average distance (in nm) between normally localized slit diaphragms in Coq2-IR versus Coq2-IR plus 5% Q₁₀ supplementation nephrocytes (*P<0.05). (F) ROS levels in normal (control) and Coq2-silenced (Coq2) nephrocytes were indicated by oxidized DHE red fluorescence in the cell nucleus. ROS levels were higher in Coq2-IR nephrocytes, and feeding Coq2-IR larvae a diet supplemented with 5% Q₁₀ reduced nephrocyte ROS levels. Dashed lines indicate cell nuclei (determined from DAPI staining, not shown). (G) Levels of DHE red nuclear fluorescence expressed relative to normal control larval nephrocytes fed a nonsupplemented diet. Coq2 gene silencing led to a 2.5–3-fold increase in ROS levels. Feeding Coq2-IR 5% Q₁₀ lowered ROS to normal levels. In each case, 30 nephrocytes (six nephrocytes from each of five larvae) were examined (*P<0.05).