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Research Article
Miro, a Rho GTPase genetically interacts with Alzheimer's disease-associated genes (Tau, Aβ42 and Appl) in Drosophila melanogaster
Komal Panchal, Anand Krishna Tiwari
Biology Open 2020 9: bio049569 doi: 10.1242/bio.049569 Published 3 September 2020

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  • Fig. 1.
    Fig. 1.

    AD related pathologies in Drosophila. (A–E) Light microscopic and (A′–E′,a–e) SEM images of eyes of 10-day-old adult flies from OregonR+ (wild-type control) (A,A′,a), GMR- GAL4/+ (experimental control) (B,B′,b), GMR-GAL4-UAS-TAUWT/+ (C,C′,c), GMR-GAL4-UAS-Aβ42(Human)/+ (D,D′,d) and GMR-GAL4/+; UAS-ApplRNAi/+ (E,E′,e). (a–e) are magnified images of SEM. Scale bar: 100 μm (A–E,A′–E′) and 20 μm (a–e). The yellow marked area shows degenerated part of eyes (C–E,C′–E′). n=50. (F) Phototaxis activity of 10-day-old control (OregonR+, GMR-GAL4/+) and AD model flies GMR-GAL4-UAS-TAUWT/+, GMR-GAL4-UAS-Aβ42(Human)/+ and GMR-GAL4/+; UAS-ApplRNAi/+. Phototaxis activity presented as a light preference index. n=100. In the box and whisker plot, the box outlines show the upper and lower quartiles. (G) Histogram showing climbing activity [expressed as % climbing (8 cm 10 s−1)] of 10-, 20- and 30-day-old adult flies of elav-Gal4C155/+;+/+;+/+ and elav-Gal4C155/+;+/+;UAS-Aβ42E693G/+. n=100. (H) Survival assay of elav-Gal4C155/+;+/+;+/+ (yellow line), elav-Gal4C155/+;+/+;UAS-Aβ42E693G/+ (blue line) and elav-Gal4C155/+;+/+;UAS-APP.C99-UAS-MAPT/+ (pink line). n=100. The Kaplan–Meier survival test was performed and significance was determined by Montel-Cox log-rank test. A table indicating data comparison between control flies versus AD model flies with median lifespan (days), Chi-Square test (χ2) and P-value (Montel-Cox log-rank test). Data comparison: life span of AD model flies such as elav-Gal4C155/+;UAS-Miro/+;UAS-Aβ42E693G/+ (P<0.0001) and elav-Gal4C155/+;UAS-Miro/+;UAS-APP.C99-UAS-MAPT/+ compared with control flies (elav-Gal4C155/+;+/+;+/+) (P<0.0001). (I) Body weight analysis of 10-, 20- and 30-day-old flies of elav-Gal4C155/+;+/+;+/+, elav- Gal4C155/+;+/+;UAS-Aβ42E693G/+ and elav-Gal4C155/+;+/+;UAS-APP.C99-UAS-MAPT/+. n=100. Error bars represent mean±s.e.m. Data significance was calculated by one-way ANOVA analysis with Tukey's test using GraphPad Prism 5.0 and is indicated as ***P<0.0001.

  • Fig. 2.
    Fig. 2.

    Miro modulates the AD-related pathologies in Drosophila. (A–D) SEM images of eyes of 10-day-old Miro overexpressing adult flies from GMR-GAL4/UAS-Miro (A), GMR-GAL4-UAS-TAUWT/UAS-Miro (B), GMR-GAL4-UAS-Aβ42(Human)/UAS-Miro (C) and GMR-GAL4/UAS-Miro; UAS-ApplRNAi/+ (D). Overexpression of Miro decreases the rough eye phenotype associated with AD model flies. (E–H) SEM images of eyes of 10-day-old Miro knockdown flies from GMR-GAL4/+;UAS-MiroRNAi/+ (E), GMR-GAL4-UAS-TauWT/+;UAS-MiroRNAi/+ (F), GMR-GAL4-UAS-Aβ42(Human)/+; UAS-MiroRNAi/+ (G) and GMR-GAL4/+; UAS-ApplRNAi/UAS-MiroRNAi (H). The knockdown of Miro enhances the rough eye phenotype associated with AD model flies. (A′–H′) are the magnified eye images of (A–H). Scale bar: 100 μm (A–H) and 20 μm (a–e). The yellow marked area showing the degenerated part of the eye. n=50. (I) Histogram showing the eye size (length and width) of GMR-GAL4/+, GMR-GAL4-UAS-TAUWT/+, GMR-GAL4-UAS-TAUWT/UAS-Miro and GMR-GAL4-UAS-TAUWT/+; UAS-MiroRNAi/+ flies. n=10. (J) Box and whisker plot showing phototaxis activity (expressed as light preference index) of 10-day-old flies of GMR-GAL4/+, GMR-GAL4/UAS-Miro, GMR-GAL4/+; UAS-MiroRNAi/+, GMR-GAL4-UAS-TauWT/+, GMR-GAL4-UAS-TauWT/UAS-Miro, GMR-GAL4-UAS-TauWT/+;UAS-MiroRNAi/+, GMR-GAL4-UAS-Aβ42(Human)/+, GMR-GAL4-UAS-Aβ42(Human)/UAS-Miro and GMR-GAL4-UAS-Aβ42(Human)/+; UAS-MiroRNAi/+. n=100. (K) Climbing activity (expressed as % climbing in 8 cm 10 s−1) of 10-, 20- and 30-day-old flies of elav-Gal4C155/+;+/+;+/+, elav-Gal4C155/+;UAS-Miro/+;+/+, elav-Gal4C155/+;+/+;UAS-Aβ42E693G/+ and elav-Gal4C155/+;UAS-Miro/+;UAS-Aβ42E693G/+. n=100. (L) Body weight analysis of 10-, 20- and 30-day-old flies of elav-Gal4C155/+;+/+;+/+, elav-Gal4C155/+;UAS-Miro/+;+/+, elav-Gal4C155/+;+/+;UAS-Aβ42E693G/+, elav-Gal4C155/+;UAS-Miro/+;UAS-Aβ42E693G/+, elav-Gal4C155/+;+/+;UAS-APP.C99.UAS-MAPT/+ and elav-Gal4C155/+;UAS-Miro/+;UAS-APP.C99.UAS-MAPT/+. n=100. Error bars represent mean±s.e.m. Data significance was calculated by one-way ANOVA analysis with Tukey's test using GraphPad Prism 5.0 and is indicated as: ns, non-significant; *P<0.05, **P<0.01, ***P<0.0001.

  • Fig. 3.
    Fig. 3.

    Lifespan analysis of Miro overexpressing AD model flies. Survival curve of elav-Gal4C155/+;+/+;+/+ (yellow line), elav-Gal4C155/+;UAS-Miro/+;+/+ (maroon line), elav-Gal4C155/+;+/+;UAS-Aβ42E693G/+ (blue line), elav-Gal4C155/+;+/+;UAS-APP.C99-UAS-MAPT/+ (pink line), elav-Gal4C155/+;UAS-Miro/+; UAS-Aβ42E693G/+ (green line) and elav-Gal4C155/+;UAS-Miro/+;UAS-APP.C99-UAS-MAPT/+ (orange line). n=100. The Kaplan–Meier survival test was performed and significance was determined by the Montel-Cox log-rank test using GraphPad Prism 5.0 software. A table indicating data comparison between all genotypes with median lifespan (days), Chi-Square test (χ2) and P-value (Montel-Cox log-rank test). Data comparison: lifespan of elav-Gal4C155/+;UAS-Miro/+;+/+ compared with elav-Gal4C155/+;+/+;+/+ (P<0.0001) and lifespan of Miro overexpressing AD model flies elav-Gal4C155/+;UAS-Miro/+;UASAβ42E693G/+ (P<0.0001) and elav-Gal4C155/+;UAS-Miro/+;UAS-APP.C99-UAS-MAPT/+ (P<0.0001) compared with AD model flies such as elav-Gal4C155/+; UAS-Miro/+;UAS-Aβ42E693G/+ and elav-Gal4C155/+;UAS-Miro/+;UAS-APP.C99-UAS-MAPT/+, respectively.

  • Fig. 4.
    Fig. 4.

    AO staining in third instar larval eye discs of Miro overexpressing/knockdown AD model flies and quantitative real time PCR analysis of the Miro gene. (A–I) Confocal images of AO stained third instar larval eye discs of GMR-GAL4/+ (A), GMR-GAL4/UAS-Miro (B), GMR-GAL4/+; UAS-MiroRNAi (C), GMR-GAL4-UAS-TAUWT/+ (D) GMR-GAL4-UAS-TAUWT/UAS-Miro (E), GMR-GAL4-UAS-TauWT/+;UAS-MiroRNAi/+ (F), GMR-GAL4-UAS- Aβ42(Human)/+ (G), GMR-GAL4-UAS-Aβ42(Human)/UAS-Miro (H), GMR-GAL4-UAS-Aβ42(Human)/+; UAS-MiroRNAi/+ (I). AO positive cells (dead cells) [posterior to the morphogenetic furrow (MF)] were indicated by white arrowheads. n=20. Scale bars: 100 µm (A–I). (J) Box and whisker plot showing average AO positive cells in third instar larval eye imaginal discs of each genotype. (K) The histogram showing quantitative real time PCR of Miro gene in 30-day-old adult flies heads of GMR-GAL4/+, GMR-GAL4-UAS-TauWT/+, GMR-GAL4-UAS-Aβ42(Human)/+ and GMR-GAL4/+;UAS-ApplRNAi/+ flies. RP49 was used as an endogenous control. The quantification of AO positive cells was done by using ImageJ software, NIH, USA. Error bar represents mean±s.e.m. Data significance was calculated by one-way ANOVA analysis with Tukey's test using GraphPad Prism 5.0 and is indicated as: ns, non-significant; *P<0.05, **P<0.01, ***P<0.0001.

  • Fig. 5.
    Fig. 5.

    Mitochondrial and cellular ROS level in Miro overexpressing AD model flies. Confocal images of third instar larval brains showing MitoSOX Red staining (A,E,I,M,Q,U), MitoTracker Green (B,F,J,N,R,V), nuclear staining with Hoechst 33342 (C,G,K,O,S,W) in elav-Gal4C155/+;+/+;+/+, elav-Gal4C155/+;UAS-Miro/+;+/+, elav-Gal4C155/+;+/+;UAS-Aβ42E693G/+, elav-Gal4C155/+;UAS-Miro/+;UAS-Aβ42E693G/+, elav-Gal4C155/+;+/+;UAS-APP.C99-UAS-MAPT/+ and elav-Gal4C155/+;UAS-Miro/+;UAS-APP.C99-UAS-MAPT/+, respectively. n=20 D, H, L, P, T, X are the merge images of A–C, E–G, I–K, M–O, Q–S and U–W, respectively. Colocalization in the merged images is shown by arrows. n=20. Scale bars: 10 µm (A–X). (a) Histogram showing average MitoSOX Red fluorescence intensity in third instar larval brains of each genotype. (b) Histogram showing cellular ROS level in 30-day-old adult flies heads of elav-Gal4C155/+;+/+;+/+, elav-Gal4C155/+;UAS-Miro/+;+/+; elav-Gal4C155/+;+/+;UAS-Aβ42E693G/+,elav-Gal4C155/+;UAS-Miro/+;UAS-Aβ42E693G/+, elav-Gal4C155/+;+/+;UAS-APP.C99-UAS-MAPT/+ and elav-Gal4C155/+;UAS-Miro/+;UAS-APP.C99-UAS-MAPT/+ flies by using DCF dye at Ex./Em. 482/527 nm and normalized to the amount of protein (µg). (c) Histogram showing the relative expression of Mn-SOD and CAT genes determined by quantitative real-time PCR in 30-day-old adult flies heads ofelav-Gal4C155/+;+/+;+/+, elav-Gal4C155/+;UAS-Miro/+;+/+; elav-Gal4C155/+;+/+;UAS-Aβ42E693G/+,elav-Gal4C155/+;UAS-Miro/+;UAS-Aβ42E693G/+, elav-Gal4C155/+;+/+;UAS-APP.C99-UAS-MAPT/+ and elav-Gal4C155/+;UAS-Miro/+;UAS-APP.C99-UAS-MAPT/+ flies. MitoSOX Red fluorescence intensity was measured by using ImageJ software (NIH, USA). Error bar represents mean±s.e.m. Data significance was calculated by one-way ANOVA analysis with Tukey's test using GraphPad Prism 5.0 and is indicated as ns: non-significant, *P<0.05, **P<0.01, ***P<0.0001.

  • Fig. 6.
    Fig. 6.

    Mitochondrial average length measurement. (A–F) Confocal microscopy images showing mitochondrial length (indicated by white arrows) in third instar larval eye discs of GMR-GAL4-MitoGFP/+ (A), GMR-GAL4-MitoGFP/UAS-Miro (B), GMR-GAL4-UAS-Mito-GFP/+; UAS-Aβ42E693G/+ (C), GMR-GAL4-UAS-Mito-GFP/UAS-Miro; UAS-Aβ42E693G/+ (D), GMR-GAL4-UAS-Mito-GFP/+;UAS-APP.C99-UAS-MAPT/+ (E) and GMR-GAL4-UAS-Mito-GFP/UAS-Miro;UAS-APP.C99-UAS-MAPT/+ (F). Scale bar: 2 µm (A–F), n=20. (G) Box whisker plot shows mitochondrial average length in third instar larval eye discs of each genotype. (H) Histogram showing the relative expression of Mitofusin (Mfn) gene determined by quantitative real-time PCR in 30-day-old adult flies heads of elav-Gal4C155/+;+/+;+/+, elav-Gal4C155/+;UAS-Miro/+;+/+; elav-Gal4C155/+;+/+;UAS-Aβ42E693G/+,elav-Gal4C155/+;UAS-Miro/+;UAS-Aβ42E693G/+, elav-Gal4C155/+;+/+;UAS-APP.C99-UAS-MAPT/+ and elav-Gal4C155/+;UAS-Miro/+;UAS-APP.C99-UAS-MAPT/+ flies. RP49 was used as an endogenous control. Error bar represents mean±s.e.m. Data significance was calculated by one-way ANOVA analysis with Tukey's test using GraphPad Prism 5.0 software and is indicated as ns: non-significant, *P<0.05, **P<0.01, ***P<0.0001.

  • Fig. 7.
    Fig. 7.

    ATP quantification of Miro overexpressing AD model flies. (A) Histogram showing ATP concentration (µM µg−1 of protein) of 30-day-old adult flies heads of elav-Gal4C155/+;+/+;+/+, elav-Gal4C155/+;UAS-Miro/+;+/+, elav-Gal4C155/+;+/+;UAS-Aβ42E693G/+, elav-Gal4C155/+;UAS-Miro/+;UAS-Aβ42E693G/+, elav Gal4C155/+;+/+;UAS-APP.C99-UAS-MAPT/+ and elav-Gal4C155/+;UAS-Miro/+;UAS-APP.C99-UAS-MAPT/+. (B) Histogram showing the relative expression of the ATP Synthase beta gene determined by quantitative real-time PCR in 30-day-old adult flies heads ofelav-Gal4C155/+;+/+;+/+, elav-Gal4C155/+;UAS-Miro/+;+/+; elav-Gal4C155/+;+/+;UAS-Aβ42E693G/+,elav-Gal4C155/+;UAS-Miro/+;UAS-Aβ42E693G/+, elav-Gal4C155/+;+/+;UAS-APP.C99-UAS-MAPT/+ and elav-Gal4C155/+;UAS-Miro/+;UAS-APP.C99-UAS-MAPT/+ flies. RP49 used as an endogenous control. Error bar represents mean±s.e.m. Data significance was calculated by one-way ANOVA analysis with Tukey's test using GraphPad Prism 5.0 software and is indicated as: ns, non-significant; *P<0.05, **P<0.01, ***P<0.0001.

  • Fig. 8.
    Fig. 8.

    Cell death and neurodegeneration analysis of Miro overexpressing AD model flies. (A–F) Confocal images showing AO staining in third instar larval brains of elav-Gal4C155/+;+/+;+/+ (A), elav-Gal4C155/+;UAS-Mio/+;+/+ (B), elav-Gal4C155/+;+/+;UAS-Aβ42E693G/+ (C), elav-Gal4C155/+;UAS-Miro/+;UAS-Aβ42E693G/+ (D), elav-Gal4C155/+;+/+;UAS-APP.C99-UAS-MAPT/+ (E), elav-Gal4C155/+;UAS-Miro/+;UAS-APP.C99-UAS-MAPT/+ (F) flies. White arrowheads indicate AO positive cells in optic lobes (OL) of third instar larval brains. Scale bar: 10 µm (A–F), n=20. (G) Box and whisker plot showing the number of AO positive cells in third instar larval brains of each genotype. (A′–F′) Confocal images showing anti-cleaved-caspase-3 staining in third instar larval brain of elav-Gal4C155/+;+/+;+/+ (A′), elav-Gal4C155/+;UAS-Mio/+;+/+ (B′), elav-Gal4C155/+;+/+;UAS-Aβ42E693G/+ (C′), elav-Gal4C155/+;UAS-Miro/+;UAS-Aβ42E693G/+ (D′), elav-Gal4C155/+;+/+;UAS-APP.C99-UAS-MAPT/+ (E′), elav-Gal4C155/+;UAS-Miro/+;UAS-APP.C99-UAS-MAPT/+ (F′) flies. Scale bar: 40 µm (A′–F′). (a′–f′) Magnified confocal images of (A′–F′). Scale bar: 20 µm (a′–f′). n=20. White arrowheads indicate caspase positive cells (A′–F′, a′–f′). (G′) Histogram showing average fluorescence intensity of cleaved caspase-3 in third instar larval brains of each genotype. Cleaved caspase-3 fluorescence intensity was measured by using ImageJ software, NIH, USA. (A″–F″) Gray scale images of H&E stained paraffin sections of 30-day-old flies brains of elav-Gal4C155/+;+/+;+/+ (A″), elav-Gal4C155/+;UAS-Mio/+;+/+ (B″), elav-Gal4C155/+;+/+;UAS-Aβ42E693G/+ (C″), elav-Gal4C155/+;UAS-Miro/+;UAS-Aβ42E693G/+ (D″), elav-Gal4C155/+;+/+;UAS-APP.C99-UAS-MAPT/+ (E″), elav-Gal4C155/+;UAS-Miro/+;UAS-APP.C99-UAS-MAPT/+ (F″). Scale bar: 50 µm (A″–F″). (a″–f″) Magnified images of central region of the adult fly brains (A″–F″). Red-colored round shape indicates vacuoles (neurodegeneration) (a″–f″). Scale bar: 20 µm (a″–f″), n=10. (G″) The histogram shows an average number of vacuoles in each genotype of adult brains. Quantification of AO positive cells and the number of vacuoles was done by using ImageJ software (NIH, USA). Error bar represents mean±s.e.m. Data significance was calculated by one-way ANOVA analysis with Tukey's test using GraphPad Prism 5.0 and is indicated as: ns, non-significant, and ***P<0.0001.

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Research Article
Miro, a Rho GTPase genetically interacts with Alzheimer's disease-associated genes (Tau, Aβ42 and Appl) in Drosophila melanogaster
Komal Panchal, Anand Krishna Tiwari
Biology Open 2020 9: bio049569 doi: 10.1242/bio.049569 Published 3 September 2020
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