Impaired mitochondrial-endoplasmic reticulum interaction and mitophagy in Miro1-mutant neurons in Parkinson's disease

Abstract

Mitochondrial Rho GTPase 1 (Miro1) protein is a well-known adaptor for mitochondrial transport and also regulates mitochondrial quality control and function. Furthermore, Miro1 was associated with mitochondrial-endoplasmic reticulum (ER) contact sites (MERCs), which are key regulators of cellular calcium homeostasis and the initiation of autophagy. Impairments of these mechanisms were linked to neurodegeneration in Parkinson's disease (PD). We recently revealed that PD fibroblasts harboring Miro1 mutations displayed dysregulations in MERC organization and abundance, affecting mitochondrial homeostasis and clearance. We hypothesize that mutant Miro1 impairs the function of MERCs and mitochondrial dynamics, altering neuronal homeostasis and integrity in PD. PD skin fibroblasts harboring the Miro1-R272Q mutation were differentiated into patient-derived neurons. Live-cell imaging and immunocytochemistry were used to study mitophagy and the organization and function of MERCs. Markers of autophagy or mitochondrial function were assessed by western blotting. Quantification of organelle juxtapositions revealed an increased number of MERCs in patient-derived neurons. Live-cell imaging results showed alterations of mitochondrial dynamics and increased sensitivity to calcium stress, as well as reduced mitochondrial clearance. Finally, western blot analysis indicated a blockage of the autophagy flux in Miro1-mutant neurons. Miro1-mutant neurons display altered ER-mitochondrial tethering compared with control neurons. This alteration likely interferes with proper MERC function, contributing to a defective autophagic flux and cytosolic calcium handling capacity. Moreover, mutant Miro1 affects mitochondrial dynamics in neurons, which may result in disrupted mitochondrial turnover and altered mitochondrial movement.

Figure 1

Miro1-R272Q does not affect mitochondrial mass, but reduces mitochondrial movement. (A) Representative western blot images of Miro1 protein and the mitochondrial marker proteins Tom20 and Hsp60 in iPSC-derived neurons. Corresponding densitometries of western blot analyses normalized to β-actin are shown at the right. Data indicated as mean ± SEM (n = 3). (B) iPSC-derived neurons were fixed and stained against Tom20. Images were obtained using a 63× objective; scale bars indicate 10 μm. (C) Quantification of mitochondrial area per cell from the Tom20 signal from images shown in B. Data indicated as mean ± SEM. Significance calculated by Mann–Whitney test (n = 4). (D) Analysis of maximum and mean speed per mitochondria from iPSC-derived neurons stained with MitoTracker Green FM. Data indicated as mean ± SEM. Significance calculated by Mann–Whitney test; ^***P < 0.001; (n = 5).

Figure 2

Miro1-R272Q impairs cytosolic calcium handling and increases sensitivity to calcium stress. (A) iPSC-derived neurons were loaded with the cytosolic calcium indicator Fluo-4 AM for live-cell imaging. During imaging, cells were treated with 5 μM ionomycin in order to increase cytosolic calcium levels. Imaging was continued for 10 min with a 2 s interval. Images were obtained with a 40× objective. (B) Analysis of mean Fluo-4 AM fluorescence intensity (F1/F0) from images shown in A. Data indicated as mean ± SEM. Significance of first time points after treatment calculated by Holm–Sidak multiple t test; ^*P < 0.05; ^**P < 0.01; (n = 4–6). (C) iPSC-derived neurons were stained with MitoTracker Green FM for live-cell imaging. Images were obtained once per minute using a 40× objective. During imaging, neurons were treated with 5 μM ionomycin and mitochondrial morphology was analyzed using ImageJ. The white boxes in the images indicate the magnified regions showing representative time points during the treatment. Scale bars indicate 20 μm in the large images, and 10 μm in the magnified images. (D) Analysis of mitochondrial fragmentation in control and mutant neurons, expressed as aspect ratio, from images shown in C. Data indicated as mean ± SEM. Significance calculated by Holm–Sidak multiple t test (n = 4–5).

Figure 3

Miro1-R272Q causes increased MERCs and increased localization of Miro1 to MERCs. (A) iPSC-derived neurons were fixed and stained with antibodies against the ER marker protein Calnexin and the mitochondrial marker protein Tom20. Images were obtained using a 63× objective; scale bars indicate 10 and 5 μm. Every field consisted of z-stacks of 0.5 μm interval. The white boxes in the merged images indicate the co-localization panels and the magnified regions where co-localizations are indicated with white arrows. (B) iPSC-derived neurons were fixed and stained with antibodies against Calnexin, Tom20 and Miro1. Images were obtained using a 63× objective; scale bars indicate 10 μm in the large images, and 5 μm in the magnified images. The white boxes in the merged images shown at the left indicate the magnified regions and the co-localization panels shown at the right. Co-localization of Miro1 signal with MERCs is indicated as white labels in the co-localization panels. (C) Quantification of co-localization events of Calnexin and Tom20 per cell, indicating the amount of MERCs, from images shown in A. (D) Quantification of ER area per cell from the Calnexin signal from images shown in A. (E) Quantification of co-localization events of Miro1 puncta with MERCs, mitochondria or the ER per cell from images shown in B. All data indicated as mean ± SEM. Significance calculated by Mann–Whitney test; ^*P < 0.05; ^***P < 0.001; (n = 4).

Figure 4

Miro1-R272Q mutant neurons show decreased mitophagy flux and mitochondrial fragmentation. (A) iPSC-derived neurons were stained with MitoTracker Green FM and LysoTracker Deep Red for live-cell imaging. Images were obtained using a 63× objective, scale bars indicate 10 μm in the large images and 5 μm in the magnified images. Every field consisted in z-stacks of 0.5 μm interval. The white boxes in the merged images indicate the magnified regions and co-localization panels shown at the right. Co-localization events of MitoTracker and LysoTracker signals are indicated as yellow labels in the co-localization panels. (B) Quantification of co-localization events of MitoTracker and LysoTracker per cell, indicating the amount of mitophagy events, from images shown in A. Data indicated as mean ± SEM. Significance calculated by Mann–Whitney test; ^**P < 0.01; ^***P < 0.001; (n = 3–6). (C) Analysis of the mean size of individual Lysotracker structures in pixels, indicating the mean lysosomal size, from images shown in A. Data indicated as mean ± SEM. Significance calculated by Mann–Whitney test; ^**P < 0.01; ^***P < 0.001; (n = 3–6). (D) Gene-edited iPSC-derived neurons expressing the ATP5C1-Rosella reporter (Mitorosella) were treated with 10 μM CCCP to induce mitophagy. The white boxes in the images on the left indicate the magnified regions showing representative time points during the treatment. Scale bars indicate 20 μm in the images of the left, and 10 μm in the magnified images. (E) Analysis of the mean size of individual mitochondria in pixels from images shown in D. Data indicated as mean ± SEM. Significance calculated by Bonferroni multiple comparisons test; ^***P < 0.001; (n = 4). (F) Quantification of only-red particles normalized to mitochondria count, indicating the amount of mitophagy events, from images shown in D. Data indicated as mean ± SEM. Significance calculated by Bonferroni multiple t test; ^***P < 0.001; (n = 4).

Figure 5

Miro1-R272Q leads to alterations of autophagy flux and Rab9 protein levels. (A) Representative western blot image of p62 and Rab9 proteins in iPSC-derived neurons after bafilomycin A1 treatment. (B) Densitometry of p62 western blot analysis normalized to β-actin. Data indicated as mean ± SEM. Significance calculated by Mann–Whitney test; ^*P < 0.05; (n = 3). (C) Densitometry of Rab9 western blot analysis normalized to β-actin. Data indicated as mean ± SEM. Significance calculated by Mann–Whitney test; ^*P < 0.05; (n = 3).