Fig 1: Quantitative Analysis of p97-Dependent Mitochondrial Proteome Remodeling and PINK1-Parkin Pathway Activity in iNeurons(A) 11-plex TMT-MS3-based proteomic analysis of iNeurons to examine total, phospho, and Ub-modified proteomes with or without inhibition of p97.(B) Volcano plots for total proteomes of iNeurons either left untreated or depolarized for 6 h in the presence of absence of the p97 inhibitors NMS-873 or CB-5083. Only proteins quantified in both p97 inhibitor dataset and also quantified with more than one peptide are represented (circle). Filled colored circles/squares indicate statistically significant hits (Welch’s t test [S0 = 0.585], corrected for multiple comparison by permutation-based FDR [1%]). MOM proteins identified by proximity biotinylation (Hung et al., 2017) are overlaid with an orange circle.(C) Relative abundance of MFN1/2 and RHOT1/2 in iNeurons either left untreated or depolarized for 6 h in the presence or absence of the p97 inhibitors NMS-873 or CB-5083. Error bars represent SEM.(D) Relative abundance (measured as ratio in the presence of AO versus untreated) of individual diGLY-containing peptides from the indicated Parkin substrates or p97 targets in iNeurons either left untreated or depolarized for 6 h in the presence of absence of the p97 inhibitor. Error bars represent SEM.(E) Relative abundance (measured as ratio in the presence of AO versus untreated) of pS65-Ub in iNeurons either left untreated or depolarized for 6 h in the presence or absence of the p97 inhibitors NMS-873 or CB-5083. Error bars represent SEM.See also Figure S4 and Table S2.
Fig 2: Miro recruits Cenp-F to mitochondria.(a) Miro is necessary for Cenp-F mitochondrial recruitment. Localization of Cenp-F (green) and mitochondria (red) in the presence and absence of Miro1, Miro2 or both. Top: Cenp-F signal only. Middle: overlay. Bottom: magnification of the boxed areas. R: Pearson coefficient colocalization analysis±s.e.m. Scale bar, 10 µm. Two to four regions were selected per cell. Number of selected regions: Miro1CRISPR: 98; siMiro2: 36; Miro1CRISPR-siMiro2: 34. The experiment was repeated at least four times. (b) Miro is sufficient for Cenp-F recruitment. Immunofluorescence of Miro-less cells overexpressing FLAG-Miro1 (blue). Green: Cenp-F, red: mitochondria. Scale bar, 10 µm. (c) Cenp-F recruitment to mitochondria by Miro1 mutants. Miro-less cells were transfected as in b with plasmids encoding the indicated mutants of Miro1. Recruitment was scored using the Pearson's R coefficient. Ø: untransfected cells. *P value <10-7 (compared with untransfected cells) from a Mann–Whitney–Wilcoxon U-test. NS, non-significant. Quantifications as in a. Number of selected regions: WT: 29; T18N: 17; E208K: 33; E328K: 28; E208K+E328K: 28; S432N: 31; f: 27. The experiment was repeated at least four times. (d) Cytosolic calcium withdrawal does not prevent Cenp-F mitochondrial recruitment. Cells treated with EGTA and BAPTA-AM to chelate extra- and intracellular calcium were imaged as in a. (e) Mapping of the Miro-interaction domain on Cenp-F. KERMIT cells were cotransfected with FLAG-Miro and GFP fusion of indicated Cenp-F fragments. Green fragments are recruited to mitochondria. Red ones are not. (f) Co-immunoprecipitation of transiently transfected Cenp-F fragment (amino acids 2375–3144) and wild-type (wt) and calcium-binding mutant (m) Miro1. * IgG shedding from the beads. (g) Yeast two-hybrid mapping of the Miro-interaction domain on Cenp-F. Green fragments are positive for interaction. Red ones are not. (h) Alignment of the Miro-binding domain of Cenp-F in chordates. The phylogenetic tree is generated by neighbour joining using percentage identity.
Fig 3: Mitochondrial protein changes following CCCP treatment in fibroblasts. (A) Mitochondrial (Mito) and cytosolic (Cyto) fractions were immunoblotted as indicated. Below: Schematic representation of our readouts. (B) Demographic and genetic information of all cell lines used in this study or described in Text. (C) Quantifications of mitochondrial protein levels. The intensity of each band in the mitochondrial fraction is normalized to that of the mitochondrial loading control VDAC from the same blot and expressed as a fraction of Mean of Healthy-1 with DMSO treatment; this control was included in every experiment. Student T Test is performed for comparing normalized band intensities within the same subject (DMSO vs. CCCP). N = 3–9 independent experiments. Please note that Healthy-2 to 12 show similar mitochondrial protein responses to CCCP as Healthy-1, previously published in Hsieh et al. (2019). (D,E) ELISA of Miro1 protein. Comparison within the same subject. Mann-Whitney U Test. (D) N = 4 with duplicates each time. (E) N = 3. (F) Intra-plate variability of ELISA shown in (E), measured by running the same fibroblast sample 4 times in the same plate. (G) The standard curve for (E) is shown. Sigmoidal 4PL is used. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. n.s.: not significant.
Fig 4: The mitochondrial network in fibroblasts, tau interaction with Miro1, and effect of Miro1 Reducer. (A) Confocal images show mitochondria stained with TMRM in fibroblasts. The mean TMRM intensity of each cell is normalized to the background intensity and quantified. N = 43 (Healthy-6), 46 (Healthy-7), and 42 (MAPT-1) cells from 4 to 5 images per coverslip from 3 coverslips. One-Way ANOVA Post Hoc Tukey Test. Scale bar: 10 µM. (B,C) Co-IP with anti-GFP from HEK cells transfected as indicated. (D) HEK cells transfected with different tau and Miro1 constructs were lysed and blotted. (E) ELISA of Miro1 protein in MAPT-1 fibroblasts treated as indicated, N = 4. Mann-Whitney U Test. *P < 0.05. n.s.: not significant.
Fig 5: Heterogeneity in mitochondrial phenotypes among Parkinson’s LRRK2R1441C patient iPSC-DA neuronal cultures. (A) Z-scores were calculated for iPSC-DA neuronal cultures derived from each LRRK2R1441C Parkinson’s patient line plotted relative to control lines to assess inter-patient variation in mitophagy index levels, mitochondrial membrane potential (??m), mitochondrial reactive oxygen species (ROS), pS65Ub levels (fold change in total pS65Ub spot area after CCCP treatment) and ubiquitinated MIRO1 induction (fold change in Ubiq.-MIRO1 after CCCP treatment as assessed by immunoblot). Line SFC869 corresponds to patient line SFC869-03-04, SFC870 corresponds to patient line SFC870-03-05 and JR207 corresponds to patient line JR207-03.
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