OMA1 mediates local and global stress responses against protein misfolding in CHCHD10 mitochondrial myopathy

Mitochondrial stress triggers a response in the cell’s mitochondria and nucleus, but how these stress responses are coordinated in vivo is poorly understood. Here, we characterize a family with myopathy caused by a dominant p.G58R mutation in the mitochondrial protein CHCHD10. To understand the disease etiology, we developed a knockin (KI) mouse model and found that mutant CHCHD10 aggregated in affected tissues, applying a toxic protein stress to the inner mitochondrial membrane. Unexpectedly, the survival of CHCHD10-KI mice depended on a protective stress response mediated by the mitochondrial metalloendopeptidase OMA1. The OMA1 stress response acted both locally within mitochondria, causing mitochondrial fragmentation, and signaled outside the mitochondria, activating the integrated stress response through cleavage of DAP3-binding cell death enhancer 1 (DELE1). We additionally identified an isoform switch in the terminal complex of the electron transport chain as a component of this response. Our results demonstrate that OMA1 was critical for neonatal survival conditionally in the setting of inner mitochondrial membrane stress, coordinating local and global stress responses to reshape the mitochondrial network and proteome.


Generation and genotyping of C10 G58R mice
A guide RNA (gRNA) near the G54 sequence in mouse C10 (mouse equivalent of G58) was used (TAGCCGTGGGCTCAGCTGTAGGG), along with a single-stranded donor oligonucleotide with a GGC → AGA substitution at G54 for repair: CTGCCGCTCCCGGCCAGCCGGGTCTTATGGCTCAGATGGCATCCACCGCCGCAG GCGTAGCCGTGAGATCAGCTGTAGGCCATGTCATGGGTAGCGCCCTGACCAGTG CCTTCAGTGGGGGAAATTCAGAGCCTGCCCAGCCTGCCGTCCAGCAGGTGAGCG GGAGGACTCAAGAAACGGAGGCAGGATTCACACATGGT. The following primers were used for genotyping: forward primer 5'-GACCCTGGAGTAGAGGGGTT-3' and reverse primer 5'-GGCCACTCCTCATTGGACTC-3'. The mouse C10 G54R mutation removes a cut site of the restriction endonuclease BanII (NEB, cat# R0119S), introducing a restriction fragment length polymorphism. Therefore, the region surrounding G54 was amplified by PCR and then subjected to a BanII restriction digest for 1 hour followed by 10 minutes of heat inactivation at 80 C, and finally run on a 2% agarose mini gel. The digested WT bands are ~150bp and the undigested G58R bands are ~300bp.

Motor function tests
C10 WT and C10 G58R littermates aged 18 weeks and C10 S59L mice aged 25 weeks were tested. Mouse forelimb grip strength was measured by pulling the mouse and recording the force generated as the mouse grips to the instrument (BIOSEB instrument with bar, cat# EB1-BIO-GS3). The mouse had to grip with both forelimbs for the trial to be scored; if the mouse used only one forelimb or did not have a proper grip, it was allowed to rest and the trial was repeated. This was repeated 3 times with 15 seconds of rest in between, and the results were averaged.
Balance and motor coordination were tested by placing the mice on a rotating rod (rotarod, Ugo Basile cat# 57624) and measuring the time to fall. The rotarod was allowed to rotate briefly to ensure that all mice faced the proper direction, and when all were facing forwards the speed was ramped from 5 rpm to 40 rpm over 120 seconds; the trial finished when all the mice had fallen off the rotarod. This was repeated 3 times, m/min, and then at 28 m/min until the mouse is fatigued. The distance run was recorded when the mice fulfilled the fatigue criterion above.
As C10 G58R mice were generally unable to turn when placed facing upwards in the pole test, the test had to be modified so that mice are placed at the top of the 50 cm pole facing downwards and allowed to descend. To train the mice, this was repeated 3 times with 1 minute of rest in between, and this was done each of the two days preceding the experiment. After the two training days, mice were placed at the top of the pole and the time needed until all four paws touch the base was recorded. This was repeated 5 times with 3 minutes of rest between each trial, and the trial results were averaged. (1 μM forward and 1 μM reverse primers in water, for a final reaction concentration of 250 nM each). The PCR was run with the following cycle: 98 C for 2 minutes, 20 x (98 C for 10 seconds, 60 C for 15 seconds, 72 C for 6 minutes and 24 seconds), 10 x (98 C for 10 seconds, 60 C for 15 seconds, 72 C for 6 minutes and 24 seconds + 20 seconds per cycle), 72 C for 5 minutes, 4 C indefinitely.
Following the PCR, the 20 μL samples were diluted in 40 μL of water and run on a 0.8% agarose gel for 1 hour at 70 Volts in TAE buffer and post-stained with SYBR Gold (Invitrogen, cat# S11494) for 30 minutes in TAE buffer. Gels were imaged on a Bio-Rad Chemidoc system.
Droplets were generated with the Automated Droplet Generator (Bio-Rad, cat# 1864101), and the reaction was run with CNV settings using the QX200 Droplet Reader (Bio-Rad, cat# 1864003). The resulting data was analyzed with QuantaSoft Analysis Pro (Bio-Rad).

Mouse COX activity stain and fiber width measurements
Mice were anesthetized with isoflurane and transcardially perfused with PBS. Tibialis and soleus muscles were collected and immediately flash-frozen in liquid-nitrogencooled isopentane and stored at -80 C until sectioning. The muscles were cryosectioned at -20 C to obtain 10 μm transverse sections. For staining, muscle sections were allowed to dry at room temperature for 1 hour and then were incubated in the incubation solution for 1 hour at 37 C.

Indirect calorimetry
The Oxymax-CLAMS setup (Columbus Instruments) was used to assess mouse metabolism. Mice were singly housed in the CLAMS chambers, received food and water ad libitum throughout the experiment, and were checked on at least twice a day throughout the 5-day duration of the experiment. Oxygen consumption, carbon dioxide production, food intake, and beam breaks (locomotion) were monitored during the experiment. The resulting data was analyzed using CalR (Mina et al., 2018).

Body composition
Non-invasive measurements of lean tissue and fat were obtained using the EchoMRI NMR machine. Un-anesthetized mice were placed in a clear plastic tube and gently restrained at the end of the tube by using a plunger with air holes. The plunger was fitted to the mouse and tightened gently to minimize movement. The tube was then inserted into the mini-spec port to a premeasured depth, and measurements were collected. The mouse was removed from the tube and returned to the home cage, and the tube was washed and sanitized after each use.

EVcouplings conservation analysis and 3D prediction
The human CHCHD10 protein sequence (Q8WYQ3) was analyzed using the EVcouplings server (https://v2.evcouplings.org/). The b0.2 cutoff yielded the most matched sequences and was used in the subsequent analysis. Outputs included assessment of amino acid frequency for each residue among the identified C10 homologs, as well as structure predictions generated using the EVfold algorithm (Marks et al., 2011 andHopf et al., 2019). The highest scoring structure was visualized using UCSF Chimera (https://www.cgl.ucsf.edu/chimera/).

Soluble/insoluble assay
This assay was performed on cultured cells or on mouse tissue. HEK293 or HeLa cells were harvested by scraping into PBS 24 hours after transfection, whereas mouse tissue was fractionated and the assay was performed on total homogenate, the cytosolic fraction, and the mitochondrial fraction. The mitochondrial and cytosolic fractions were treated with 1% TX-100 in 50 mM Tris, and after 21300 g centrifugation for 10 minutes the supernatant was used as the soluble fraction and the pellet was treated again with 2% SDS in 50 mM Tris buffer. After centrifugation, the supernatant was used as the insoluble fraction. Laemmli buffer was added to both fractions to obtain an equal volume of 1X buffer with 2.5% (v/v) 2-mercaptoethanol. Lysates were boiled at 98 C, separated by SDS-PAGE, and analyzed by immunoblotting.

Mouse histochemistry
Mice were anesthetized with isoflurane and transcardially perfused with 25 mL PBS.
The heart midventricular region and tibialis muscle were dissected and postfixed in 4% PFA for 1 hour (heart) or 30 minutes (tibialis) at room temperature, with the heart additionally fixed overnight at 4 C. After fixation, the tissue was washed for 5 minutes in PBS 3 times. Some heart specimens were sent to Histoserv (Germantown, MD) for paraffin embedding and microtome sectioning to obtain 4 μm sections. H&E and Masson's trichrome stains were additionally performed by Histoserv.
35 μm free-floating sections of heart and muscle were obtained using the Compresstome VF-300-0Z (Precisionary). The sections were permeabilized and blocked in 0.4% Triton-X (Sigma, X100) and 4% BSA fraction V (MP Biomedicals, cat# 02160069) in PBS for 2 hours at room temperature. Afterwards, the sections underwent primary antibody incubation in 0.3% Triton-X and 1% BSA in PBS overnight at 4 C (note: samples from the PDH/C10 experiment from Figure S4E were incubated in primary antibodies for 3 days). The samples were washed for 10 minutes in PBS 3 times, and then incubated in secondary antibodies in PBS (1:500). Afterwards, the samples were washed for 10 minutes in PBS 3 times, mounted on a slide with mounting medium (KPL, cat# 71-00-16), coverslipped (Thorlabs, cat# CG15KH), and sealed with nail polish.

Proteinase K protection assay
About 15 μg of mitochondria was lysed in a buffer containing 250 mM sucrose, 10 mM HEPES, 10 mM KCl, 2 mM MgCl2, and 1 mM EDTA (pH 7.4). According to different treatment conditions, lysates were added with or without different percentages of digitonin or 1% Triton X-100. After sitting on ice for 10 minutes the proteinase K was added for a final concentration of 50 μg/mL and the lysates were kept on ice for another 15 minutes. Finally, 3 mM PMSF was added to stop the proteinase K digestion. The samples were mixed with 4X Laemmli sample buffer and 2-mercaptoethanol and analyzed by western blotting.

Transmission electron microscopy
Human pectoralis muscle was snap frozen in liquid-nitrogen-cooled isopentane and subsequently processed for electron microscopy according to standard protocols.
Mouse heart and skeletal muscle TEM was performed as previously described (Liu et al., 2020). For the serial TEM sections, the samples were prepared with a modified protocol for heavy membrane staining. Heart samples were obtained and fixed with glutaraldehyde as above. Densitometry measurements were performed using Fiji (NIH) and Image Studio (LI-COR). OMA1-cleaved S-OPA1 bands were calculated by measuring the maximum intensity of each of the five bands in a linescan of the optical density of the five OPA1 bands on the blot using Fiji. After subtracting background intensity, the peaks of the c and e bands were summed and divided by the sum of the five bands (a-e) to obtain the percentage of OMA1-generated S-OPA1 from total OPA1.

Measurements of mitochondrial membrane potential and ROS
To assess mitochondrial potential stability, Tet-inducible HEK293 C10 WT, G58R, or S59L cells also expressing mito-mEmerald were either untreated or treated with 1 μg/mL doxycycline for 48 hours. The cells were then stained with 40 nM TMRE for at least 15 minutes and imaged live using an Olympus FLUOVIEW FV3000 confocal microscope in galvano mode to visualize membrane potential fluctuations. Images were obtained every ~9.5 seconds for at least 90 seconds. Using the mito-mEmerald channel, 20 mitochondria per FOV (60 mitochondria total from 3 biological replicates) were randomly selected and their TMRE/mito-mEm intensities were tracked for 10 frames. The TMRE/mito-mEm signal for each frame for each mitochondrion was normalized by the respective mitochondrion's 10-frame TMRE/mito-mEm average. The normalized TMRE/mito-mEm signals are plotted in Figure 6I, and the 10-frame normalized TMRE/mito-mEm standard deviation for each mitochondrion is represented in Figure 6H.
To assess ΔΨm by flow cytometry, Tet-inducible HEK293 C10 G58R cells were treated overnight with DMSO or 1 μg/mL doxycycline. The cells were trypsinized and counted.
Equal numbers of cells were incubated with 5 nM TMRE and 200 nM MitoTracker green for 15 minutes in a round bottom plate kept in the 37 C tissue culture incubator. After incubation the plate was spun at 400 g for 3 min, the cells were resuspended in a cell sorting buffer described previously (Liu et al., 2020), and their membrane potential was measured using a CellStream (Luminex) flow cytometer. For each sample, TMRE was measured separately for BFP+ cells (which contain the C10 WT or C10 G58R expression cassette) and a small BFP-negative population (which does not contain the expression cassette). The TMRE signal of BFP-cells was used for normalization. For Figure 6E, the stably transduced HEK 293 WT and C2/C10 DKO cells lines were stained with only 5 nM TMRE and the TMRE signal from GFP-cells was used for normalization. For ROS assessment, Tet-inducible HEK293 C10 G58R cells were treated overnight with DMSO or 1 μg/mL doxycycline. The cells were then trypsinized, counted, and equal numbers were incubated in media containing 5 μM MitoSOX or 10 μM H2DCFDA for 30 minutes in suspension. The cells were pelleted by centrifugation at 400 g for 3 minutes and then resuspended in a cell sorting buffer. MitoSOX or H2DCFDA signal intensity was measured using the CellStream flow cytometer. All cell lines were incubated at 37 C with 5% CO2 and ambient O2.

Generation of primary fibroblasts
WT and G58R fibroblast cells were generated from P1 newborn pups. The pups were placed in Dulbecco's modified Eagle medium (DMEM) + 1% L-glutamine + 15% fetal bovine serum (FBS) + MEM non-essential amino acid solution (MEM-NEAA) + 1% sodium pyruvate + 1X penicillin/streptomycin (pen/strep) + 1X gentamicin +1X amphotericin B. Skin from the pups was cut into thin 1-2 mm slices using sterile scalpels. The tissues were then transferred to a plate coated with 1% gelatin and containing the dissection media and covered with sterile cover slips. High-glucose DMEM + sodium pyruvate + 10% FBS + pen/strep was used for primary fibroblasts after the first passage.

Mitochondrial isolation
The method was adapted from a previously-published protocol (Frezza et al., 2007).
Mice were anesthetized with isoflurane, transcardially perfused with 25 mL PBS, and the heart was dissected and placed in a dish with cold PBS. The heart was then transferred into a tube with 10 mM EDTA in PBS and was minced into small pieces with scissors. The sample was centrifuged for 30 seconds at 5000 g and was washed twice with PBS/EDTA solution. After the washes, PBS/10 mM EDTA/0.05% trypsin solution was added and the tube was placed on a rocker for 30 minutes at 4 C. The tube was centrifuged at 200 g for 5 minutes and the supernatant was discarded. The tissue was weighed and 10 times the volume of the IBm1 buffer (6.7 mL of 1 M sucrose, 5 mL of 1 M KCI, 5 mL of 1 M Tris/HCI, 1 mL of 1 M EDTA, 2 mL of 10% BSA with water to make 100 mL of buffer, pH 7.4) was added before tissue was homogenized. The homogenate was centrifuged at 700 g for 10 minutes and the supernatant was collected, and it was then centrifuged at 8000 g for 10 minutes. The cytosolic fraction (supernatant) was discarded and the pellet was resuspended in the IBm2 buffer (25 mL of 1 M sucrose, 1 mL of 1 M Tris/HCl, 3 mL of 0.1 M EGTA/Tris with water to make 100 mL of buffer, pH 7.4) and centrifuged again at 8000 g for 10 minutes. The pellet was washed once more in IBm2 buffer before it was resuspended in RIPA buffer with protease inhibitor and phosphatase inhibitor. The sample was sonicated and spun at the highest speed (21130 g) for 10 minutes and the supernatant was collected and processed for BCA analysis.

Mouse transmission electron microscopy
Mice were anesthetized with isoflurane and transcardially perfused with 25 mL of PBS.
Hearts and tibialis muscles were rapidly dissected and a 1 mm x

Oxygen consumption
Seahorse Extracellular Flux Analyzer XF (Agilent) was used to measure the oxygen consumption rates (OCR) of 293 WT or OMA1 KO cells stably transduced with the pCIG3-IRES-EGFP empty vector or C10 G58R. 30,000 cells per well were seeded in XF96 cell culture microplates coated with poly-lysine and incubated for 24 hours at 37 C with 5% CO2 in standard complete high glucose DMEM. One hour before the assay, the cells were placed in DMEM lacking bicarbonate (XF DMEM medium pH 7.4 (Agilent cat# 103575-100)) and supplemented with 10 mM glucose, 1 mM pyruvate, and 2 mM glutamine. The culture plates were moved to a 37 C incubator with atmospheric CO2 to degas for 1 hour prior to the assay. 2-2.5 μM oligomycin, 2 μM FCCP and 0.5 μM of rotenone and antimycin were added during the assay as the OCR was being measured.
The data was normalized by protein concentration using the BCA assay.

RNA microarray analysis
In Transcriptome Analysis Console, data were summarized using the gene-level signal space transformation -robust multiple-array average normalization (SST-RMA) method.
DEGs were required to have a genome-wide q-value of < 0.01 or 0.05 (as specified)  , 2005). A separate GSEA was performed using reactome pathways plus a custom "G58R Upregulated" category of genes that were upregulated with an FDR q-value < 0.01 in the C10 G58R ; OMA1 +/vs C10 WT ; OMA1 +/comparison.

Proteomics and analysis
Mitochondria were isolated from newly harvested mouse hearts as described above.
Protein concentration of the mitochondrial fraction was determined using a BCA kit. 86 μg of the mitochondrial fraction was solubilized in 1% digitonin and complexes were separated on a blue native (BN)-PAGE gel with bovine heart mitochondria used as a molecular weight standard. One gel band was cut for each lane. In-gel proteins were For the complex IV monomer vs supercomplexes experiment, the BN-PAGE gel was cut into 4 slices as in Figure S14E and proteomics analysis was performed on each slice as above.

BN-PAGE
The mitochondrial fraction was resuspended in 10 mM of HEPES pH 7.6 and 0.5 M sucrose. Protein concentration was measured using the BCA assay. The mitochondrial fraction was solubilized in 1% n-Dodecyl-β-D-Maltoside (DDM) and 1X NativePAGE sample buffer. The NativePAGE Novex Bis-Tris Gel System (Thermo Fisher Scientific) was used according to the manufacturer's instructions with the following modifications: 20 μg of the mitochondrial fraction was loaded, only Light Blue Cathode Buffer was used, and electrophoresis was performed at 150 V for 1 hour then 250 V for 2 hours.
Polyvinylidene fluoride (PVDF) was used as the membrane for immunoblotting, and transfer was performed at 130 V for 1 hour at 4 C. The membrane was then washed with 10% acetic acid for 20 minutes and air-dried. Afterwards, the membrane was washed 5 times with methanol to remove residual Coomassie Blue dye, blocked with 5% milk in TBST for 1 hour at room temperature and blotted for proteins of interest.      Aggregate area (μm²)                  foldchange of complex IV subunits from the C10 G58R vs C10 WT heart mitochondrial proteomics experiment in Figure 11A.