Disruption of mitochondrial homeostasis and permeability transition pore opening in OPA1 iPSC-derived retinal ganglion cells

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Harvey, Paul E. Sladen, Giada Becchi, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5153627/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Feb, 2025 Read the published version in Acta Neuropathologica Communications → Version 1 posted 10 You are reading this latest preprint version Abstract Dominant optic atrophy (DOA) is the most common inherited optic neuropathy, characterised by the selective loss of retinal ganglion cells (RGCs). Over 60% of DOA cases are caused by pathogenic variants in the OPA1 gene, which encodes a dynamin-related GTPase protein. OPA1 plays a key role in the maintenance of the mitochondrial network, mitochondrial DNA integrity and bioenergetic function. However, our current understanding of how OPA1 dysfunction contributes to vision loss in DOA patients has been limited by access to disease-relevant, patient-derived RGCs. Here, we used induced pluripotent stem cell (iPSC)-RGCs to study how OPA1 affects cellular homeostasis in human RGCs, the most vulnerable cell type in DOA. iPSCs derived from OPA1 DOA patients and isogenic CRISPR-Cas9-corrected iPSCs were differentiated to iPSC-RGCs. Defects in mitochondrial networks and increased levels of reactive oxygen species were observed in iPSC-RGCs carrying OPA1 pathogenic variants. Ultrastructural analyses also revealed changes in mitochondrial shape and cristae structure, with decreased endoplasmic reticulum (ER):mitochondrial contact length in DOA iPSC-RGCs. Mitochondrial membrane potential was reduced and its maintenance was also impaired following inhibition of the F1Fo-ATP synthase with oligomycin, suggesting that mitochondrial membrane potential is maintained in DOA iPSC-RGCs through reversal of the ATP synthase and ATP hydrolysis. These impairments in mitochondrial structure and function were associated with defects in cytosolic calcium buffering following ER calcium release and store-operated calcium entry, and following stimulation with the excitatory neurotransmitter glutamate. In response to mitochondrial calcium overload, DOA iPSC-RGCs exhibited increased sensitivity to opening of the mitochondrial permeability transition pore. These data reveal novel aspects of DOA pathogenesis in patient-derived RGCs. The findings suggest a mechanism in which primary defects in mitochondrial network dynamics disrupt core mitochondrial functions, including bioenergetics, calcium homeostasis, and opening of the permeability transition pore, which may contribute to vision loss in DOA patients. Dominant optic atrophy OPA1 iPSCs retinal ganglion cells neurodegeneration mitochondrial networks calcium homeostasis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Dominant optic atrophy (DOA) is the most common inherited optic neuropathy, with an estimated minimum prevalence of 1 in 25,000 [ 1 ]. Clinically, DOA usually presents in the first two decades of life, characterised by bilateral progressive loss of central vision, dyschromatopsia and the development of optic disc pallor [ 2 ]. Optical coherence tomography (OCT) studies have demonstrated thinning of the retinal nerve fibre layer (RNFL), in particular the papillomacular bundle, highlighting the preferential loss of retinal ganglion cells (RGCs) in DOA pathogenesis [ 3 ]. Around 60% of people with DOA have pathogenic OPA1 variants [ 4 ]. OPA1 is a dynamin-related GTPase protein that localises to the inner mitochondrial membrane (IMM) [ 5 ]. OPA1 is ubiquitously expressed, and whilst loss of OPA1 function appears to primarily affect RGC function in DOA, approximately 20% of DOA patients exhibit a more severe syndromic ‘DOA+’ phenotype, characterised by multisystem neurodegeneration. These patients exhibit a broad range of neurological defects, including sensorineural hearing loss, ataxia, peripheral neuropathy, and myopathy [ 6 , 7 ]. At the molecular level, DOA + is often associated with missense variants in the GTPase domain and this could be due to a dominant negative effect in which dysfunctional, pathogenic OPA1 variants impair WT OPA1 protein function, driving a more severe clinical phenotype [ 7 ]. OPA1 maintains mitochondrial network dynamics by facilitating fusion of the IMM, possibly through a mechanism involving cardiolipin [ 8 ]. As such, loss of OPA1 function has been associated with defects in mitochondrial network morphology [ 9 , 10 ]. OPA1 also maintains mitochondrial cristae shape, and loss of OPA1 function has been correlated with aberrant cristae architecture [ 11 ] and cytochrome c release [ 12 ], highlighting a possible pathogenic mechanism underpinning RGC cell death in DOA. Reduced OPA1 function causes impaired function of the electron transport chain (ETC), with reduced oxygen consumption [ 13 – 15 ], and a reduced mitochondrial membrane potential (MMP) [ 12 , 16 , 17 ]. These bioenergetic defects may indirectly sensitise RGCs to cell death in DOA via autophagic [ 18 , 19 ] or excitotoxic [ 20 , 21 ] mechanisms, possibly via ATP depletion. OPA1 also participates in the maintenance of mitochondrial DNA (mtDNA) integrity, with a postulated role in anchoring mtDNA molecules to the IMM [ 22 ], however, distinct effects of attenuated OPA1 dysfunction on mtDNA have been reported across different tissues/cell types [ 23 , 24 ]. OPA1 dysfunction may impact calcium homeostasis in a cell type-dependent manner. Reports that OPA1 downregulation enhanced mitochondrial calcium uptake in patient-derived fibroblasts [ 25 ] conflicted with reports that siRNA-mediated OPA1 knock-down reduced mitochondrial calcium uptake in HeLa cells [ 20 ]. Whilst pro-opiomelanocortin neurons in which OPA1 protein levels were experimentally knocked down displayed unaltered cytosolic, but attenuated mitochondrial calcium transients [ 26 ], siRNA-mediated OPA1 knockdown in rat RGCs led to delayed calcium deregulation (DCD) in response to excitotoxic stress, an event that preceded cell death [ 20 ]. Furthermore, reduced OPA1 function increased basal cytosolic calcium levels, leading to cell death in mouse RGCs and Caenorhabditis elegans GABAergic motor neurons transfected with plasmids containing pathogenic variants of OPA1 [ 27 ]. These findings highlight the importance of studying the effect of OPA1 dysfunction on calcium homeostasis in human RGCs to better understand pathophysiological mechanisms in DOA patients. Here, we used DOA patient-derived and CRISPR-Cas9-corrected iPSC-RGCs to study the effect of OPA1 dysfunction in human RGCs. Changes in mitochondrial structure, network morphology and reactive oxygen species were assessed. The effect on calcium homeostasis was also investigated, as well as the mechanism of maintenance of the MMP, and the threshold for opening of the mitochondrial permeability transition pore (mPTP) in response to mitochondrial calcium overload. Materials and methods Generation, maintenance and differentiation of iPSC lines Patient-derived fibroblasts carrying the c.1334 G > A (p. R445H) substitution were reprogrammed to iPSCs and isogenic controls were created using CRISPR-Cas9 gene editing as described previously [ 28 ]. iPSCs were maintained on Geltrex-coated plates in mTeSR media (Stem Cell Technologies) at 37°C 5% CO 2 . For passaging, iPSC colonies were manually dissociated twice weekly. iPSCs were differentiated to iPSC-RGCs as previously described using a 42 day directed differentiation protocol [ 13 ]. Immunocytochemistry To measure mitochondrial network dynamics, 25 nM Mitotracker Orange (Thermo Fisher Scientific) was loaded for 30 min at 37°C 5% CO 2 in recording buffer (150 mM NaCl, 4.25 mM KCl, 4 mM NaHCO 3 , 1.25 mM NaH 2 PO 4 , 1.2 mM CaCl 2 , 1.2 mM MgCl 2 , 10 mM D-glucose, and 10 mM HEPES at pH 7.4). Neurons were washed, then fixed in 4% paraformaldehyde for 15 min. When assessing network dynamics in neuronal cell bodies, cells were washed and immediately mounted onto a glass slide with Fluoromount (Dako). Mitotracker Orange was excited at 555 nm and a ≥ 580 nm emission filter was used. Leica Lightning software was used for image deconvolution. ImageJ plugin MINA Version 3 ( https://imagej.net/plugins/mina ) was used for quantification. Mitochondrial footprint = volume of the mitochondrial signal; branch length mean = mean length of all the lines used to represent the mitochondrial structure; summed branch length mean = sum of all branch lengths divided by the number of independent skeletons; network branches mean = mean number of attached lines used to represent each structure. To assay mitochondrial length/distribution in neurites, after fixation and washing, neurons were incubated in blocking solution (10% normal donkey serum, 1% bovine serum albumin) for 1h, then incubated overnight at 4°C with rabbit anti-TUJ1 antibodies (Abcam) in 50% blocking buffer diluted in PBS. Anti-rabbit AlexaFluor 488 secondary antibodies (Thermo Fisher Scientific) were incubated for 2h at room temperature, before mounting the cells onto a glass slide. Images were acquired on a Stellaris 8 confocal microscope equipped with a 40x oil objective. Detection of reactive oxygen species iPSC-RGCs were plated in 96 well black-walled plates (Thermo Fisher Scientific). After washing with recording buffer, neurons were stained with 5 µM dihydroethidium (DHE; Thermo Fisher Scientific) or 5 µM MitoSOX (Thermo Fisher Scientific) for 30 min at 37°C 5% CO 2 in recording buffer. Assessment of DHE/MitoSOX fluorescence was performed on a Cytation 10 microplate reader (Agilent). DHE was excited at 518 +/- 20 nm, emission 606 +/- 20 nm. MitoSOX was excited at 535 +/- 20 nm, emission 585 +/- 20 nm. Hoechst dye was added at 10 µg/mL for 10 min at the end of the experiment to normalise DHE/MitoSOX signals to total cell number. Hoechst was excited at 350 +/- 20 nm, emission 450 +/- 20 nm. Western blotting Western blotting iPSC-RGCs were lysed in RIPA buffer (1% NP-40, 20 mM Tris-HCl, 5 mM sodium pyrophosphate, 5 mM EDTA) with 2% protease inhibitor cocktail (Sigma Aldrich). 5 µg of protein was loaded into 10% polyacrylamide gels, resolved at 80V for 2h, then transferred onto nitrocellulose membranes at 90V for 90 min. Membranes were blocked with 5% milk powder (diluted in PBS 0.2% Tween-20) (Sigma Aldrich) for 1h, then incubated with primary antibodies (Santa Cruz sc-17767 mouse anti-SOD1; 1:100 dilution, Abcam 18207 rabbit anti-TUJ1 1:5,000 dilution, or Proteintech 60004-1 mouse anti-GAPDH 1:8,000 dilution) overnight at 4°C. Membranes were incubated with HRP-conjugated goat anti-mouse secondaries for 1h at room temperature, bands were visualised with ECL Clarity Substrate (Biorad) and a Chemidoc imaging system (Biorad). Quantification of band intensity was performed in ImageLab (BioRad). Uncropped blots are available in Supplementary Figures. Live cell confocal imaging experiments For live imaging assessments, iPSC-RGCs were plated on 35 mm Fluorodishes (World Precision Instruments). All live imaging experiments were performed with cells incubated in recording buffer, with two exceptions: for glutamate stimulation, MgCl 2 was removed to maximise activation of N-methyl D-aspartate (NMDA) receptors; for ER calcium release/SOCE, cells were kept in calcium-free recording buffer until the introduction of 1.2 mM CaCl 2 at the end of the experiment. iPSC-RGCs were maintained at 37°C 5% CO 2 throughout image acquisition. All confocal imaging was performed on a Stellaris 8 microscope equipped with a 20x dry or 40x oil objective. For measurement of cytosolic calcium levels, iPSC-RGCs were loaded with 1.5 µM Fluo4 AM (Thermo Fisher Scientific) for 30 min at 37°C 5% CO 2, then washed twice to remove any residual Fluo4 AM. iPSC-RGCs were exposed to 1 µM thapsigargin (Selleck Chemicals), 1.2 mM CaCl 2 , 5 µM glutamate (Sigma Aldrich), or 10 µM ionomycin (Sigma Aldrich) at the indicated time points. To assay MMP, iPSC-RGCs were loaded with 5 nM tetramethylrhodamine ethyl ester (TMRE; Thermo Fisher Scientific) for 30 min at 37°C 5% CO 2 . 5 nM TMRE was kept in the recording buffer throughout the experiment. iPSC-RGCs were incubated with 1.5 µM oligomycin (Sigma Aldrich) or ascending doses (2.5–17.5 µM; 2.5 µM each step) of ferutinin (Sigma Aldrich) at the indicated time points. Fluo4 was excited at 488 nm, collecting light longer than 520 nm, whilst TMRE was excited at 555 nm, collecting light longer than 580 nm. These experiments were performed at 0.1% laser strength to minimise the effect of photobleaching/oxidative damage. Analysis of Fluo4/TMRE fluorescence was performed in ImageJ. Transmission electron microscopy imaging Cells were fixed by adding 4% PFA and 4% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.4 to the cell culture media at 1:1 and left for 1 h at room temperature. The cells were washed in 0.1 M cacodylate buffer before incubating in 1% osmium tetroxide and 1.5% potassium ferrocyanide in distilled water for 1 h in the dark at 4°C. En bloc staining was performed by incubating the cells in UA-Zero (Agar Scientific, Stansted, UK) for 1 h in the dark at room temperature. Subsequently, the cells were dehydrated in increasing concentrations of ethanol (70%, 90%, and 100%) followed by a mixture of propylene oxide:epon (1:1) overnight at room temperature. The propylene oxide:epon was replaced with two changes of epon every 3 h at room temperature before embedding in epon overnight at 60°C. 100nm sections were cut and imaged on a JEOL 1400Plus EM (JEOL ltd, Tokyo, Japan) fitted with an Advanced Microscopy Technologies (AMT) NanoSprint12 (AMT Imaging Direct, Woburn, MA, USA) camera. Analysis of TEM imaging was performed in ImageJ. Mito/cristae circularity = 4π*area/perimeter^2. Results Disturbed mitochondrial structures in DOA iPSC-RGCs OPA1 participates in the maintenance of mitochondrial network morphology [ 11 ]. We sought to establish whether OPA1 dysfunction was associated with changes in the structure of the mitochondrial network in iPSC-RGC cell bodies, and mitochondrial length in iPSC-RGC neurites, using Mitotracker dyes to delineate mitochondrial structure (Fig. 1 a). Neuronal soma with the R445H pathogenic OPA1 variant (derived from a DOA + patient) displayed a 1.5-fold increased mitochondrial footprint compared to corrected isogenic controls (Fig. 1 b). Conversely, mean branch length, mean summed branch length, and mean network branches were decreased in RGCs carrying the R445H pathogenic variant compared to isogenic iPSC-RGCs (Fig. 1 c-e), suggesting fragmentation of mitochondrial networks is associated with OPA1 dysfunction in human RGCs. To extend these data, mitochondrial ultrastructure was assessed with transmission electron microscopy (TEM) imaging (Fig. 1 f). The area and length of individual mitochondria were reduced, whilst roundness was increased in R445H iPSC-RGCs compared to isogenic controls (Fig. 1 g-i). These data again support fragmentation of mitochondrial structures in cells with pathogenic OPA1 variants. Mitochondrial cristae architecture was also altered between R445H and isogenic control iPSC-RGCs. In R445H neurons, mitochondrial cristae more commonly displayed a vesicular architecture compared to isogenic control cells. In support of this, cristae area was reduced in R445H cells, whilst circularity was increased compared to isogenic control samples (Fig. 1 j & k) We then tested if OPA1 dysfunction was associated with changes in mitochondria:ER membrane contact (MERC) length in our iPSC-RGC model, reasoning that similar defects have been observed across a range of neuropathologies [ 29 ], and observed that contact lengths were reduced in R445H vs. isogenic control iPSC-RGCs (Fig. 1 l). iPSC-RGC neurite mitochondrial morphology (Fig. 1 m) was investigated, and it was observed that R445H neurite mitochondrial length was decreased compared to isogenic control cells (Fig. 1 n). A statistically significant change was not observed between genotypes in the distribution of mitochondria in iPSC-RGC neurites (Fig. 1 o); however, mitochondrial area in TUJ1 + neurites was significantly lower in R445H iPSC-RGCs compared to isogenic controls (Fig. 1 p). Consistent with the role of OPA1 as a pro-fusion factor, collectively these data demonstrate fragmentation of mitochondrial networks and highlight potential changes in mitochondrial cristae and MERCs in DOA iPSC-RGCs. Oxidative stress and MMP defects in DOA iPSC-RGCs We previously reported a bioenergetic defect in iPSC-RGCs harbouring pathogenic OPA1 variants [ 13 ]. Mitochondrial dysfunction in neurodegenerative disease is often associated with increased generation of reactive oxygen species (ROS) [ 30 ], in particular mitochondrial superoxide production arising from ETC dysfunction [ 31 ]. Oxidative stress sensitises mitochondria to opening of the mPTP, an event that leads to cell death [ 32 ]. We therefore investigated whether superoxide levels and corresponding antioxidant defence mechanisms were affected by OPA1 dysfunction in iPSC-RGCs. DHE fluorescence was 1.7-fold higher in R445H compared to isogenic control neurons (Fig. 2 a). Similarly, MitoSOX fluorescence was 1.2-fold higher in cells harbouring the pathogenic OPA1 variant (Fig. 2 b). These results suggest that oxidative stress arises from mitochondrial dysfunction in OPA1-DOA. We also examined antioxidant defence mechanisms by measuring SOD1 protein levels by Western blotting. SOD1 levels (which localises to the cytosol and mitochondrial intermembrane space (IMS)) were not significantly different between genotypes (Fig. 2 c & Supplementary Fig. 1). Depolarisation of MMP may result from dysfunction of the ETC, which usually pumps protons into the IMS to sustain the electrochemical gradient that drives ATP synthesis. As there is a bioenergetic defect in DOA iPSC-RGCs [ 13 ], we measured MMP in iPSC-RGCs using TMRE, a cationic fluorescent dye that localises to the mitochondrial matrix in response to the potential difference (Fig. 2 d). iPSC-RGCs harbouring the R445H variant showed a decrease in TMRE fluorescence compared with isogenic control neurons, suggesting partial depolarisation of MMP (Fig. 2 e). ATP synthase utilises the electrochemical gradient generated by the ETC to synthesise ATP during oxidative phosphorylation [ 33 ]. However, if the free energy available from the membrane potential fails to balance the free energy available from the ATP/ADP ratio, the ATP synthase will reverse to act as a proton pumping ATPase, consuming (glycolytic) ATP and maintaining a membrane potential [ 34 ]. As such, ATP hydrolysis has been proposed as a mechanism that maintains MMP in bioenergetically compromised neurons and has been described in a range of mitochondrial disease models [ 35 – 37 ]. We therefore tested whether OPA1 dysfunction affects ATP hydrolysis in iPSC-RGCs using TMRE and the ATP synthase inhibitor oligomycin (Fig. 2 f). In isogenic control neurons, oligomycin treatment elicited an increase in TMRE fluorescence over time, indicating gradual hyperpolarisation of MMP as expected (Fig. 2 g). By contrast, in R445H iPSC-RGCs TMRE fluorescence decayed in response to ATP synthase inhibition (Fig. 2 h). At the endpoint of the experiment, TMRE fluorescence was decreased by 43% in R445H compared to isogenic control neurons (Fig. 2 i), showing that MMP is partially maintained by ATP hydrolysis in DOA iPSC-RGCs. These findings also highlight ETC dysfunction (i.e. reduced proton pumping by complexes I, III and IV) associated with OPA1 dysfunction in accordance with our previous data [ 13 ]. Altered cytosolic calcium handling in DOA iPSC-RGCs in response to ER calcium release Calcium signalling plays a key role in neuronal function, including regulation of gene expression [ 38 ], synaptic function [ 39 ], and mitochondrial metabolism [ 40 ]. To study calcium signalling dynamics in iPSC-RGCs, calcium uptake into the ER was inhibited with thapsigargin, an irreversible inhibitor of sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase (SERCA), in calcium-free recording buffer. As this results in depletion of ER stores, reintroduction of calcium into the recording buffer enables the assessment of store-operated calcium entry (SOCE), in which stromal interaction molecules (STIM1/2) and ORAI channel (ORAI1-ORAI3) complexes, and transient receptor potential channels (TRPC1-TRPC7) at the plasma membrane facilitate extracellular calcium influx to the cytosol in an attempt to refill ER calcium stores [ 41 ]. After treatment with thapsigargin, R445H iPSC-RGCs displayed an elevated initial slope (2.9-fold increase) and amplitude (3.7-fold increase) of Fluo4 fluorescence compared to isogenic control neurons (Fig. 3 a-e). The rate of peak Fluo4 fluorescence recovery to baseline levels was higher in R445H iPSC-RGCs compared to isogenic controls (Fig. 3 a-c & f), indicating faster calcium clearance after reaching peak levels. These data suggest that OPA1 dysfunction is associated with altered cytosolic calcium handling upon SERCA blockade and the release of calcium from the ER. After store depletion, reintroduction of calcium to the recording buffer led to a 1.9-fold elevation of amplitude of Fluo4 fluorescence in R445H iPSC-RGCs compared to isogenic controls (Fig. 3 a-c & g & h), indicating higher levels of SOCE associated with OPA1 dysfunction. The rate of Fluo4 fluorescence decay after the Ca 2+ influx was also increased in R445H iPSC-RGCs compared to isogenic controls (Fig. 3 a-c & i), similar to the pattern seen after thapsigargin treatment, indicating faster calcium clearance after reaching peak levels. Altered cytosolic calcium handling in DOA iPSC-RGCs in response to physiological glutamate challenge We then tested the hypothesis that OPA1 dysfunction would lead to changes in calcium handling following stimulation with the excitatory neurotransmitter glutamate. Glutamate activates the influx of extracellular calcium into the cytosol, whereby the opening of ionotropic NMDA receptors in particular mediates a significant increase in intracellular calcium levels [ 42 ]. Mitochondria act to buffer elevations in cytosolic calcium, utilising the electrochemical gradient generated by the ETC to draw calcium ions into the matrix via the mitochondrial calcium uniporter (MCU) [ 43 ]. R445H iPSC-RGCs exhibited an elevated initial slope (2.6-fold) and amplitude (1.9-fold) of Fluo4 fluorescence upon glutamate challenge compared to isogenic controls, suggesting that this excitatory pathway may lead to a faster accumulation and higher level of calcium in the cytosol (Fig. 4 a-e). The rate of Fluo4 fluorescence decay was lower in R445H compared to isogenic neurons. Whilst isogenic control iPSC-RGCs displayed a gradual decrease in Fluo4 fluorescence after the peak, suggesting restoration of cytosolic calcium homeostasis, R445H neuron Fluo4 fluorescence remained largely at peak levels, suggesting cytosolic calcium homeostasis could not be restored (Fig. 4 a-c & f). These data suggest OPA1 dysfunction limits the capacity of human RGCs to restore calcium homeostasis in response to excitotoxic stress. In summary, these data suggest OPA1 dysfunction in iPSC-RGCs leads to perturbations in cytosolic calcium handling in response to SERCA blockade and SOCE, and after treatment with glutamate. Increased sensitivity to opening of the mPTP in DOA iPSC-RGCs Mitochondria play a key role in regulating intracellular calcium signalling under normal conditions, but excessive mitochondrial calcium uptake may contribute to pathology by facilitating the opening of the mPTP, a process thought to be exacerbated by oxidative stress [ 44 ]. mPTP opening is associated with the collapse of MMP and cessation of ATP synthesis, and is therefore a catastrophic event for neurons that depend mainly on oxidative phosphorylation to meet their energy requirements [ 32 ]. To test the threshold for mPTP opening, we challenged R445H and isogenic control iPSC-RGCs with ascending doses of ferutinin, an electrogenic calcium ionophore that increases mitochondrial calcium levels, eventually leading to mitochondrial calcium overload and mPTP opening [ 35 ]. The collapse of MMP was measured as a readout for mPTP opening, and the concentration of ferutinin required to induce a rapid loss of MMP was significantly lower in R445H iPSC-RGCs compared to isogenic controls (Fig. 5 a-d; R445H, ~ 12.5 µM vs isogenic, 15.0-17.5 µM ferutinin). These data suggest that the threshold for mPTP opening may be reduced by OPA1 dysfunction in iPSC-RGCs. Discussion The results presented in this study suggest that OPA1 dysfunction leads to defects in mitochondrial structure, ETC function, MMP, calcium signalling, and mPTP opening in human RGCs, highlighting pathophysiological mechanisms that could contribute to vision loss in DOA patients. Consistent with the role of OPA1 in facilitating fusion of the IMM [ 45 ], OPA1 neurons displayed fragmented mitochondrial networks in RGC somas and shorter neurite mitochondria compared to isogenic control neurons, data which were corroborated by EM ultrastructural analysis. OPA1 dysfunction was also associated with increased mitochondrial footprint in RGC somas and decreased neurite mitochondrial area, but not a statistically significant change in neurite mitochondria distribution, which may have been mediated by differences in mitochondrial anterograde/retrograde transport [ 46 ], or mitochondrial quality control mechanisms [ 47 ]. We also detected increased levels of ROS generation, specifically superoxide anions, in DOA iPSC-RGCs. These results are in accordance with previous reports of the effect of OPA1 dysfunction in fibroblasts, in which galactose was used in place of glucose in the cell media to force ATP generation by oxidative phosphorylation [ 48 , 49 ]. A recent study reported increased superoxide levels arising from attenuated OPA1 function in primary vascular cells, with reduced SOD1 protein levels [ 50 ], however, we did not detect a statistically significant decrease in SOD1 protein levels in iPSC-RGCs, likely reflecting the different cell types under investigation in the respective studies. We previously showed bioenergetic defects, in iPSC-RGCs harbouring pathogenic OPA1 variants suggesting dysfunction of the ETC [ 13 ]. In this study, we identified morphological changes in mitochondrial cristae, specifically, the appearance of vesicular cristae in R445H iPSC-RGCs. This structural deficit was associated with a partial depolarisation and maintenance of MMP by ATP hydrolysis in R445H vs. isogenic control iPSC-RGCs, a process that consumes ATP and, therefore, likely reduces the energy supply available for other cellular functions. It remains to be determined whether inhibition of ATP hydrolysis (whilst maintaining ATP synthesis) could be utilised as a potential therapeutic tool in DOA as described in other mitochondrial disease models [ 36 ], or if it would accelerate mitochondrial depolarisation and dysfunction. Cytosolic calcium handling is mediated by a range of mechanisms, including buffering into/release from intracellular stores and influx/efflux mechanisms that operate at the cell surface [ 38 – 40 ]. We observed that neurons with the R445H pathogenic variant exhibited increased calcium release when ER calcium uptake was inhibited, and increased levels of cytosolic calcium after initiation of SOCE. Differences in resting ER calcium levels and/or the activity of the inositol 1,4,5-triphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) which mediate calcium release from the ER could explain these results, however, buffering of cytosolic calcium into the (depolarised) mitochondrial matrix may also have played a role. Given we observed reduced MERC length in DOA iPSC-RGCs, it is possible that changes in calcium transfer between mitochondria and ER compartments contribute to calcium deregulation in DOA. OPA1 neurons demonstrated upregulated SOCE which could reflect an attempt to refill ER calcium stores, a pathway that has been described in other neurodegenerative disease models [ 51 – 53 ]. We observed an increased rate of Fluo4 fluorescence decay in R445H compared to corrected iPSC-RGCs following both thapsigargin and CaCl 2 treatment, suggesting an increase in cytosolic calcium clearance. As this effect is unlikely to be explained by increased ER/mitochondrial calcium uptake, this may reflect the activity of other cytosolic calcium efflux mechanisms, such as Ca 2+ /Na + exchangers (NCX) and/or plasma membrane Ca 2+ ATPases (PMCA) operating at the cell membrane [ 54 ]. Mitochondria serve as buffers to increased cytosolic calcium levels, a process that is dependent on the MMP and MCU activity [ 43 ]. As such, the elevation of cytosolic calcium in DOA iPSC-RGCs observed after ER store release and SOCE may be partially explained by reduced uptake of calcium into the mitochondrial matrix, possibly due to MMP depolarisation. In support of this, R445H iPSC-RGCs demonstrated increased Fluo4 fluorescence initial slopes and amplitudes compared to isogenic controls after treatment with glutamate, which might be a consequence of reduced buffering of cytosolic calcium by dysfunctional mitochondria. It is possible that changes in NMDA receptor subunit expression, which have been suggested in previous reports on OPA1 dysfunction [ 21 ], could also explain the differences we observed. In contrast to the patterns evident after ER calcium release and SOCE, Fluo4 fluorescence remained elevated in R445H iPSC-RGCs after reaching peak levels. This suggests that calcium clearance is attenuated after glutamate challenge in human RGCs with OPA1 dysfunction, in agreement with previous reports detailing the induction of DCD in OPA1 mouse RGCs after glutamate treatment [ 20 ]. In summary, these investigations into calcium handling identified defects associated with OPA1 dysfunction in iPSC-RGCs, in which both the ER and mitochondrial cellular compartments could play a role. mPTP opening is associated with the collapse of MMP and cessation of ATP synthesis. We challenged cells with ascending doses of ferutinin, an electrogenic calcium ionophore, to induce mitochondrial calcium overload and assayed loss of MMP as a biomarker for mPTP opening. We found that ferutinin induced mPTP formation at lower concentrations in R445H iPSC-RGCs compared to isogenic control neurons. The threshold for mPTP opening is known to decrease when matrix calcium and ROS levels are high [ 44 ]. Given that we have identified changes in cytosolic calcium handling and increased oxidative stress in DOA iPSC-RGCs, it is possible that OPA1 dysfunction sensitises RGCs to mPTP opening via mitochondrial calcium deregulation and increased superoxide production. Conclusions In summary, this study identified several structural and functional defects associated with OPA1 dysfunction in human RGCs. OPA1 neurons exhibited fragmented mitochondrial networks, oxidative stress and MMP defects. Cytosolic calcium handling was also affected, and the threshold for mPTP opening was reduced. Collectively, these mechanisms may contribute to progressive vision loss in DOA patients. Abbreviations ATP adenosine triphosphate CRISPR clustered regularly interspaced short palindromic repeats DCD delayed calcium deregulation DHE dihydroethidium DOA dominant optic atrophy DOA+ dominant optic atrophy plus ECL enhanced chemiluminescence ER endoplasmic reticulum ETC electron transfer chain GABA gamma aminobutyric acid GTP guanosine triphosphate HEPES2 (4–(2–hydroxyethyl)–1–piperazinyl)–ethanesulfonic acid HRP horse radish peroxidase IMM inner mitochondrial membrane IMS inter membrane space iPSC induced pluripotent stem cell IP3R inositol 1,4,5–trisphosphate receptor MCU mitochondrial calcium uniporter MERC mitochondria:endoplasmic reticulum contact MMP mitochondrial membrane potential mtDNA mitochondrial DNA NCX sodium calcium exchanger NCLX mitochondrial sodium calcium exchanger NMDA N–methyl D–aspartic acid OCT optical coherence tomography OPA1 optic atrophy 1 ORAI calcium release–activated calcium modulator 1 PBS phosphate buffered saline PMCA plasma membrane calcium ATPase RGC retinal ganglion cell RNFL retinal nerve fibre layer ROS reactive oxygen species RyR ryanodine receptors SERCA sarcoplasmic endoplasmic reticulum siRNA small interfering RNA SOD1 superoxide dismutase SOCE store operated calcium entry STIM stromal interaction molecule TMRE tetramethylrhodamine ethyl ester TRPC transient receptor potential channel TUJ1 β3–tubulin Declarations Ethics approval and consent to participate For the use of patient-derived fibroblasts in this study, informed consent was obtained following the tenets of the Declaration of Helsinki. Ethical approval was granted by the Yorkshire and The Humber-Leeds Bradford Research Ethics Committee (REC reference: 13/YH/0310). Consent for publication Not applicable Competing Interests PYWM is a consultant for GenSight Biologics, Stoke Therapeutics, PYC Therapeutics and has received research support from GenSight Biologics and Chiesi. MEC is a consultant for Prime Medicine. Funding MW was supported by a grant from Moorfields Eye Charity (to MEC, YJS and PYWM), and the National Institute for Health and Care Research Biomedical Research Centre at Moorfields Eye Hospital and UCL Institute of Ophthalmology. TB receives funding from MRC Career Development Award grant (MR/X020827/1). MEC received funding from Wellcome Trust Investigator Award (205041), Foundation Fighting Blindness (USA), Fight for Sight, Moorfields Eye Charity, the Macular Society and the NIHR Moorfields Biomedical Research Centre. PYWM is supported by an NIHR Advanced Fellowship (NIHR301696), NIHR Cambridge Biomedical Research Centre (NIHR203312), NIHR Biomedical Research Centre based at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology (NIHR203322), and the LifeArc Centre for Rare Mitochondrial Diseases. Author Contribution MW designed the study, conducted the majority of the experimental work and analysis, and drafted the manuscript. PES established the iPSC lines and differentiation protocol. JPH and GB assisted with the experimental work. TB processed samples for TEM imaging. TB, YJS, KS, MD, PYWM and MEC helped design the study, and provided feedback on data analysis and interpretation. MEC, YJS and PYWM secured funding for the research. All co-authors provided feedback on the manuscript. Acknowledgement The authors would like to acknowledge the UCL Institute of Ophthalmology Imaging Department for their assistance with the TEM imaging. Availability of data and material The datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request. References Harvey JP, Sladen PE, Yu-Wai-Man P, Cheetham ME (2022) Induced Pluripotent Stem Cells for Inherited Optic Neuropathies - Disease Modeling and Therapeutic Development. J. Neuro-Ophthalmology Yu-Wai-Man P, Griffiths PG, Gorman GS, Lourenco CM, Wright AF, Auer-Grumbach M et al (2010) Multi-system neurological disease is common in patients with OPA1 mutations. Brain 133:771–786 Asanad S, Tian JJ, Frousiakis S, Jiang JP, Kogachi K, Felix CM et al (2019) Optical Coherence Tomography of the Retinal Ganglion Cell Complex in Leber’s Hereditary Optic Neuropathy and Dominant Optic Atrophy. Curr Eye Res. ;44 Newman NJ, Yu-Wai-Man P, Biousse V, Carelli V (2023) Understanding the molecular basis and pathogenesis of hereditary optic neuropathies: towards improved diagnosis and management. Lancet Neurol Olichon A, Emorine LJ, Descoins E, Pelloquin L, Brichese L, Gas N et al (2002) The human dynamin-related protein OPA1 is anchored to the mitochondrial inner membrane facing the inter-membrane space. FEBS Lett. ;523 Yu-Wai-Man P, Chinnery PF (2013) Dominant optic atrophy: Novel OPA1 mutations and revised prevalence estimates. Ophthalmology Yu-Wai-Man P, Griffiths PG, Gorman GS, Lourenco CM, Wright AF, Auer-Grumbach M et al (2010) Multi-system neurological disease is common in patients with OPA1 mutations. Brain 133:771–786 Ban T, Ishihara T, Kohno H, Saita S, Ichimura A, Maenaka K et al (2017) Molecular basis of selective mitochondrial fusion by heterotypic action between OPA1 and cardiolipin. Nat Cell Biol. ;19 Zanna C, Ghelli A, Porcelli AM, Karbowski M, Youle RJ, Schimpf S et al (2008) OPA1 mutations associated with dominant optic atrophy impair oxidative phosphorylation and mitochondrial fusion. 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EMBO J. ;27 White KE, Davies VJ, Hogan VE, Piechota MJ, Nichols PP, Turnbull DM et al (2009) Opa1 deficiency associated with increased autophagy in retinal ganglion cells in a murine model of dominant optic atrophy. Investig Ophthalmol Vis Sci. ;50 Kushnareva YE, Gerencser AA, Bossy B, Ju WK, White AD, Waggoner J et al (2013) Loss of OPA1 disturbs cellular calcium homeostasis and sensitizes for excitotoxicity. Cell Death Differ. ;20 Nguyen D, Alavi MV, Kim KY, Kang T, Scott RT, Noh YH et al (2011) A new vicious cycle involving glutamate excitotoxicity, oxidative stress and mitochondrial dynamics. Cell Death Dis. ;2 Elachouri G, Vidoni S, Zanna C, Pattyn A, Boukhaddaoui H, Gaget K et al (2011) OPA1 links human mitochondrial genome maintenance to mtDNA replication and distribution. Genome Res. ;21 Chen L, Liu T, Tran A, Lu X, Tomilov AA, Davies V et al (2012) OPA1 mutation and late-onset cardiomyopathy: mitochondrial dysfunction and mtDNA instability. 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Arch Biochem Biophys Lhuissier C, Desquiret-Dumas V, Girona A, Alban J, Faure J, Cassereau J et al (2024) Mitochondrial F0F1-ATP synthase governs the induction of mitochondrial fission. iScience [Internet]. ;27. https://doi.org/10.1016/j.isci.2024.109808 Ludtmann MHR, Angelova PR, Horrocks MH, Choi ML, Rodrigues M, Baev AY et al (2018) α-synuclein oligomers interact with ATP synthase and open the permeability transition pore in Parkinson’s disease. Nat Commun. ;9 Acin-Perez R, Benincá C, Fernandez del Rio L, Shu C, Baghdasarian S, Zanette V et al (2023) Inhibition of ATP synthase reverse activity restores energy homeostasis in mitochondrial pathologies. EMBO J. ;42 McKenzie M, Liolitsa D, Akinshina N, Campanella M, Sisodiya S, Hargreaves I et al (2007) Mitochondrial ND5 gene variation associated with encephalomyopathy and mitochondrial ATP consumption. J Biol Chem. ;282 West AE, Chen WG, Dalva MB, Dolmetsch RE, Kornhauser JM, Shaywitz AJ et al (2001) Calcium regulation of neuronal gene expression. Proc Natl Acad Sci U S A. ;98 Dolphin AC, Lee A (2020) Presynaptic calcium channels: specialized control of synaptic neurotransmitter release. Nat Rev Neurosci Duchen MR (2012) Mitochondria, calcium-dependent neuronal death and neurodegenerative disease. Pflugers Arch Eur J Physiol Hogan PG, Rao A (2015) Store-operated calcium entry: Mechanisms and modulation. Biochem Biophys Res Commun Dong XX, Wang Y, Qin ZH (2009) Molecular mechanisms of excitotoxicity and their relevance to pathogenesis of neurodegenerative diseases. Acta Pharmacol Sin Vais H, Payne R, Paudel U, Li C, Foskett JK (2020) Coupled transmembrane mechanisms control MCU-mediated mitochondrial Ca2 + uptake. Proc Natl Acad Sci U S A. ;117 Halestrap AP (2009) What is the mitochondrial permeability transition pore? J Mol Cell Cardiol Burté F, Carelli V, Chinnery PF, Yu-Wai-Man P (2015) Disturbed mitochondrial dynamics and neurodegenerative disorders. Nat Rev Neurol Sun S, Erchova I, Sengpiel F, Votruba M (2020) Opa1 deficiency leads to diminished mitochondrial bioenergetics with compensatory increased mitochondrial motility. Investig Ophthalmol Vis Sci. ;61 Liao C, Ashley N, Diot A, Morten K, Phadwal K, Williams A et al (2017) Dysregulated mitophagy and mitochondrial organization in optic atrophy due to OPA1 mutations. Neurology. ;88 Lee H, Smith SB, Sheu SS, Yoon Y (2020) The short variant of optic atrophy 1 (OPA1) improves cell survival under oxidative stress. J Biol Chem. ;295 Quintana-Cabrera R, Manjarrés-Raza I, Vicente-Gutiérrez C, Corrado M, Bolaños JP, Scorrano L (2021) Opa1 relies on cristae preservation and ATP synthase to curtail reactive oxygen species accumulation in mitochondria. Redox Biol. ;41 Robert P, Nguyen PMC, Richard A, Grenier C, Chevrollier A, Munier M et al (2021) Protective role of the mitochondrial fusion protein OPA1 in hypertension. FASEB J. ;35 Oulès B, Del Prete D, Greco B, Zhang X, Lauritzen I, Sevalle J et al (2012) Ryanodine receptor blockade reduces amyloid-β load and memory impairments in Tg2576 mouse model of alzheimer disease. J Neurosci. ;32 Ye J, Yin Y, Yin Y, Zhang H, Wan H, Wang L et al (2020) Tau-induced upregulation of C/EBPβ-TRPC1-SOCE signaling aggravates tauopathies: A vicious cycle in Alzheimer neurodegeneration. Aging Cell. ;19 de León A, Gibon J, Barker PA (2022) APP Genetic Deficiency Alters Intracellular Ca21 Homeostasis and Delays Axonal Degeneration in Dorsal Root Ganglion Sensory Neurons. J Neurosci. ;42 Ivannikov MV, Sugimori M, Llinás RR (2010) Calcium clearance and its energy requirements in cerebellar neurons. Cell Calcium. ;47 Additional Declarations Competing interest reported. PYWM is a consultant for GenSight Biologics, Stoke Therapeutics, PYC Therapeutics and has received research support from GenSight Biologics and Chiesi. MEC is a consultant for Prime Medicine. Supplementary Files Whiteheadetal2024OPA1iPSCRGCsupplementarymaterial.pdf Cite Share Download PDF Status: Published Journal Publication published 13 Feb, 2025 Read the published version in Acta Neuropathologica Communications → Version 1 posted Editorial decision: Revision requested 06 Dec, 2024 Reviews received at journal 06 Dec, 2024 Reviews received at journal 06 Dec, 2024 Reviewers agreed at journal 15 Nov, 2024 Reviewers agreed at journal 14 Nov, 2024 Reviewers agreed at journal 30 Sep, 2024 Reviewers invited by journal 30 Sep, 2024 Editor assigned by journal 27 Sep, 2024 Submission checks completed at journal 27 Sep, 2024 First submitted to journal 25 Sep, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5153627","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":387289374,"identity":"dc4cf12d-5b70-4074-aa4d-679bc37e6aa9","order_by":0,"name":"Michael Whitehead","email":"","orcid":"","institution":"UCL Institute of Ophthalmology","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Whitehead","suffix":""},{"id":387289376,"identity":"b5263f94-f8a3-44fb-9ad8-74efeff578ef","order_by":1,"name":"Joshua P. 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Scale bar, 10 μm. (b-e) MINA analysis of mitochondrial networks. Isogenic, n=37; R445H, n=34 cells, sampled from three independent differentiations. (b) and (c), ** = p\u0026lt;0.01, Mann-Whitney tests, (d) and (e), *** = p\u0026gt;0.001, Welch's t-tests. Box plots show median (middle line), 25th-75th percentile (box) and min/max values (whiskers). (f) Representative TEM images of mitochondrial structures in isogenic control and R445H iPSC-RGCs. Scale bar, 2000 nm. (g-i) Quantification of mitochondrial area, roundness, and length, performed in ImageJ. Isogenic, n=483; R445H, n=510 ROIs. **** = p\u0026lt;0.0001, Mann-Whitney tests. Box plots show median (middle line), 25th-75th percentile (box) and min/max values (whiskers). (j \u0026amp; k) Quantification of mitochondrial cristae shape, performed in ImageJ. Isogenic, n=292; R445H n=280 ROIs. **** = p\u0026lt;0.0001, Mann-Whitney tests. Box plots show median (middle line), 25th-75th percentile (box) and min/max values (whiskers). (l) Quantification of mitochondria:ER contact sites, performed in ImageJ. Isogenic, n=350; R445H n=419 ROIs. **** = p\u0026lt;0.0001, Mann-Whitney tests. Box plots show median (middle line), 25th-75th percentile (box) and min/max values (whiskers). (m) Representative images of Mitotracker-stained mitochondria in TUJ1+ neurites. Scale bar, 5 μm. (n) Quantification of mitochondria length, performed in ImageJ. Isogenic, n=1,241 ROIs; R445H, n=625 ROIs, sampled from three independent experiments. **** = p\u0026lt;0.0001, Mann-Whitney test. Box plot shows median (middle line), 25th-75th percentile (box) and min/max values (whiskers). (o) Quantification of mitochondria distribution in TUJ1+ neurites. Isogenic/R445H, n=14 images acquired from three independent experiments. No significant difference observed between genotypes, p=0.358, Mann-Whitney test. Scatter plot shows the mean values +/- SD. (p) Quantification of mitochondrial area in TUJ1+ neurites. Isogenic, n=1,209 ROIs; R445H, n=590 ROIs, sampled from three independent experiments. **** = p\u0026lt;0.0001, Mann-Whitney test. Box plot shows median (middle line), 25th-75th percentile (box) and min/max values (whiskers).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5153627/v1/1f75a400a3b7c019c586aba7.png"},{"id":72207264,"identity":"52b88fe6-3423-4a73-ace8-12d52b33f05c","added_by":"auto","created_at":"2024-12-23 16:52:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1491519,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOxidative stress and MMP defects in OPA1 iPSC-RGCs. \u003c/strong\u003e(a) DHE fluorescence signal normalised to cell number (Hoechst fluorescence).\u0026nbsp; Isogenic/R445H, n=18 wells sampled from three independent differentiations.\u0026nbsp; **** = p\u0026lt;0.0001, Mann-Whitney test. Scatter plot shows the mean values +/- SD. (b) MitoSOX fluorescence signal normalised to cell number (Hoechst fluorescence).\u0026nbsp; Isogenic/R445H, n=18 wells sampled from three independent differentiations.\u0026nbsp; *** = p\u0026lt;0.001, unpaired t-test.\u0026nbsp; Scatter plot shows the mean values +/- SD. (c) Quantification of SOD1 Western blots.\u0026nbsp; Isogenic/R445H, n=12 sampled from three independent differentiations.\u0026nbsp; No significant differences were observed between genotypes, p=0.671, Mann-Whitney test.\u0026nbsp; Scatter plot shows the mean values +/- SD.\u0026nbsp; Western blot data is available in Supplementary Figure 1. (d) Representative live cell confocal images of MMP in TMRE-stained isogenic control and R445H iPSC-RGCs at baseline and after FCCP treatment.\u0026nbsp; Scale bar, 10 μm. (e) Quantification of MMP expressed as the fold-change in TMRE fluorescence relative to isogenic control samples.\u0026nbsp; FCCP was used as a negative staining control to subtract non-mitochondrial TMRE fluorescence.\u0026nbsp; Isogenic, n=11; R445H, n=10 images sampled from three independent differentiations.\u0026nbsp; **** = p\u0026lt;0.0001, unpaired t-test.\u0026nbsp; Scatter plot shows the mean values +/- SD. (f) Representative live cell confocal images of TMRE-stained isogenic control and R445H iPSC-RGCs at baseline, 10 and 20 min after addition of 1.5 μM oligomycin, and following FCCP treatment.\u0026nbsp; Scale bar, 10 μm. (g \u0026amp; h) Quantification of fold-change in TMRE fluorescence over time in isogenic control and R445H neurons.\u0026nbsp; Fluorescence intensity was normalised to baseline values (F\u003csub\u003e0\u003c/sub\u003e).\u0026nbsp; 1.5 μM oligomycin and 1 μM FCCP were added at the indicated time points.\u0026nbsp; FCCP was used as a negative staining control to subtract non-mitochondrial TMRE fluorescence.\u0026nbsp; Grey lines show individual cells TMRE fluorescence intensity from a representative experiment, blue/red lines the mean average normalised TMRE fluorescence intensity. (i) Quantification of endpoint TMRE fluorescence in isogenic control and R445H iPSC-RGCs prior to FCCP addition.\u0026nbsp; Isogenic/R445H, n=4 images acquired from three independent differentiations.\u0026nbsp; ** = p\u0026lt;0.01, unpaired t-test.\u0026nbsp; Scatter plot shows the mean values +/- SD.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5153627/v1/c231660dbef5ce0447c28165.png"},{"id":72206531,"identity":"7fc60dd5-14f8-4167-9525-be8bfaf4d29c","added_by":"auto","created_at":"2024-12-23 16:44:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1663444,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDysregulation of cytosolic calcium homeostasis in OPA1 iPSC-RGCs in response to ER calcium release. \u003c/strong\u003e(a) Representative live cell confocal images of cytosolic calcium levels in isogenic control and R445H neurons stained with Fluo4 at baseline, and after treatment with 1.5 μM thapsigargin and 1.2 mM CaCl\u003csub\u003e2\u003c/sub\u003e.\u0026nbsp; Scale bar, 20 μm. (b \u0026amp; c) Quantification of fold-change in Fluo4 fluorescence intensity over time normalised to baseline values, denoted F\u003csub\u003e0\u003c/sub\u003e.\u0026nbsp; 1.5 μM thapsigargin and 1.2 mM CaCl\u003csub\u003e2\u003c/sub\u003e were added at the indicated time points.\u0026nbsp; Neurons were maintained in calcium-free recording buffer until addition of CaCl\u003csub\u003e2\u003c/sub\u003e.\u0026nbsp; Grey lines show Fluo4 fluorescence intensity in individual cells from a representative experiment, blue/red lines the mean average normalised Fluo4 fluorescence intensity. (d-i) Quantification of initial slope, amplitude and decay slope of Fluo4 fluorescence intensity in response to 1.5 μM thapsigargin and 1.2 mM CaCl\u003csub\u003e2\u003c/sub\u003e in isogenic control R445H and iPSC-RGCs.\u0026nbsp; Isogenic control, n=140; R445H, n=190 cells sampled from three independent differentiations.\u0026nbsp; * = p\u0026lt;0.05; **** = p\u0026lt;0.0001, Mann-Whitney tests.\u0026nbsp; Box plots show median (middle line), 25th-75th percentile (box) and min/max values (whiskers).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5153627/v1/095b86c235864daa36b6e528.png"},{"id":72206533,"identity":"34580510-fb0f-4011-b0f4-f547fab84323","added_by":"auto","created_at":"2024-12-23 16:44:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1977075,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCytosolic calcium homeostasis is dysregulated in response to the excitatory neurotransmitter glutamate in OPA1 iPSC-RGCs. \u003c/strong\u003e(a) Representative live cell confocal images of cytosolic calcium levels in isogenic control and R445H neurons stained with Fluo4 at baseline, and after stimulation with 5 µM glutamate and 10 µM ionomycin.\u0026nbsp; Scale bar, 20 μm. (b \u0026amp; c) Quantification of fold-change in Fluo4 fluorescence intensity over time normalised to baseline values, denoted F\u003csub\u003e0\u003c/sub\u003e.\u0026nbsp; 5 μM glutamate and 10 µM ionomycin were added at the indicated time points.\u0026nbsp; Neurons were maintained in magnesium-free recording buffer throughout the experiment.\u0026nbsp; Grey lines show Fluo4 fluorescence intensity in individual cells from a representative experiment, blue/red lines the mean average normalised Fluo4 fluorescence intensity. (d-f) Quantification of initial slope, amplitude and decay slope of Fluo4 fluorescence intensity in response to 5 µM glutamate in isogenic control R445H and iPSC-RGCs.\u0026nbsp; Isogenic control, n=167; R445H, n=210 cells sampled from three independent differentiations.\u0026nbsp; **** = p\u0026lt;0.0001, Mann-Whitney tests.\u0026nbsp; Box plots show median (middle line), 25th-75th percentile (box) and min/max values (whiskers).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5153627/v1/39ae12791766b2bea6950948.png"},{"id":72207263,"identity":"0fc5a817-8953-4b11-93c0-999713875c89","added_by":"auto","created_at":"2024-12-23 16:52:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3426635,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIncreased sensitivity to opening of the mPTP in OPA1 iPSC-RGCs in response to mitochondrial calcium overload. \u003c/strong\u003e(a) Representative live cell confocal images of the MMP in Mitotracker-stained isogenic control and R445H iPSC-RGCs at baseline and after treatment with 12.5 µM and 20 µM ferutinin.\u0026nbsp; Scale bar, 20 μm. (b \u0026amp; c) Quantification of fold-change in Mitotracker fluorescence over time in response to mitochondrial calcium overload in isogenic control and R445H neurons.\u0026nbsp; Fluorescence intensity was normalised to baseline values (F\u003csub\u003e0\u003c/sub\u003e).\u0026nbsp; Ferutinin concentration was increased in 2.5 µM increments at the indicated time points indicated by the arrows.\u0026nbsp; Loss of Mitotracker fluorescence indicates mitochondrial depolarisation due to opening of the mPTP (grey box). (d) Quantification of ferutinin concentration required to open the mPTP.\u0026nbsp; Isogenic control/R445H, n=7 experiments sampled from four independent differentiations.\u0026nbsp; ** = p\u0026lt;0.01, unpaired t-test.\u0026nbsp; Scatter plot shows the mean values +/- SD.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5153627/v1/6f9ff92bdcd23bca87cd050d.png"},{"id":76487708,"identity":"320760cc-5687-443a-9b33-11642f58562e","added_by":"auto","created_at":"2025-02-17 16:11:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10311591,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5153627/v1/a5c169e3-e178-48fb-9767-c4df1d95a412.pdf"},{"id":72206534,"identity":"78d3b82e-bd9a-41d3-bc87-d288075832eb","added_by":"auto","created_at":"2024-12-23 16:44:02","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":2044601,"visible":true,"origin":"","legend":"","description":"","filename":"Whiteheadetal2024OPA1iPSCRGCsupplementarymaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5153627/v1/dae91f6a51f829700cbad2ed.pdf"}],"financialInterests":"Competing interest reported. PYWM is a consultant for GenSight Biologics, Stoke Therapeutics, PYC Therapeutics and has received research support from GenSight Biologics and Chiesi. MEC is a consultant for Prime Medicine.","formattedTitle":"Disruption of mitochondrial homeostasis and permeability transition pore opening in OPA1 iPSC-derived retinal ganglion cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDominant optic atrophy (DOA) is the most common inherited optic neuropathy, with an estimated minimum prevalence of 1 in 25,000 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Clinically, DOA usually presents in the first two decades of life, characterised by bilateral progressive loss of central vision, dyschromatopsia and the development of optic disc pallor [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Optical coherence tomography (OCT) studies have demonstrated thinning of the retinal nerve fibre layer (RNFL), in particular the papillomacular bundle, highlighting the preferential loss of retinal ganglion cells (RGCs) in DOA pathogenesis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAround 60% of people with DOA have pathogenic \u003cem\u003eOPA1\u003c/em\u003e variants [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. OPA1 is a dynamin-related GTPase protein that localises to the inner mitochondrial membrane (IMM) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. OPA1 is ubiquitously expressed, and whilst loss of OPA1 function appears to primarily affect RGC function in DOA, approximately 20% of DOA patients exhibit a more severe syndromic \u0026lsquo;DOA+\u0026rsquo; phenotype, characterised by multisystem neurodegeneration. These patients exhibit a broad range of neurological defects, including sensorineural hearing loss, ataxia, peripheral neuropathy, and myopathy [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. At the molecular level, DOA\u0026thinsp;+\u0026thinsp;is often associated with missense variants in the GTPase domain and this could be due to a dominant negative effect in which dysfunctional, pathogenic OPA1 variants impair WT OPA1 protein function, driving a more severe clinical phenotype [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOPA1 maintains mitochondrial network dynamics by facilitating fusion of the IMM, possibly through a mechanism involving cardiolipin [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. As such, loss of OPA1 function has been associated with defects in mitochondrial network morphology [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. OPA1 also maintains mitochondrial cristae shape, and loss of OPA1 function has been correlated with aberrant cristae architecture [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and cytochrome \u003cem\u003ec\u003c/em\u003e release [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], highlighting a possible pathogenic mechanism underpinning RGC cell death in DOA. Reduced OPA1 function causes impaired function of the electron transport chain (ETC), with reduced oxygen consumption [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and a reduced mitochondrial membrane potential (MMP) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. These bioenergetic defects may indirectly sensitise RGCs to cell death in DOA via autophagic [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] or excitotoxic [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] mechanisms, possibly via ATP depletion. OPA1 also participates in the maintenance of mitochondrial DNA (mtDNA) integrity, with a postulated role in anchoring mtDNA molecules to the IMM [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], however, distinct effects of attenuated OPA1 dysfunction on mtDNA have been reported across different tissues/cell types [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOPA1 dysfunction may impact calcium homeostasis in a cell type-dependent manner. Reports that OPA1 downregulation enhanced mitochondrial calcium uptake in patient-derived fibroblasts [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] conflicted with reports that siRNA-mediated \u003cem\u003eOPA1\u003c/em\u003e knock-down reduced mitochondrial calcium uptake in HeLa cells [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Whilst pro-opiomelanocortin neurons in which OPA1 protein levels were experimentally knocked down displayed unaltered cytosolic, but attenuated mitochondrial calcium transients [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], siRNA-mediated OPA1 knockdown in rat RGCs led to delayed calcium deregulation (DCD) in response to excitotoxic stress, an event that preceded cell death [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Furthermore, reduced OPA1 function increased basal cytosolic calcium levels, leading to cell death in mouse RGCs and \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e GABAergic motor neurons transfected with plasmids containing pathogenic variants of \u003cem\u003eOPA1\u003c/em\u003e [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. These findings highlight the importance of studying the effect of OPA1 dysfunction on calcium homeostasis in human RGCs to better understand pathophysiological mechanisms in DOA patients.\u003c/p\u003e \u003cp\u003eHere, we used DOA patient-derived and CRISPR-Cas9-corrected iPSC-RGCs to study the effect of OPA1 dysfunction in human RGCs. Changes in mitochondrial structure, network morphology and reactive oxygen species were assessed. The effect on calcium homeostasis was also investigated, as well as the mechanism of maintenance of the MMP, and the threshold for opening of the mitochondrial permeability transition pore (mPTP) in response to mitochondrial calcium overload.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGeneration, maintenance and differentiation of iPSC lines\u003c/h2\u003e \u003cp\u003ePatient-derived fibroblasts carrying the c.1334 G\u0026thinsp;\u0026gt;\u0026thinsp;A (p. R445H) substitution were reprogrammed to iPSCs and isogenic controls were created using CRISPR-Cas9 gene editing as described previously [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. iPSCs were maintained on Geltrex-coated plates in mTeSR media (Stem Cell Technologies) at 37\u0026deg;C 5% CO\u003csub\u003e2\u003c/sub\u003e. For passaging, iPSC colonies were manually dissociated twice weekly. iPSCs were differentiated to iPSC-RGCs as previously described using a 42 day directed differentiation protocol [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunocytochemistry\u003c/h3\u003e\n\u003cp\u003eTo measure mitochondrial network dynamics, 25 nM Mitotracker Orange (Thermo Fisher Scientific) was loaded for 30 min at 37\u0026deg;C 5% CO\u003csub\u003e2\u003c/sub\u003e in recording buffer (150 mM NaCl, 4.25 mM KCl, 4 mM NaHCO\u003csub\u003e3\u003c/sub\u003e, 1.25 mM NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 1.2 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 1.2 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 10 mM D-glucose, and 10 mM HEPES at pH 7.4). Neurons were washed, then fixed in 4% paraformaldehyde for 15 min. When assessing network dynamics in neuronal cell bodies, cells were washed and immediately mounted onto a glass slide with Fluoromount (Dako). Mitotracker Orange was excited at 555 nm and a\u0026thinsp;\u0026ge;\u0026thinsp;580 nm emission filter was used. Leica Lightning software was used for image deconvolution. ImageJ plugin MINA Version 3 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://imagej.net/plugins/mina\u003c/span\u003e\u003cspan address=\"https://imagej.net/plugins/mina\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used for quantification. Mitochondrial footprint\u0026thinsp;=\u0026thinsp;volume of the mitochondrial signal; branch length mean\u0026thinsp;=\u0026thinsp;mean length of all the lines used to represent the mitochondrial structure; summed branch length mean\u0026thinsp;=\u0026thinsp;sum of all branch lengths divided by the number of independent skeletons; network branches mean\u0026thinsp;=\u0026thinsp;mean number of attached lines used to represent each structure. To assay mitochondrial length/distribution in neurites, after fixation and washing, neurons were incubated in blocking solution (10% normal donkey serum, 1% bovine serum albumin) for 1h, then incubated overnight at 4\u0026deg;C with rabbit anti-TUJ1 antibodies (Abcam) in 50% blocking buffer diluted in PBS. Anti-rabbit AlexaFluor 488 secondary antibodies (Thermo Fisher Scientific) were incubated for 2h at room temperature, before mounting the cells onto a glass slide. Images were acquired on a Stellaris 8 confocal microscope equipped with a 40x oil objective.\u003c/p\u003e\n\u003ch3\u003eDetection of reactive oxygen species\u003c/h3\u003e\n\u003cp\u003eiPSC-RGCs were plated in 96 well black-walled plates (Thermo Fisher Scientific). After washing with recording buffer, neurons were stained with 5 \u0026micro;M dihydroethidium (DHE; Thermo Fisher Scientific) or 5 \u0026micro;M MitoSOX (Thermo Fisher Scientific) for 30 min at 37\u0026deg;C 5% CO\u003csub\u003e2\u003c/sub\u003e in recording buffer. Assessment of DHE/MitoSOX fluorescence was performed on a Cytation 10 microplate reader (Agilent). DHE was excited at 518 +/- 20 nm, emission 606 +/- 20 nm. MitoSOX was excited at 535 +/- 20 nm, emission 585 +/- 20 nm. Hoechst dye was added at 10 \u0026micro;g/mL for 10 min at the end of the experiment to normalise DHE/MitoSOX signals to total cell number. Hoechst was excited at 350 +/- 20 nm, emission 450 +/- 20 nm.\u003c/p\u003e\n\u003ch3\u003eWestern blotting\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eWestern blotting\u003c/div\u003e \u003cp\u003eiPSC-RGCs were lysed in RIPA buffer (1% NP-40, 20 mM Tris-HCl, 5 mM sodium pyrophosphate, 5 mM EDTA) with 2% protease inhibitor cocktail (Sigma Aldrich). 5 \u0026micro;g of protein was loaded into 10% polyacrylamide gels, resolved at 80V for 2h, then transferred onto nitrocellulose membranes at 90V for 90 min. Membranes were blocked with 5% milk powder (diluted in PBS 0.2% Tween-20) (Sigma Aldrich) for 1h, then incubated with primary antibodies (Santa Cruz sc-17767 mouse anti-SOD1; 1:100 dilution, Abcam 18207 rabbit anti-TUJ1 1:5,000 dilution, or Proteintech 60004-1 mouse anti-GAPDH 1:8,000 dilution) overnight at 4\u0026deg;C. Membranes were incubated with HRP-conjugated goat anti-mouse secondaries for 1h at room temperature, bands were visualised with ECL Clarity Substrate (Biorad) and a Chemidoc imaging system (Biorad). Quantification of band intensity was performed in ImageLab (BioRad). Uncropped blots are available in Supplementary Figures.\u003c/p\u003e\n\u003ch3\u003eLive cell confocal imaging experiments\u003c/h3\u003e\n\u003cp\u003eFor live imaging assessments, iPSC-RGCs were plated on 35 mm Fluorodishes (World Precision Instruments). All live imaging experiments were performed with cells incubated in recording buffer, with two exceptions: for glutamate stimulation, MgCl\u003csub\u003e2\u003c/sub\u003e was removed to maximise activation of N-methyl D-aspartate (NMDA) receptors; for ER calcium release/SOCE, cells were kept in calcium-free recording buffer until the introduction of 1.2 mM CaCl\u003csub\u003e2\u003c/sub\u003e at the end of the experiment. iPSC-RGCs were maintained at 37\u0026deg;C 5% CO\u003csub\u003e2\u003c/sub\u003e throughout image acquisition. All confocal imaging was performed on a Stellaris 8 microscope equipped with a 20x dry or 40x oil objective.\u003c/p\u003e \u003cp\u003eFor measurement of cytosolic calcium levels, iPSC-RGCs were loaded with 1.5 \u0026micro;M Fluo4 AM (Thermo Fisher Scientific) for 30 min at 37\u0026deg;C 5% CO\u003csub\u003e2,\u003c/sub\u003e then washed twice to remove any residual Fluo4 AM. iPSC-RGCs were exposed to 1 \u0026micro;M thapsigargin (Selleck Chemicals), 1.2 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 5 \u0026micro;M glutamate (Sigma Aldrich), or 10 \u0026micro;M ionomycin (Sigma Aldrich) at the indicated time points. To assay MMP, iPSC-RGCs were loaded with 5 nM tetramethylrhodamine ethyl ester (TMRE; Thermo Fisher Scientific) for 30 min at 37\u0026deg;C 5% CO\u003csub\u003e2\u003c/sub\u003e. 5 nM TMRE was kept in the recording buffer throughout the experiment. iPSC-RGCs were incubated with 1.5 \u0026micro;M oligomycin (Sigma Aldrich) or ascending doses (2.5\u0026ndash;17.5 \u0026micro;M; 2.5 \u0026micro;M each step) of ferutinin (Sigma Aldrich) at the indicated time points. Fluo4 was excited at 488 nm, collecting light longer than 520 nm, whilst TMRE was excited at 555 nm, collecting light longer than 580 nm. These experiments were performed at 0.1% laser strength to minimise the effect of photobleaching/oxidative damage. Analysis of Fluo4/TMRE fluorescence was performed in ImageJ.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eTransmission electron microscopy imaging\u003c/h2\u003e \u003cp\u003eCells were fixed by adding 4% PFA and 4% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.4 to the cell culture media at 1:1 and left for 1 h at room temperature. The cells were washed in 0.1 M cacodylate buffer before incubating in 1% osmium tetroxide and 1.5% potassium ferrocyanide in distilled water for 1 h in the dark at 4\u0026deg;C. En bloc staining was performed by incubating the cells in UA-Zero (Agar Scientific, Stansted, UK) for 1 h in the dark at room temperature. Subsequently, the cells were dehydrated in increasing concentrations of ethanol (70%, 90%, and 100%) followed by a mixture of propylene oxide:epon (1:1) overnight at room temperature. The propylene oxide:epon was replaced with two changes of epon every 3 h at room temperature before embedding in epon overnight at 60\u0026deg;C. 100nm sections were cut and imaged on a JEOL 1400Plus EM (JEOL ltd, Tokyo, Japan) fitted with an Advanced Microscopy Technologies (AMT) NanoSprint12 (AMT Imaging Direct, Woburn, MA, USA) camera. Analysis of TEM imaging was performed in ImageJ. Mito/cristae circularity\u0026thinsp;=\u0026thinsp;4π*area/perimeter^2.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eDisturbed mitochondrial structures in DOA iPSC-RGCs\u003c/h2\u003e \u003cp\u003eOPA1 participates in the maintenance of mitochondrial network morphology [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. We sought to establish whether OPA1 dysfunction was associated with changes in the structure of the mitochondrial network in iPSC-RGC cell bodies, and mitochondrial length in iPSC-RGC neurites, using Mitotracker dyes to delineate mitochondrial structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Neuronal soma with the R445H pathogenic OPA1 variant (derived from a DOA\u0026thinsp;+\u0026thinsp;patient) displayed a 1.5-fold increased mitochondrial footprint compared to corrected isogenic controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Conversely, mean branch length, mean summed branch length, and mean network branches were decreased in RGCs carrying the R445H pathogenic variant compared to isogenic iPSC-RGCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec-e), suggesting fragmentation of mitochondrial networks is associated with OPA1 dysfunction in human RGCs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo extend these data, mitochondrial ultrastructure was assessed with transmission electron microscopy (TEM) imaging (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). The area and length of individual mitochondria were reduced, whilst roundness was increased in R445H iPSC-RGCs compared to isogenic controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg-i). These data again support fragmentation of mitochondrial structures in cells with pathogenic \u003cem\u003eOPA1\u003c/em\u003e variants. Mitochondrial cristae architecture was also altered between R445H and isogenic control iPSC-RGCs. In R445H neurons, mitochondrial cristae more commonly displayed a vesicular architecture compared to isogenic control cells. In support of this, cristae area was reduced in R445H cells, whilst circularity was increased compared to isogenic control samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ej \u0026amp; k)\u003c/p\u003e \u003cp\u003eWe then tested if OPA1 dysfunction was associated with changes in mitochondria:ER membrane contact (MERC) length in our iPSC-RGC model, reasoning that similar defects have been observed across a range of neuropathologies [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], and observed that contact lengths were reduced in R445H vs. isogenic control iPSC-RGCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003el).\u003c/p\u003e \u003cp\u003eiPSC-RGC neurite mitochondrial morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003em) was investigated, and it was observed that R445H neurite mitochondrial length was decreased compared to isogenic control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003en). A statistically significant change was not observed between genotypes in the distribution of mitochondria in iPSC-RGC neurites (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eo); however, mitochondrial area in TUJ1\u0026thinsp;+\u0026thinsp;neurites was significantly lower in R445H iPSC-RGCs compared to isogenic controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ep). Consistent with the role of OPA1 as a pro-fusion factor, collectively these data demonstrate fragmentation of mitochondrial networks and highlight potential changes in mitochondrial cristae and MERCs in DOA iPSC-RGCs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eOxidative stress and MMP defects in DOA iPSC-RGCs\u003c/h2\u003e \u003cp\u003eWe previously reported a bioenergetic defect in iPSC-RGCs harbouring pathogenic \u003cem\u003eOPA1\u003c/em\u003e variants [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Mitochondrial dysfunction in neurodegenerative disease is often associated with increased generation of reactive oxygen species (ROS) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], in particular mitochondrial superoxide production arising from ETC dysfunction [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Oxidative stress sensitises mitochondria to opening of the mPTP, an event that leads to cell death [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. We therefore investigated whether superoxide levels and corresponding antioxidant defence mechanisms were affected by OPA1 dysfunction in iPSC-RGCs. DHE fluorescence was 1.7-fold higher in R445H compared to isogenic control neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Similarly, MitoSOX fluorescence was 1.2-fold higher in cells harbouring the pathogenic OPA1 variant (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). These results suggest that oxidative stress arises from mitochondrial dysfunction in OPA1-DOA. We also examined antioxidant defence mechanisms by measuring SOD1 protein levels by Western blotting. SOD1 levels (which localises to the cytosol and mitochondrial intermembrane space (IMS)) were not significantly different between genotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec \u0026amp; Supplementary Fig.\u0026nbsp;1).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDepolarisation of MMP may result from dysfunction of the ETC, which usually pumps protons into the IMS to sustain the electrochemical gradient that drives ATP synthesis. As there is a bioenergetic defect in DOA iPSC-RGCs [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], we measured MMP in iPSC-RGCs using TMRE, a cationic fluorescent dye that localises to the mitochondrial matrix in response to the potential difference (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). iPSC-RGCs harbouring the R445H variant showed a decrease in TMRE fluorescence compared with isogenic control neurons, suggesting partial depolarisation of MMP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003eATP synthase utilises the electrochemical gradient generated by the ETC to synthesise ATP during oxidative phosphorylation [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. However, if the free energy available from the membrane potential fails to balance the free energy available from the ATP/ADP ratio, the ATP synthase will reverse to act as a proton pumping ATPase, consuming (glycolytic) ATP and maintaining a membrane potential [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. As such, ATP hydrolysis has been proposed as a mechanism that maintains MMP in bioenergetically compromised neurons and has been described in a range of mitochondrial disease models [\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. We therefore tested whether OPA1 dysfunction affects ATP hydrolysis in iPSC-RGCs using TMRE and the ATP synthase inhibitor oligomycin (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). In isogenic control neurons, oligomycin treatment elicited an increase in TMRE fluorescence over time, indicating gradual hyperpolarisation of MMP as expected (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg). By contrast, in R445H iPSC-RGCs TMRE fluorescence decayed in response to ATP synthase inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh). At the endpoint of the experiment, TMRE fluorescence was decreased by 43% in R445H compared to isogenic control neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei), showing that MMP is partially maintained by ATP hydrolysis in DOA iPSC-RGCs. These findings also highlight ETC dysfunction (i.e. reduced proton pumping by complexes I, III and IV) associated with OPA1 dysfunction in accordance with our previous data [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAltered cytosolic calcium handling in DOA iPSC-RGCs in response to ER calcium release\u003c/h2\u003e \u003cp\u003eCalcium signalling plays a key role in neuronal function, including regulation of gene expression [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], synaptic function [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], and mitochondrial metabolism [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. To study calcium signalling dynamics in iPSC-RGCs, calcium uptake into the ER was inhibited with thapsigargin, an irreversible inhibitor of sarcoplasmic/endoplasmic reticulum Ca\u003csup\u003e2+\u003c/sup\u003e-ATPase (SERCA), in calcium-free recording buffer. As this results in depletion of ER stores, reintroduction of calcium into the recording buffer enables the assessment of store-operated calcium entry (SOCE), in which stromal interaction molecules (STIM1/2) and ORAI channel (ORAI1-ORAI3) complexes, and transient receptor potential channels (TRPC1-TRPC7) at the plasma membrane facilitate extracellular calcium influx to the cytosol in an attempt to refill ER calcium stores [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAfter treatment with thapsigargin, R445H iPSC-RGCs displayed an elevated initial slope (2.9-fold increase) and amplitude (3.7-fold increase) of Fluo4 fluorescence compared to isogenic control neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-e). The rate of peak Fluo4 fluorescence recovery to baseline levels was higher in R445H iPSC-RGCs compared to isogenic controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-c \u0026amp; f), indicating faster calcium clearance after reaching peak levels. These data suggest that OPA1 dysfunction is associated with altered cytosolic calcium handling upon SERCA blockade and the release of calcium from the ER.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter store depletion, reintroduction of calcium to the recording buffer led to a 1.9-fold elevation of amplitude of Fluo4 fluorescence in R445H iPSC-RGCs compared to isogenic controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-c \u0026amp; g \u0026amp; h), indicating higher levels of SOCE associated with OPA1 dysfunction. The rate of Fluo4 fluorescence decay after the Ca\u003csup\u003e2+\u003c/sup\u003e influx was also increased in R445H iPSC-RGCs compared to isogenic controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-c \u0026amp; i), similar to the pattern seen after thapsigargin treatment, indicating faster calcium clearance after reaching peak levels.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAltered cytosolic calcium handling in DOA iPSC-RGCs in response to physiological glutamate challenge\u003c/h2\u003e \u003cp\u003eWe then tested the hypothesis that OPA1 dysfunction would lead to changes in calcium handling following stimulation with the excitatory neurotransmitter glutamate. Glutamate activates the influx of extracellular calcium into the cytosol, whereby the opening of ionotropic NMDA receptors in particular mediates a significant increase in intracellular calcium levels [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Mitochondria act to buffer elevations in cytosolic calcium, utilising the electrochemical gradient generated by the ETC to draw calcium ions into the matrix via the mitochondrial calcium uniporter (MCU) [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eR445H iPSC-RGCs exhibited an elevated initial slope (2.6-fold) and amplitude (1.9-fold) of Fluo4 fluorescence upon glutamate challenge compared to isogenic controls, suggesting that this excitatory pathway may lead to a faster accumulation and higher level of calcium in the cytosol (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-e). The rate of Fluo4 fluorescence decay was lower in R445H compared to isogenic neurons. Whilst isogenic control iPSC-RGCs displayed a gradual decrease in Fluo4 fluorescence after the peak, suggesting restoration of cytosolic calcium homeostasis, R445H neuron Fluo4 fluorescence remained largely at peak levels, suggesting cytosolic calcium homeostasis could not be restored (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-c \u0026amp; f). These data suggest OPA1 dysfunction limits the capacity of human RGCs to restore calcium homeostasis in response to excitotoxic stress. In summary, these data suggest OPA1 dysfunction in iPSC-RGCs leads to perturbations in cytosolic calcium handling in response to SERCA blockade and SOCE, and after treatment with glutamate.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eIncreased sensitivity to opening of the mPTP in DOA iPSC-RGCs\u003c/h2\u003e \u003cp\u003eMitochondria play a key role in regulating intracellular calcium signalling under normal conditions, but excessive mitochondrial calcium uptake may contribute to pathology by facilitating the opening of the mPTP, a process thought to be exacerbated by oxidative stress [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. mPTP opening is associated with the collapse of MMP and cessation of ATP synthesis, and is therefore a catastrophic event for neurons that depend mainly on oxidative phosphorylation to meet their energy requirements [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. To test the threshold for mPTP opening, we challenged R445H and isogenic control iPSC-RGCs with ascending doses of ferutinin, an electrogenic calcium ionophore that increases mitochondrial calcium levels, eventually leading to mitochondrial calcium overload and mPTP opening [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe collapse of MMP was measured as a readout for mPTP opening, and the concentration of ferutinin required to induce a rapid loss of MMP was significantly lower in R445H iPSC-RGCs compared to isogenic controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-d; R445H, ~\u0026thinsp;12.5 \u0026micro;M vs isogenic, 15.0-17.5 \u0026micro;M ferutinin). These data suggest that the threshold for mPTP opening may be reduced by OPA1 dysfunction in iPSC-RGCs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe results presented in this study suggest that OPA1 dysfunction leads to defects in mitochondrial structure, ETC function, MMP, calcium signalling, and mPTP opening in human RGCs, highlighting pathophysiological mechanisms that could contribute to vision loss in DOA patients.\u003c/p\u003e \u003cp\u003eConsistent with the role of OPA1 in facilitating fusion of the IMM [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], OPA1 neurons displayed fragmented mitochondrial networks in RGC somas and shorter neurite mitochondria compared to isogenic control neurons, data which were corroborated by EM ultrastructural analysis. OPA1 dysfunction was also associated with increased mitochondrial footprint in RGC somas and decreased neurite mitochondrial area, but not a statistically significant change in neurite mitochondria distribution, which may have been mediated by differences in mitochondrial anterograde/retrograde transport [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], or mitochondrial quality control mechanisms [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe also detected increased levels of ROS generation, specifically superoxide anions, in DOA iPSC-RGCs. These results are in accordance with previous reports of the effect of OPA1 dysfunction in fibroblasts, in which galactose was used in place of glucose in the cell media to force ATP generation by oxidative phosphorylation [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. A recent study reported increased superoxide levels arising from attenuated OPA1 function in primary vascular cells, with reduced SOD1 protein levels [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], however, we did not detect a statistically significant decrease in SOD1 protein levels in iPSC-RGCs, likely reflecting the different cell types under investigation in the respective studies.\u003c/p\u003e \u003cp\u003eWe previously showed bioenergetic defects, in iPSC-RGCs harbouring pathogenic \u003cem\u003eOPA1\u003c/em\u003e variants suggesting dysfunction of the ETC [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In this study, we identified morphological changes in mitochondrial cristae, specifically, the appearance of vesicular cristae in R445H iPSC-RGCs. This structural deficit was associated with a partial depolarisation and maintenance of MMP by ATP hydrolysis in R445H vs. isogenic control iPSC-RGCs, a process that consumes ATP and, therefore, likely reduces the energy supply available for other cellular functions. It remains to be determined whether inhibition of ATP hydrolysis (whilst maintaining ATP synthesis) could be utilised as a potential therapeutic tool in DOA as described in other mitochondrial disease models [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], or if it would accelerate mitochondrial depolarisation and dysfunction.\u003c/p\u003e \u003cp\u003eCytosolic calcium handling is mediated by a range of mechanisms, including buffering into/release from intracellular stores and influx/efflux mechanisms that operate at the cell surface [\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. We observed that neurons with the R445H pathogenic variant exhibited increased calcium release when ER calcium uptake was inhibited, and increased levels of cytosolic calcium after initiation of SOCE. Differences in resting ER calcium levels and/or the activity of the inositol 1,4,5-triphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) which mediate calcium release from the ER could explain these results, however, buffering of cytosolic calcium into the (depolarised) mitochondrial matrix may also have played a role. Given we observed reduced MERC length in DOA iPSC-RGCs, it is possible that changes in calcium transfer between mitochondria and ER compartments contribute to calcium deregulation in DOA. OPA1 neurons demonstrated upregulated SOCE which could reflect an attempt to refill ER calcium stores, a pathway that has been described in other neurodegenerative disease models [\u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. We observed an increased rate of Fluo4 fluorescence decay in R445H compared to corrected iPSC-RGCs following both thapsigargin and CaCl\u003csub\u003e2\u003c/sub\u003e treatment, suggesting an increase in cytosolic calcium clearance. As this effect is unlikely to be explained by increased ER/mitochondrial calcium uptake, this may reflect the activity of other cytosolic calcium efflux mechanisms, such as Ca\u003csup\u003e2+\u003c/sup\u003e/Na\u003csup\u003e+\u003c/sup\u003e exchangers (NCX) and/or plasma membrane Ca\u003csup\u003e2+\u003c/sup\u003e ATPases (PMCA) operating at the cell membrane [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMitochondria serve as buffers to increased cytosolic calcium levels, a process that is dependent on the MMP and MCU activity [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. As such, the elevation of cytosolic calcium in DOA iPSC-RGCs observed after ER store release and SOCE may be partially explained by reduced uptake of calcium into the mitochondrial matrix, possibly due to MMP depolarisation. In support of this, R445H iPSC-RGCs demonstrated increased Fluo4 fluorescence initial slopes and amplitudes compared to isogenic controls after treatment with glutamate, which might be a consequence of reduced buffering of cytosolic calcium by dysfunctional mitochondria. It is possible that changes in NMDA receptor subunit expression, which have been suggested in previous reports on OPA1 dysfunction [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], could also explain the differences we observed. In contrast to the patterns evident after ER calcium release and SOCE, Fluo4 fluorescence remained elevated in R445H iPSC-RGCs after reaching peak levels. This suggests that calcium clearance is attenuated after glutamate challenge in human RGCs with OPA1 dysfunction, in agreement with previous reports detailing the induction of DCD in OPA1 mouse RGCs after glutamate treatment [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In summary, these investigations into calcium handling identified defects associated with OPA1 dysfunction in iPSC-RGCs, in which both the ER and mitochondrial cellular compartments could play a role.\u003c/p\u003e \u003cp\u003emPTP opening is associated with the collapse of MMP and cessation of ATP synthesis. We challenged cells with ascending doses of ferutinin, an electrogenic calcium ionophore, to induce mitochondrial calcium overload and assayed loss of MMP as a biomarker for mPTP opening. We found that ferutinin induced mPTP formation at lower concentrations in R445H iPSC-RGCs compared to isogenic control neurons. The threshold for mPTP opening is known to decrease when matrix calcium and ROS levels are high [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Given that we have identified changes in cytosolic calcium handling and increased oxidative stress in DOA iPSC-RGCs, it is possible that OPA1 dysfunction sensitises RGCs to mPTP opening via mitochondrial calcium deregulation and increased superoxide production.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, this study identified several structural and functional defects associated with OPA1 dysfunction in human RGCs. OPA1 neurons exhibited fragmented mitochondrial networks, oxidative stress and MMP defects. Cytosolic calcium handling was also affected, and the threshold for mPTP opening was reduced. Collectively, these mechanisms may contribute to progressive vision loss in DOA patients.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eATP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eadenosine triphosphate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCRISPR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eclustered regularly interspaced short palindromic repeats\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDCD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edelayed calcium deregulation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDHE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edihydroethidium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDOA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edominant optic atrophy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDOA+\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edominant optic atrophy plus\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eECL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eenhanced chemiluminescence\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eER\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eendoplasmic reticulum\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eETC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eelectron transfer chain\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGABA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003egamma aminobutyric acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGTP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eguanosine triphosphate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHEPES2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e(4\u0026ndash;(2\u0026ndash;hydroxyethyl)\u0026ndash;1\u0026ndash;piperazinyl)\u0026ndash;ethanesulfonic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHRP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehorse radish peroxidase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIMM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003einner mitochondrial membrane\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIMS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003einter membrane space\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eiPSC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003einduced pluripotent stem cell\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIP3R\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003einositol 1,4,5\u0026ndash;trisphosphate receptor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMCU\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emitochondrial calcium uniporter\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMERC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emitochondria:endoplasmic reticulum contact\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMMP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emitochondrial membrane potential\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003emtDNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emitochondrial DNA\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNCX\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esodium calcium exchanger\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNCLX\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emitochondrial sodium calcium exchanger\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNMDA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eN\u0026ndash;methyl D\u0026ndash;aspartic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOCT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eoptical coherence tomography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOPA1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eoptic atrophy 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eORAI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecalcium release\u0026ndash;activated calcium modulator 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ephosphate buffered saline\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePMCA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eplasma membrane calcium ATPase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRGC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eretinal ganglion cell\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRNFL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eretinal nerve fibre layer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eROS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ereactive oxygen species\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRyR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eryanodine receptors\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSERCA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esarcoplasmic endoplasmic reticulum\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003esiRNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esmall interfering RNA\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSOD1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esuperoxide dismutase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSOCE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003estore operated calcium entry\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSTIM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003estromal interaction molecule\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTMRE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etetramethylrhodamine ethyl ester\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTRPC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etransient receptor potential channel\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTUJ1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eβ3\u0026ndash;tubulin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003eFor the use of patient-derived fibroblasts in this study, informed consent was obtained following the tenets of the Declaration of Helsinki. Ethical approval was granted by the Yorkshire and The Humber-Leeds Bradford Research Ethics Committee (REC reference: 13/YH/0310).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable\u003c/p\u003e \u003c/p\u003e\u003cp\u003e\u003ch2\u003eCompeting Interests\u003c/h2\u003e\u003cp\u003ePYWM is a consultant for GenSight Biologics, Stoke Therapeutics, PYC Therapeutics and has received research support from GenSight Biologics and Chiesi. MEC is a consultant for Prime Medicine.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eMW was supported by a grant from Moorfields Eye Charity (to MEC, YJS and PYWM), and the National Institute for Health and Care Research Biomedical Research Centre at Moorfields Eye Hospital and UCL Institute of Ophthalmology. TB receives funding from MRC Career Development Award grant (MR/X020827/1). MEC received funding from Wellcome Trust Investigator Award (205041), Foundation Fighting Blindness (USA), Fight for Sight, Moorfields Eye Charity, the Macular Society and the NIHR Moorfields Biomedical Research Centre. PYWM is supported by an NIHR Advanced Fellowship (NIHR301696), NIHR Cambridge Biomedical Research Centre (NIHR203312), NIHR Biomedical Research Centre based at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology (NIHR203322), and the LifeArc Centre for Rare Mitochondrial Diseases.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eMW designed the study, conducted the majority of the experimental work and analysis, and drafted the manuscript. PES established the iPSC lines and differentiation protocol. JPH and GB assisted with the experimental work. TB processed samples for TEM imaging. TB, YJS, KS, MD, PYWM and MEC helped design the study, and provided feedback on data analysis and interpretation. MEC, YJS and PYWM secured funding for the research. All co-authors provided feedback on the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors would like to acknowledge the UCL Institute of Ophthalmology Imaging Department for their assistance with the TEM imaging.\u003c/p\u003e\u003ch2\u003eAvailability of data and material\u003c/h2\u003e \u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHarvey JP, Sladen PE, Yu-Wai-Man P, Cheetham ME (2022) Induced Pluripotent Stem Cells for Inherited Optic Neuropathies - Disease Modeling and Therapeutic Development. J. 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Cell Calcium. ;47\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"acta-neuropathologica-communications","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"anec","sideBox":"Learn more about [Acta Neuropathologica Communications](https://actaneurocomms.biomedcentral.com/)","snPcode":"40478","submissionUrl":"https://submission.springernature.com/new-submission/40478/3","title":"Acta Neuropathologica Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Dominant optic atrophy, OPA1, iPSCs, retinal ganglion cells, neurodegeneration, mitochondrial networks, calcium homeostasis","lastPublishedDoi":"10.21203/rs.3.rs-5153627/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5153627/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDominant optic atrophy (DOA) is the most common inherited optic neuropathy, characterised by the selective loss of retinal ganglion cells (RGCs). Over 60% of DOA cases are caused by pathogenic variants in the \u003cem\u003eOPA1\u003c/em\u003e gene, which encodes a dynamin-related GTPase protein. OPA1 plays a key role in the maintenance of the mitochondrial network, mitochondrial DNA integrity and bioenergetic function. However, our current understanding of how OPA1 dysfunction contributes to vision loss in DOA patients has been limited by access to disease-relevant, patient-derived RGCs. Here, we used induced pluripotent stem cell (iPSC)-RGCs to study how OPA1 affects cellular homeostasis in human RGCs, the most vulnerable cell type in DOA. iPSCs derived from OPA1 DOA patients and isogenic CRISPR-Cas9-corrected iPSCs were differentiated to iPSC-RGCs. Defects in mitochondrial networks and increased levels of reactive oxygen species were observed in iPSC-RGCs carrying \u003cem\u003eOPA1\u003c/em\u003e pathogenic variants. Ultrastructural analyses also revealed changes in mitochondrial shape and cristae structure, with decreased endoplasmic reticulum (ER):mitochondrial contact length in DOA iPSC-RGCs. Mitochondrial membrane potential was reduced and its maintenance was also impaired following inhibition of the F1Fo-ATP synthase with oligomycin, suggesting that mitochondrial membrane potential is maintained in DOA iPSC-RGCs through reversal of the ATP synthase and ATP hydrolysis. These impairments in mitochondrial structure and function were associated with defects in cytosolic calcium buffering following ER calcium release and store-operated calcium entry, and following stimulation with the excitatory neurotransmitter glutamate. In response to mitochondrial calcium overload, DOA iPSC-RGCs exhibited increased sensitivity to opening of the mitochondrial permeability transition pore. These data reveal novel aspects of DOA pathogenesis in patient-derived RGCs. The findings suggest a mechanism in which primary defects in mitochondrial network dynamics disrupt core mitochondrial functions, including bioenergetics, calcium homeostasis, and opening of the permeability transition pore, which may contribute to vision loss in DOA patients.\u003c/p\u003e","manuscriptTitle":"Disruption of mitochondrial homeostasis and permeability transition pore opening in OPA1 iPSC-derived retinal ganglion cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-23 16:43:57","doi":"10.21203/rs.3.rs-5153627/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-12-06T22:23:05+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-06T22:19:30+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-06T08:41:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"250532897772833859510381865276136492246","date":"2024-11-15T07:40:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"47884780069184516679707074728356677060","date":"2024-11-15T04:18:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"277399959985540980459968717801882516607","date":"2024-09-30T10:07:29+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-09-30T06:46:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-09-27T14:41:59+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-09-27T09:17:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"Acta Neuropathologica Communications","date":"2024-09-25T16:45:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"acta-neuropathologica-communications","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"anec","sideBox":"Learn more about [Acta Neuropathologica Communications](https://actaneurocomms.biomedcentral.com/)","snPcode":"40478","submissionUrl":"https://submission.springernature.com/new-submission/40478/3","title":"Acta Neuropathologica Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"970adf23-0888-442f-b732-ed348417a36f","owner":[],"postedDate":"December 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-02-17T16:06:35+00:00","versionOfRecord":{"articleIdentity":"rs-5153627","link":"https://doi.org/10.1186/s40478-025-01942-z","journal":{"identity":"acta-neuropathologica-communications","isVorOnly":false,"title":"Acta Neuropathologica Communications"},"publishedOn":"2025-02-13 15:57:46","publishedOnDateReadable":"February 13th, 2025"},"versionCreatedAt":"2024-12-23 16:43:57","video":"","vorDoi":"10.1186/s40478-025-01942-z","vorDoiUrl":"https://doi.org/10.1186/s40478-025-01942-z","workflowStages":[]},"version":"v1","identity":"rs-5153627","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5153627","identity":"rs-5153627","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}