Oxidative stress imbalance and cellular damage mediated by the ND4 G11778A mutation

Abstract

The mechanisms underlying cellular damage induced by the mitochondrially encoded NADH dehydrogenase subunit 4 (MT-ND4) with R340H mutation caused by the variant m.11778G > A in complex I are intricate. Numerous studies indicate that mitochondria play a primary role in cellular death due to this mutation. However, the detailed pathological effects remain incompletely elucidated. To decipher the specific impacts of this mutation on cellular death, mitochondrial dysfunction was investigated in 661 W cells expressing exogenous Mut-ND4 (m.G11778A). Importantly, the oxygen consumption rate (OCR) assessed by Seahorse XF analyzer exhibited a significant decrease under galactose conditions and an excessive production of reactive oxygen species (ROS). Conversely, the activity levels of catalase (CAT), superoxide dismutase (SOD), and total glutathione (T-GSH) were decreased, leading to increased cell death in cells expressing Mut-ND4 (m.G11778A) under galactose conditions. In addition, structural disruptions in the optic nerves of mice subjected to Mut-ND4-AAV infection were revealed. These findings suggest that Mut-ND4 (m.G11778A) contributes to cellular injury and an oxidative stress imbalance, characterized by decreased mitochondrial oxygen consumption, increased oxidative products, and reduced antioxidant capacity.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-40061-0.

Introduction

Leber Hereditary Optic Neuropathy (LHON) is an inherited optic nerve disease that predominantly results in blindness in young adult males^1,2. The diagnosis of LHON relies primarily on the pathogenic variants in mitochondrial DNA (mtDNA). Among LHON patients, the mtDNA variant with the G-to-A transition at mitochondrial nucleotide 11,778 encoding the NADH dehydrogenase subunit 4 (ND4) of complex I accounts for more than half of all cases³. Additionally, the variant m.11778G > A (p.R340H) is associated with significant visual loss and poor recovery⁴. As a result, numerous studies focus on ND4 with the m.11778G > A variant⁵. While promising preclinical results from animal models of LHON have prompted valuable and crucial clinical trials for cases caused by the m.11778G > A pathogenic variant, the detailed pathological process and mechanisms of ND4 with the m.11778G > A variant remain incompletely elucidated^6–8. The R340H mutation encoded by the variant m.11778G > A in mtDNA is a key pathological factor for LHON, and mitochondrial dysfunction due to this mutation is central to the disease. Investigating early events, such as electron transport chain (ETC) dysfunction, oxidative stress, and the antioxidant enzyme response, may provide insights into potential treatments for LHON. Therefore, in this paper, we would focus on the imbalance of oxidative stress and cellular damages in the injury induced by the R340H mutation.

Materials and methods

Materials

Dulbecco’s modified Eagle’s medium (DMEM, high glucose, BIOEXPLORER, Cat#B1101-001); DMEM glucose-free medium (Basalmedia, Cat#L160KJ); Fetal bovine serum (FBS, Capricorn, Cat#F12A); Penicillin and streptomycin (Gibco, Cat#15140122); Galactose (Sigma-Aldrich, Cat#G5388); Puromycin (Basalmedia, Cat#S250J0); Cell-counting kit 8 (CCK-8) (Biosharp, Cat#BS350A); ROS assay kit (Solarbio, Beijing, China, Cat#CA1410); Superoxide Dismutase Assay Kit with WST-8 (Beyotime, Cat#S0101S); Catalase (CAT); Assay Kit (Solarbio, Cat#BC0205); GSH and GSSG Assay Kit (Beyotime, Cat#S0053); One-step TUNEL Apoptosis Kit (Elabscience, Cat#E-CK-A322); DAPI (Beyotime, Cat#C1005); Hoechst 33,342 (Solarbio, Cat#C0030); Seahorse XFe24 microplates (Agilent, Cat#02340); Agilent Seahorse XF DMEM medium (Agilent, Cat#103015); Sodium pyruvate (Agilent, Cat#103578); Glutamine (Agilent, Cat#103579); D-glucose (Agilent, Cat#103577); BCA protein assay kit (Beyotime, Cat#P0010); Mitochondrial Extraction Kit ( solarbio, Cat: SM0020); Blue/Clear Native-PAGE Gel Quick Preparation Kit (Meilunbio, MA0470); Acetylcysteine ( MedChemExpress, Cat#HY-B0215 ).

661w cells culture^9,10

661 W cells from Aolu Biotechnology (Shanghai, China) were cultured in high-glucose DMEM supplemented with 10% FBS, 100 µg/mL streptomycin and 100 U/mL penicillin. For selection using galactose, the cells were cultured in glucose-free DMEM supplemented with 5 mM galactose, 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% FBS for three days. The cells were cultured in a humidified atmosphere of 95% air and 5% CO₂ at 37 °C.

Stable expression of exogenous ND4 gene in 661w cells

The following plasmids were used for lentiviral production by Hanbio (Shanghai, China), HBVL-WT-Null-PURO, HBVL-Mut-Null-PURO. As previously reported by Dr John Guy¹¹, the exogenous ND4 gene was engineered to include a mitochondrial targeting sequence and an epitope tag to facilitate mitochondrial import and detection. The gene was synthesized using overlapping 80-mer oligonucleotide pairs that were designed to cover the entire 1377-nucleotide sequence of the nuclear-encoded ND4 gene. To establish stable 661 W cells expressing exogenous ND4, cells at approximately 50% to 70% confluence were infected with 5.0 × 10⁸ lentiviral particles carrying ND4 gene with the m.11778G > A (Mut-ND4 gene) or wild type ND4 gene (WT-ND4 gene), both of which have an ATPc targeting sequence appended to the N terminus, which directs proteins to the mitochondria following nuclear expression, and a 3xHA sequence at the C terminus. In brief, 661 W cells were infected with lentiviruses containing the target DNA. 72 h after infection, the cells were selected in the medium with 0.3 µg/mL puromycin for 2 weeks. Expression of exogenous ND4 was confirmed by assessing cell viability under a biological microscope (Leica DM500).

Immunostaining

The immunostaining procedures were adapted from previous reports¹². Cell slides were blocked with a blocking buffer and then incubated overnight at 4 °C with primary antibodies: Anti-TOMM20 (1:1000, CAT#: ab56783, Abcam), HA-Tag (1:1000, CAT#:C29F4; Cell Signaling Technology). After washing, the coverslips were incubated with secondary antibodies for 1 h in the dark: anti-mouse IgG, Alexa Fluor^® 488 (1:1000, CAT#: 4408; Cell Signaling Technology), anti-mouse IgG, Alexa Fluor^® 594 (1:1000, CAT#: 8890; Cell Signaling Technology), anti-rabbit IgG, Alexa Fluor^® 488 (1:1000, CAT#: 4412; Cell Signaling Technology), or anti-rabbit IgG, Alexa Fluor^® 594 (1:1000, CAT#: 8889; Cell Signaling Technology). Nuclei were counterstained with DAPI (CAT#: C1005; Beyotime) for 3–5 min. Fluorescence images were acquired using confocal microscopy (TCS SP8; Leica, Germany) and analyzed using ImageJ software (Fiji, Image J 1.47, NIH, Bethesda, MD, USA).

Mitochondrial respiration assessment¹³

Mitochondrial function in 661 W cells was assessed using a Seahorse XFe24 analyzer (Agilent XFe24) by measuring the oxygen consumption rate (OCR). First, 661 W cells expressing either Mut-ND4 or WT-ND4 were seeded at 8 × 10³ cells/well in Seahorse XFe24 plates and incubated in either high-glucose or glucose-free medium supplemented with galactose for 24 h. Second, in a non-CO₂ incubator, the sensor cartridges of the analyzer in Seahorse XF calibrant were hydrated at 37 °C overnight. Following hydration, the culture medium was then replaced with Agilent Seahorse XF DMEM medium containing 2 mM glutamine, 1 mM D-glucose, and 1 mM sodium pyruvate, and the cells were incubated at 37 °C for 1 h prior to OCR measurement. During the assay, these substances of FCCP (1.5 µM), Rot/AA (0.5 µM), and oligomycin (1.5 µM), were subsequently injected to measure the OCR. Finally, protein concentrations of cells from different groups were measured using a BCA protein assay, and the OCR was normalized based on total protein levels. The entire experiments were performed at 37 °C.

Measurement of ROS¹²

661 W cells expressing different exogenous ND4 variants were respectively seeded into 12-well plates. After various treatments, the cells were stained with 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH-DA) and incubated at 37 °C for half an hour. The nuclei were stained with Hoechst 33,342 for 15 min. After washing with PBS, ROS levels were then visualized and quantified using a fluorescence microscope.

Detection of oxidative markers (SOD, CAT, and T-GSH)¹² and CCK8 assay

According to the manufacturer’s instructions, the activity of SOD was measured using a Total Superoxide Dismutase Assay Kit. CAT activity was detected using a CAT Assay Kit. The level of GSSG was evaluated using a GSH and GSSG Assay Kit. The cells were seeded in a 96-well plate and incubated in a 5% CO₂ incubator. After 3 days of culture, the cells were washed with Phosphate Buffered Saline (PBS) twice. CCK-8 reagent was added to each well, and the cells were incubated at 37 °C for 2 h. A multimode reader (SpectraMax i3X, Molecular Devices) was used to measure OD at 450 nm for each experimental well.

Mitochondrial extraction and blue native PAGE analysis

Cultured cells were digested, washed with PBS, and collected by centrifugation at 800 g for 5–10 min. After cell counting, a total of 5 × 10⁷ cells were resuspended in 1.0 mL of ice-cold Lysis Buffer and homogenized with 30–40 strokes in a small-volume glass homogenizer on ice. The homogenate was centrifuged at 1,000 g for 5 min at 4 °C. The supernatant was collected and subjected to a second centrifugation under the same conditions. The resulting supernatant was then centrifuged at 12,000 g for 10 min at 4 °C to separate the cytoplasmic fraction (supernatant) from the mitochondrial pellet. The mitochondrial pellet was washed by resuspension in 0.5 mL of Wash Buffer, followed by centrifugation at 1,000 g for 5 min at 4 °C. The supernatant from this step was subsequently centrifuged at 12,000 g for 10 min at 4 °C. The final mitochondrial pellet was obtained after discarding the supernatant. Mitochondria were gently lysed with 0.2% Triton X-100 for 20 min. The mitochondrial lysates were analyzed by Blue Native PAGE using a 4–16% gradient gel, with both sample and electrophoresis buffers being SDS-free. The Blue Native PAGE procedure was based on the method described byIlka Wittig et al.¹⁴.

Western blot

Samples were collected using RIPA lysis buffer (CAT#: P0013B; Beyotime). Proteins were extracted by centrifugation at 4 °C and denatured at 100 °C for 10 min, and separated using Tris-glycine gels. The proteins were then transferred to polyvinylidene difluoride (PVDF) membranes (CAT#: IPVH00010; Millipore). The membranes were blocked with 5% dry milk (CAT#: 9999; Cell Signaling Technology) in Tris-buffered saline with tween 20 (TBST) for 2 h at room temperature. Following blocking, the membranes were incubated with primary antibodies against Bcl-2 (1:1000, CAT#:15071, Cell Signaling Technology), β-Actin (1:1000, CAT#: GB15001; Servicebio), HA tag (1:1000, CAT#: 51064-2-AP, proteintech), ND4 (1:1000, CAT#:26736-1-AP, proteintech) and NDUFB8 (1:2000, CAT#: ET7108-25, HUABIO) at 4 °C overnight. After three 10-min washes with TBST, the membranes were incubated with the horseradish peroxidase (HRP)-conjugated goat anti-mouse (1:2500, CAT#: AP123P; Merck Millipore) or anti-rabbit (1:2500, CAT#: AP132P; Merck Millipore) secondary antibodies for 1 h at room temperature. Immunoblots were visualized using enhanced chemiluminescence imaging and then quantified using Fiji software.

TUNEL assay

To evaluate the influence of 661 W cells expressing different exogenous ND4 on cell apoptosis, the cells cultured in different media for 3 days were subjected to TUNEL assay using a One-step TUNEL Apoptosis Kit. The cells were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100 for 15 min and incubated with TdT equilibration buffer for 30 min at 37 °C. The cells were then treated with labeling working solution for 2 h in the dark. The 661 W cells were counterstained with DAPI for 3 min, and images were captured using a fluorescence microscope (Leica DMi8). The apoptotic cells were quantified using ImageJ, and the ratio of apoptotic cells to overall cells was calculated.

Animals and injection procedures

Adult male DBA1/J mice (10–12 weeks old) were purchased from GemPharmatech Co. Ltd and maintained under standard 12-hour light/12-hour dark laboratory conditions. All procedures were approved by the Fujian Medical University Animal Care and Veterinary Service (IACUC FJMU 2022 − 0477) and complied with the ARRIVE guidelines. All experiments were performed in accordance with ARRIVE guidelines and regulations. Male mice between the ages of 10 and 12 weeks were used for these studies (N = 20). Animals were injected intravitreally in the right eye with 1 µL of 10¹² VG/mL of AAV2/2 with mutation ND4. (i.e., 1 × 10⁹ total viral particles) and an equal dose of AVV2/2 WT-ND4 in the left eye. At 4.5 months after injection, mice were euthanized for tissue collection as described below. Eyes and optic nerves were then sampled.

Tissue collection and preparation of optic nerves for transmission electron microscopy¹⁵

Mice were anesthetized with 5% isoflurane in an induction chamber for 5 min. Once a deep sedation state was confirmed (no reflexes to pinching, slow respiration), transcardial perfusion was performed. The perfusion procedure was adapted from previous reports¹⁶. Briefly, the thoracic cavity was opened, the right atrium was incised, and perfusion was initiated through the left ventricle with 0.9% saline followed by a fixative solution containing 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Euthanasia was performed by transcardial perfusion under deep anesthesia, the last heartbeats immediately followed the fixative injection. Following perfusion, both eyes with intact optic nerves were carefully enucleated. Optic nerve was isolated and fixed overnight in 1.5% paraformaldehyde and 3% glutaraldehyde in 0.1 M PBS (pH 7.2), then incubated with 1% osmium acid-1.5% potassium ferrocyanide at 4 °C for 2 h. The samples were dehydrated through a gradient of ethanol solutions ranging from 50% to 100% ethanol (50%, 70%, 80%,90% and 100% for 10 min), then processed in a 1:1 ratio of acetone to EPON618 for 1.5 h. The samples were gradually embedded in EPON618 at 35 °C for 24 h, at 45 °C for 12 h, at 60 °C for 3 days. The samples were sectioned at thickness of 80 nm using a Leica UC7 ultramicrotome system (Leica Microsystems, Germany) and stained with 1% uranyl acetate for 20 min, followed by lead citrate staining for 5–10 min. Transmission electron microscopy (Tecnai G2 20 S-TWIN TEM) was used to capture images of the optic nerve ultrastructure, focusing on the central region of the optic nerve for ultrastructural analysis of both left and right optic nerves.

Axon counts

Axon counting was performed as described¹⁶. For each sample, images were captured from five randomly selected fields under a 100x objective lens. One picture in each set was excluded based on highest degree of longitudinally arranged axonal fibers. The nerves in the four remaining pictures were manually counted.

Statistical analysis

All experiments were carried out at least three replicates. Data are presented as mean ± standard error of the mean. Statistical analysis was performed using GraphPad Prism 10.0 (San Diego, CA, USA). Differences between the two groups were analyzed using Student’s t-test, while differences among three or more groups were analyzed using one-way analysis of variance (ANOVA) with the least significant difference test, accepting a significance level of p < 0.05.

Result

Puromycin-based purification of 661W cells with high expression of exogenous ND4

To enrich for 661 W cells expressing exogenous ND4, the cells were infected with lentivirus carrying the pac gene for puromycin selection. Two weeks after infection and selection, double immunofluorescent staining was performed to confirm expression of exogenous ND4 in mitochondria. As shown in Fig. 1, co-localized puncta of HA and TOM20 were observed in 661 W cells expressing mutant ND4 (m.11778G > A) and wild-type ND4, indicating successful expression of the exogenous ND4 and its mutants in the mitochondria.

Fig. 1.

661 W cells with high expression of exogenous ND4. Exogenous ND4 fused with HA tag was visualized. TOMM20 was specifically labeled for mitochondria. Scale bar = 20 μm.

Mutant ND4 impairs mitochondrial respiration

ND4, a critical subunit of complex I in mitochondria, plays a pivotal role in mitochondrial respiration. To investigate the impact of the R340H mutation on mitochondrial respiration, oxygen consumption rate (OCR), a key parameter of mitochondrial respiration, was assessed using a Seahorse XFe24 extracellular flux analyzer. Real-time measurement of OCR was performed in different groups with inhibitors, oligomycin (an ATP synthase inhibitor), cyanide p-trifluoromethoxy-phenyl-hydrazone (FCCP, a proton ionophore), and rotenone/antimycin A (electron transport chain inhibitors) used to determine basal or maximal OCR. As shown in Fig. 2, the basal and maximal OCR were the lowest in cells expressing the R340H variant (Mut-ND4 gene) under galactose condition. This suggests that the R340H mutation exacerbates mitochondrial dysfunction, particularly impairing mitochondrial OXPHOS activity in galactose condition. The R340H mutation thus disrupts complex I function, leading to impaired mitochondrial function and reduced the activation of OXPHOS.

Fig. 2.

Mitochondrial respiration in 661 W cells. The cells expressing different exogenous ND4 (with mutant ND4 gene or with wild-type ND4 gene) were cultured in medium with high glucose or galactose. (A and B) representative Oxygen Consumption Rates (OCR) profiles; (C) the basal OCR; (P = 0.0005, P = 0.0258, n = 5 per group); (D) mitochondrial ATP production in the different groups(P = 0.039, n = 5 per group); (E) maximal OCR (P<0.0001, P = 0.0038, n = 5 per group) ; (F) space capacity for mitochondria (P = 0.0096, n = 5 per group). Data are presented as means ± SEM, n = 5, one-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001; ****P < 0.0001.

Mutant ND4 increases oxidative stress

The deficiency in oxidative phosphorylation (OXPHOS) is closely linked to the downstream cascade reactions involving oxidative stress. To elucidate the oxidation reactions, the levels of reactive oxygen species (ROS)–byproducts of electron transport chain reactions in mitochondria –were assessed. Elevated ROS levels are indicative of mitochondrial dysfunction and can lead to further cellular damage. As depicted in Fig. 3A and B, the group of 661 W cells expressing mutant ND4 (m.11778G > A) in a galactose medium exhibited the highest ROS levels compared to other groups of 661 W cells expressing wild-type ND4 or those cultured in galactose medium. These suggest that the detrimental effect of ND4 (m.11778G > A mutation) was exacerbated under galactose conditions in contrast to high glucose conditions. This is consistent with Lambiri et al., in which mitochondrial DNA mutations affecting complex I lead to elevated ROS production, triggering axonal degeneration through mechanisms involving H2O2-mediated oxidative stress propagation¹⁷. Furthermore, treatment with the exogenous antioxidant N-acetylcysteine (NAC; 2 mM for 48 h) effectively suppressed the mutation-induced rise in ROS levels¹⁸.These findings indicate that the ND4 R340H mutation exacerbates oxidative stress when cells are forced to rely on mitochondrial respiration.

Fig. 3.

Mutant ND4 led to an imbalance of oxidative stress. The 661 W cells cultured in galactose or high glucose DMEM were stably expressing exogenous ND4. The levels of ROS (A, B) were measured. Under galactose conditions, an increase in ROS was evident in 661 W cells. Furthermore, cells expressing exogenous m.11778G > A genes displayed exacerbated changes. (P = 0.0212, P<0.0001, n = 6 per group). Scale bar = 100 μm. Data is presented as means ± SEM, n = 4, one-way ANOVA. ****P < 0.001.

Mutant ND4 reduces antioxidative enzyme activities

Cells possess an antioxidant enzyme system that regulates the cellular redox balance, including CAT, SOD, and GSSG, which can neutralize excess ROS. To elucidate the antioxidation reactions, the activity of catalase (CAT), superoxide dismutase (SOD), and total glutathione (T-GSH) were assessed. As illustrated in Fig. 4A–C, the activity of these antioxidant enzymes, CAT (4 A), SOD (4B), and T-GSH (4 C), were lower in cells with mutant mtND4 cultured in galactose medium compared to any other group of 661 W cells with wild-type ND4 or mutant ND4 cultured in high glucose medium. The reduction in antioxidative enzyme activity highlights an impaired defense mechanism against oxidative stress in cells with the ND4 R340H mutation, particularly under energy-limited conditions such as galactose culture. The combination of increased ROS (Fig. 3) and diminished antioxidant enzyme activity suggests a pronounced oxidative imbalance, contributing to cellular injury and dysfunction driven by the ND4 mutation, especially when the mitochondrial energy supply is restricted.

Fig. 4.

Mutant ND4 led to a decrease of antioxidative stress. The 661 W cells cultured in galactose or high glucose DMEM were stably expressing exogenous ND4. The activity of the enzymes SOD (P = 0.0079, P<0.0001, n = 6 per group) (A) and T-GSH (P = 0.0044, n = 4 per group) (B), and the level of CAT (P = 0.0442, n = 4 per group) (C) were measured. Under high-glucose conditions, the activity of SOD and T-GSH were significantly reduced in 661 W cells. Under galactose conditions, the activity of SOD was significantly reduced in 661 W cells. Furthermore, cells expressing exogenous m.11778G > A genes displayed exacerbated changes. Scale bar = 100 μm. Data is presented as box and whiskers, one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.

Mutant ND4 decreases cell survival

Given that the ND4 mutation can result in OXPHOS disruption and oxidative imbalance, we aimed to ascertain whether the mutant ND4 could provoke the demise of 661 W cells (photoreceptor cells) through morphology analysis and CCK8 assay. As depicted in Fig. 5A, 661 W cells expressing exogenous wild-type or m.11778G > A mutant ND4 were cultured in DMEM with high glucose or galactose for 72 h and then examined under light microscopy. Our findings revealed that cells grown in high-glucose maintained normal density, regardless of whether 661 W cells exogenously expressed wild type or mutant ND4. In contrast, in galactose medium, cell density was decreased, with lowest density in cells expressing mutant ND4, indicating a marked reduction in cell survival under conditions that limit energy supply. To further validate cell viability, the CCK-8 cell assay was conducted at three temporal intervals (24, 48, and 72 h). As shown in Fig. 5B, the cells cultured in the galactose medium had a lower OD compared to those in glucose medium, reflecting reduced cell viability. The group expressing mutant ND4 in galactose medium exhibited the lowest OD value, confirming that the ND4 mutation exacerbates cell death when oxidative stress and energy supply are compromised. In order to verify the apoptosis of the cells, we measured the Bcl-2 expression levels at 72 h, as shown in Fig. 5C,D. This suggests that mut-ND4, particularly under energy-deprived conditions, negatively affects 661 W cell survival through impaired mitochondrial function and increased susceptibility to oxidative damage.

Fig. 5.

The influence of mutant ND4 on 661 W cells survival. 661 W cells expressing exogenous wild type ND4 or m.11778G > A mutant was cultured in high glucose medium or galactose medium respectively. The cells were subjected to different treatments and examined under light microscopy (A). The cell viability at 3 different time points was evaluated by the CCK8 assay (24 h: P = 0.0122, P = 0.0175; 48 h: P = 0.0248, P = 0.0096; 72 h: P = 0.0196, P < 0.0001; n = 6 per group) (B). Bcl-2 protein was detected by western blot and the ratio of Bcl-2 to β-actin were analyzed (P = 0.0357) (C, D). Original blots are presented in Supplementary Fig. S2. The images were analyzed using ImageJ software. Scale bar = 100 μm. Data is presented as means ± SEM, n ≥ 3, one-way ANOVA. *P < 0.05; **P < 0.01; ****P < 0.00010.

Mutant ND4 promotes apoptosis

Considering the role of mitochondrial dysfunction in apoptosis, we investigated whether the ND4 mutation (m.11778G > A) induces cellular damage through apoptotic pathways. Apoptosis was assessed using TUNEL staining in 661 W cells exogenously expressing either wild-type or m.11778G > A mutant ND4, cultured in DMEM with high glucose or galactose for 72 h. As depicted in Fig. 6A,B, cells cultured in high-glucose medium exhibited relatively lower apoptotic cells, regardless of ND4 expression. However, a significant increase in apoptotic cells was observed in the galactose medium group, particularly in cells expressing mutant ND4. Interestingly, the group expressing mutant ND4 in galactose medium has the highest number of apoptotic cells, correlating with the observed reduction in cell density from light microscopy examination and viability results obtained from CCK8 assay. This suggests that the m.11778G > A ND4 mutation aggravates cellular apoptosis, especially under conditions of metabolic stress, further contributing to mitochondrial dysfunction and cell death.

Fig. 6.

The influence of mutant ND4 on cellular apoptosis. The apoptotic cells were detected through TUNEL staining, and the results were presented in statistical form (P < 0.0001, P = 0.0005 n = 6 per group) (A, B). Scale bar = 100 μm. Data is presented as means ± SEM, n ≥ 4, one-way ANOVA. ***P < 0.001; ****P < 0.0001.

Mutant ND4 induces optic nerve damage in mice

To evaluate the in vivo impact of ND4 mutation, AAV2/2 vectors expressing mutant ND4 with an N-terminal mitochondrial targeting sequence and a C-terminal HA tag (AAV2.mND4 HA) were delivered via intravitreal injection into the ipsilateral eye of mice, while the contralateral eye received AAV2 carrying wild-type ND4 HA as a control. Four and a half months post-injection of ND4-AAV, the ultrastructure of the optical nerve was examined using transmission electron microscopy. Axon count analysis demonstrated a substantial reduction in AAV-mut-ND4, compared to the AAV-wt-ND4 group (Fig. 7A,B). These ultrastructural abnormalities in the optic nerve confirm that the R340H ND4 mutation causes and axonal damage in vivo, recapitulating key features of LHON.

Fig. 7.

Transmission electron microscopy (TEM) reveals structural abnormalities in the optic nerves of AAV-mut-ND4-treated mice. Mice infected with AAV-mut-ND4 exhibited a significant reduction in axon counts compared with the AAV-wt-ND4 groups (P = 0.0006; n = 4 mice per group) (A, B). Scale bars = 5 μm. Insets show detailed images of optic nerves. Scale bars = 2 μm. Data is presented as means ± SEM, student’s t-test. ***P < 0.001.

Discussion

Mitochondria play a pivotal role as the cell’s powerhouses, generating ATP through oxidative phosphorylation coupled with respiration¹⁹. The mitochondrial Electron Transport Chain (ETC) employs oxidation-reduction reactions to shuttle electrons between protein components, producing the necessary energy to convert ADP to ATP^20–22. This process fuels essential cellular functions, particularly in energy-demanding tissues like the optic nerve and brain. However, mitochondrial DNA (mtDNA) is particularly susceptible to mutations due to its relatively limited repair mechanisms in comparison to nuclear DNA^23,24.

Mutations in mtDNA can result in mitochondrial dysfunction, a hallmark of several neurodegenerative diseases, including Leber’s hereditary optic neuropathy (LHON). LHON is predominantly caused by mitochondrial DNA mutations, including the R340H substitution in the ND4 subunit, a core component of respiratory complex I. The function and assembly of this complex are governed by diverse regulatory mechanisms, one of which involves the mitochondrial protein TMEM126A, a product of a disease gene associated with recessive optic atrophy²⁵.

Notably, notable therapeutic advances have been made, with clinical studies showing that gene therapy approaches (e.g., GS010, lenadogene nolparvovec) can lead to substantial visual improvement in affected patients^26,27. However, comprehending the exact mechanisms underlying oxidative stress imbalance in LHON necessitates further exploration. To model LHON, we utilized allotopic expression of the mutant human R340H (G11778A) ND4 or wild-type ND4 subunit of complex I in 661 W cells. Allotopic expression involves expressing a nuclear-encoded version of a gene typically encoded by mitochondrial DNA in the cytoplasm, from which it is subsequently imported into the mitochondria. The ATPc mitochondrial targeting sequence (MTS) directed the allotopically expressed ND4 genes (R340H or wild type ND4) fused with HA polypeptide into the mitochondria^11,16,28,29. In addition, we also proved that in the site of complex I the proteins of HA tag, ND4 and NDUFB8 were detected, as shown in supplementary Fig. S1.

Our study demonstrates that culturing cells in galactose medium—a metabolic condition that induces mitochondrial stress by limiting glycolysis-dependent ATP synthesis—reveals a critical role for the R340H mutant ND4 in disrupting electron transport chain (ETC) function. This impairment manifests as reduced mitochondrial oxygen consumption rates and oxidative phosphorylation efficiency. This indicates that the R340H mutant ND4 detrimentally affects ETC function. Under specific environmental stressors such as hypoxia, hypoglycemia, or ischemia, these mitochondrial dysfunctions are exacerbated.

As mitochondria are a major source of intracellular reactive oxygen species (ROS) due to oxidative phosphorylation, the rise in ROS levels observed under galactose conditions aligns with ETC dysfunction measured by the Seahorse XF24 Cell Mito Stress Test. Maintaining cellular redox balance necessitates a proper equilibrium between oxidants and antioxidants. Our results also reveal a decline in antioxidants such as CAT, SOD, and T-GSH in cells expressing ND4 R340H under galactose conditions, contrasting with elevated ROS levels. These findings indicate that the R340H mutant ND4 disrupts the ETC, causing an imbalance between oxidants and antioxidants under stressful conditions. The resulting energy deficits can explain the neurodegenerative nature of LHON.

Oxidative stress is implicated in a broad spectrum of diseases, contributing to cellular damage through various mechanisms. Our study showcases cellular damage in 661 W cells expressing ND4 R340H under galactose conditions, as evidenced through morphological analysis, CCK8 assay, and TUNEL staining. Structural disruptions were also exhibited in the optic nerves with the R340H mutant ND4. The R340H mutation, under these stress conditions, induced pronounced cellular damage and apoptosis by impeding mitochondrial ETC function and disrupting the oxidant-antioxidant equilibrium, potentially contributing to pathogenesis of LHON.

While this study provides important mechanistic insights into the ND4 R340H mutation, we acknowledge certain limitations inherent to the modeling approach. Primarily, the allotopic expression system itself introduces pathological artifacts. Though the allotopic strategy is designed as a gene therapy tool to rescue Complex I function by supplying wild-type protein, using it here to express the mutant protein does not replicate the natural genetics of the disease. The system necessarily does not replicate mtDNA heteroplasmy (the mixture of mutant and wild-type mtDNA), which is a key factor governing disease penetrance and severity in vivo. Furthermore, allotopic expression of highly hydrophobic proteins like ND4 in mammalian cells has been previously shown to be challenging, often leading to proteins that decorate the periphery of mitochondria or aggregate in the cytoplasm rather than fully and correctly assembling into Complex I, which may lead to artifacts like loss of mitochondrial membrane potential or cell death, independent of the mutation’s native effect^30–33. In addition, the reliance on galactose medium to induce metabolic stress, while a standard method to force reliance on oxidative phosphorylation, is a specific, non-physiological stressor. Published work notes that the use of galactose can significantly alter the overall metabolic phenotype and might exaggerate the effect of the mitochondrial mutation more severely than conditions found in vivo^{30,31,34–36}.

In conclusion, as shown in Fig. 8, our study provides important insights into the pathogenic mechanisms of LHON, highlighting the mutant R340H ND4 as a pivotal factor in LHON, instigating cellular damage and apoptosis through ETC dysfunction and oxidative stress imbalance. These findings identify potential therapeutic targets for addressing mitochondrial dysfunction in LHON, offering potential targets for restoring ETC function, mitigating oxidative stress, and preventing LHON in affected individuals.

Fig. 8.

Mut-ND4 (m.G11778A) contributes to an oxidative stress imbalance and cellular damage. The upper panel illustrates cellular damage in 661 W cells expressing ND4 R340H. The lower panel compares normal intracellular physiological processes (left) with the effects in cells harboring the ND4 R340H (right), where antioxidants such as CAT, SOD, and GSH, are reduced, and ROS levels are increased. The ND4 R340H mutation disrupts ETC function, resulting in impaired mitochondrial oxygen consumption and reduced oxidative phosphorylation.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

1. Lijun Fang: wrote the main manuscript and performed research. 2. Kangyue Fu, Mengyu Yang, Yiwen Xu and Ezeugwu Sussan Ukamaka: analyzed data and performed research. 3. Dianbo Qu: wrote the manuscript and analyzed data. 4. Tianwen Huang and Jianzhang Hu: wrote the main manuscript text, performed research and designed research. All the authors reviewed the manuscript.

Funding

This research received generous support from Joint Funds for the Innovation of Science and Technology, Fujian Province under grant No. 2024Y9336 and No. 2023Y9185. Additionally, it was made possible through the sponsorship of the Fujian Province Natural Science Foundation under grant No. 2022J01739 and the Fujian Provincial Health Technology Project under grant No. 2022GGA018.

Data availability

The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.