Mitochondria are highly dynamic organelles that undergo structural remodeling in a well-regulated manner. Mitochondrial quality control (MQC) including dynamics and mitophagy is the important process for maintenance of mitochondrial function and structure (1, 2). Especially, mitophagy, the selective mitochondrial autophagy, is pivotal in controlling metabolic homeostasis and maintaining mitochondrial quality and function. These roles are critical in molecular events, such as oxidative stress, that lead to age-related dysfunctions development and subsequent disease progression (3, 4). Impaired mitophagy and cumulative mitochondrial dysfunction are closely linked to multiple human diseases, as supported by considerable evidence, such as age-related neurodegenerative diseases, metabolic diseases, muscle dystrophy, and liver disease (4, 5). The initial stage of mitophagy is stimulated by kinases such as 5’-AMP-activated protein kinase (AMPK), unc-51 like autophagy activating kinase 1 (Ulk1), and PTEN-induced putative kinase 1 (Pink1). The kinases promote each other and stimulate a downstream regulation of autophagy (6, 7). Consequently, Pink1–Parkin-mediated mitophagy, receptor-mediated mitophagy, or adaptor protein-mediated mitophagy is triggered (8). The polyubiquitination of various mitochondrial outer membrane proteins by Parkin induces the recognition of several adaptor molecules and microtubule‐associated protein 1 light chain 3 (LC3) and the recruitment of autophagic machinery (6, 8, 9). The mitophagy-enhancing process facilitates the elimination of damaged mitochondria and is the basis for developing therapeutic strategies for several diseases. However, the molecular mechanism governing mitophagy remains unclear.

Current studies have demonstrated that mitoNEET, a protein exposed at the cytosolic surface of the mitochondria, controls the formation of intermitochondrial junctions and mitochondrial network morphology (10). Therefore, the deletion of mitoNEET alters the integrity of inter-mitochondrial junctions (11). Previous studies showed that mitoNEET is concerned in the regulation of autophagy signaling and it prevents autophagy (12, 13). In cells with reduced mitoNEET expression, proteins involved in autophagy accumulate; additionally, electron microscopy revealed autophagosome accumulation (12, 14). Furthermore, mitoNEET, along with other integral mitochondrial proteins in the outer mitochondrial membrane, undergoes ubiquitination during mitophagy. Subsequently, the integral mitochondrial proteins containing mitoNEET are phosphorylated by Pink1 and reduced by E3 ubiquitin protein ligase (Parkin) recruitment during the early stages of mitophagy (7, 15, 16). These studies demonstrated that mitoNEET acts as a direct substrate of Parkin in the recruitment of autophagy adaptors and the elimination of mitoNEET is necessary for mitophagy initiation (16, 17). However, whether targeting mitoNEET enhances mitophagy has not been studied yet. Herein, we showed that mitoNEET inhibition promotes the accumulation of mitophagy-linked proteins and activation of the crosstalk between lysosomes and mitochondria during CCCP-induced mitochondrial damage. Thus, mitoNEET plays a conclusive role in mitophagy regulation, which has been clearly demonstrated in the results of the previous studies. Therefore, we hypothesized that mitoNEET is a potential molecular therapeutic target for several diseases through the elimination of damaged mitochondria and maintenance of mitochondrial quality control.

CCCP-mediated mitochondrial depolarization induced mitoNEET expression in macrophages

Mitochondrial depolarization due to environmental stress such as CCCP, a mitochondrial oxidative phosphorylation uncoupler, induces mitophagy-mediated clearance of damaged mitochondria or activates apoptosis. Therefore, CCCP is an indirect mitophagy and apoptosis inducer (4). In the present study, we hypothesized that mitoNEET contributes to faulty mitophagy in mitochondrial dysfunction diseases. To elucidate the role of mitoNEET in autophagy function during CCCP-induced selective autophagy of the mitochondria, we assayed mitoNEET expression during CCCP treatment. We harvested total protein at 0.5, 1, 3, 6, 9, and 12 hours after CCCP administration in RAW264.7 cells (Fig. 1A). MitoNEET protein expression was significantly increased at 1 and 3 hours prior to the conversion of LC3-I to LC3-Ⅱ protein. Additionally, 6 hours after CCCP treatment, LC3-Ⅱ protein levels increased, with decreased mitoNEET protein levels. These results indicated that mitoNEET is involved in the initial stages of autophagy. To study the function of mitoNEET in autophagy signaling, we analyzed the expression of autophagosomal marker LC3-Ⅱ and autophagy adaptor protein p62/SQSTM in mitoNEET inhibition by inhibitor NL-1. We found that NL-1 enhanced the expression levels of LC3-Ⅱ and p62 protein in cells exposed to CCCP (Fig. 1B), followed by a rapid decrease at 12 hours. To examine whether CCCP-induced expression of mitoNEET alters autophagy responses, we generated mitoNEET shRNA- or control shRNA-expressing cells. Expression levels of LC3-II and p62 protein in cells of shRNA-mediated knock-down of mitoNEET were higher than those in control cells (Fig. 1C), and then decreased rapidly at 12 hours. These results demonstrated that the elimination of mitoNEET is necessary for autophagy activation. Moreover, mitoNEET inhibition using inhibitor or shRNA could activate the CCCP-induced autophagy response. To verify that the inhibition of mitoNEET induces mitophagy, we assessed colocalization of lysosomes and mitochondria by confocal microscopy using MitoTracker Deep Red, and LysoTracker Green. (Fig. 1D, E). Confocal microscopy images showed that NL-1 or mitoNEET shRNA increased CCCP-induced colocalization of mitochondria and lysosomes when compared with CCCP alone. Taken together, these results indicated that inhibiting mitoNEET induces recruitment of autophagosomes and the selective degradation of mitochondria during CCCP-induced mitochondrial depolarization.

Inhibition of mitoNEET activated Pink1–Parkin-mediated mitophagy during CCCP-induced mitochondrial depolarization

Prior studies demonstrated that CCCP induces protein kinase (AMPK) activation and promotes mitochondrial fission, manifesting as heightened Pink1 and Parkin E3 ligase activity toward mitoNEET (7, 17). However, whether targeting of mitoNEET plays a role as an activator of Pink1–Parkin-mediated mitophagy remains unknown. To confirm that CCCP induces AMPK and mitophagy-associated molecules Pink1 and Parkin, we examined protein levels using western blot analysis in CCCP-treated RAW264.7 cells (Fig. 2A). Phosphate AMPKα protein expression levels were significantly increased in the early stages compared with the corresponding expression levels in the vehicle, followed by a rapid decrease. In addition, Pink1 protein levels began to increase 1 hour following CCCP administration, and there was a corresponding sequential increase in Parkin expression. We showed that mitoNEET inhibition using inhibitor or shRNA augments autophagy marker expression and colocalization of lysosomes and mitochondria following CCCP treatment (Fig. 1). To verify whether mitoNEET inhibition activates CCCP-induced Pink1–Parkin-mediated mitophagy, we examined the expression levels of phosphate AMPKα, Pink1, and Parkin protein in CCCP- treated RAW264.7 cells in the presence or absence of NL-1 (Fig. 2B). We found that NL-1 enhanced the expression levels of phosphate AMPKα, Pink1, and Parkin proteins. Consistent with this, AMPKα, Pink1, and Parkin protein levels in cells of shRNA-mediated knock-down of mitoNEET were higher than the corresponding levels in control cells (Fig. 2C). These results demonstrated that mitoNEET inhibition using inhibitor or shRNA activated mitophagy through the upregulation of mitophagy-associated genes in the Pink1–Parkin pathway during CCCP-induced mitochondrial depolarization. CCCP is a strong inducer of AMPK through inhibition of ATP (adenosine triphosphate) synthesis (7, 18). Additionally, mitoNEET function is associated with ATP synthase, and pyruvate dehydrogenase, enzymes involved in the β-oxidation of fatty acids (19). We hypothesized that acti-vated phosphate AMPKα in mitoNEET inhibition is generated by suppressing ATP synthesis during CCCP treatment, and assessed intracellular ATP levels using an ATP Assay Kit. We found that NL-1 reduced the intracellular ATP levels in cells treated to CCCP for 1 hour (Fig. 2D). Consistent with this, intracellular ATP levels in cells expressing mitoNEET shRNA were decreased in comparison with the corresponding levels in the control shRNA-expressing cells in the presence of CCCP (Fig. 2E). These results indicated that inhibiting mitoNEET in RAW264.7 cells promotes CCCP-mediated inhibition of ATP synthesis and enhances the phosphate AMPKα levels. This leads to stimulation of Pink1–Parkin during CCCP-induced mitochondrial damage. Taken together, these results indicate that targeting mitoNEET stimulates Pink1–Parkin, which acts as a downstream regulator of mitophagy, via phosphate AMPKα during CCCP-induced mitochondrial damage.

Inhibition of mitoNEET attenuated CCCP-induced mitochondrial ROS

CCCP induces superoxide production through mitochondrial depolarization (20). It has been postulated that mitochondrial dysfunction-induced reactive oxygen species (ROS) govern the pathogenesis of various diseases. Mitophagy activated in response to mitochondrial depolarization decreases ROS generation through the removal of damaged mitochondria (21). We evaluated whether mitophagy enhanced by inhibiting mitoNEET attenuates CCCP-induced mitochondrial ROS. Superoxide levels by CCCP (70 μM) were analyzed using flow cytometry and decreased by NL-1 administration at 9 hours (Fig. 3A). Additionally, mitoSOX levels were assessed by the mitoSOX mitochondrial superoxide indicator. The CCCP-induced mitoSOX level decreased following mitoNEET inhibitor, NL-1 (Fig. 3B). Furthermore, CCCP-induced superoxide and mitoSOX levels were reduced in mitoNEET shRNA expressing RAW264.7 cells (Fig. 3C, D). In addition, confocal microscopy clearly demonstrated that mitoNEET inhibition suppressed cytosolic ROS following treatment of CCCP (Fig. 3E, F).

To confirm the antioxidant effect of NL-1, during CCCP-induced ROS production, we examined the expression of PGC-1α (peroxisome proliferator-activated receptor gamma coactivator 1-alpha), as well as SOD2 (superoxide dismutase 2), in the presence or absence of NL-1. We found that mitoNEET inhibitor, NL-1, enhanced the mRNA (Fig. 3G, H) and protein (Fig. 3K) expression levels of PGC-1α and SOD2 in RAW264.7 cells. Consistent with this, PGC-1α, SOD2 mRNA (Fig. 3I, J), and protein (Fig. 3L) levels in cell of shRNA-mediated knock-down of mitoNEET were higher than those in the control cells. These results demonstrated that NL-1 or mitoNEET shRNA may attenuate CCCP-induced mitochondrial ROS production through the upregulation of the expression of antioxidant defense genes with enhanced mitophagy. Furthermore, PGC-1α, as a major regulator of mitochondrial biogenesis, maintains a network of healthy mitochondria by balancing the generation of new organelles and the removal of damaged mitochondria (8, 22). We did another experiment to demonstrate that mitoNEET inhibition induces the activation of mitochondrial biogenesis via the increase of PGC-1α and the activation of mitophagy via increase of Drp-1 (a fission protein) and decrease of mitofusin 2 (Mfn2; a fusion protein) in the presence with CCCP by western blot analysis. (Supplementary Fig. 1). These data indicated that by targeting mitoNEET, it is possible to sustain mitochondrial quality by maintaining a network of healthy mitochondria during the enhancement of mitophagy following CCCP treatment.

Inhibition of mitoNEET hampered CCCP-induced mitochondrial apoptosis

Mitochondria play key roles in activating apoptotic cell death in mammalian cells (23). Mitochondrial depolarization by CCCP induces ROS-mediated cell death (20). We showed that CCCP-induced mitochondrial ROS was attenuated by mitoNEET inhibition (Fig. 3). We assessed the effects of mitoNEET inhibition during CCCP-induced cell death using the Annexin V (AV) / Propidium Iodide (PI) Apoptosis Detection Kit. CCCP-induced apoptosis was rescued by mitoNEET inhibitor, NL-1, or mitoNEET shRNA at 12 hours (Fig. 4A, B) Moreover, mitoNEET inhibition decreased the levels of lactate dehydrogenase (LDH), the release of which serves as a cell-death marker (Fig. 4C, D). We treated cells with CCCP and then performed a cell-viability assay. Cell viability was evaluated by the MTT assay using the Ez-Cytox Cell viability Assay Kit. CCCP-induced cell death results indicated that inhibiting mitoNEET bestows a protective effect against CCCP-induced apoptosis through the attenuation of mitochondrial ROS via mitophagy. CCCP directly interferes with mitochondrial function and induces apoptosis (20). We hypothesized that cell death regulated by mitoNEET inhibition during CCCP-induced mitochondrial damage is mitochondrial apoptosis. We isolated protein from cells at 12, 24, and 36 hours after CCCP (10 μM) or NL-1 administration. Next, levels of mitochondrial apoptosis marker protein were determined using western blotting. CCCP-induced cleavage of caspase-9 (cysteine-aspartic proteases-9), caspase-3 (cysteine-aspartic proteases-3), and PARP-1 (Poly [ADP-ribose] polymerase 1) proteins were decreased in the response of NL-1 in RAW264.7 cells (Fig. 4G) and in mitoNEET shRNA expressing RAW264.7 cells (Fig. 4H). Taken together, these results demonstrated that mitoNEET serves as a key molecule in mediating mitochondrial cell death through the regulation of mitochondrial quality including the activation of mitophagy and PGC-1α, the antioxidant defense gene, following CCCP treatment.

Mitophagy, selective autophagy of mitochondria, is an important mitochondrial quality control mechanism that removes damaged mitochondria (1). In the current research study, defective mitophagy is emerging as a potential therapeutic target for the treatment of human diseases (4, 24). Early studies suggested that mitoNEET, a mitochondrial outer membrane protein, plays a role in the intermitochondrial junction and in the regulation of autophagy or mitophagy (11, 12, 16). In the present study, we proposed that targeting mitoNEET is a potential enhancer of mitophagy in mitochondrial dysfunctional diseases. Our results showed that mitoNEET protein levels are induced prior to the elevation of LC3-Ⅱ and p62 levels, which are autophagy-related proteins, during CCCP treatment in RAW264.7 cells (Fig. 1). These results demonstrated that mitoNEET is operative in the early stage of the autophagic response. Prior studies reported that mitoNEET is significantly reduced in response to E3 ubiquitin protein ligase (Parkin) recruitment in CCCP-induced mitophagy (17). However, whether the targeting of mitoNEET plays a role as a mitophagy activator has not been investigated yet. Interestingly, our data showed that mitoNEET inhibition using inhibitor or shRNA augments autophagy-related proteins, in addition to the colocalization of mitochondria and lysosomes (Fig. 1). Mitophagy mediated by AMPK activation in response to stresses that deplete intracellular ATP in early time recovers ATP production and mitochondrial function (7, 25). In our results, with the early reduction of ATP, mitophagy-associated protein levels of phosphate AMPKα, Pink1, and Parkin were enhanced by mitoNEET inhibition (Fig. 2). Therefore, our results showed that inhibiting mitoNEET, which acts as a regulator of the intermitochondrial junction, could enhance Pink1–Parkin-mediated mitophagy. Recent studies have reported that mitoNEET is associated with mitochondrial dysfunction in disease progression. Mitochondrial dysfunction could induce mitophagy as a feedback mechanism to remove damaged mitochondria. A prior study advanced an opinion that mitoNEET is a mitophagy-activating molecule as suggested by accumulating autophagosomes caused by mitoNEET-mediated mitochondrial dysfunction (26). However, in several human diseases, inefficient mitophagy results in the accumulation of dysfunctional mitochondria and mitophagosome due to the decreased fusion with lysosomes and consequent failed mitochondrial quality control (4). In our study, we showed that LC3-II protein levels raised with the attenuation of mitoNEET protein levels, which were initially increased. In addition, mitoNEET inhibition promoted the accumulation of mitophagy-linked proteins and the activation of mitochondria– lysosome crosstalk. These findings demonstrated that mitoNEET-mediated dysfunctional mitochondria generation under stress conditions needs to be suppressed for the enhancement and initiation of mitophagy (27). Furthermore, mitoNEET inhibition using inhibitor or shRNA attenuated CCCP-induced ROS or superoxide (Fig. 3) and increased PGC-1α and SOD2 mRNA and protein levels (Fig. 4). Particularly, as PGC-1α is a major regulator of mitochondrial biogenesis, the balance of elevated PGC-1α and mitophagy assures maintenance of the mitochondrial contents (8, 22). CCCP-induced ROS, disequilibrium of mitochondrial homeostasis cause deterioration of mitochondrial function and cell death (22, 23). Our results showed that mitoNEET inhibition recovers CCCP-induced cell death (Fig. 4). These results indicated that targeting mitoNEET exerts a protective effect against cell stress through mitochondrial quality control with elevated mitophagy and the replacement by mitochondrial biogenesis. Therefore, our results delineated a new paradigm for the role of mitoNEET in the mitophagic process and demonstrated that mitoNEET is a potential therapeutic target for the elimination of irreversibly damaged mitochondria and maintaining mitochondrial quality control in a wide range of diseases.

Details on the used methods are provided in the expanded Materials and Methods section in the online data supplement.

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education to S.W.C. (NRF-2014R1A6A1030318 and NRF-2021R1A2C1094382) and the KRIBB and KIST institutional Program to S.J.L. (KGM5322214 and 2E31700-22-P005).

The authors have no conflicting interests.

Conceptualization, S.W.C., S-J.L.; Methodology, Seunghee Lee; Validation, Seunghee Lee, Sangguk Lee and S-J.L., S.W.C.; Formal Analysis, Seunghee Lee, Sangguk Lee; Investigation, Seunghee Lee, Sangguk Lee; Writing Original Draft Preparation, S.W.C., Seunghee Lee; Writing Review & Editing, S.W.C., Seunghee Lee; Visualization, Seunghee Lee; Supervision, S.W.C.; Project Administration, S.W.C.; Funding Acquisition, S.W.C., S-J.L.