Mitochondrial Recruitment Research Articles

Ginsenoside Rg5 increases cardiomyocyte resistance to ischemic injury through regulation of mitochondrial hexokinase-II and dynamin-related protein 1

Hexokinase-II (HK-II) and dynamin-related protein 1 (Drp1) regulate mitochondrial function differently. This study was designed to investigate the cardioprotective effect of ginsenoside Rg5 (Rg5) with emphasis on the regulation of mitochondrial HK-II and Drp1. Saturated acid palmitate (PA) stimulation increased lactate accumulation and induced cellular acidification by impairing the activity of pyruvate dehydrogenase (PDH) in cardiomyocytes, leading to HK-II dissociation from mitochondria. Rg5 improved PDH activity and prevented cellular acidification by combating fatty-acid oxidation, contributing to protecting mitochondrial HK-II. HK-II binding to mitochondria prevented mitochondrial Drp1 recruitment, whereas Drp1 activation decreased the content of mitochondrial HK-II, demonstrating the reciprocal control for binding to mitochondria. Rg5 promoted Akt translocation to mitochondria and increased HK-II binding to mitochondria while coordinately suppressing Drp1 recruitment and mitochondrial fission. Akt inhibitor triciribine or knockdown of Akt with small interfering RNA diminished the effects of Rg5, indicating that Rg5 inhibited Drp1 activation and promoted HK-II mitochondrial binding through Akt activation. Rg5 prevented the opening of mitochondrial permeability transition pore and increased ATP production, resultantly increasing cardiomyocyte resistance to hypoxia/reoxygenation injury. Meanwhile, Rg5 prevented cell apoptosis with increased HK-II binding and reduced Drp1 recruitment to mitochondria in isoproterenol-induced ischemic heart of mice. Taken together, these findings not only established a previously unrecognized role of ginsenosides in cardioprotection but also suggest that mitochondrial HK-II binding and Drp1 recruitment could be targeted therapeutically to prevent ischemic injury in the heart.

Multifunctional Mitochondrial Epac1 Controls Myocardial Cell Death.

Although the second messenger cyclic AMP (cAMP) is physiologically beneficial in the heart, it largely contributes to cardiac disease progression when dysregulated. Current evidence suggests that cAMP is produced within mitochondria. However, mitochondrial cAMP signaling and its involvement in cardiac pathophysiology are far from being understood. To investigate the role of MitEpac1 (mitochondrial exchange protein directly activated by cAMP 1) in ischemia/reperfusion injury. We show that Epac1 (exchange protein directly activated by cAMP 1) genetic ablation (Epac1-/-) protects against experimental myocardial ischemia/reperfusion injury with reduced infarct size and cardiomyocyte apoptosis. As observed in vivo, Epac1 inhibition prevents hypoxia/reoxygenation-induced adult cardiomyocyte apoptosis. Interestingly, a deleted form of Epac1 in its mitochondrial-targeting sequence protects against hypoxia/reoxygenation-induced cell death. Mechanistically, Epac1 favors Ca2+ exchange between the endoplasmic reticulum and the mitochondrion, by increasing interaction with a macromolecular complex composed of the VDAC1 (voltage-dependent anion channel 1), the GRP75 (chaperone glucose-regulated protein 75), and the IP3R1 (inositol-1,4,5-triphosphate receptor 1), leading to mitochondrial Ca2+ overload and opening of the mitochondrial permeability transition pore. In addition, our findings demonstrate that MitEpac1 inhibits isocitrate dehydrogenase 2 via the mitochondrial recruitment of CaMKII (Ca2+/calmodulin-dependent protein kinase II), which decreases nicotinamide adenine dinucleotide phosphate hydrogen synthesis, thereby, reducing the antioxidant capabilities of the cardiomyocyte. Our results reveal the existence, within mitochondria, of different cAMP-Epac1 microdomains that control myocardial cell death. In addition, our findings suggest Epac1 as a promising target for the treatment of ischemia-induced myocardial damage.

Mitochondria in the context of Parkinson's disease.

Mitochondria in the context of Parkinson's disease.

PINK1-dependent phosphorylation of PINK1 and Parkin is essential for mitochondrial quality control.

Mitochondrial dysfunction has been linked to the pathogenesis of a large number of inherited diseases in humans, including Parkinson's disease, the second most common neurodegenerative disorder. The Parkinson's disease genes pink1 and parkin, which encode a mitochondrially targeted protein kinase, and an E3 ubiquitin ligase, respectively, participate in a key mitochondrial quality-control pathway that eliminates damaged mitochondria. In the current study, we established an in vivo PINK1/Parkin-induced photoreceptor neuron degeneration model in Drosophila with the aim of dissecting the PINK1/Parkin pathway in detail. Using LC-MS/MS analysis, we identified Serine 346 as the sole autophosphorylation site of Drosophila PINK1 and found that substitution of Serine 346 to Alanine completely abolished the PINK1 autophosphorylation. Disruption of either PINK1 or Parkin phosphorylation impaired the PINK1/Parkin pathway, and the degeneration phenotype of photoreceptor neurons was obviously alleviated. Phosphorylation of PINK1 is not only required for the PINK1-mediated mitochondrial recruitment of Parkin but also induces its kinase activity toward Parkin. In contrast, phosphorylation of Parkin by PINK1 is dispensable for its translocation but required for its activation. Moreover, substitution with autophosphorylation-deficient PINK1 failed to rescue pink1 null mutant phenotypes. Taken together, our findings suggest that autophosphorylation of PINK1 is essential for the mitochondrial translocation of Parkin and for subsequent phosphorylation and activation of Parkin.

Myofibrillogenesis Regulator 1 Rescues Renal Ischemia/Reperfusion Injury by Recruitment of PI3K-Dependent P-AKT to Mitochondria

To investigate whether myofibrillogenesis regulator 1 (MR-1) attenuates renal ischemia/reperfusion (I/R) injury via inhibiting phosphorylated Akt (p-Akt) mitochondrial translocation-mediated opening of the mitochondrial permeability transition pore (mPTP), we injected adenovirus containing MR-1 gene or its siRNAs to the left kidney subcapsular areas of Sprague-Dawley rats, which subsequently underwent experimental renal I/R injury. Renal functions and the severity of the tubular injury were evaluated by the serum creatinine and blood urea nitrogen levels and the pathological scores. We also examined the mitochondrial morphology and functions. Total/p-Akt were assessed by western blot using the mitochondrial and the cytosolic fractions of cortex of renal tissue, respectively. We found that mitochondrial and cytosolic MR-1 levels and mitochondrial p-Akt decreased, and cytosolic p-Akt increased after reperfusion. Subcapsular injection of adenovirus led to higher MR-1 expression in the mitochondria/cytosol, inhibited mPTP opening, and alleviated renal I/R injury; adenovirus injection also upregulated mitochondrial total and p-Akt levels more prominently compared with the normal saline (NS) group. Subcapsular injection of MR-1 siRNAs significantly lowered MR-1 expression and induced renal injury, with increased mPTP opening and mitochondrial damage, similar to I/R injury. MR-1 interacted with Akt in renal cortex homogenate. Wortmannin, a phosphatidylinositol 3 kinase (PI3K) inhibitor, abolished both mitochondrial p-Akt recruitment and the protective effect of MR-1 overexpression on I/R injury. To conclude, MR-1 protects kidney against I/R injury through inhibiting mPTP opening and maintaining mitochondrial integrity, through the recruitment of PI3K-dependent p-Akt to the mitochondria. MR-1 could be a new therapeutic strategy for renal I/R injury.

Niemann-Pick type C2 deficiency impairs autophagy-lysosomal activity, mitochondrial function, and TLR signaling in adipocytes

In this study, we investigated the role and mechanism of Niemann-Pick type C (NPC)2 in regulating lysosomal activity, mitophagy, and mitochondrial function in adipocytes. We found that knocking down NPC2 impaired lysosomal activity, as evidenced by the reduced mature cathepsin B, the increased accumulation of light chain 3 (LC3) and p62, and the decreased autophagic flux. In NPC2-knockdown (kd) adipocytes, the starvation-induced conversion of LC3-I to LC3-II was abolished. More interestingly, the majority of NPC2 was found in the mitochondrial fraction, and NPC2 deficiency led to impaired autophagic flux and decreased induction of LC3-II in the mitochondrial fraction during mitochondrial stress. Moreover, cellular respiration profiling revealed that NPC2-kd adipocytes had significantly decreased basal/maximal respiration and mitochondrial gene expression compared with scrambled cells, suggesting mitochondrial dysfunction. Additionally, we found that the mitochondrial recruitment of LC3-II induced by lipopolysaccharide (LPS), but not TNFα, was blunted in NPC2-kd adipocytes. Most intriguingly, NPC2-kd selectively diminished LPS-induced NFκB and ERK1/2 phosphorylation and the expression of pro-inflammatory genes, indicating that toll-like receptor signaling activation is impaired in the absence of NPC2. Our results suggest that NPC2 is in a mitochondrially associated autophagosome and plays an important role in regulating mitophagy, mitochondrial quality control, and mitochondrial function.

Actin filaments as dynamic reservoirs for Drp1 recruitment.

Drp1 is a dynamin-family GTPase recruited to mitochondria and peroxisomes, where it oligomerizes and drives membrane fission. Regulation of mitochondrial Drp1 recruitment is not fully understood. We previously showed that Drp1 binds actin filaments directly, and actin polymerization is necessary for mitochondrial Drp1 oligomerization in mammals. Here we show the Drp1/actin interaction displays unusual properties that are influenced by several factors. At saturation, only a fraction Drp1 binds actin filaments, and the off-rate of actin-bound Drp1 is significantly increased by unbound Drp1. GDP and GTP accelerate and decelerate Drp1/actin binding dynamics, respectively. Actin has a biphasic effect on Drp1 GTP hydrolysis, increasing at low actin:Drp1 ratio but returning to baseline at high ratio. Drp1 also bundles filaments. Bundles have reduced dynamics but follow the same trends as single filaments. Drp1 preferentially incorporates into bundles at higher ionic strength. We measure Drp1 concentration to be ∼0.5 μM in U2OS cell cytosol, suggesting the actin-binding affinity measured here (Kd = 0.6 μM) is in the physiologically relevant range. The ability of Drp1 to bind actin filaments in a highly dynamic manner provides potential for actin filaments to serve as reservoirs of oligomerization-competent Drp1 that can be accessed for mitochondrial fission.

Amitriptyline induces mitophagy that precedes apoptosis in human HepG2 cells.

Systemic treatments for hepatocellular carcinoma (HCC) have been largely unsuccessful. This study investigated the antitumoral activity of Amitriptyline, a tricyclic antidepressant, in hepatoma cells. Amitriptyline-induced toxicity involved early mitophagy activation that subsequently switched to apoptosis. Amitriptyline induced mitochondria dysfunction and oxidative stress in HepG2 cells. Amitriptyline specifically inhibited mitochondrial complex III activity that is associated with decreased mitochondrial membrane potential (∆Ψm) and increased reactive oxygen species (ROS) production. Transmission electron microscopy (TEM) studies revealed structurally abnormal mitochondria that were engulfed by double-membrane structures resembling autophagosomes. Consistent with mitophagy activation, fluorescence microscopy analysis showed mitochondrial Parkin recruitment and colocalization of mitochondria with autophagosome protein markers.Pharmacological or genetic inhibition of autophagy exacerbated the deleterious effects of Amitriptyline on hepatoma cells and led to increased apoptosis. These results suggest that mitophagy acts as an initial adaptive mechanism of cell survival. However persistent mitochondrial damage induced extensive and lethal mitophagy, autophagy stress and autophagolysome permeabilization leading eventually to cell death by apoptosis. Amitriptyline also induced cell death in hepatoma cells lines with mutated p53 and non-sense p53 mutation.Our results support the hypothesis that Amitriptyline-induced mitochondrial dysfunction can be a useful therapeutic strategy for HCC treatment, especially in tumors showing p53 mutations and/or resistant to genotoxic treatments.

The Salmonella effector SopB prevents ROS-induced apoptosis of epithelial cells by retarding TRAF6 recruitment to mitochondria

Microbial pathogens enter host cells by injecting effector proteins of the Type III secretion system (T3SS), which facilitate pathogen translocation across the host cell membrane. These effector proteins exert their effects by modulating a variety of host innate immune responses, thereby facilitating bacterial replication and systemic infection. Salmonella enterica serovar typhimurium (S.typhimurium) is a clinically important pathogen that causes food poisoning and gastroenteritis. The SopB effector protein of S. typhimurium, encoded by Salmonella pathogenicity islands (SPI)-1 T3SS, protects host epithelial cells from infection-induced apoptosis. However, how SopB influences apoptosis induction remains unclear. Here, we investigated the mechanism of SopB action in host cells. We found that SopB inhibits infection-induced apoptosis by attenuating the production of reactive oxygen species (ROS) in mitochondria, the crucial organelles for apoptosis initiation. Further investigation revealed that SopB binds to cytosolic tumor necrosis factor receptor associated factor 6 (TRAF6) and forms a trap preventing the mitochondrial recruitment of TRAF6, an essential event for ROS generation within mitochondria. By studying the response of Traf6+/+ and Traf6−/−mouse embryonic fibroblasts to S. typhimurium infection, we found that TRAF6 promoted apoptosis by increasing ROS accumulation, which led to increased Bax/Bcl-2 ratio, Bax recruitment to mitochondrial membrane, and release of Cyt c into the cytoplasm. These findings show that SopB suppresses host cell apoptosis by binding to TRAF6 and preventing mitochondrial ROS generation.

MOAP-1 Mediates Fas-Induced Apoptosis in Liver by Facilitating tBid Recruitment to Mitochondria.

Fas apoptotic signaling regulates diverse physiological processes. Acute activation of Fas signaling triggers massive apoptosis in liver. Upon Fas receptor stimulation, the BH3-only protein Bid is cleaved into the active form, tBid. Subsequent tBid recruitment to mitochondria, which is facilitated by its receptor MTCH2 at the outer mitochondrial membrane (OMM), is a critical step for commitment to apoptosis via the effector proteins Bax or Bak. MOAP-1 is a Bax-binding protein enriched at the OMM. Here, we show that MOAP-1-deficient mice are resistant to Fas-induced hepatocellular apoptosis and lethality. In the absence of MOAP-1, mitochondrial accumulation of tBid is markedly impaired. MOAP-1 binds to MTCH2, and this interaction appears necessary for MTCH2 to engage tBid. These findings reveal a role for MOAP-1 in Fas signaling in the liver by promoting MTCH2-mediated tBid recruitment to mitochondria.

A novel de novo dominant negative mutation in DNM1L impairs mitochondrial fission and presents as childhood epileptic encephalopathy

DNM1L encodes dynamin-related protein 1 (DRP1/DLP1), a key component of the mitochondrial fission machinery that is essential for proper functioning of the mammalian brain. Previously reported probands with de novo missense mutations in DNM1L presented in the first year of life with severe encephalopathy and refractory epilepsy, with several dying within the first several weeks after birth. In contrast, we report identical novel missense mutations in DNM1L in two unrelated probands who experienced normal development for several years before presenting with refractory focal status epilepticus and subsequent rapid neurological decline. We expand the phenotype of DNM1L-related mitochondrial fission defects, reveal common unique clinical characteristics and imaging findings, and compare the cellular impact of this novel mutation to the previously reported A395D lethal variant. We demonstrate that our R403C mutation, which resides in the assembly region of DRP1, acts by a dominant-negative mechanism and reduces oligomerization, mitochondrial fission activity, and mitochondrial recruitment of DRP1, but to a lesser extent compared to the A395D mutation. In contrast to the initial report of neonatal lethality resulting from DNM1L mutation and DRP1 dysfunction, our results show that milder DRP1 impairment is compatible with normal early development and subsequently results in a distinct set of neurological findings. In addition, we identify a common pathogenic mechanism whereby DNM1L mutations impair mitochondrial fission. © 2016 Wiley Periodicals, Inc.

Drp1-Dependent Mitochondrial Fission Mediates Toxicity of Positively Charged Graphene in Microglia.

The unique physicochemical properties of graphene and its derivatives enable their application in the diagnostics and therapy of central nervous system (CNS) diseases. However, the potential impacts of surface properties of functionalized graphene on microglia remain poorly understood. Herein, we used graphene oxides (GO), polyethylene glycol (PEG)- and polyethylenimine (PEI)-functionalized GO, which possess different surface charges, to investigate their effects on microglia by focusing on mitochondrial dynamics. The positively charged GO-PEI was found to promote mitochondrial fission as observed in BV-2 cells with mitochondria labeled by DsRed2-mito, indicating that alterations in mitochondrial dynamics depend on the surface properties of graphene. Concurrent to mitochondrial fragmentation, treatment with positively charged GO-PEI induced an increase in mitochondrial recruitment of dynamin-related protein (Drp1). Additionally, GO-PEI treatment also led to apoptotic and autophagic cell death. However, Drp1 silencing by small interfering RNA (siRNA) could effectively attenuate GO-PEI-induced apoptotic and autophagic cell death, indicating that mitochondrial fragmentation occurs upstream of GO-PEI-mediated toxicity in microglia. Overall, our study indicated that positively charged GO-PEI might cause deleterious influence on the central immune homeostasis by Drp1-dependent mitochondrial fragmentation, and provide the strategies for the rational design of graphene-based materials in neuroscience.

SUMO-regulated mitochondrial function in Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder characterized by cardinal motor signs such as rigidity, bradykinesia or rest tremor that arise from a significant death of dopaminergic neurons. Non-dopaminergic degeneration also occurs and it seems to induce the deficits in olfactory, emotional, and memory functions that precede the classical motor symptoms in PD. Despite the majority of PD cases being sporadic, several genes have previously been associated with the hereditary forms of the disease. The proteins encoded by some of these genes, including α-synuclein, DJ-1, and parkin, are modified by small ubiquitin-like modifier (SUMO), a post-translational modification that regulates a variety of cellular processes. Among the several pathogenic mechanisms proposed for PD is mitochondrial dysfunction. Recent studies suggest that SUMOylation can interfere with mitochondrial dynamics, which is essential for neuronal function, and may play a pivotal role in PD pathogenesis. Here, we present an overview of recent studies on mitochondrial disturbance in PD and the potential SUMO-modified proteins and pathways involved in this process. SUMOylation, a post-translational modification, interferes with mitochondrial dynamics, and may play a pivotal role in Parkinson's disease (PD). SUMOylation maintains α-synuclein (α-syn) in a soluble form and activates DJ-1, decreasing mitochondrial oxidative stress. SUMOylation may reduce the amount of parkin available for mitochondrial recruitment and decreases mitochondrial biogenesis through suppression of peroxisomal proliferator-activated receptor-γ co-activator 1 α (PGC-1α). Mitochondrial fission can be regulated by dynamin-related protein 1 SUMO-1- or SUMO-2/3-ylation. A fine balance for the SUMOylation/deSUMOylation of these proteins is required to ensure adequate mitochondrial function in PD.

Mitochondrial Recruitment of Drp1 by Mff is Opposed by the Variable Domain

Mitochondria are critical cellular components and form a dynamically interconnected network that responds to several stimuli such as energy demands, apoptotic stimuli, and mitochondrial quality control. Concomitantly, the processes of mitochondrial fission and fusion are highly regulated in order to maintain a population of healthy organelles, and disruption of either process leads to mitochondrial dysfunction. In fact, a detailed mechanistic understanding of the fission process is not known. Dynamin related protein 1 (Drp1) is known to be the master regulator of mitochondrial fission. This large cytosolic protein is comprised of several domains, including the GTPase, middle, variable, and GTPase effector domains (GED). The role of the variable domain is largely uncharacterized and inherently disordered. Recently, the variable domain has been implicated in interactions between Drp1 and mitochondrial lipids, and we have shown that it regulates Drp1 self-assembly. This oligomerization forms a larger complex capable of generating mechanical force using energy from GTP hydrolysis. At the surface of mitochondria, Drp1 is recruited by various partner proteins anchored in the outer membrane. Mitochondrial fission factor (Mff) is a small protein with a C-terminal transmembrane helix that is critical for recruitment of Drp1 and subsequent mitochondrial fission. The direct Mff-Drp1 interaction has been investigated using transmission electron microscopy, a novel lipid scaffold, GTPase assays, and various biophysical methods. Interestingly, the variable domain of Drp1 limits its interaction with Mff. Removal of this domain allows for formation of a stable Drp1-Mff complex, and results in elevated Drp1 GTPase activity in the presence of Mff. Further characterization of Drp1, Mff, and various mutants of these proteins in MEFs lacking the endogenous protein will build on our current knowledge to refine the role of the variable domain and oligomerization in Drp1 interactions with Mff.

Nestin regulates proliferation and invasion of gastrointestinal stromal tumor cells by altering mitochondrial dynamics.

Nestin is widely expressed in numerous tumors and has become a diagnostic and prognostic indicator. However, the exact mechanism by which nestin contributes to tumor malignancy remains poorly understood. Here, we found marked upregulation of nestin expression in highly proliferative and invasive gastrointestinal stromal tumor (GIST) specimens. Nestin knockdown in GIST cells reduced the proliferative and invasive activity owing to a decrease of mitochondrial intracellular reactive oxygen species (ROS) generation. Furthermore, nestin was co-localized with mitochondria, and knockdown of nestin increased mitochondrial elongation and influenced the mitochondrial function, including oxygen consumption rates, ATP generation and mitochondrial membrane potential and so on. In exploring the underlying mechanism, we demonstrated nestin knockdown inhibited the mitochondrial recruitment of Dynamin-related protein1 and induced the change of mitochondrial dynamics. Thus, nestin may have an important role in GIST malignancy by regulating mitochondrial dynamics and altering intracellular ROS levels. The findings provide new clues to reveal mechanisms by which nestin mediates the proliferation and invasion of GISTs.

Kinases Mst1 and Mst2 positively regulate phagocytic induction of reactive oxygen species and bactericidal activity.

SummaryMitochondria need to be juxtaposted to phagosomes to synergistically produce ample reactive oxygen species (ROS) in phagocytes for pathogens killing. However, how phagosomes transmit signal to recruit mitochondria remains unclear. Here, we report that the kinases Mst1 and Mst2 function to control ROS production by regulating mitochondrial trafficking and mitochondrion-phagosome juxtaposition. Mst1 and Mst2 activate Rac GTPase to promote Toll-like receptor (TLR)-triggered assembly of the TRAF6-ECSIT complex that is required for mitochondrial recruitment to phagosomes. Inactive forms of Rac, including the human Rac2D57N mutant, disrupt the TRAF6-ECSIT complex by sequestering TRAF6, and severely dampen ROS production and greatly increase susceptibility to bacterial infection. These findings demonstrate the TLR-Mst1-Mst2-Rac signalling axis to be critical for effective phagosome-mitochondrion function and bactericidal activity.

COX assembly factor ccdc56 regulates mitochondrial morphology by affecting mitochondrial recruitment of Drp1

Mitochondria are dynamic organelles that alter their morphology in response to cellular signaling and differentiation through balanced fusion and fission. In this study, we found that the mitochondrial inner membrane ATPase ATAD3A interacted with ccdc56/MITRAC12/COA3, a subunit of the cytochrome oxidase (COX)-assembly complex. Overproduction of ccdc56 in HeLa cells resulted in fragmented mitochondrial morphology, while mitochondria were highly elongated in ccdc56-repressed cells by the defective recruitment of the fission factor Drp1. We also found that mild and chronic inhibition of COX led to mitochondrial elongation, as seen in ccdc56-repressed cells. These results indicate that ccdc56 positively regulates mitochondrial fission via regulation of COX activity and the mitochondrial recruitment of Drp1, and thus, suggest a novel relationship between COX assembly and mitochondrial morphology.

Optogenetic apoptosis: light-triggered cell death.

An optogenetic Bax has been designed that facilitates light-induced apoptosis. We demonstrate that mitochondrial recruitment of a genetically encoded light-responsive Bax results in the release of mitochondrial proteins, downstream caspase-3 cleavage, changes in cellular morphology, and ultimately cell death. Mutagenesis of a key phosphorylatable residue or modification of the C-terminus mitigates background (dark) levels of apoptosis that result from Bax overexpression. The mechanism of optogenetic Bax-mediated apoptosis was explored using a series of small molecules known to interfere with various steps in programmed cell death. Optogenetic Bax appears to form a mitochondrial apoptosis-induced channel analogous to that of endogenous Bax.

Optogenetic Apoptosis: Light‐Triggered Cell Death

AbstractAn optogenetic Bax has been designed that facilitates light‐induced apoptosis. We demonstrate that mitochondrial recruitment of a genetically encoded light‐responsive Bax results in the release of mitochondrial proteins, downstream caspase‐3 cleavage, changes in cellular morphology, and ultimately cell death. Mutagenesis of a key phosphorylatable residue or modification of the C‐terminus mitigates background (dark) levels of apoptosis that result from Bax overexpression. The mechanism of optogenetic Bax‐mediated apoptosis was explored using a series of small molecules known to interfere with various steps in programmed cell death. Optogenetic Bax appears to form a mitochondrial apoptosis‐induced channel analogous to that of endogenous Bax.

Thyroid hormone induction of mitochondrial activity is coupled to mitophagy via ROS-AMPK-ULK1 signaling

Currently, there is limited understanding about hormonal regulation of mitochondrial turnover. Thyroid hormone (T3) increases oxidative phosphorylation (OXPHOS), which generates reactive oxygen species (ROS) that damage mitochondria. However, the mechanism for maintenance of mitochondrial activity and quality control by this hormone is not known. Here, we used both in vitro and in vivo hepatic cell models to demonstrate that induction of mitophagy by T3 is coupled to oxidative phosphorylation and ROS production. We show that T3 induction of ROS activates CAMKK2 (calcium/calmodulin-dependent protein kinase kinase 2, β) mediated phosphorylation of PRKAA1/AMPK (5′ AMP-activated protein kinase), which in turn phosphorylates ULK1 (unc-51 like autophagy activating kinase 1) leading to its mitochondrial recruitment and initiation of mitophagy. Furthermore, loss of ULK1 in T3-treated cells impairs both mitophagy as well as OXPHOS without affecting T3 induced general autophagy/lipophagy. These findings demonstrate a novel ROS-AMPK-ULK1 mechanism that couples T3-induced mitochondrial turnover with activity, wherein mitophagy is necessary not only for removing damaged mitochondria but also for sustaining efficient OXPHOS.