bims-mitlys 2021-04-04 papers

bims-mitlys

Biomed News

on Mitochondria and Lysosomes

Issue of 2021–04–04
sixteen papers selected by
Nicoletta Plotegher, University of Padova

Mitochondrial and Autophagic Regulation of Adult Neurogenesis in the Healthy and Diseased Brain.
Mitochondrial quality control: from molecule to organelle.
Cell Death and Inflammation: The Role of Mitochondria in Health and Disease.
Emerging Views of OPTN (optineurin) Function in the Autophagic Process Associated with Disease.
Interactions of Lipid Droplets with the Intracellular Transport Machinery.
Targeting the Mitochondria-Proteostasis Axis to Delay Aging.
Lysosomal Calcium Channels in Autophagy and Cancer.
Stimulation of mTOR-independent autophagy and mitophagy by rilmenidine exacerbates the phenotype of transgenic TDP-43 mice.
Transfer of mitochondria and endosomes between cells by gap junction internalization.
Gene Expression-Related Changes in Morphologies of Organelles and Cellular Component Organization in Mucopolysaccharidoses.
Lysosomal Functions in Glia Associated with Neurodegeneration.
Alzheimer's Disease Pathogenesis: Role of Autophagy and Mitophagy Focusing in Microglia.
ATG4 family proteins drive phagophore growth independently of the LC3/GABARAP lipidation system.
AMPK-dependent and -independent coordination of mitochondrial function and muscle fiber type by FNIP1.
Synaptic activity controls autophagic vacuole motility and function in dendrites.
Mitochondrial Dysfunction Induced by Callyspongiolide Promotes Autophagy-Dependent Cell Death.

Int J Mol Sci. 2021 Mar 24. pii: 3342. [Epub ahead of print]22(7):

Mitochondrial and Autophagic Regulation of Adult Neurogenesis in the Healthy and Diseased Brain.

Hansruedi Büeler.

  Adult neurogenesis is a highly regulated process during which new neurons are generated from neural stem cells in two discrete regions of the adult brain: the subventricular zone of the lateral ventricle and the subgranular zone of the dentate gyrus in the hippocampus. Defects of adult hippocampal neurogenesis have been linked to cognitive decline and dysfunction during natural aging and in neurodegenerative diseases, as well as psychological stress-induced mood disorders. Understanding the mechanisms and pathways that regulate adult neurogenesis is crucial to improving preventative measures and therapies for these conditions. Accumulating evidence shows that mitochondria directly regulate various steps and phases of adult neurogenesis. This review summarizes recent findings on how mitochondrial metabolism, dynamics, and reactive oxygen species control several aspects of adult neural stem cell function and their differentiation to newborn neurons. It also discusses the importance of autophagy for adult neurogenesis, and how mitochondrial and autophagic dysfunction may contribute to cognitive defects and stress-induced mood disorders by compromising adult neurogenesis. Finally, I suggest possible ways to target mitochondrial function as a strategy for stem cell-based interventions and treatments for cognitive and mood disorders.

Keywords:  adult neurogenesis; autophagy/mitophagy; cognitive dysfunction; hippocampus; mitochondrial dynamics; mitochondrial metabolism; mood disorders; neurodegeneration; psychological stress; reactive oxygen species (ROS)

DOI:  https://doi.org/10.3390/ijms22073342
Cell Mol Life Sci. 2021 Mar 29.

Mitochondrial quality control: from molecule to organelle.

Alba Roca-Portoles, Stephen W G Tait.

  Mitochondria are organelles central to myriad cellular processes. To maintain mitochondrial health, various processes co-operate at both the molecular and organelle level. At the molecular level, mitochondria can sense imbalances in their homeostasis and adapt to these by signaling to the nucleus. This mito-nuclear communication leads to the expression of nuclear stress response genes. Upon external stimuli, mitochondria can also alter their morphology accordingly, by inducing fission or fusion. In an extreme situation, mitochondria are degraded by mitophagy. Adequate function and regulation of these mitochondrial quality control pathways are crucial for cellular homeostasis. As we discuss, alterations in these processes have been linked to several pathologies including neurodegenerative diseases and cancer.

Keywords:  ISR; Mitochondrial diseases; Mitochondrial dysfunction; Mitochondrial fission; Mitochondrial fusion; Mitophagy; PINK1; Parkin; UPRmt

DOI:  https://doi.org/10.1007/s00018-021-03775-0
Cells. 2021 Mar 03. pii: 537. [Epub ahead of print]10(3):

Cell Death and Inflammation: The Role of Mitochondria in Health and Disease.

Anna Picca, Riccardo Calvani, Hélio José Coelho-Junior, Emanuele Marzetti.

  Mitochondria serve as a hub for a multitude of vital cellular processes. To ensure an efficient deployment of mitochondrial tasks, organelle homeostasis needs to be preserved. Mitochondrial quality control (MQC) mechanisms (i.e., mitochondrial dynamics, biogenesis, proteostasis, and autophagy) are in place to safeguard organelle integrity and functionality. Defective MQC has been reported in several conditions characterized by chronic low-grade inflammation. In this context, the displacement of mitochondrial components, including mitochondrial DNA (mtDNA), into the extracellular compartment is a possible factor eliciting an innate immune response. The presence of bacterial-like CpG islands in mtDNA makes this molecule recognized as a damaged-associated molecular pattern by the innate immune system. Following cell death-triggering stressors, mtDNA can be released from the cell and ignite inflammation via several pathways. Crosstalk between autophagy and apoptosis has emerged as a pivotal factor for the regulation of mtDNA release, cell's fate, and inflammation. The repression of mtDNA-mediated interferon production, a powerful driver of immunological cell death, is also regulated by autophagy-apoptosis crosstalk. Interferon production during mtDNA-mediated inflammation may be exploited for the elimination of dying cells and their conversion into elements driving anti-tumor immunity.

Keywords:  apoptosis; damage-associated molecular patterns (DAMPs); immunogenic cell death; innate immunity; mitochondrial dynamics; mitochondrial dysfunction; mitochondrial quality control (MQC); mitophagy; oxidative stress; reactive oxygen species (ROS)

DOI:  https://doi.org/10.3390/cells10030537
Autophagy. 2021 Mar 30.

Emerging Views of OPTN (optineurin) Function in the Autophagic Process Associated with Disease.

Yueping Qiu, Jincheng Wang, Hui Li, Bo Yang, Jiajia Wang, Qiaojun He, Qinjie Weng.

  Macroautophagy/autophagy is a highly conserved process in eukaryotic cells. It plays a critical role in cellular homeostasis by delivering cytoplasmic cargos to lysosomes for selective degradation. OPTN (optineurin), a well-recognized autophagy receptor, has received considerable attention due to its multiple roles in the autophagic process. OPTN is associated with many human disorders that are closely related to autophagy, such as rheumatoid arthritis, osteoporosis, and nephropathy. Here, we review the function of OPTN as an autophagy receptor at different stages of autophagy, focusing on cargo recognition, autophagosome formation, autophagosome maturation, and lysosomal quality control. OPTN tends to be protective in most autophagy associated diseases, though the molecular mechanism of OPTN regulation in these diseases is not well understood. A comprehensive review of the function of OPTN in autophagy provides valuable insight into the pathogenesis of human diseases related to OPTN and facilitates the discovery of potential key regulators and novel therapeutic targets for disease intervention in patients with autophagic diseases.

Keywords:  autophagosome formation; autophagy; cargo recognition; diseases; lysosomal quality control; mitophagy; optineurin (OPTN)

DOI:  https://doi.org/10.1080/15548627.2021.1908722
Int J Mol Sci. 2021 Mar 09. pii: 2776. [Epub ahead of print]22(5):

Interactions of Lipid Droplets with the Intracellular Transport Machinery.

Selma Yilmaz Dejgaard, John F Presley.

  Historically, studies of intracellular membrane trafficking have focused on the secretory and endocytic pathways and their major organelles. However, these pathways are also directly implicated in the biogenesis and function of other important intracellular organelles, the best studied of which are peroxisomes and lipid droplets. There is a large recent body of work on these organelles, which have resulted in the introduction of new paradigms regarding the roles of membrane trafficking organelles. In this review, we discuss the roles of membrane trafficking in the life cycle of lipid droplets. This includes the complementary roles of lipid phase separation and proteins in the biogenesis of lipid droplets from endoplasmic reticulum (ER) membranes, and the attachment of mature lipid droplets to membranes by lipidic bridges and by more conventional protein tethers. We also discuss the catabolism of neutral lipids, which in part results from the interaction of lipid droplets with cytosolic molecules, but with important roles for both macroautophagy and microautophagy. Finally, we address their eventual demise, which involves interactions with the autophagocytotic machinery. We pay particular attention to the roles of small GTPases, particularly Rab18, in these processes.

Keywords:  autophagy; biogenesis; endoplasmic reticulum; lipid droplet; microautophagy; mitochondria

DOI:  https://doi.org/10.3390/ijms22052776
Front Cell Dev Biol. 2021 ;9 656201

Targeting the Mitochondria-Proteostasis Axis to Delay Aging.

Andreas Zimmermann, Corina Madreiter-Sokolowski, Sarah Stryeck, Mahmoud Abdellatif.

  Human life expectancy continues to grow globally, and so does the prevalence of age-related chronic diseases, causing a huge medical and economic burden on society. Effective therapeutic options for these disorders are scarce, and even if available, are typically limited to a single comorbidity in a multifaceted dysfunction that inevitably affects all organ systems. Thus, novel therapies that target fundamental processes of aging itself are desperately needed. In this article, we summarize current strategies that successfully delay aging and related diseases by targeting mitochondria and protein homeostasis. In particular, we focus on autophagy, as a fundamental proteostatic process that is intimately linked to mitochondrial quality control. We present genetic and pharmacological interventions that effectively extend health- and life-span by acting on specific mitochondrial and pro-autophagic molecular targets. In the end, we delve into the crosstalk between autophagy and mitochondria, in what we refer to as the mitochondria-proteostasis axis, and explore the prospect of targeting this crosstalk to harness maximal therapeutic potential of anti-aging interventions.

Keywords:  aging; anti-aging targets; autophagy; mitochondria; proteostasis

DOI:  https://doi.org/10.3389/fcell.2021.656201
Cancers (Basel). 2021 Mar 15. pii: 1299. [Epub ahead of print]13(6):

Lysosomal Calcium Channels in Autophagy and Cancer.

Yi Wu, Peng Huang, Xian-Ping Dong.

  Ca2+ is pivotal intracellular messenger that coordinates multiple cell functions such as fertilization, growth, differentiation, and viability. Intracellular Ca2+ signaling is regulated by both extracellular Ca2+ entry and Ca2+ release from intracellular stores. Apart from working as the cellular recycling center, the lysosome has been increasingly recognized as a significant intracellular Ca2+ store that provides Ca2+ to regulate many cellular processes. The lysosome also talks to other organelles by releasing and taking up Ca2+. In lysosomal Ca2+-dependent processes, autophagy is particularly important, because it has been implicated in many human diseases including cancer. This review will discuss the major components of lysosomal Ca2+ stores and their roles in autophagy and human cancer progression.

Keywords:  autophagy; calcium; cancer; ion channel; lysosome

DOI:  https://doi.org/10.3390/cancers13061299
Neurobiol Dis. 2021 Mar 30. pii: S0969-9961(21)00108-X. [Epub ahead of print] 105359

Stimulation of mTOR-independent autophagy and mitophagy by rilmenidine exacerbates the phenotype of transgenic TDP-43 mice.

Nirma D Perera, Doris Tomas, Nayomi Wanniarachchillage, Brittany Cuic, Sophia J Luikinga, Valeria Rytova, Bradley J Turner.

  Autophagy, which mediates delivery of cytoplasmic substrates to the lysosome for degradation, is essential for maintaining proper cell homeostasis in physiology, ageing and disease. There is increasing evidence that autophagy is defective in neurodegenerative disorders, including motor neurons affected in amyotrophic lateral sclerosis (ALS). Restoring impaired autophagy in motor neurons may therefore represent a rational approach for ALS. Here, we demonstrate autophagy impairment in spinal cords of mice expressing mutant TDP-43Q331K or co-expressing TDP-43WTxQ331K transgenes. The clinically approved anti-hypertensive drug rilmenidine was used to stimulate mTOR-independent autophagy in double transgenic TDP-43WTxQ331K mice to alleviate impaired autophagy. Although rilmenidine treatment induced robust autophagy in spinal cords, this exacerbated the phenotype of TDP-43WTxQ331K mice shown by truncated lifespan, accelerated motor neuron loss and pronounced nuclear TDP-43 clearance. Importantly, rilmenidine significantly promoted mitophagy in spinal cords TDP-43WTxQ331K mice, evidenced by reduced mitochondrial markers and load in spinal motor neurons. These results suggest that autophagy induction accelerates the phenotype of this TDP-43 mouse model of ALS, most likely through excessive mitochondrial clearance in motor neurons. These findings also emphasise the importance of balancing autophagy stimulation with the potential negative consequences of hyperactive mitophagy in ALS and other neurodegenerative diseases.

Keywords:  ALS; Autophagy; Motor neuron; Rilmenidine; TDP-43

DOI:  https://doi.org/10.1016/j.nbd.2021.105359
Traffic. 2021 Apr 02.

Transfer of mitochondria and endosomes between cells by gap junction internalization.

Rachael P Norris.

  Intercellular organelle transfer has been documented in several cell types and has been proposed to be important for cell-cell communication and cellular repair. However, the mechanisms by which organelle transfer occurs are uncertain. Recent studies indicate that the gap junction protein, connexin 43 (Cx43), is required for mitochondrial transfer but its specific role is unknown. Using three-dimensional electron microscopy and immunogold labeling of Cx43, this report shows that whole organelles including mitochondria and endosomes are incorporated into double-membrane vesicles, called connexosomes or annular gap junctions, that form as a result of gap junction internalization. These findings demonstrate a novel mechanism for intercellular organelle transfer mediated by Cx43 gap junctions. This article is protected by copyright. All rights reserved.

Keywords:  Gap junction; internalization; mitochondria; organelle transfer

DOI:  https://doi.org/10.1111/tra.12786
Int J Mol Sci. 2021 Mar 09. pii: 2766. [Epub ahead of print]22(5):

Gene Expression-Related Changes in Morphologies of Organelles and Cellular Component Organization in Mucopolysaccharidoses.

Lidia Gaffke, Karolina Pierzynowska, Estera Rintz, Zuzanna Cyske, Izabela Giecewicz, Grzegorz Węgrzyn.

  Mucopolysaccharidoses (MPS) are inherited metabolic diseases characterized by accumulation of incompletely degraded glycosaminoglycans (GAGs) in lysosomes. Although primary causes of these diseases are mutations in genes coding for enzymes involved in lysosomal GAG degradation, it was demonstrated that storage of these complex carbohydrates provokes a cascade of secondary and tertiary changes affecting cellular functions. Potentially, this might lead to appearance of cellular disorders which could not be corrected even if the primary cause of the disease is removed. In this work, we studied changes in cellular organelles in MPS fibroblasts relative to control cells. All 11 types and subtypes of MPS were included into this study to obtain a complex picture of changes in organelles in this group of diseases. Two experimental approaches were employed, transcriptomic analyses and electron microscopic assessment of morphology of organelles. We analyzed levels of transcripts of genes grouped into two terms included into the QuickGO database, 'Cellular component organization' (GO:0016043) and 'Cellular anatomical entity' (GO:0110165), to find that number of transcripts with significantly changed levels in MPS fibroblasts vs. controls ranged from 109 to 322 (depending on MPS type) in GO:0016043, and from 70 to 208 in GO:0110165. This dysregulation of expression of genes crucial for proper structures and functions of various organelles was accompanied by severe changes in morphologies of lysosomes, nuclei, mitochondria, Golgi apparatus, and endoplasmic reticulum. Interestingly, some observed changes occurred in all/most MPS types while others were specific to particular disease types/subtypes. We suggest that severe changes in organelles in MPS cells might arise from dysregulation of expression of a battery of genes involved in organelles' structures and functions. Intriguingly, normalization of GAG levels by using recombinant human enzymes specific to different MPS types corrected morphologies of some, but not all, organelles, while it failed to improve regulation of expression of selected genes. These results might suggest reasons for inability of enzyme replacement therapy to correct all MPS symptoms, particularly if initiated at advanced stages of the disease.

Keywords:  electron microscopy; mucopolysaccharidoses; organelles; transcriptomic analyses

DOI:  https://doi.org/10.3390/ijms22052766
Biomolecules. 2021 Mar 09. pii: 400. [Epub ahead of print]11(3):

Lysosomal Functions in Glia Associated with Neurodegeneration.

Conlan Kreher, Jacob Favret, Malabika Maulik, Daesung Shin.

  Lysosomes are cellular organelles that contain various acidic digestive enzymes. Despite their small size, they have multiple functions. Lysosomes remove or recycle unnecessary cell parts. They repair damaged cellular membranes by exocytosis. Lysosomes also sense cellular energy status and transmit signals to the nucleus. Glial cells are non-neuronal cells in the nervous system and have an active role in homeostatic support for neurons. In response to dynamic cues, glia use lysosomal pathways for the secretion and uptake of regulatory molecules, which affect the physiology of neighboring neurons. Therefore, functional aberration of glial lysosomes can trigger neuronal degeneration. Here, we review lysosomal functions in oligodendrocytes, astrocytes, and microglia, with emphasis on neurodegeneration.

Keywords:  astrocytes; autophagy; glia; lysosomes; microglia; neurodegenerative diseases; oligodendrocytes; synapse

DOI:  https://doi.org/10.3390/biom11030400
Int J Mol Sci. 2021 Mar 24. pii: 3330. [Epub ahead of print]22(7):

Alzheimer's Disease Pathogenesis: Role of Autophagy and Mitophagy Focusing in Microglia.

Mehdi Eshraghi, Aida Adlimoghaddam, Amir Mahmoodzadeh, Farzaneh Sharifzad, Hamed Yasavoli-Sharahi, Shahrokh Lorzadeh, Benedict C Albensi, Saeid Ghavami.

  Alzheimer's disease (AD) is a debilitating neurological disorder, and currently, there is no cure for it. Several pathologic alterations have been described in the brain of AD patients, but the ultimate causative mechanisms of AD are still elusive. The classic hallmarks of AD, including amyloid plaques (Aβ) and tau tangles (tau), are the most studied features of AD. Unfortunately, all the efforts targeting these pathologies have failed to show the desired efficacy in AD patients so far. Neuroinflammation and impaired autophagy are two other main known pathologies in AD. It has been reported that these pathologies exist in AD brain long before the emergence of any clinical manifestation of AD. Microglia are the main inflammatory cells in the brain and are considered by many researchers as the next hope for finding a viable therapeutic target in AD. Interestingly, it appears that the autophagy and mitophagy are also changed in these cells in AD. Inside the cells, autophagy and inflammation interact in a bidirectional manner. In the current review, we briefly discussed an overview on autophagy and mitophagy in AD and then provided a comprehensive discussion on the role of these pathways in microglia and their involvement in AD pathogenesis.

Keywords:  Alzheimer’s; autophagy; inflammation; microglia; mitochondria; mitophagy; neurodegeneration; neuroinflammation

DOI:  https://doi.org/10.3390/ijms22073330
Mol Cell. 2021 Mar 25. pii: S1097-2765(21)00169-6. [Epub ahead of print]

ATG4 family proteins drive phagophore growth independently of the LC3/GABARAP lipidation system.

Thanh Ngoc Nguyen, Benjamin Scott Padman, Susanne Zellner, Grace Khuu, Louise Uoselis, Wai Kit Lam, Marvin Skulsuppaisarn, Runa S J Lindblom, Emily M Watts, Christian Behrends, Michael Lazarou.

  The sequestration of damaged mitochondria within double-membrane structures termed autophagosomes is a key step of PINK1/Parkin mitophagy. The ATG4 family of proteases are thought to regulate autophagosome formation exclusively by processing the ubiquitin-like ATG8 family (LC3/GABARAPs). We discover that human ATG4s promote autophagosome formation independently of their protease activity and of ATG8 family processing. ATG4 proximity networks reveal a role for ATG4s and their proximity partners, including the immune-disease protein LRBA, in ATG9A vesicle trafficking to mitochondria. Artificial intelligence-directed 3D electron microscopy of phagophores shows that ATG4s promote phagophore-ER contacts during the lipid-transfer phase of autophagosome formation. We also show that ATG8 removal during autophagosome maturation does not depend on ATG4 activity. Instead, ATG4s can disassemble ATG8-protein conjugates, revealing a role for ATG4s as deubiquitinating-like enzymes. These findings establish non-canonical roles of the ATG4 family beyond the ATG8 lipidation axis and provide an AI-driven framework for rapid 3D electron microscopy.

Keywords:  ATG4; ATG9a; FIB-SEM; LRBA; PINK1; Parkin; autophagosome; autophagy; mitochondria; mitophagy

DOI:  https://doi.org/10.1016/j.molcel.2021.03.001
PLoS Genet. 2021 Mar 29. 17(3): e1009488

AMPK-dependent and -independent coordination of mitochondrial function and muscle fiber type by FNIP1.

Liwei Xiao, Jing Liu, Zongchao Sun, Yujing Yin, Yan Mao, Dengqiu Xu, Lin Liu, Zhisheng Xu, Qiqi Guo, Chenyun Ding, Wanping Sun, Likun Yang, Zheng Zhou, Danxia Zhou, Tingting Fu, Wenjing Zhou, Yuangang Zhu, Xiao-Wei Chen, John Zhong Li, Shuai Chen, Xiaoduo Xie, Zhenji Gan.

Mitochondria are essential for maintaining skeletal muscle metabolic homeostasis during adaptive response to a myriad of physiologic or pathophysiological stresses. The mechanisms by which mitochondrial function and contractile fiber type are concordantly regulated to ensure muscle function remain poorly understood. Evidence is emerging that the Folliculin interacting protein 1 (Fnip1) is involved in skeletal muscle fiber type specification, function, and disease. In this study, Fnip1 was specifically expressed in skeletal muscle in Fnip1-transgenic (Fnip1Tg) mice. Fnip1Tg mice were crossed with Fnip1-knockout (Fnip1KO) mice to generate Fnip1TgKO mice expressing Fnip1 only in skeletal muscle but not in other tissues. Our results indicate that, in addition to the known role in type I fiber program, FNIP1 exerts control upon muscle mitochondrial oxidative program through AMPK signaling. Indeed, basal levels of FNIP1 are sufficient to inhibit AMPK but not mTORC1 activity in skeletal muscle cells. Gain-of-function and loss-of-function strategies in mice, together with assessment of primary muscle cells, demonstrated that skeletal muscle mitochondrial program is suppressed via the inhibitory actions of FNIP1 on AMPK. Surprisingly, the FNIP1 actions on type I fiber program is independent of AMPK and its downstream PGC-1α. These studies provide a vital framework for understanding the intrinsic role of FNIP1 as a crucial factor in the concerted regulation of mitochondrial function and muscle fiber type that determine muscle fitness.

DOI: https://doi.org/10.1371/journal.pgen.1009488
J Cell Biol. 2021 Jun 07. pii: e202002084. [Epub ahead of print]220(6):

Synaptic activity controls autophagic vacuole motility and function in dendrites.

Vineet Vinay Kulkarni, Anip Anand, Jessica Brandt Herr, Christina Miranda, Maria Chalokh Vogel, Sandra Maday.

Macroautophagy (hereafter "autophagy") is a lysosomal degradation pathway that is important for learning and memory, suggesting critical roles for autophagy at the neuronal synapse. Little is known, however, about the molecular details of how autophagy is regulated with synaptic activity. Here, we used live-cell confocal microscopy to define the autophagy pathway in primary hippocampal neurons under various paradigms of synaptic activity. We found that synaptic activity regulates the motility of autophagic vacuoles (AVs) in dendrites. Stimulation of synaptic activity dampens AV motility, whereas silencing synaptic activity induces AV motility. Activity-dependent effects on dendritic AV motility are local and reversible. Importantly, these effects are compartment specific, occurring in dendrites and not in axons. Most strikingly, synaptic activity increases the presence of degradative autolysosomes in dendrites and not in axons. On the basis of our findings, we propose a model whereby synaptic activity locally controls AV dynamics and function within dendrites that may regulate the synaptic proteome.

DOI: https://doi.org/10.1083/jcb.202002084
BMB Rep. 2021 Apr 01. pii: 5323. [Epub ahead of print]

Mitochondrial Dysfunction Induced by Callyspongiolide Promotes Autophagy-Dependent Cell Death.

Soohyun Lee, Yoonjeong Jeong, Jae-Seok Roe, Hoyoung Huh, Sang Hoon Paik, Jaewhan Song.

Callyspongiolide is a marine macrolide known to induce caspase-independent cancer cell death. While its toxic effects have been known, the mechanism leading to cell death is yet to be identified. We report that Callyspongiolide R form at C-21 (cally2R) causes mitochondrial dysfunction by inhibiting-mitochondrial complex I or II, leading to a disruption of mitochondrial membrane potential and a deprivation of cellular energy. Subsequently, we observed, using electron microscopy, a drastic formation of autophagosome and mitophagy. Supporting these data, LC3, an autophagosome marker, was shown to co-localize with LAMP2, a lysosomal protein, showing autolysosome formation. RNA sequencing results indicated the induction of hypoxia and blocking of EGF-dependent pathways, which could be caused by induction of autophagy. Furthermore, mTOR and AKT pathways preventing autophagy were repressed while AMPK was upregulated, supporting autophagosome progress. Finally, the combination of cally2R with known anti-cancer drugs, such as gefitinib, sorafenib, and rapamycin, led to synergistic cell death, implicating potential therapeutic applications of callyspongiolide for future treatments.