Cerebral ischemia is a prevalent cerebrovascular disorder, with the restoration of blocked blood vessels serving as the current standard clinical treatment. However, reperfusion can exacerbate neuronal damage and neurological dysfunction, resulting in cerebral ischemia-reperfusion (I/R) injury. Presently, clinical treatment strategies for cerebral I/R injury are limited, creating an urgent need to identify new effective therapeutic targets. The PI3K/AKT signaling pathway, a pro-survival pathway associated with cerebral I/R injury, has garnered significant attention. We conducted a comprehensive review of the literature on the PI3K/AKT pathway in the context of cerebral I/R. Our findings indicate that activation of the PI3K/AKT signaling pathway following cerebral I/R can alleviate oxidative stress, reduce endoplasmic reticulum stress (ERS), inhibit inflammatory responses, decrease neuronal apoptosis, autophagy, and pyroptosis, mitigate blood-brain barrier (BBB) damage, and promote neurological function recovery. Consequently, this pathway ultimately reduces neuronal death, alleviates brain tissue damage, decreases the volume of cerebral infarction, and improves behavioral impairments. These results suggest that the PI3K/AKT signaling pathway is a promising therapeutic target for further research and drug development, holding significant potential for the treatment of cerebral I/R injury.

Liver cancer is the third most lethal and prevalent cancer in the Asia‑Pacific regions. Despite the use of tyrosine kinase inhibitors as first‑ and second‑line therapies, the overall survival rate for advanced liver cancer remains dismal and has not improved over the past decade. The present study, through high‑throughput screening, identified and demonstrated that PD161570, a new tyrosine kinase inhibitor, inhibited cell growth and proliferation in liver cancer cells. Mechanistically, PD161570 induced autophagy and enhanced autophagic flux in an autophagy‑related gene (ATG5)‑dependent and mammalian target of rapamycin kinase‑independent manner. Furthermore, when combined with chloroquine treatment, PD161570 not only suppressed cell proliferation but also increased cell apoptosis due to autophagy inhibition. RNA sequencing analysis revealed 1,121 differentially expressed genes in liver cancer cells following PD161570 treatment under autophagy inhibition via ATG5 knockdown. Notably, key molecules involved in autophagy (such as Damage Regulated Autophagy Modulator 1) and apoptosis regulators (including HRK, CTSS, BIRC3, BBC3, DDIT3 and GADD45B), were identified. Functional enrichment analyses, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), demonstrated enrichment in apoptotic and cell death signaling pathways, highlighting the critical role of the mitogen‑activated protein kinases signaling pathway. In conclusion, PD161570 elicited an ATG5‑dependent autophagic process in liver cancer cells, while simultaneously enhancing apoptosis under conditions of autophagy inhibition.

Fucosyltransferase 2 (FUT2) is an enzyme that adds fucose to proteins or lipids via α-1,2-fucosylation in the intestinal mucosa. While FUT2 deficiency is linked to increased susceptibility to inflammatory bowel disease (IBD), its role in colorectal cancer (CRC) is unclear, and the molecular mechanisms involved remain largely unknown. We established an azoxymethane (AOM) and dextran sulfate sodium (DSS) model to induce CRC. FUT2 expression was assessed in human CRC tissues, AOM/DSS-induced mouse models, and CRC cell lines using qRT-PCR, western blotting, and UEA-I staining. FUT2 knockout (FUT2△IEC) mice were treated with AOM/DSS, and FUT2-overexpressing CRC cells were created to evaluate the effects of FUT2 on apoptosis in both in vitro and in vivo settings through Western blot analyses and functional assays. N-glycoproteomics, UEA-I chromatography, and co-immunoprecipitation were utilized to identify regulatory mechanisms and target fucosylated proteins. FUT2 expression and α-1,2-fucosylation were significantly decreased in CRC. FUT2 deficiency worsened AOM/DSS-induced CRC and reduced tumor apoptosis, while FUT2 overexpression induced apoptosis and inhibited proliferation in CRC cells and xenografts. Mechanistically, FUT2 appears to suppress autophagy by impairing lysosomal function and directly targeting and fucosylating LAMP1, contributing to lysosomal dysfunction. Our study reveals a fucosylation-dependent antitumor mechanism of FUT2 in CRC, suggesting potential therapeutic strategies for CRC treatment.

Upon inflammation, the bone marrow (BM) landscape undergoes significant architectural and functional modifications. Stimulation of the hematopoietic niche triggers a series of lightning events, which begin with stem/progenitor blood elements mobilization and culminates with the activation of immune responses. Ageing partially mirrors this process, albeit with a propensity towards chronic inflammation and immune dysfunction. Age-related chronic inflammation disrupts bone homeostasis and accompanies impaired tissue regeneration. Thus, focusing on the bone marrow's dynamics during inflammatory bone diseases could lay the way for the development of novel therapeutic platforms aimed at niche reprogramming. Herein, we summarize inflammatory and age-induced processes in multiple BM compartments, with particular reference to hematopoietic, stromal stem/progenitor cells, and mature immunocytes. Finally, we focus on autophagy and its potential to clinically re-modulate the pathological "flogistic" bias, possibly by restoring functional phenotypes within the bone marrow niche elements.

Parkinson's Disease is characterized by selective degeneration of dopaminergic neurons, primarily in the substantia nigra pars compacta, as well as accumulation of alpha-synuclein enriched protein aggregates within neurons. The pathogenesis of PD is still not completely understood, and no treatments exist that alter disease progression. Obvious genetic causes are detected in only a small number of PD patients (5-10 %), suggesting that environmental factors play a significant role the development of PD. Correlative studies suggest that the microbiota could be an important environmental modifier of neurodegeneration. We identified a microbiotal isolate, Actinomyces viscosus, that reduced neurodegeneration in C. elegans expressing a pathological mutant form (G2019S) of leucine-rich repeat kinase 2 (LRRK2) in dopaminergic neurons. A. viscosus also suppressed autophagic dysfunction in these animals and reduced alpha-synuclein aggregation in a synucleinopathy model. Global gene expression analysis revealed increased expression of aspartic cathepsins in response to A. viscosus. Consistent with the involvement of these proteins in neuroprotection, we found that reducing aspartic cathepsin function increased neurodegeneration in the LRRK2 transgenic model. Our findings contribute to the current understanding of how the gut microbiota may influence PD, elucidating one potential mechanism of microbiota-mediated neuroprotection.

Herpes simplex virus 1 (HSV-1) establishes latent infections in sensory neurons, from which HSV sporadically reactivates due to external stress and other stimuli. Latency and reactivation are studied through in vivo models in a variety of hosts, as well as in vitro models using primary neurons, and neurons derived from pluripotent stem cells (iPSCs). These systems behave disparately, but the reasons remain unknown. The interferon (IFN)-based neuronal innate immune response is critical in controlling HSV-1 replication and HSV-1 counters these responses in part through infectedcell protein 34.5 (ICP34.5). ICP34.5 also promotes neurovirulence by preventing host translational shutoff and interfering with host cell autophagy. Here we demonstrate in a human iPSC neuronal model that sustaining host translation is the key activity of ICP34.5 for enhancement of reactivation. Specifically, our data shows that ICP34.5 was key for maintenance of HSV-1 latency. While interaction of ICP34.5 with the autophagy regulator Beclin 1 was important for maintaining latency, this was not due to modulation of bulk autophagy. Our work from primary mouse neurons suggested that the major effect of ICP34.5 on latency maintenance occurs in an IRF3/7-dependent manner. Notably, the role of ICP34.5 in regulating latency and reactivation differs between neurons derived from human iPSCs (iNeurons) and primary mouse trigeminal (TG) neurons. This highlights the importance of selecting an appropriate neuronal model and validating experimental outcomes in multiple models.

Hexavalent chromium [Cr(VI)], due to its high solubility and permeability, is significantly more toxic than trivalent chromium [Cr(III)] as it generates reactive oxygen species (ROS) during cellular reduction. Industrial discharges have led to increasing Cr(VI) contamination in surface and groundwater, posing serious environmental and public health concerns. In our previous study, we demonstrated that exposure to 5 ppm Cr(VI) for 4 and 8 months adversely affected body weight, water consumption, and liver function in Swiss albino mice. Histological analyses revealed tissue alterations, disrupted DNA repair gene expression in liver tissue, and a marked increase in apoptotic gene expression after 8 months of exposure. Building on these findings, we employed the same Cr(VI) concentration (5 ppm via drinking water) over 4 and 8 months in the present study. Our results showed a significant increase in ROS generation in the liver, brain, and kidney tissues at both time intervals. Additionally, the presence of autophagolysosomes was markedly elevated after chronic Cr(VI) exposure in each tissue. We also observed altered expression patterns of key autophagy-related genes (Atg5, Beclin1, and Lc3) and mTor in these tissues. Immunohistochemical analysis further confirmed a significant increase in LC3B expression after 4 months of exposure. Our findings suggest that heightened intracellular oxidative stress triggers a protective autophagy response, mediated via mTOR signaling, to maintain cellular integrity. However, prolonged toxic insult and ROS accumulation may eventually shift pro-survival autophagy toward apoptotic cell death in the liver and brain tissues.

Bovine viral diarrhoea (BVD), a condition triggered by bovine viral diarrhoea virus (BVDV), is recognized globally as a prevalent pathogen among ruminants and markedly affects the economics of animal husbandry. MicroRNAs, a class of small noncoding RNAs, play pivotal roles in regulating a myriad of biological processes.The ATG7-LC3 pathway, a canonical autophagy mechanism, is integral in defending against pathogenic invasion and maintaining cellular homeostasis.

In this study, we observed significant downregulation of bta-miR-221 in cells infected with BVDV. We further established that overexpression of bta-miR-221 markedly attenuated BVDV replication in Madin‒Darby bovine kidney (MDBK) cells. Through bioinformatics prediction analysis, we identified ATG7, an autophagy-related gene, as a direct downstream target of bta-miR-221. However, the intricate relationships among bta-miR-221, the ATG7-LC3 pathway, and BVDV infection remained unclear. Our study revealed that ATG7 expression was significantly elevated in BVDV-infected cells, whereas bta-miR-221 mimics repressed both endogenous and exogenous ATG7 expression. Following BVDV infection, we noted a decrease in LC3I expression, its conversion to LC3II, a significant increase in ATG7 expression, and a notable decrease in SQSTM1/p62 expression. By employing laser confocal microscopy and immunoprecipitation assays, we elucidated the regulation of the ATG7-LC3 pathway by bta-miR-221 in MDBK cells. Our findings recealed that BVDV infection enhanced the ATG7-LC3 interaction, inducing autophagy through the suppression of bta-miR-221 in MDBK cells. Consequently, bta-miR-221 emerged as a potent inhibitor of BVDV, impacting its proliferation and replication within the host.

This research sheds light on novel aspects of virus-host interactions and lays a foundation for the development of antiviral therapeutics.

Transmembrane B cell lymphoma 2-associated X protein inhibitor motif-containing (TMBIM) 6, also known as Bax Inhibitor-1 (BI-1), has been heavily researched for its cytoprotective functions. TMBIM6 functional diversity includes modulating cell survival, stress, metabolism, cytoskeletal dynamics, organelle function, regulating cytosolic acidification, calcium, and reactive oxygen species (ROS). Clinical research shows TMBIM6 plays a key role in many of the world's top diseases/injuries (i.e., Alzheimer's, Parkinson's, diabetes, obesity, brain injury, liver disease, heart disease, aging, etc.), including cancer, where TMBIM6 expression impacts patient survival, chemoresistance, cancer progression, and metastasis. We show TMBIM6 is activated by, and undergoes, different conformational changes that dictate its function following a significant change in the cell's IntraCellular Environment (ICE). TMBIM6 agonism, following ICE change, can help the cell overcome multiple stresses including toxin exposure, viral infection, wound healing, and excitotoxicity. However, in cancer cells TMBIM6 agonism results in rapid paraptotic induction irrespective of the cancer type, sub-type, genotype or phenotype. Furthermore, the level of TMBIM6 expression in cancer did not dictate the level of paraptotic induction; however, it did dictate the rate at which paraptosis occurred. TMBIM6 agonism did not induce paraptosis in cancer via canonical routes involving p38 MAPK, JNK, ERK, UPR, autophagy, proteasomes, or Caspase-9. Instead, TMBIM6 agonism in cancer upregulates cytosolic Ca2+ and ROS, activates lysosome biogenesis, and induces paraptosis via ERAD II mechanisms. In xenograft models, we show TMBIM6 agonism induces rapid cancer cell death with no toxicity, even at high doses of TMBIM6 agonist (>450 mg/kg). In summary, this study shows TMBIM6's functional diversity is only activated by severe ICE change in diseased/injured cells, highlighting its transformative potential as a therapeutic target across various diseases and injuries, including cancer.

Impaired mitophagy results in the accumulation of defective mitochondria that are unable to be cleared effectively in Alzheimer's disease (AD). Aloe-emodin (AE), a key component of the traditional Chinese medicine Rhubarb, exhibits neuroprotective effects against Alzheimer's disease, though the underlying mechanism remains unclear. Studying aloe-emodin's role in enhancing mitophagy is vital for improving cognitive function and reducing neuronal damage in Alzheimer's disease.

The APP/PS1 double transgenic mice were adopted as models for AD to assess the effects of aloe-emodin upon cognitive function and its neuroprotective impact on hippocampal neurons. Additionally, we investigated the regulatory mechanisms of proteins within the aforementioned pathway, and the morphological characteristics of mitophagy-related proteins. An AD hippocampal neuron model was developed using Aβ25-35 to evaluate the mitochondrial function, the protein expression of such a pathway and the mitophagy. This approach aims to elucidate the effects and underlying mechanisms of aloe-emodin in relation to AD.

AE activates mitophagy in neurons, improves cognitive dysfunction, reduces hippocampal damage, and alleviates AD symptoms in model mice. AE activates the expression of AMPK, PGC-1α and SIRT3. Increased expression of SIRT3 in mitochondria promotes mitophagy and regulates the function of mitochondrial proteins. When mitochondrial autophagy is enhanced, the expression of Beclin1, LC3, P62, Parkin, and PINK1-related proteins changes. Further in vitro experiments showed that AE can enhance mitochondrial function in Alzheimer's disease cell models. The mitochondrial membrane potential, GSH, ROS and Ca2+ levels gradually recover, alleviating the pathological manifestations of AD. Knocking down SIRT3 leads to increased mitochondrial damage and a reduction in mitophagy in HT22 cells.

Experimental results show that AE can activate mitophagy through AMPK/PGC-1α/SIRT3 pathway, alleviate cognitive dysfunction in AD, and reduce damage to hippocampal neurons.

In plant cells, autophagy is an essential quality control process by forming a double-membrane structure named the autophagosome, which envelopes and transports the cargoes to the vacuole for degradation/recycling. Autophagy-related (ATG) 8, a key regulator in autophagy, exerts multifunctional roles during autophagy. ATG8 anchors on the phagophore membrane through the ATG8 conjugation system and participates in different steps during autophagosome formation. Accumulating evidence has demonstrated that ATG8 cooperates with other ATG or non-ATG proteins in autophagosome biogenesis. Meanwhile, ATG8 plays an important role in cargo recognition, which is mainly attributed by the specific interactions between ATG8 and the selective autophagy receptors (SARs) or cargos for selective autophagy. Emerging roles of ATG8 in non-canonical autophagy have been recently reported in plants for different stress adaptations. Here, we review the diverse functions of ATG8 in plants, focusing on autophagosome biogenesis and cargo recognition in canonical and non-canonical autophagy.

This study aimed to evaluate the influence of back-fat thickness (BF), at mating of sows, on autophagy in placenta and the potential mechanism. The sows were divided into two groups according to their BF at mating: BFI (15-20 mm, n = 14) and BFII (21-27 mm, n = 14) as the maternal obesity group. The placental samples used for investigating autophagic function and fatty acid profiles were obtained by vaginal delivery. Our results demonstrated that autophagy defects were observed in placenta from BFII sows along with altered circulating and placental fatty acid profiles. Indicative of impaired autophagy, reduced autophagic vesicles as well as LC3-positive puncta were linked to decreased mRNA or protein expression of autophagy-related genes, including ATG5, ATG7, Beclin1, ATG12, LC3, LAMP1 and LAMP2 in the placenta of BFII sows (p < 0.05). Meanwhile, we found reduced conversion of LC3-I to LC3-II and up-regulated protein content of p62 in the placenta from BFII group (p < 0.05). Furthermore, excessive back-fat was also associated with increased activation of AKT/mTOR signaling and decreased mRNA content of transcription factors regulating the autophagic pathway, including PPARα and PGC1α, but increased mRNA expression of NcoR1 in placenta. Together, these findings indicate that maternal obesity incites autophagy injury in pig term placenta, which may contribute to augmented placental lipid accumulation and therefore impaired placental function.

Hyperphosphorylation and aggregation of MAPT (microtubule-associated protein tau) is a pathogenic hallmark of tauopathies and a defining feature of Alzheimer disease (AD). Pathological MAPT/tau is targeted by macroautophagy/autophagy for clearance after being sequestered within autophagosomes, but autophagy dysfunction is indicated in tauopathy. While mitochondrial bioenergetic deficits have been shown to precede MAPT/tau pathology in tauopathy brains, it is unclear whether energy metabolism deficiency is involved in the pathogenesis of autophagy defects. Here, we reveal that stimulation of anaplerotic metabolism restores defective oxidative phosphorylation (OXPHOS) in tauopathy neurons which, strikingly, leads to pronounced MAPT/tau clearance by boosting autophagy functionality through enhancements of mitochondrial biosynthesis and supply of phosphatidylethanolamine for autophagosome biogenesis. Furthermore, early anaplerotic stimulation of OXPHOS elevates autophagy activity and attenuates MAPT/tau pathology, thereby counteracting memory impairment in tauopathy mice. Taken together, our study sheds light on a pivotal role of mitochondrial bioenergetic deficiency in tauopathy-related autophagy defects and suggests a new therapeutic strategy to prevent the buildup of pathological MAPT/tau in AD and other tauopathy diseases.Abbreviation: AA: antimycin A; AD, Alzheimer disease; ATP, adenosine triphosphate; AV, autophagosome/autophagic vacuole; AZ, active zone; Baf-A1: bafilomycin A1; CHX, cycloheximide; COX, cytochrome c oxidase; DIV, days in vitro; DRG, dorsal root ganglion; ETN, ethanolamine; FRET, Förster/fluorescence resonance energy transfer; FTD, frontotemporal dementia; Gln, glutamine; HA: hydroxylamine; HsMAPT/Tau, human MAPT; IMM, inner mitochondrial membrane; LAMP1, lysosomal-associated membrane protein 1; LIs, lysosomal inhibitors; MDAV, mitochondria-derived autophagic vacuole; MmMAPT/Tau, murine MAPT; NFT, neurofibrillary tangle; OCR, oxygen consumption rate; Omy: oligomycin; OXPHOS, oxidative phosphorylation; PPARGC1A/PGC-1alpha: peroxisome proliferative activated receptor, gamma, coactivator 1 alpha; PE, phosphatidylethanolamine; phospho-MAPT/tau, hyperphosphorylated MAPT; PS, phosphatidylserine; PISD, phosphatidylserine decarboxylase;SQSTM1/p62, sequestosome 1; STX1, syntaxin 1; SYP, synaptophysin; Tg, transgenic; TCA, tricarboxylic acid; TEM, transmission electron microscopy.

Iron plays critical roles in many cellular functions, including energy production, metabolism, and cell proliferation. However, the role of iron in maintaining oocyte quality remains unclear. In this study, DMT1 was identified as a key iron transporter during porcine oocyte maturation. The results demonstrated that iron deficiency in porcine oocyte led to aberrant meiotic progression, accompanied by increased gene expression of DMT1. Inhibition of DMT1 resulted in the failure of cumulus cell expansion and oocyte maturation, along by the abnormal actin and microtubule assembly. Furthermore, loss of DMT1 function caused disruption in mitochondrial function and dynamics, resulting in oxidative stress and Ca2+ dyshomeostasis. Additionally, the absence of DMT1 function activated PINK1/Parkin-dependent mitophagy in porcine oocyte. These findings suggested that DMT1 played a crucial role in safeguarding oocyte quality by protecting against iron-deficiency-induced mitochondrial dysfunction and autophagy. This study provided compelling evidence that DMT1 and iron homeostasis were crucial for maintaining the capacity of porcine oocyte maturation. Moreover, the results hinted at the potential of DMT1 as a novel therapeutic target for treating iron deficiency-related female reproductive disorders.

Macropinocytosis is reported to fuel tumor growth and drug resistance by allowing cancer cells to scavenge extracellular macromolecules. However, accurately defining the role of macropinocytosis in cancer depends on our ability to selectively block this process. 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) is widely used to inhibit macropinocytosis but affects multiple Na+/H+ exchangers (NHE) that regulate cytoplasmic and organellar pH. Consistent with this, we report that EIPA slows proliferation to a greater extent than can be accounted for by macropinocytosis inhibition and triggers conjugation of ATG8 to single membranes (CASM). Knocking down only NHE1 would not avoid macropinocytosis-independent effects on pH. Moreover, contrary to published reports, NHE1 loss did not block macropinocytosis in multiple cell lines. Knocking down CARMIL1 with CRISPR-Cas9 editing limited macropinocytosis, but only by 50%. In contrast, expressing the CARMIL1-AA mutant inhibits macropinocytosis induced by a wide range of macropinocytic stimuli to a similar extent as EIPA. CARMIL1-AA expression did not inhibit proliferation, highlighting the shortcomings of EIPA as a macropinocytosis inhibitor. Importantly, autophagy, another actin dependent, nutrient-producing process, was not affected by CARMIL1-AA expression. In sum, constitutive or inducible CARMIL1-AA expression reduced macropinocytosis without affecting proliferation, RAC activation, or autophagy, other processes that drive tumor initiation and progression.

The severity, frequency, and duration of extreme events, in the context of global warming, have placed many marine ecosystems at high risk. Therefore, the application of methods that can mediate the impacts of global warming on marine organisms seems to be an emerging necessity in the near term. In this context, enhancing the thermal resilience of marine organisms may be crucial for their sustainability. It has been shown that the repeated time-limited exposure of an organism to an environmental stimulus modifies its response mode, thus enhancing resilience and allowing adaptation of the physiological and developmental phenotype to environmental stress. In the present study, we investigated the "stress memory" effect caused by heat hardening on Mytilus galloprovincialis cellular pathways to identify the underlying biochemical mechanisms that enhance mussel thermal tolerance. Heat hardening resulted in increased ETS activity and ATP production and increased autophagic performance at all elevated temperatures (24 °C, 26 °C, and 28 °C). Furthermore, at these increased temperatures, apoptosis and inflammation remain at significantly lower levels in pregnant individuals than in nonhardened individuals. Autophagy, as a negative regulator of apoptosis, may lead to decreased damage to surrounding cells, which in turn alleviates inflammatory effects. In conclusion, the exposure of mussels to heat hardening seems to provide a physiological response that enhances heat tolerance and increases cell survival through increased energy production and reduced cell death and inflammatory responses. The latter can be utilized for the management and conservation of aquatic species of economic value or endangered status.

Autophagy is an intracellular degradation process that sequesters cytoplasmic components in double-membrane vesicles known as autophagosomes, which are degraded upon fusion with lysosomes. This pathway maintains the integrity of proteins and organelles while providing energy and nutrients to cells, particularly under nutrient deprivation. Deregulation of autophagy can cause genomic instability, low protein quality, and DNA damage, all of which can contribute to cancer. Autophagy can also be overactivated in cancer cells to aid in cancer cell survival and drug resistance. Emerging evidence indicates that autophagy has functions beyond cargo degradation, including roles in tumor immunity and cancer stem cell survival. Additionally, autophagy can also influence the tumor microenvironment. This feature warrants further investigation of the role of autophagy in cancer, in which autophagy manipulation can improve cancer therapies, including cancer immunotherapy. This review discusses recent findings on the regulation of autophagy and its role in cancer therapy and drug resistance.

Effective management of cellular components is essential for maintaining brain health, and studies have identified several crucial biological processes in the brain. Among these, autophagy and the role of exosomes in cellular communication are critical for brain health and disease. The interaction between autophagy and exosomes in the nervous system, as well as their contributions to brain damage, have garnered significant attention. This review summarizes that exosomes and their cargoes have been implicated in the autophagy process in the pathophysiology of nervous system diseases. Furthermore, the onset and progression of neurological disorders may be affected by autophagy regulation of the secretion and release of exosomes. These findings may provide new insights into the potential mechanism by which autophagy mediates different exosome secretion and release, as well as the valuable biomedical applications of exosomes in the prevention and treatment of various brain diseases by targeting autophagy.

Autophagy is a cellular recycling process that is associated with tumor growth, anti-tumor immune responses, and therapy resistance in colorectal cancer (CRC). In this report, we identify the small GTPase Rab27B to control the autophagy process in CRC. Depletion of Rab27B showed an abnormal accumulation of autophagy vesicles and increased autophagy markers in CRC cells, indicating autophagy dysregulation. Image analysis indicated that autophagy flux is blocked at the autophagosome/lysosome fusion step when Rab27B is lost. While Rab27B deficient cells are proficient at growth under 2D in vitro conditions, cell growth was significantly impacted in both in vitro 3D growth and in vivo tumorigenesis studies. Together, these results demonstrate a new role of Rab27B in the autophagy trafficking process in CRC and identify Rab27B as a potential therapeutic target for CRC.

Unlike mild DNA damage exposure, DNA damage repair (DDR) is reported to be ineffective in full-grown mammalian oocytes exposed to moderate or severe DNA damage. The underlying mechanisms of this weakened DDR are unknown. Here, we show that moderate DNA damage in full-grown oocytes leads to aneuploidy. Our data reveal that DNA-damaged oocytes have an altered, closed, chromatin state, and suggest that the failure to repair damaged DNA could be due to the inability of DDR proteins to access damaged loci. Our data also demonstrate that, unlike somatic cells, mouse and porcine oocytes fail to activate autophagy in response to DNA double-strand break-inducing treatment, which we suggest may be the cause of the altered chromatin conformation and inefficient DDR. Importantly, autophagy activity is further reduced in maternally aged oocytes (which harbor severe DNA damage), and its induction is correlated with reduced DNA damage in maternally aged oocytes. Our findings provide evidence that reduced autophagy activation contributes to weakened DDR in oocytes, especially in those from aged females, offering new possibilities to improve assisted reproductive therapy in women with compromised oocyte quality.

High salt (HS) consumption is an independent risk factor for neurodegenerative diseases such as dementia, stroke, and cerebral small vessel disease related to cognitive decline. Recently, Alzheimer's disease-like pathology changes have been reported as consequences of a HS diet in wild-type (wt) mice. However, it has not been revealed how HS diets accelerate the progress of Alzheimer's disease (AD) in APP/PS1 mice. Here, we fed APP/PS1 mice a HS diet or normal diet (ND) for six months; the effects of the HS/ND on wt mice were also observed. The results of our behavior test reveal that the HS diet exacerbates anxiety, β-amyloid overload, neuron loss, and synapse damage in the hippocampi of APP/PS1 mice; this was not observed in HS-treated wt mice. RNA sequencing shows that nearly all serpin family members were increased in the hippocampus of HS-treated APP/PS1 mice. Gene function analysis showed that a HS diet induces neurodegeneration, including axon dysfunction and neuro-ligand-based dysfunction, and regulates serine protein inhibitor activities. The mRNA and protein levels of Serpina3n were dramatically increased. Upregulated Serpina3n may be the key for β-amyloid aggregation and neuronal loss in the hippocampus of HS-treated APP/PS1 mice. Serpina3n inhibition attenuated the anxiety and increased the number of neurons in the hippocampal CA1(cornu ammonis) region of APP/PS1 mice. Our study provides novel insights into the mechanisms by which excessive HS diet deteriorates anxiety in AD mice. Therefore, decreasing daily dietary salt consumption constitutes a pivotal public health intervention for mitigating the progression of neuropathology, especially for old patients and those with neurodegenerative disease.

GABARAP is a member of the ATG8 family of ubiquitin-like autophagy related proteins. It was initially discovered as a facilitator of GABA-A receptor translocation to the plasma membrane and has since been shown to promote the intracellular transport of a variety of other proteins under non-autophagic conditions. We and others have shown that GABARAP interacts with the Type II phosphatidylinositol 4-kinase, PI4K2A, and that this interaction is important for autophagosome-lysosome fusion. Here, we identify a 7-amino acid segment within the PI4K2A catalytic domain that contains the GABARAP interaction motif (GIM). This segment resides in an exposed loop that is not conserved in the other mammalian Type II PI 4-kinase, PI4K2B, explaining the specificity of GABARAP binding to the PI4K2A isoform. Mutation of the PI4K2A GIM inhibits GABARAP binding and PI4K2A-mediated recruitment of cytosolic GABARAP to subcellular organelles. We further show that GABARAP binds to mono-phosphorylated phosphoinositides, PI3P, PI4P, and PI5P, raising the possibility that these lipids contribute to the binding energies that drive GABARAP-protein interactions on membranes.

Autophagy is a complex cellular process that can either promote or inhibit cancer progression and development, depending on the context and molecular regulation involved. This study investigates how LSD1 regulates autophagy in ovarian cancer by interacting with the autophagy protein LC3B. Utilizing the bioinformatic analysis of TCGA, CPTAC, and GEO datasets, as well as immunohistochemistry in ovarian cancer patients, we explored the expression association between LSD1 and LC3B. Molecular mechanisms were further analyzed using Western blotting, immunoprecipitation, and GST pull-down assays. Our findings reveal that LSD1 binds to LC3B via its SWIRM domain, and high levels of LSD1 are closely associated with aggressive ovarian cancer and poor patient outcomes. Mechanistically, LSD1 demethylates LC3B, leading to decreased LC3B stability. The observed inverse correlation between LSD1 expression and LC3B protein levels in clinical samples underscores the need for further investigation to elucidate how reduced LC3B protein levels induced by LSD1 demethylation may contribute to ovarian cancer.

The splice variant of retinal G-protein-coupled receptor deletion (RGR-d) is a persistent component of drusen and may be involved in the pathogenesis of dry age-related macular degeneration (AMD). Increasing evidence has demonstrated the critical role of autophagy in AMD. In this study, we investigated whether RGR-d disrupts autophagy in early dry AMD in vivo and in vitro.

Fundus imaging and fluoroscopy were performed on RGR-d mice created by multiplex gene editing. The retina microstructure was evaluated by performing hematoxylin and eosin (H&E) staining as well as transmission electron microscopy (TEM). Retinal function was assessed by full-field electroretinography (ERG). After lentivirus transfection and stimulation, the permeability, phagocytosis, and tight junctions of ARPE-19 cells were evaluated. Western blotting of ATG5, Beclin-1, LC3II/I, and P62 was performed to detect the changes in autophagy pathways.

Atrophy and patchy penetrating hyperfluorescent foci, consistent with early AMD-like defects, were observed in the fundus of 12-month-old RGR-d mice. H&E staining of retinal tissues indicated thinning of each layer of the retinal structure. H&E staining of retinal tissues indicated thinning of each layer of the retinal structure. TEM analysis showed some diffuse granular deposits. And the morphology of choroidal microvascular endothelial cells was degraded and distorted. The morphology of the photoreceptor outer segments showed structural damage, and Bruch's membrane was thickened. ERG indicated that the photoreceptor of RGR-d mice were dysfunctional. Changes in autophagy-related protein expression were observed in the retinal pigment epithelium and retinal neurepithelium, and autophagy regulation was decreased. Palmitic acid (PA) stimulation caused permeability, phagocytosis, and tight junction dysfunction in cells overexpressing RGR-d. Beclin-1 and LC3II/I expression levels were significantly decreased and that of P62 was elevated in RGR-d cells after PA stimulation.

RGR-d disrupts the autophagy pathway, causing the development of an early AMD-like pathophysiology.

Roburic acid (ROB) is a newly discovered tetracyclic triterpene acid extracted from oak galls, which has anti-inflammatory effects, but the mechanism of its anticancer effect is not clear. Our study focuses on exploring the potential mechanism of action of ROB in the treatment of lung cancer using a combination of network pharmacological prediction, molecular docking technique and experimental validation.

A network pharmacology approach was used to screen the protein targets of ROB and lung cancer, and PPI network analysis and enrichment analysis were performed on the intersecting genes. The tissue and organ distribution of the targets was also evaluated based on the BioGPS database. To ensure the reliability of the network pharmacology prediction results, we proceeded to use molecular docking technique to determine the relationship between drugs and targets. Finally, in vitro experiments with cell lines were performed to further reveal the potential mechanism of ROB for the treatment of lung cancer.

A total of 83 potential targets of ROB in lung cancer were collected and further screened by using Cytoscape software, and 7 targets of PTGS2, CYP19A1, PTGS1, AR, CYP17A1, PTGES and SRD5A1 were obtained as hub genes and 7 hub targets had good binding energy with ROB. GO and KEGG analysis showed that ROB treatment of lung cancer mainly involves Arachidonic acid metabolism, Notch signaling pathway, cancer pathway and PPAR signaling pathway. The results of in vitro experiments indicated that ROB may inhibit the proliferation and metastasis of lung cancer cells and activate the PPARγ signaling pathway, as well as induce cellular autophagy.

The results of this study comprehensively elucidated the potential targets and molecular mechanisms of ROB for the treatment of lung cancer, providing new ideas for further lung cancer therapy.

Polyglutamine (polyQ)-mediated spinocerebellar ataxia (SCA), including SCA1, 2, 3, 6, 7, and 17, are caused by mutant genes with expanded CAG repeats, leading to the intracellular accumulation of aggregated proteins, the production of reactive oxygen species, and cell death. Among SCA, SCA3 is caused by a mutation in the ATXN3 (ataxin-3) gene. In a circumstance of polyQ aggregation, the autophagic pathway is induced to degrade the aggregated proteins, thereby suppressing downstream deleterious effects and promoting neuronal survival. In this study, we tested the effects of synthetic indole (NC009-1, -2, -3, -6) and coumarin (LM-022, -031) derivatives as chemical chaperones to assist mutant ATXN3-Q75 folding, as well as autophagy inducers to clear aggregated protein. Among the tested compounds, NC009-1, -2, and -6 and LM-031 interfered with Escherichia coli-derived ATXN3-Q75 aggregation in thioflavin T binding and filter trap assays. In SH-SY5Y cells expressing GFP-fused ATXN3-Q75, these compounds displayed aggregation-inhibitory and neurite growth-promoting potentials compared to untreated cells. Furthermore, these compounds activated autophagy by increasing the phosphatidylethanolamine-conjugated LC3 (microtubule associated protein 1 light chain 3)-II:cytosolic LC3-I ratio in these cells. A biochemical co-immunoprecipitation assay by using a mixture of HEK 293T cell lysates containing recombinant ATXN3-Q75-Venus-C-terminus (VC) or Venus-N-terminus (VN)-LC3 protein indicated that NC009-1 and -2 and LM-031 served as an autophagosome-tethering compound (ATTEC) to interact with ATXN3-Q75 and LC3, and the interaction was further confirmed by bimolecular fluorescence complementation analysis in cells co-expressing both ATXN3-Q75-VC and VN-LC3 proteins. The study results suggest the potential of NC009-1 and -2 and LM-031 as an ATTEC in treating SCA3 and, probably, other polyQ diseases.

Autophagy induction in cancer is involved in cancer progression and the acquisition of resistance to anticancer agents. Therefore, autophagy is considered a potential therapeutic target in cancer therapy. In this study, we found that long-chain fatty acids (LCFAs) have inhibitory effects on Atg4B, which is essential for autophagosome formation, through screening based on the pharmacophore of 21f, a recently developed Atg4B inhibitor. Among these fatty acids, docosahexaenoic acid (DHA), a polyunsaturated fatty acid, exhibited the most potent Atg4B inhibitory activity. DHA inhibited autophagy induced by androgen receptor signaling inhibitors (ARSI) in LNCaP and 22Rv1 prostate cancer cells and significantly increased apoptotic cell death. Furthermore, we investigated the effect of DHA on resistance to ARSI by establishing darolutamide-resistant prostate cancer 22Rv1 (22Rv1/Dar) cells, which had developed resistance to darolutamide, a novel ARSI. At baseline, 22Rv1/Dar cells showed a higher autophagy level than parental 22Rv1 cells. DHA significantly suppressed Dar-induced autophagy and sensitized 22Rv1/Dar cells by inducing apoptotic cell death through mitochondrial dysfunction. These results suggest that DHA supplementation may improve prostate cancer therapy with ARSI.

Maintenance of the mitochondrial thiol redox state is essential for cell survival. However, we lack a comprehensive understanding of the redox response to mitochondrial glutathione depletion. We developed a mitochondria-penetrating peptide, mtCDNB, to specifically deplete mitochondrial glutathione. A genome-wide CRISPR/Cas9 screen in tandem with mtCDNB treatment was employed to uncover regulators of the redox response to mitochondrial glutathione depletion. We identified nucleoside diphosphate kinase 3 (NME3) as a regulator of mitochondrial dynamics. We show that NME3 is recruited to the mitochondrial outer membrane when under redox stress. In the absence of NME3, there is impaired mitophagy, which leads to the accumulation of dysfunctional mitochondria. NME3 knockouts depleted of mitochondrial glutathione have increased mitochondrial ROS production, accumulate mtDNA lesions, and present a senescence-associated secretory phenotype. Our findings suggest a novel role for NME3 in selecting mitochondria for degradation through mitophagy under conditions of mitochondrial redox stress.

Here, we present a protocol to comprehensively quantify autophagy initiation using the readout of the microtubule associated protein 1 light chain 3 beta (LC3B) Förster's resonance energy transfer (FRET) biosensor. We describe steps for cell seeding, transfection, FRET/FLIM (fluorescence lifetime imaging microscopy) imaging, and image analysis. This protocol can be useful in any physiology- or disease-related paradigm where the LC3B biosensor can be expressed to determine whether autophagy has been initiated or is stalled. The analysis pipeline presented here can be applied to any other genetically encoded FRET sensor imaged using FRET/FLIM. For complete details on the use and execution of this protocol, please refer to Gökerküçük et al.1.

The evolutionarily conserved ATG4 cysteine proteases regulate macroautophagy/autophagy through the priming and deconjugation of the Atg8-family proteins. In mammals there are four ATG4 family members (ATG4A, ATG4B, ATG4C, ATG4D) but ATG4D has been relatively understudied. Heightened interest in ATG4D has been stimulated by recent links to human disease. Notably, genetic variations in human ATG4D were implicated in a heritable neurodevelopmental disorder. Genetic analyses in dogs, along with loss-of-function zebrafish and mouse models, further support a neuroprotective role for ATG4D. Here we discuss the evidence connecting ATG4D to neurological diseases and other pathologies and summarize its roles in both autophagy-dependent and autophagy-independent cellular processes.Abbrevation: ATG: autophagy related; BafA1: bafilomycin A1; BCL2: BCL2 apoptosis regulator; BH3: BCL2 homology region 3; CASP3: caspase 3; EV: extracellular vesicle; GABA: gamma aminobutyric acid; GABARAP: GABA type A receptor-associated protein; GABARAPL1: GABA type A receptor associated protein like 1; GABARAPL2: GABA type A receptor associated protein like 2; GFP: green fluorescent protein; LIR: LC3-interacting region; MAP1LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; MYC: MYC proto-oncogene, bHLH transcription factor; PE: phosphatidylethanolamine; PS: phosphatidylserine; QKO: quadruple knockout; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel; SQSTM1: sequestosome 1.

Autophagy, a highly conserved catabolic process that targets various types of cellular cargoes to lysosomal degradation, is one of the most important biological mechanisms critical for cellular homeostasis. Components of these cellular cargoes can range from individual proteins to invading pathogens, and degrading these materials is important for maintaining organismal health and survival. The process of autophagy is carried out by complex molecular mechanisms, and a growing body of evidence indicates that these mechanisms intersect with those involved in the cell death pathways. In this review, we examine several emerging studies elucidating the role of autophagy in RIP1-mediated cell death signaling, with particular emphasis on impaired autophagy caused by ATG16L1 deficiency. We also discuss how autophagy in RIP1-mediated cell death affects intestinal homeostasis in preclinical models, and the implications of the intersection between RIP1 and autophagy for understanding the intestinal pathologies associated with inflammatory bowel disease (IBD). Finally, we highlight the potential benefits of therapeutic targeting of RIP1 and autophagy proteins, while also proposing areas of research that will likely elucidate new links between autophagy and cell death signaling.

Autophagosome formation initiated on the endoplasmic reticulum (ER)-associated omegasome requires LC3. Translational regulation of LC3 biosynthesis is unexplored. Here we demonstrate that LC3 mRNA is recruited to omegasomes by directly binding to the ER transmembrane Sigma-1 receptor (S1R). Cell-based and in vitro reconstitution experiments show that S1R interacts with the 3' UTR of LC3 mRNA and ribosomes to promote LC3 translation. Strikingly, the 3' UTR of LC3 is also required for LC3 protein lipidation, thereby linking the mRNA-3' UTR to LC3 function. An autophagy-defective S1R mutant responsible for amyotrophic lateral sclerosis cannot bind LC3 mRNA or induce LC3 translation. We propose a model wherein S1R de-represses LC3 mRNA via its 3' UTR at the ER, enabling LC3 biosynthesis and lipidation. Because several other LC3-related proteins use the same mechanism, our data reveal a conserved pathway for localized translation essential for autophagosome biogenesis with insights illuminating the molecular basis of a neurodegenerative disease.

ATG8 proteins form a family of small ubiquitin-like modifiers, well-known for their importance in both macroautophagy and autophagy-independent processes. A unique feature of this protein family is their conjugation to membrane lipids through the covalent attachment of a glycine residue at the C-terminus of ATG8 proteins. Notably, most ATG8 proteins are expressed with additional amino acids at their C-terminus, shielding the key glycine residue. Consequently, lipidation requires the activation of the ATG8 precursors through proteolytic cleavage, known as priming. ATG4 proteases catalyze this priming process, and under physiological conditions, unprimed forms of ATG8 are not detected. This raises the question about the purpose of the C-terminal extension of ATG8 proteins. While the roles of lipidated and free, primed ATG8 proteins have been extensively studied, the potential function of their precursor form or the priming process itself remains largely unexplored. Here, we summarize information from existing literature and our own experiments to contribute to the understanding of these neglected amino acids.

Autophagy engulfs cellular components in double-membrane-bound autophagosomes for clearance and recycling after fusion with lysosomes. Thus, autophagy is a key process for maintaining proteostasis and a powerful cell-intrinsic host defense mechanism, protecting cells against pathogens by targeting them through a specific form of selective autophagy known as xenophagy. In this context, ubiquitination acts as a signal of recognition of the cargoes for autophagic receptors, which direct them towards autophagosomes for subsequent breakdown. Nevertheless, autophagy can carry out a dual role since numerous viruses including members of the Orthoherpesviridae family can either inhibit or exploit autophagy for its own benefit and to replicate within host cells. There is growing evidence that Herpes simplex virus type 1 (HSV-1), a highly prevalent human pathogen that infects epidermal keratinocytes and sensitive neurons, is capable of negatively modulating autophagy. Since the effects of HSV-1 infection on autophagic receptors have been poorly explored, this study aims to understand the consequences of HSV-1 productive infection on the levels of the major autophagic receptors involved in xenophagy, key proteins in the recruitment of intracellular pathogens into autophagosomes. We found that productive HSV-1 infection in human neuroglioma cells and keratinocytes causes a reduction in the total levels of Ub conjugates and decreases protein levels of autophagic receptors, including SQSTM1/p62, OPTN1, NBR1, and NDP52, a phenotype that is also accompanied by reduced levels of LC3-I and LC3-II, which interact directly with autophagic receptors. Mechanistically, we show these phenotypes are the result of xenophagy activation in the early stages of productive HSV-1 infection to limit virus replication, thereby reducing progeny HSV-1 yield. Additionally, we found that the removal of the tegument HSV-1 protein US11, a recognized viral factor that counteracts autophagy in host cells, enhances the clearance of autophagic receptors, with a significant reduction in the progeny HSV-1 yield. Moreover, the removal of US11 increases the ubiquitination of SQSTM1/p62, indicating that US11 slows down the autophagy turnover of autophagy receptors. Overall, our findings suggest that xenophagy is a potent host defense against HSV-1 replication and reveals the role of the autophagic receptors in the delivery of HSV-1 to clearance via xenophagy.

Membrane-anchored proteins play critical roles in cell signaling, cellular architecture, and membrane biology. Hydrophilic proteins are post-translationally modified by a diverse range of lipid molecules such as phospholipids, glycosylphosphatidylinositol, and isoprenes, which allows their partition and anchorage to the cell membrane. In this review article, we discuss the biochemical basis of isoprenoid synthesis, the mechanisms of isoprene conjugation to proteins, and the functions of prenylated proteins in the neural retina. Recent discovery of novel prenyltransferases, prenylated protein chaperones, non-canonical prenylation-target motifs, and reversible prenylation is expected to increase the number of inherited systemic and blinding diseases with aberrant protein prenylation. Recent important investigations have also demonstrated the role of several unexpected regulators (such as protein charge, sequence/protein-chaperone interaction, light exposure history) in the photoreceptor trafficking of prenylated proteins. Technical advances in the investigation of the prenylated proteome and its application in vision research are discussed. Clinical updates and technical insights into known and putative prenylation-associated retinopathies are provided herein. Characterization of non-canonical prenylation mechanisms in the retina and retina-specific prenylated proteome is fundamental to the understanding of the pathogenesis of protein prenylation-associated inherited blinding disorders.

Autophagy, an intracellular self-degradation process, is governed by a complex interplay of signaling pathways and interactions between proteins and organelles. Its fundamental purpose is to efficiently clear and recycle cellular components that are damaged or redundant. Central to this process are autophagic vesicles, specialized structures that encapsulate targeted cellular elements, playing a pivotal role in autophagy. Despite growing interest in the molecular components of autophagic machinery and their regulatory mechanisms, capturing the detailed ultrastructural dynamics of autophagosome formation continues to present significant challenges. However, recent advancements in microscopy, particularly in electron microscopy, have begun to illuminate the dynamic regulatory processes underpinning autophagy. This review endeavors to provide an exhaustive overview of contemporary research on the ultrastructure of autophagic processes. By synthesizing observations from diverse technological methodologies, this review seeks to deepen our understanding of the genesis of autophagic vesicles, their membrane origins, and the dynamic alterations that transpire during the autophagy process. The aim is to bridge gaps in current knowledge and foster a more comprehensive comprehension of this crucial cellular mechanism.

Pancreatic ductal adenocarcinoma (PDAC), the most prevalent type of pancreatic cancer, is one of the deadliest forms of cancer with limited therapy options. Overexpression of the heat shock protein 70 (HSP70) is a hallmark of cancer that is strongly associated with aggressive disease and worse clinical outcomes. However, the underlying mechanisms by which HSP70 allows tumor cells to thrive under conditions of continuous stress have not been fully described. Here, we report that PDAC has the highest expression of HSP70 relative to normal tissue across all cancers analyzed. Furthermore, HSP70 expression is associated with tumor grade and is further enhanced in metastatic PDAC. We show that genetic or therapeutic ablation of HSP70 alters mitochondrial subcellular localization, impairs mitochondrial dynamics, and promotes mitochondrial swelling to induce apoptosis. Mechanistically, we find that targeting HSP70 suppresses the PTEN-induced kinase 1 (PINK1) mediated phosphorylation of dynamin-related protein 1 (DRP1). Treatment with the HSP70 inhibitor AP-4-139B was efficacious as a single agent in primary and metastatic mouse models of PDAC. In addition, we demonstrate that HSP70 inhibition promotes the AMP-activated protein kinase (AMPK) mediated phosphorylation of Beclin-1, a key regulator of autophagic flux. Accordingly, we find that the autophagy inhibitor hydroxychloroquine (HCQ) enhances the ability of AP-4-139B to mediate anti-tumor activity in vivo. Collectively, our results suggest that HSP70 is a multi-functional driver of tumorigenesis that orchestrates mitochondrial dynamics and autophagy. Moreover, these findings support the rationale for concurrent inhibition of HSP70 and autophagy as a novel therapeutic approach for HSP70-driven PDAC.

Glioblastoma (GBM), one of the most malignant brain tumors in the world, has limited treatment options and a dismal survival rate. Effective and safe disease-modifying drugs for glioblastoma are urgently needed. Here, we identified a small molecule, Molephantin (EM-5), effectively penetrated the blood-brain barrier (BBB) and demonstrated notable antitumor effects against GBM with good safety profiles both in vitro and in vivo. Mechanistically, EM-5 not only inhibits the proliferation and invasion of GBM cells but also induces cell apoptosis through the reactive oxygen species (ROS)-mediated PI3K/Akt/mTOR pathway. Furthermore, EM-5 causes mitochondrial dysfunction and blocks mitophagy flux by impeding the fusion of mitophagosomes with lysosomes. It is noteworthy that EM-5 does not interfere with the initiation of autophagosome formation or lysosomal function. Additionally, the mitophagy flux blockage caused by EM-5 was driven by the accumulation of intracellular ROS. In vivo, EM-5 exhibited significant efficacy in suppressing tumor growth in a xenograft model. Collectively, our findings not only identified EM-5 as a promising, effective, and safe lead compound for treating GBM but also uncovered its underlying mechanisms from the perspective of apoptosis and mitophagy.