Environmental metal mixtures can cause combined neurotoxicity, but the underlying mechanism remains unclear. Mitochondria are crucial for energy metabolism in the nervous system, and their dysfunction leads to neurodegeneration. Zinc (Zn) is a coenzyme of many mitochondrial enzymes that controls mitochondrial function. This study investigated the role of Zn in the neurotoxicity induced by Mn + Pb and Pb + As mixtures. Zn supplementation improved the survival rate and learning ability of Caenorhabditis elegans following their exposure to mixtures of Mn + Pb and Pb + As by enhancing their mitochondrial morphology, membrane potential, and respiratory chain. Similarly, in HT22 cells, Zn mitigated the decrease in cellular activity and increase in apoptosis induced by the Mn + Pb and Pb + As mixtures by improving mitochondrial morphology and function. Mechanistically, Zn activated the PINK1 and MFN-2/OPA-1 pathways, promoting mitophagy and mitochondrial fusion. However, inhibition of mitophagy reversed the protective effect of Zn, indicating its reliance on mitophagy for neuroprotection. Our study demonstrated that Zn alleviates the combined neurotoxicity of Mn + Pb and Pb + As mixtures by enhancing mitophagy and mitochondrial fusion, suggesting that Zn supplementation is a potential treatment for metal-induced neurotoxicity.

Alzheimer's disease (AD) is currently the seventh leading cause of death worldwide. In this study, we explored the critical role of autophagy in AD pathology using a streptozotocin (STZ)-induced AD model in Wistar rats. The experimental groups included sham, STZ-induced AD, and STZ + MCC950-treated animals. Our findings revealed that administering two doses of STZ (3 mg/kg) intracerebroventricular at the interval of 48 h (on days 0 and 2), triggered autophagy, as evidenced by elevated levels of autophagy markers such as LC3II, ULK1, Beclin1, Ambra1, Cathepsin B, and a reduction in p62 levels. Behavioral assessments, including the water maze and novel object recognition tests, confirmed cognitive deficits and memory impairment, while the open-field test indicated increased anxiety in STZ-induced AD rats. In particular, treating the STZ-induced AD group with MCC950 (50 mg/kg) decreased the overexpression of autophagy-related proteins, which was consistent with better behavioral outcomes and lower anxiety. Overall, this study highlights new insights into AD pathophysiology and suggests potential therapeutic avenues.

Selective autophagy plays a crucial role in innate antiviral immunity by targeting essential viral components and host factors necessary for virus propagation. Among these factors, the nonstructural protein 9 (Nsp9) of Porcine Epidemic Diarrhea Virus (PEDV) is required for viral replication. However, the host factors regulating Nsp9 have remained elusive. In our study, we discovered that Nsp9 undergoes degradation through selective autophagy. Using coimmunoprecipitation combined with mass spectrometry analysis, we identified Toll-interacting protein (TOLLIP) as an autophagy cargo receptor binding to Nsp9 and facilitating its autophagic degradation. Additionally, we found that TOLLIP interacts with LC3A, LC3C, and GABARAPL1. Further investigations revealed that Nsp9 specifically enhances the binding of TOLLIP to LC3A, rather than LC3C or GABARAPL1. Importantly, TOLLIP promotes the engulfment of Nsp9 by LC3A-coated autophagosomes and mediates Nsp9 trafficking to lysosomes, ultimately leading to LC3A-dependent degradation of Nsp9. Consequently, TOLLIP suppresses PEDV replication. Overall, our findings highlight the role of TOLLIP in connecting viral proteins to LC3A-dependent autophagosome, emphasizing its significance in combating viruses through selective autophagy.

Cytoplasmic K63-linked polyubiquitin signals have well-established roles in endocytosis and selective autophagy. However, how these signals help to direct different cargos to different intracellular trafficking routes is unclear. Here we report that, when the K63-polyubiquitin signal is blocked by intracellular expression of a high-affinity sensor (named Vx3), many proteins originating from the plasma membrane are found trapped in clusters of small vesicles that colocalize with ATG9A, a transmembrane protein that plays an essential role in autophagy. Importantly, whereas ATG9A is required for cluster formation, other core autophagy machinery as well as selective autophagy cargo receptors are not required. Although the cargos are sequestered in the vesicular clusters in an ATG9-dependent manner, additional signals are needed to induce LC3 conjugation. Upon removal of the Vx3 block, K63-polyubiquitylated cargos are rapidly delivered to lysosomes. These observations suggest that ATG9A plays an unexpected role in the trafficking of K63-polyubiquitin-modified membrane proteins.

The 5‑year survival rate of patients with esophageal squamous cell carcinoma (ESCC) is <20%, highlighting the need for the development of novel therapeutic targets. Neuregulin‑1 (NRG1), a transmembrane protein involved in cell proliferation and survival signaling, has unclear biological functions and clinical value in ESCC. The present study investigated the association between NRG1 expression and ESCC by analyzing data from both patients with ESCC and The Cancer Genome Atlas database. Reverse transcription‑quantitative PCR and immunohistochemistry staining were used to determine the levels of gene and protein in the tissue. The findings revealed that NRG1 gene and protein levels were significantly higher in tumor tissues compared with the normal tissues. Elevated expression of NRG1 was associated with poor outcomes, particularly in patients with advanced ESCC. Silencing NRG1 decreased both its mRNA and protein levels, disrupting key signaling pathways, such as phosphorylated (p‑)AKT and cellular rapidly accelerated fibrosarcoma (p‑cRAF), which led to decreased cancer cell proliferation, migration and tumor sphere formation, along with increased cell death. High expression levels of NRG1 and cRAF were significantly associated with poor prognosis. Additionally, silencing NRG1 promoted autophagosome and autolysosome formation, decreasing LC3B levels. The use of the autophagy inhibitor chloroquine significantly enhanced cell death induced by NRG1 silencing, suggesting that autophagy functions as a survival mechanism in ESCC cells in which NRG1 is silenced. Furthermore, high co‑expression of NRG1 and LC3B was associated with a worse prognosis. On the whole, the present study demonstrated that targeting NRG1 with autophagy inhibitors may serve as a potential therapeutic strategy for ESCC.

Obesity during pregnancy increases the risk of obesity, insulin resistance, diabetes, and the development and progression of chronic kidney disease (CKD) in later life in offspring. Impaired renal autophagic process is linked to kidney dysfunction in the setting of increased renal lipid accumulation. The aim of this study was to elucidate the effect of maternal obesity on kidney injury related to impaired renal autophagic process in the offspring.

Maternal obesity model was conducted using female C57BL/6 mice fed with high-fat diet (HFD) for 8 weeks before mating. HFD was consecutively maintained throughout gestation and lactation. Male offspring were selected for investigation after weaning. Metabolic parameters and kidney morphology were performed. Renal insulin signaling, lipid metabolism, lipid accumulation, fibrosis and autophagy were determined.

Male offspring of HFD fed mothers developed obesity with insulin resistance, hyperglycemia, hyperlipidemia and consequently promoted kidney injury. Maternal obesity increased CD36, FAS, SREBP1c and Perilipin-2 while suppressed PPARα and CPT1A. The reduction of AMPK, SIRT1, Beclin-1, LC3B, and LAMP2 and the elevation of mTOR and SQSTM1/P62 were observed. These findings indicated the impairment of autophagy and renal lipid metabolism exaggerating renal lipid accumulation in the offspring of maternal obesity.

This study demonstrated that long-term HFD consumption in mothers promoted obesity with insulin resistance related kidney injury through the impairment of autophagic process and renal lipid metabolism in the offspring. These circumstances accelerated kidney injury and contributed to an increased susceptibility to CKD in male offspring of maternal obesity.

Mitochondria, as central regulators of cellular processes such as energy production, apoptosis, and metabolic homeostasis, are essential to cellular function and health. The maintenance of mitochondrial integrity, especially through mitophagy-the selective removal of impaired mitochondria-is crucial for cellular homeostasis. Dysregulation of mitochondrial function, dynamics, and biogenesis is linked to neurodegenerative and metabolic diseases, notably Alzheimer's disease (AD), which is increasingly recognized as a metabolic disorder due to its shared pathophysiologic features: insulin resistance, oxidative stress, and chronic inflammation. In this review, we highlight recent advancements in pharmacological interventions, focusing on agents that modulate mitophagy, mitochondrial uncouplers that reduce oxidative phosphorylation, compounds that directly scavenge reactive oxygen species to alleviate oxidative stress, and molecules that ameliorate amyloid beta plaque accumulation and phosphorylated tau pathology. Additionally, we explore dietary and lifestyle interventions-MIND and ketogenic diets, caloric restriction, physical activity, hormone modulation, and stress management-that complement pharmacological approaches and support mitochondrial health. Our review underscores mitochondria's central role in the pathogenesis and potential treatment of neurodegenerative and metabolic diseases, particularly AD. By advocating for an integrated therapeutic model that combines pharmacological and lifestyle interventions, we propose a comprehensive approach aimed at mitigating mitochondrial dysfunction and improving clinical outcomes in these complex, interrelated diseases.

SQSTM1/p62 serves as an autophagy receptor that binds to ubiquitinated misfolded proteins and delivers them to the phagophores for removal. This function can be augmented by autophagy adaptors, such as UXT. Here, by in silico structural homology modeling, we demonstrated that UXT can potentially form a hexameric structure to bind to misfolded proteins. Importantly, the UXT hexamer can assemble into a high-order oligomer via β hairpins positioned outside of each hexamer, facilitating the formation and efficient removal of protein aggregates. Consistently, the high-order oligomer of UXT was found to be essential for inducing the efficient clearance of SOD1(A4V) aggregates, in both in vitro and in vivo. Collectively, our research emphasizes the crucial importance of UXT oligomerization in its role as an autophagy adaptor and explains why the structurally and functionally similar prefoldin, which lacks such high-order oligomerization capacity, is employed for the refolding of individual misfolded proteins, but not autophagy.

Exosomes are multivesicular body-derived extracellular vesicles that are secreted by metazoan cells. Exosomes have utility as disease biomarkers, and exosome-mediated miRNA secretion has been proposed to facilitate tumor growth and metastasis. Previously, we demonstrated that the Lupus La protein (La) mediates the selective incorporation of miR-122 into metastatic breast cancer-derived exosomes; however, the mechanism by which La itself is sorted into exosomes remains unknown. Using unbiased proximity labeling proteomics, biochemical fractionation, superresolution microscopy and genetic tools, we establish that the selective autophagy receptor p62 sorts La and miR-122 into exosomes. We then performed small RNA sequencing and found that p62 depletion reduces the exosomal secretion of tumor suppressor miRNAs and results in their accumulation within cells. Our data indicate that p62 is a quality control factor that modulates the miRNA composition of exosomes. Cancer cells may exploit p62-dependent exosome cargo sorting to eliminate tumor suppressor miRNAs and thus to promote cell proliferation.

Sorafenib has demonstrated great efficacy in liver cancer, however, its application as first-line treatment has been hampered due to the emerging drug resistance. This study is aimed to investigate the mechanism underlying acquired sorafenib resistance in liver cancer. Based on GSE109211 and TCGA datasets, bioinformatics analysis was conducted to find the potential genes implicated in the sorafenib resistance in liver cancer. mCherry-/eGFP-LC3B dual-fluorescent system was used to assess autophagic state. Wild and mutant types of HA-labeled ubiquitin (K27, K29, K33, K48, K63, K29R and K48R) were used to identify the type of polyubiquitin chains added to p27 by CUL1. Herein, we identified that F-box protein (SCF) ubiquitin ligase complexes (CUL1 and SKP2) and NEDD8 were highly expressed in sorafenib-resistant tissues using both the public data and clinical samples. NEDD8-mediated CUL1 neddylation enhanced SCF ubiquitin ligase complex to target p27 and subsequently linked K29-linked polyubiquitin chains to p27. Furthermore, NBR1 facilitated the degradation of ubiquitinated p27 protein by enhancing autophagy flux. Knocking down of CUL1 could prevent ubiquitination- and autophagy-mediated p27 protein degradation. The resistance to sorafenib was suppressed with CUL1 knockdown both in vitro and in vivo. In conclusion, our findings indicated that blocking neddylation or autophagy can restore drug sensitivity, thus providing a potential strategy for overcoming sorafenib resistance in the future.

The mechanisms of adipose tissue activation and inactivation have been a hot topic of research in the last decade, from which countermeasures have been attempted to be found against obesity as well as other lipid metabolism-related diseases, such as type 2 diabetes mellitus and non-alcoholic fatty liver disease. Autophagy has been shown to be closely related to the regulation of adipocyte activity, which is involved in the whole process including white adipocyte differentiation/maturation and brown or beige adipocyte generation/activation. Dysregulation of autophagy in adipose tissue has been demonstrated to be associated with obesity. On this basis, we summarize the pathways and mechanisms of autophagy involved in the regulation of lipid metabolism and present a review of its pathophysiological roles in lipid metabolism-related diseases, in the hope of providing ideas for the treatment of these diseases.

Therapeutic administration of mitochondria has been increasingly explored. However, how these administered mitochondria impact immune response remains to be fully addressed. In this proof-of-concept study, we show that extracellularly added mitochondria to cultured peritoneal macrophages increase phagocytosis and recycling exocytosis that amplifies neuroplasticity mediated by recycled mitochondria transfer. Macrophage activation markers such as Nos2, Arg1, and Cd163 were unchanged at 3 h post-treatment with mitochondria, but whole mitochondria or delivery of mRNAs extracted from whole mitochondria appeared to increase SQSTM1 protein and activate Nrf2-mediated phagocytosis in macrophages, whereas mitochondria treatment did not change the ability of phagocytosis in cultured microglia or astrocytes. Notably, the once engulfed mitochondria in macrophages appear to be released via Rab27a-mediated recycling pathway that were favorably incorporated in mechanically damaged neurons compared with healthy neurons, resulting in accelerating neurite extension in damaged neurons in a direct co-culture model. Altogether, these findings uncover unappreciated mechanisms that mitochondria-treated macrophages upregulate phagocytosis and recycling exocytosis, implicating that engineering mitochondria delivery to macrophages is a new therapeutic intervention to promote neurorecovery in CNS disorders.

Liquid storage of semen is a widely used technology for promoting genetic improvement in goat breeding. The short shelf life of spermatozoa greatly limits the application of liquid storage, which urgently needs to explore the underlying regulatory factors. Autophagy as a cellular catabolic process plays critical roles in eliminating damaged material, that thus protects the function and fertilizing ability of spermatozoa. Nevertheless, the regulatory mechanisms of autophagy in goat spermatozoa under liquid storage remain unclear. In this study, the typical morphologic abnormalities and ultrastructural changes in goat spermatozoa, such as plasma membrane swollen and shrunken, acrosome exfoliation, and axoneme exposure, were observed after liquid storage at 4°C. Moreover, assessment of the formation of autophagy in liquid-stored goat spermatozoa was performed by a morphological "gold standard" of electron microscopy. Notably, a large number of vesicles with double-membrane structure indicating autophagosome were found to surround the aberrant spermatozoa, suggesting the activation of autophagy. Several proteins, such as LC3, ATG5, and p62, exhibited differential expression after liquid storage, which further validated the occurrence of autophagy in liquid-stored goat spermatozoa. Furthermore, chloroquine treatment was used to inhibit the autophagy of spermatozoa, which caused a significantly decrease in the quality of liquid-stored spermatozoa, including motility, viability, plasma membrane integrity, and acrosome integrity. Significant increase in ROS and MDA levels of spermatozoa and significant decrease in Ca2+ influx and protein tyrosine phosphorylation of spermatozoa were also detected after chloroquine-induced autophagy inhibition. The ultrastructural observation of double-membrane autophagosome provides strong evidences for the activation of autophagy in goat spermatozoa under liquid storage. The inhibition of autophagy mediated by chloroquine indicated that autophagy plays vital roles in the survival of spermatozoa. These results facilitate understanding the activation of autophagy in spermatozoa and provide valuable references for uncovering the underlying regulatory mechanisms of liquid storage of goat spermatozoa.

F-prostanoid receptor (FP receptor), a receptor for Prostaglandin F2α(PGF2α), is involved in the process of tissue fibrosis, but its exact role in the aging heart remains unclear.

We investigated cardiac function, myocardial fibrosis levels, autophagy levels and related mechanistic pathways in different groups of mice using gene silencing. At the cellular level, we simulated the senescence process of cardiac fibroblasts and investigated the related mechanisms using relevant inhibitors.

In aging mice, FP receptor and PI3K/AKT/mTOR pathways are increased and autophagy levels are decreased, ultimately leading to cardiac fibrosis. FP receptor gene silencing slows down the above process. We found similar changes at the cellular level.

FP receptor could activate PI3K/AKT/mTOR pathway and inhibit cardiac autophagy, which resulted in aging-related cardiac fibrosis. Thus, the inhibition of FP receptor could improve aging-related cardiac remodeling, implicating its potential therapeutic application to treat cardiovascular diseases associated with aging.

p62/SQSTM1 (p62) and neighbor of BRCA1 gene 1 (NBR1) are two important cargo receptors involved in selective autophagy. While p62 is known to safeguard cells against the toxic effects of the environmental toxicant methylmercury (MeHg), the specific functions of p62 and NBR1 in MeHg-exposed cells remain unclear. In this study, we aimed to elucidate the distinct roles of p62 and NBR1 in conferring protection against cytotoxicity induced by MeHg. We found that MeHg increased both the mRNA and protein levels of p62 while decreasing those of NBR1. Upon exposure to MeHg, p62-knockout (KO) cells exhibited an approximately 30 % reduction in cell viability compared to wild-type (WT) cells; however, no such reduction was observed in NBR1KO cells. Additionally, p62KO cells exhibited a 1.5-fold increase in intracellular mercury (Hg) concentration compared to the WT following MeHg exposure, whereas NBR1KO cells had Hg levels comparable to those of WT cells. Upon exposure to MeHg, Nrf2 signaling activation was significantly reduced in p62KO cells compared to that in WT cells, whereas NBR1KO cells displayed Nrf2 activation levels similar to those of WT cells. Overall, these results suggest that p62, rather than NBR1, plays a crucial role in mitigating MeHg-induced cytotoxicity by reducing intracellular Hg levels through the activation of the Nrf2 signaling pathway.

Parkinson's disease (PD) is characterized by the loss of neuromelanin (NM)-containing dopaminergic (DA) neurons in the substantia nigra (SN) pars compacta (SNpc) and the buildup of α-synuclein (α-syn) inclusions, called Lewy bodies. To investigate the roles of NM and α-syn in DA neuron degeneration, we modeled PD by inducing NM accumulation in a humanized α-syn mouse model (Snca-; PAC-Tg(SNCAWT)) via the expression of human tyrosinase in the SN. We found that this mouse strain develops naturally progressive motor dysfunction and dopaminergic neuronal loss in the SN with aging. Upon tyrosinase injection, NM-containing neurons developed p62 and ubiquitin inclusions. Furthermore, the upregulation of genes associated with microglial activation in the midbrain indicated a role of pro-inflammatory factors in neurodegeneration. Midbrain RNA sequencing confirmed the microglial response and identified Fkbp5 as one of the more dysregulated genes. Next, we showed that FKBP51(51 kDa) was significantly upregulated with aging and in PD human brains. Pharmacological treatment with SAFit2, a potent FKBP51 inhibitor, led to a reduction in ubiquitin-positive inclusions, prevention of neurodegeneration in the SNpc, and improved motor function in NM-SNCAWT mice. These results highlight the critical role of FKBP51 in PD and propose SAFit2 as a promising therapeutic candidate for reducing neurodegeneration in PD.

Pulmonary hypertension (PH) is heterogeneous diseases that can lead to death due to progressive right heart failure. Emerging evidence suggests that, in addition to its role in ATP production, changes in mitochondrial play a central role in their pathogenesis, regulating integrated metabolic and signal transduction pathways. This review focuses on the basic principles of mitochondrial redox status in pulmonary vascular and right ventricular disorders, a series of dysfunctional processes including mitochondrial quality control (mitochondrial biogenesis, mitophagy, mitochondrial dynamics, mitochondrial unfolded protein response) and mitochondrial redox homeostasis. In addition, we will summarize how mitochondrial renewal and dynamic changes provide innovative insights for studying and evaluating PH. This will provide us with a clearer understanding of the initial signal transmission of mitochondria in PH, which would further improve our understanding of the pathogenesis of PH.

Paclitaxel (PTX) binds to spindle microtubules and inhibits mitotic division leading to cell death. However, its wide distribution, high absorption, and less selectively, minimize its application in cancer clinics. In this study, isolated arabinoxylans were used to encapsulate PTX, and then both were covered by polyethylene glycol conjugated to folic acid (FA), to strengthen its specificity to cancerous cells. Beclin-1 and P21 were significantly overexpressed by (79.6 ± 0.97 %, p ≤ 0.00001, &62.2 ± 1.15 % p ≤ 0.0001 in MCF-7 cells) and (74.8 ± 8.04 %, p ≤ 0.0001 &75.3 ± 2.3 %, p ≤ 0.0001, in NSCLCs) respectively after their incubation for 48 h with nano-targeted PTX NPs. Similarly, P53 and GSK3 beta-pSer9 were significantly increased by (63.5 ± 1 % p ≤ 0.0001, & 87 ± 1 % p ≤ 0.0001, in MCF) respectively and (81.5 ± 6 % p ≤ 0.01, & 84.8 ± 3.8 % p ≤ 0.001, in A549) respectively. The findings confirmed the activation of acidic/neutral autophagosomes in acridine orange/ethidium bromide (AO/EB) and nuclear fragmentation was clearly shown by 4', 6-diamidino-2 phenylindole (DAPI) nuclear stains. The findings provide the basis for the potential application of arabinoxylans as a great vehicle for the encapsulation of chemotherapeutic agents such as PTX for target delivery, and also as an immune mediator.

Mitochondria play a key role in the regulation of energy homeostasis and ATP production in cardiac cells. Mitochondrial dysfunction can trigger several pathological events that contribute to the development and progression of cardiovascular diseases. These mechanisms include the induction of oxidative stress, dysregulation of intracellular calcium cycling, activation of the apoptotic pathway, and alteration of lipid metabolism. This review focuses on the role of mitochondria in intracellular signaling associated with cardiovascular diseases, emphasizing the contributions of reactive oxygen species production and mitochondrial dynamics. Indeed, mitochondrial dysfunction has been implicated in every aspect of cardiovascular disease and is currently being evaluated as a potential target for therapeutic interventions. To treat cardiovascular diseases and improve overall heart health, it is important to better understand these biochemical systems. These findings allow the achievement of targeted therapies and preventive measures. Therefore, this review investigates different studies that demonstrate how changes in mitochondrial dynamics like fusion, fission, and mitophagy contribute to the development or worsening of disorders related to heart diseases by summarizing current research on their role.

Cells undergo metabolic reprogramming to adapt to changes in nutrient availability, cellular activity, and transitions in cell states. The balance between glycolysis and mitochondrial respiration is crucial for energy production, and metabolic reprogramming stipulates a shift in such balance to optimize both bioenergetic efficiency and anabolic requirements. Failure in switching bioenergetic dependence can lead to maladaptation and pathogenesis. While cellular degradation is known to recycle precursor molecules for anabolism, its potential role in regulating energy production remains less explored. The bioenergetic switch between glycolysis and mitochondrial respiration involves transcription factors and organelle homeostasis, which are both regulated by the cellular degradation pathways. A growing body of studies has demonstrated that both stem cells and differentiated cells exhibit bioenergetic switch upon perturbations of autophagic activity or endolysosomal processes. Here, we highlighted the current understanding of the interplay between degradation processes, specifically autophagy and endolysosomes, transcription factors, endolysosomal signaling, and mitochondrial homeostasis in shaping cellular bioenergetics. This review aims to summarize the relationship between degradation processes and bioenergetics, providing a foundation for future research to unveil deeper mechanistic insights into bioenergetic regulation.

Trametinib, an inhibitor of mitogen-activated extracellular signal-regulated kinases 1/2 (MEK1/2), is used to treat BRAFV600E/K melanoma and non-small-cell lung cancer (NSCLC). Mutant Kirsten rat sarcoma viral oncogene homolog (KRAS) promotes glutamine utilization, therefore, in the present study we investigated the anti-cancer effects of trametinib in combination with V-9302, a glutamine transporter inhibitor, in NSCLC with KRAS mutations. Trametinib in combination with V-9302 exhibited a potent synergistic antitumor effect, inducing cell cycle arrest and pyroptosis. Mechanistically, combination treatment triggered caspase-3 activation and gasdermin E (GSDME) cleavage, as well as elevated lactate dehydrogenase (LDH) and IL-1β levels. Meanwhile, combination treatment reduced cyclin D1 and p-Rb levels and increased p27 expression. Moreover, this combination increased forkhead box class O3a (FOXO3a) levels and decreased forkhead box M1 (FOXM1) expression by regulating the phosphorylation of ERK, Akt, AMPK, and c-Jun N-terminal kinase (JNK). Trametinib in combination with V-9302 increased reactive oxygen species (ROS) generation and reduced glutathione (GSH) synthesis and ATP levels. Furthermore, V-9302 in combination with trametinib inhibited the trametinib-induced autophagy, thereby enhancing pyroptosis in cancer cells. In vivo, the co-administration of trametinib and V-9302 remarkably inhibited tumor growth in a xenograft mouse model compared to each drug alone. Taken together, the combination of trametinib and V-9302 resulted in increased pyroptosis and cell cycle arrest compared to each single agent through regulation of the FOXO3a/FOXM1 axis and autophagy and significantly enhanced antitumor efficacy in vivo. Our results suggest a potential new therapeutic strategy for KRAS-mutant NSCLC using trametinib in combination with glutamine restriction.

Macroautophagy is a catabolic process that maintains cellular homeostasis by recycling intracellular material through the use of double-membrane vesicles called autophagosomes. In turn, autophagosomes fuse with vacuoles (in yeast and plants) or lysosomes (in metazoans), where resident hydrolases degrade the cargo. Given the conservation of autophagy, Saccharomyces cerevisiae is a valuable model organism for deciphering molecular details that define macroautophagy pathways. In yeast, macroautophagic pathways fall into two subclasses: selective and nonselective (bulk) autophagy. Bulk autophagy is predominantly upregulated following TORC1 inhibition, triggered by nutrient stress, and degrades superfluous random cytosolic proteins and organelles. In contrast, selective autophagy pathways maintain cellular homeostasis when TORC1 is active by degrading damaged organelles and dysfunctional proteins. Here, selective autophagy receptors mediate cargo delivery to the vacuole. Now, two groups have discovered a new hybrid autophagy mechanism, coined cargo hitchhiking autophagy (CHA), that uses autophagic receptor proteins to deliver selected cargo to phagophores built in response to nutrient stress for the random destruction of cytosolic contents. In CHA, various autophagic receptors link their cargos to lipidated Atg8, located on growing phagophores. In addition, the sorting nexin heterodimer Snx4-Atg20 assists in the degradation of cargo during CHA, possibly by aiding the delivery of cytoplasmic cargos to phagophores and/or by delaying the closure of expanding phagophores. This review will outline this new mechanism, also known as Snx4-assisted autophagy, that degrades an assortment of cargos in yeast, including transcription factors, glycogen, and a subset of ribosomal proteins.

Macroautophagy/autophagyis a lysosomal-regulated degradation process that participates incellular stress and then promotes cell survival or triggers celldeath. Ferroptosis was initially described as anautophagy-independent, iron-regulated, nonapoptotic cell death.However, recent studies have revealed that autophagy is positivelyassociated with sensitivity to ferroptosis. Nonetheless, themolecular mechanisms by which these two types of regulated cell death(RCD) modulate each other remain largely unclear. Here, we screened85 deubiquitinating enzymes (DUBs) and found that overexpression ofUSP13 (ubiquitin specific peptidase 13) could significantlyupregulate NFE2L2/NRF2 (NFE2 like bZIP transcription factor 2)protein levels. In addition, in 39 cases of KRAS-mutated lungadenocarcinoma (LUAD), we found that approximately 76% of USP13overexpression is positively correlated with NFE2L2 overexpression.USP13 interacts with and catalyzes the deubiquitination of thetranscription factor NFE2L2. Additionally, USP13 depletion promotesan autophagy-to-ferroptosis switch invitro andin xenograft tumor mouse models, through the activation of theNFE2L2-SQSTM1/p62 (sequestosome 1)-KEAP1 axis in KRAS mutant cellsand tumor tissues. Hence, targeting USP13 effectively switchedautophagy-to-ferroptosis, thereby inhibiting KRAS (KRASproto-oncogene, GTPase) mutant LUAD, suggesting the therapeuticpromise of combining autophagy and ferroptosis in the KRAS-mutantLUAD.Abbreviation: ACSL4: acyl-CoA synthetase long-chain family member 4; ACTB: actin beta; AL: autolysosomes; AP: autophagosomes; BCL2L1/BCL-xL: BCL2 like 1; CCK8: Cell Counting Kit-8; CQ: chloroquine; CUL3: cullin 3; DMSO: dimethyl sulfoxide; DOX: doxorubicin; DUB: deubiquitinating enzyme; Ferr-1: ferrostatin-1; GPX4: glutathione peroxidase 4; GSEA: gene set enrichment analysis; 4HNE: 4-hydroxynonenal; IKE: imidazole ketone erastin; KEAP1: kelch like ECH associated protein 1; KRAS: KRAS proto-oncogene, GTPase; LCSC: lung squamous cell carcinoma; IF: immunofluorescence; LUAD: lung adenocarcinoma; Lys05: Lys01 trihydrochloride; MAPK1/ERK2/p42: mitogen-activated protein kinase 1; MAPK3/ERK1/p44; MTOR: mechanistic target of rapamycin kinase; NFE2L2/NRF2: NFE2 like bZIP transcription factor, 2; NQO1: NAD(P)H quinone dehydrogenase 1; PG: phagophore; RCD: regulated cell death; RAPA: rapamycin; ROS: reactive oxygen species; SLC7A11/xCT: solute carrier family 7 member 11; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TUBB/beta-tubulin: tubulin, beta; UPS: ubiquitin-proteasome system; USP13: ubiquitin specific peptidase 13.

Mitophagy is a process by which cells selectively eliminate damaged or dysfunctional mitochondria through the autophagy-lysosome pathway, thereby maintaining mitochondrial quality and cellular homeostasis. This process is closely linked to the onset and progression of various heart diseases. Modern pharmacological research has demonstrated that phytochemicals can regulate mitochondrial homeostasis in cardiomyocytes through multiple mechanisms, influencing mitophagy and protecting cardiomyocytes, which in turn exerts anti-cardiovascular effects. However, the underlying mechanisms of these effects are not yet fully understood. This study summarizes the pharmacological effects and molecular mechanisms of mitophagy in heart diseases, aiming to provide reference for the research and treatment of phytochemicals targeting mitophagy against heart diseases. The results indicated that phytochemicals (such as Berberine, Ginsenoside Rg1, Quercetin, Resveratrol, Baicalein, and so on) can exert preventive and therapeutic effects on heart diseases (such as cardiac toxicity or damage, myocardial ischemia/reperfusion injury, heart failure, heart aging, cardiac hypertrophy, cardiomyopathy, and so on.) via regulating the PINK1/Parkin and FUNDC1-dependent mitophagy pathway. These compounds mainly exert their effects by regulating mitochondrial homeostasis, mitochondrial dynamics, mitochondrial oxidative stress, mitochondrial apoptosis, and mitochondrial energy metabolism. This study provides a reference that phytochemicals have effect on anti-cardiovascular effects by regulating mitophagy. However, further in-depth mechanistic and clinical research are needed in the future.

Monosodium glutamate (MSG) is one of the most commonly used food additives, known for its adverse health effects. Alogliptin (ALO) is a highly selective dipeptidyl peptidase-4 inhibitor, but its role in male reproductive function remains debated. The study was designed to evaluate and compare the potential of ALO in mitigating MSG-induced testicular toxicity in juvenile and adult male rats. Juvenile and adult male rats were treated with either MSG or pretreated with ALO before MSG administration. The rats then received ALO and MSG concurrently for 28 days. Testicular tissues were isolated and subjected to histo-biochemical and molecular assessments. Our results demonstrated that ALO reversed MSG-induced testicular injury, as evidenced by the restoration of reproductive hormone balance (increased serum luteinizing hormone and testosterone concentrations), suppression of oxidative stress injury (decreased testicular malondialdehyde, increased superoxide dismutase activity, and minimal 8-hydroxy-2'-deoxyguanosine immunoreactivity), inflammation (reduced testicular tumor necrosis factor-alpha levels), and fibrosis (decreased testicular collagen fiber deposition). Additionally, ALO impeded apoptosis and activated autophagy by decreasing caspase-3 activity, stimulating the AMPK/mTOR pathway, downregulating Bax and SQSTM-1/p62 expression, upregulating Bcl2 and Beclin 1, promoting testicular proliferation (increased number of proliferating cell nuclear antigen-positive cells in the testis), restoring glycogen content in the testis (mild to moderate periodic acid-Schiff reaction), and preserving testicular architecture. MSG induced more severe adverse testicular effects in juvenile rats, while ALO pretreatment was more protective in adult rats. ALO's anti-inflammatory, antioxidant, antiapoptotic, pro-autophagic, antifibrotic, and proliferative actions in the testis suggest its promising potential for combating male reproductive dysfunction.

Ultraviolet B (UVB) radiation can distort cellular homeostasis and predispose the skin to carcinogenesis. Amongst the deteriorating effects of the sun's UVB radiation on cellular homeostasis is the formation of DNA photoproducts. These photoproducts can cause significant changes in the structure and conformation of DNA, inducing gene mutations which may accumulate to trigger the formation of skin cancer. Photoproducts are typically repaired by nucleotide excision repair. Notwithstanding, when the repair mechanism fails, apoptosis ensues to prevent the accumulation of mutations and to restore cellular homeostasis. This present study reports that T-cell protein tyrosine phosphatase (TC-PTP) can increase UVB-induced apoptosis by inhibiting autophagy-mediated cell survival of damaged keratinocytes. TC-PTP deficiency in 3PC mouse keratinocytes led to the formation of autophagic vacuoles and increased expression of LC3-II. We established human TC-PTP-deficient (TC-PTP/KO) HaCaT cells using the CRISPR/Cas9 system. TC-PTP/KO HaCaT cells exhibited increased cell survival upon UVB exposure, which was accompanied by increased expression of LC3-II and decreased expression of p62 compared to control cells. Pretreatment of TC-PTP/KO HaCaT cells with early-phase autophagy inhibitor, 3-methyladenine significantly decreased the expression of LC3-II and reduced cell survival in response to UVB irradiation in comparison with untreated TC-PTP/KO cells. Pretreatment of TC-PTP/KO HaCaT cells with late-phase inhibitor, chloroquine also significantly reduced cell viability with increased accumulation of LC3-II after UVB irradiation compared to untreated counterpart cells. While UVB significantly increased apoptosis in the engineered (Mock) cells, this was not observed in similarly treated TC-PTP/KO HaCaT cells. However, chloroquine treatment increased apoptosis in TC-PTP/KO HaCaT cells. Examination of human squamous cell carcinomas (SCCs) revealed that TC-PTP expression was inversely correlated with LC3 expression. Our findings suggest that TC-PTP negatively regulates autophagy-mediated survival of damaged cells following UVB exposure, which can contribute to remove damaged keratinocytes via apoptosis.

Mitochondrial homeostasis, closely associated with mitophagy and antioxidant mechanisms, is essential for proper meiotic spindle assembly and chromosome segregation during oocyte maturation. SIRT5, known to modulate mitochondrial function under various conditions, has been shown to impact oocyte quality when inhibited, however, the precise mechanisms linking SIRT5 to mitochondrial homeostasis during meiotic progression remain unclear. In this study, we demonstrate that SIRT5 localizes predominantly at the periphery of the meiotic spindle and is enriched on chromosomes during oocyte maturation. Inhibition of SIRT5 led to significant meiotic defects, including disrupted spindle organization and chromosome misalignment. These defects were associated with increased histone acetylation, which impaired kinetochore-microtubule attachments. Moreover, SIRT5 inhibition resulted in mitochondrial dysfunction, subsequently elevating ROS levels and triggering oxidative stress, which further exacerbated meiotic abnormalities. Mechanistically, SIRT5 inhibition disrupted the balance of Parkin-dependent mitophagy by inducing ULK phosphorylation. Additionally, it activated the PI3K/Akt signaling pathway, which increased NADPH consumption and reduced GSH levels. Collectively, these findings reveal that SIRT5 plays dual roles in maintaining mitochondrial homeostasis during oocyte maturation: (1) by regulating Parkin-dependent mitophagy to prevent excessive mitochondrial clearance, and (2) by preserving the NADPH/GSH antioxidant system to ensure redox balance. These insights provide potential targets for improving oocyte quality and addressing mitochondrial dysfunction-related reproductive disorders in females.

The phase separation of the cargo receptor sequestome-1/p62 (SQSTM1/p62) is a critical mechanism for assembling signaling complexes in autophagy. During this process, p62 undergoes phase separation upon binding to polyubiquitin chains, concentrating ubiquitinated substrates within p62 droplets. These droplets further gather membrane sources and core autophagy machineries to facilitate autophagosome formation. The dynamics of p62 droplets are finely tuned in response to autophagy signals triggered by cellular stresses. Recent studies have revealed new regulatory mechanisms that highlight the significance of p62 phase separation in regulating autophagy. This review summarizes and discusses the molecular mechanisms of p62 phase separation and its roles in autophagy, with particular emphasis on the regulation of p62 droplets and their interaction modes with autophagic membranes.

The CGG repeat expansions in the 5'-UTR regions of certain genes have been implicated in various neurodegenerative and muscular disorders. However, the underlying pathogenic mechanisms are not well understood. In this study, we explore the role of the small molecular chaperone HSPB1 in counteracting neurodegeneration induced by poly-glycine (poly-G) aggregates. Employing a reporter system, we demonstrate that CGG repeat expansions within the 5'-UTR of the GIPC1 gene produce poly-G proteins, by repeat-associated non-AUG (RAN) translation. Through proximity labeling and subsequent mass spectrometry analysis, we characterize the composition of poly-G insoluble aggregates and reveal that these aggregates sequester key macroautophagy/autophagy receptors, SQSTM1/p62 and TOLLIP. This sequestration disrupts MAP1LC3/LC3 recruitment and impairs autophagosome formation, thereby compromising the autophagic pathway. Importantly, we show that HSPB1 facilitates the dissociation of these receptors from poly-G aggregates and consequently restores autophagic function. Overexpressing HSPB1 alleviates poly-G-induced neurodegeneration in mouse models. Taken together, these findings highlight a mechanistic basis for the neuroprotective effects of HSPB1 and suggest its potential as a therapeutic target in treating poly-G-associated neurodegenerative diseases.Abbreviations: AD: Alzheimer disease; AIF1/Iba1: allograft inflammatory factor 1; Baf A1: bafilomycin A1; BFP: blue fluorescent protein; CQ: chloroquine; EIF2A/eIF-2α: eukaryotic translation initiation factor 2A; FRAP: fluorescence recovery after photobleaching; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFAP: glial fibrillary acidic protein; GFP: green fluorescent protein; HSPB1: heat shock protein family B (small) member 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; NOTCH2NLC: notch 2 N-terminal like C; PD: Parkinson disease; PFA: paraformaldehyde; poly-A: poly-alanine; poly-G: poly-glycine; poly-R: poly-arginine; RAN translation: repeat-associated non-AUG translation; RBFOX3/NeuN: RNA binding fox-1 homolog 3; STED: stimulated emission depletion; TARDBP/TDP-43: TAR DNA binding protein; TG: thapsigargin; TOLLIP: toll interacting protein.

Brain-machine interface performance can be affected by neuroinflammatory responses due to blood-brain barrier (BBB) damage following intracortical microelectrode implantation. Recent findings suggest that certain gut bacterial constituents might enter the brain through damaged BBB. Therefore, we hypothesized that damage to the BBB caused by microelectrode implantation could facilitate microbiome entry into the brain. In our study, we found bacterial sequences, including gut-related ones, in the brains of mice with implanted microelectrodes. These sequences changed over time. Mice treated with antibiotics showed a reduced presence of these bacteria and had a different inflammatory response, which temporarily improved microelectrode recording performance. However, long-term antibiotic use worsened performance and disrupted neurodegenerative pathways. Many bacterial sequences found were not present in the gut or in unimplanted brains. Together, the current study established a paradigm-shifting mechanism that may contribute to chronic intracortical microelectrode recording performance and affect overall brain health following intracortical microelectrode implantation.

Autophagy activation contributes to chemotherapy resistance in several cancers, including ovarian cancer. Hydroxychloroquine (HCQ) is an autophagy inhibitor inhibiting the fusion of the autophagosome with the lysosome and has been repurposed as an anti-cancer agent. In this randomized phase II study, we used HCQ in combination with standard chemotherapy in platinum sensitive relapsed ovarian cancer (PSROC) patients.

Patients were randomized in a 1:1 ratio to receive standard chemotherapy (carboplatin with paclitaxel/gemcitabine) with or without HCQ. Those randomized to receive HCQ received additional HCQ 200mg orally twice daily. The primary endpoint was the overall response rate (ORR). Other endpoints included survival outcomes, changes in autophagy biomarkers, toxicity, and quality of life.

A total of 59 patients were enrolled- chemotherapy + HCQ (N = 28), chemotherapy alone (N = 31), and 56 were evaluable ( received ≥ 3 cycles treatment). The ORR was not superior with the addition of HCQ [85% (22/26) in the experimental arm as compared to 80% (24/30)  in the control arm, chi-square test, P = 0.65]. The median progression-free survival was 12 (95% CI, 9.75-14.24) months for the experimental arm and 11 (95% CI, 5.25-16.74) months for the control arm (P = 0.56) , and the median overall survival was 16 (95% CI, 8.54-23.45) months vs. 21 (95% CI, 11.70-30.59 ) months (P = 0.49) respectively. HCQ was well tolerated, with no excess adverse events [21 (75%) in the experimental arm vs. 22 (71%) in the control arm]. There were no substantial differences in the reduction of autophagy biomarker levels and QOL between the control and experimental arms.

Adding HCQ to chemotherapy failed to improve response rates or survival in patients with PSROC. Conducting biomarker-stratified clinical trials might show the potential benefit of HCQ. Trial registration number (TRN): The trial was registered in the Clinical Trial Registry of India ( www.ctri.nic.in ; CTRI/2020/06/025790) on 17th June 2020.

The multidomain protein cargo adaptor p62, also known as sequestosome 1, serves as a shuttling factor and adaptor for the degradation of substrates via the proteasome and autophagy pathways. Regarding its structure, p62 is composed of several functional domains, including the N-terminal Phox1 and Bem1p domains, a ZZ-type zinc finger domain, a LIM protein-binding domain that contains the tumor necrosis factor receptor-associated factor 6 (TRAF6) binding region, two nuclear localization signals (NLS 1/2), a nuclear export signal (NES), the LC3-interacting region (LIR), a Kelch-like ECH-associated protein 1 (KEAP1)-interacting region, and a ubiquitin-associated (UBA) domain. Recent studies have highlighted the critical role of p62 in the development and progression of various malignancies. Overexpression and/or impaired degradation of p62 are linked to the initiation and progression of numerous cancers. While p62 is primarily localized in the cytosol and often considered a cytoplasmic protein, most of the existing literature focuses on its cytoplasmic functions, leaving its nuclear roles less explored. However, an increasing body of research has uncovered p62's involvement in the cellular response to DNA damage. In this review, we summarize the current understanding of p62's molecular functions in malignancies, with particular emphasis on its role in DNA damage repair, highlighting the latest advances in this field.

Macroautophagy/autophagy plays a crucial role in maintaining cellular and organismal health, making the measurement of autophagy flux in vivo essential for its study. Current tools often depend on the overexpression of autophagy probes. In this study, we developed a knock-in mouse model, termed tfLC3-KI, by inserting a tandem fluorescent tag coding sequence into the native Map1lc3b gene locus. We found that tfLC3-KI mice exhibit optimal expression of mRFP-eGFP-LC3B, allowing for convenient measurement of autophagic structures and flux at single-cell resolution, both in vivo and in primary cell cultures. Additionally, we compared autophagy in neurons and glial cells across various brain regions between tfLC3-KI mice and CAG-tfLC3 mice, the latter overexpressing the probe under the strong CMV promoter. Finally, we used tfLC3-KI mice to map the spatial and temporal dynamics of basal autophagy activity in the reproductive system. Our findings highlight the value of the tfLC3-KI mouse model for investigating autophagy flux in vivo and demonstrate the feasibility of tagging endogenous proteins to visualize autophagic structures and flux in both bulk and selective autophagy research in vivo.Abbreviation: BafA1: bafilomycin A1; CQ: chloroquine; EBSS: Earle's balanced salt solution; Es: elongating spermatids; HPF: hippocampalformation; HY: hypothalamus; LCs: leydig cells; OLF: olfactory areas; PepA: pepstatin A; Rs: round spermatids; SCs: sertoli cells; Spc: spermatocytes; Spg: spermatogonia; tfLC3: tandem fluorescently tagged mRFP-eGFP-LC3; TH: thalamus.

Induction of autophagy is an ancient function of the cyclic GMP-AMP (cGAMP) synthase (cGAS)-stimulator of interferon genes (STING) pathway through which autophagic cargoes are delivered to lysosomes for degradation. However, whether lysosome function is also modulated by the cGAS-STING pathway remains unknown. Here, we discovered that the cGAS-STING pathway upregulated lysosomal activity by stimulating lysosome biogenesis independently of the downstream protein kinase TANK-binding kinase 1 (TBK1). STING activation enhanced lysosome biogenesis through inducing the nuclear translocation of transcription factor EB (TFEB) as well as its paralogs transcription factor E3 (TFE3) and microphthalmia-associated transcription factor (MITF). STING-induced lipidation of GABA type A receptor-associated protein (GABARAP), an autophagy-related protein, on STING vesicles was responsible for TFEB activation. Membrane-bound GABARAP sequestered the GTPase-activating protein folliculin (FLCN) and FLCN-interacting protein (FNIP) complex to block its function toward the Rag GTPases Ras-related GTP-binding C and D (RagC and RagD), abolishing mechanistic target of rapamycin (mTOR) complex 1 (mTORC1)-dependent phosphorylation and inactivation of TFEB. Functionally, STING-induced lysosome biogenesis within cells facilitated the clearance of cytoplasmic DNA and invading pathogens. Thus, our findings reveal that induction of lysosome biogenesis is another important function of the cGAS-STING pathway.

Chronic kidney disease-mineral and bone disorder (CKD-MBD) presents with extra-skeletal calcification and renal osteodystrophy (ROD). However, the pathophysiology of ROD remains unclear. Here we examine the hypothesis that stalled mitophagy within osteocytes of CKD-MBD mouse models contributes to bone loss. RNA-seq analysis revealed an altered expression of genes associated with mitophagy and mitochondrial function in tibia of CKD-MBD mice. The expression of mitophagy regulators, p62/SQSTM1, ATG7 and LC3, was inconsistent with functional mitophagy, and in mito-QC reporter mice with ROD, there was a two- to three-fold increase in osteocyte mitolysosomes. To determine if uremic toxins were potentially responsible for these observations, treatment of cultured osteoblasts with uremic toxins revealed increased mitolysosome number and mitochondria with distorted morphology. Membrane potential and oxidative phosphorylation were also decreased, and oxygen-free radical production increased. The altered p62/SQSTM1 and LC3-II expression was consistent with impaired mitophagy machinery, and the effects of uremic toxins were reversible by rapamycin. A causal link between uremic toxins and the development of mitochondrial abnormalities and ROD was established by showing that a mitochondria-targeted antioxidant (MitoQ) and the charcoal adsorbent AST-120 were able to mitigate the uremic toxin-induced mitochondrial changes and improve bone health. Overall, our study shows that impaired clearance of damaged mitochondria may contribute to the ROD phenotype. Targeting uremic toxins, oxygen-free radical production and the mitophagy process may offer novel routes for intervention to preserve bone health in patients with CKD-MBD. This would be timely as our current armamentarium of anti-fracture medications for patients with severe CKD-MBD is limited.

With the exacerbation of global population aging, sarcopenia has become an increasingly recognized public health issue. Sarcopenia, characterized by a progressive decline in skeletal muscle mass, strength, and function, significantly impacts the quality of life in the elderly. Herein, we explore the role of chroniclow-gradeinflammation in the development of sarcopenia and its underlying molecular mechanisms, including chronic inflammation-associated signaling pathways, immunosenescence, obesity and lipid infiltration, gut microbiota dysbiosis and intestinal barrier disruption, and the decline of satellite cells. The interplay and interaction of these molecular mechanisms provide new perspectives on the complexity of the pathogenesis of sarcopenia and offer a theoretical foundation for the development of future therapeutic strategies.

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.

Genetic mutations in retinol dehydrogenase 5 (RDH5), a rate-limiting enzyme of the visual cycle, is associated with nyctalopia, age-related macular disease, and stationary congenital fundus albipunctatus (FA). A majority of these mutations impair RDH5 protein expression and intracellular localization. However, the regulatory mechanisms underlying RDH5 metabolism remain unclear. Here, we find that RDH5 undergoes degradation via the autophagy-lysosomal pathway, and its stability is regulated by interacting with HSP90. Deletion of HSP90α or HSP90β by CRISPR-Cas9 or inhibition of HSP90 activity by IPI-504 downregulates RDH5 protein level, but not its mRNA expression, and this downregulation is restored by autophagic inhibitors (3-MA, CQ, and Baf-A1) and siRNA of ATG5 or ATG7, but not by the proteasome inhibitor MG132. RDH5 can physically interact with SQSTM1/P62, and this interaction is enhanced in HSP90-deficient cells as well as in CQ-treated cells. Knocking down SQSTM1/P62 by siRNA induces RDH5 protein accumulation. Moreover, HSP90, RDH5, and Calnexin form a complex through intermolecular interactions. Deficiency of HSP90α or HSP90β dissociates RDH5 from Calnexin and increases RDH5 translocation from the endoplasmic reticulum to the cytosol. Taken together, we propose that dysfunction of HSP90 leads to RDH5 release from Calnexin in the endoplasmic reticulum into the cytosol, where it binds to the adaptor SQSTM1/P62 for degradation in the autolysosome. RDH5 is a novel client candidate of HSP90. The downregulation of RDH5 may be responsible for the nyctalopia side effect noted in cancer patients receiving HSP90 inhibitor treatment currently in the clinical trial.

While moderately activated microglia in Alzheimer's disease (AD) are pivotal in clearing amyloid beta (Aβ), hyperactivated microglia perpetuate neuroinflammation. Prior investigations reported that the elimination of ~80% of microglia through inhibition of the colony-stimulating factor 1 receptor (CSF1R) during the advanced stage of neuroinflammation in 5xFamilial AD (5xFAD) mice mitigates synapse loss and neurodegeneration. Furthermore, prolonged CSF1R inhibition diminished the development of parenchymal plaques. Nonetheless, the effects of short-term CSF1R inhibition during the early stages of neuroinflammation on residual microglia are unknown. Therefore, we investigated the effects of 10-day CSF1R inhibition using PLX5622 in three-month-old female 5xFAD mice, a stage characterized by the onset of neuroinflammation and minimal Aβ plaques. We observed ~65% microglia depletion in the hippocampus and cerebral cortex. The leftover microglia displayed a noninflammatory phenotype with reduced NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome complexes. Moreover, plaque-associated microglia were reduced with diminished Clec7a expression. Additionally, phosphorylated S6 ribosomal protein and the protein sequestosome 1 analysis suggested reduced mechanistic targets of rapamycin (mTOR) signaling and autophagy in microglia and neurons within the hippocampus and cerebral cortex. Biochemical assays validated the inhibition of NLRP3 inflammasome activation, decreased mTOR signaling in the hippocampus and cerebral cortex, and enhanced autophagy in the hippocampus. However, short-term CSF1R inhibition did not influence Aβ plaques, soluble Aβ-42 levels, astrocyte hypertrophy, or hippocampal neurogenesis. Thus, short-term CSF1R inhibition during the early stages of neuroinflammation in 5xFAD mice promotes the retention of homeostatic microglia with diminished inflammasome activation and mTOR signaling, alongside increased autophagy.