Regulated cell death is a form of cell death that is actively controlled by biomolecules. Several studies have shown that regulated cell death plays a key role after spinal cord injury. Pyroptosis and ferroptosis are newly discovered types of regulated cell deaths that have been shown to exacerbate inflammation and lead to cell death in damaged spinal cords. Autophagy, a complex form of cell death that is interconnected with various regulated cell death mechanisms, has garnered significant attention in the study of spinal cord injury. This injury triggers not only cell death but also cellular survival responses. Multiple signaling pathways play pivotal roles in influencing the processes of both deterioration and repair in spinal cord injury by regulating pyroptosis, ferroptosis, and autophagy. Therefore, this review aims to comprehensively examine the mechanisms underlying regulated cell deaths, the signaling pathways that modulate these mechanisms, and the potential therapeutic targets for spinal cord injury. Our analysis suggests that targeting the common regulatory signaling pathways of different regulated cell deaths could be a promising strategy to promote cell survival and enhance the repair of spinal cord injury. Moreover, a holistic approach that incorporates multiple regulated cell deaths and their regulatory pathways presents a promising multi-target therapeutic strategy for the management of spinal cord injury.

Platinum-based antitumor drugs, such as cisplatin and carboplatin, are well-known for their severe ototoxicity. The ototoxic effects of these drugs are primarily attributed to oxidative stress induced damage within cochlear hair cells (HCs), leading to cell death and subsequent irreversible hearing loss. Over the past decade, studies have demonstrated that upregulating autophagy levels in HCs can greatly alleviate the death of cochlear HCs as part of the oxidative damage induced by ototoxic drugs. However, the molecular mechanisms by which platinum-based drugs affect autophagy and ultimately lead to HCs death remain unclear. In the present study, we investigated the effects of cisplatin on the mTOR signaling pathway, a critical regulator of autophagy, in cochlear explants of mice. Our results indicated that while cisplatin enhances autophagy activity initially, it also activates mTOR Complex1 (mTORC1) within HCs. The persistent activation of mTORC1 inhibits autophagy in HCs, resulting in the accumulation of reactive oxygen species and leading to cell death. Further pharmacological experiments confirmed the protective role of rapamycin, a specific mTORC1 inhibitor, highlighting the importance of autophagy in combating cisplatin-induced ototoxicity. Our findings suggest that modulating the mTOR signaling pathway to regulate autophagy could be an effective strategy for preventing cisplatin-induced ototoxic damage.

Nerve regeneration following traumatic peripheral nerve injuries and neuropathies is a complex process modulated by diverse factors and intricate molecular mechanisms. Past studies have focused on factors that stimulate axonal outgrowth and myelin regeneration. However, recent studies have highlighted the pivotal role of autophagy in peripheral nerve regeneration, particularly in the context of traumatic injuries. Consequently, autophagy-targeting modulation has emerged as a promising therapeutic approach to enhancing peripheral nerve regeneration. Our current understanding suggests that activating autophagy facilitates the rapid clearance of damaged axons and myelin sheaths, thereby enhancing neuronal survival and mitigating injury-induced oxidative stress and inflammation. These actions collectively contribute to creating a favorable microenvironment for structural and functional nerve regeneration. A range of autophagy-inducing drugs and interventions have demonstrated beneficial effects in alleviating peripheral neuropathy and promoting nerve regeneration in preclinical models of traumatic peripheral nerve injuries. This review delves into the regulation of autophagy in cell types involved in peripheral nerve regeneration, summarizing the potential drugs and interventions that can be harnessed to promote this process. We hope that our review will offer novel insights and perspectives on the exploitation of autophagy pathways in the treatment of peripheral nerve injuries and neuropathies.

Hypertension poses a great health threat globally. We probed the mechanisms of long non-coding RNA plasmacytoma variant translocation 1 (lncRNA PVT1) mediating ventricular remodeling (VR) in spontaneously hypertensive rats (SHR).

PVT1 was down-regulated or miR-30a was inhibited in SHR in vivo. Hypertensive injury model was established in vitro. VR, fibrosis and autophagy-related indicators were detected by echocardiography, HE/WGA/Masson staining, ELISA, and immunohistochemistry. Cell viability, fibrosis markers, autophagy-related markers, and lncRNA PVT1 and miR-30a levels were assessed. Interactions between PVT1, Beclin-1 and miR-30a were verified.

PVT1 was up-regulated in myocardial tissues of SHR. PVT1 knockdown alleviated VR and myocardial fibrosis (MF) in SHR, as evidenced by decreased systolic blood pressure, left ventricular end-systolic diameter, left ventricular end-systolic diameter, and heart weight index, boosted left ventricular fractional shortening and left ventricular ejection fraction, abated inflammatory infiltration of myocardial tissues, decreased myocardial hypertrophy and interstitial fibrosis, reduced serum angiotensin II (Ang II) and atrial natriuretic peptide, and downregulated collagen I, collagen II, α-smooth muscle actin, and fibronectin protein. PVT1 knockdown down-regulated Beclin 1 and LC3B-II/LC3B-I and up-regulated p62 protein. In vitro, PVT1 knockdown improved fibrosis by inhibiting Ang II-induced cardiomyocyte autophagy. PVT1 acted as a competitive endogenous RNA to competitively bind to miR-30a to target Beclin-1 expression. PVT1 targeted the miR-30a/Beclin-1 axis to mediate autophagy to affect VR and MF in SHR.

LncRNA PVT1 promotes cellular autophagy by targeting the miR-30a/Beclin-1 axis, thereby promoting VR and MF in SHR. Knockdown of lncRNA PVT1 attenuates VR and MF in SHR.

Selective autophagy plays a crucial role in maintaining cellular homeostasis by specifically targeting unwanted cargo labeled with "autophagy cues" signals for autophagic degradation. In this study, we identify Rab GTPases as a class of such autophagy cues signals involved in selective autophagy. Through biochemical and imaging screens, we reveal that human Rab GTPases are common autophagy substrates. Importantly, we confirm the conservation of Rab GTPase autophagic degradation in different model organisms. Rab GTPases translocate to damaged mitochondria, lipid droplets, and invading Salmonella-containing vacuoles (SCVs) to serve as degradation signals. Furthermore, they facilitate mitophagy, lipophagy, and xenophagy, respectively, by recruiting receptors. This interplay between Rab GTPases and receptors may ensure the de novo synthesis of isolation membranes around Rab-GTPase-labeled cargo, thereby mediating selective autophagy. These processes are further influenced by upstream regulators such as LRRK2, GDIs, and RabGGTase. In conclusion, this study unveils a conserved mechanism involving Rab GTPases as autophagy cues signals and proposes a model for the spatiotemporal control of selective autophagy.

With age, our metabolic systems undergo significant alterations, which can lead to a cascade of adverse effects that are implicated in both metabolic disorders, such as diabetes, and in the body's ability to respond to acute stress and trauma. To elucidate the metabolic imbalances arising from aging, we introduce the concept of "metabolaging." This framework encompasses the broad spectrum of metabolic disruptions associated with the hallmarks of aging, including the functional decline of key metabolically active organs, like the adipose tissue. By examining how these organs interact with essential nutrient-sensing pathways, "metabolaging" provides a more comprehensive view of the systemic metabolic imbalances that occur with age. This concept extends to understanding how age-related metabolic disturbances can influence the response to acute stressors, like burn injuries, highlighting the interplay between metabolic dysfunction and the ability to handle severe physiological challenges. Finally, we propose potential interventions that hold promise in mitigating the effects of metabolaging and its downstream consequences.

Acute myeloid leukemia (AML) is the most prevalent kind of acute leukemia in adults. Despite the availability of new targeted therapies, AML remains connected with a poor prognosis and decreased rate of survival. Tubeimoside I (TBMS1), a critical compound extracted from Bolbostemma paniculatum, has demonstrated potential anticancer effects in lung and colorectal cancers. Nevertheless, the TBMS1 anticancer pathway against AML is still elusive. This study aimed to explore the potential role of TBMS1 in anti-AML and its molecular mechanism. In vitro, TBMS1 treatment suppressed AML cells proliferation, induced apoptosis, and mitochondrial damage, and elevated ROS levels. Network pharmacological analysis suggested, and subsequent studies confirmed, that TBMS1 induced mitophagy in AML cells by modulating the PINK1/Parkin/Mfnh2 signaling pathway, an effect that was effectively reversed following PINK1 knockdown. In vivo, TBMS1 treatment suppressed the proliferation of AML cells after 21 days, improved the survival rates of nude mice, and showed no evident organ toxicity. These evidences suggest that TBMS1 may have significant therapeutic potential in treating AML.

Breast cancer (BC) is the most common malignant tumor and the leading cause of cancer-related deaths among women worldwide. Great progress has been recently achieved in controlling breast cancer; however, mortality from breast cancer remains a substantial challenge, and new treatment mechanisms are being actively sought. Programmed cell death (PCD) is associated with the progression and treatment of many types of human cancers. PCD can be divided into multiple pathways including autophagy, apoptosis, mitotic catastrophe, necroptosis, ferroptosis, pyroptosis, and anoikis. Ubiquitination is a post-translational modification process in which ubiquitin, a 76-amino acid protein, is coupled to the lysine residues of other proteins. Ubiquitination is involved in many physiological events and promotes cancer development and progression. This review elaborates the role of ubiquitin-specific protease (USP) in programmed cell death, which is common in breast cancer cells, and lays the foundation for tumor diagnosis and targeted therapy.

Alzheimer's disease (AD) is a neurological disorder that affects cognition and behavior, accounting for 60-70 % of dementia cases. Its mechanisms involve amyloid aggregates, hyperphosphorylated tau tangles, and loss of neural connections. Current treatments have limited efficacy due to a lack of specific targets. Recently, microRNAs (miRNAs) have emerged as key modulators in AD, regulating gene expression through interactions with mRNA. Dysregulation of specific miRNAs contributes to disease progression by disrupting clearance pathways. Antisense oligonucleotide (ASO)-based therapies show promise for AD treatment, particularly when combined with miRNA mimics or antagonists, targeting complex regulatory networks. However, miRNAs can interact with each other, complicating cellular processes and potentially leading to side effects. Our review emphasizes the role of miRNAs in regulating amyloid-beta (Aβ) clearance and highlights their potential as therapeutic targets and early biomarkers for AD, underscoring the need for further research to enhance their efficacy and safety.

The root of aging is attributed to kidney essence insufficiency and gradual loss of kidney function. The combination of Epimedii Folium and Ligustri Lucidi Fructus (ELL) is traditionally recognized to tonify kidney yin and yang and has significant efficacy in delaying aging and aging-related diseases, but little is known about the exact mechanism.

The research focuses on the mechanism of delaying renal aging by which ELL regulates mitophagy through serine/threonine kinase AMP-activated protein kinase (AMPK)/UNC-51- like autophagy activating kinase 1 (ULK1)/B-cell lymphoma-2-like protein 13 (Bcl2L13) in vivo.

We employed a rat model of natural aging, using rats of different ages as dynamic controls, and a natural aging mouse model to evaluate the effects of ELL on delaying renal aging via AMPK/ULK1/Bcl2L13. The assessment included renal histopathology, oxidative stress, cell senescence, mitochondrial dynamics, mitophagy, and the AMPK/ULK1/Bcl2L13 signaling pathway. In the aging rat model, network pharmacology and proteomics were combined to dissect the renal aging process, and a Multilayer Perceptron (MLP) -artificial neural networks (ANN) model was used to evaluate the effects of ELL comprehensively. In the aging mouse model, the AMPK inhibitor dorsomorphin was applied to assess whether the AMPK signaling pathway was involved in ELL-induced mitophagy.

Compared with the young rats, the kidney exhibited signs of degenerative pathologies and increased oxidative stress in 17-month-old rats. A thorough analysis identified the mitochondrial protein Bcl2L13 as a crucial biomarker associated with renal aging. The AMPK/ULK1/Bcl2L13 pathway significantly regulated mitochondrial function and mitophagy, which were potential mechanisms underlying renal aging. In contrast to aged rats, the renal pathological changes and cell senescence in rats treated with ELL were significantly mitigated, the antioxidant capacity, mitochondrial dynamics, and mitophagy were improved, and the expression of AMPK/ULK1/Bcl2L13 was upregulated. After the application of AMPK inhibitor dorsomorphin, the effects of ELL were reversed. It appears that ELL modulates the AMPK/ULK1/Bcl2L13 signaling pathway, and upregulates mitophagy to potentially decelerate renal aging.

The findings indicate that aging kidneys display mitochondrial dysfunction, disorganization of mitochondria, and a decrease in mitophagy. Concurrently, ELL significantly regulates mitochondrial dynamics and mitophagy via AMPK/ULK1/Bcl2L13. This regulation helps mitigate mitochondrial dysfunction, suggesting ELL as a promising herbal remedy for delaying renal aging and age-related kidney diseases.

Obstructive sleep apnea syndrome is closely associated with tumor growth. Chronic intermittent hypoxia promotes autophagy and is related to malignant tumor development. However, the role of autophagy in obstructive sleep apnea syndrome progression remains unclear.

obstructive sleep apnea syndrome datasets (GSE135917 and GSE38792) from Gene Expression Omnibus were analyzed to identify differentially expressed genes and autophagy-related differentially expressed genes. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and gene set enrichment analysis were conducted, and a protein-protein interaction network identified hub genes. Colorectal cancer datasets from The Cancer Genome Atlas were used for differential expression and survival analyses, along with gene set enrichment analysis and immune infiltration analysis. Chronic intermittent hypoxia-induced autophagy and oxidative stress were investigated in Sprague-Dawley rats using reactive oxygen species assays. Hub genes were validated in rats and obstructive sleep apnea syndrome patient samples.

Gene set enrichment analysis revealed significant differences in autophagy-related gene expression among obstructive sleep apnea syndrome patients. Hub genes ATG5, CASP1, MAPK8, EIF4G1, and TANK-binding kinase 1 were identified, with ATG5 and TANK-binding kinase 1 validated. Autophagy-related differentially expressed genes were predominantly upregulated in colorectal cancer tissues. TANK-binding kinase 1 expression in colorectal cancer patients was associated with enhanced sensitivity to immunotherapy and CD8 + T cell, macrophage, and regulatory T cell infiltration, potentially influencing the immune microenvironment. The animal experiments showed that chronic intermittent hypoxia increased reactive oxygen species levels, suggesting that chronic intermittent hypoxia plays a role in autophagy. TANK-binding kinase 1 expression was significantly higher in obstructive sleep apnea syndrome patients than in controls, and continuous positive airway pressure did not alter TANK-binding kinase 1 levels.

This study is the first to describe the potential contribution of TANK-binding kinase 1 to the development of obstructive sleep apnea syndrome and its potential as a novel biomarker and potential therapeutic target for obstructive sleep apnea syndrome.

LAMP2 is a ubiquitously expressed protein critical for autophagy. Alternative splicing gives rise to three isoforms. However, the roles of major LAMP2 isoforms in the heart are not known. To address this knowledge gap, we generated lamp2a and lamp2b knockout (KO) mice to investigate the role of these isoforms in heart function and autophagy. Deletion of either Lamp2a or Lamp2b did not alter cardiac structure or function. Lack of all LAMP2 isoforms led to increased cardiac fibrosis and reduced survival during pressure overload, which were not observed in lamp2a or lamp2b KO mice. Also, LAMP2B loss did not affect levels of the autophagy markers LC3-II and SQSTM1/p62. Conversely, LAMP2A was upregulated in hearts lacking LAMP2B, potentially preserving autophagy and cardiac function. Reintroducing LAMP2A in lamp2 KO mice effectively reduced autophagosome accumulation and improved cardiac function. Overall, these data support LAMP2 isoform functional redundancy in the myocardium under pathological conditions.Abbreviations: AAV: adeno-associated virus; ACTA2: actin alpha 2, smooth muscle, aorta; CMA: chaperone-mediated autophagy; KO: knockout; LAMP2: lysosomal-associated membrane protein 2; LV: Left ventricle; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; NPPA: natriuretic peptide type A; NPPB: natriuretic peptide type B; SQSTM1/p62: sequestosome 1; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; TAC: transverse aortic constriction; WT: wild type.

Histamine H3 receptor (H3R) antagonists regulate histamine release that modulates neuronal activity and cognitive function. Although H3R is elevated in Alzheimer's disease (AD) patients, whether H3R antagonists can rescue AD-associated neural impairments and cognitive deficits remains unknown. Pitolisant is a clinically approved H3R antagonist/inverse agonist that treats narcolepsy. Here, we find that pitolisant reverses AD-like pathophysiology and cognitive impairments in an AD mouse model. Behavioral assays and in vivo wide-field Ca2+ imaging revealed that recognition memory, learning flexibility, and slow-wave impairment were all improved following the 15-day pitolisant treatment. Improved recognition memory was tightly correlated with slow-wave coherence, suggesting slow waves serve as a biomarker for treatment response and for AD drug screening. Furthermore, pitolisant reduced amyloid-β deposition and dystrophic neurites surrounding plaques, and enhanced neuronal lysosomal activity, inhibiting which blocked cognitive and slow-wave restoration. Our findings identify pitolisant as a potential therapeutic agent for AD treatments.

The prevalence and spread of canine influenza virus (CIV) pose a threat to the health of dogs and humans. Some studies have shown that autophagy is closely related to virus replication, but the exact relationship between CIV replication and autophagy is still unclear. Therefore, this study investigated the effects of autophagy on CIV replication in vitro and in vivo. The data showed that CIV infection significantly caused respiratory tract damage in mice, upregulated the mRNA/protein levels of CIV replication-related genes and autophagy-related genes. In addition, the activation of autophagy by rapamycin (Rapa) significantly intensified the CIV replication and the respiratory tract damage of mice, while the inhibition of autophagy by 3-Methyladenine (3-MA) significantly alleviated these effects. Data of MDCK cells also demonstrated that CIV promoted self-replication through activating autophagy, and the upregulation of AKT/mTOR by insulin significantly inhibited the CIV replication. In summary, this study showed that CIV could promote self-replication by activating AKT/mTOR mediated autophagy, which provides new ideas for the prevention and treatment of canine influenza.

Pacsin1 is a crucial protein involved in vesicle formation and transport, and its role in neuronal development and cytosolic dynamics has been extensively studied. However, its involvement in immune regulation still needs to be better understood. In this study, we show that pacsin1 exerts a negative regulatory effect on RLR-mediated signaling pathways activated by SCRV or poly(I:C), thereby inhibiting MITA-mediated antiviral responses. Mechanistically, pacsin1 facilitates the degradation of MITA, thus impeding immune signaling. Additionally, overexpression of pacsin1 promotes the conversion of LC3B-I to LC3B-II, while treatment with the autophagy inhibitor ammonium chloride results in the accumulation of LC3B-II and prevents pacsin1-mediated MITA degradation. Our findings suggest that pacsin1 targets MITA for autophagic degradation, thereby suppressing the innate antiviral response in fish.

Various forms of tissue damage can lead to fibrosis, an abnormal reparative reaction. In the industrialized countries, 45% of deaths are attributable to fibrotic disorders. Autophagy is a highly preserved process. Lysosomes break down organelles and cytoplasmic components during autophagy. The cytoplasm is cleared of pathogens and dysfunctional organelles, and its constituent components are recycled. With the growing body of research on autophagy, it is becoming clear that autophagy and its associated mechanisms may have a role in the development of numerous fibrotic disorders. However, a comprehensive understanding of autophagy in fibrosis is still lacking and the progression of fibrotic disease has not yet been thoroughly investigated in relation to autophagy‑associated processes. The present review focused on the latest findings and most comprehensive understanding of macrophage autophagy, endoplasmic reticulum stress‑mediated autophagy and autophagy‑mediated endothelial‑to‑mesenchymal transition in the initiation, progression and treatment of fibrosis. The article also discusses treatment strategies for fibrotic diseases and highlights recent developments in autophagy‑targeted therapies.

Endometriosis affects about 10 % of women of reproductive age, leading to a disabling gynecologic condition. Chronic pain, inflammation, and oxidative stress have been identified as the molecular pathways involved in the progression of this disease, although its precise etiology remains uncertain. Although mitochondria are considered crucial organelles for cellular activity, their dysfunction has been linked to the development of this disease. The purpose of this review is to examine the functioning of the mitochondrion in endometriosis: in particular, we focused on the mitochondrial dynamics of biogenesis, fusion, and fission. Since excessive mitochondrial activity is reported to affect cell proliferation, we also considered mitophagy as a mechanism involved in limiting disease development. To better understand mitochondrial activity, we also considered alterations in circadian rhythms, the gut microbiome, and estrogen receptors: indeed, these mechanisms are also involved in the development of endometriosis. In addition, we focused on recent research about the impact of numerous substances on mitochondrial activity; some of them may offer a future breakthrough in endometriosis treatment by acting on mitochondria and inhibiting cell proliferation.

ATG9A is a transmembrane protein essential for macroautophagy/autophagy that drives autophagosome formation by delivering essential proteins and lipids to the phagophore through vesicle trafficking. Here, we demonstrate that the atypical Rho GTPase RHOD is required for ATG9A trafficking and stimulates autophagosome formation. Upon starvation, RHOD interacted with ATG9A and accompanied ATG9A trafficking from the Golgi toward phagophores. In addition, starvation-induced high levels of RHOD resulted in Golgi fragmentation to further promote ATG9A vesicle export from the trans-Golgi network to the peripheral region. Loss of RHOD suppressed ATG9A trafficking and reduced the distribution of ATG9A on the phagophore. Moreover, WHAMM (WASP homolog associated with actin, golgi membranes and microtubules) forms a complex with RHOD and participates in this process in a RHOD-dependent manner. Importantly, RHOD mutants, which lack the exon II-containing effector region motif that is required for ATG9A binding or lack the CAAX box that is responsible for membrane targeting, fail to stimulate ATG9A trafficking and autophagosome formation. Furthermore, RHOD plays a distinct suppressor role in tumor development, partly associated with its regulatory effect on autophagy. These findings reveal the important roles of RHOD in autophagy and tumor development.Abbreviation: ATG9A: autophagy related 9A; BafA1: bafilomycin A1; BiFC: bimolecular fluorescence complementation; co-IP: co-immunoprecipitation; EBSS: Earle's balanced salt solution; FM: full culture medium; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PUP-IT: pupylation-based interaction tagging; RHOD: ras homolog family member D; SQSTM1: sequestosome 1; TGN: trans-Golgi network; VC: Venus C-terminal; VN: Venus N-terminal; WHAMM: WASP homolog associated with actin, golgi membranes and microtubules; WIPI2: WD repeat domain, phosphoinositide interacting 2; WT: wild-type; 3-MA: phosphatidylinositol 3-kinase (PtdIns3K) inhibitor 3-methyladenine.

Necroptosis is a programmed form of cell death that has gained significant attention in the field of cardiovascular research due to its involvement in myocardial infarction (MI) and myocardial ischaemia-reperfusion (I/R) injury. Unlike apoptosis, necroptosis elicits a pro-inflammatory response, contributing to myocardial injury, fibrosis, and adverse remodelling. This review aims to provide an overview of the molecular mechanisms underlying necroptosis, with a particular focus on its role in myocardial I/R injury. Key regulatory proteins such as Receptor-interacting protein kinase 3 (RIPK3) and Mixed lineage kinase domain-like protein (MLKL) are central to the necroptotic process, mediating cell death and inflammation. The review discusses the potential of targeting necroptosis as a therapeutic strategy for managing cardiovascular diseases, particularly post-MI. The RIPK3-CaMKII-mitochondrial permeability transition pore (mPTP) pathway is identified as a critical signalling axis in necroptosis and its inhibition may offer protective benefits in myocardial injury. The review also considers the role of natural and chemical inhibitors and other genes in necroptosis regulation. Overall, targeting necroptosis represents a promising avenue for therapeutic intervention to mitigate cardiac injury, promote recovery, and improve long-term patient outcomes in cardiovascular diseases.

Autophagy, a critical intracellular degradation and recycling pathway mediated by lysosomes, is essential for maintaining cellular homeostasis through the quality control of proteins and organelles. Our research focused on the role of proximal tubular autophagy in the pathophysiology of aging, obesity, and diabetes. Using a novel method to monitor autophagic flux in kidney tissue, we revealed that age-associated high basal autophagy supports mitochondrial quality control and delays kidney aging. However, an impaired ability to upregulate autophagy under additional stress accelerates kidney aging. In obesity induced by a high-fat diet, lysosomal dysfunction disrupts autophagy, leading to renal lipotoxicity. Although autophagy is initially activated to repair organelle membranes and maintain proximal tubular cell integrity, this demand overwhelms lysosomes, resulting in "autophagic stagnation" characterized by phospholipid accumulation. Similar lysosomal phospholipid accumulation was observed in renal biopsies from elderly and obese patients. We identified TFEB-mediated lysosomal exocytosis as a mechanism to alleviate lipotoxicity by expelling accumulated phospholipids. Therapeutically, interventions such as the SGLT2 inhibitor empagliflozin and eicosapentaenoic acid restore lysosomal function and autophagic activity. Based on these findings, we propose a novel disease concept, "Obesity-Related Proximal Tubulopathy." This study underscores autophagic stagnation as a key driver of kidney disease progression in aging and obesity, offering insights into the pathophysiology of kidney diseases and providing a foundation for targeted therapeutic strategies.

Rheumatoid arthritis (RA) is a chronic autoimmune disease in which autophagy is pivotal in its pathogenesis. This study aims to identify autophagy-related genes associated with RA and investigate their functional roles.

We performed mRNA sequencing to identify differentially expressed genes (DEGs) between RA and osteoarthritis (OA) and intersected these with autophagy-related genes to obtain autophagy-related DEGs (ARDEGs) in RA. Bioinformatics and machine learning approaches were used to identify key biomarkers. Functional experiments, including real-time cellular analysis (RTCA), scratch healing, and flow cytometry, were conducted to examine the effects of gene silencing on the proliferation and migration of MH7A cells.

A total of 37 ARDEGs were identified in RA. Through bioinformatics analysis, interferon regulatory factor 4 (IRF4) emerged as a key hub gene, with its high expression confirmed in RA synovial tissues and RA FLS cells. IRF4 knockdown inhibited the proliferation and migration and promoted the death of MH7A cells.

IRF4 is an autophagy-related diagnostic biomarker for RA. Targeting IRF4 could serve as a potential diagnostic and therapeutic strategy for RA, although further clinical studies are required to validate its effectiveness.

Osteoarthritis (OA) is closely related to aging, and autophagy is implicated in the retardation of aging. Activated synoviocytes play important roles in OA; the synoviocytes could produce osteopontin (OPN) and its main receptors CD44 and integrin, which are all involved in OA. The purpose of this study is to investigate whether OPN has an effect on autophagy in osteoarthritic synoviocytes.

We cultured human OA fibroblast-like synoviocytes (FLS) and treated them with rhOPN and antibodies against CD44 and CD51/61 (αvβ3 integrin) or isotype IgG to block the interaction between receptors and ligands. Infection with lentivirus mRFP-GFP-LC3, laser confocal imaging and Western blotting were used to determine changes in the expression of autophagy markers, and cell proliferation of FLS was assessed with a CCK-8 assay.

Our results showed the expression level of autophagy marker protein LC3 II and the mRFP-GFP-LC3 puncta were significantly decreased after treatment with rhOPN when compared with the control group, when the FLS were incubated with antibodies against CD44 or CD51/61 (αvβ3 integrin) or with control isotype IgG for 1 h, followed by rhOPN treatment for 48 h, rhOPN could suppress the relative expression of LC3 II and Beclin1 via integrin and CD44 in the FLS, CCK-8 assay also showed that rhOPN significantly increased the cell proliferation and viability of FLS.

OPN could inhibit autophagy via CD44 and αvβ3 integrin and promote the proliferation of FLS, playing an important role in OA synovitis.

Catalpol, classified as an iridoid glucoside, is recognized for its significant role in medicine, particularly in the treatment of various conditions such as diabetes mellitus, neuronal disorders, and inflammatory diseases. This review aims to evaluate the biological implications of catalpol and the mechanisms underlying its diverse pharmacological effects. A thorough exploration of existing literature was conducted utilizing the keyword "Catalpol" across prominent public domains like Google Scholar, PubMed, and EKB. Catalpol has demonstrated a diverse array of pharmacological effects in experimental models, showcasing its anti-diabetic, cardiovascular-protective, neuroprotective, anticancer, hepatoprotective, anti-inflammatory, and antioxidant properties. In summary, catalpol manifests a spectrum of biological effects through a myriad of mechanisms, prominently featuring its anti-inflammatory and antioxidant capabilities. Its diverse pharmacological profile underscores its potential for therapeutic applications across a range of conditions. Further research is warranted to fully elucidate the clinical implications of catalpol and optimize its use in medical practice.

Skeletal muscle plays a central role in the storage, synthesis, and breakdown of nutrients, yet little research has explored temporal responses of this human tissue, especially with concurrent measures of systemic biomarkers of metabolism.

To characterize temporal profiles in skeletal muscle expression of genes involved in carbohydrate metabolism, lipid metabolism, circadian clocks, and autophagy and descriptively relate them to systemic metabolites and hormones during a controlled laboratory protocol.

Ten healthy adults (9M/1F, [mean ± SD] age 30 ± 10 years; BMI 24.1 ± 2.7 kg·m-2) rested in the laboratory for 37 hours with all data collected during the final 24 hours (08:00-08:00 hours). Participants ingested hourly isocaloric liquid meal replacements alongside appetite assessments during waking before a sleep opportunity from 22:00 to 07:00 hours. Blood samples were collected hourly for endocrine and metabolite analyses, with muscle biopsies occurring every 4 hours from 12:00 to 08:00 hours the following day to quantify gene expression.

Plasma insulin displayed diurnal rhythmicity peaking at 18:04 hours. Expression of skeletal muscle genes involved in carbohydrate metabolism (Name, Acrophase [hours]: GLUT4, 14:40; PPARGC1A, 16:13; HK2, 18:24) and lipid metabolism (FABP3, 12:37; PDK4, 05:30; CPT1B, 12:58) displayed 24-hour rhythmicity that reflected the temporal rhythm of insulin. Equally, circulating glucose (00:19 hours), nonesterified fatty acids (04:56), glycerol (04:32), triglyceride (23:14), urea (00:46), C-terminal telopeptide (05:07), and cortisol (22:50) concentrations also all displayed diurnal rhythmicity.

Diurnal rhythms were present in human skeletal muscle gene expression as well systemic metabolites and hormones under controlled diurnal conditions. The temporal patterns of genes relating to carbohydrate and lipid metabolism alongside circulating insulin are consistent with diurnal rhythms being driven in part by the diurnal influence of cyclic feeding and fasting.

Cancer immunotherapy has become a revolutionary strategy in oncology, utilizing the host immune system to fight malignancies. Notwithstanding major progress, obstacles such as immune evasion by tumors and the development of resistance still remain. This manuscript examines the function of chaperone-mediated autophagy (CMA) in cancer biology, focusing on its effects on tumor immunotherapy response and resistance. CMA is a selective degradation mechanism for cytosolic proteins, which is crucial for sustaining cellular homeostasis and regulating immune responses. By degrading specific proteins, CMA can either facilitate tumor progression in stressful conditions, or promote tumor suppression by removing oncogenic factors. This double-edged sword highlights the complexity of CMA in cancer progression and its possible effect on treatment results. Here we clarify the molecular mechanisms by which CMA can regulate the immune response and its possible role as a therapeutic target for improving the effectiveness of cancer immunotherapy.

Multiple myeloma (MM), a plasma cell-derived malignant hematological disease, is often treated with bortezomib, a highly effective first-generation proteasome inhibitor. However, resistance to bortezomib is a common occurrence. Profilin 1 (PFN1), a cytoskeleton-related gene known to promote autophagy in MM, induces this resistance to bortezomib, but it is unclear why. The aim of this study was to uncover the molecular mechanisms involved in bortezomib resistance, considering not only PFN1, but also CD138, a transmembrane proteoglycan that is a hallmark of plasma and MM cells. We detected CD138 and PFN1 in the bone marrow of patients with MM immunohistochemically. We also studied the Gene Expression Omnibus (GEO) data and found that CD138 was associated with PFN1 and autophagy. We then evaluated their expression in an MM cell line via western blot analysis, immunofluorescence assay, and flow cytometry; constructed PFN1-overexpressing and -knockdown cell lines; and detected ubiquitinated CD138 in the cells of both cell lines. Overexpression of PFN1 or PFN1-induced autophagy downregulated CD138 expression. Owing to the stemness and resistance of CD138- MM cells, inhibition of autophagy or CD138 overexpression reversed the resistance of PFN1-overexpressing MM cells to bortezomib, as indicated in our clonogenic, apoptosis, and CCK-8 assays. These results indicated that CD138 plays an important role in the resistance of MM cells to bortezomib. Targeting PFN1, CD138, or autophagic pathways may provide a promising therapeutic strategy for overcoming PFN1-induced drug resistance in MM.

Diabetic kidney disease (DKD) is one of the complications of diabetes mellitus, which triggers kidney fibrosis and eventually develops into end-stage renal disease. Nuciferine (NF) is one of the most important functional components in lotus leaves (LL), but its role and mechanism for the treatment of DKD are unclear. A high-fat-diet (HFD)-induced DKD model in KK-AY mice was established in this study. NF treatment significantly improved blood glucose and blood biochemical indices in DKD mice. Furthermore, NF reduced the levels of mALB, UCRE, Scr, and BUN in mice urine. Further, the extent of renal lesions in the mice in this study was at stage IV according to the Mogensen staging method. NF treatment was effective in ameliorating renal injury during this period. Concurrently, the protein levels of FN, N-cadherin, TGFβ, p-Smad3, p-PI3K, p-AKT, p-mTOR, and p62 were decreased. In contrast, the level of expression of Beclin-1 was increased. In the high glucose-exposed HK-2 cell model, the expression of p-PI3K, p-AKT, and p-mTOR was all downregulated, and autophagy proteins were increased after NF intervention. In addition, HK-2 cells were treated with high glucose in combination with Wortmannin and 3-MA, respectively. The results demonstrated that NF inhibited the expression of TGFβ and p-Smad3 by regulating autophagy through the PI3K-AKT-mTOR pathway, thereby ameliorating renal fibrosis at stage IV in mice. Therefore, LL can be used as a dietary component for the prevention of renal fibrosis in DKD patients.

Numerous drugs have been developed to meet the critical demand for treatments inhibiting cardiac remodeling following cardiovascular disease. Acacetin is a flavonoid with potential therapeutic effects against various cardiovascular diseases.

This study investigated the effect of acacetin on pressure overload-induced cardiac remodeling and its underlying molecular regulatory mechanisms.

We simulated pressure overload-induced cardiac remodeling in male C57BL/6 mice by constricting the thoracic aortic arch and assessed the effect of acacetin on cardiac remodeling.

Acacetin significantly ameliorated cardiac remodeling by downregulating ubiquitin-specific peptidase 10 (USP10) protein expression and reducing autophagy levels in cardiomyocytes. These findings confirm that acacetin improves cardiac remodeling by suppressing cardiomyocyte autophagy and highlight the crucial role of USP10 in the Beclin 1 ubiquitination degradation-mediated inhibition of the cardiomyocyte autophagy signaling pathway.

These results suggest that acacetin is a promising candidate drug for the treatment of cardiac remodeling induced by pressure overload.

Histone deacetylase 6 (HDAC6) is a promising target for treating neurodegenerative disorders, several cancer types and viral infections. Unique among HDACs, the HDAC6 isoform possesses a zinc finger ubiquitin-binding domain (UBD) crucial for managing misfolded protein aggregates and facilitating viral infection. HDAC6 binds aggregated polyubiquitinated proteins through its UBD, mediating their transport to the aggresome and subsequent removal via autophagy. Despite the importance of the UBD in proteostasis and viral infection, its pharmacological inhibition has been minimally explored thus far, with research largely focused on the deacetylase domain. We synthesized a diverse library of new compounds designed to target the HDAC6-UBD, termed HZUBi, with varied core structures including quinazolinone, oxindole and tetrahydrothiopyrano[4,3-b]indole, aimed at enhancing UBD interaction and extending into the side pocket. New structure-activity relationships were established, computational docking and molecular dynamics studies were performed and the functional impact of selected inhibitors was assessed in the context of multiple myeloma and viral infection. Several new HZUBi could displace a ubiquitin peptide from HDAC6-UBD in a differential manner, although to a lower extent than the literature reference compound HZUBi-3e. Despite exhibiting in vitro target engagement, neither HZUBi-3e nor its ester prodrug HZUBi-1e enhanced proteasome inhibitor-mediated multiple myeloma cell killing. Finally, none of the screened HZUBi triggered anti-viral activity.

Autophagy plays an important role in the physiology and pathology of the liver. Several negative autophagy regulators have been discovered, including epidermal growth factor receptor (EGFR), mediated by activation of the PI3K/Akt/mTOR signaling pathway. Disabled-1 (Dab1) is one of the mediating adaptor factors of PI3K/Akt/mTOR signaling pathways. We investigated the potential impact of Dab1 on autophagy-related markers (LC3B, LAMP2A, HSC70, and GRP78) in the developing liver by using a model of yotari mice and compared it with autophagy marker expression in human liver development. Mouse embryos were obtained at gestation days 13.5 and 15.5 (E13.5 and E15.5), and a total of 5 normal human conceptuses were obtained between gestation days 5 and 10. Histological sections were analyzed by immunohistochemistry. The highest expression of the early endosome-forming factor LC3B and the microautophagy factor LAMP2a was observed at the transition from embryonic to early fetal phase, whereas the expression of the chaperones HSC 70 and GRP78 was highest at embryonic phase. The expression patterns of three of these factors in mouse liver were different from those in human liver: the expression of LC3B was high at E13.5, that of HSC 70 at 15.5, whereas the expression of GRP78 did not change significantly. On the other hand, the expression pattern of LAMP2a was similar to that in human development and was higher at E15.5 than at E13.5. Moreover, knockout of Dab1 resulted in significantly lower expression of LC3B and LAMP2a in mouse embryo livers (at E13.5), indicating a possible role of Dab1 in regulating autophagy during embryonic development in the liver.

Exercise activates autophagy and lysosome system in skeletal muscle, which are known to play an important role in metabolic adaptation. However, the mechanism of exercise-activated autophagy and lysosome system in obese insulin resistance remains covert. In this study, we investigated the role of exercise-induced activation of autophagy and lysosome system in improving glucose metabolism of skeletal muscle. Male C57BL/6 mice were randomly divided into five groups: the chow diet (CD) group, the high-fat diet (HFD) group, the high-fat diet plus exercise (HFD-E) group and the HFD-E treated with calcineurin inhibitor FK506 (HFD-E-F) or saline (HFD-E-S) groups. The mice in exercise groups (HFD-E, HFD-E-F and HFD-E-S) were subjected to aerobic treadmill exercise (speed at 12 m/min for 1 h per session, 0° slope, 5 days per week for 12 weeks). Mice of HFD-E-F group were intraperitoneally administered FK506 (1 mg/kg), once each day for 2 weeks before the end of exercise. Expressions pTFEB, T-TFEB and autophagy-lysosome markers, including Beclin1, LC3, ULK1, SQSTM1, LAMP1, CTSD and CTSL proteins in gastrocnemius muscle were analysed. We demonstrated that HFD induced insulin resistance and decreased autophagy-lysosomal proteins and the exercise significantly increased transcription factor EB (TFEB) translocation from the cytoplasm to the nucleus, restored the impaired autophagy-lysosomal-related protein expressions, and improved glucose metabolism. The increase in TFEB nuclear translocation was partly blocked by the calcineurin inhibitor FK506. Our results suggest that exercise promotes autophagy and lysosome restoration by regulating calcineurin-mediated TFEB nuclear translocation, ultimately alleviating HFD-induced insulin resistance in mice skeletal muscle.

Skeletal muscle dysfunction is a common extrapulmonary comorbidity of chronic obstructive pulmonary disease (COPD) and is associated with decreased quality-of-life and survival in patients. The autophagy lysosome pathway is one of the proteolytic systems that significantly affect skeletal muscle structure and function. Intriguingly, both promoting and inhibiting autophagy have been observed to improve COPD skeletal muscle dysfunction, yet the mechanism is unclear. This paper first reviewed the effects of macroautophagy and mitophagy on the structure and function of skeletal muscle in COPD, and then explored the mechanism of autophagy mediating the dysfunction of skeletal muscle in COPD. The results showed that macroautophagy- and mitophagy-related proteins were significantly increased in COPD skeletal muscle. Promoting macroautophagy in COPD improves myogenesis and replication capacity of muscle satellite cells, while inhibiting macroautophagy in COPD myotubes increases their diameters. Mitophagy helps to maintain mitochondrial homeostasis by removing impaired mitochondria in COPD. Autophagy is a promising target for improving COPD skeletal muscle dysfunction, and further research should be conducted to elucidate the specific mechanisms by which autophagy mediates COPD skeletal muscle dysfunction, with the aim of enhancing our understanding in this field.

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.

Autophagy is a conserved and unique degradation system in eukaryotic cells, which plays crucial roles in the growth, development and pathogenesis of Fungi. Despite that, it is poorly understood in Fusarium graminearum currently. Here, we identified an autophagy gene FgAtg27 from F. graminearum, and investigated its possible roles in regulating morphogenesis and pathogenicity. Results showed that FgAtg27 is homologous to Saccharomyces cerevisiae Atg27 and with an active signal peptide at N-terminal. Then, the ΔFgAtg27 mutant was generated and gene deletion did not change growth and sporulation, whereas significantly decreased pathogenicity. FgAtg27 showed subcellular localization at pre-autophagosomal structure (PAS). After starvation induction, amount of autophagosomes in ΔFgAtg27 was significantly less than wild type and complemented strain, indicating that FgAtg27 deletion affects the autophagosome formation in F. graminearum. Meanwhile, under high Ca2+ concentration conditions, ΔFgAtg27 exhibited slowed growth, confirming that FgAtg27 also involved in F. graminearum's hyperosmotic reaction to Ca2+ concentration stress. In addition, yeast two-hybrid experiments, revealed that FgAtg27 interacts with the autophagy key protein FgAtg9. Collectively, we found that the deletion of FgAtg27 did not impact the growth phenotype of F. graminearum, whereas significantly reduced its pathogenicity and Ca2+ stress through affecting autophagic process.

Oral submucous fibrosis (OSF) is a chronic, progressive, and potentially malignant disease of the oral cavity. A previous study by our team found that the aberrant expression of tRNA-derived small RNA (tsRNA) was involved in the development of OSF, with tiRNA-Val-CAC-002 showing the most significant difference. This study aimed to investigate the effect of tiRNA-Val-CAC-002 on fibroblast activation and its underlying mechanisms, elucidate the pathogenesis of OSF, and explore new effective targets for OSF prevention and treatment.

RT-PCR was used to detect tiRNA-Val-CAC-002 expression in OSF and arecoline-treated fibroblasts. Western blotting, MDC staining, and transmission electron microscopy validated the effects of arecoline and 002 on fibroblast autophagy. Western blotting was used to explore the signaling pathways related to tiRNA-Val-CAC-002 in OSF.

Arecoline promotes fibroblast (FB) activation by upregulating tiRNA-Val-CAC-002. Arecoline stimulation and tiRNA-Val-CAC-002 overexpression activated fibroblasts by promoting autophagy. tiRNA-Val-CAC-002 regulates PI3K/AKT by mediating ITGB3 expression.

Arecoline upregulates tiRNA-Val-CAC-002 expression in fibroblasts. Moreover, tiRNA-Val-CAC-002 may activate the autophagy of fibroblasts in OSF by ITGB3/PI3K/AKT pathway regulation, promoting the expression of collagen fibers.

The calcium-activated phosphatase PPP3/calcineurin dephosphorylates TFEB (transcription factor EB) to trigger its nuclear translocation and the activation of macroautophagic/autophagic targets. However, the detailed molecular mechanism regulating TFEB activation remains poorly understood. Here, we highlighted the importance of SMURF1 (SMAD specific E3 ubiquitin protein ligase 1) in the activation of TFEB for lysosomal homeostasis. SMURF1 deficiency prevents the calcium-triggered ubiquitination of the catalytic subunit of PPP3/calcineurin in a manner consistent with defective autophagic degradation of damaged lysosomes. Mechanically, PPP3CB/CNA2 plays a bridging role in the recruitment of SMURF1 by LGALS3 (galectin 3) upon lysosome damage. Importantly, PPP3CB increases the dissociation of the N-terminal tail (NT) and C-terminal carbohydrate-recognition domain (CRD) of LGALS3, which may promote the formation of open conformers in a PPP3CB dephosphorylation activity-dependent manner. In addition, PPP3CB is ubiquitinated at lysine 146 by the recruited SMURF1 in response to intracellular calcium stimulation. The K63-linked ubiquitination of PPP3CB enhances the recruitment of TFEB. Moreover, TFEB directly interacts with both PPP3CB and the regulatory subunit PPP3R1 which facilitate the conformational correction of TFEB for its activation for the transcription of TFEB-targeted genes. Altogether, our results highlighted a critical mechanism for the regulation of PPP3/calcineurin activity via its ubiquitin ligase SMURF1 in response to lysosomal membrane damage, which may account for a potential target for the treatment of stress-related diseases.Abbreviation AID: autoinhibitory domain; ATG: autophagy related; CD: catalytic domain; CRD: carbohydrate-recognition domain; CsA: cyclosporin A; DMSO: dimethyl sulfoxide; ESCRT: endosomal sorting complexes required for transport; GSK3B: glycogen synthase kinase 3 beta; LAMP1: lysosomal associated membrane protein 1; LGALS3: galectin 3; LLOMe: L-leucyl-L-leucine methyl ester; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; ML-SA1: mucolipin synthetic agonist 1; MTORC1: mechanistic target of rapamycin kinase complex 1; NT: N-terminal tail; PPP3CB: protein phosphatase 3 catalytic subunit beta; PPP3R1: protein phosphatase 3 regulatory subunit B, alpha; SMURF1: SMAD specific E3 ubiquitin protein ligase 1; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; VCP/p97: valosin containing protein; YWHA/14-3-3: tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein.

Iron accumulation and mitochondrial dysfunction in astroglia are reported in Parkinson's disease (PD). Astroglia control iron availability in neurons in which dopamine (DA) synthesis is affected in PD. Despite their intimate relationship the role of DA in astroglial iron homeostasis is limited. Here we show that DA degrades iron storage protein ferritin in astroglial cells involving lysosomal proteolysis. Lysosomal ferritinophagy is mainly associated with macroautophagy; however, we revealed the involvement of chaperone-mediated autophagy (CMA) in DA-induced ferritin degradation. In CMA, cytosolic proteins containing a specific pentapeptide motif bind with HSC70 to be transported to lysosome mediated by LAMP2A. We identified the conserved pentapeptide motif in ferritin-H (Ft-H), mutations of which resulted loss of its interaction with HSC70. Pharmacological inhibitors of HSC70 or LAMP2/2A knockdown blocks DA-induced Ft-H degradation. DA also induces cytosolic cargo NCOA4 for ferritinophagy. We further reveal that DA promotes cathepsin B to lysis ferritin within the lysosome. Inhibitor of cathepsin B, knocking down of LAMP2, or HSC70 inhibitor attenuate DA-induced elevated mitochondrial iron level. Our results establish a direct role of DA on astroglial iron homeostasis and novel involvement of CMA in ferritin degradation in response to a biological stimulus. These results also may help in better understanding iron dyshomeostasis and mitochondrial dysfunction reported in PD.

Sirtuin 5 (SIRT5) in mitochondria possesses a strong capacity for lysine desuccinylation, involving in various biological processes. Our previous research demonstrated that NH3 regulated autophagy dependent on SIRT5 in bovine mammary epithelial cells (bMECs). Interestingly, we discovered that SIRT5 reduced the content of NH3 and glutamate by inhibiting GLS activity in bMECs, the ratio of ADP/ATP also declined. In this study, we identified that SIRT5 interacted with endogenous GLS and GDH through Co-IP assay, but had no effect on endogenous GLS and GDH expression. SIRT5 made the succinylation levels of GLS and GDH significantly declined and resulted in the reduction of GLS and GDH activity. Next, the content of ammonia and glutamate, as well as the related autophagy markers were measured, we found that SIRT5 affected the glutamine metabolism, which attenuated ammonia release in MAC-T cells, accompanying with cellular autophagy decline, reducing the formation of autophagosome. Deletion of SIRT5 gene in MAC-T cells by means of CRISPR-cas9, we found the content of NH3 and glutamate increased, as well as autophagy promoted, which could be alleviated by SIRT5 overexpression. SIRT5 KO also resulted in increase of succinylation of GLS and GDH, as well as autophagy response in bMECs. Furthermore, SIRT5 promoted the maintenance of mitochondria homeostasis. Mechanistically, SIRT5 reduced ammonia release by modulating the succinylation levels and enzymatic activities of GLS and GDH in mitochondria and promoted the maintenance of mitochondria homeostasis, as well as further attenuated ammonia-stimulated autophagy in bovine mammary epithelial cells.

Neurofibromatosis type 1 (NF1) is a genetic disorder caused by mutation of the NF1 gene that is associated with various symptoms, including the formation of benign tumors, called neurofibromas, within nerves. Drug treatments are currently limited. The mitogen-activated protein kinase kinase (MEK) inhibitor selumetinib is used for a subset of plexiform neurofibromas (PNs) but is not always effective and can cause side effects. Therefore, there is a clear need to discover new drugs to target NF1-deficient tumor cells. Using a Drosophila cell model of NF1, we performed synthetic lethal screens to identify novel drug targets. We identified 54 gene candidates, which were validated with variable dose analysis as a secondary screen. Pathways associated with five candidates could be targeted using existing drugs. Among these, chloroquine (CQ) and bafilomycin A1, known to target the autophagy pathway, showed the greatest potential for selectively killing NF1-deficient Drosophila cells. When further investigating autophagy-related genes, we found that 14 out of 30 genes tested had a synthetic lethal interaction with NF1. These 14 genes are involved in multiple aspects of the autophagy pathway and can be targeted with additional drugs that mediate the autophagy pathway, although CQ was the most effective. The lethal effect of autophagy inhibitors was conserved in a panel of human NF1-deficient Schwann cell lines, highlighting their translational potential. The effect of CQ was also conserved in a Drosophila NF1 in vivo model and in a xenografted NF1-deficient tumor cell line grown in mice, with CQ treatment resulting in a more significant reduction in tumor growth than selumetinib treatment. Furthermore, combined treatment with CQ and selumetinib resulted in a further reduction in NF1-deficient cell viability. In conclusion, NF1-deficient cells are vulnerable to disruption of the autophagy pathway. This pathway represents a promising target for the treatment of NF1-associated tumors, and we identified CQ as a candidate drug for the treatment of NF1 tumors.

Autophagy is a ubiquitous process of organelle interaction in eukaryotic cells, in which various organelles or proteins are recycled and operated through the autophagy pathway to ensure nutrient and energy homeostasis. Although numerous fluorescent probes have been developed to image autophagy, these environment-responsive probes suffer from inherent deficiencies such as inaccuracy and limited versatility. Here, we present a modular macrocyclic amphiphile Förster Resonance Energy Transfer (FRET) platform (SC6A12C/NCM, SN), constructed through the amphiphilic assembly of sulfonatocalix[6]arene (SC6A12C) with N-cetylmorpholine (NCM) for lysosome targeting. The hydrophobic fluorophore BPEA (FRET donor) was entrapped within the inner hydrophobic phase and showed strong fluorescence emission. Attributed to the broad-spectrum encapsulation of SC6A12C, three commercially available organelle probes (Mito-Tracker Red, ER Tracker Red, and RhoNox-1) were selected as SC6A12C guests (FRET acceptors). During autophagy process, the formation of intracellular host-guest complexes leads to strong FRET signal, allowing us to visualize the fusion of mitochondria, endoplasmic reticulum, and Golgi apparatus with lysosomes, respectively. This study provides a versatile and accessible platform for imaging organelle autophagy.