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Autophagy is a dynamic intracellular recycling process where cargo of interest is sequestered into double membrane vesicles and degraded down to its basic building blocks. These building blocks are then reused by the cells as nutrients and/or energy to proliferate and promote cell survival. Dysregulation of autophagy is highly implicated in many diseases such as cancer, neurodegeneration, and metabolic disorders, and for this reason, small molecules that modulate the autophagy pathway are promising therapeutics. In the realm of small molecule characterization aimed at identifying favorable candidates, the conventional techniques employed to assess autophagy protein levels after drug administration exhibit limitations in their capacity to quantitatively measure protein expression with high temporal resolution. Furthermore, these methods do not allow for the distinct measurement of rates pertaining to various stages of the pathway. Consequently, crucial information about the time-dependent impact on specific pathway steps are lost, making it challenging to discern the mechanism of action of the small molecule of interest. Through the utilization of a dynamic live-cell fluorescent imaging protocol as developed by Beesabathuni et al. (2022) [1], we conducted quantitative and temporal analyses of two distinct small molecules, each in a different biological context. Specifically, we first employed this methodology to assess the inhibitory effects of an ULK-1 (unc-51 like autophagy initiating kinase 1) inhibitor, MRT-68921, on the autophagy response, unraveling novel insights. Through our approach of quantitative and temporalmeasurements, we discovered that MRT-68921 exhibited a drastically reduced degradation rate of cargo—3 times less over a 12-hour period compared to negative control cells. Furthermore, in the presence MRT-68921 coupled with mTORC-1 (mammalian target of rapamycin 1)-inhibition, a stimulus known to induce autophagy, the treatment led to an 8X decrease in cargo degradation compared to the negative control. To facilitate the identification of morphological features beyond vesicle numbers that exhibited significant temporal variance, we used imagebased profiling tools as developed by Beesabathuni et al. (2023)[2]. Through this approach, we discovered intriguing phenotypes such as increased cellular and autophagosome areas postMRT-68921 treatment. Furthermore, by employing dimensionality reduction techniques on extracted features, we successfully clustered treatment conditions that exhibited similar cargo degradation rates. This strategy not only extends to a broader spectrum of small molecules but also serves as a foundation for a compiling a robust training dataset, to drive the prediction of cargo degradation for uncharacterized small molecules relative to those experimentally resolved within the training set.In the second context, by collaborating with the Chang-il Hwang lab at UCD, we quantified the autophagy rates following treatment with the small molecule BET inhibitor, JQ1 to quantitatively investigate the role of the pathway in facilitating synthetic lethality in Brca2 deficient murine PDAC cells [3]. Through the resulting rate measurements, we depicted that BRCA2-deficient PDAC cells exhibited a heightened basal autophagy flux compared to control PDAC cells, which was further augmented by JQ1 treatment.Ultimately, these insightful revelations hold great potential in screening small molecules to precisely modulate the autophagy response. By integrating these findings with experimental tools like biosensor-based target activity tracking and proteomic profiling of cargo contents, we can dive deeper into unraveling the intricate mechanisms that underlie the dynamic autophagy response. Additionally, we can comprehend the consequences of these behaviors in both assisting the disease state and identifying potential treatments for such states.

Here we discuss the developmental and homeostatic conditions necessary for Hydra regeneration. Hydra is characterized by populations of adult stem cells paused in the G2 phase of the cell cycle, ready to respond to injury signals. The body column can be compared to a blastema-like structure, populated with multifunctional epithelial stem cells that show low sensitivity to proapoptotic signals, and high inducibility of autophagy that promotes resistance to stress and starvation. Intact Hydra polyps also exhibit a dynamic patterning along the oral-aboral axis under the control of homeostatic organizers whose activity results from regulatory loops between activators and inhibitors. As in bilaterians, injury triggers the immediate production of reactive oxygen species (ROS) signals that promote wound healing and contribute to the reactivation of developmental programs via cell death and the de novo formation of new organizing centers from somatic tissues. In aging Hydra, regeneration is rapidly lost as homeostatic conditions are no longer pro-regenerative.

Periodontitis is an oral chronic inflammatory disease. Inhibiting tissue destruction and promoting tissue regeneration are important means for the treatment of periodontitis. Metformin not only has hypoglycemic effect but also has anti-inflammatory effect. Metformin has been shown to inhibit oxidative stress and activate autophagy through AMPK/mTOR pathway. High mobility group box 1 (HMGB1) has been implicated in the pathogenesis of many inflammatory diseases including periodontitis, it can participate in the induction of oxidative stress. HMGB1 is an autophagy regulator under oxidative stress, which can activate mTOR pathway. However, it is not clear whether metformin is related to HMGB1 and its mechanism in the process of periodontitis. Cell viability and expression of inflammatory cytokines were clarified by Cell Counting Kit-8, real-time PCR and enzyme-linked immunosorbent assay. Western blot and immunofluorescence were conducted to determine HMGB1 intracellular localization and expression of autophagy-associated proteins in vitro. Experimental periodontitis mice model was induced by administering a ligature. Immunohistochemistry was performed to detect the expression and localization of HMGB1 in vivo. The results of CCK-8, real-time PCR, enzyme-linked immunosorbent assay, Western blot and immunofluorescence showed lipopolysaccharide (LPS) treatment inhibited cell viability, and increased HMGB1 expression at a dose-independent manner. Metformin can reduce the effect of LPS. It also improves autophagy pathway inhibited by LPS and down-regulates mTOR expression. In addition, metformin attenuated alveolar bone resorption induced by ligation. This study provides new evidence for that metformin is a potential drug for the treatment of periodontitis and HMGB1 may be a potential target for periodontal intervention.

ObjectivesDetermine the role of apolipoprotein M (ApoM) in anthracycline (Dox) cardiotoxicity.BackgroundApoM binds the cardioprotective sphingolipid sphingosine-1-phosphate (S1P). Circulating ApoM is inversely associated with mortality in human heart failure (HF).MethodsIn the Penn HF Study (PHFS), we tested the relationship between ApoM and mortality in a subset with anthracycline-induced cardiomyopathy. We measured ApoM in humans and mice treated with Dox and utilized hepatic ApoM transgenic (ApomTG), ApoM knockout (ApomKO), ApoM knock-in mice with impaired S1P binding, and S1P receptor 3 (S1PR3) knockout mice in Dox cardiotoxicity. We assayed autophagy in left ventricular tissue from anthracycline-induced HF patients versus donor controls.ResultsApoM was inversely associated with mortality in PHFS, and Dox reduced circulating ApoM in mice and breast cancer patients.ApomTGmice were protected from Dox-induced cardiac dysfunction and loss of left ventricular mass.ApomTGattenuated Dox-induced impairment in autophagic flux in vivo and accumulation of insoluble p62, which was also observed in the myocardium of patients with anthracycline-induced HF. In vehicle-treated mice, ApoM negatively regulated transcription factor EB (TFEB), a master regulator of autophagy and lysosomal biogenesis. The effect of ApoM on TFEB required both S1P binding and S1PR3. In the presence of Dox, ApoM preserved TFEB and cardiomyocyte lysosomal abundance assessed as lysosomal associated membrane protein 1 positive structures in vivo, while S1P mimetic pretreatment of cardiomyocytes prevented Dox-induced changes in lysosomal pH.ConclusionsApoM attenuates Dox cardiotoxicity via the autophagy-lysosome pathway. The association between ApoM and reduced mortality may be explained by its role in sustaining autophagy.HighlightsCirculating ApoM is inversely associated with survival in human anthracycline-induced cardiomyopathyAnthracycline treatment reduces circulating ApoM in humans and miceIncreasing ApoM attenuates doxorubicin cardiotoxicity, lysosomal injury and preserves myocardial autophagic flux, but does not impact doxorubicin anti-neoplastic efficacyAutophagic impairment is characteristic of human anthracycline cardiomyopathy

Loss of proteostasis and failure to effectively degrade cellular waste through the lysosome are common features among neurodegenerative diseases, including Alzheimer’s disease (AD) and Huntington’s disease (HD). Dysregulation of autophagy, the process of trafficking waste proteins to the lysosome for degradation, may lead to accumulation of toxic misfolded waste proteins and ultimately contribute to cell death. A single genetic mutation, a CAG repeat expansion within the HD gene, causes HD. Late-onset AD is sporadic and polygenic, but the APOE4 allele has emerged as the strongest genetic risk factor. Understanding what role wild-type HTT and APOE may play in autophagy pathways and how autophagy in turn contributes to clearance of these proteins may lead to a clearer understanding of disease pathogenesis and possibly opportunities for therapeutic intervention. This thesis describes my findings that APOE is degraded by autophagy, and that autophagic trafficking of APOE4 is increased in cellular models, causing lysosomal swelling and reduced overall autophagic flux. Knockout or knockdown of HTT modulates the contents of lysosomes, stimulating the lysosomal degradation of some autophagic substrates including APOE in hepatic cells. I investigated whether the combination of aberrant lysosomal trafficking of APOE4 with lysosomal trafficking induced by reduced HTT levels may cause toxicity in vivo. Preliminary studies of peripheral knockdown of HTT in APOE4-expressing mice have revealed toxicity in liver tissue, a major source of APOE expression. Ongoing and future studies of the interaction between these two neurodegenerative disease-associated proteins, and their function in and degradation by autophagy, may have implications for possible therapeutic strategies for HD involving peripheral total-HTT lowering, and for development of disease-modifying strategies for AD.

Mouse molars resemble human teeth, as their roots develop and stop growing. In contrast, mouse incisors grow and erupt continuously due to the presence of a persistent stem cell niche identified as the labial cervical loop, which actively gives rise to dentin-forming odontoblasts to support tooth homeostasis and injury repair. Autophagy (ATG), a cellular degradation process whereby macromolecules and organelles are sequestered to be recycled, has been shown to play a role in mineralized tissue-forming cells including odontoblasts. However, the precise role of ATG in odontoblasts in vivo remains unclear. Beclin1 is an indispensable ATG-related protein involved in ATG initiation. Therefore, we conditionally deleted Beclin1 in odontoblast-specific manner and generated Becn1f/f;Dmp1Cre/+ (Becn1 cKO) mice. From these mice, molars and incisors were evaluated radiographically and histologically. MicroCT scanning was employed to observe dentin formation and measure pulp tissue volume, histological analysis to examine dentin formation and dental pulp cells, and RNAscope in situ hybridization to identify odontoblast differentiation in the incisor stem cell niche and mesenchymal tissue in molars. We further evaluated the mRNA expression of FGF signaling genes in human dental pulp stem cells in vitro using Becn1 knocked down dental pulp cells (DPCs). Becn1 cKO mice were born at the expected Mendalian ratio. However, Becn1 cKO mice at the 6th month mark exhibited increased obliteration of the pulp chamber in both molar and incisors. Interestingly, both Becn1 wildtype (WT) and Becn1 ckO mice a normal laCL histology. When Becn1 was knocked down in DPCs, Etv4, a downstream target gene of FGF signaling pathway, was significantly upregulated. Our study highlights the possible role of Beclin1 in pulp calcification of molars, as Beclin1 KO mice molars were characterized by a completely obliteration and calcification of the pulp space, and suggests that Beclin1 may play a role in developed teeth, but not in developing teeth.

It is necessary to develop a new strategy for treatment of lung cancer since it is the main cause of cancer death. Tanshinone IIA (Tan IIA), an active ingredient of a commonly used traditional Chinese herb Salvia miltiorrhiza, provides a new direction to develop a new strategy for the treatment. It has been found that Tan IIA could inhibit lung cancer in vitro and in vivo by inducing autophagic apoptosis. Tan IIA increased apoptotic cells and the expression of cleaved caspase 3 and cleaved caspase 9, decreased B-cell lymphoma-2 (Bcl-2)/Bcl-2 associated X protein (Bax) ratio in human non-small cell lung cancer (NSCLC) cell lines, which was promoted by an autophagy activator Rapamycin, and weaken by autophagy inhibitor 3-methyladenine (3-MA). In addition, Tan IIA induced more autophagosomes, up-regulated light chain 3β (LC-3B) I and LC-3B II conversion and less sequestosome 1 (SQSTM1/p62) expression, which cannot be weakened by the caspase 3 antagonist. Moreover, overexpression of LC-3B gene (LC3B) and downregulation of autophagic gene 5 (ATG5) further confirmed that Tan IIA induced autophagic apoptosis in NSCLC cell lines. Beclin-1 gene (BECN1) overexpression and silence attenuated the effects of Tan IIA, suggesting autophagic apoptosis that Tan IIA induced was dependent on Beclin-1. Overall, our study demonstrated a new treatment mechanism of Tan IIA and suggested that Tan IIA is a potential new anti-cancer therapeutic option.

The Golgi reassembly stacking proteins (GRASPs) have fascinated the scientific community due to their myriad functions throughout the cell. While originally identified as required factors for in vitro stacking of Golgi cisternae,1,2 GRASPs have also been implicated in autophagy and linked to the ER-Golgi interface, although the underlying mechanisms and biological significance have not been fully delineated. To better understand mammalian GRASP55, I leveraged genetic inhibition of GRASP55 and found that loss of GRASP55 was associated with both increased early-stage autophagy and impaired late-stage autophagy, suggesting that GRASP55 both inhibits and promotes autophagy at different steps. I also found that loss of GRASP55 phenocopied the effects of starvation on increasing colocalization of early secretory markers, suggesting that GRASP55 negatively regulates coalescence of the early secretory pathway. Interestingly, GRASP55 was not degraded during starvation, but the presence of GRASP55 dimers was enhanced by starvation, raising the possibility that the dimerization status of GRASP55 directs its functions in autophagy and secretory marker colocalization during starvation. Proximity-dependent biotinylation combined with mass spectrometry led us to identify a GRASP55 proximal interactome that was enriched in ER-Golgi interface proteins. Overall, these findings shed light on the intersections between GRASP55, autophagy, and the ER-Golgi interface.

Lichens, algae and fungi-based symbiotic associations, are sources of many important secondary metabolites, such as antibiotics, anti-inflammatory, antioxidants, and anticancer agents. Wide range of experiments based on in vivo and in vitro studies revealed that lichens are a rich treasure of anti-cancer compounds. Lichen extracts and isolated lichen compounds can interact with all biological entities currently identified to be responsible for tumor development. The critical ways to control the cancer development include induction of cell cycle arrests, blocking communication of growth factors, activation of anti-tumor immunity, inhibition of tumor-friendly inflammation, inhibition of tumor metastasis, and suppressing chromosome dysfunction. Also, lichen-based compounds induce the killing of cells by the process of apoptosis, autophagy, and necrosis, that inturn positively modulates metabolic networks of cells against uncontrolled cell division. Many lichen-based compounds have proven to possess potential anti-cancer activity against a wide range of cancer cells, either alone or in conjunction with other anti-cancer compounds. This review primarily emphasizes on an updated account of the repository of secondary metabolites reported in lichens. Besides, we discuss the anti-cancer potential and possible mechanism of the most frequently reported secondary metabolites derived from lichens.

Although the classic activities of p53 including induction of cell-cycle arrest, senescence, and apoptosis are well accepted as critical barriers to cancer development, accumulating evidence suggests that loss of these classic activities is not sufficient to abrogate the tumor suppression activity of p53. Numerous studies suggest that metabolic regulation contributes to tumor suppression, but the mechanisms by which it does so are not completely understood. Cancer cells rewire cellular metabolism to meet the energetic and substrate demands of tumor development. It is well established that p53 suppresses glycolysis and promotes mitochondrial oxidative phosphorylation through a number of downstream targets against the Warburg effect. The role of p53-mediated metabolic regulation in tumor suppression is complexed by its function to promote both cell survival and cell death under different physiological settings. Indeed, p53 can regulate both pro-oxidant and antioxidant target genes for complete opposite effects. In this review, we will summarize the roles of p53 in the regulation of glucose, lipid, amino acid, nucleotide, iron metabolism, and ROS production. We will highlight the mechanisms underlying p53-mediated ferroptosis, AKT/mTOR signaling as well as autophagy and discuss the complexity of p53-metabolic regulation in tumor development.

Noncoding RNAs (ncRNAs) regulation of various diseases including cancer has been extensively studied. Reactive oxidative species (ROS) elevated by oxidative stress are associated with cancer progression and drug resistance, while autophagy serves as an ROS scavenger in cancer cells. However, the regulatory effects of ncRNAs on autophagy and ROS in various cancer cells remains complex. Here, we explore how currently investigated ncRNAs, mainly miRNAs and lncRNAs, are involved in ROS production through modulating antioxidant genes. The regulatory effects of miRNAs and lncRNAs on autophagy-related (ATG) proteins to control autophagy activity in cancer cells are discussed. Moreover, differential expression of ncRNAs in tumor and normal tissues of cancer patients are further analyzed using The Cancer Genome Atlas (TCGA) database. This review hypothesizes links between ATG genes- or antioxidant genes-modulated ncRNAs and ROS production, which might result in tumorigenesis, malignancy, and cancer recurrence. A better understanding of the regulation of ROS and autophagy by ncRNAs might advance the use of ncRNAs as diagnostic and prognostic markers as well as therapeutic targets in cancer therapy.

Tumour-promoting inflammation is a critical hallmark in cancer development, and inflammasomes are well-known regulators of inflammatory processes within the tumour microenvironment. Different inflammasome components along with the adaptor, apoptosis-associated speck-like protein containing caspase activation and recruitment domain (ASC), and the effector, caspase-1, have a significant influence on tumorigenesis but in a tissue-specific and stage-dependent manner. The downstream products of inflammasome activation, that is the proinflammatory cytokines such as IL-1β and IL-18, regulate tissue homeostasis and induce antitumour immune responses, but in contrast, they can also favour cancer growth and proliferation by directing various oncogenic signalling pathways in cancer cells. Moreover, different epigenetic mechanisms, including DNA methylation, histone modification and noncoding RNAs, control inflammasomes and their components by regulating gene expression during cancer progression. Furthermore, autophagy, a master controller of cellular homeostasis, targets inflammasome-induced carcinogenesis by maintaining cellular homeostasis and removing potential cancer risk factors that promote inflammasome activation in support of tumorigenesis. Here, in this review, we summarize the effect of inflammasome activation in cancers and discuss the role of epigenetic and autophagic regulatory mechanisms in controlling inflammasomes. A proper understanding of the interactions among these key processes will be useful for developing novel therapeutic regimens for targeting inflammasomes in cancer.

Hand, foot and mouth disease (HFMD) is a serious threat to the health of infants, which can be caused by enterovirus 71 (EV71). The clinical symptoms are mostly self-limited, but some of them develop into aseptic meningitis with poor prognosis and even death. In this study, we screened Urolithin A (UroA), an intestinal metabolite of ellagic acid, significantly inhibited the replication of EV71 in cells. Further evaluation showed that UroA was better than ribavirin in CC50, IC50 and selection index (SI). Moreover, we found that UroA inhibits the proliferation of EV71 by promoting autophagy and apoptosis of infected cells. Therefore, UroA is a candidate drug for the treatment of EV71 infection.

Background: The most prevalent malignant tumor in women is breast cancer (BC). As autophagic therapies have been identified to contribute to BC cell death, the potential prognostic role of long non-coding RNA (lncRNA) related to autophagy in patients with BC was examined. Methods: The lncRNAs expression profiles were derived from The Cancer Genome Atlas (TCGA) database. Throughout univariate Cox regression and multivariate Cox regression test, lncRNA with BC prognosis have been differentially presented. We then defined the optimal cut-off point between high and low-risk groups. The receiver operating characteristic (ROC) curves were drawn to test this signature. In order to examine possible signaling mechanisms linked to these lncRNAs, the Gene Set Enrichment Analysis (GSEA) has been carried out. Results: Based on the lncRNA expression profiles for BC, a 9 lncRNA signature associated with autophagy was developed. The optimal cut-off value for high-risk and low-risk groups was used. The high-risk group had less survival time than the low-risk group. The result of this lncRNA signature was highly sensitive and precise. GSEA study found that the gene sets have been greatly enriched in many cancer pathways. Conclusions: Our signature of 9 lncRNAs related to autophagy has prognostic value for BC, and these lncRNAs related to autophagy may play an important role in BC biology.

The high mortality in larval-to-juvenile transition is a big problem in aquaculture which is related to the shifting from endogenous to exogenous feeding. However, the underlying causes remain poorly understood. Autophagy, an evolutionary regulated cellular mechanism, is highly conserved in eukaryotic organisms to maintain energy homeostasis against stress including starvation. To investigate whether autophagy plays a role during larval-to-juvenile transition, we generated atg7 and beclin1 zebrafish mutant lines using CRISPR/Cas9 gene editing technology. In this study, both atg7 and beclin1 null zebrafish died during larval-to-juvenile transition because atg7 and beclin1 mutants were unable to cope with the metabolic stress after yolk absorption and fail to activate autophagy in response to nutrient restriction. Meanwhile, dramatic defects in the intestine architecture and metabolic functions in the liver of these mutants were observed even though refeeding them. Treatment with rapamycin, an activator of autophagy, could effectively extend the survival time of both atg7 and beclin1 null zebrafish through decreasing the metabolic rate while it couldn't activate autophagy in mutants via the canonical pathway. Thus, our results revealed that autophagy played a crucial role in zebrafish ontogeny during larval-to-juvenile transition, and it could be considered as one of the most important endogenous factors judging the survival rate of the developing embryos during this period.

Activating transcription factors, ATFs, are a group of bZIP transcription factors that act as homodimers or heterodimers with a range of other bZIP factors. In general, ATFs respond to extracellular signals, indicating their important roles in maintaining homeostasis. The ATF family includes ATF1, ATF2, ATF3, ATF4, ATF5, ATF6, and ATF7. Consistent with the diversity of cellular processes reported to be regulated by ATFs, the functions of ATF are also diverse. ATFs play an important role in cell proliferation, apoptosis, differentiation and inflammation-related pathological processes. The expression and phosphorylation status of ATFs are also related to neurodegenerative diseases and polycystic kidney disease. Various miRNAs target ATF to regulate cancer proliferation, apoptosis, autophagy, sensitivity and resistance to radiotherapy and chemotherapy. Moreover, ATFs are necessary to maintain cell redox homeostasis. Therefore, deepening our understanding of the regulation and function of ATFs will provide insights into the basic regulatory mechanisms that influence how cells integrate extracellular and intracellular signals into genomic responses through transcription factors. Under pathological conditions, especially in cancer biology and response to treatment, the characterization of ATF dysfunction is important for understanding how to therapeutically utilize ATF2 or other pathways controlled by transcription factors. In this review, we will demonstrate how ATF1, ATF2, ATF3, ATF4, ATF5, ATF6, and ATF7 function in promoting or suppressing cancer development and identify their roles in tumour immunotherapy.