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Mitophagy degrades damaged mitochondria, but we show here that it can also target functional mitochondria. This latter scenario occurs during programmed mitophagy and involves the mitophagy receptors NIX and BNIP3. Although AMP-activated protein kinase (AMPK), the energy-sensing protein kinase, can influence damaged-induced mitophagy, its role in programmed mitophagy is unclear. We found that AMPK directly inhibits NIX-dependent mitophagy by triggering 14-3-3-mediated sequestration of ULK1, via ULK1 phosphorylation at two sites: Ser556 and an additional identified site, Ser694. By contrast, AMPK activation increases Parkin phosphorylation and enhances the rate of depolarization-induced mitophagy, independently of ULK1. We show that this happens both in cultured cells and tissues in vivo , using the mito -QC mouse model. Our work unveils a mechanism whereby AMPK activation downregulates mitophagy of functional mitochondria but enhances that of dysfunctional/damaged ones. [Display omitted] • AMPK activation blocks NIX-dependent mitophagy • AMPK phosphorylates ULK1 to trigger inhibitory 14-3-3 binding • NIX-dependent mitophagy can target functioning mitochondria • Parkin-dependent mitophagy is enhanced by AMPK independently of ULK1 Under energetic stress, it is essential to balance dysfunctional versus healthy mitochondrial turnover. Longo and Bishnu et al. uncover a mechanism by which AMPK activation achieves this. They find that although AMPK enhances Parkin-mediated mitophagy upon mitochondrial damage, it blocks NIX-mediated mitophagy, which they now show can target functioning mitochondria. [ABSTRACT FROM AUTHOR]

BACKGROUND: Studies have shown that short-chain fatty acids (SCFAs) are a potential regulator of skeletal muscle energy metabolism, but the exact mechanism is unclear. OBJECTIVE: To observe the effect of SCFAs on aged rats with sarcopenia and to explore the underlying mechanism. METHODS: Sprague-Dawley rats were randomly divided into control group, sarcopenia group, and sarcopenia + SCFAs group (SCFAs group). In the latter two groups, rat models of sarcopenia were established using ovariectomized rats injected with 5 mg/kg dexamethasone for 7 days. In the control group, the abdominal cavity was only exposed but not removed, and then sutured. Rats in the SCFAs group were administered drinking water containing 150 mmol/L short-chain fatty acids, 600 mg/kg sodium acetate, 200 mg/kg sodium propionate, and 200 mg/kg sodium butyrate for 4 weeks. Rats in the control and sarcopenia groups were given the same volume of normal saline. Successful modeling was assessed by measuring the bilateral gastrocnemius muscle mass and body mass to calculate the gastrocnemius index after modeling. Food intake, body mass and grip strength of rats were measured at 0, 1, 2 and 4 weeks after successful modeling; morphological changes of gastrocnemius muscle were observed by hematoxylin-eosin staining; and the expression of p-AMPK and p-ULK1 proteins in gastrocnemius muscle was detected by western blot. RESULTS AND CONCLUSION: Compared with the control group, the sarcopenia group showed significantly decreased body mass, food intake, forelimb grip strength (P < 0.05), wet mass of gastrocnemius muscle (P < 0.05), and protein levels of p-AMPK and p-ULK1 (P < 0.05). Compared with the sarcopenia group, the SCFAs group showed a significant increase in food intake, body mass, grip strength, wet mass of gastrocnemius muscle, and protein levels of p-AMPK and p-ULK1 in gastrocnemius muscle (P < 0.05). All these findings indicate that SCFAs improve symptoms of sarcopenia in the elderly and may be associated with the upregulation of AMPK and ULK1 proteins in skeletal muscle. [ABSTRACT FROM AUTHOR]

Signal transducer and activator of transcription 1 (STAT1) plays a critical role in immune response, human STAT1 as a transcriptional suppressor of autophagy genes and autophagic activity. Classical swine fever virus (CSFV)-infected induce autophagy, leading to immune evasion. However, there are limited reports on the function of porcine STAT1 in autophagy during CSFV infection. There is also lack of suitable in vitro models for studying porcine STAT1. The objective of this study was to establish porcine PK-15 STAT1-/- and 3D4/21 STAT1-/- cell lines using the CRISPR/Cas9 system to investigate the function of the STAT1 in autophagy. The PK-15STAT1-/- and 3D4/21STAT1-/- cell lines, featuring homozygous knockout of STAT1 gene were successfully constructed using the CRISPR/Cas9 editing system. The knockout efficiency determined to be 82.4% and 81.1%, respectively. Infection with CSFV in porcine PK-15STAT1-/- and 3D4/21STAT1-/- cells led to an observable increase in autophagosomes as evidenced by transmission electron microscope. Additionally, STAT1 knockout (STAT1-/-) by the CRISPR/Cas9 system upregulated the expression of ULK1, Beclin1, and LC3 genes, thereby enhancing autophagy during CSFV infection. Conversely, overexpression of STAT1 downregulated the expression of ULK1, Beclin1, and LC3 genes, leading to inhibition of autophagy during CSFV infection.The application of an autophagy dual-fluorescent-tracking plasmid demonstrated that STAT1 knockout enhanced autophagy accumulation during CSFV infection, while STAT1 overexpression inhibited it. Moreover, the 3D4/21STAT1-/- cell line proved to be a more suitable in vitro model compared to the PK-15STAT1-/- cell line for elucidating the involvement of STAT1 in autophagy during CSFV infection. [ABSTRACT FROM AUTHOR]

Ischemia reperfusion (I/R) was considered as one of main causes of acute kidney injury (AKI). However, the exact mechanism remains unclear. Here, this study aimed to investigate the role and mechanism of the m6A demethylase fat mass and obesity‐associated (FTO) protein in I/R‐induced AKI. HK‐2 cells and SD rats were utilized to establish hypoxia/reoxygenation (H/R) or I/R induced AKI models. The changes of RNAs and proteins were quantified using RT‐qPCR, western blot, and immunofluorescence assays, respectively. Cell proliferation and apoptosis were assessed by CCK‐8 and flow cytometry. Interactions between molecules were investigated using RIP, ChIP, Co‐IP, RNA pull‐down, and dual luciferase reporter assays. Global m6A quantification was evaluated by kits. TUNEL and HE staining were employed for histopathological examinations. Oxidative stress‐related indicators and renal function were determined using ELISA assays. The FTO expression was downregulated in H/R‐induced HK‐2 cells and renal tissues from I/R‐induced rats. Overexpression of FTO improved the cell viability but repressed apoptosis and oxidative stress in H/R‐treated HK‐2 cells, as well as enhanced renal function and alleviated kidney injury in I/R rats. Notably, the FTO overexpression significantly increased autophagy‐related LC3 and ULK1 levels. When autophagy was inhibited, the protective effects of FTO in AKI were diminished. Notably, Ambra1, a crucial regulator of autophagy, was repressed in H/R‐induced HK‐2 cells. However, the FTO overexpression restored the Ambra1 expression by reducing m6A modification of its mRNA. SP1, acting as an upstream transcription factor, directly interacts with the FTO promoter to enhance FTO expression. Knockdown of SP1 or Ambra1 suppressed the beneficial effects of FTO upregulation on autophagy and oxidative stress injury in H/R‐stimulated cells. FTO, transcriptionally activated by SP1, promoted autophagy by upregulating Ambra1/ULK1 signaling, thereby inhibiting oxidative stress and kidney injury. These findings may provide some novel insights for AKI treatment. [ABSTRACT FROM AUTHOR]

Background: Autophagy dysfunction in glial cells is implicated in the pathogenesis of Parkinson's disease (PD). The previous study reported that α-synuclein (α-Syn) disrupted autophagy in cultured microglia. However, the mechanism of microglial autophagy dysregulation is poorly understood. Methods: Two α-Syn-based PD models were generated via AAV-mediated α-Syn delivery into the mouse substantia nigra and striatal α-Syn preformed fibril (PFF) injection. The levels of microglial UNC-51-like kinase 1 (Ulk1) and other autophagy-related genes in vitro and in PD mice, as well as in the peripheral blood mononuclear cells of PD patients and healthy controls, were determined via quantitative PCR, western blotting and immunostaining. The regulatory effect of signal transducer and activator of transcription 1 (STAT1) on Ulk1 transcription was determined via a luciferase reporter assay and other biochemical studies and was verified through Stat1 knockdown or overexpression. The effect of α-Syn on glial STAT1 activation was assessed by immunohistochemistry and western blotting. Changes in microglial status, proinflammatory molecule expression and dopaminergic neuron loss in the nigrostriatum of PD and control mice following microglial Stat1 conditional knockout (cKO) or treatment with the ULK1 activator BL-918 were evaluated by immunostaining and western blotting. Motor behaviors were determined via open field tests, rotarod tests and balance beam crossing. Results: The transcription of microglial ULK1, a kinase that controls autophagy initiation, decreased in both in vitro and in vivo PD mouse models. STAT1 plays a critical role in suppressing Ulk1 transcription. Specifically, Stat1 overexpression downregulated Ulk1 transcription, while Stat1 knockdown increased ULK1 expression, along with an increase in LC3II and a decrease in the SQSTM1/p62 protein. α-Syn PFF caused toll-like receptor 4-dependent activation of STAT1 in microglia. Ablation of Stat1 alleviated the decrease in microglial ULK1 expression and disruption of autophagy caused by α-Syn PFF. Importantly, the ULK1 activator BL-918 and microglial Stat1 cKO attenuated neuroinflammation, dopaminergic neuronal damage and motor defects in PD models. Conclusions: These findings reveal a novel mechanism by which α-Syn impairs microglial autophagy and indicate that targeting STAT1 or ULK1 may be a therapeutic strategy for PD. [ABSTRACT FROM AUTHOR]

Autophagy, a highly conserved form of cellular recycling, is essential for cellular homeostasis. Its dysregulation has been linked to neurodegenerative diseases and various cancers, including breast cancer. We set out to determine if the RNA‐binding protein (RBP) YBX3 regulates autophagy mRNAs, as previous findings suggest YBX3 depletion reduces distinct autophagy transcripts. We found that YBX3 interacts with and stabilizes the mRNA of the autophagy initiation factor ATG13 in HeLa, which in turn increases ATG13 protein expression. We have shown that this requires the 3′ untranslated region (UTR) of ATG13 and occurs in other human cell lines, including HEK293, HepG2, and HCT116. Together, our data suggest a novel regulatory role for YBX3 of autophagy initiation through posttranscriptional control of ATG13 mRNA stability. [ABSTRACT FROM AUTHOR]

Breast cancer, the most prevalent and aggressive tumor affecting women, requires identification of disease determinants to facilitate the development of effective therapeutic strategies. Transient receptor potential vanilloid 2 (TRPV2), an ion channel highly permeable for calcium (Ca2+), is implicated in physiological and pathological processes. Nevertheless, the role of TRPV2 in breast cancer remains poorly elucidated. In this study, we found high levels of TRPV2 expression associated with advanced malignancy, thereby suggesting its potential as a biomarker for breast cancer staging. We demonstrated that TRPV2 activation promotes breast cancer cell proliferation, migration, and invasion, while silencing of TRPV2 suppresses breast cancer progression, highlighting the oncogenic role of TRPV2. Moreover, we reveal that TRPV2 facilitates cancer progression by modulating the CaMKKβ/AMPK/ULK1-autophagic axis through mediating calcium influx, providing new insights into TRPV2 as a novel therapeutic target for breast cancer treatment. [ABSTRACT FROM AUTHOR]

BACKGROUND: After peripheral facial nerve injury, glial cell-derived neurotrophic factor (GDNF) can play a protective role in facial neurons. It has been found that GDNF can regulate the level of autophagy through mammalian target of rapamycin (mTOR), but it is unclear whether it can regulate facial neurons through the adenylate-activated protein kinase/Unc-51-like kinase 1 (AMPK/ULK1) signaling pathway after facial nerve injury. OBJECTIVE: To establish a facial nerve injury model in Sprague-Dawley rats and explore the role of autophagy in facial nerve regeneration and the mechanism by which the GDNF/AMPK/ULK1 signaling pathway promotes facial nerve repair after injury. METHODS: Seventy-two Sprague-Dawley rats were randomly divided into sham group, model group and autophagy inhibitor 3-methyladenine (3-MA) group, with 24 rats in each group. Only the main trunk of the facial nerve was exposed in the sham group, while the remaining two groups were modeled for the compression injury of the facial nerve trunk. After successful modeling, the model group was given intraperitoneal injection of normal saline (15 mg/kg), and the 3-MA group was given intraperitoneal injection of 3-MA (15 mg/kg), both once daily for 7 days. The rats in each group were scored on the Simone 10-point behavioral scale at 1, 4, 7, 14, 21 and 28 days after surgery. Nissl staining was performed to observe the morphology and number of facial neuron cells at 7, 14, 21, and 28 days. The expression levels of p-AMPK, p-ULK1, Beclin1 and GDNF in the facial neuron tissues of rats were detected by western blot assay. RESULTS AND CONCLUSION: Behavioral scoring showed that the improvement of facial paralysis symptoms in the 3-MA group was worse and later than that in the model group (P < 0.05). Nissl staining showed that the morphology and number of Nissl bodies in facial neurons in the 3-MA group recovered poorly and the number was less than that in the model group (P < 0.05). Western blot detection results showed that the expression of p-AMPK and Beclin1 in the model group was higher than that in the 3-MA group and the sham group (P < 0.05). The protein expression of p-ULK1 in the model group was lower than that in the 3-MA group and the sham group (P < 0.05). To conclude, autophagy inhibitor delays nerve repair after facial nerve injury, which may be related to downregulation of GDNF expression, inactivation of AMPK, and phosphorylation of ULK1, thereby inhibiting neuronal autophagy levels. [ABSTRACT FROM AUTHOR]

The molecular mechanism underlying abnormal osteoclastogenesis triggering subchondral bone remodeling in osteoarthritis (OA) is still unclear. Here, single‐cell and bulk transcriptomics sequencing analyses are performed on GEO datasets to identify key molecules and validate them using knee joint tissues from OA patients and rat OA models. It is found that the catalytic subunit of protein phosphatase 2A (PP2Ac) is highly expressed during osteoclastogenesis in the early stage of OA and is correlated with autophagy. Knockdown or inhibition of PP2Ac weakened autophagy during osteoclastogenesis. Furthermore, the ULK1 expression of the downstream genes is significantly increased when PP2Ac is knocked down. PP2Ac‐mediated autophagy is dependent on ULK1 phosphorylation activity during osteoclastogenesis, which is associated with enhanced dephosphorylation of ULK1 Ser637 residue regulating at the post‐translational level. Additionally, mTORC1 inhibition facilitated the expression level of PP2Ac during osteoclastogenesis. In animal OA models, decreasing the expression of PP2Ac ameliorated early OA progression. The findings suggest that PP2Ac is also a promising therapeutic target in early OA. [ABSTRACT FROM AUTHOR]

Background: Deubiquitinating enzymes (DUBs) are pivotal in maintaining cell homeostasis by regulating substrate protein ubiquitination in both healthy and cancer cells. Ubiquitin-specific protease 10 (USP10) belongs to the DUB family. In this study, we investigated the clinical and pathological significance of USP10 and Unc-51-like autophagy activating kinase 1 (ULK1) in osteosarcoma (OS), as well as the mechanism of USP10 action in ULK1-mediated autophagy and disease progression. Results: The analysis of OS and adjacent normal tissues demonstrated that USP10 and ULK1 were significantly overexpressed in OS, and a positive association between their expression and malignant properties was observed. USP10 knockdown in OS cells reduced ULK1 mRNA and protein expression, whereas USP10 overexpression increased ULK1 mRNA and protein expression. In vitro experiments showed that USP10 induced autophagy, cell proliferation, and invasion by enhancing ULK1 expression in OS cell lines. Furthermore, we found that the regulation of ULK1-mediated autophagy, cell proliferation, and invasion in OS by USP10 was dependent on glycogen synthase kinase 3β (GSK3β) activity. Mechanistically, USP10 promoted ULK1 transcription by interacting with and stabilising GSK3β through deubiquitination, which, in turn, increased the activity of the ULK1 promoter, thereby accelerating OS progression. Using a xenograft mouse model, we showed that Spautin-1, a small-molecule inhibitor targeting USP10, significantly reduced OS development, with its anti-tumour activity significantly enhanced when combined with the chemotherapeutic agent cisplatin. Conclusion: Collectively, we demonstrated that the USP10-GSK3β-ULK1 axis promoted autophagy, cell proliferation, and invasion in OS. The findings imply that targeting USP10 may offer a promising therapeutic avenue for treating OS. [ABSTRACT FROM AUTHOR]

Abstract Background Autophagy dysfunction in glial cells is implicated in the pathogenesis of Parkinson’s disease (PD). The previous study reported that α-synuclein (α-Syn) disrupted autophagy in cultured microglia. However, the mechanism of microglial autophagy dysregulation is poorly understood. Methods Two α-Syn-based PD models were generated via AAV-mediated α-Syn delivery into the mouse substantia nigra and striatal α-Syn preformed fibril (PFF) injection. The levels of microglial UNC-51-like kinase 1 (Ulk1) and other autophagy-related genes in vitro and in PD mice, as well as in the peripheral blood mononuclear cells of PD patients and healthy controls, were determined via quantitative PCR, western blotting and immunostaining. The regulatory effect of signal transducer and activator of transcription 1 (STAT1) on Ulk1 transcription was determined via a luciferase reporter assay and other biochemical studies and was verified through Stat1 knockdown or overexpression. The effect of α-Syn on glial STAT1 activation was assessed by immunohistochemistry and western blotting. Changes in microglial status, proinflammatory molecule expression and dopaminergic neuron loss in the nigrostriatum of PD and control mice following microglial Stat1 conditional knockout (cKO) or treatment with the ULK1 activator BL-918 were evaluated by immunostaining and western blotting. Motor behaviors were determined via open field tests, rotarod tests and balance beam crossing. Results The transcription of microglial ULK1, a kinase that controls autophagy initiation, decreased in both in vitro and in vivo PD mouse models. STAT1 plays a critical role in suppressing Ulk1 transcription. Specifically, Stat1 overexpression downregulated Ulk1 transcription, while Stat1 knockdown increased ULK1 expression, along with an increase in LC3II and a decrease in the SQSTM1/p62 protein. α-Syn PFF caused toll-like receptor 4-dependent activation of STAT1 in microglia. Ablation of Stat1 alleviated the decrease in microglial ULK1 expression and disruption of autophagy caused by α-Syn PFF. Importantly, the ULK1 activator BL-918 and microglial Stat1 cKO attenuated neuroinflammation, dopaminergic neuronal damage and motor defects in PD models. Conclusions These findings reveal a novel mechanism by which α-Syn impairs microglial autophagy and indicate that targeting STAT1 or ULK1 may be a therapeutic strategy for PD.

Abstract Background Deubiquitinating enzymes (DUBs) are pivotal in maintaining cell homeostasis by regulating substrate protein ubiquitination in both healthy and cancer cells. Ubiquitin-specific protease 10 (USP10) belongs to the DUB family. In this study, we investigated the clinical and pathological significance of USP10 and Unc-51-like autophagy activating kinase 1 (ULK1) in osteosarcoma (OS), as well as the mechanism of USP10 action in ULK1-mediated autophagy and disease progression. Results The analysis of OS and adjacent normal tissues demonstrated that USP10 and ULK1 were significantly overexpressed in OS, and a positive association between their expression and malignant properties was observed. USP10 knockdown in OS cells reduced ULK1 mRNA and protein expression, whereas USP10 overexpression increased ULK1 mRNA and protein expression. In vitro experiments showed that USP10 induced autophagy, cell proliferation, and invasion by enhancing ULK1 expression in OS cell lines. Furthermore, we found that the regulation of ULK1-mediated autophagy, cell proliferation, and invasion in OS by USP10 was dependent on glycogen synthase kinase 3β (GSK3β) activity. Mechanistically, USP10 promoted ULK1 transcription by interacting with and stabilising GSK3β through deubiquitination, which, in turn, increased the activity of the ULK1 promoter, thereby accelerating OS progression. Using a xenograft mouse model, we showed that Spautin-1, a small-molecule inhibitor targeting USP10, significantly reduced OS development, with its anti-tumour activity significantly enhanced when combined with the chemotherapeutic agent cisplatin. Conclusion Collectively, we demonstrated that the USP10-GSK3β-ULK1 axis promoted autophagy, cell proliferation, and invasion in OS. The findings imply that targeting USP10 may offer a promising therapeutic avenue for treating OS.

The activation of hepatic stellate cells (HSCs) is central to the occurrence and development of liver fibrosis. Our previous studies showed that autophagy promotes HSC activation and ultimately accelerates liver fibrosis. Unc-51-like autophagy activating kinase 1 (ULK1) is an autophagic initiator in mammals, and N 6-methyladenosine (m 6A) modification is closely related to autophagy. In this study, we find that the m 6A demethylase fat mass and obesity-associated protein (FTO), which is the m 6A methylase with the most significant difference in expression, is upregulated during HSC activation and bile duct ligation (BDL)-induced hepatic fibrosis. Importantly, we identify that FTO overexpression aggravates HSC activation and hepatic fibrosis via autophagy. Mechanistically, compared with other autophagy-related genes, ULK1 is a target of FTO because FTO mainly mediates the m 6A demethylation of ULK1 and upregulates its expression, thereby enhancing autophagy and the activation of HSCs. Notably, the m 6A reader YTH domain-containing protein 2 (YTHDC2) decreases ULK1 mRNA level by recognizing the m 6A binding site and ultimately inhibiting autophagy and HSC activation. Taken together, our findings highlight m 6A-dependent ULK1 as an essential regulator of HSC autophagy and reveal that ULK1 is a novel potential therapeutic target for hepatic fibrosis treatment.

Abstract Background High expression of ubiquitin ligase MDM2 is a primary cause of p53 inactivation in many tumors, making it a promising therapeutic target. However, MDM2 inhibitors have failed in clinical trials due to p53-induced feedback that enhances MDM2 expression. This underscores the urgent need to find an effective adaptive genotype or combination of targets. Methods Kinome-wide CRISPR/Cas9 knockout screen was performed to identify genes that modulate the response to MDM2 inhibitor using TP53 wild type cancer cells and found ULK1 as a candidate. The MTT cell viability assay, flow cytometry and LDH assay were conducted to evaluate the activation of pyroptosis and the synthetic lethality effects of combining ULK1 depletion with p53 activation. Dual-luciferase reporter assay and ChIP-qPCR were performed to confirm that p53 directly mediates the transcription of GSDME and to identify the binding region of p53 in the promoter of GSDME. ULK1 knockout / overexpression cells were constructed to investigate the functional role of ULK1 both in vitro and in vivo. The mechanism of ULK1 depletion to activate GSMDE was mainly investigated by qPCR, western blot and ELISA. Results By using high-throughput screening, we identified ULK1 as a synthetic lethal gene for the MDM2 inhibitor APG115. It was determined that deletion of ULK1 significantly increased the sensitivity, with cells undergoing typical pyroptosis. Mechanistically, p53 promote pyroptosis initiation by directly mediating GSDME transcription that induce basal-level pyroptosis. Moreover, ULK1 depletion reduces mitophagy, resulting in the accumulation of damaged mitochondria and subsequent increasing of reactive oxygen species (ROS). This in turn cleaves and activates GSDME via the NLRP3-Caspase inflammatory signaling axis. The molecular cascade makes ULK1 act as a crucial regulator of pyroptosis initiation mediated by p53 activation cells. Besides, mitophagy is enhanced in platinum-resistant tumors, and ULK1 depletion/p53 activation has a synergistic lethal effect on these tumors, inducing pyroptosis through GSDME directly. Conclusion Our research demonstrates that ULK1 deficiency can synergize with MDM2 inhibitors to induce pyroptosis. p53 plays a direct role in activating GSDME transcription, while ULK1 deficiency triggers upregulation of the ROS-NLRP3 signaling pathway, leading to GSDME cleavage and activation. These findings underscore the pivotal role of p53 in determining pyroptosis and provide new avenues for the clinical application of p53 restoration therapies, as well as suggesting potential combination strategies.

Background: High expression of ubiquitin ligase MDM2 is a primary cause of p53 inactivation in many tumors, making it a promising therapeutic target. However, MDM2 inhibitors have failed in clinical trials due to p53-induced feedback that enhances MDM2 expression. This underscores the urgent need to find an effective adaptive genotype or combination of targets. Methods: Kinome-wide CRISPR/Cas9 knockout screen was performed to identify genes that modulate the response to MDM2 inhibitor using TP53 wild type cancer cells and found ULK1 as a candidate. The MTT cell viability assay, flow cytometry and LDH assay were conducted to evaluate the activation of pyroptosis and the synthetic lethality effects of combining ULK1 depletion with p53 activation. Dual-luciferase reporter assay and ChIP-qPCR were performed to confirm that p53 directly mediates the transcription of GSDME and to identify the binding region of p53 in the promoter of GSDME. ULK1 knockout / overexpression cells were constructed to investigate the functional role of ULK1 both in vitro and in vivo. The mechanism of ULK1 depletion to activate GSMDE was mainly investigated by qPCR, western blot and ELISA. Results: By using high-throughput screening, we identified ULK1 as a synthetic lethal gene for the MDM2 inhibitor APG115. It was determined that deletion of ULK1 significantly increased the sensitivity, with cells undergoing typical pyroptosis. Mechanistically, p53 promote pyroptosis initiation by directly mediating GSDME transcription that induce basal-level pyroptosis. Moreover, ULK1 depletion reduces mitophagy, resulting in the accumulation of damaged mitochondria and subsequent increasing of reactive oxygen species (ROS). This in turn cleaves and activates GSDME via the NLRP3-Caspase inflammatory signaling axis. The molecular cascade makes ULK1 act as a crucial regulator of pyroptosis initiation mediated by p53 activation cells. Besides, mitophagy is enhanced in platinum-resistant tumors, and ULK1 depletion/p53 activation has a synergistic lethal effect on these tumors, inducing pyroptosis through GSDME directly. Conclusion: Our research demonstrates that ULK1 deficiency can synergize with MDM2 inhibitors to induce pyroptosis. p53 plays a direct role in activating GSDME transcription, while ULK1 deficiency triggers upregulation of the ROS-NLRP3 signaling pathway, leading to GSDME cleavage and activation. These findings underscore the pivotal role of p53 in determining pyroptosis and provide new avenues for the clinical application of p53 restoration therapies, as well as suggesting potential combination strategies. [ABSTRACT FROM AUTHOR]

Individuals with genetic elimination of MLKL (mixed lineage kinase domain like pseudokinase) exhibit an increased susceptibility to neurodegenerative diseases like Alzheimer disease (AD). However, the mechanism is not yet fully understood. Here, we observed significant compromise in macroautophagy/autophagy in the brains of mlkl knockout (KO) mice, as evidenced by the downregulation of BECN1/Beclin1 and ULK1 (unc-51 like autophagy activating kinase 1). We identified UBA52 (ubiquitin A-52 residue ribosomal protein fusion product 1) as the binding partner of MLKL under physiological conditions. Loss of Mlkl induced a decrease in ubiquitin levels by preventing UBA52 cleavage. Furthermore, we demonstrated that the deubiquitinase (DUB) USP7 (ubiquitin specific peptidase 7) mediates the processing of UBA52, which is regulated by MLKL. Moreover, our results indicated that the reduction of BECN1 and ULK1 upon Mlkl loss is attributed to a decrease in their lysine 63 (K63)-linked polyubiquitination. Additionally, single-nucleus RNA sequencing revealed that the loss of Mlkl resulted in the disruption of multiple neurodegenerative disease-related pathways, including those associated with AD. These results were consistent with the observation of cognitive impairment in mlkl KO mice and exacerbation of AD pathologies in an AD mouse model with mlkl deletion. Taken together, our findings demonstrate that MLKL-USP7-UBA52 signaling is required for autophagy in brain through maintaining ubiquitin homeostasis, and highlight the contribution of Mlkl loss-induced ubiquitin deficits to the development of neurodegeneration. Thus, the maintenance of adequate levels of ubiquitin may provide a novel perspective to protect individuals from multiple neurodegenerative diseases through regulating autophagy.Abbreviations: 4HB: four-helix bundle; AAV: adeno-associated virus; AD: Alzheimer disease; AIF1: allograft inflammatory factor 1; APOE: apolipoprotein E; APP: amyloid beta precursor protein; Aβ: amyloid β; BECN1: beclin 1; co-IP: co-immunoprecipitation; DEGs: differentially expressed genes; DLG4: discs large MAGUK scaffold protein 4; DUB: deubiquitinase; EBSS: Earle’s balanced salt solution; GFAP: glial fibrillary acidic protein; HRP: horseradish peroxidase; IL1B: interleukin 1 beta; IL6: interleukin 6; IPed: immunoprecipitated; KEGG: Kyoto Encyclopedia of Genes and Genomes; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MLKL: mixed lineage kinase domain like pseudokinase; NSA: necrosulfonamide; OPCs: oligodendrocyte precursor cells; PFA: paraformaldehyde; PsKD: pseudo-kinase domain; SYP: synaptophysin; UB: ubiquitin; UBA52: ubiquitin A-52 residue ribosomal protein fusion product 1; UCHL3: ubiquitin C-terminal hydrolase L3; ULK1: unc-51 like autophagy activating kinase 1; UMAP: uniform manifold approximation and projection; UPS: ubiquitin-proteasome system; USP7: ubiquitin specific peptidase 7; USP9X: ubiquitin specific peptidase 9 X-linked. [ABSTRACT FROM AUTHOR]

Macroautophagy/autophagy enables lysosomal degradation of a diverse array of intracellular material. This process is essential for normal cellular function and its dysregulation is implicated in many diseases. Given this, there is much interest in understanding autophagic mechanisms of action in order to determine how it can be best targeted therapeutically. In mitophagy, the selective degradation of mitochondria via autophagy, mitochondria first need to be primed with signals that allow the recruitment of the core autophagy machinery to drive the local formation of an autophagosome around the target mitochondrion. To determine how the recruitment of different core autophagy components can drive mitophagy, we took advantage of the mito-QC mitophagy assay (an outer mitochondrial membrane-localized tandem mCherry-GFP tag). By tagging autophagy proteins with an anti-mCherry (or anti-GFP) nanobody, we could recruit them to mitochondria and simultaneously monitor levels of mitophagy. We found that targeting ULK1, ATG16L1 and the different Atg8-family proteins was sufficient to induce mitophagy. Mitochondrial recruitment of ULK1 and the Atg8-family proteins induced a conventional mitophagy pathway, requiring RB1CC1/FIP200, PIK3C3/VPS34 activity and ATG5. Surprisingly, the mitophagy pathway upon recruitment of ATG16L1 proceeded independently of ATG5, although it still required RB1CC1 and PIK3C3/VPS34 activity. In this latter pathway, mitochondria were alternatively delivered to lysosomes via uptake into early endosomes.Abbreviation: aGFP: anti-GFP nanobody; amCh: anti-mCherry nanobody; ATG: autophagy related; ATG16L1: autophagy related 16 like 1; AUTAC/AUTOTAC: autophagy-targeting chimera; BafA1: bafilomycin A1; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CCCP: carbonyl cyanide m-chlorophenylhydrazone; COX4/COX IV: cytochrome c oxidase subunit 4; DFP: deferiprone; DMSO: dimethyl sulfoxide; GABARAP: GABA type A receptor-associated protein; GABARAPL1: GABA type A receptor associated protein like 1; HSPD1/HSP60: heat shock protein family D (Hsp60) member 1; HRP: horseradish peroxidase; HTRA2/OMI: HtrA serine peptidase 2; IB: immunoblotting; IF: immunofluorescence; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; NBR1: NBR1 autophagy cargo receptor; OMM: outer mitochondrial membrane; OPA1: OPA1 mitochondrial dynamin like GTPase; OPTN: optineurin; (D)PBS: (Dulbecco’s) phosphate-buffered saline; PD: Parkinson disease; PFA: paraformaldehyde; POI: protein of interest; PtdIns3K: class III phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; RAB: RAB, member RAS oncogene family; RB1CC1/FIP200: RB1 inducible coiled-coil 1; SQSTM1: sequestosome 1; TAX1BP1: Tax1 binding protein 1; ULK: unc-51 like autophagy activating kinase 1; VPS: vacuolar protein sorting; WIPI: WD repeat domain, phosphoinositide interacting. [ABSTRACT FROM AUTHOR]

Objective To evaluate the impact of microRNAs (miRNAs) on lung cancer and elucidate the specific mechanism, providing theoretical basis for the development of anti-cancer biological agents. Methods Differential expression of miRNAs in lung cancer tissues and normal lung tissues was identified by sequencing. The relationship between miRNAs and lung cancer was validated using data from The Cancer Genome Atlas (TCGA) database. Cell viability, invasion, migration, and anti-apoptotic capacity were assessed using Cell Counting Kit-8 (CCK-8), transwell, and flow cytometry assays after miRNA intervention. Bioinformatics analysis of miRNA target genes and functional validation were conducted. Results Sequencing revealed decreased expression of miR-139-5 p in lung cancer tissues compared to normal lung tissues. TCGA data confirmed the role of miR-139-5 p in lung cancer, suggesting its potential as a tumor suppressor. Functional assays demonstrated that miR-139-5 p inhibited the viability, invasion, and anti-apoptotic capacity of lung cancer cells. Mechanistic studies revealed the involvement of autophagy in the development of lung cancer, and miR-139-5 p directly targeted and inhibited the autophagy gene unc-51-like kinase 1 (ULK1), thereby suppressing autophagy in lung cancer. Downregulation of ULK1 reduced the viability, invasion, and anti-apoptotic capacity of lung cancer cells, while upregulation of ULK1 had the opposite effects. Conclusion MiR-139-5 p attenuates autophagy in non-small cell lung cancer by targeting ULK1, thereby weakening the carcinogenic effects of autophagy. [ABSTRACT FROM AUTHOR]

Distal hereditary motor neuropathy (dHMN) is a progressive neurological disease characterized by distal limb muscle weakness and amyotrophy. Sigma 1 receptor (σ1R), a gene product of SIGMAR1, mutations have been reported to induce dHMN, but its mechanism remains unknown. This study aims to explore the effect of C238T and 31_50del mutations in σ1R on neuronal SH-SY5Y cell functions. The SH-SY5Y cells that overexpressed σ1R, C238T mutant σ1R (σ1RC238T) or 31_50del mutant σ1R (σ1R31_50del) were constructed by pEGFPN1 vectors. We used Western blot (WB) and immunofluorescence (IF) staining to detect the expression of σ1R and green fluorescent proteins (GFP). Then, we evaluated the impact of σ1R mutation on apoptosis, autophagy, endoplasmic reticulum stress, and the involvement of the unfolded protein response (UPR) pathway in SH-SY5Y cells. We found that σ1RC238T and σ1R31_50del downregulated σ1R and promoted the apoptosis of SH-SY5Y cells. σ1RC238T and σ1R31_50del increased p-PERK, p-eIF2α, p-JNK, BIP, ATF4, CHOP, ATF6, XBP1, Caspase3, Caspase12 expressions and Ca2+ concentration, whereas decreased ATP content in SH-SY5Y cells. Besides, the expressions of LC3B, Lamp1, ATG7, Beclin-1 and phosphorylation of AMPK and ULK1 were increased, while the p62 level decreased after C238T or 31_50del mutation of σ1R. Additionally, AMPK knockdown abolished the apoptosis mediated by σ1RC238T or σ1R31_50del in SH-SY5Y cells. Our results indicated that C238T or 31_50del mutation in σ1R promoted motor neuron apoptosis through the AMPK/ULK1 pathway in dHMN. This study shed light on a better understanding of the neurons pathological mechanisms mediated by σ1R C238T and σ1R 31-50del in dHMN. [ABSTRACT FROM AUTHOR]

UNC-51-like kinase 1( ULK1) is an important factor involved in regulating the initiation of autophagy. ULK1 regulates inflammatory cytokines through autophagy and mitochondrial oxidative stress, and is involved in the pathological processes of various diseases. ULK1 and its complexes are regulated by rapamycin( mTOR) and AMP-activated protein kinase( AMPK) to initiate autophagy, thereby exerting differential effects on a variety of inflammatory diseases. In inflammatory diseases, mitochondrial oxidative stress can induce ULK1 into the nucleus to accelerate apoptosis. Therefore, ULK1 plays different important roles in inflammatory diseases. For example, ULK1 initiates airway epithelial mitochondrial autophagy in asthma, participates in mitochondrial oxidative stress in acute liver failure, affects related inflammatory factors in atherosclerosis, and modulates beneficial effects of autophagy in diabetes. This article reviews the biological function of ULK1, its impact on inflammatory diseases and the research progress of targeted drugs. [ABSTRACT FROM AUTHOR]

The β-thalassemias are inherited genetic disorders affecting the hematopoietic system. In β-thalassemias, more than 350 mutations of the adult β-globin gene cause the low or absent production of adult hemoglobin (HbA). A clinical parameter affecting the physiology of erythroid cells is the excess of free α-globin. Possible experimental strategies for a reduction in excess free α-globin chains in β-thalassemia are CRISPR-Cas9-based genome editing of the β-globin gene, forcing "de novo" HbA production and fetal hemoglobin (HbF) induction. In addition, a reduction in excess free α-globin chains in β-thalassemia can be achieved by induction of the autophagic process. This process is regulated by the Unc-51-like kinase 1 (Ulk1) gene. The interplay with the PI3K/Akt/TOR pathway, with the activity of the α-globin stabilizing protein (AHSP) and the involvement of microRNAs in autophagy and Ulk1 gene expression, is presented and discussed in the context of identifying novel biomarkers and potential therapeutic targets for β-thalassemia. [ABSTRACT FROM AUTHOR]

Signal transducer and activator of transcription 1 (STAT1) plays a critical role in immune response, human STAT1 as a transcriptional suppressor of autophagy genes and autophagic activity. Classical swine fever virus (CSFV)-infected induce autophagy, leading to immune evasion. However, there are limited reports on the function of porcine STAT1 in autophagy during CSFV infection. There is also lack of suitable in vitro models for studying porcine STAT1. The objective of this study was to establish porcine PK-15 STAT1-/- and 3D4/21 STAT1-/- cell lines using the CRISPR/Cas9 system to investigate the function of the STAT1 in autophagy. The PK-15STAT1-/- and 3D4/21STAT1-/- cell lines, featuring homozygous knockout of STAT1 gene were successfully constructed using the CRISPR/Cas9 editing system. The knockout efficiency determined to be 82.4% and 81.1%, respectively. Infection with CSFV in porcine PK-15STAT1-/- and 3D4/21STAT1-/- cells led to an observable increase in autophagosomes as evidenced by transmission electron microscope. Additionally, STAT1 knockout (STAT1-/-) by the CRISPR/Cas9 system upregulated the expression of ULK1, Beclin1, and LC3 genes, thereby enhancing autophagy during CSFV infection. Conversely, overexpression of STAT1 downregulated the expression of ULK1, Beclin1, and LC3 genes, leading to inhibition of autophagy during CSFV infection.The application of an autophagy dual-fluorescent-tracking plasmid demonstrated that STAT1 knockout enhanced autophagy accumulation during CSFV infection, while STAT1 overexpression inhibited it. Moreover, the 3D4/21STAT1-/- cell line proved to be a more suitable in vitro model compared to the PK-15STAT1-/- cell line for elucidating the involvement of STAT1 in autophagy during CSFV infection.

Abstract The molecular mechanism underlying abnormal osteoclastogenesis triggering subchondral bone remodeling in osteoarthritis (OA) is still unclear. Here, single‐cell and bulk transcriptomics sequencing analyses are performed on GEO datasets to identify key molecules and validate them using knee joint tissues from OA patients and rat OA models. It is found that the catalytic subunit of protein phosphatase 2A (PP2Ac) is highly expressed during osteoclastogenesis in the early stage of OA and is correlated with autophagy. Knockdown or inhibition of PP2Ac weakened autophagy during osteoclastogenesis. Furthermore, the ULK1 expression of the downstream genes is significantly increased when PP2Ac is knocked down. PP2Ac‐mediated autophagy is dependent on ULK1 phosphorylation activity during osteoclastogenesis, which is associated with enhanced dephosphorylation of ULK1 Ser637 residue regulating at the post‐translational level. Additionally, mTORC1 inhibition facilitated the expression level of PP2Ac during osteoclastogenesis. In animal OA models, decreasing the expression of PP2Ac ameliorated early OA progression. The findings suggest that PP2Ac is also a promising therapeutic target in early OA.

The five-year survival rate for patients with hepatocellular carcinoma (HCC) is only 20 %, highlighting the urgent need to identify new therapeutic targets and develop potential therapeutic options to improve patient prognosis. One promising approach is inhibiting autophagy as a strategy for HCC treatment. In this study, we established a virtual docking conformation of the autophagy promoter ULK1 binding XST-14 derivatives. Based on this conformation, we designed and synthesized four series of derivatives. By evaluating their affinity and anti-HCC effects, we confirmed that these compounds exert anti-HCC activity by inhibiting ULK1. The structure-activity relationship was summarized, with derivative A4 showing 10 times higher activity than XST-14 and superior efficacy to sorafenib against HCC. A4 has excellent effect on reducing tumor growth and enhancing sorafenib activity in HepG2 and HCCLM3 cells. Moreover, we verified the therapeutic effect of A4 in sorafenib-resistant HCC cells both in vivo and in vitro. These results suggest that inhibiting ULK1 to regulate autophagy may become a new treatment method for HCC and that A4 will be used as a lead drug for HCC in further research. Overall, A4 shows good drug safety and efficacy, offering hope for prolonging the survival of HCC patients.

Acute lung injury (ALI) is a serious condition characterized by damage to the lungs. Recent research has revealed that activation of the NLRP3 inflammasome in alveolar macrophages, a type of immune cell in the lungs, plays a key role in the development of ALI. This process, known as pyroptosis, contributes significantly to ALI pathogenesis. Researchers have conducted comprehensive bioinformatics analyses and identified 15 key genes associated with alveolar macrophage pyroptosis in ALI. Among these, NLRP3 has emerged as a crucial regulator. This study further reveal that the ULK1 protein diminishes the expression of NLRP3, thereby reducing the immune response of alveolar macrophages and mitigating ALI. Conversely, TRAF3, another protein, is found to inhibit ULK1 through a process called ubiquitination, leading to increased activation of the NLRP3 inflammasome and exacerbation of ALI. This TRAF3-mediated suppression of ULK1 and subsequent activation of NLRP3 are confirmed through various in vitro and in vivo experiments. The presence of abundant M0 and M1 alveolar macrophages in the ALI tissue samples further support these findings. This research highlights the TRAF3-ULK1-NLRP3 regulatory axis as a pivotal pathway in ALI development and suggests that targeting this axis could be an effective therapeutic strategy for ALI treatment.

Summary: SMARCA4 is a gene traditionally considered a tumor suppressor. Recent research has however found that SMARCA4 likely promotes cancer growth and is a good target for cancer treatment. The drug carbamazepine, an autophagy inducer, was used on colorectal cancer cell lines, HCT1116 and Hke3 (KRAS mutant and wildtype). Our study finds that Carbamazepine affects SMARCA4 levels and that this effect is different depending on the KRAS mutation status. This study analyzes the effect of carbamazepine on early-stage autophagy via ULK1 as well as simulates the docking of carbamazepine on KRAS, depending on the mutation status. Our study highlights the therapeutic uses of carbamazepine on cancer, and we propose that carbamazepine in conjunction with other chemotherapies may prove useful in targeting KRAS-mutated colorectal cancer. [ABSTRACT FROM AUTHOR]

The mineralocorticoid receptor (MR) is a transcription factor for genes mediating diverse, cell-specific functions, including trophic effects as well as promoting fluid/electrolyte homeostasis. It was reported that in intercalated cells, phosphorylation of the MR at serine 843 (S843) by Unc-51–like kinase (ULK1) inhibits MR activation and that phosphorylation of ULK1 by mechanistic target of rapamycin (mTOR) inactivates ULK1, and thereby prevents MR inactivation. We extended these findings with studies in M1 mouse cortical collecting duct cells stably expressing the rat MR and a reporter gene. Pharmacological inhibition of ULK1 dose-dependently increased ligand-induced MR transactivation, while ULK1 activation had no effect. Pharmacological inhibition of mTOR and CRISPR/gRNA gene knockdown of rapamycin-sensitive adapter protein of mTOR (Raptor) or rapamycin-insensitive companion of mTOR (Rictor) decreased phosphorylated ULK1 and ligand-induced activation of the MR reporter gene, as well as transcription of endogenous MR-target genes. As predicted, ULK1 inhibition had no effect on aldosterone-mediated transcription in M1 cells with the mutated MR-S843A (alanine cannot be phosphorylated). In contrast, mTOR inhibition dose-dependently decreased transcription in the MR-S843A cells, though not as completely as in cells with the wild-type MR-S843. mTOR, Raptor, and Rictor coprecipitated with the MR and addition of aldosterone increased their phosphorylated, active state. These results suggest that mTOR significantly regulates MR activity in at least 2 ways: by suppressing MR inactivation by ULK1, and by a yet ill-defined mechanism that involves direct association with MR. They also provide new insights into the diverse functions of ULK1 and mTOR, 2 key enzymes that monitor the cell's energy status. [ABSTRACT FROM AUTHOR]

Abstract Background Pancreatic ductal adenocarcinoma (PDAC) is characterized by rapid progression and poor prognosis. Understanding the genetic mechanisms that affect cancer properties and reprogram tumor immune microenvironment will develop new strategies to maximize the benefits for cancer therapies. Methods Gene signatures and biological processes associated with advanced cancer and unfavorable outcome were profiled using bulk RNA sequencing and spatial transcriptome sequencing, Caprin-1 was identified as an oncogenesis to expedite pancreatic cancer growth by activating autophagy. The mechanism of Caprin-1 inducing autophagy activation was further explored in vitro and in vivo. In addition, higher level of Caprin-1 was found to manipulate immune responses and inflammatory-related pathways. The immune profiles associated with increased levels of Caprin-1 were identified in human PDAC samples. The roles of CD4+T cells, CD8+T cells and tumor associated macrophages (TAMs) on clinical outcomes prediction were investigated. Results Caprin-1 was significantly upregulated in advanced PDAC and correlated with poor prognosis. Caprin-1 interacted with both ULK1 and STK38, and manipulated ULK1 phosphorylation which activated autophagy and exerted pro-tumorigenic phenotypes. Additionally, the infiltrated CD4+T cells and tumor associated macrophages (TAMs) were increased in Caprin-1High tissues. The extensive CD4+T cells determined poor clinical outcome in Caprin-1high patients, arguing that highly expressed Caprin-1 may assist cancer cells to escape from immune surveillance. Conclusions Our findings establish causal links between the upregulated expression of Caprin-1 and autophagy activation, which may manipulate immune responses in PDAC development. Our study provides insights into considering Caprin-1 as potential therapeutic target for PDAC treatment.

Preeclampsia (PE) is a serious hypertensive disorder affecting 4–5% of pregnancies globally, leading to maternal and perinatal morbidity and mortality and reducing life expectancy in surviving women post-gestation. Late-onset PE (LO-PE) is a clinical type of PE diagnosed after 34 weeks of gestation, being less severe than the early-onset PE (EO-PE) variant, although both entities have a notable impact on the placenta. Despite the fact that most studies have focused on EO-PE, LO-PE does not deserve less attention since its prevalence is much higher and little is known about the role of the placenta in this pathology. Via RT-qPCR and immunohistochemistry methods, we measured the gene and protein expressions of several macroautophagy markers in the chorionic villi of placentas from women who underwent LO-PE (n = 68) and compared them to normal pregnancies (n = 43). We observed a markedly distinct expression pattern, noticing a significant drop in NUP62 expression and a considerable rise in the gene and protein expressions of ULK1, ATG9A, LC3, ATG5, STX-17, and LAMP-1 in the placentas of women with LO-PE. A major induction of autophagic processes was found in the placental tissue of patients with LO-PE. Abnormal signaling expression of these molecular patterns in this condition aids in the understanding of the complexity of pathophysiology and proposes biomarkers for the clinical management of these patients. [ABSTRACT FROM AUTHOR]

Type 2 diabetes mellitus (T2DM) is an epidemiological risk factor for dementia and has been implicated in multifactorial pathologies, including neuroinflammation. In the present study, we aimed to elucidate the potential anti-inflammatory effects of imeglimin, a novel antidiabetic agent, on high-glucose (HG)-stimulated microglia. Mouse microglial BV2 cells were stimulated with HG in the presence or absence of imeglimin. We examined the effects of imeglimin on the levels of proinflammatory cytokines, intracellular reactive oxygen species (ROS), mitochondrial integrity, and components related to the inflammasome or autophagy pathways in these cells. Our results showed that imeglimin suppressed the HG-induced production of interleukin-1beta (IL-1β) by reducing the intracellular ROS levels, ameliorating mitochondrial dysfunction, and inhibiting the activation of the thioredoxin-interacting protein (TXNIP)–NOD-like receptor family pyrin domain containing 3 (NLRP3) axis. Moreover, the inhibitory effects of imeglimin on the TXNIP–NLRP3 axis depended on the imeglimin-induced activation of ULK1, which also exhibited novel anti-inflammatory effects without autophagy induction. These findings suggest that imeglimin exerted novel suppressive effects on HG-stimulated microglia through the ULK1–TXNIP–NLRP3 axis, and may, thereby, contribute to the development of innovative strategies to prevent T2DM-associated cognitive impairment. [ABSTRACT FROM AUTHOR]

MHY1485 is an mTOR activator that inhibits the autophagy process by inhibiting the fusion between autophagosomes and lysosomes. This study aimed to explore the role and mechanism of MHY1485 in hepatocellular carcinoma (HCC) and to provide an in-depth understanding of the mechanisms of autophagy regulation in relation to adriamycin (ADM) resistance, as well as the development of a molecularly targeted autophagy-modulating approach. Here, ADM was used to treat HepG2 cells and construct an ADM-resistant cell model. The HepG2/ADM cell line and HepG2 cells were treated with MHY1485 and ADM, respectively, and the proliferation and apoptosis of HCC cells were detected using CCK8, clone formation, flow cytometry, and 5-ethynyl-2′-deoxyuridine staining assays. Ki-67, mTOR phosphorylation, and LC3A expression were detected by IF staining; the expression or phosphorylation levels of autophagy-related proteins (i.e., GLUT1, PGI, PFK, END, and MTHFD2) and apoptosis-related proteins (caspase-3, caspase-8, and caspase-9) were detected by qPCR and western blotting. The number of autophagosomes was determined by monodansylcadaverine staining. Our results showed that MHY1485 can inhibit the proliferation and growth of liver cancer cells, and that MHY1485 combined with ADM can effectively inhibit the tolerance of HepG2/ADM cells to ADM and enhance the efficacy of ADM. The results of the detection of the autophagy-related protein LC3A also indicated that MHY1485 activates mTOR and can affect the phosphorylation level of ULK1, inhibit autophagy, and enhance the sensitivity of liver cancer cells to adriamycin. In summary, MHY1485 can enhance the sensitivity of adriamycin-resistant cells to adriamycin by activating mTOR and blocking the autophagy process in cells; therefore, mTOR may become a potential target for the treatment of liver cancer. [ABSTRACT FROM AUTHOR]

The accumulation of protein aggregates defines distinct, yet overlapping pathologies such as Alzheimer's disease (AD), dementia with Lewy bodies (DLB), and frontotemporal dementia (FTD). In this study, we investigated ATG5, UBQLN2, ULK1, and LC3 concentrations in 66 brain specimens and 120 plasma samples from AD, DLB, FTD, and control subjects (CTRL). Protein concentration was measured with ELISA kits in temporal, frontal, and occipital cortex specimens of 32 AD, 10 DLB, 10 FTD, and 14 CTRL, and in plasma samples of 30 AD, 30 DLB, 30 FTD, and 30 CTRL. We found alterations in ATG5, UBQLN2, ULK1, and LC3 levels in patients; ATG5 and UBQLN2 levels were decreased in both brain specimens and plasma samples of patients compared to those of the CTRL, while LC3 levels were increased in the frontal cortex of DLB and FTD patients. In this study, we demonstrate alterations in different steps related to ATG5, UBQLN2, and LC3 autophagy pathways in DLB and FTD patients. Molecular alterations in the autophagic processes could play a role in a shared pathway involved in the pathogenesis of neurodegeneration, supporting the hypothesis of a common molecular mechanism underlying major neurodegenerative dementias and suggesting different potential therapeutic targets in the autophagy pathway for these disorders. [ABSTRACT FROM AUTHOR]

TRIM proteins are characterized by their conserved N-terminal RING, B-box, and coiled-coil domains. These proteins are efficient regulators of autophagy, apoptosis, and innate immune responses and confer immunity against viruses and bacteria. TRIMs function as receptors or scaffold proteins that target substrates for autophagy-mediated degradation. Most TRIMs interact with the BECN1-ULK1 complex to form TRIMosomes, thereby efficiently targeting substrates to autophagosomes. They regulate the functions of ATG proteins through physical interactions or ubiquitination. TRIMs affect the lipidation of MAP1LC3B1 to form MAP1LC3B2, which is a prerequisite for phagophore and autophagosome formation. In addition, they regulate MTOR kinase and TFEB, thereby regulating the expression of ATG genes. TRIM proteins are efficient regulators of apoptosis and are crucial for regulating cell proliferation and tumor formation. Many TRIM proteins regulate intrinsic and extrinsic apoptosis via the cell surface receptors TGFBR2, TNFRSF1A, and FAS. Mitochondria modulate the anti- and proapoptotic functions of BCL2, BAX, BAK1, and CYCS. These proteins use a multipronged approach to regulate the intrinsic and extrinsic apoptotic pathways, culminating in coordinated activation or inhibition of the initiator and executor CASPs. Furthermore, TRIMs can have a dual effect in determining cell fate and are therefore crucial for cellular homeostasis. In this review, we discuss mechanistic insights into the role of TRIM proteins in regulating autophagy and apoptosis, which can be used to better understand cellular physiology. These findings can be used to develop therapeutic interventions to prevent or treat multiple genetic and infectious diseases. [ABSTRACT FROM AUTHOR]

Autophagy is a cellular mechanism that utilizes lysosomes to degrade its own components and is performed using Atg5 and other molecules originating from the endoplasmic reticulum membrane. On the other hand, we identified an alternative type of autophagy, namely, Golgi membrane-associated degradation (GOMED), which also utilizes lysosomes to degrade its own components, but does not use Atg5 originating from the Golgi membranes. The GOMED pathway involves Ulk1, Wipi3, Rab9, and other molecules, and plays crucial roles in a wide range of biological phenomena, such as the regulation of insulin secretion and neuronal maintenance. We here describe the overview of GOMED, methods to detect autophagy and GOMED, and to distinguish GOMED from autophagy. [ABSTRACT FROM AUTHOR]

Introduction: Metabolism-associated fatty liver disease (MAFLD) is a global health concern because of its association with obesity, insulin resistance, and other metabolic abnormalities. Methylsulfonylmethane (MSM), an organic sulfur compound found in various plants and animals, exerts antioxidant and anti-inflammatory effects. Here, we aimed to assess the anti-obesity activity and autophagy-related mechanisms of Methylsulfonylmethane. Method: Human hepatoma (HepG2) cells treated with palmitic acid (PA) were used to examine the effects of MSM on autophagic clearance. To evaluate the anti-obesity effect of MSM, male C57/BL6 mice were fed a high-fat diet (HFD; 60% calories) and administered an oral dose of MSM (200 or 400mg/kg/day). Moreover, we investigated the AMP-activated protein kinase (AMPK)/mechanistic target of rapamycin complex 1 (mTORC1)/UNC-51-like autophagy-activating kinase 1 (ULK1) signaling pathway to further determine the underlying action mechanism of MSM. Results: Methylsulfonylmethane treatment significantly mitigated PA-induced protein aggregation in human hepatoma HepG2 cells. Additionally, Methylsulfonylmethane treatment reversed the PA-induced impairment of autophagic flux. Methylsulfonylmethane also enhanced the insulin sensitivity and significantly suppressed the HFD-induced obesity and hepatic steatosis in mice. Western blotting revealed that Methylsulfonylmethane improved ubiquitinated protein clearance in HFD-induced fatty liver. Remarkably, Methylsulfonylmethane promoted the activation of AMPK and ULK1 and inhibited mTOR activity. Conclusion: Our study suggests that MSM ameliorates hepatic steatosis by enhancing the autophagic flux via an AMPK/mTOR/ULK1-dependent signaling pathway. These findings highlight the therapeutic potential of MSM for obesity-related MAFLD treatment. [ABSTRACT FROM AUTHOR]

Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by rapid progression and poor prognosis. Understanding the genetic mechanisms that affect cancer properties and reprogram tumor immune microenvironment will develop new strategies to maximize the benefits for cancer therapies. Methods: Gene signatures and biological processes associated with advanced cancer and unfavorable outcome were profiled using bulk RNA sequencing and spatial transcriptome sequencing, Caprin-1 was identified as an oncogenesis to expedite pancreatic cancer growth by activating autophagy. The mechanism of Caprin-1 inducing autophagy activation was further explored in vitro and in vivo. In addition, higher level of Caprin-1 was found to manipulate immune responses and inflammatory-related pathways. The immune profiles associated with increased levels of Caprin-1 were identified in human PDAC samples. The roles of CD4+T cells, CD8+T cells and tumor associated macrophages (TAMs) on clinical outcomes prediction were investigated. Results: Caprin-1 was significantly upregulated in advanced PDAC and correlated with poor prognosis. Caprin-1 interacted with both ULK1 and STK38, and manipulated ULK1 phosphorylation which activated autophagy and exerted pro-tumorigenic phenotypes. Additionally, the infiltrated CD4+T cells and tumor associated macrophages (TAMs) were increased in Caprin-1High tissues. The extensive CD4+T cells determined poor clinical outcome in Caprin-1high patients, arguing that highly expressed Caprin-1 may assist cancer cells to escape from immune surveillance. Conclusions: Our findings establish causal links between the upregulated expression of Caprin-1 and autophagy activation, which may manipulate immune responses in PDAC development. Our study provides insights into considering Caprin-1 as potential therapeutic target for PDAC treatment. [ABSTRACT FROM AUTHOR]

T‐lymphokine‐activated killer cell‐originated protein kinase (TOPK) is a serine–threonine kinase that is overexpressed in gastric cancer (GC) and promotes tumor progression. Polyphyllin VII (PPVII), a pennogenin isolated from the rhizomes of Paris polyphylla, shows anticancer effects. Here, we explored the antitumor activity and mechanism of PPVII in GC. Ferroptosis was detected by transmission electron microscope, malondialdehyde, and iron determination assays. Autophagy and its upstream signaling pathway were detected by Western blot, and gene alterations. The binding of PPVII and TOPK was examined through microscale thermophoresis and drug affinity responsive target stability assays. An in vivo mouse model was performed to evaluate the therapeutic of PPVII. PPVII inhibits GC by inducing autophagy‐mediated ferroptosis. PPVII promotes the degradation of ferritin heavy chain 1, which is responsible for autophagy‐mediated ferroptosis. PPVII activates the Unc‐51‐like autophagy‐activating kinase 1 (ULK1) upstream of autophagy. PPVII inhibits the activity of TOPK, thereby weakening the inhibition of downstream ULK1. PPVII stabilizes the dimer of the inactive form of TOPK by direct binding. PPVII inhibits tumor growth without causing obvious toxicity in vivo. Collectively, this study suggests that PPVII is a potential agent for the treatment of GC by targeting TOPK to activate autophagy‐mediated ferroptosis. [ABSTRACT FROM AUTHOR]

Introduction: Autophagy is an important mechanism of cell homeostasis maintenance. As essential serine/threonine-protein kinases, casein kinase I family members affect tumorigenesis by regulating a variety of cellular progression. However, the mechanism by which they regulate autophagy remains unclear. Materials and methods: We silenced CK1δ/ε in cancer cells and observed cell morphology, the expression of autophagy-related genes, and its impact on cancer cell growth and viability. By inhibiting CK1δ/ε-induced upregulation of autophagy genes, we profiled the regulatory mechanism of CK1δ/ε on autophagy and cancer cell growth. The impact of CK1δ/ε inhibition on tumor cell growth was also assessed in vivo. Results: Here, we found that CK1δ/ε played an important role in ULK1-mediated autophagy regulation in both lung cancer and melanoma cells. Mechanically, silencing CK1δ/ε increased ULK1 expression with enhanced autophagic flux and suppressed cancer cell proliferation, while ULK1 knockdown blocked the activation of autophagy caused by CK1δ/ε inhibition. By silencing CK1δ/ε in syngeneic mouse model bearing LLC1 murine lung cancer cells in vivo, we observed tumor growth suppression mediated by CK1δ/ε inhibition. Conclusion: Our results provide evidence for the role of CK1δ/ε in the regulation of tumorigenesis via the ULK1-mediated autophagy, and also suggest the impact of CK1δ/ε inhibition on tumor growth and its significance as a potential therapeutic target.

Epithelial cells transport substances through the cellular and paracellular pathways. The last one depends on tight junctions, particularly on claudins, the family of integral membrane proteins responsible for the permeability and selectivity of these junctions. 300 nM ouabain (OUA) induces endocytosis and lysosomal degradation of claudin-2 and -4 in an Src and ERK1/2 kinases-dependent manner. Here we investigate whether OUA-induced lysosomal degradation of claudins implicates autophagy in renal epithelial Madin-Darby canine kidney cells. During autophagy, LC3 protein binds phosphatidylethanolamine and incorporates, together with protein p62, into the phagophore. Subsequently, the autolysosome degrades both LC3 and p62 proteins. OUA’s occupancy of its site in the Na⁺/K⁺ATPase (300 nM, 10 h) increases autophagic flux because of degradation of LC3 and p62 and an increase in the number of autophagosomes, as detected by fluorescent LC3 and p62 puncta and the rise in autolysosomes seen by the GFP-LC3-RFP probe. Finally, OUA increases the colocalisation of claudin-1, -2, or -4 with p62 in these puncta. OUA induces autophagy increasing reactive oxygen species generation that activates AMP-activated protein kinase, phosphorylating ULK1 at S555. The autophagy inducer rapamycin causes a degradation of the studied claudins comparable to the one generated by OUA. Furthermore, the autophagy inhibitor dorsomorphin blocks OUA-induced autophagy and claudin-1, -2, and -4 degradation. These results demonstrated that OUA induces claudin-1, -2, and -4 autophagy through oxidative stress. Abbreviations: AMP: adenosine monophosphate; AMPK: AMP-activated protein kinase; ATP: Adenosine triphosphate; DM: dorsomorphin; EGFR: epidermal growth factor receptor; ERK: extracellular signal-regulated kinase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; LC3: microtubule-associated protein 1A/1B-light chain 3; MDCK: Madin-Darby canine kidney; mTOR: mammalian target of rapamycin; NAC: N-acetylcysteine; OUA: ouabain; PCC: Pearson’s correlation coefficient; PE: phosphatidylethanolamine, Rapa: rapamycin; ROS: reactive oxygen species; SNK: Student–Newman–Keuls; TER: transepithelial electrical resistance; TJs: tight junctions; ULK1: Unc-51-like kinase 1.

YKT6 is a SNARE (Soluble N-ethylmaleimide-Sensitive Fusion Protein Attachment Protein Receptor) protein governing membrane fusion events of several cellular organelles. In autophagy, YKT6 is involved in early phagophore formation as well as directly in the fusion process between autophagosomes and the lytic compartment. Recently we showed in yeast, mammalian cells, and nematodes that the function of YKT6 in autophagy can be regulated by phosphorylation. Atg1/ULK1 (Unc-51-like kinase 1)-dependent phosphorylation of YKT6 results in autophagy defects during both early (autophagosome formation) and late (autophagosome-lysosome fusion) steps, ultimately resulting in decreased survival of mammalian cells due to defective stress-induced autophagy. These findings show that not only the function but also the regulation of YKT6 is conserved across species.

The β-thalassemias are hereditary monogenic diseases characterized by a low or absent production of adult hemoglobin and excess in the content of α-globin. This excess is cytotoxic for the erythroid cells and responsible for the β-thalassemia-associated ineffective erythropoiesis. Therefore, the decrease in excess α-globin is a relevant clinical effect for these patients and can be realized through the induction of fetal hemoglobin, autophagy, or both. The in vivo effects of sirolimus (rapamycin) and analogs on the induction of fetal hemoglobin (HbF) are of key importance for therapeutic protocols in a variety of hemoglobinopathies, including β-thalassemias. In this research communication, we report data showing that a decrease in autophagy-associated p62 protein, increased expression of ULK-1, and reduction in excess α-globin are occurring in erythroid precursors (ErPCs) stimulated in vitro with low dosages of sirolimus. In addition, increased ULK-1 mRNA content and a decrease in α-globin content were found in ErPCs isolated from β-thalassemia patients recruited for the NCT03877809 clinical trial and treated with 0.5–2 mg/day sirolimus. Our data support the concept that autophagy, ULK1 expression, and α-globin chain reduction should be considered important endpoints in sirolimus-based clinical trials for β-thalassemias. [ABSTRACT FROM AUTHOR]

The β-thalassemias are inherited genetic disorders affecting the hematopoietic system. In β-thalassemias, more than 350 mutations of the adult β-globin gene cause the low or absent production of adult hemoglobin (HbA). A clinical parameter affecting the physiology of erythroid cells is the excess of free α-globin. Possible experimental strategies for a reduction in excess free α-globin chains in β-thalassemia are CRISPR-Cas9-based genome editing of the β-globin gene, forcing “de novo” HbA production and fetal hemoglobin (HbF) induction. In addition, a reduction in excess free α-globin chains in β-thalassemia can be achieved by induction of the autophagic process. This process is regulated by the Unc-51-like kinase 1 (Ulk1) gene. The interplay with the PI3K/Akt/TOR pathway, with the activity of the α-globin stabilizing protein (AHSP) and the involvement of microRNAs in autophagy and Ulk1 gene expression, is presented and discussed in the context of identifying novel biomarkers and potential therapeutic targets for β-thalassemia.

Cerebral ischemic stroke (CIS) has become the second leading cause of death worldwide, which is largely related to cerebral ischemia reperfusion injury (CIRI). Surgical intervention is a reliable treatment for CIS, which predictably causes cerebral reperfusion. Therefore, the choice of anesthetic drugs has important clinical significance. Isoflurane (ISO), one of the most used anesthetics, attenuates cognitive impairment and has brain protective effects. However, the role of isoflurane in regulating autophagy and its regulatory mechanism on inflammation in CIRI are still unclear. The middle cerebral artery occlusion (MCAO) method was used to establish a rat model of CIRI. After 24 h of reperfusion, all rats were evaluated by mNSS scoring and dark avoidance experiment. Western blotting and immunofluorescence were used to examine the expression of key proteins. Compared with the sham group, the MCAO group showed increased neurobehavioral scores and decreased cognitive memory function (P < 0.05). As for the ISO-treated MCAO rats, the neurobehavioral score was significantly decreased, the expression of AMPK, ULK1, Beclin1, and LC3B was significantly increased, and the cognitive and memory functions were also significantly improved (P < 0.05). After inhibition of autophagy pathway or key protein AMPK in autophagy, neurobehavioral scores and protein expression of NLRP3, IL-1β, and IL-18 were significantly increased (P < 0.05). Isoflurane post-treatment may enhance autophagy by activating the AMPK/ULK1 signaling pathway and further inhibit the release of inflammatory factors from NLRP3 inflammasomes, thereby ameliorating neurological function and cognitive impairment and exerting a protective effect on the brain in CIRI rats. [ABSTRACT FROM AUTHOR]

NRF2 is a transcription factor responsible for antioxidant stress responses that is usually regulated in a redox‐dependent manner. p62 bodies formed by liquid–liquid phase separation contain Ser349‐phosphorylated p62, which participates in the redox‐independent activation of NRF2. However, the regulatory mechanism and physiological significance of p62 phosphorylation remain unclear. Here, we identify ULK1 as a kinase responsible for the phosphorylation of p62. ULK1 colocalizes with p62 bodies, directly interacting with p62. ULK1‐dependent phosphorylation of p62 allows KEAP1 to be retained within p62 bodies, thus activating NRF2. p62S351E/+ mice are phosphomimetic knock‐in mice in which Ser351, corresponding to human Ser349, is replaced by Glu. These mice, but not their phosphodefective p62S351A/S351A counterparts, exhibit NRF2 hyperactivation and growth retardation. This retardation is caused by malnutrition and dehydration due to obstruction of the esophagus and forestomach secondary to hyperkeratosis, a phenotype also observed in systemic Keap1‐knockout mice. Our results expand our understanding of the physiological importance of the redox‐independent NRF2 activation pathway and provide new insights into the role of phase separation in this process. Synopsis: NRF2 is a master transcription factor for antioxidant stress that is usually regulated by redox states of KEAP1. This study shows that when ULK1 phosphorylates phase‐separated p62 bodies, they bind KEAP1, leading to NRF2‐activation in a redox‐independent manner. ULK1 phosphorylates p62 bodies formed by liquid–liquid phase separation.KEAP1 is retained into these phosphorylated p62 bodies, leading to NRF2 activation.Persistent activation of NRF2 by the redox‐independent pathway causes hyperkeratosis in vivo.The resulting esophageal and forestomach obstruction causes severe growth retardation due to malnutrition. [ABSTRACT FROM AUTHOR]

Advanced bladder cancer (BC) (local invasive and/or metastatic) is not curable even with cytotoxic chemotherapy, immune checkpoint inhibitors, and targeted treatment. Targeting GSK-3β is a promising novel approach in advanced BC. The induction of autophagy is a mechanism of secondary resistance to various anticancer treatments. Our objectives are to investigate the synergistic effects of GSK-3β in combination with autophagy inhibitors to evade GSK-3β drug resistance. Small molecule GSK-3β inhibitors and GSK-3β knockdown using siRNA promote the expression of autophagy-related proteins. We further investigated that GSK-3β inhibition induced the nucleus translocation of transcription factor EB (TFEB). Compared to the GSK-3β inhibition alone, its combination with chloroquine (an autophagy inhibitor) significantly reduced BC cell growth. These results suggest that targeting autophagy potentiates GSK-3β inhibition-induced apoptosis and retarded proliferation in BC cells. [ABSTRACT FROM AUTHOR]

Background: The roles of STK33 in renal cell carcinoma (RCC) remain unclear. This study was designed to investigate the interaction between STK33 and the autophagy in the RCC. Methods and results: STK33 was knocked down in 786-O and CAKI-1 cells. Then CCK8, clony formation assay, wound healing assay and Transwell assay were performed to analyze the proliferation, migration and invasion of the cancer cells. In addition, the activation of autophagy was determined using fluorescence, followed by investigating the potential signaling pathways in this process. After STK33 knockdown, the proliferation and migration of cell lines were inhibited, and the apoptosis of renal cancer cells was promoted. Autophagy fluorescence experiment showed that after STK33 knockdown, green LC3 protein fluorescence particles could be seen in the cells. Western blot analysis showed that after STK33 knockdown, there was significant down-regulation in P62 and p-mTOR, as well as significant up-regulation of Beclin1, LC3 and p-ULK1. Conclusions: STK33 affected autophagy in RCC cells by activating mTOR/ ULK1pathway. [ABSTRACT FROM AUTHOR]