Novel insights into autophagy in gastrointestinal pathologies, mechanisms in metabolic dysfunction-associated fatty liver disease and acute liver failure.

In this editorial, we comment on three articles published in a recent issue of World Journal of Gastroenterology. There is a pressing need for new research on autophagy's role in gastrointestinal (GI) disorders, and also novel insights into some liver conditions, such as metabolic dysfunction-associated fatty liver disease (MAFLD) and acute liver failure (ALF). Despite advancements, understanding autophagy's intricate mechanisms and implications in these diseases remains incomplete. Moreover, MAFLD's pathogenesis, encompassing hepatic steatosis and metabolic dysregulation, require further elucidation. Similarly, the mechanisms underlying ALF, a severe hepatic dysfunction, are poorly understood. Innovative studies exploring the interplay between autophagy and GI disorders, as well as defined mechanisms of MAFLD and ALF, are crucial for identifying therapeutic targets and enhancing diagnostic and treatment strategies to mitigate the global burden of these diseases.

More on the interplay between gut microbiota, autophagy, and inflammatory bowel disease is needed.

The concept of inflammatory bowel disease (IBD), which encompasses Crohn's disease and ulcerative colitis, represents a complex and growing global health concern resulting from a multifactorial etiology. Both dysfunctional autophagy and dysbiosis contribute to IBD, with their combined effects exacerbating the related inflammatory condition. As a result, the existing interconnection between gut microbiota, autophagy, and the host's immune system is a decisive factor in the occurrence of IBD. The factors that influence the gut microbiota and their impact are another important point in this regard. Based on this initial perspective, this manuscript briefly highlighted the intricate interplay between the gut microbiota, autophagy, and IBD pathogenesis. In addition, it also addressed the potential targeting of the microbiota and modulating autophagic pathways for IBD therapy and proposed suggestions for future research within a more specific and expanded context. Further studies are warranted to explore restoring microbial balance and regulating autophagy mechanisms, which may offer new therapeutic avenues for IBD management and to delve into personalized treatment to alleviate the related burden.

Lipid homeostasis in diabetic kidney disease.

Lipid homeostasis is crucial for proper cellular and systemic functions. A growing number of studies confirm the importance of lipid homeostasis in diabetic kidney disease (DKD). Lipotoxicity caused by imbalance in renal lipid homeostasis can further exasperate renal injury. Large lipid deposits and lipid droplet accumulation are present in the kidneys of DKD patients. Autophagy plays a critical role in DKD lipid homeostasis and is involved in the regulation of lipid content. Inhibition or reduction of autophagy can lead to lipid accumulation, which in turn further affects autophagy. Lipophagy selectively recognizes and degrades lipids and helps to regulate cellular lipid metabolism and maintain intracellular lipid homeostasis. Therefore, we provide a systematic review of fatty acid, cholesterol, and sphingolipid metabolism, and discuss the responses of different renal intrinsic cells to imbalances in lipid homeostasis. Finally, we discuss the mechanism by which autophagy, especially lipophagy, maintains lipid homeostasis to support the development of new DKD drugs targeting lipid homeostasis.

Biomimetic nanoparticles with enhanced rapamycin delivery for autism spectrum disorder treatment via autophagy activation and oxidative stress modulation.

Rationale: Autism spectrum disorder (ASD) represents a complex neurodevelopmental condition lacking specific pharmacological interventions. Given the multifaced etiology of ASD, there exist no effective treatment for ASD. Rapamycin (RAPA) can activate autophagy by inhibiting the mTOR pathway and has exhibited promising effects in treating central nervous system disorders; however, its limited ability to cross the blood-brain barrier (BBB) has hindered its clinical efficacy, leading to substantial side effects. Methods: To address this challenge, we designed a drug delivery system utilizing red blood cell membrane (CM) vesicles modified with SS31 peptides to enhance the brain penetration of RAPA for the treatment of autism. Results: The fabricated SCM@RAPA nanoparticles, with an average diameter of 110 nm, exhibit rapid release of RAPA in a pathological environment characterized by oxidative stress. In vitro results demonstrate that SCM@RAPA effectively activate cellular autophagy, reduce intracellular ROS levels, improve mitochondrial function, thereby ameliorating neuronal damage. SS31 peptide modification significantly enhances the BBB penetration and rapid brain accumulation of SCM@RAPA. Notably, SCM@RAPA nanoparticles demonstrate the potential to ameliorate social deficits, improve cognitive function, and reverse neuronal impairments in valproic acid (VPA)-induced ASD models. Conclusions: The therapeutic potential of SCM@RAPA in managing ASD signifies a paradigm shift in autism drug treatment, holding promise for clinical interventions in diverse neurological conditions.

Endosome-microautophagy targeting chimera (eMIATAC) for targeted proteins degradation and enhance CAR-T cell anti-tumor therapy.

Rationale: Since oncogene expression products often exhibit upregulation or abnormally activated activity, developing a technique to regulate abnormal protein levels represent a viable approach for treating tumors and protein abnormality-related diseases. Methods: We first screened out eMIATAC components with high targeted degradation efficiency and explored the mechanism by which eMIATAC induced target protein degradation, and verified the degradation efficiency of the target protein by protein imprinting and flow cytometry. Next, we recombined eMIATAC with some controllable elements to verify the regulatable degradation performance of the target protein. Subsequently, we constructed eMIATAC that can express targeted degradation of AKT1 and verified its effect on GBM cell development in vitro and in vivo. Finally, we concatenated eMIATAC with CAR sequences to construct CAR-T cells with low BATF protein levels and verified the changes in their anti-tumor efficacy. Results: we developed a system based on the endosome-microautophagy-lysosome pathway for degrading endogenous proteins: endosome-MicroAutophagy TArgeting Chimera (eMIATAC), dependent on Vps4A instead of lysosomal-associated membrane protein 2A (LAMP2A) to bind to the chaperone Hsc70 and the protein of interest (POI). The complex was then transported to the lysosome by late endosomes, where degradation occurred similarly to microautophagy. The eMIATACs demonstrated accuracy, efficiency, reversibility, and controllability in degrading the target protein EGFP. Moreover, eMIATAC exhibited excellent performance in knocking down POI when targeting endogenous proteins in vivo and in vitro. Conclusions: The eMIATACs could not only directly knock down abnormal proteins for glioma treatment but also enhance the therapeutic effect of CAR-T cell therapy for tumors by knocking down T cell exhaustion-related proteins. The newly developed eMIATAC system holds promise as a novel tool for protein knockdown strategies. By enabling direct control over endogenous protein levels, eMIATAC has the potential to revolutionize treatment for cancer and genetic diseases.

Cell Death in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a severe pulmonary vascular disease characterized by increased pulmonary vascular resistance because of vascular remodeling and vasoconstriction. Subsequently, PAH leads to right ventricular hypertrophy and heart failure. Cell death mechanisms play a significant role in development and tissue homeostasis, and regulate the balance between cell proliferation and differentiation. Several basic and clinical studies have demonstrated that multiple mechanisms of cell death, including pyroptosis, apoptosis, autophagy, ferroptosis, anoikis, parthanatos, and senescence, are closely linked with the pathogenesis of PAH. This review summarizes different cell death mechanisms involved in the death of pulmonary artery smooth muscle cells (PASMCs) and pulmonary artery endothelial cells (PAECs), the primary target cells in PAH. This review summarizes the role of these cell death mechanisms, associated signaling pathways, unique effector molecules, and various pro-survival or reprogramming mechanisms. The aim of this review is to summarize the currently known molecular mechanisms underlying PAH. Further investigations of the cell death mechanisms may unravel new avenues for the prevention and treatment of PAH.

The mechanism of lovastatin in suppressing the proliferation of esophageal squamous cell carcinoma based on proteomics.

BACKGROUND: Lovastatin, a type of statin usually considered as a lipid-lowering drug that lowers blood cholesterol and low-density lipoprotein cholesterol levels, has been rediscovered to have anticancer activity. Fewer studies exist regarding the effect of lovastatin on esophageal squamous cell carcinoma (ESCC). METHODS: Here, we report that lovastatin shows anticancer effect on ESCC By affecting the mitochondrial autophagy pathway. Moreover, based on proteomics and computer molecular simulations found that RAB38 and RAB27A may be a target of lovastatin. RESULTS: We observed that autophagy of mitochondria is inhibited by lovastatin, affecting esophageal squamous cell proliferation. There is a possible link between the expression of RAB38, RAB27A and immune cell invasion in esophageal cancer. CONCLUSIONS: These results demonstrate the huge potential of lovastatin as an RAB38, RAB27A inhibitor in esophageal cancer chemotherapy and chemoprevention.

CTLA-4 expression on CD4+ lymphocytes in patients with sepsis-associated immunosuppression and its relationship to mTOR mediated autophagic-lysosomal disorder.

Background: The aim of this study was to clarify the relationship between expression level of CTLA-4 on CD4+ T cells and sepsis-associated immunosuppression (SAI), and to elucidate the possible mechanism of mTOR pathway mediated autophagic-lysosomal disorder in regulating CTLA-4 expression. Methods: We enrolled 63 sepsis patients admitted to our ICU between January 1 and June 30, 2023. Peripheral blood mononuclear cells were isolated from the patients within 24 hours of recruitment. Expression levels of mTOR, P62, LC3II, and CTLA-4 on circulating CD4+ T lymphocytes were quantitated using flow cytometry. The association of these markers and relationship between CTLA-4 expression and the incidence of SAI and 28-day mortality were comprehensively analyzed. Results: Compared with non-immunosuppressed patients with sepsis, patients with SAI had a higher 28-day mortality rate (37.5% vs 13.0%, P=0.039) and higher CTLA-4 mean fluorescence intensity (MFI) on CD4+ T cells (328.7 versus 78.7, P<0.0001). CTLA-4 MFI on CD4+ cells was independently associated with the occurrence of SAI (95% confidence interval: 1.00-1.14, P=0.044). In patients with sepsis and SAI, non-survivors had higher CTLA-4 expression than survivors (sepsis: 427.5 versus 130.6, P=0.002; and SAI: 506.7 versus 225.2, P<0.0001). The sensitivity and specificity of CTLA-4 MFI at predicting 28-day mortality in patients with SAI was 100% and 80% respectively with the cutoff value of 328.7 and the area under the curve of 0.949. The MFI of mTOR, P62, and LC3II on CD4+ T cells were statistically higher in patients with SAI than in non-immunosuppressed patients (267.2 versus 115.9, P<0.0001; 314.8 versus 173.7, P<0.0001; and 184.7 versus 1123.5, P=0.012, respectively); P62 and LC3II were markedly higher in non-survivors than in survivors of sepsis (302.9 versus 208.9, P=0.039; and 244.3 versus 122.8, P<0.0001 respectively). The expression of CTLA-4 statistically correlated with that of LC3II in patients with sepsis, patients with SAI, and patients with SAI who did not survive (correlation coefficient: 0.69, 0.68, and 0.73, respectively, P<0.0001). Conclusions: CTLA-4 overexpression on CD4+ T cells was markedly associated with the incidence of SAI and had great relevance to 28-day mortality. mTOR pathway mediated autophagic-lysosomal disorder showed significant association with CTLA-4 expression.

Research progress of T cell autophagy in autoimmune diseases.

T cells, as a major lymphocyte population involved in the adaptive immune response, play an important immunomodulatory role in the early stages of autoimmune diseases. Autophagy is a cellular catabolism mediated by lysosomes. Autophagy maintains cell homeostasis by recycling degraded cytoplasmic components and damaged organelles. Autophagy has a protective effect on cells and plays an important role in regulating T cell development, activation, proliferation and differentiation. Autophagy mediates the participation of T cells in the acquired immune response and plays a key role in antigen processing as well as in the maintenance of T cell homeostasis. In autoimmune diseases, dysregulated autophagy of T cells largely influences the pathological changes. Therefore, it is of great significance to study how T cells play a role in the immune mechanism of autoimmune diseases through autophagy pathway to guide the clinical treatment of diseases.

mTOR Deletion Alleviates CD4+ T-Cell Dysfunction in Sepsis through Reducing CTLA4 Accumulation Mediated by Rescuing Autophagy.

Sepsis has been the leading cause of death in ICU patients. CD4+ T cells are the mainstay of the body's immune system, and the depletion of CD4+ T cells in sepsis is of great concern. Cytotoxic T lymphocyte-associated protein 4 (CTLA4) is a negative immunomodulator for T cell activation and degradation through the autophagy-lysosome pathway. Mammalian target of rapamycin (mTOR) is the most classical upstream regulator of autophagy. With a mouse model of sepsis through cecal ligation and puncture (CLP), T cell specific-mTOR/tuberous sclerosis complex 1 (TSC1)-knockout mice, and bafilomycin A1, a specific autophagosome-lysosome (A-L) fusion inhibitor, we primarily proved that mTOR could modulate the expression and accumulation of CTLA4 by regulating the onset process of autophagy such as A-L fusion. Given such a regulatory relationship, targeting mTOR could provide new light to improve immune function in sepsis, and the prospect of using rapamycin in the clinic would be worth exploring further.

Metabolism: Secretory autophagy balances nutrient supply and demand.

The hormone leptin is critical for regulation of food intake, energy expenditure and overall metabolism. However, the mechanisms that promote leptin secretion from adipocytes in response to nutrient surplus and limit its secretion during nutrient scarcity are unclear. New work reveals that the autophagy protein Atg8/LC3 has a bidirectional role in leptin secretion, both facilitating and limiting its release.

SIGMAR1 targets AMPK/ULK1 pathway to inhibit SH-SY5Y cell apoptosis by regulating endoplasmic reticulum stress and autophagy.

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.

The connection between autophagy and ferroptosis in AKI: recent advances regarding selective autophagy.

Acute kidney injury (AKI) is a significant issue in public health, displaying a high occurrence rate and mortality rate. Ferroptosis, a form of programmed cell death (PCD), is characterized by iron accumulation and intensified lipid peroxidation. Recent studies have demonstrated the pivotal significance of ferroptosis in AKI caused by diverse stimuli, including ischemia-reperfusion injury (IRI), sepsis and toxins. Autophagy, a multistep process that targets damaged organelles and macromolecules for degradation and recycling, also plays an essential role in AKI. Previous research has demonstrated that autophagy deletion in proximal tubules could aggravate tubular injury and renal function loss, indicating the protective function of autophagy in AKI. Consequently, finding ways to stimulate autophagy has become a crucial therapeutic strategy. The recent discovery of the role of selective autophagy in influencing ferroptosis has identified new therapeutic targets for AKI and has highlighted the importance of understanding the cross-talk between autophagy and ferroptosis. This study aims to provide an overview of the signaling pathways involved in ferroptosis and autophagy, focusing on the mechanisms and functions of selective autophagy and autophagy-dependent ferroptosis. We hope to establish a foundation for future investigations into the interaction between autophagy and ferroptosis in AKI as well as other diseases.

NCAPH, ubiquitinated by TRIM21, promotes cell proliferation by inhibiting autophagy of cervical cancer through AKT/mTOR dependent signaling.

Autophagy is closely related to the occurrence and development of human malignancies; however, the detailed mechanisms underlying autophagy in cervical cancer require further investigation. Previously, we found that the ectopic expression of NCAPH, a regulatory subunit of condensed protein complexes, significantly enhanced the proliferation of tumor cells; however, the underlying mechanisms were unclear. Here, we revealed that NCAPH is a novel autophagy-associated protein in cervical cancer that promotes cell proliferation by inhibiting autophagosome formation and reducing autophagy, with no effect on the cell cycle, apoptosis, or aging. Tripartite motif-containing protein 21 (TRIM21) is well known to be involved in inflammation, autoimmunity and cancer, mainly via its E3 ubiquitin ligase activity. Mass spectrometry and immunoprecipitation assays showed that TRIM21 interacted with NCAPH and decreased the protein stability of NCAPH via ubiquitination at the K11 lysine residue. Structural domain mutation analysis revealed that TRIM21 combined with NCAPH through its PRY/SPRY and CC domains and accelerated the degradation of NCAPH through the RING domain. Furthermore, TRIM21 promoted autophagosome formation and reduced cell proliferation by inhibiting NCAPH expression and the downstream AKT/mTOR pathway in cervical cancer cells. Immunohistochemical staining revealed that the protein expression of TRIM21 was negatively correlated with that of NCAPH and positively correlated with that of beclin-1 in cervical cancer tissues. Therefore, we provide evidence for the role of the TRIM21-NCAPH axis in cervical cancer autophagy and proliferation and the involvement of the AKT/mTOR signaling pathway in this process. These results deepen our understanding of the carcinogenesis of cervical cancer, broaden the understanding of the molecular mechanisms of TRIM21 and NCAPH, and provide guidance for individualized treatment of cervical cancer in the future.

Methods to Monitor Nucleophagy in Yeast.

The selective degradation of nuclear components via autophagy, termed nucleophagy, is an essential process observed from yeasts to mammals and crucial for maintaining nucleus homeostasis and regulating nucleus functions. In the budding yeast Saccharomyces cerevisiae, nucleophagy occurs in two different manners: one involves autophagosome formation for the sequestration and vacuolar transport of nucleus-derived vesicles (NDVs), and the other proceeds with the invagination of the vacuolar membrane for the uptake of NDVs into the vacuole, termed macronucleophagy and micronucleophagy, respectively. This chapter describes methods to analyze and quantify activities of these nucleophagy pathways in yeast.

Synthetic Dimerization Approaches for In Vivo Studies in Saccharomyces cerevisiae.

Synthetic tethering approaches induced by chemical means offer precise control over protein interactions in cells. They enable the manipulation of when, where, and how proteins interact, making it possible to study their functions, dynamics, and cellular consequences at a molecular level. These methods are versatile, reversible, and adaptable, allowing the dissection of complex cellular processes and the engineering of cellular functions. Here, we describe two chemically induced dimerization systems in the model organism Saccharomyces cerevisiae. Using the autophagy pathway as an example, we show how these approaches can be used to dissect molecular events in cells.

Fluorescence Microscopy and Immunoblotting for Mitophagy in Budding Yeast.

Selective removal of excess or damaged mitochondria is an evolutionarily conserved process that contributes to mitochondrial quality and quantity control. This catabolic event relies on autophagy, a membrane trafficking system that sequesters cytoplasmic constituents into double membrane-bound autophagosomes and delivers them to lysosomes (vacuoles in yeast) for hydrolytic degradation and is thus termed mitophagy. Dysregulation of mitophagy is associated with various diseases, highlighting its physiological relevance. In budding yeast, the pro-mitophagic single-pass membrane protein Atg32 is upregulated under prolonged respiration or nutrient starvation, anchored on the surface of mitochondria, and activated to recruit the autophagy machinery for the formation of autophagosomes surrounding mitochondria. In this chapter, we provide protocols to assess Atg32-mediated mitophagy using fluorescence microscopy and immunoblotting.

Analysis of Mitophagy Reporters in Drosophila.

Mitophagy is the degradation of mitochondria via the autophagy-lysosome system, disruption of which has been linked to multiple neurodegenerative diseases. As a flux process involving the identification, tagging, and degradation of subcellular components, the analysis of mitophagy benefits from the microscopy analysis of fluorescent reporters. Studying the pathogenic mechanisms of disease also benefits from analysis in animal models in order to capture the complex interplay of molecular and cell biological phenomena. Here, we describe protocols to analyze mitophagy reporters in Drosophila by light microscopy.

In Vivo Monitoring of Nucleophagy in Caenorhabditis elegans.

The autophagy-lysosomal pathway enables the controlled degradation of cellular contents. Nucleophagy is the selective autophagic recycling of nuclear components upon delivery to the lysosome. Although methods to monitor and quantify autophagy as well as selective types of autophagy have been developed and implemented in cells and in vivo, methods monitoring nucleophagy remain scarce. Here, we describe a procedure to monitor the autophagic engagement of an endogenous nuclear envelope component, i.e., ANC-1, the nematode homologue of the mammalian Nesprins in vivo, utilizing super-resolution microscopy.

Methods for Monitoring ER-Phagy.

The endoplasmic reticulum (ER) serves as a central hub for protein synthesis, folding, and lipid biosynthesis in eukaryotic cells. Maintaining ER homeostasis is essential for optimal cellular function, and one mechanism that has garnered attention is endoplasmic reticulum-specific autophagy, or ER-phagy. ER-phagy selectively removes specific ER portions, playing a pivotal role in cellular health and adaptation to environmental stressors. ER-phagy can be induced by diverse cellular conditions such as amino acid starvation, disruption of ER quality control mechanisms, and accumulation of misfolded ER protein, highlighting cellular adaptability and the significance of ER-phagy in stress responses. Clinically relevant mutations in ER-phagy receptors are implicated in various diseases, underlining the fundamental importance of ER-phagy in ER homeostasis. Here, we provide comprehensive protocols and general considerations while investigating ER-phagy using three fundamental techniques-Western blotting, immunofluorescence, and flow cytometry-commonly used in ER-phagy detection and quantitation.