Deciphering the neuroprotective mechanisms of RACK1 in cerebral ischemia-reperfusion injury: Pioneering insights into mitochondrial autophagy and the PINK1/Parkin axis.

INTRODUCTION: Cerebral ischemia-reperfusion injury (CIRI) is a common and debilitating complication of cerebrovascular diseases such as stroke, characterized by mitochondrial dysfunction and cell apoptosis. Unraveling the molecular mechanisms behind these processes is essential for developing effective CIRI treatments. This study investigates the role of RACK1 (receptor for activated C kinase 1) in CIRI and its impact on mitochondrial autophagy. METHODS: We utilized high-throughput transcriptome sequencing and weighted gene co-expression network analysis (WGCNA) to identify core genes associated with CIRI. In vitro experiments used human neuroblastoma SK-N-SH cells subjected to oxygen and glucose deprivation (OGD) to simulate ischemia, followed by reperfusion (OGD/R). RACK1 knockout cells were created using CRISPR/Cas9 technology, and cell viability, apoptosis, and mitochondrial function were assessed. In vivo experiments involved middle cerebral artery occlusion/reperfusion (MCAO/R) surgery in rats, evaluating neurological function and cell apoptosis. RESULTS: Our findings revealed that RACK1 expression increases during CIRI and is protective by regulating mitochondrial autophagy through the PINK1/Parkin pathway. In vitro, RACK1 knockout exacerbated cell apoptosis, while overexpression of RACK1 reversed this process, enhancing mitochondrial function. In vivo, RACK1 overexpression reduced cerebral infarct volume and improved neurological deficits. The regulatory role of RACK1 depended on the PINK1/Parkin pathway, with RACK1 knockout inhibiting PINK1 and Parkin expression, while RACK1 overexpression restored them. CONCLUSION: This study demonstrates that RACK1 safeguards against neural damage in CIRI by promoting mitochondrial autophagy through the PINK1/Parkin pathway. These findings offer crucial insights into the regulation of mitochondrial autophagy and cell apoptosis by RACK1, providing a promising foundation for future CIRI treatments.

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.

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.

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.

Enhancing adipose tissue functionality in obesity: senotherapeutics, autophagy and cellular senescence as a target.

Obesity, a global health crisis, disrupts multiple systemic processes, contributing to a cascade of metabolic dysfunctions by promoting the pathological expansion of visceral adipose tissue (VAT). This expansion is characterized by impaired differentiation of pre-adipocytes and an increase in senescent cells, leading to a pro-inflammatory state and exacerbated oxidative stress. Particularly, the senescence-associated secretory phenotype (SASP) and adipose tissue hypoxia further impair cellular function, promoting chronic disease development. This review delves into the potential of autophagy modulation and the therapeutic application of senolytics and senomorphics as novel strategies to mitigate adipose tissue senescence. By exploring the intricate mechanisms underlying adipocyte dysfunction and the emerging role of natural compounds in senescence modulation, we underscore the promising horizon of senotherapeutics in restoring adipose health. This approach not only offers a pathway to combat the metabolic complications of obesity, but also opens new avenues for enhancing life quality and managing the global burden of obesity-related conditions. Our analysis aims to bridge the gap between current scientific progress and clinical application, offering new perspectives on preventing and treating obesity-induced adipose dysfunction.

ATG10-dependent autophagy is required for DDX10 to regulate cell proliferation, apoptosis and stemness in colorectal cancer.

Colorectal cancer (CRC) remains a highly prevalent gastrointestinal neoplasm, presenting significant prevalence and lethality rate. DEAD/H box RNA helicase 10 (DDX10) has been proposed as a potential oncogene in CRC, the specific action mechanism by which DDX10 modulates the aggressive biological cellular events in CRC remains implicitly elucidated, however. During this study, DDX10 expression was detected via RT-qPCR and Western blotting. Cell proliferation was estimated via EDU staining. TUNEL staining and Western blotting appraised cell apoptosis. Cell stemness was evaluated by sphere formation assay, RT-qPCR, Western blotting as well as immunofluorescence staining. Relevant assay kit examined aldehyde dehydrogenase (ALDH) activity. Western blotting and immunofluorescence staining also detected autophagy. DDX10 was hyper-expressed in CRC cells. Down-regulation of DDX10 hampered cell proliferation, aggravated the apoptosis while eliminated the ability to form spheroid cells in CRC. In addition, DDX10 deletion improved ATG10 expression and therefore activated autophagy in CRC cells. Consequently, ATG10 depletion or treatment with autophagy inhibitor 3-Methyladenine (3-MA) partially compensated the influences of DDX10 silencing on the proliferation, apoptosis and stemness of CRC cells. Accordingly, DDX10 deficiency may aggravate autophagy mediated by ATG10 to impede cell proliferation, stemness and facilitate cell apoptosis, hence blocking the progression of CRC.

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.

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.

Fluorescence Methods to Measure Pexophagy.

We outline our approach for studying the selective autophagy of peroxisomes (pexophagy), using fluorescence microscopy in tissue cell culture models. Ratiometric reporters, which specifically localize to peroxisomes, allow a quantitative assessment of pexophagy in fixed and live cells, as well as whole organisms. We discuss chemical and physiological inducers of pexophagy and any overlap with the induction of mitophagy.

Microscopy Analysis of Autophagosomal Structures in Plant Cells.

Macroautophagy, hereafter autophagy, plays a crucial role in the degradation of harmful or unwanted cellular components through a double-membrane autophagosome. Upon autophagosome fusion with the vacuole, the degraded materials are subsequently recycled to generate macromolecules, contributing to cellular homeostasis, metabolism, and stress tolerance in plants. A hallmark during autophagy is the formation of isolation membrane structure named as phagophore, which undergoes multiple steps to become as a complete double-membrane autophagosome. Methodologies have been developed in recent years to observe and quantify the autophagic process, which greatly advance knowledge of autophagosome biogenesis in plant cells. In this chapter, we will introduce two methods to dissect the autophagosome-related structures in the Arabidopsis plant cells, including the correlative light and electron microscopy, to map the ultrastructural feature of autophagosomal structures, and time-lapse imaging to monitor the temporal recruitment of autophagy machinery during autophagosome formation.

Studying Autophagy and Senescence of Arabidopsis Stigmatic Cells.

The pistil is the most important organ for fertilization in flowering plants, and the stigmatic papilla cells are responsible for pollen acceptance and pollen tube germination. Arabidopsis plants possess dry stigmas exhibiting high selectivity for compatible pollen. When compatible pollens are recognized and accepted by stigmatic papilla cells, water and nutrients are then transported from the stigma to pollen grains through the secretory pathway. Here, we present light microscopy-based methods for investigating autophagy and senescence of stigmatic papilla cells. These methods include the assessment of viability of stigmatic papilla cells using dual staining with fluorescein diacetate/propidium iodide, as well as the examination of vacuolar-accumulated proteins during stigma senescence. These methods can be used to understand the functions of the stigma tissue from a subcellular perspective.

The pivotal role of dysregulated autophagy in the progression of non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is a clinicopathologic syndrome characterized by excessive fat deposition in hepatocytes and a major cause of end-stage liver disease. Autophagy is a metabolic pathway responsible for degrading cytoplasmic products and damaged organelles, playing a pivotal role in maintaining the homeostasis and functionality of hepatocytes. Recent studies have shown that pharmacological intervention to activate or restore autophagy provides benefits for liver function recovery by promoting the clearance of lipid droplets (LDs) in hepatocytes, decreasing the production of pro-inflammatory factors, and inhibiting activated hepatic stellate cells (HSCs), thus improving liver fibrosis and slowing down the progression of NAFLD. This article summarizes the physiological process of autophagy, elucidates the close relationship between NAFLD and autophagy, and discusses the effects of drugs on autophagy and signaling pathways from the perspectives of hepatocytes, kupffer cells (KCs), and HSCs to provide assistance in the clinical management of NAFLD.

Spatial Activation of Autophagy in Human Placenta-Related Tissue During Labor: A Possible Mechanism for Labor Onset.

INTRODUCTION: To explore the mechanisms of labor by investigating the autophagy of placental and fetal membranes tissue in normal pregnant women. METHODS: Placenta and fetal membranes were collected from women with singleton pregnancies without any medical complications and from women who delivered vaginally (labor-initiated group; L group) or by caesarean section (labor-noninitiated group; NL group). Autophagosomes were observed by transmission electron microscopy (TEM). Immunofluorescence and western blotting (WB) were used to detect protein levels of the autophagy markers LC3A and LC3B. TEM, immunohistochemistry (IHC), and WB were used to compare autophagy in different parts of the placenta and fetal membranes in the L and NL groups. The expression of LC3B/LC3A, ROCK1, and ROCK2 in the placenta of nonpregnant and pregnant rats was detected by WB and IHC. RESULTS: TEM and IHC results showed an increase in the number of autophagosomes and autophagic lysosomes in the L group, and WB results indicated an increase in the LC3B/A ratio between the placenta and fetal membranes in the L group. Autophagy was significantly increased on the maternal side of the placenta in the L group, and the level of autophagy became higher near rupture in the fetal membranes and near the point where the umbilical cord joins the placenta in the L group. The LC3B/A ratio increased and ROCK1 and ROCK2 levels decreased in postnatal rats. DISCUSSION: Autophagy can occur in the placenta and fetal membranes and its activity is higher at the onset of labor, suggesting a role in labor.

Palmitoylation of ULK1 by ZDHHC13 plays a crucial role in autophagy.

Autophagy is a highly conserved process from yeast to mammals in which intracellular materials are engulfed by a double-membrane organelle called autophagosome and degrading materials by fusing with the lysosome. The process of autophagy is regulated by sequential recruitment and function of autophagy-related (Atg) proteins. Genetic hierarchical analyses show that the ULK1 complex comprised of ULK1-FIP200-ATG13-ATG101 translocating from the cytosol to autophagosome formation sites as a most upstream ATG factor; this translocation is critical in autophagy initiation. However, how this translocation occurs remains unclear. Here, we show that ULK1 is palmitoylated by palmitoyltransferase ZDHHC13 and translocated to the autophagosome formation site upon autophagy induction. We find that the ULK1 palmitoylation is required for autophagy initiation. Moreover, the ULK1 palmitoylated enhances the phosphorylation of ATG14L, which is required for activating PI3-Kinase and producing phosphatidylinositol 3-phosphate, one of the autophagosome membrane's lipids. Our results reveal how the most upstream ULK1 complex translocates to the autophagosome formation sites during autophagy.

OTULIN's influence on neuroinflammation and pain modulation in trigeminal neuralgia.

INTRODUCTION: Trigeminal neuralgia (TN), marked by chronic pain from neural damage, is closely associated with inflammation. The role of OTULIN, a key regulator in inflammation and autophagy, is not fully understood in TN. The regulatory mechanism of OTULIN, a key protein involved in modulating inflammatory responses and autophagy processes, remains incompletely elucidated, particularly in the context of TN and neuroinflammation. METHODS: An infraorbital nerve ligation-induced rat model of TN was used. OTULIN's expression was modulated using adenovirus vectors and short hairpin RNA. The impact on pain and inflammatory responses was assessed via quantitative real-time polymerase chain reaction, western blot, immunofluorescence, and transcriptomic analysis. RESULTS: Enhanced OTULIN expression significantly increased head withdrawal thresholds and reduced pain sensitivity and neuroinflammatory markers in the model. Conversely, silencing OTULIN exacerbated pain and inflammation. Transcriptomic data revealed OTULINs influence on both inflammatory and autophagy pathways, specifically in suppressing NLR family pyrin domain containing 3 (NLRP3) inflammasome and promoting autophagy. In vitro experiments demonstrated OTULIN's inhibition of inflammatory markers in microglia and neurons. CONCLUSION: OTULIN is crucial in modulating TN, reducing neuropathic pain and neuroinflammation by activating the autophagy pathway and inhibiting the NLRP3 inflammasome.

Novel perspectives on autophagy-oxidative stress-inflammation axis in the orchestration of adipogenesis.

Adipose tissue, an indispensable organ, fulfils the pivotal role of energy storage and metabolism and is instrumental in maintaining the dynamic equilibrium of energy and health of the organism. Adipocyte hypertrophy and adipocyte hyperplasia (adipogenesis) are the two primary mechanisms of fat deposition. Mature adipocytes are obtained by differentiating mesenchymal stem cells into preadipocytes and redifferentiation. However, the mechanisms orchestrating adipogenesis remain unclear. Autophagy, an alternative cell death pathway that sustains intracellular energy homeostasis through the degradation of cellular components, is implicated in regulating adipogenesis. Furthermore, adipose tissue functions as an endocrine organ, producing various cytokines, and certain inflammatory factors, in turn, modulate autophagy and adipogenesis. Additionally, autophagy influences intracellular redox homeostasis by regulating reactive oxygen species, which play pivotal roles in adipogenesis. There is a growing interest in exploring the involvement of autophagy, inflammation, and oxidative stress in adipogenesis. The present manuscript reviews the impact of autophagy, oxidative stress, and inflammation on the regulation of adipogenesis and, for the first time, discusses their interactions during adipogenesis. An integrated analysis of the role of autophagy, inflammation and oxidative stress will contribute to elucidating the mechanisms of adipogenesis and expediting the exploration of molecular targets for treating obesity-related metabolic disorders.

Non-coding RNAs as regulators of autophagy in chondrocytes: Mechanisms and implications for osteoarthritis.

Osteoarthritis (OA) is a chronic degenerative joint disease with multiple causative factors such as aging, mechanical injury, and obesity. Autophagy is a complex dynamic process that is involved in the degradation and modification of intracellular proteins and organelles under different pathophysiological conditions. Autophagy, as a cell survival mechanism under various stress conditions, plays a key role in regulating chondrocyte life cycle metabolism and cellular homeostasis. Non-coding RNAs (ncRNAs) are heterogeneous transcripts that do not possess protein-coding functions, but they can act as effective post-transcriptional and epigenetic regulators of gene and protein expression, thus participating in numerous fundamental biological processes. Increasing evidence suggests that ncRNAs, autophagy, and their crosstalk play crucial roles in OA pathogenesis. Therefore, we summarized the complex role of autophagy in OA chondrocytes and focused on the regulatory role of ncRNAs in OA-associated autophagy to elucidate the complex pathological mechanisms of the ncRNA-autophagy network in the development of OA, thus providing new research targets for the clinical diagnosis and treatment of OA.

Mapping the landscape: A bibliometric perspective on autophagy in spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) is a severe condition that often leads to persistent damage of nerve cells and motor dysfunction. Autophagy is an intracellular system that regulates the recycling and degradation of proteins and lipids, primarily through lysosomal-dependent organelle degradation. Numerous publications have highlighted the involvement of autophagy in the secondary injury of SCI. Therefore, gaining a comprehensive understanding of autophagy research is crucial for designing effective therapies for SCI. METHODS: Dates were obtained from Web of Science, including articles and article reviews published from its inception to October 2023. VOSviewer, Citespace, and SCImago were used to visualized analysis. Bibliometric analysis was conducted using the Web of Science data, focusing on various categories such as publications, authors, journals, countries, organizations, and keywords. This analysis was aimed to summarize the knowledge map of autophagy and SCI. RESULTS: From 2009 to 2023, the number of annual publications in this field exhibited wave-like growth, with the highest number of publications recorded in 2020 (44 publications). Our analysis identified Mei Xifan as the most prolific author, while Kanno H emerged as the most influential author based on co-citations. Neuroscience Letters was found to have published the largest number of papers in this field. China was the most productive country, contributing 232 publications, and Wenzhou Medical University was the most active organization, publishing 39 papers. CONCLUSION: We demonstrated a comprehensive overview of the relationship between autophagy and SCI utilizing bibliometric tools. This article could help to enhance the understanding of the field about autophagy and SCI, foster collaboration among researchers and organizations, and identify potential therapeutic targets for treatment.