The role of autophagy in Parkinson's disease: a gender difference overview.

Recent studies have demonstrated dysregulation of the autophagy pathway in patients with Parkinson's disease (PD) and in animal models of PD, highlighting its emerging role in disease. In particular, several studies indicate that autophagy, which is an essential degradative process for the damaged protein homeostasis and the management of cell balance, can manifest significant variations according to gender. While some evidence suggests increased autophagic activation in men with PD, women may have distinct regulatory patterns. In this review, we examined the existing literature on gender differences in PD-associated autophagic processes, focusing on the autophagy related proteins (ATGs) and leucine rich repeat kinase 2 (LRRK2) genes. Also, this review would suggest that an in-depth understanding of these gender differences in autophagic processes could open new perspectives for personalized therapeutic strategies, promoting more effective and targeted management of PD.

Korean Red Ginseng exerts anti-inflammatory and autophagy-promoting activities in aged mice.

BACKGROUND: Korean Red Ginseng (KRG) is a traditional herb that has several beneficial properties including anti-aging, anti-inflammatory, and autophagy regulatory effects. However, the mechanisms of these effects are not well understood. In this report, the underlying mechanisms of anti-inflammatory and autophagy-promoting effects were investigated in aged mice treated with KRG-water extract (WE) over a long period. METHODS: The mechanisms of anti-inflammatory and autophagy-promoting activities of KRG-WE were evaluated in kidney, lung, liver, stomach, and colon of aged mice using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), quantitative RT-PCR (qRT-PCR), and western blot analysis. RESULTS: KRG-WE significantly suppressed the mRNA expression levels of inflammation-related genes such as interleukin (IL)-1ß, IL-8, tumor necrosis factor (TNF)-α, monocyte chemoattractant protein-1 (MCP-1), and IL-6 in kidney, lung, liver, stomach, and colon of the aged mice. Furthermore, KRG-WE downregulated the expression of transcription factors and their protein levels associated with inflammation in lung and kidney of aged mice. KRG-WE also increased the expression of autophagy-related genes and their protein levels in colon, liver, and stomach. CONCLUSION: The results suggest that KRG can suppress inflammatory responses and recover autophagy activity in aged mice.

Targeting autophagy using small-molecule compounds to improve potential therapy of Parkinson's disease.

Parkinson's disease (PD), known as one of the most universal neurodegenerative diseases, is a serious threat to the health of the elderly. The current treatment has been demonstrated to relieve symptoms, and the discovery of new small-molecule compounds has been regarded as a promising strategy. Of note, the homeostasis of the autolysosome pathway (ALP) is closely associated with PD, and impaired autophagy may cause the death of neurons and thereby accelerating the progress of PD. Thus, pharmacological targeting autophagy with small-molecule compounds has been drawn a rising attention so far. In this review, we focus on summarizing several autophagy-associated targets, such as AMPK, mTORC1, ULK1, IMPase, LRRK2, beclin-1, TFEB, GCase, ERRα, C-Abelson, and as well as their relevant small-molecule compounds in PD models, which will shed light on a clue on exploiting more potential targeted small-molecule drugs tracking PD treatment in the near future.

Cholesterol-associated lysosomal disorder triggers cell death of hematological malignancy: Dynamic analysis on cytotoxic effects of LW-218.

The integrity of lysosomes is of vital importance to survival of tumor cells. We demonstrated that LW-218, a synthetic flavonoid, induced rapid lysosomal enlargement accompanied with lysosomal membrane permeabilization in hematological malignancy. LW-218-induced lysosomal damage and lysosome-dependent cell death were mediated by cathepsin D, as the lysosomal damage and cell apoptosis could be suppressed by depletion of cathepsin D or lysosome alkalization agents, which can alter the activity of cathepsins. Lysophagy, was initiated for cell self-rescue after LW-218 treatment and correlated with calcium release and nuclei translocation of transcription factor EB. LW-218 treatment enhanced the expression of autophagy-related genes which could be inhibited by intracellular calcium chelator. Sustained exposure to LW-218 exhausted the lysosomal capacity so as to repress the normal autophagy. LW-218-induced enlargement and damage of lysosomes were triggered by abnormal cholesterol deposition on lysosome membrane which caused by interaction between LW-218 and NPC intracellular cholesterol transporter 1. Moreover, LW-218 inhibited the leukemia cell growth in vivo. Thus, the necessary impact of integral lysosomal function in cell rescue and death were illustrated.

Natural compounds modulate the autophagy with potential implication of stroke.

Stroke is considered a leading cause of mortality and neurological disability, which puts a huge burden on individuals and the community. To date, effective therapy for stroke has been limited by its complex pathological mechanisms. Autophagy refers to an intracellular degrading process with the involvement of lysosomes. Autophagy plays a critical role in maintaining the homeostasis and survival of cells by eliminating damaged or non-essential cellular constituents. Increasing evidence support that autophagy protects neuronal cells from ischemic injury. However, under certain circumstances, autophagy activation induces cell death and aggravates ischemic brain injury. Diverse naturally derived compounds have been found to modulate autophagy and exert neuroprotection against stroke. In the present work, we have reviewed recent advances in naturally derived compounds that regulate autophagy and discussed their potential application in stroke treatment.

Multi-omics approaches identify SF3B3 and SIRT3 as candidate autophagic regulators and druggable targets in invasive breast carcinoma.

Autophagy is a critical cellular homeostatic mechanism, and its dysfunction is linked to invasive breast carcinoma (BRCA). Recently, several omics methods have been applied to explore autophagic regulators in BRCA; however, more reliable and robust approaches for identifying crucial regulators and druggable targets remain to be discovered. Thus, we report here the results of multi-omics approaches to identify potential autophagic regulators in BRCA, including gene expression (EXP), DNA methylation (MET) and copy number alterations (CNAs) from The Cancer Genome Atlas (TCGA). Newly identified candidate genes, such as SF3B3, TRAPPC10, SIRT3, MTERFD1, and FBXO5, were confirmed to be involved in the positive or negative regulation of autophagy in BRCA. SF3B3 was identified firstly as a negative autophagic regulator, and siRNA/shRNA-SF3B3 were shown to induce autophagy-associated cell death in in vitro and in vivo breast cancer models. Moreover, a novel small-molecule activator of SIRT3, 1-methylbenzylamino amiodarone, was discovered to induce autophagy in vitro and in vivo. Together, these results provide multi-omics approaches to identify some key candidate autophagic regulators, such as the negative regulator SF3B3 and positive regulator SIRT3 in BRCA, and highlight SF3B3 and SIRT3 as new druggable targets that could be used to fill the gap between autophagy and cancer drug development.

Recommendations for the use of the acetaminophen hepatotoxicity model for mechanistic studies and how to avoid common pitfalls.

Acetaminophen (APAP) is a widely used analgesic and antipyretic drug, which is safe at therapeutic doses but can cause severe liver injury and even liver failure after overdoses. The mouse model of APAP hepatotoxicity recapitulates closely the human pathophysiology. As a result, this clinically relevant model is frequently used to study mechanisms of drug-induced liver injury and even more so to test potential therapeutic interventions. However, the complexity of the model requires a thorough understanding of the pathophysiology to obtain valid results and mechanistic information that is translatable to the clinic. However, many studies using this model are flawed, which jeopardizes the scientific and clinical relevance. The purpose of this review is to provide a framework of the model where mechanistically sound and clinically relevant data can be obtained. The discussion provides insight into the injury mechanisms and how to study it including the critical roles of drug metabolism, mitochondrial dysfunction, necrotic cell death, autophagy and the sterile inflammatory response. In addition, the most frequently made mistakes when using this model are discussed. Thus, considering these recommendations when studying APAP hepatotoxicity will facilitate the discovery of more clinically relevant interventions.

Natural compounds modulate the autophagy with potential implication of stroke

Stroke is considered a leading cause of mortality and neurological disability, which puts a huge burden on individuals and the community. To date, effective therapy for stroke has been limited by its complex pathological mechanisms. Autophagy refers to an intracellular degrading process with the involvement of lysosomes. Autophagy plays a critical role in maintaining the homeostasis and survival of cells by eliminating damaged or non-essential cellular constituents. Increasing evidence support that autophagy protects neuronal cells from ischemic injury. However, under certain circumstances, autophagy activation induces cell death and aggravates ischemic brain injury. Diverse naturally derived compounds have been found to modulate autophagy and exert neuroprotection against stroke. In the present work, we have reviewed recent advances in naturally derived compounds that regulate autophagy and discussed their potential application in stroke treatment.

Multi-omics approaches identify

Autophagy is a critical cellular homeostatic mechanism, and its dysfunction is linked to invasive breast carcinoma (BRCA). Recently, several omics methods have been applied to explore autophagic regulators in BRCA; however, more reliable and robust approaches for identifying crucial regulators and druggable targets remain to be discovered. Thus, we report here the results of multi-omics approaches to identify potential autophagic regulators in BRCA, including gene expression (EXP), DNA methylation (MET) and copy number alterations (CNAs) from The Cancer Genome Atlas (TCGA). Newly identified candidate genes, such as

Recommendations for the use of the acetaminophen hepatotoxicity model for mechanistic studies and how to avoid common pitfalls

Acetaminophen (APAP) is a widely used analgesic and antipyretic drug, which is safe at therapeutic doses but can cause severe liver injury and even liver failure after overdoses. The mouse model of APAP hepatotoxicity recapitulates closely the human pathophysiology. As a result, this clinically relevant model is frequently used to study mechanisms of drug-induced liver injury and even more so to test potential therapeutic interventions. However, the complexity of the model requires a thorough understanding of the pathophysiology to obtain valid results and mechanistic information that is translatable to the clinic. However, many studies using this model are flawed, which jeopardizes the scientific and clinical relevance. The purpose of this review is to provide a framework of the model where mechanistically sound and clinically relevant data can be obtained. The discussion provides insight into the injury mechanisms and how to study it including the critical roles of drug metabolism, mitochondrial dysfunction, necrotic cell death, autophagy and the sterile inflammatory response. In addition, the most frequently made mistakes when using this model are discussed. Thus, considering these recommendations when studying APAP hepatotoxicity will facilitate the discovery of more clinically relevant interventions.

Cholesterol-associated lysosomal disorder triggers cell death of hematological malignancy: Dynamic analysis on cytotoxic effects of LW-218

The integrity of lysosomes is of vital importance to survival of tumor cells. We demonstrated that LW-218, a synthetic flavonoid, induced rapid lysosomal enlargement accompanied with lysosomal membrane permeabilization in hematological malignancy. LW-218-induced lysosomal damage and lysosome-dependent cell death were mediated by cathepsin D, as the lysosomal damage and cell apoptosis could be suppressed by depletion of cathepsin D or lysosome alkalization agents, which can alter the activity of cathepsins. Lysophagy, was initiated for cell self-rescue after LW-218 treatment and correlated with calcium release and nuclei translocation of transcription factor EB. LW-218 treatment enhanced the expression of autophagy-related genes which could be inhibited by intracellular calcium chelator. Sustained exposure to LW-218 exhausted the lysosomal capacity so as to repress the normal autophagy. LW-218-induced enlargement and damage of lysosomes were triggered by abnormal cholesterol deposition on lysosome membrane which caused by interaction between LW-218 and NPC intracellular cholesterol transporter 1. Moreover, LW-218 inhibited the leukemia cell growth

Autosis: A New Target to Prevent Cell Death.

Excessive autophagy induces a defined form of cell death called autosis, which is characterized by unique morphological features, including ballooning of perinuclear space and biochemical features, including sensitivity to cardiac glycosides. Autosis is observed during the late phase of reperfusion after a period of ischemia and contributes to myocardial injury. This review discusses unique features of autosis, the involvement of autosis in myocardial injury, and the molecular mechanism of autosis. Because autosis promotes myocardial injury under some conditions, a better understanding of autosis may lead to development of novel interventions to protect the heart against myocardial stress.

Secretory Autophagy and Its Relevance in Metabolic and Degenerative Disease.

Proteins to be secreted through so-called "conventional mechanisms" are characterized by the presence of an N-terminal peptide that is a leader or signal peptide, needed for access to the endoplasmic reticulum and the Golgi apparatus for further secretion. However, some relevant cytosolic proteins lack of this signal peptides and should be secreted by different unconventional or "non-canonical" processes. One form of this unconventional secretion was named secretory autophagy (SA) because it is specifically associated with the autophagy pathway. It is defined by ATG proteins that regulate the biogenesis of the autophagosome, its representative organelle. The canonical macroautophagy involves the fusion of the autophagosomes with lysosomes for content degradation, whereas the SA pathway bypasses this degradative process to allow the secretion. ATG5, as well as other factors involved in autophagy such as BCN1, are also activated as part of the secretory pathway. SA has been recognized as a new mechanism that is becoming of increasing relevance to explain the unconventional secretion of a series of cytosolic proteins that have critical biological importance. Also, SA may play a role in the release of aggregation-prone protein since it has been related to the autophagosome biogenesis machinery. SA requires the autophagic pathway and both, secretory autophagy and canonical degradative autophagy are at the same time, integrated and highly regulated processes that interact in ultimate cross-talking molecular mechanisms. The potential implications of alterations in SA, its cargos, pathways, and regulation in human diseases such as metabolic/aging pathological processes are predictable. Further research of SA as potential target of therapeutic intervention is deserved.

Targeting autophagy-related protein kinases for potential therapeutic purpose.

Autophagy, defined as a scavenging process of protein aggregates and damaged organelles mediated by lysosomes, plays a significant role in the quality control of macromolecules and organelles. Since protein kinases are integral to the autophagy process, it is critically important to understand the role of kinases in autophagic regulation. At present, intervention of autophagic processes by small-molecule modulators targeting specific kinases has becoming a reasonable and prevalent strategy for treating several varieties of human disease, especially cancer. In this review, we describe the role of some autophagy-related kinase targets and kinase-mediated phosphorylation mechanisms in autophagy regulation. We also summarize the small-molecule kinase inhibitors/activators of these targets, highlighting the opportunities of these new therapeutic agents.

RNA-binding protein, human antigen R regulates hypoxia-induced autophagy by targeting ATG7/ATG16L1 expressions and autophagosome formation.

Autophagy, a prosurvival mechanism offers a protective role during acute kidney injury. We show novel findings on the functional role of RNA binding protein, HuR during hypoxia-induced autophagy in renal proximal tubular cells-2 (HK-2). HK-2 cells showed upregulated expressions of HuR and autophagy-related proteins such as autophagy related 7 (ATG7), autophagy related 16 like 1 (ATG16L1), and LC3II under hypoxia. Increased autophagosome formation was visualized as LC3 puncta in hypoxic cells. Further, short hairpin-RNA-mediated loss of HuR function in HK-2 cells significantly decreased ATG7 and ATG16L1 protein expressions. Bioinformatics prediction revealed HuR motif binding on the coding region of ATG7 and AU-rich element at 3'UTR ATG16L1 messnger RNA (mRNA). The RNA immunoprecipitation study showed that HuR was predominantly associated with ATG7 and ATG16L1 mRNAs under hypoxia. In addition, HuR enhanced autophagosome formation by regulating LC3II expressions. These results show that HuR regulates ATG7 and ATG16L1 expressions and thereby mediate autophagy in HK-2 cells. Importantly, HuR knockdown cells underwent apoptosis during hypoxia as observed through the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Collectively, these findings show the crucial role of HuR under hypoxia by regulating autophagy and suppressing apoptosis in renal tubular cells.

Impaired glucose tolerance attenuates bone accrual by promoting the maturation of osteoblasts: Role of Beclin1-mediated autophagy.

Patients with type 2 diabetes mellitus (T2DM) experience a 1.5-3.5 fold increase in fracture risk, but the mechanisms responsible for these alterations in bone biomechanical properties remain elusive. Macroautophagy, often referred to as autophagy, is regulated by signaling downstream of the insulin receptor. Metabolic changes associated with the progression of glucose intolerance have been shown to alter autophagy in various tissues, but limited information is available in relation to bone cells. The aim of this study was to (a) investigate whether autophagy is altered in bone tissue during impaired glucose tolerance, and (b) determine how autophagy impacts osteoblast differentiation, activity, and maturation. Four-week-old, male C57BL/6 mice were fed a control (Con) or high fat (HF) diet for 2, 8, or 16 wks. Mice on the HF diet demonstrated elevated fasting blood glucose and impaired glucose tolerance. Reduced trabecular bone in the femoral neck was evident in the mice on the HF diet by 8 wks compared to Con mice. Histological evaluation of the tibia suggested that the high fat diet promoted terminal differentiation of the osteoblast to an osteocyte. This shift of the osteoblasts towards a non-mineralizing, osteocyte phenotype appears to be coordinated by Beclin1-mediated autophagy. Consistent with these changes in the osteoblast in vivo, the induction of autophagy was able to direct MC3T3-E1 cells towards a more mature osteoblast phenotype. Although these data are somewhat observational, further investigation is warranted to determine if Beclin1-mediated autophagy is essential for the terminal differentiation of the osteoblasts and whether autophagy is having a protective or deleterious effect on bone in T2DM.

Role of farnesoid X receptor and bile acids in alcoholic liver disease.

Alcoholic liver disease (ALD) is one of the major causes of liver morbidity and mortality worldwide. Chronic alcohol consumption leads to development of liver pathogenesis encompassing steatosis, inflammation, fibrosis, cirrhosis, and in extreme cases, hepatocellular carcinoma. Moreover, ALD may also associate with cholestasis. Emerging evidence now suggests that farnesoid X receptor (FXR) and bile acids also play important roles in ALD. In this review, we discuss the effects of alcohol consumption on FXR, bile acids and gut microbiome as well as their impacts on ALD. Moreover, we summarize the findings on FXR, FoxO3a (forkhead box-containing protein class O3a) and PPARα (peroxisome proliferator-activated receptor alpha) in regulation of autophagy-related gene transcription program and liver injury in response to alcohol exposure.

TNFAIP3 promotes survival of CD4 T cells by restricting MTOR and promoting autophagy.

Autophagy plays important roles in metabolism, differentiation, and survival in T cells. TNFAIP3/A20 is a ubiquitin-editing enzyme that is thought to be a negative regulator of autophagy in cell lines. However, the role of TNFAIP3 in autophagy remains unclear. To determine whether TNFAIP3 regulates autophagy in CD4 T cells, we first analyzed Tnfaip3-deficient naïve CD4 T cells in vitro. We demonstrated that Tnfaip3-deficient CD4 T cells exhibited reduced MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) puncta formation, increased mitochondrial content, and exaggerated reactive oxygen species (ROS) production. These results indicate that TNFAIP3 promotes autophagy after T cell receptor (TCR) stimulation in CD4 T cells. We then investigated the mechanism by which TNFAIP3 promotes autophagy signaling. We found that TNFAIP3 bound to the MTOR (mechanistic target of rapamycin) complex and that Tnfaip3-deficient cells displayed enhanced ubiquitination of the MTOR complex and MTOR activity. To confirm the effects of enhanced MTOR activity in Tnfaip3-deficient cells, we analyzed cell survival following treatment with Torin1, an MTOR inhibitor. Tnfaip3-deficient CD4 T cells exhibited fewer cell numbers than the control cells in vitro and in vivo. In addition, the impaired survival of Tnfaip3-deficient cells was ameliorated with Torin1 treatment in vitro and in vivo. The effect of Torin1 was abolished by Atg5 deficiency. Thus, enhanced MTOR activity regulates the survival of Tnfaip3-deficient CD4 T cells. Taken together, our findings illustrate that TNFAIP3 restricts MTOR signaling and promotes autophagy, providing new insight into the manner in which MTOR and autophagy regulate survival in CD4 T cells.

Identification of a lung cancer cell line deficient in atg7-dependent autophagy.

Autophagy is a major cellular process for bulk degradation of proteins and organelles in order to maintain metabolic homeostasis, and it represents an emerging target area for cancer. Initially proposed to be a cancer-restricting process for tumor initiation, recent studies suggest that autophagy can also promote cell survival in established tumors. ATG7 is an essential autophagy gene that encodes the E1 enzyme necessary for the lipidation of the LC3 family of ubiquitin-like proteins and autophagosome formation. In this study we identified a rare case of a cancer cell line, H1650 lung adenocarcinoma, which has lost ATG7 expression due to a focal biallelic deletion within the ATG7 locus. These cells displayed no evidence of ATG7 pathway activity; however, reconstituting the cells with wild-type ATG7 restored both LC3 lipidation and downstream autophagic consumption of autophagy substrates such as the SQSTM1/p62 protein. We characterized several phenotypes reported to be influenced by autophagy, and observed an ATG7-dependent increase in cell growth and clearance of proteasome-inhibitor induced protein aggregates. Cellular changes in mitochondrial metabolism or response to nutrient starvation were unaffected by ATG7 expression. In addition, parental H1650 cells that lacked ATG7 were still able to consume autophagy substrates SQSTM1, NBR1 and TAX1BP1 via a bafilomycin A1-sensitive pathway, suggesting that these proteins were not exclusively degraded by autophagy. Overall, these findings highlight a unique outlier instance of complete loss of ATG7-dependent autophagy in a cancer cell line. The H1650 cell line may be a useful system for future studies to further understand the role of autophagy in tumorigenesis and potential redundant pathways that allow cells to circumvent the loss of ATG7-dependent autophagy in cancer.

Divergent roles of BECN1 in LC3 lipidation and autophagosomal function.

BECN1/Beclin 1 is regarded as a critical component in the class III phosphatidylinositol 3-kinase (PtdIns3K) complex to trigger autophagy in mammalian cells. Despite its significant role in a number of cellular and physiological processes, the exact function of BECN1 in autophagy remains controversial. Here we created a BECN1 knockout human cell line using the TALEN technique. Surprisingly, the complete loss of BECN1 had little effect on LC3 (MAP1LC3B/LC3B) lipidation, and LC3B puncta resembling autophagosomes by fluorescence microscopy were still evident albeit significantly smaller than those in the wild-type cells. Electron microscopy (EM) analysis revealed that BECN1 deficiency led to malformed autophagosome-like structures containing multiple layers of membranes under amino acid starvation. We further confirmed that the PtdIns3K complex activity and autophagy flux were disrupted in BECN1(-/-) cells. Our results demonstrate the essential role of BECN1 in the functional formation of autophagosomes, but not in LC3B lipidation.