Cholesteryl Hemiazelate Present in Cardiovascular Disease Patients Causes Lysosome Dysfunction in Murine Fibroblasts.

There is growing evidence supporting the role of fibroblasts in all stages of atherosclerosis, from the initial phase to fibrous cap and plaque formation. In the arterial wall, as with macrophages and vascular smooth muscle cells, fibroblasts are exposed to a myriad of LDL lipids, including the lipid species formed during the oxidation of their polyunsaturated fatty acids of cholesteryl esters (PUFA-CEs). Recently, our group identified the final oxidation products of the PUFA-CEs, cholesteryl hemiesters (ChE), in tissues from cardiovascular disease patients. Cholesteryl hemiazelate (ChA), the most prevalent lipid of this family, is sufficient to impact lysosome function in macrophages and vascular smooth muscle cells, with consequences for their homeostasis. Here, we show that the lysosomal compartment of ChA-treated fibroblasts also becomes dysfunctional. Indeed, fibroblasts exposed to ChA exhibited a perinuclear accumulation of enlarged lysosomes full of neutral lipids. However, this outcome did not trigger de novo lysosome biogenesis, and only the lysosomal transcription factor E3 (TFE3) was slightly transcriptionally upregulated. As a consequence, autophagy was inhibited, probably via mTORC1 activation, culminating in fibroblasts' apoptosis. Our findings suggest that the impairment of lysosome function and autophagy and the induction of apoptosis in fibroblasts may represent an additional mechanism by which ChA can contribute to the progression of atherosclerosis.

Spatiotemporal roles of AMPK in PARP-1- and autophagy-dependent retinal pigment epithelial cell death caused by UVA.

BACKGROUND: Although stimulating autophagy caused by UV has been widely demonstrated in skin cells to exert cell protection, it remains unknown the cellular events in UVA-treated retinal pigment epithelial (RPE) cells. METHODS: Human ARPE-19 cells were used to measure cell viability, mitochondrial reactive oxygen species (ROS), mitochondrial membrane potential (MMP), mitochondrial mass and lysosomal mass by flow cytometry. Mitochondrial oxygen consumption rate (OCR) was recorded using Seahorse XF flux analyzer. Confocal microscopic images were performed to indicate the mitochondrial dynamics, LC3 level, and AMPK translocation after UVA irradiation. RESULTS: We confirmed mitochondrial ROS production and DNA damage are two major features caused by UVA. We found the cell death is prevented by autophagy inhibitor 3-methyladenine and gene silencing of ATG5, and UVA induces ROS-dependent LC3II expression, LC3 punctate and TFEB expression, suggesting the autophagic death in the UVA-stressed RPE cells. Although PARP-1 inhibitor olaparib increases DNA damage, ROS production, and cell death, it also blocks AMPK activation caused by UVA. Interestingly we found a dramatic nuclear export of AMPK upon UVA irradiation which is blocked by N-acetylcysteine and olaparib. In addition, UVA exposure gradually decreases lysosomal mass and inhibits cathepsin B activity at late phase due to lysosomal dysfunction. Nevertheless, cathepsin B inhibitor, CA-074Me, reverses the death extent, suggesting the contribution of cathepsin B in the death pathway. When examining the role of EGFR in cellular events caused by UVA, we found that UVA can rapidly transactivate EGFR, and treatment with EGFR TKIs (gefitinib and afatinib) enhances the cell death accompanied by the increased LC3II formation, ROS production, loss of MMP and mass of mitochondria and lysosomes. Although AMPK activation by ROS-PARP-1 mediates autophagic cell death, we surprisingly found that pretreatment of cells with AMPK activators (A769662 and metformin) reverses cell death. Concomitantly, both agents block UVA-induced mitochondrial ROS production, autophagic flux, and mitochondrial fission without changing the inhibition of cathepsin B. CONCLUSION: UVA exposure rapidly induces ROS-PARP-1-AMPK-autophagic flux and late lysosomal dysfunction. Pre-inducing AMPK activation can prevent cellular events caused by UVA and provide a new protective strategy in photo-oxidative stress and photo-retinopathy.

The Combination of Gold and Silver Food Nanoparticles with Gluten Peptides Alters the Autophagic Pathway in Intestinal Crypt-like Cells.

Metallic nanoparticles (mNPs) are widely used as food additives and can interact with gliadin triggering an immune response, but evaluation of the effects on crypts, hypertrophic in celiac subjects, is still lacking. This study evaluated the effects of gold and silver mNPs in combination with gliadin on crypt-like cells (HIEC-6). Transmission electron microscopy (TEM) was used to evaluate gliadin-mNP aggregates in cells. Western blot and immunofluorescence analysis assessed autophagy-related molecule levels (p62, LC3, beclin-1, EGFR). Lysosome functionality was tested with acridine orange (AO) and Magic Red assays. TEM identified an increase in autophagic vacuoles after exposure to gliadin + mNPs, as also detected by significant increments in LC3-II and p62 expression. Immunofluorescence confirmed the presence of mature autophagosomes, showing LC3 and p62 colocalization, indicating an altered autophagic flux, further assessed with EGFR degradation, AO and Magic Red assays. The results showed a significant reduction in lysosomal enzyme activity and a modest reduction in acidity. Thus, gliadin + mNPs can block the autophagic flux inducing a lysosomal defect. The alteration of this pathway, essential for cell function, can lead to cell damage and death. The potential effects of this copresence in food should be further characterized to avoid a negative impact on celiac disease subjects.

The Pathological Mechanism of Neuronal Autophagy-Lysosome Dysfunction After Ischemic Stroke.

The abnormal initiation of autophagy flux in neurons after ischemic stroke caused dysfunction of autophagy-lysosome, which not only led to autophagy flux blockage, but also resulted in autophagic death of neurons. However, the pathological mechanism of neuronal autophagy-lysosome dysfunction did not form a unified viewpoint until now. In this review, taking the autophagy lysosomal dysfunction of neurons as a starting point, we summarized the molecular mechanisms that led to neuronal autophagy lysosomal dysfunction after ischemic stroke, which would provide theoretical basis for the clinical treatment of ischemic stroke.

Oxidized cholesteryl ester induces exocytosis of dysfunctional lysosomes in lipidotic macrophages.

A key event in atherogenesis is the formation of lipid-loaded macrophages, lipidotic cells, which exhibit irreversible accumulation of undigested modified low-density lipoproteins (LDL) in lysosomes. This event culminates in the loss of cell homeostasis, inflammation, and cell death. Nevertheless, the exact chemical etiology of atherogenesis and the molecular and cellular mechanisms responsible for the impairment of lysosome function in plaque macrophages are still unknown. Here, we demonstrate that macrophages exposed to cholesteryl hemiazelate (ChA), one of the most prevalent products of LDL-derived cholesteryl ester oxidation, exhibit enlarged peripheral dysfunctional lysosomes full of undigested ChA and neutral lipids. Both lysosome area and accumulation of neutral lipids are partially irreversible. Interestingly, the dysfunctional peripheral lysosomes are more prone to fuse with the plasma membrane, secreting their undigested luminal content into the extracellular milieu with potential consequences for the pathology. We further demonstrate that this phenotype is mechanistically linked to the nuclear translocation of the MiT/TFE family of transcription factors. The induction of lysosome biogenesis by ChA appears to partially protect macrophages from lipid-induced cytotoxicity. In sum, our data show that ChA is involved in the etiology of lysosome dysfunction and promotes the exocytosis of these organelles. This latter event is a new mechanism that may be important in the pathogenesis of atherosclerosis.

Cell-autonomous lipid-handling defects in Stargardt iPSC-derived retinal pigment epithelium cells.

Stargardt retinopathy is an inherited form of macular degeneration caused by mutations in gene ABCA4 and characterized by the accumulation of lipid-rich deposits in the retinal pigment epithelium (RPE), RPE atrophy, and photoreceptor cell death. Inadequate mechanistic insights into pathophysiological changes occurring in Stargardt RPE have hindered disease treatments. Here, we show that ABCA4 knockout and induced pluripotent stem cell-derived RPE (STGD1-iRPE) from patients with Stargardt differentiate normally but display intracellular lipid and ceramide deposits reminiscent of the disease phenotype. STGD1-iRPE also shows defective photoreceptor outer segment (POS) processing and reduced cathepsin B activity-indicating higher lysosomal pH. Lipid deposits in STGD1-iRPE are lowered by increasing the activity of ABCA1, a lipid transporter, and ABCA4 ortholog. Our work suggests that ABCA4 is involved in POS and lipid handling in RPE cells and provides guidance for ongoing gene therapy approaches to target both RPE and photoreceptor cells for an effective treatment.

Gold nanoparticles targeting the autophagy-lysosome system to combat the inflammation-compromised osteogenic potential of periodontal ligament stem cells: From mechanism to therapy.

Although substantial data indicate that the osteogenic potential of periodontal ligament stem cells (PDLSCs) is compromised under inflammatory conditions, the underlying mechanism remains largely unexplored. In this study, we found that both the autophagy levels and autophagic flux levels were decreased in PDLSCs incubated under inflammatory conditions (I-PDLSCs). Based on the increased expression of LC3 II (at an autophagy level) and decreased accumulation of LC3 II (at an autophagic flux level) in I-PDLSCs, we speculated that the disruption of I-PDLSC autophagy arose from dysfunction of the cellular autophagy-lysosome system. Subsequently, our hypothesis was demonstrated by inhibited autophagosome-lysosome fusion, damaged lysosomal function, and suppressed activation of transcription factor EB (TFEB, a master regulator of the autophagy-lysosome system) in I-PDLSCs and verified by TFEB overexpression in I-PDLSCs. We found that gold nanoparticle (Au NP) treatment rescued the osteogenic potential of I-PDLSCs by restoring the inflammation-compromised autophagy-lysosome system. In this context, Au NP ceased to be effective when TFEB was knocked down in PDLSCs. Our data demonstrate the crucial role of the autophagy-lysosome system in cellular osteogenesis under inflammatory conditions and suggest a new target for rescuing inflammation-induced cell dysfunction using nanomaterials to aid cell biology and tissue regeneration.

Time-dependent changes in autophagy, mitophagy and lysosomes in skeletal muscle during denervation-induced disuse.

Deficits in skeletal muscle mitochondrial content and quality are observed following denervation-atrophy. This is due to alterations in the biogenesis of new mitochondria as well as their degradation via mitophagy. The regulation of autophagy and mitophagy over the course of denervation (Den) remains unknown. Further, the time-dependent changes in lysosome content, the end-stage organelle for mitophagy, remain unexplored. Here, we studied autophagic as well as mitophagic pre-lysosomal flux in subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria from rat muscle subjected to Den for 1, 3 or 7 days. We also assessed flux at 1 day post-denervation in transgenic mt-keima mice. Markers of mitochondrial content were reduced at 7 days following Den, and Den further resulted in rapid decrements in mitochondrial respiration, along with increased ROS emission. Pre-lysosomal autophagy flux was upregulated at 1 and 3 days post-Den but was reduced compared to time-matched sham-operated controls at 7 days post-Den. Similarly, pre-lysosomal mitophagy flux was enhanced in SS mitochondria as early as 1 and 3 days of Den but decreased in both SS and IMF subfractions following 7 days of Den. Lysosome protein content and transcriptional regulators TFEB and TFE3 were progressively enhanced with Den, an adaptation designed to enhance autophagic capacity. However, evidence for lysosome dysfunction was apparent by 7 days, which may limit degradation capacity. This may contribute to an inability to clear dysfunctional mitochondria and increased ROS signalling, thereby accelerating muscle atrophy. Thus, therapeutic targeting of lysosome function may help to maintain autophagy and muscle health during conditions of muscle disuse or denervation. KEY POINTS: Denervation is an experimental model of peripheral neuropathies as well as muscle disuse, and it helps us understand some aspects of the sarcopenia of ageing. Muscle disuse is associated with reduced mitochondrial content and function, leading to metabolic impairments within the tissue. Although the processes that regulate mitochondrial biogenesis are understood, those that govern mitochondrial breakdown (i.e. mitophagy) are not well characterized in this context. Autophagy and mitophagy flux, measured up to the point of the lysosome (pre-lysosomal flux rates), were increased in the early stages of denervation, along with mitochondrial dysfunction, but were reduced at later time points when the degree of muscle atrophy was highest. Denervation led to progressive increases in lysosomal proteins to accommodate mitophagy flux, yet evidence for lysosomal impairment at later stages may limit the removal of dysfunctional mitochondria, stimulate reactive oxygen species signalling, and reduce muscle health as denervation time progresses.

Cholesteryl hemiazelate causes lysosome dysfunction impacting vascular smooth muscle cell homeostasis.

In atherosclerotic lesions, vascular smooth muscle cells (VSMCs) represent half of the foam cell population, which is characterized by an aberrant accumulation of undigested lipids within lysosomes. Loss of lysosome function impacts VSMC homeostasis and disease progression. Understanding the molecular mechanisms underlying lysosome dysfunction in these cells is, therefore, crucial. We identify cholesteryl hemiazelate (ChA), a stable oxidation end-product of cholesteryl-polyunsaturated fatty acid esters, as an inducer of lysosome malfunction in VSMCs. ChA-treated VSMCs acquire a foam-cell-like phenotype, characterized by enlarged lysosomes full of ChA and neutral lipids. The lysosomes are perinuclear and exhibit degradative capacity and cargo exit defects. Lysosome luminal pH is also altered. Even though the transcriptional response machinery and autophagy are not activated by ChA, the addition of recombinant lysosomal acid lipase (LAL) is able to rescue lysosome dysfunction. ChA significantly affects VSMC proliferation and migration, impacting atherosclerosis. In summary, this work shows that ChA is sufficient to induce lysosomal dysfunction in VSMCs, that, in ChA-treated VSMCs, neither lysosome biogenesis nor autophagy are triggered, and, finally, that recombinant LAL can be a therapeutic approach for lysosomal dysfunction.

Torin 1 alleviates impairment of TFEB-mediated lysosomal biogenesis and autophagy in TGFBI (p.G623_H626del)-linked Thiel-Behnke corneal dystrophy.

Thiel-Behnke corneal dystrophy (TBCD) is an epithelial-stromal TGFBI dystrophy caused by mutations in the TGFBI (transforming growth factor beta induced) gene, though the underlying mechanisms and pathogenesis of TBCD are still obscure. The study identifies a novel mutation in the TGFBI gene (p.Gly623_His626del) in a TBCD pedigree. Characteristics of the typical vacuole formation, irregular corneal epithelial thickening and thinning, deposition of eosinophilic substances beneath the epithelium, and involvement of the anterior stroma were observed in this pedigree via transmission electron microscopy (TEM) and histological staining. Tgfbi-p.Gly623_Tyr626del mouse models of TBCD were subsequently generated via CRISPR/Cas9 technology, and the above characteristics were further verified via TEM and histological staining. Lysosomal dysfunction and downregulation of differential expression protein CTSD (cathepsin D) were observed using LysoTracker Green DND-26 and proteomic analysis, respectively. Hence, lysosomal dysfunction probably leads to autophagic flux obstruction in TBCD; this was supported by enhanced LC3-II and SQSTM1 levels and decreased CTSD. TFEB (transcription factor EB) was prominently decreased in TBCD corneal fibroblasts and administration of ATP-competitive MTOR inhibitor torin 1 reversed this decline, resulting in the degradation of accumulated mut-TGFBI (mutant TGFBI protein) via the ameliorative lysosomal function and autophagic flux owing to elevated TFEB activity as measured by western blot, confocal microscopy, and flow cytometry. Transfected HEK 293 cells overexpressing human full-length WT-TGFBI and mut-TGFBI were generated to further verify the results obtained in human corneal fibroblasts. Amelioration of lysosome dysfunction may therefore have therapeutic efficacy in the treatment of TBCD.Abbreviations AS-OCT: anterior segment optical coherence tomography; ATP: adenosine triphosphate; Cas9: CRISPR-associated protein 9; CLEAR: coordinated lysosomal expression and regulation; CRISPR: clustered regularly interspaced short palindromic repeats; CTSB: cathepsin B; CTSD: cathepsin D; CTSF: cathepsin F; CTSL: cathepsin L; DNA: deoxyribonucleic acid; ECM: extracellular matrix; Fas1: fasciclin 1; FC: flow cytometry; GAPDH: glyceraldeyde-3-phosphate dehydrogenase; GCD2: granular corneal dystrophy type 2; HE: hematoxylin and eosin; LAMP2: lysosomal-associated membrane protein; MT: mutation type; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; mut-TGFBI: mutant TGFBI protein; SD: standard deviation; TBCD: Thiel-Behnke corneal dystrophy; TEM: transmission electron microscopy; TFEB: transcription factor EB; TGFBI: transforming growth factor beta induced; WT: wild type.

Lysosome (Dys)function in Atherosclerosis-A Big Weight on the Shoulders of a Small Organelle.

Atherosclerosis is a progressive insidious chronic disease that underlies most of the cardiovascular pathologies, including myocardial infarction and ischemic stroke. The malfunctioning of the lysosomal compartment has a central role in the etiology and pathogenesis of atherosclerosis. Lysosomes are the degradative organelles of mammalian cells and process endogenous and exogenous substrates in a very efficient manner. Dysfunction of these organelles and consequent inefficient degradation of modified low-density lipoproteins (LDL) and apoptotic cells in atherosclerotic lesions have, therefore, numerous deleterious consequences for cellular homeostasis and disease progression. Lysosome dysfunction has been mostly studied in the context of the inherited lysosomal storage disorders (LSDs). However, over the last years it has become increasingly evident that the consequences of this phenomenon are more far-reaching, also influencing the progression of multiple acquired human pathologies, such as neurodegenerative diseases, cancer, and cardiovascular diseases (CVDs). During the formation of atherosclerotic plaques, the lysosomal compartment of the various cells constituting the arterial wall is under severe stress, due to the tremendous amounts of lipoproteins being processed by these cells. The uncontrolled uptake of modified lipoproteins by arterial phagocytic cells, namely macrophages and vascular smooth muscle cells (VSMCs), is the initial step that triggers the pathogenic cascade culminating in the formation of atheroma. These cells become pathogenic "foam cells," which are characterized by dysfunctional lipid-laden lysosomes. Here, we summarize the current knowledge regarding the origin and impact of the malfunctioning of the lysosomal compartment in plaque cells. We further analyze how the field of LSD research may contribute with some insights to the study of CVDs, particularly how therapeutic approaches that target the lysosomes in LSDs could be applied to hamper atherosclerosis progression and associated mortality.

Lysosome dysfunction as a cause of neurodegenerative diseases: Lessons from frontotemporal dementia and amyotrophic lateral sclerosis.

Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are fatal neurodegenerative disorders that are thought to exist on a clinical and pathological spectrum. FTD and ALS are linked by shared genetic causes (e.g. C9orf72 hexanucleotide repeat expansions) and neuropathology, such as inclusions of ubiquitinated, misfolded proteins (e.g. TAR DNA-binding protein 43; TDP-43) in the CNS. Furthermore, some genes that cause FTD or ALS when mutated encode proteins that localize to the lysosome or modulate endosome-lysosome function, including lysosomal fusion, cargo trafficking, lysosomal acidification, autophagy, or TFEB activity. In this review, we summarize evidence that lysosomal dysfunction, caused by genetic mutations (e.g. C9orf72, GRN, MAPT, TMEM106B) or toxic-gain of function (e.g. aggregation of TDP-43 or tau), is an important pathogenic disease mechanism in FTD and ALS. Further studies into the normal function of many of these proteins are required and will help uncover the mechanisms that cause lysosomal dysfunction in FTD and ALS. Mutations or polymorphisms in genes that encode proteins important for endosome-lysosome function also occur in other age-dependent neurodegenerative diseases, including Alzheimer's (e.g. APOE, PSEN1, APP) and Parkinson's (e.g. GBA, LRRK2, ATP13A2) disease. A more complete understanding of the common and unique features of lysosome dysfunction across the spectrum of neurodegeneration will help guide the development of therapies for these devastating diseases.

Characterization of Fluid Biomarkers Reveals Lysosome Dysfunction and Neurodegeneration in Neuronopathic MPS II Patients.

Mucopolysaccharidosis type II is a lysosomal storage disorder caused by a deficiency of iduronate-2-sulfatase (IDS) and characterized by the accumulation of the primary storage substrate, glycosaminoglycans (GAGs). Understanding central nervous system (CNS) pathophysiology in neuronopathic MPS II (nMPS II) has been hindered by the lack of CNS biomarkers. Characterization of fluid biomarkers has been largely focused on evaluating GAGs in cerebrospinal fluid (CSF) and the periphery; however, GAG levels alone do not accurately reflect the broad cellular dysfunction in the brains of MPS II patients. We utilized a preclinical mouse model of MPS II, treated with a brain penetrant form of IDS (ETV:IDS) to establish the relationship between markers of primary storage and downstream pathway biomarkers in the brain and CSF. We extended the characterization of pathway and neurodegeneration biomarkers to nMPS II patient samples. In addition to the accumulation of CSF GAGs, nMPS II patients show elevated levels of lysosomal lipids, neurofilament light chain, and other biomarkers of neuronal damage and degeneration. Furthermore, we find that these biomarkers of downstream pathology are tightly correlated with heparan sulfate. Exploration of the responsiveness of not only CSF GAGs but also pathway and disease-relevant biomarkers during drug development will be crucial for monitoring disease progression, and the development of effective therapies for nMPS II.

Width-Consistent Mesoporous Silica Nanorods with a Precisely Controlled Aspect Ratio for Lysosome Dysfunctional Synergistic Chemotherapy/Photothermal Therapy/Starvation Therapy/Oxidative Therapy.

Although differently shaped mesoporous silica is widely studied, the formation of width-consistent mesoporous silica nanorods (MSNRs) with a precisely controlled aspect ratio (AR: length/width) is challenging and has not been reported. Herein, width-consistent (100 nm) MSNRs with ARs of 2, 3, 4, 6, 8, and 10 were obtained by increasing the concentrations while maintaining the molar ratio of cetyltrimethylammonium bromide (CTAB) and tetraethyl orthosilicate (TEOS). The results demonstrated that the as-prepared MSNR with an AR of 6 (AR6) possesses high cellular-uptake efficiency and drug-loading capacity. Thus, AR6-based cancer-cell-targeting nanosystems were designed. These nanosystems encapsulated doxorubicin (DOX) into the porous channel of AR6, adsorbed glucose oxidase (GOx), and then formed a polydopamine (PDA) layer for Siramesine (Siram, a lysosome dysfunctional drug) adsorption and folic acid modification. In this design, the PDA shell could prevent the leakage of loading components and keep the activity of GOx during delivery while achieving an on-demand drug release in the targeted location and photothermal therapy under near-infrared irradiation. The increase in temperature was highly beneficial for elevating the catalytic efficiency of GOx, accelerating the consumption of intracellular glucose, and generating a relatively high level of cytotoxic H2O2, all of which enhanced starvation and oxidative therapies. Siram was employed to inhibit lysosomal metabolism and accompany GOx to reach a dual-enhanced starvation therapy effect. In addition, DOX entered the nucleus and altered DNA for chemotherapy. The results showed that the nanosystems have superior therapeutic efficacy against cancer cells and not much toxicity to normal cells. Therefore, this study provides a novel strategy for lysosome dysfunctional synergistic chemotherapy/photothermal therapy/starvation therapy/oxidative therapy based on MSNR.

Autophagy, lysosome dysfunction and mTOR inhibition in MNU-induced photoreceptor cell damage.

Progressive photoreceptor death is the main cause of retinal degeneration diseases. Determining the underlying mechanism of this process is essential for therapy improvement. Autophagy has long been considered to be involved in neuronal degeneration diseases, and the regulation of autophagy is thought to have potential implications for neurodegenerative disease therapies. However, whether autophagy is protective or destructive varies among diseases and is controversial. In the present study, we established an N-methyl-N-nitrosourea (MNU)-induced photoreceptor cell damage model in vitro that faithfully replicated photoreceptor cell death in retinal degeneration diseases. Cell viability was tested by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assays. Reactive oxygen species (ROS) levels were assessed through 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence. Autophagy was confirmed by observing autophagosomes using transmission electron microscopy (TEM). A lysosome tracker was used to identify acidic lysosomes in cells. We also measured the expression of some proteins related to autophagy, apoptosis and lysosomal degradation by western blot and immunofluorescence assays. We found that MNU could decrease photoreceptor cell viability in a time- and dose-dependent manner, and this change was accompanied by concomitant increases in ROS and the expression of the apoptosis-inducing protein cleaved caspase-3. Moreover, autophagy was activated by MNU treatment during this process. Inhibition of autophagy with 3-methyladenine accelerated cell damage. Lysosome dysfunction was confirmed by autophagosome enlargement and increased cathepsin expression, which was accompanied by mTOR dephosphorylation. In conclusion, autophagy was activated through inhibition of the PI3K/mTOR pathway in the context of MNU-induced photoreceptor cell death. Prolonged mTOR dephosphorylation and autophagy activation resulted in autophagic vacuole accumulation, as indicated by inefficient degradation in lysosomes, and further led to apoptosis.

UBL4A inhibits autophagy-mediated proliferation and metastasis of pancreatic ductal adenocarcinoma via targeting LAMP1.

BACKGROUND: Ubiquitin-like protein 4A (UBL4A) plays a significant role in protein metabolism and the maintenance of cellular homeostasis. In cancer, UBL4A represses tumorigenesis and is involved in various signaling pathways. Pancreatic ductal adenocarcinoma (PDAC) is still a major cause of cancer-related death and the underlying molecular mechanism of UBL4A and PDAC remains unknown. METHODS: First, the prognostic role of UBL4A and its expression in human PDAC patients and in pancreatic cancer cell lines were detected by survival analysis and qRT-PCR, western blotting, and immunohistochemistry. Next, the effects of UBL4A on proliferation and metastasis in pancreatic cancer were evaluated by functional assays in vitro and in vivo. In addition, chloroquine was introduced to determine the role of autophagy in UBL4A-related tumor proliferation and metastasis. Ultimately, coimmunoprecipitation was used to confirm the interaction between UBL4A and lysosome associated membrane protein-1 (LAMP1), and western blotting was performed to explore the UBL4A mechanism. RESULTS: We found that UBL4A was decreased in PDAC and that high levels of UBL4A correlated with a favorable prognosis. We observed that UBL4A inhibited tumor proliferation and metastasis through suppression of autophagy, a critical intracellular catabolic process that reportedly protects cells from nutrient starvation and other stress conditions. UBL4A caused impaired autophagic degradation in vitro, a crucial process in autophagy, by disturbing the function of lysosomes and contributing to autophagosome accumulation. We found a positive correlation between UBL4A and LAMP1. Furthermore, UBL4A caused lysosomal dysfunction by directly interacting with LAMP1, and LAMP1 overexpression reversed the antitumor effects of UBL4A in pancreatic cancer. In addition, we demonstrated that UBL4A suppressed tumor growth and metastasis in a pancreatic orthotopic tumor model. CONCLUSIONS: These findings suggest that UBL4A exerts an antitumor effect on autophagy-related proliferation and metastasis in PDAC by directly targeting LAMP1. Herein, we describe a novel mechanism of UBL4A that suppresses the progression of pancreatic cancer. UBL4A might be a promising target for the treatment and prognostication of PDAC.

Lipid and Non-lipid Factors Affecting Macrophage Dysfunction and Inflammation in Atherosclerosis.

Atherosclerosis is a chronic inflammatory disease and a leading cause of human mortality. The lesional microenvironment contains a complex accumulation of variably oxidized lipids and cytokines. Infiltrating monocytes become polarized in response to these stimuli, resulting in a broad spectrum of macrophage phenotypes. The extent of lipid loading in macrophages influences their phenotype and consequently their inflammatory status. In response to excess atherogenic ligands, many normal cell processes become aberrant following a loss of homeostasis. This can have a direct impact upon the inflammatory response, and conversely inflammation can lead to cell dysfunction. Clear evidence for this exists in the lysosomes, endoplasmic reticulum and mitochondria of atherosclerotic macrophages, the principal lesional cell type. Furthermore, several intrinsic cell processes become dysregulated under lipidotic conditions. Therapeutic strategies aimed at restoring cell function under disease conditions are an ongoing coveted aim. Macrophages play a central role in promoting lesional inflammation, with plaque progression and stability being directly proportional to macrophage abundance. Understanding how mixtures or individual lipid species regulate macrophage biology is therefore a major area of atherosclerosis research. In this review, we will discuss how the myriad of lipid and lipoprotein classes and products used to model atherogenic, proinflammatory immune responses has facilitated a greater understanding of some of the intricacies of chronic inflammation and cell function. Despite this, lipid oxidation produces a complex mixture of products and with no single or standard method of derivatization, there exists some variation in the reported effects of certain oxidized lipids. Likewise, differences in the methods used to generate macrophages in vitro may also lead to variable responses when apparently identical lipid ligands are used. Consequently, the complexity of reported macrophage phenotypes has implications for our understanding of the metabolic pathways, processes and shifts underpinning their activation and inflammatory status. Using oxidized low density lipoproteins and its oxidized cholesteryl esters and phospholipid constituents to stimulate macrophage has been hugely valuable, however there is now an argument that only working with low complexity lipid species can deliver the most useful information to guide therapies aimed at controlling atherosclerosis and cardiovascular complications.

Parkinson's disease pathogenesis from the viewpoint of small fish models.

Parkinson's disease is a neurodegenerative disorder that involves movement discloses, degeneration of dopaminergic neurons, and presence of cytoplasmic inclusion bodies. Various animal models have been developed and small fish including zebrafish and medaka fish have recently been employed as a new model for Parkinson disease. In this review, we summarize fish models of Parkinson's disease mainly using our own findings and explain two major hypotheses of PD: lysosome dysfunction theory and mitochondrial dysfunction theory. Finally, we discuss the potential for future application of small fish model.

The Importance of Mitophagy in Maintaining Mitochondrial Function in U373MG Cells. Bafilomycin A1 Restores Aminochrome-Induced Mitochondrial Damage.

Aminochrome, an orthoquinone formed during the dopamine oxidation of neuromelanin, is neurotoxic because it induces mitochondria dysfunction, protein degradation dysfunction (both autophagy and proteasomal systems), α-synuclein aggregation to neurotoxic oligomers, neuroinflammation, and oxidative and endoplasmic reticulum stress. In this study, we investigated the relationship between aminochrome-induced autophagy/lysosome dysfunction and mitochondrial dysfunction in U373MGsiGST6 cells. Aminochrome (75 µM) induces mitochondrial dysfunction as determined by (i) a significant decrease in ATP levels (70%; P < 0.001) and (ii) a significant decrease in mitochondrial membrane potential (P < 0.001). Interestingly, the pretreatment of U373MGsiGST6 cells with 100 nM bafilomycin-A1, an inhibitor of lysosomal vacuolar-type H+-ATPase, restores ATP levels, mitochondrial membrane potential, and mitophagy, and decreases cell death. These results reveal (i) the importance of macroautophagy/the lysosomal degradation system for the normal functioning of mitochondria and for cell survival, and (ii) aminochrome-induced lysosomal dysfunction depends on the aminochrome-dependent inactivation of the vacuolar-type H+-ATPase, which pumps protons into the lysosomes. This study also supports the proposed protective role of glutathione transferase mu2-2 (GSTM2) in astrocytes against aminochrome toxicity, mediated by mitochondrial and lysosomal dysfunction.

Glutathione transferase mu 2 protects glioblastoma cells against aminochrome toxicity by preventing autophagy and lysosome dysfunction.

U373MG cells constitutively express glutathione S-transferase mu 2 (GSTM2) and exhibit (3)H-dopamine uptake, which is inhibited by 2 µM of nomifensine and 15 µM of estradiol. We generated a stable cell line (U373MGsiGST6) expressing an siRNA against GSTM2 that resulted in low GSTM2 expression (26% of wild-type U373MG cells). A significant increase in cell death was observed when U373MGsiGST6 cells were incubated with 50 µM purified aminochrome (18-fold increase) compared with wild-type cells. The incubation of U373MGsiGST6 cells with 75 µM aminochrome resulted in the formation of autophagic vacuoles containing undigested cellular components, as determined using transmission electron microscopy. A significant increase in autophagosomes was determined by measuring endogenous LC3-II, a significant decrease in cell death was observed in the presence of bafilomycin A 1, and a significant increase in cell death was observed in the presence of trehalose. A significant increase in LAMP2 immunostaining was observed, a significant decrease in bright red fluorescence of lysosomes with acridine orange was observed, and bafilomycin A 1 pretreatment reduced the loss of lysosome acidity. A significant increase in cell death was observed in the presence of lysosomal protease inhibitors. Aggregation of TUBA/α-tubulin (tubulin, α) and SQSTM1 protein accumulation were also observed. Moreover, a significant increase in the number of lipids droplets was observed compared with U373MG cells with normal expression of GSTM2. These results support the notion that GSTM2 is a protective enzyme against aminochrome toxicity in astrocytes and that aminochrome cell death in U373MGsiGST6 cells involves autophagic-lysosomal dysfunction.