Enhancement Of Autophagic Flux Research Articles

Harnessing Nanochaperone-Mediated Autophagy for Selective Clearance of Pathogenic Tau Protein in Alzheimer's Disease.

Accumulation of pathological tau is a hallmark of Alzheimer's disease (AD), which correlates more closely with cognitive impairment than does the amyloid-β (Aβ) burden. Autophagy is a powerful process for the clearance of toxic proteins including aberrant tau. However, compromised autophagy is demonstrated in neurodegeneration including AD, and current autophagy inducers remain enormously challenging due to inability of restoring autophagy pathway and lack of targeting specificity. Here, pathogenic tau-specific autophagy based on customized nanochaperone is developed for AD treatment. In this strategy, the nanochaperone can selectively bind to pathogenic tau and maintain tau homeostasis, thereby ensuring microtubule stability which is important for autophagy pathway. Meanwhile, the bound pathogenic tau can be sequestered in autophagosomes by in situ autophagy activation of nanochaperone. Consequently, autophagosomes wrapping with pathogenic tau are able to be trafficked along the stabilized microtubule to achieve successful fusion with lysosomes, resulting in the enhancement of autophagic flux and pathologic tau clearance. After treatment with this nanochaperone-mediated autophagy strategy, the tau burden, neuron damages, and cognitive deficits of AD mice are significantly alleviated in the brain. Therefore, this work represents a promising candidate for AD-targeted therapy and provides new insights into future design of anti-neurodegeneration drugs.

Molecular Mechanism of Calycosin Inhibited Vascular Calcification.

Vascular calcification (VC) is a pathological condition frequently observed in cardiovascular diseases. Primary factors contributing to VC are osteogenic differentiation of vascular smooth muscle and hydroxyapatite deposition. Targeted autophagy (a lysosome-mediated mechanism for degradation/recycling of unnecessary cellular components) is a useful approach for inhibiting VC and promoting vascular cell health. Calycosin has been shown to alleviate atherosclerosis by enhancing macrophage autophagy, but its therapeutic effect on VC has not been demonstrated. Using an in vitro model (rat thoracic aortic smooth muscle cell line A7r5), we demonstrated effective inhibition of VC using calycosin (the primary flavonoid component of astragalus), based on the enhancement of autophagic flux. Calycosin treatment activated AMPK/mTOR signaling to induce initiation of autophagy and restored mTORC1-dependent autophagosome-lysosome fusion in late-stage autophagy by promoting soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation, thereby preventing stoppage of autophagy in calcified cells. Calycosin substantially reduced degrees of both osteogenic differentiation and calcium deposition in our VC cell model by enhancing autophagy. The present findings clarify the mechanism whereby calycosin mitigates autophagy stoppage in calcified smooth muscle cells and provide a basis for effective VC treatment via autophagy enhancement.

Design, synthesis, and biological evaluation of carbonyl-hydrazine-1-carboxamide derivatives as anti-hepatic fibrosis agents targeting Nur77

Hepatic fibrosis remains a great challenge clinically. The orphan nuclear receptor Nur77 is recently suggested as the critical regulator of transforming growth factor-β (TGF-β) signaling, which plays a central role in multi-organic fibrosis. Herein, we optimized our previously reported Nur77-targeted compound 9 h for attempting to develop effective and safe anti-hepatic fibrosis agents. The critical pharmacophore scaffold of pyridine-carbonyl-hydrazine-1-carboxamide was retained, while the naphthalene ring was replaced with an aromatic ring containing pyridyl or indole groups. Four series of derivatives were thus generated, among which the compound 16f had excellent binding activity toward Nur77-LBD (KD = 470 nM) with the best inhibitory activity against the TGF- β 1 activation of hepatic stellate cells (HSCs) and low cytotoxicity to normal mice liver AML-12 cells (IC50 > 80 μM). In mice, 16f displayed potent activity against CCl4-induced liver fibrosis with improved liver function. Mechanistically, 16f-mediated inactivation of HSC and suppression of liver fibrosis were associated with its enhancement of autophagic flux in a Nur77-dependent manner. Together, 16f was identified as a potential anti-liver fibrosis agent. Our study suggests that Nur77 may serve as a critical anti-hepatic fibrosis target.

Glyphosate-induced mitochondrial reactive oxygen species overproduction activates parkin-dependent mitophagy to inhibit testosterone synthesis in mouse leydig cells

Glyphosate (GLY), one of the most extensively used herbicides in the world, has been shown to inhibit testosterone synthesis in male animals. Mitochondria are crucial organelles for testosterone synthesis and its dysfunction has been demonstrated to induce the inhibition of testosterone biosynthesis. However, whether low-dose GLY exposure targets mitochondria to inhibit testosterone synthesis and its underlying mechanism remains unclear. Here, an in vitro model of 10 μM GLY-exposed mouse Leydig (TM3) cells was established to elucidate this issue. Data firstly showed that mitochondrial malfunction, mainly manifested by ultrastructure damage, disturbance of mitochondrial dynamics and mitochondrial reactive oxygen species (mtROS) overproduction, was responsible for GLY-decreased protein levels of steroidogenic enzymes, which leads to the inhibition of testosterone synthesis. Enhancement of autophagic flux and activation of mitophagy were shown in GLY-treated TM3 cells, and further studies have revealed that GLY-activated mitophagy is parkin-dependent. Notably, GLY-inhibited testosterone production was significantly improved by parkin knockdown. Finally, data showed that treatment with mitochondria-targeted antioxidant Mito-TEMPO (M-T) markedly reversed GLY-induced mitochondrial network fragmentation, activation of parkin-dependent mitophagy and consultant testosterone reduction. Overall, these findings demonstrate that GLY induces mtROS overproduction to activate parkin-dependent mitophagy, which contributes to the inhibition of testosterone synthesis. This study provides a potential mechanistic explanation for how GLY inhibits testosterone synthesis in mouse Leydig cells.

Progranulin Promotes Functional Recovery in Rats with Acute Spinal Cord Injury via Autophagy-Induced Anti-inflammatory Microglial Polarization.

Since microglia-associated neuroinflammation plays a critical role in the progression of acute spinal cord injury, modulation of microglial activation has been suggested as a potential therapeutic strategy. Progranulin has been reported to exert neuroprotective effects by attenuating neuroinflammation, but whether these effects are due to the modulation of microglial polarization and the underlying mechanism remain unclear. Here, we investigated the effect of progranulin on microglial polarization and analyzed the crosstalk between microglial autophagy and polarization. We found that progranulin could reduce proinflammatory cytokine production at the lesion site and promote locomotor functional recovery after acute spinal cord injury. In vitro, we found that progranulin could activate microglia to acquire an anti-inflammatory phenotype and express IL-10. Moreover, progranulin-mediated enhancement of anti-inflammatory microglial polarization was attributed to the protection of lysosomal function and the enhancement of autophagic flux. Above all, progranulin exerts anti-inflammatory effects by protecting lysosomal function to enhance microglial autophagy, induce M2 microglial polarization, and ultimately improve neurological function after acute spinal cord injury. These results suggest that targeting the autophagy-lysosomal pathway to modulate microglial polarization and reduce neuroinflammation is a potential treatment for spinal cord injury.

Protective effects of 5-heptadecylresorcinol against adipocyte mitochondrial dysfunction through upregulation of Sirt3-mediated autophagy

5-heptadecylresorcinol (AR-C17), a natural phenolic lipid component and biomarker of whole grain rye consumption, has been widely reported to confer multiple health benefits. However, the molecular mechanism underlying its potential protective effect against obesity and other related diseases is still unclear. In the present study, we investigated the potential role and mechanism of AR-C17 in mitigating mitochondrial dysfunction and lipid disturbance of adipocytes both in vitro and in vivo. AR-C17 treatment alleviated inflammatory conditioned medium (CM) induced adipocyte lipolysis and mitochondrial damage, accompanied by attenuated mitochondrial reactive oxygen species production and mitochondrial membrane depolarization. Moreover, we observed improved mitochondrial content, upregulated expression of mitochondrial biogenesis related transcription factors and increased oxygen consumption rate. Meanwhile, AR-C17 attenuated CM-mediated adipocyte mitochondria dysfunction by promoting autophagy, followed by the enhancement of autophagic flux and related protein expression, such as LC3B-II/I, Beclin1, Atg5, PINK1 and Parkin. Further analysis showed that the protective effect of AR-C17 against mitochondrial dysfunction was depended on the upregulation of Sirt3-mediated autophagy. At the whole animal level, AR-C17 administration ameliorated high-fat diet induced C57BL/6J mice obesity and its associated adipose tissue macrophage infiltration and mitochondrial dysfunction. Activation of Sirt3-mediated autophagy in adipose tissue was also observed in AR-C17-treated mice. Together, these findings indicate that Sirt3-mediated autophagy plays an essential role in alleviating adipocyte mitochondrial dysfunction by AR-C17 and suggest AR-C17 as a potential dietary bioactive ingredient for obesity prevention and treatment.

Anti-Apoptosis and Autophagy Effects of Melatonin Protect Rat Chondrocytes against Oxidative Stress via Regulation of AMPK/Foxo3 Pathways

Emerging evidence has indicated that excessive reactive oxygen species (ROS) have detrimental effects on osteoarthritis (OA). This study aimed to elucidate the effects of melatonin (MT), an antioxidant indolamine secreted from the pineal gland, on chondrocyte senescence and cartilage degeneration, thereby clarifying the underlying mechanisms of ROS-induced OA pathogenesis. Hydrogen peroxide (H2O2) was used to induce oxidative stress in rat chondrocytes. ROS levels were evaluated using cytometry and immunofluorescence. Cell viability was detected using the Cell Counting Kit-8 (CCK-8) assay. Western blotting and qPCR (Quantiative Real-Time Polymerase Chain Reaction) were used to examine apoptosis and autophagy. For in vivo experiments, male Sprague-Dawley rats were randomly divided into a sham-operated group, DMM (destabilization of the medial meniscus) surgery group, and surgery groups that received melatonin. Knee joints were collected and stained for histological analysis. The data demonstrated that melatonin treatment significantly suppressed H2O2-induced matrix degradation and apoptosis, and maintained mitochondrial redox homeostasis. In addition, an enhancement of autophagic flux was observed through western blotting. These findings corresponded with activation of the AMPK/Foxo3 signaling pathways upon melatonin treatment. Histological staining and transmission electron microscopy (TEM) micrographs also demonstrated that melatonin alleviated cartilage ossification and chondrocyte hypertrophy in vivo. Our results indicated that melatonin protected chondrocytes via mitochondrial redox homeostasis and autophagy. The effects of melatonin on senescence may apply to other age-related diseases. Thus, melatonin may have multiple potential therapeutic applications.

Filamin C Cardiomyopathy Variants Cause Protein and Lysosome Accumulation.

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MicroRNA-21 suppresses ox-LDL-induced human aortic endothelial cells injuries in atherosclerosis through enhancement of autophagic flux: Involvement in promotion of lysosomal function

Atherosclerosis is a common pathological basis of cardiovascular disease and remains the leading cause of mortality. Endothelial cell (EC) injury and autophagy dysfunction have been proved to contribute to the development of atherosclerosis. Recently, accumulating evidence confirms that microRNAs (miRNAs) have emerged as vital regulators and fine-tuners of various pathophysiological cellular impacts and molecular signaling pathways involved in atherosclerosis. Herein, the objective of the present study was to explore the biological function of miR-21 in oxidized low-density lipoprotein (ox-LDL)-induced human aortic endothelial cells (HAECs) injury and the underlying molecular mechanism. The results showed that ox-LDL treatment significantly decreased HAECs viability, increased caspase-3 activity, apoptosis ratio and Bax protein expression, and reduced Bcl-2 protein expression resulting in EC injuries. Simultaneously, ox-LDL treatment obviously reduced miR-21 level in a time-and dose-dependent manner. Notably, ox-LDL-induced EC injuries were abolished by miR-21 mimics transfection. In addition, miR-21 mimics alleviated ox-LDL-induced impaired autophagic flux as illustrated by the increases in LC3-II/LC3-I ratio and Beclin-1 protein expression, and the decrease in p62 protein expression in HAECs. Moreover, ox-LDL suppressed the expressions of lysosomal membrane protein (LAMP1) and cathepsin D proteins, and attenuated cathepsin D activity in HAECs, leading to lysosomal dysfunction, while these effects were also blocked by miR-21 mimics. These findings indicated that miR-21 restored impaired autophagic flux and lysosomal dysfunction, thereby attenuating ox-LDL-induced HAECs injuries.

Dendrobium nobile Lindl alkaloid, a novel autophagy inducer, protects against axonal degeneration induced by Aβ25-35 in hippocampus neurons in vitro.

Axonal degeneration is a pathological symbol in the early stage of Alzheimer's disease (AD), which can be triggered by amyloid-β (Aβ) peptide deposition. Growing evidence indicates that deficit of autophagy eventually leads to the axonal degeneration. Our previous studies have shown that Dendrobium nobile Lindl alkaloid (DNLA) had protective effect on neuron impairment in vivo and in vitro; however, the underlying mechanisms is still unclear. We exposed cultured hippocampus neurons to Aβ25-35 to investigate the effect of DNLA in vitro. Axonal degeneration was evaluated by immunofluorescence staining and MTT assay. Neurons overexpressing GFP-LC3B were used to measure the formation of autophagosome. Autophagosome-lysosome fusion, the lysosomal pH, and cathepsin activity were assessed to reflect autophagy process. Proteins of interest were analyzed by Western blot. DNLA pretreatment significantly inhibited axonal degeneration induced by Aβ25-35 peptide in vitro. Further studies revealed DNLA treatment increased autophagic flux through promoting formation and degradation of autophagosome in hippocampus neurons. Moreover, enhancement of autophagic flux was responsible for the protective effects of DNLA on axonal degeneration. DNLA prevents Aβ25-35 -induced axonal degeneration via activation of autophagy process and could be a novel therapeutic target.

The Temporal Pattern, Flux, and Function of Autophagy in Spinal Cord Injury

Previous studies have indicated that autophagy plays a critical role in spinal cord injury (SCI), including traumatic spinal cord injury (TSCI) and ischemia-reperfusion spinal cord injury (IRSCI). However, while the understanding of mechanisms underlying autophagy in SCI has progressed, there remain several controversial points: (1) temporal pattern results of autophagic activation after SCI are not consistent across studies; (2) effect of accumulation of autophagosomes due to the blockade or enhancement of autophagic flux is uncertain; (3) overall effect of enhanced autophagy remains undefined, with both beneficial and detrimental outcomes reported in SCI literature. In this review, the temporal pattern of autophagic activation, autophagic flux, autophagic cell death, relationship between autophagy and apoptosis, and pharmacological intervention of autophagy in TSCI (contusion injury, compression injury and hemisection injury) and IRSCI are discussed. Types of SCI and severity appear to contribute to differences in outcomes regarding temporal pattern, flux, and function of autophagy. With future development of specific strategies on autophagy intervention, autophagy may play an important role in improving functional recovery in patients with SCI.

NDP52 associates with phosphorylated tau in brains of an Alzheimer disease mouse model

We previously showed that NDP52 (also known as calcoco2) plays a role as an autophagic receptor for phosphorylated tau facilitating its clearance via autophagy. Here, we examined the expression and association of NDP52 with autophagy-regulated gene (ATG) proteins including LC3, as well as phosphorylated tau and amyloid-beta (Aβ) in brains of an AD mouse model. NDP52 was expressed not only in neurons, but also in microglia and astrocytes. NDP52 co-localized with ATGs and phosphorylated tau as expected since it functions as an autophagy receptor for phosphorylated tau in brain. Compared to wild-type mice, the number of autophagic vesicles (AVs) containing NDP52 in both cortex and hippocampal regions was significantly greater in AD model mice. Moreover, the protein levels of NDP52 and phosphorylated tau together with LC3-II were also significantly increased in AD model mice, reflecting autophagy impairment in the AD mouse model. By contrast, a significant change in p62/SQSTM1 level was not observed in this AD mouse model. NDP52 was also associated with intracellular Aβ, but not with the extracellular Aβ of amyloid plaques. We conclude that NDP52 is a key autophagy receptor for phosphorylated tau in brain. Further our data provide clear evidence for autophagy impairment in brains of AD mouse model, and thus strategies that result in enhancement of autophagic flux in AD are likely to be beneficial.

Ferulic Acid Induces Mammalian Target of Rapamycin Inactivation in Cultured Mammalian Cells

Ferulic acid (FA), a naturally occurring polyphenol abundant in vegetables and rice bran, is known to possess a potent antioxidant activity, thereby protecting cells from oxidative stress. In the present study, we show that in addition to its known anti-oxidant activity, ferulic acid exerts substantial inhibitory activity on cellular mammalian target of rapamycin (mTor)-signaling pathways. In HeLa cells and mouse primary hepatocytes cultured with conventional nutrient-rich media, ferulic acid (1 mM) elicited dephosphorylation of S6 kinase and its substrate ribosomal S6. The dephosphorylating activity of ferulic acid was almost comparable to that of rapamycin, an established mTor inhibitor (TORC1). We next investigated the effect of ferulic acid on autophagy, a major cellular degradative process, which significantly contributes to the maintenance of cell homeostasis. Using a conventional green fluorescent protein-microtubule-associated protein IA/IB light chain 3 (GFP-LC3) dot assay to evaluate autophagy flux, we showed that ferulic acid caused a significant increase in GFP-LC3 dots under serum-rich conditions in HeLa cells. The enhancement of autophagic flux by ferulic acid was almost equivalent to that of rapamycin. Furthermore, ferulic acid significantly enhanced autophagic degradation of (14)C-leucine-labeled long-lived proteins of cultured mouse hepatocytes under nutrient-rich conditions, but not nutrient-deprived conditions. These results indicate that ferulic acid is almost the equivalent of rapamycin in the ability to inhibit mTor (TORC1), which makes it a potent activator of basal autophagy.

Enhancement of Autophagic Flux after Neonatal Cerebral Hypoxia-Ischemia and Its Region-Specific Relationship to Apoptotic Mechanisms

The multiplicity of cell death mechanisms induced by neonatal hypoxia-ischemia makes neuroprotective treatment against neonatal asphyxia more difficult to achieve. Whereas the roles of apoptosis and necrosis in such conditions have been studied intensively, the implication of autophagic cell death has only recently been considered. Here, we used the most clinically relevant rodent model of perinatal asphyxia to investigate the involvement of autophagy in hypoxic-ischemic brain injury. Seven-day-old rats underwent permanent ligation of the right common carotid artery, followed by 2 hours of hypoxia. This condition not only increased autophagosomal abundance (increase in microtubule-associated protein 1 light chain 3-11 level and punctuate labeling) but also lysosomal activities (cathepsin D, acid phosphatase, and beta-N-acetylhexosaminidase) in cortical and hippocampal CA3-damaged neurons at 6 and 24 hours, demonstrating an increase in the autophagic flux. In the cortex, this enhanced autophagy may be related to apoptosis since some neurons presenting a high level of autophagy also expressed apoptotic features, including cleaved caspase-3. On the other hand, enhanced autophagy in CA3 was associated with a more purely autophagic cell death phenotype. In striking contrast to CA3 neurons, those in CA1 presented only a minimal increase in autophagy but strong apoptotic characteristics. These results suggest a role of enhanced autophagy in delayed neuronal death after severe hypoxia-ischemia that is differentially linked to apoptosis according to the cerebral region.