Ya-Ping Yang

Application and interpretation of current autophagy inhibitors and activators

Autophagy is the major intracellular degradation system, by which cytoplasmic materials are delivered to and degraded in the lysosome. As a quality control mechanism for cytoplasmic proteins and organelles, autophagy plays important roles in a variety of human diseases, including neurodegenerative diseases, cancer, cardiovascular disease, diabetes and infectious and inflammatory diseases. The discovery of ATG genes and the dissection of the signaling pathways involved in regulating autophagy have greatly enriched our knowledge on the occurrence and development of this lysosomal degradation pathway. In addition to its role in degradation, autophagy may also promote a type of programmed cell death that is different from apoptosis, termed type II programmed cell death. Owing to the dual roles of autophagy in cell death and the specificity of diseases, the exact mechanisms of autophagy in various diseases require more investigation. The application of autophagy inhibitors and activators will help us understand the regulation of autophagy in human diseases, and provide insight into the use of autophagy-targeted drugs. In this review, we summarize the latest research on autophagy inhibitors and activators and discuss the possibility of their application in human disease therapy.

Paeoniflorin, a potent natural compound, protects PC12 cells from MPP+ and acidic damage via autophagic pathway

ETHNOPHARMACOLOGICAL RELEVANCEnPaeoniflorin (PF) is the principal bioactive component of Radix Paeoniae alba, which is widely used in Traditional Chinese Medicine for the treatment of neurodegenerative disorders such as Parkinsons disease (PD).nnnAIM OF THE STUDYnTo evaluate the neuroprotective effects of PF on MPP(+)- or acid- (pH 5.0) induced injury in cultured PC12 cells and to investigate the activity of autophagy-lysosome pathway (ALP). Amiloride (Ami), a non-selective blocker of acid-sensing ion channels (ASICs), as a positive control drug, since it is neuroprotective in rodent models of PD.nnnMATERIALS AND METHODSnThe cell viability was analyzed with MTT assay. The cell injury was assessed by lactate dehydrogenase (LDH) assay. Flow cytometry and Western blot analysis were used to study the apoptotic, calcium influx and autophagic mechanisms.nnnRESULTSnAmi (100 microM) and PF (50 microM) both protected PC12 cells against MPP(+)- or acid-induced injury as assessed by MTT assay, lactate dehydrogenase release, and apoptosis rate. The concentrations of cytosolic free Ca(2+) were raised after exposure to MPP(+) or acidosis, while Ami and PF both reduced the influx of Ca(2+). More importantly, we found that the mechanisms of neuroprotective effects of Ami and PF were closely associated with the upregulation of LC3-II protein, which is specifically associated with autophagic vacuole membranes. Furthermore, application of MPP(+) or acid induced the overexpression of LAMP2a, which is directly correlated with the activity of the chaperone-mediated autophagy pathway. However, Ami and PF inhibited the overexpression of LAMP2a.nnnCONCLUSIONSnOur data provide the first experimental evidence that PF modulates autophagy in models of neuron injury, as well as providing the first indication of a relationship between ASICs and ALP.

Protective role of autophagy in AGE-induced early injury of human vascular endothelial cells.

Advanced glycation end-products (AGEs) contribute to the pathogenesis of diabetes mellitus and atherosclerosis by promoting vascular endothelial cell proliferation, migration, damage and death. In this study, we examined the role of autophagy in HUVECs exposed to AGE-modified bovine serum albumin (AGE-BSA). HUVECs incubated with AGE-BSA for 6 h showed an increase in the formation of acidic vesicular organelles and autophagosomes. AGE-BSA-induced upregulation of microtubule associated protein 1 light chain 3-II (LC3-II), a marker of autophagy, was abolished by pretreatment with the autophagy inhibitor 3-methyladenine (3-MA), and was increased by rapamycin, an autophagy inducer. The increase of lactate dehydrogenase (LDH) leakage induced by AGE-BSA was increased by 3-MA, but not rapamycin. An oxidative inhibitor, α-tocopherol, decreased not only the AGE-BSA-induced increase of reactive oxygen species, but also the upregulation of LC3-II protein levels. These results suggest that AGE-BSA increases the level of autophagy, which is protective against HUVEC injury, and that ROS play a role in this activation of autophagy.

dl-3-n-Butylphthalide prevents oxidative damage and reduces mitochondrial dysfunction in an MPP+-induced cellular model of Parkinson's disease

The aim of the present study was to explore the neuroprotective effects and mechanisms of action of dl-3-n-butylphthalide (NBP) in a 1-methyl-4-phenylpyridiniumion (MPP(+))-induced cellular model of Parkinsons disease (PD). NBP was extracted from seeds of Apium graveolens Linn. (Chinese celery). MPP(+) treatment of PC12 cells caused reduced viability, formation of reactive oxygen, and disruption of mitochondrial membrane potential. Our results indicated that NBP reduced the cytotoxicity of MPP(+) by suppressing the mitochondrial permeability transition, reducing oxidative stress, and increasing the cellular GSH content. NBP also reduced the accumulation of alpha-synuclein, the main component of Lewy bodies. Given that NBP is safe and currently used in clinical trials for stroke patients, NBP will likely be a promising chemical for the treatment of PD.

HDAC6 regulates aggresome-autophagy degradation pathway of α-synuclein in response to MPP+-induced stress.

J. Neurochem. (2011) 117, 112–120.

ASICs mediate the modulatory effect by paeoniflorin on alpha-synuclein autophagic degradation

Acid-sensing ion channels (ASICs) are ligand-gated cation channels that respond to acidic stimuli. They are expressed throughout the mammalian nervous system. Complex subunit combinations and lack of specific blockers of native receptors result in the difficulty of resolving the functions of ASICs. In this study, we showed that rat pheochromocytoma cells (PC12 cells) functionally express ASICs with the activity of endogenous proton-gated conductance. PF is the principal active ingredient extracted from the root of Paeoniae alba, a Chinese herb commonly used to treat neurodegenerative disorders, especially PD. It was found that PF significantly up regulated the expression of LC3-II, which is specifically associated with autophagic vacuole membranes. PF also reduced the MPP(+) and acidosis-induced accumulation of α-synuclein, the major component of Lewy bodies. Moreover, PF was highly efficacious in modulating ASICs activity and protein expression. In addition, the data showed that PF was able to protect PC12 cells against MPP(+) and acidosis-induced cytotoxicity. In summary, these findings demonstrate for the first time that PF could enhance the autophagic degradation of α-synuclein by regulating the expression and activity of ASICs and thus produces protective effects against cytotoxicity. It also offers the experimental evidence for the potential role of ASICs in the pathogenesis of PD.

Effects of IL-6 secreted from astrocytes on the survival of dopaminergic neurons in lipopolysaccharide-induced inflammation

The role of astrocytes in microglia-induced neuronal death remains controversial. In this study, astrocytes and astrocyte-derived conditioned media (ACM) supported the survival of dopaminergic neurons, and the former was more effective than the latter. In the presence of astrocytes, low concentrations of LPS enhanced the survival of dopaminergic neurons, while high concentrations attenuated survival. LPS dramatically induced astrocytes to secrete IL-6 in a dose-dependent manner with no effect on secretion of GDNF. Neuron-astrocyte cultures had highest secretion of GDNF, followed by ACM-treated neuron-enriched cultures. After neuron-astrocyte cultures treated with IL-6-neutralizing antibody, both effects of the enhanced and attenuated survival of dopaminergic neurons were abolished. Our results indicate that astrocytes play a protective role in the LPS-induced damage of dopaminergic neurons in certain circumstances, and the interaction between astrocytes and dopaminergic neurons may enhance the protective effect of astrocytes. Suitable activation of astrocytes increases the protective effect while excessive activation attenuates it, and IL-6 might mediate this dual action. The underlying mechanisms related to the secretion of GDNF and proinflammatory factors warrant further investigation.

Crosstalk between the proteasome system and autophagy in the clearance of α-synuclein.

Aim:A growing body of evidence suggests that α-synuclein accumulation may play an important role in the pathogenesis of Parkinsons disease. The aim of this study was to investigate the roles of the proteasome and autophagy pathways in the clearance of wild-type and mutant α-synuclein in PC12 cells.Methods:PC12 cells overexpressing either wild-type or A30P mutant α-synuclein were treated with the proteasome inhibitor epoxomicin, the macroautophagy inhibitor 3-MA and the macroautophagy activator rapamycin alone or in combination. The cell viability was assessed using MTT assay. Immunofluorescence and Western blot analysis were used to detect the level of α-synuclein, LAMP-2A, E1 activase, and E2 ligase in the cells. Chymotrypsin-like proteasomal activity was measured using a commercial kit.Results:When the proteasome and macroautophagy in the wild-type and mutant cells were inhibited with epoxomicin and 3-MA, respectively, the cell viability was significantly decreased, and the α-synuclein level was increased. Both epoxomicin and 3-MA activated the chaperone-mediated autophagy (CMA) by increasing the level of the CMA-limiting enzyme LAMP-2A. Furthermore, 3-MA or epoxomicin significantly decreased chymotrypsin-like proteasomal activity. 3-MA or epoxomicin did not change E1 activase expression in either mutant or wild-type cells, but increased E2 ligase expression, especially when used together. Macroautophagy inducer rapamycin increased the cell viability and reduced epoxomicin-induced α-synuclein accumulation. Interestingly, CMA was also activated by rapamycin.Conclusion:Our results demonstrate the existence of complex crosstalk between different forms of autophagy and between autophagy and the proteasome pathway in the clearance of α-synuclein in PC12 cells.

TNF compromises lysosome acidification and reduces α-synuclein degradation via autophagy in dopaminergic cells

Tumor necrosis factor-α (TNF) is increasingly implicated as a critical pro-inflammatory cytokine involved in chronic inflammation and neurodegeneration of Parkinsons disease (PD). However, the cellular and molecular events that lead to dopaminergic neuron degeneration are not fully understood. In this study, we demonstrated that microglia-released and recombinant TNF disrupted α-synuclein (α-SYN) degradation and caused its accumulation in PC12 cells and midbrain neurons. At subtoxic doses, recombinant TNF was found to increase the number of LC3 puncta dots and LC3II protein level, associated with the increases of P62 protein level. Inhibition of lysosomal degradation with Bafilomycin A1 pretreatment abrogated the TNF-induced elevation in LC3II protein level whereas autophagy inhibitor 3-methyladenine did not affect it. Moreover, TNF led to a marked increase in the number of yellow LC3 dots with a marginal elevation in red-only dots in RFP-GFP-tandem fluorescent LC3 (tf-LC3) transfected PC12 cells, implying the impairment in autophagic flux. Furthermore, TNF treatment reduced lysosomal acidification, as LysoTracker Red fluorescence and LysoSensor fluorescence shift from blue to yellow was markedly decreased in TNF-treated PC12 cells. Co-treatment with mammalian target of rapamycin kinase complex 1 (mTORC1) inhibitor PP242, which activated transcription factor EB (TFEB) signaling and lysosome biogenesis, partially rescued the accumulation of α-SYN in PC12 cells and midbrain neurons. Taken together, our results demonstrated that at subtoxic levels, TNF was able to impair autophagic flux and result in α-SYN accumulation by compromising lysosomal acidification in dopaminergic cells. This may represent a novel mechanism for TNF-induced dopaminergic neuron degeneration in PD.

A pivotal role of FOS-mediated BECN1/Beclin 1 upregulation in dopamine D2 and D3 receptor agonist-induced autophagy activation

Autophagy dysfunction is implicated in the pathogenesis of Parkinson disease (PD). BECN1/Beclin 1 acts as a critical regulator of autophagy and other cellular processes; yet, little is known about the function and regulation of BECN1 in PD. In this study, we report that dopamine D2 and D3 receptor (DRD2 and DRD3) activation by pramipexole and quinpirole could enhance BECN1 transcription and promote autophagy activation in several cell lines, including PC12, MES23.5 and differentiated SH-SY5Y cells, and also in tyrosine hydroxylase positive primary midbrain neurons. Moreover, we identified a novel FOS (FBJ murine osteosarcoma viral oncogene homolog) binding sequence (5′-TGCCTCA-3′) in the rat and human Becn1/BECN1 promoter and uncovered an essential role of FOS binding in the enhancement of Becn1 transcription in PC12 cells in response to the dopamine agonist(s). In addition, we demonstrated a critical role of intracellular Ca2+ elevation, followed by the enhanced phosphorylation of CAMK4 (calcium/calmodulin-dependent protein kinase IV) and CREB (cAMP responsive element binding protein) in the increases of FOS expression and autophagy activity. More importantly, pramipexole treatment ameliorated the SNCA/α-synuclein accumulation in rotenone-treated PC12 cells that overexpress wild-type or A53T mutant SNCA by promoting autophagy flux. This effect was also demonstrated in the substantia nigra and the striatum of SNCAA53T transgenic mice. The inhibition of SNCA accumulation by pramipexole was attenuated by cotreatment with the DRD2 and DRD3 antagonists and Becn1 siRNAs. Thus, our findings suggest that DRD2 and DRD3 agonist(s) may induce autophagy activation via a BECN1-dependent pathway and have the potential to reduce SNCA accumulation in PD.