Removal Of Protein Aggregates Research Articles

Targeting Autophagy for the Treatment of Alzheimer's Disease: Challenges and Opportunities.

Alzheimer’s disease (AD) is the most common type of dementia which characterized by a progressive loss of memory and cognitive function due to degeneration of synapses and axons. Currently, there is no cure for AD. Deposition of extracellular amyloid-β (Aβ) plaques and intracellular tau neurofibrillary tangles (NFTs) are two hallmark pathologic changes in the brains of Alzheimer’s patients. Autophagy is the major mechanism in cells responsible for removing protein aggregates. Accumulation of immature autophagic vacuoles (AVs) in dystrophic neurites of Alzheimer patients’ brains suggests that autophagy process is disrupted. Till now, it is far from clear what role autophagy plays in AD, a causative role, a protective role, or just a consequence of the disease process itself. To design more effective therapeutic strategies towards this devastating disorder, it is essential to understand the exact role of autophagy played during different stages of AD.

Abstract 909: Determining the Role of the Co-chaperone BAG3 at the Cardiac Myofilament

Bcl-2-associated athanogene 3 (BAG3) is an adaptor protein often associated with protein quality control. In the heart, decreased BAG3 expression is linked to dilated cardiomyopathy. Mutations to BAG3 can result in myofibrillar disarray, indicating a possible role at the sarcomere. One previous study found BAG3 has a role in maintaining sarcomere structure, however this was in neonatal myocytes. Whether BAG3 has any role in the myofilament of the adult myocyte is unknown. To determine if BAG3 is present in the cardiac sarcomere and identify the myofilament-BAG3 interactome, we used co-immunoprecipitation of the myofilament fraction from mouse LV and mass spectrometry. This revealed a robust interaction of BAG3 with the myofilament. Furthermore, BAG3 interacted with a heat shock protein (HSPB8) known to assist in the removal of protein aggregates, which are abundant after MI. To determine the mechanistic role of BAG3, we overexpressed the protein in mice hearts using adeno-associated virus 9. At baseline, overexpression caused a slight decrease in skinned myocyte calcium sensitivity compared to WT. This effect may be due to BAG3’s interaction with β-adrenergic receptors, which can cause PKA phosphorylation of thin filament proteins known to reduce calcium sensitivity. Preliminary data support this. We then exposed these mice to left anterior descending coronary artery ligation to induce MI. Compared to shams, MI decreased maximum calcium activated force (F max ) in the WT mice, but BAG3 overexpression restored F max to near sham levels. We also studied the functional impact of a known disease-causing mutation discovered in humans, P209L. Transgenic overexpression of P209L BAG3 had no effect on F max , calcium sensitivity, or cooperativity, suggesting the mutation may not interfere with domains in BAG3 required for its myofilament role. This work is the first to show a functional role for BAG3 in the adult myofilament. However, our findings reveal a complex interaction in which the effect of altered BAG3 expression varies with context. The P209L mutation findings suggest that not all BAG3 mutations have shared phenotypes, a conclusion supported by recent clinical work. Further research is necessary to understand BAG3’s myofilament role in health and disease.

Abstract 349: Beclin1 as a Critical Regulator of the Endosomal Degradation Pathway

Effective cellular quality control is crucial for cellular homeostasis and cardiac health. Defects in the pathways that regulate the removal of damaged proteins and organelles contribute to heart failure. Autophagy is well known for its role in removing protein aggregates and organelles; however, we recently found that the small GTPase Rab5 and early endosomes also participate in Parkin-mediated mitochondrial clearance. Beclin1 is a scaffolding protein which can form distinct PI3K protein complexes to regulate different processes such as autophagy. Here, we have investigated the role of Beclin1 in regulating the endosomal degradation pathway in mice. We have generated inducible cardiac-specific Beclin1 KO mice to investigate the in vivo role of Beclin1 in myocytes. We found significant contractile dysfunction as early as one week after deletion of Beclin1, which led to increased mortality. Loss of Beclin1 led to rapid development of cardiac hypertrophy as characterized by increased Myh7, Nppa, Nppb , myocyte size, and heart weight/body weight. Furthermore, we found that loss of Beclin1 led to increased inflammatory markers Il6 and Tgfb1 , as well as increased DNA damage/apoptosis in the heart. Ultrastructural analysis revealed Beclin1 deficiency led to significant myocardial disarray, demonstrating the importance of Beclin1 for proper myocyte alignment. These mice also had reduced autophagic flux but increased IGFR-beta receptor protein levels, indicating defects in both the autophagy and endosomal degradation pathways. Additionally, immunofluorescence analysis revealed that Beclin1 colocalizes with Rab5 at the early endosome in cells, and coimmunoprecipitation experiments confirmed their interaction. Together, these data establish the essential role of Beclin1 in cellular homeostasis as a central regulator of endosomal activity.

Transcription Factor EB Activation Rescues Advanced αB-Crystallin Mutation-Induced Cardiomyopathy by Normalizing Desmin Localization.

BackgroundMutations in αB‐crystallin result in proteotoxic cardiomyopathy with desmin mislocalization to protein aggregates. Intermittent fasting (IF) is a novel approach to activate transcription factor EB (TFEB), a master regulator of the autophagy‐lysosomal pathway, in the myocardium. We tested whether TFEB activation can be harnessed to treat advanced proteotoxic cardiomyopathy.Methods and ResultsMice overexpressing the R120G mutant of αB‐crystallin in cardiomyocytes (Myh6‐CryABR120G) were subjected to IF or ad‐lib feeding, or transduced with adeno‐associated virus–TFEB or adeno‐associated virus—green fluorescent protein after development of advanced proteotoxic cardiomyopathy. Adeno‐associated virus–short hairpin RNA–mediated knockdown of TFEB and HSPB8 was performed simultaneously with IF. Myh6‐CryABR120G mice demonstrated impaired autophagic flux, reduced lysosome abundance, and mammalian target of rapamycin activation in the myocardium. IF resulted in mammalian target of rapamycin inhibition and nuclear translocation of TFEB with restored lysosome abundance and autophagic flux; and reduced aggregates with normalized desmin localization. IF also attenuated left ventricular dilation and myocardial hypertrophy, increased percentage fractional shortening, and increased survival. Adeno‐associated virus–TFEB transduction was sufficient to rescue cardiomyopathic manifestations, and resulted in reduced aggregates and normalized desmin localization in Myh6‐CryABR120G mice. CryABR120G‐expressing hearts demonstrated increased interaction of desmin with αB‐crystallin and reduced interaction with chaperone protein, HSPB8, compared with wild type, which was reversed by both IF and TFEB transduction. TFEB stimulated autophagic flux to remove protein aggregates and transcriptionally upregulated HSPB8, to restore normal desmin localization in CryABR120G‐expressing cardiomyocytes. Short hairpin RNA–mediated knockdown of TFEB and HSPB8 abrogated IF effects, in vivo.ConclusionsIF and TFEB activation are clinically relevant therapeutic strategies to rescue advanced R120G αB‐crystallin mutant‐induced cardiomyopathy by normalizing desmin localization via autophagy‐dependent and autophagy‐independent mechanisms.

MicroRNA-29 enhances autophagy and cleanses exogenous mutant αB-crystallin in retinal pigment epithelial cells

Retinal pigment epithelial cells (RPEs), a pigmented cell layer in the outer retina, are constantly exposed to photo-oxidative stress. Autophagy relieves the stress by removing oxidative protein adducts, protein aggregates, and damaged mitochondria. We previously found that miR-29 is downregulated in choroid/RPE tissue in a model of exudative age-related macular degeneration (AMD), suggesting that miR-29 deficiency may contribute to autophagy inhibition and AMD progression. Here we wanted to test whether overexpression of miR-29 in RPEs could enhance autophagy, thereby facilitating removal of drusen components. Indeed, overexpression of miR-29 in the RPEs increased autophagy, assessed by decreased protein levels of p62, increased lipid form of microtubule-associated protein light chain (LC3-II), and elevated autophagy flux. Furthermore, overexpression of miR-29 mitigated the formation of mutant αB-crystallin (R120G) protein aggregates. In probing the mechanism, we demonstrated that miR-29 post-transcriptionally repressed LAMPTOR1/p18 via targeting its 3’-UTRs of messenger RNA. MiR-29 overexpression and knockdown of LAMPTOR1/p18 led to limited mTORC1 recruitment to lysosomes and inhibition of mTORC1 activity. Altogether, miR-29 enhances autophagy which aids in removal of protein aggregates. These findings reveal a novel role of miR-29, which has the potential of being a therapeutic strategy for rescuing RPE degeneration in ocular disorders.

Abstract 577: Beclin1 Regulates UVRAG and Rab5-Mediated Endosomal Degradation Pathway

Effective cellular quality control is crucial for cellular homeostasis and cardiac health. Defects in the pathways that regulate the removal of damaged proteins and organelles contribute to heart failure. Autophagy is well known for its role in removing protein aggregates and organelles; however, we recently found that the small GTPase Rab5 and early endosomes also participate in Parkin-mediated mitochondrial clearance. Beclin1 is a scaffolding protein which can form distinct PI3K protein complexes to regulate different processes. Here we have investigated the role of Beclin1 in regulating the endosomal degradation pathway in cells. Utilizing Beclin1-deficient mouse embryonic fibroblasts (MEFs), we assessed the effect of Beclin1 deficiency on endosomal activity. We found that the number of Rab5+ early endosomes were decreased, while EGF receptor levels were increased in Beclin1-/- MEFs compared to WT. This suggests that endosomal-mediated degradation is reduced in the absence of Beclin1. Moreover, the constitutively active Rab5Q79L is unable to hydrolyze bound GTP and its overexpression leads to oversized endosomes in WT cells. Interestingly, Rab5Q79L protein was unstable in Beclin1-/- MEFs and failed to induce formation of enlarged endosomes. Beclin1 has also been reported to form a complex with UVRAG, but the exact function of the Beclin1-UVRAG complex is unclear. We found that Beclin1-/- MEFs had significantly decreased UVRAG protein levels. Unexpectedly, restoration of UVRAG, but not Beclin1, restored Rab5Q79L stability in Beclin1-/- MEFs. Endogenous Rab5 levels were unaffected in Beclin1-/- MEFs after UVRAG restoration, indicating that UVRAG is required to stabilize the GTP bound active Rab5. Finally, to examine the function of Beclin1 in myocytes in vivo , we generated inducible cardiac-specific Beclin1 KO mice. Within one week of deleting Beclin1 in the adult heart, we observed significant contractile dysfunction and increased mortality. These mice also had reduced autophagic flux and significantly decreased UVRAG protein levels, but increased IGFR-beta receptor protein levels. Together, these data demonstrate the critical role of Beclin1 in cellular homeostasis as a regulator of not only autophagy but also endosomal activities.

Lactoferrin is a natural inhibitor of plasminogen activation

The plasminogen system is essential for dissolution of fibrin clots, and in addition, it is involved in a wide variety of other physiological processes, including proteolytic activation of growth factors, cell migration, and removal of protein aggregates. On the other hand, uncontrolled plasminogen activation contributes to many pathological processes (e.g. tumor cells' invasion in cancer progression). Moreover, some virulent bacterial species (e.g. Streptococci or Borrelia) bind human plasminogen and hijack the host's plasminogen system to penetrate tissue barriers. Thus, the conversion of plasminogen to the active serine protease plasmin must be tightly regulated. Here, we show that human lactoferrin, an iron-binding milk glycoprotein, blocks plasminogen activation on the cell surface by direct binding to human plasminogen. We mapped the mutual binding sites to the N-terminal region of lactoferrin, encompassed also in the bioactive peptide lactoferricin, and kringle 5 of plasminogen. Finally, lactoferrin blocked tumor cell invasion in vitro and also plasminogen activation driven by Borrelia Our results explain many diverse biological properties of lactoferrin and also suggest that lactoferrin may be useful as a potential tool for therapeutic interventions to prevent both invasive malignant cells and virulent bacteria from penetrating host tissues.

DNA-damage inducible protein 1 is a conserved metacaspase substrate that is cleaved and further destabilized in yeast under specific metabolic conditions.

Metacaspases, distant relatives of metazoan caspases, have been shown to participate in programmed cell death in plants and in progression of the cell cycle and removal of protein aggregates in unicellular eukaryotes. However, since natural proteolytic substrates have scarcely been identified to date, their roles in these processes remain unclear. Here, we report that the DNA-damage inducible protein 1 (Ddi1) represents a conserved protein substrate for metacaspases belonging to divergent unicellular eukaryotes (trypanosomes and yeasts). We show that although the recognized cleavage sequence is not identical among the different model organisms tested, in all of them the proteolysis consequence is the removal of the ubiquitin-associated domain (UBA) present in the protein. We also demonstrate that Ddi1 cleavage is tightly regulated in vivo as it only takes place in yeast when calcium increases but under specific metabolic conditions. Finally, we show that metacaspase-mediated Ddi1 cleavage reduces the stability of this protein which can certainly impact on the many functions ascribed for it, including shuttle to the proteasome, cell cycle control, late secretory pathway regulation, among others.

Aberrant subcellular localization of SQSTM1/p62 contributes to increased vulnerability to proteotoxic stress recovery in Huntington's disease

Proteotoxic stress plays an important role in the pathogenesis of Huntington's disease (HD). Autophagy is proposed as a compensatory mechanism to remove protein aggregates under proteotoxic stress by up-regulating p62 expression. In the present study, we investigated the molecular action of p62 to proteotoxic stress in HD cells. Using two different HD cellular models, STHdhQ7 and STHdhQ111 cells derived from wild type and HD knock-in mice and human fibroblasts from healthy and HD patients, we found that HD cells are more vulnerable to cell death under proteotoxic stress and during stress recovery. We further showed that P62 was up-regulated in both STHdhQ7 and STHdhQ111 cells in response to the stress with distinct subcellular localization patterns. While dispersed p62 puncti were found in STHdhQ7 cells, p62 bodies were initially present in the lysosomes and accumulated to the juxtanuclear regions of STHdhQ111 cells as MG132 incubation continued. Unlike in STHdhQ7 cells, p62 puncti were not associated with K48-linked polyubiquitinated protein aggregates or proteasomal components in STHdhQ111. Interestingly, addition of cysteine during MG132 incubation rescued cell death in STHdhQ111 cells caused by stress recovery and altered the subcellular distribution of p62. Our data suggest that aberrant positioning of p62 affects the proteasomal clearance of protein aggregates and may contribute to the increased vulnerability to proteotoxic stress-induced cell death in HD cells.

Hepatitis C Virus Induces the Localization of Lipid Rafts to Autophagosomes for Its RNA Replication.

Autophagy plays important roles in maintaining cellular homeostasis. It uses double- or multiple-membrane vesicles termed autophagosomes to remove protein aggregates and damaged organelles from the cytoplasm for recycling. Hepatitis C virus (HCV) has been shown to induce autophagy to enhance its own replication. Here we describe a procedure that combines membrane flotation and affinity chromatography for the purification of autophagosomes from cells that harbor an HCV subgenomic RNA replicon. The purified autophagosomes had double- or multiple-membrane structures with a diameter ranging from 200 nm to 600 nm. The analysis of proteins associated with HCV-induced autophagosomes by proteomics led to the identification of HCV nonstructural proteins as well as proteins involved in membrane trafficking. Notably, caveolin-1, caveolin-2, and annexin A2, which are proteins associated with lipid rafts, were also identified. The association of lipid rafts with HCV-induced autophagosomes was confirmed by Western blotting, immunofluorescence microscopy, and immunoelectron microscopy. Their association with autophagosomes was also confirmed in HCV-infected cells. The association of lipid rafts with autophagosomes was specific to HCV, as it was not detected in autophagosomes induced by nutrient starvation. Further analysis indicated that the autophagosomes purified from HCV replicon cells could mediate HCV RNA replication in a lipid raft-dependent manner, as the depletion of cholesterol, a major component of lipid rafts, from autophagosomes abolished HCV RNA replication. Our studies thus demonstrated that HCV could specifically induce the association of lipid rafts with autophagosomes for its RNA replication.IMPORTANCE HCV can cause severe liver diseases, including cirrhosis and hepatocellular carcinoma, and is one of the most important human pathogens. Infection with HCV can lead to the reorganization of membrane structures in its host cells, including the induction of autophagosomes. In this study, we developed a procedure to purify HCV-induced autophagosomes and demonstrated that HCV could induce the localization of lipid rafts to autophagosomes to mediate its RNA replication. This finding provided important information for further understanding the life cycle of HCV and its interaction with the host cells.

P.80 - Identification of therapies for myofibrillar myopathy

Myofibrillar myopathies exhibit progressive muscle weakness and are characterized by structural failure of the muscle at the Z-disk and the formation of cytoplasmic protein aggregates. Mutations in the co-chaperone, and autophagy regulator, BAG3 are known to cause myofibrillar myopathy, with the first identified and most frequent mutation being BAG3P209L. We have previously demonstrated that expression of BAG3P209L is sufficient to cause formation of the characteristic protein aggregates but it is the loss of BAG3 that results in fibre disintegration. To further investigate the biology underlying the disease and investigate potential therapies we have generated zebrafish models of BAG3P209L myofibrillar myopathy, which conditionally express fluorescently tagged, human wildtype BAG3, or BAG3P209L in the muscle and a BAG3 loss of function model through CRISPR/Cas9 genome editing. The transgenic BAG3P209L fish exhibit the protein aggregation and the BAG3-/- fish demonstrate exercise dependent fibre disintegration, reduced swimming activity, and have a significant reduction in autophagic activity. To identify drugs that may be effective in treating BAG3 myofibrillar myopathy we conducted a screen of 76 autophagy promoting compounds using an automated aggregate quantification system to identify compounds that significantly reduce the number of aggregates. We identified nine compounds effective at removing protein aggregates, including two that are currently FDA approved. Further evaluation of these compounds demonstrated that not only are they able to remove the protein aggregates but they are also able to rescue the swimming deficit observed in the BAG3-/- fish. Here we will describe the work examining pathology in the zebrafish disease models and demonstrating the action of the potential therapies for BAG3 myofibrillar myopathy.

Autophagy in diabetic kidney disease: regulation, pathological role and therapeutic potential.

Diabetic kidney disease, a leading cause of end-stage renal disease, has become a serious public health problem worldwide and lacks effective therapies. Autophagy is a highly conserved lysosomal degradation pathway that removes protein aggregates and damaged organelles to maintaincellular homeostasis. As important stress-responsive machinery, autophagy is involved in the pathogenesis of various diseases. Emerging evidence has suggested that dysregulated autophagy may contribute to both glomerular and tubulointerstitial pathologies in kidneys under diabetic conditions. This review summarizes the recent findings regarding the role of autophagy in the pathogenesis of diabetic kidney disease and highlights the regulation of autophagy by the nutrient-sensing pathways and intracellular stress signaling in this disease. The advances in our understanding of autophagy in diabetic kidney disease will facilitate the discovery of a new therapeutic target for the prevention and treatment of this life-threatening diabetes complication.

Constitutive over-expression of rice ClpD1 protein enhances tolerance to salt and desiccation stresses in transgenic Arabidopsis plants

Caseinolytic proteases (Clps) perform the important role of removing protein aggregates from cells, which can otherwise prove to be highly toxic. ClpD system is a two-component protease complex composed of a regulatory ATPase module ClpD and a proteolytic component ClpP. Under desiccation stress condition, rice ClpD1 (OsClpD1) gene encoding for the regulatory subunit, was represented by four variant transcripts differing mainly in the expanse of their N-terminal amino acids. These transcripts were expressed in a differential manner in response to salt, mannitol and polyethylene glycol stresses in rice. Purified OsClpD1.3 protein exhibited intrinsic chaperone activity, shown using citrate synthase as substrate. Arabidopsis (Col-0) plants over-expressing OsClpD1.3 open reading frame downstream to CaMV35S promoter (ClpD1.3 plants) showed higher tolerance to salt and desiccation stresses as compared to wild type plants. ClpD1.3 seedlings also showed enhanced growth during the early stages of seed germination under unstressed, control conditions. The free proline levels and starch breakdown activities were higher in the ClpD1.3 seedlings as compared to the wild type Arabidopsis seedlings. It thus emerges that increasing the potential of ClpD1 chaperoning activity may be of advantage in protection against abiotic stresses.

How Is Proteinuric Diabetic Nephropathy Caused by Disturbed Proteostasis and Autophagy in Podocytes?

Progression of diabetic nephropathy (DN) is commonly defined by an increase in albuminuria from normoalbuminuria to microalbuminuria and from microalbuminuria to macroalbuminuria. Although many therapeutic interventions, including reducing hyperglycemia and intraglomerular pressure, have been shown to slow down the progression of DN, many patients still develop end-stage renal disease. A major difficulty in inducing remission in patients with early DN is the identification of biomarkers that could help to identify patients more likely to progress to end-stage renal disease. Traditional risk factors, such as albuminuria, do not effectively predict DN progression, and other predictors of DN have yet to be characterized and validated. The need for discovering sensitive and robust biomarkers to monitor the decline in renal function and to separate progressors from nonprogressors of DN is therefore of paramount importance. Next to mesangial extracellular matrix deposition and a thickening of basement membranes, progressive loss of glomerular pericytes and “podocytes” and microvascular alterations appear to most closely correlate with the functional renal decline in DN (1–3). Autophagy (“self-eating” in Greek) is a highly regulated lysosomal protein degradation pathway that removes protein aggregates and damaged or excess organelles in order to maintain intracellular homeostasis and cell integrity (4–6). This process was first described in 1957 by Sam Clark Jr. (7), but the term “autophagy” was coined in 1963 by Christian de Duve (8). Autophagy process is well conserved in the evolution from yeast to mammals in various cell types in many organs (9,10). The formation of autophagosomes depends on several …

Trehalose, an mTOR-Independent Inducer of Autophagy, Inhibits Human Cytomegalovirus Infection in Multiple Cell Types.

Human cytomegalovirus (HCMV) is the major viral cause of birth defects and a serious problem in immunocompromised individuals and has been associated with atherosclerosis. Previous studies have shown that the induction of autophagy can inhibit the replication of several different types of DNA and RNA viruses. The goal of the work presented here was to determine whether constitutive activation of autophagy would also block replication of HCMV. Most prior studies have used agents that induce autophagy via inhibition of the mTOR pathway. However, since HCMV infection alters the sensitivity of mTOR kinase-containing complexes to inhibitors, we sought an alternative method of inducing autophagy. We chose to use trehalose, a nontoxic naturally occurring disaccharide that is found in plants, insects, microorganisms, and invertebrates but not in mammals and that induces autophagy by an mTOR-independent mechanism. Given the many different cell targets of HCMV, we proceeded to determine whether trehalose would inhibit HCMV infection in human fibroblasts, aortic artery endothelial cells, and neural cells derived from human embryonic stem cells. We found that in all of these cell types, trehalose induces autophagy and inhibits HCMV gene expression and production of cell-free virus. Treatment of HCMV-infected neural cells with trehalose also inhibited production of cell-associated virus and partially blocked the reduction in neurite growth and cytomegaly. These results suggest that activation of autophagy by the natural sugar trehalose or other safe mTOR-independent agents might provide a novel therapeutic approach for treating HCMV disease. HCMV infects multiple cell types in vivo, establishes lifelong persistence in the host, and can cause serious health problems for fetuses and immunocompromised individuals. HCMV, like all other persistent pathogens, has to finely tune its interplay with the host cellular machinery to replicate efficiently and evade detection by the immune system. In this study, we investigated whether modulation of autophagy, a host pathway necessary for the recycling of nutrients and removal of protein aggregates, misfolded proteins, and pathogens, could be used to target HCMV. We found that autophagy could be significantly increased by treatment with the nontoxic, natural disaccharide trehalose. Importantly, trehalose had a profound inhibitory effect on viral gene expression and strongly impaired viral spread. These data constitute a proof-of-concept for the use of natural products targeting host pathways rather than the virus itself, thus reducing the risk of the development of resistance to treatment.

Abstract 322: Reversal of Intramuscular Lipotoxicity is Mediated by a Novel p62-lipophagy Pathway

Background and Hypothesis: In the mammalian heart, autophagy is considered an adaptive mechanism promoting the removal of protein aggregates or damaged cell organelles. A common feature of non-ischemic heart failure is lipid accumulation. We hypothesize that autophagy is impaired in the muscle in chronic lipid overload. Therefore, we investigated whether activation of autophagy protects myocytes from lipotoxicity. Methods: We incubated rat L6 myocytes over a period of 6 days with or without long chain fatty acids (equimolar mixture of oleate and palmitate, 1.0mM). At day 6, an autophagic inhibitor (bafilomycin A1, 200 nM), or an autophagic activator (rapamycin, 1 μM), or both, were added for 48 hours. Following the pharmacologic treatments, glucose uptake was measured using [3H] 2-Deoxy-D-glucose, and insulin sensitivity was assessed. Intracellular triglyceride (TG) accumulation was assessed by Oil Red O staining and immunofluorescence and was quantified enzymatically. Protein markers of autophagic flux (LC3 and p62) and cell death (Caspase 3 cleavage) were measured by immunoblotting. Results: Inhibition of autophagy using bafilomycin increased TG accumulation and also increased fatty acid-mediated cell death. Conversely, activation of autophagy using rapamycin reduced both intracellular lipid accumulation and cell death. Unexpectedly, treatment with both rapamycin and bafilomycin resulted in a decrease in lipid accumulation. Immunoblotting indicated p62 degradation (autophagic flux), while immunofluorescence revealed the colocalization of p62 with lipid droplets in these cells. These findings indicate the potential association of p62 with lipid droplet turnover, which is a novel pathway for the breakdown of lipid droplets in muscle cells. In the same cells, rapamycin treatment increased glucose uptake, in response to insulin (100mM). Conclusions: Autophagy promotes the clearance of lipids from myocytes, improves insulin sensitivity, and switches to an alternative p62 mediated pathway of lipophagy in the context of chronic lipid overload. Moreover, lipophagy promotes metabolic adaptation and myocyte survival.

Abstract 174: The BH3-Only Protein BNIP3 Induces Mitochondrial Clearance via Multiple Pathways

Autophagy plays an important role in cellular quality control and is responsible for removing protein aggregates and dysfunctional organelles. BNIP3 is an atypical BH3-only protein which is known to cause mitochondrial dysfunction and cell death in the myocardium. Interestingly, BNIP3 can also protect against cell death by promoting removal of dysfunctional mitochondria via autophagy (mitophagy). We have previously reported that BNIP3 is a potent inducer of mitophagy in cardiac myocytes and that BNIP3 contains an LC3 Interacting Region (LIR) that binds to LC3 on the autophagosome, tethering the mitochondrion to the autophagosome for engulfment. However, the molecular mechanism(s) underlying BNIP3-mediated mitophagy are still unclear. In this study, we discovered that BNIP3 can mediate mitochondrial clearance in cells even in the absence of a functional autophagy pathway. We found that overexpression of BNIP3 led to significant clearance of mitochondria in both wild type (WT) and autophagy deficient Atg5-/- MEFs. BNIP3 caused an increase in LC3II levels in WT MEFs, indicating increased formation of autophagosomes. In contrast, LC3II was undetectable in Atg5-/- MEFs. Furthermore, we found that BNIP3-mediated clearance in WT and Atg5-/- MEFs did not require the presence of Parkin, an E3 ubiquitin ligase which plays a critical role in clearing dysfunctional mitochondria in cells. Also, overexpression of Parkin did not enhance BNIP3-mediated mitochondrial clearance. When investigating activation of alternative cellular degradation pathways, we found that BNIP3 induced activation of the endosomal-lysosomal pathway in both WT and Atg5-/- MEFs. Mutating the LC3 binding site in BNIP3 did not interfere with the activation of the endosomal pathway and clearance of mitochondria in Atg5-/- MEFs. Thus, these findings suggest that BNIP3 can promote clearance of mitochondria via multiple pathways in cells. The role of autophagy in removing mitochondria is already well established and we are currently exploring the roles of the endosomal and alternative autophagy pathways in BNIP3-mediated mitochondrial clearance in myocytes.

Hepatitis C virus and autophagy.

Autophagy is a catabolic process by which cells remove protein aggregates and damaged organelles for recycling. It can also be used by cells to remove intracellular microbial pathogens, including viruses, in a process known as xenophagy. However, many viruses have developed mechanisms to subvert this intracellular antiviral response and even use this pathway to support their own replications. Hepatitis C virus (HCV) is one such virus and is an important human pathogen that can cause severe liver diseases. Recent studies indicated that HCV could activate the autophagic pathway to support its replication. This review summarizes the current knowledge on the interplay between HCV and autophagy and how this interplay affects HCV replication and host innate immune responses.

The divergent roles of autophagy in ischemia and preconditioning

Autophagy is an evolutionarily conserved and lysosome-dependent process for degrading and recycling cellular constituents. Autophagy is activated following an ischemic insult or preconditioning, but it may exert dual roles in cell death or survival during these two processes. Preconditioning or lethal ischemia may trigger autophagy via multiple signaling pathways involving endoplasmic reticulum (ER) stress, AMPK/TSC/mTOR, Beclin 1/BNIP3/SPK2, and FoxO/NF-κB transcription factors, etc. Autophagy then interacts with apoptotic and necrotic signaling pathways to regulate cell death. Autophagy may also maintain cell function by removing protein aggregates or damaged mitochondria. To date, the dual roles of autophagy in ischemia and preconditioning have not been fully clarified. The purpose of the present review is to summarize the recent progress in the mechanisms underlying autophagy activation during ischemia and preconditioning. A better understanding of the dual effects of autophagy in ischemia and preconditioning could help to develop new strategies for the preventive treatment of ischemia.

62-OR

Objectives Autophagy is a lysosomal degradation pathway that removes protein aggregates and damaged organelles maintaining the cellular integrity. It is an inducible process that responds to the environment and it seems to be essential for cell survival during stress, starvation, hypoxia and consequently to the placenta implantation and development. Studies on the role of autophagy in placenta from pregnant women with preeclampsia are scarce and still unclear. The aim of this study was to compare the expression of autophagy-related proteins, LC3-II, beclin-1 as well as mTOR protein in placental tissue from pregnant women with preeclampsia and normotensive pregnant women. Methods The expression of LC3-II and beclin-1 proteins involved in the autophagosome formation as well as mTOR protein was evaluated in 18 placentas of pregnant women with preeclampsia and in 20 of normotensive pregnant women who had elective cesarean delivery before the onset of labor. Fragments of the placenta were obtained immediately after delivery and the samples were prepared for real-time quantitative polymerase chain reaction (RT-qPCR), immunohistochemistry and transmission electron microscopy analysis. Results LC3-II expression as detected by RT-qPCR was significant higher in placentas of normotensive pregnant women compared with preeclamptic women. No significant difference in beclin-1 and mTOR expression was seen between these two groups. LC3-II, beclin-1 and mTOR proteins were detected by immunohistochemistry in syncytiotrophoblast and cytotrophoblast in both groups, and the presence of autophagic vacuoles containing intracytoplasmatic organelles was confirmed with electron microscopy. Conclusions The results of the impaired autophagy demonstrated by decrease of LC3- II expression in placenta of preeclamptic patients might play a role in the pathophysiology of preeclampsia. Financial support: FAPESP 2013/00535-1 and 2012/24697-8. Disclosures I.C. Weel: None. V.R. Ribeiro: None. M. Romao: None. M.L. Matias: None. V.T. Borges: None. J.C. Peracoli: None. J.P. Araujo Jr: None. M.T. Peracoli: None.