Autophagy regulation: RNF2 targets AMBRA1

The phosphatidylinositol 3-kinase complex I (PI3K complex I) is a crucial regulator of autophagy, which contains Beclin 1 (or ATG6), ATG14L, VPS34 (or the class III phosphatidylinositol 3-kinase and its adaptor VPS15) and AMBRA1, and controls autophagosome formation. In a paper recently published in Cell Research, Xia et al. report that during nutrient deprivation the ubiquitin E3 ligase RNF2 is recruited to the PI3K complex I, and ubiquitinates AMBRA1 to trigger its degradation and downregulate autophagy.Macroautophagy (hereafter called autophagy) is a lysosomal degradation pathway for cytoplasmic components¹. The ubiquitin-proteasome pathway is also critical during the process of autophagy. The formation of autophagosomes (double-membrane bound vacuoles that sequester cargo in bulk or in a selective manner before their delivery to the lysosomal compartment) depends on ubiquitination-like activity². The selective removal of cargo (e.g., protein aggregates, organelles) by autophagy is dependent in many cases on the ubiquitination of the cargo³. Recent studies also indicate that ubiquitination regulates the activity of autophagy proteins that comprise autophagosomes⁴.Autophagosome formation is dependent on evolutionarily conserved ATG (autophagy-related) proteins initially identified in yeast². These proteins function in complexes or functional modules on a membrane known as the phagophore that matures into the autophagosome via stages of initiation, elongation, and sealing. Phosphatidylinositol 3-phosphate kinase complex I (PI3K complex I) plays a key role in the initiation step. In this complex, Beclin 1 (the mammalian homolog of yeast ATG6) interacts with ATG14L, AMBRA1, and class III PI3K or VPS34² (Figure 1). The activity of this complex, which produces the lipid phosphatidylinositol 3-phosphate (PI3P), is crucial to recruitment of ATG, which is required for the elongation and sealing of the autophagosomal membrane.Open in a separate windowFigure 1Role of RNF2 and WASH during autophagosome formation. Upon autophagy stimulation by nutrient deprivation, Beclin 1 forms a complex with VPS34 and its adaptor VPS15, ATG14L, and AMBRA1. In this complex, Beclin 1 is K63-polyubiquitylated (green circles) by the E3 ligase AMBRA1 to activate the production of PI3P by VPS34. The production of PI3P at the phagophore recruits WIPI proteins to trigger the ATG machinery to elongate and seal the membrane to form an autophagosome. After the induction of autophagy, WASH and RNF2 are recruited to downregulate the autophagy pathway by inhibiting the VPS34 activity. WASH negatively regulates autophagy through suppression of Beclin 1 K63-linked polyubiquitination whereas RNF2 is recruited to the complex via the Beclin 1 interactor WASH. RNF2 catalyzes K48-linked polyubiquitination (orange circles) of AMBRA1 to mediate its proteasomal degradation.Recently, Xia and colleagues⁵ demonstrate that RNF2 (also called Ring1B) regulates autophagosome formation in response to nutrient starvation by influencing the ubiquitination of AMBRA1. RNF2 is a member of the RING-domain ubiquitin E3 ligase family⁶. In a series of experiments involving two-hybrid screens with RNF2 as bait, Xia and colleagues⁵ showed that RNF2 interacts with AMBRA1 and that this interaction is enhanced upon autophagy stimulation in cells cultured in the absence of serum and amino acids. Deletion of RNF2 robustly stimulates autophagy in response to starvation whereas restoration of RNF2 in RNF2^−/− mouse embryo fibroblasts (MEFs) reduces autophagy. An RNF2 mutant with no E3 ligase activity does not impair autophagy in RNF2^−/− MEFs. The authors further showed that RNF2 downregulates autophagy by promoting the degradation of AMBRA1. RNF2 catalyzes the K48-linked polyubiquitination of AMBRA1 which mediates its proteasomal degradation. The crucial site for AMBRA1 K48-linked polyubiquitination is lysine 45, and a K45R AMBRA1 mutant is not sensitive to RNF2-mediated ubiquitination and is able to sustain VPS34 activity and autophagy. Beclin 1 exists in different complexes involved in different steps of the autophagic pathway². In the current study, the authors showed that RNF2 is associated with the PI3K complex 1 with Beclin 1 ATG14 and AMBRA1, a complex involved in early stage of autophagosome formation. Interestingly, in the absence of RNF2, the association of Beclin 1 with VPS34 in the PI3K complex 1 is enhanced.Recently, Fan''s group reported that AMBRA1-DDB1-CUL4A is the E3 ligase that mediates K63-linked ubiquitination of Beclin 1 to enhance its binding to VPS34 in response to starvation⁷. In the present study⁵, the group showed that the K63-linked ubiquitination of Beclin 1 is inhibited by RNF2. A screen identified WASH as an interactor of RNF2. WASH is part of a complex that promotes actin polymerization to facilitate endosomal protein sorting⁸ and has been recently shown to interact with Beclin 1 and to be associated with autophagosomal membrane⁷. This interaction impairs the AMBRA1-mediated K63 polyubiquitination of Beclin 1. WASH recruits RNF2 to AMBRA1 and the PI3K complex 1 in response to starvation. The absence of WASH abolishes the K48-linked polyubiquitination of AMBRA1. In addition, WASH overexpression partially impairs the interaction of AMBRA1 with Beclin1 to block its K63-linked polyubiquitination⁷. The study by Xia et al. reveals a novel layer of regulation: WASH recruits RNF2 to promote AMBRA1 degradation to impair Beclin 1 ubiquitination⁵. This work points to the complex role of ubiquitination in the regulation of the early stage of autophagy with a balance between activating K63-linked polyubiquitination of Beclin 1 and inhibitory K48-linked polyubiquitination of AMBRA1. These post-translational modifications depend on the activity of two E3 ligases, AMBRA1 and RNF2. This study also stresses the importance of AMBRA1 in stabilization of proteins engaged in the early stage of autophagosome formation via its E3 ligase activity with DDB1-CUL4A to enhance Beclin 1 association with VPS34⁷ and with TRAF6 to stabilize ULK1, which acts in a complex upstream of the PI3K complex 1 in autophagy⁹. Finally, the study by Xia et al. emphasizes that autophagy must be tightly regulated to avoid deleterious effects on cell homeostasis.The present study raises several questions regarding how and when WASH and RNF2 are recruited to downregulate autophagy in response to starvation. Recently it has been shown that WASH is a positive modulator of autophagosome biogenesis in mammalian cells through regulation of endosomal trafficking of ATG9A¹⁰. Altogether, these findings suggest that the role of WASH in autophagy is dependent on its subcellular localization and its partners in intracellular membranes.     

Distinct requirements of Autophagy-related genes in programmed cell death

Although most programmed cell death (PCD) during animal development occurs by caspase-dependent apoptosis, autophagy-dependent cell death is also important in specific contexts. In previous studies, we established that PCD of the obsolete Drosophila larval midgut tissue is dependent on autophagy and can occur in the absence of the main components of the apoptotic pathway. As autophagy is primarily a survival mechanism in response to stress such as starvation, it is currently unclear if the regulation and mechanism of autophagy as a pro-death pathway is distinct to that as pro-survival. To establish the requirement of the components of the autophagy pathway during cell death, we examined the effect of systematically knocking down components of the autophagy machinery on autophagy induction and timing of midgut PCD. We found that there is a distinct requirement of the individual components of the autophagy pathway in a pro-death context. Furthermore, we show that TORC1 is upstream of autophagy induction in the midgut indicating that while the machinery may be distinct the activation may occur similarly in PCD and during starvation-induced autophagy signalling. Our data reveal that while autophagy initiation occurs similarly in different cellular contexts, there is a tissue/function-specific requirement for the components of the autophagic machinery.There is a fundamental requirement for multicellular organisms to remove excess, detrimental, obsolete and damaged cells by programmed cell death (PCD).^{1, 2} In the majority of cases caspase-dependent apoptosis is the principle pathway of PCD; however, there are other modes of cell death with important context-specific roles, such as autophagy.^{3, 4} Defects in autophagy have significant adverse consequences to normal cellular functions and contribute to the pathogenesis of numerous human diseases. This is particularly evident in cancer where depending on the context autophagy can have tumour-suppressing or -promoting roles. Given the number of clinical trials targeting autophagy in cancer therapy, it will be critically important to understand the context-specific regulation and functions of autophagy.⁵Autophagy is a highly conserved multi-step catabolic process characterised by the encapsulation of part of the cytoplasm inside a double-membrane vesicle called the autophagosome. Autophagosomes then fuse with lysosomes and the components are subsequently degraded by acidic lysosomal hydrolases.⁶ The process of autophagy can be functionally divided into four groups: (1) serine/threonine kinase Atg1 (ULK1 in mammals) complex and its regulators responsible for the induction of autophagy; (2) the class III phosphatidylinositol 3-kinase (PI3K) complex, which involves Atg6 and functions in the nucleation of the autophagosome; (3) the Atg8 and Atg12 conjugation systems, which involves several Autophagy-related (Atg) proteins essential for the expansion of autophagosome; and (4) Atg9 and its associated proteins including Atg2 and Atg18, which aids the recycling of lipid and proteins.⁷ In addition, several of the Atg proteins can function in multiple steps. For example, Atg1 interacts with proteins with different functions (e.g. Atg8, Atg18 and others), suggesting that it is not only required for initiation but also participates in the formation of autophagosomes.⁸ It is yet to be fully established if the context-specific functions of autophagy have distinct requirements for select components of the autophagy pathway.High levels of autophagy are induced in response to stress, such as nutrient deprivation, intracellular stress, high temperature, high culture density, hormones and growth factor deprivation.^{9, 10} The target of rapamycin (TOR) pathway is a central mediator in regulating the response to nutrients and growth signalling. TOR functions in two distinct complexes, with regulatory associated protein of TOR (Raptor) in TOR complex 1 (TORC1) or with rapamycin insensitive companion of TOR (Rictor) in TOR complex 2 (TORC2).^{11, 12, 13, 14, 15} Of these, TORC1 regulates autophagy; in nutrient-rich conditions, TORC1 activity inhibits the Atg1 complex preventing autophagy and cellular stress such as starvation leads to inactivation of TORC1 promoting a dramatic increase in autophagy. TORC2 can also negatively regulate autophagy via the FoxO3 complex in specific context.¹⁶Most direct in vivo evidence for a role of autophagy in cell death has emerged from studies in Drosophila.⁵ Developmentally regulated removal of the Drosophila larval midgut can occur in the absence of canonical apoptosis pathway, whereas inhibiting autophagy delays the process.^{17, 18} Also, inhibition of autophagy leads to delayed degradation of larval salivary glands in Drosophila.¹⁹ Genetic studies have shown that many of the Atg genes known to be involved in starvation-induced autophagy in the Drosophila fat body are also involved in autophagy-dependent degradation of salivary glands and midgut.^{5, 20, 21} However, systematic studies to test whether starvation-induced autophagy and autophagy required for PCD require identical components have not been carried out, and there are some observations suggesting that there may be distinctions. For example, in Atg7-null mutants autophagy is perturbed but the larval–adult midgut transition proceeds normally.²² In addition, a novel Atg7- and Atg3-independent autophagy pathway is required for cell size reduction during midgut removal.²³ Here we show that downregulation of TORC1 activity is required for induction of autophagy during midgut removal. Surprisingly, however, the requirement of part of the autophagy machinery during midgut degradation was found to be distinct to that which is required during autophagy induced by starvation. We report that Atg genes required for autophagy initiation, Atg8a and recycling are all essential for autophagy-dependent midgut removal, whereas other components of the elongation and nucleation steps are not essential.     

Inhibition of Target of Rapamycin Signaling and Stress Activate Autophagy in Chlamydomonas reinhardtii

Autophagy is a catabolic membrane-trafficking process whereby cells recycle cytosolic proteins and organelles under stress conditions or during development. This degradative process is mediated by autophagy-related (ATG) proteins that have been described in yeast, animals, and more recently in plants. In this study, we report the molecular characterization of autophagy in the unicellular green alga Chlamydomonas reinhardtii. We demonstrate that the ATG8 protein from Chlamydomonas (CrATG8) is functionally conserved and may be used as a molecular autophagy marker. Like yeast ATG8, CrATG8 is cleaved at the carboxyl-terminal conserved glycine and is associated with membranes in Chlamydomonas. Cell aging or different stresses such as nutrient limitation, oxidative stress, or the accumulation of misfolded proteins in the endoplasmic reticulum caused an increase in CrATG8 abundance as well as the detection of modified forms of this protein, both landmarks of autophagy activation. Furthermore, rapamycin-mediated inhibition of the Target of Rapamycin signaling pathway, a major regulator of autophagy in eukaryotes, results in identical effects on CrATG8 and a relocalization of this protein in Chlamydomonas cells similar to the one observed upon nutrient limitation. Thus, our findings indicate that Chlamydomonas cells may respond to stress conditions by inducing autophagy via Target of Rapamycin signaling modulation.Protein turnover is essential for the adaptation of cells to variable environmental conditions. Similar to other eukaryotes, plants have developed two distinct mechanisms to regulate protein degradation, a selective ubiquitin/26S proteasome pathway (Vierstra, 2009) and macroautophagy (hereafter referred to as autophagy), a nonselective membrane-trafficking process (Bassham, 2009). During autophagy, a large number of cytosolic components, including entire organelles, organelle fragments, and protein complexes, are enclosed in bulk within a double-membrane structure known as the autophagosome and delivered to the vacuole/lysosome for degradation to recycle needed nutrients or degrade toxic components (Xie and Klionsky, 2007; Nakatogawa et al., 2009). The autophagosomes appear to arise from isolation membranes usually observed in close proximity to the vacuole called the preautophagosomal structure (PAS). These membranes expand and fuse to encircle portions of the cytoplasm, generating an autophagosome that is targeted to the vacuole. The outer membrane of the autophagosome then fuses with the vacuole membrane, and the remaining vesicle, known as the autophagic body, is finally released to the vacuole for its degradation (Xie and Klionsky, 2007).The evolutionary conservation of autophagy among eukaryotes indicates that structural and regulatory components of this cellular process must be also conserved. Accordingly, a significant number of autophagy-related (ATG) genes that participate in autophagy regulation and autophagosome formation have been identified, initially through genetic approaches in yeast and subsequently in higher eukaryotes, including mammals, insects, protozoa, and plants (Bassham et al., 2006; Bassham, 2007; Meijer et al., 2007). In yeast, two protein conjugation systems composed of the ubiquitin-like proteins ATG8 and ATG12 and the three enzymes ATG3, ATG7, and ATG10 play an essential role in autophagosome formation and seem to be conserved through evolution (Geng and Klionsky, 2008). ATG8 becomes modified with the lipid molecule phosphatidylethanolamine (PE) by the consecutive action of the ATG7 and ATG3 enzymes in a process mechanistically similar to ubiquitination (Ichimura et al., 2000). Prior to this modification, ATG8 must be cleaved by the Cys protease ATG4 to expose a C-terminal Gly residue that is conjugated to PE (Kirisako et al., 2000; Kim et al., 2001). ATG12 becomes covalently attached to the ATG5 protein in a conjugation reaction that is catalyzed by ATG7 and ATG10 (Mizushima et al., 1998). ATG8-PE and ATG12-ATG5 conjugates localize to autophagy-related membranes and are required for the initiation and expansion of autophagosomal membrane and hemifusion of this membrane with the vacuolar membrane (Hanada et al., 2007; Nakatogawa et al., 2007, 2009; Fujita et al., 2008; Geng and Klionsky, 2008; Xie et al., 2008).Our understanding of the autophagy process has substantially increased with the development of specific markers for autophagy. In plants, two markers for autophagosomes have been described, the monodansylcadaverine dye and GFP-ATG8 fusion protein (Yoshimoto et al., 2004; Contento et al., 2005; Thompson et al., 2005). As in other species, binding of ATG8 to autophagosomes has been used to monitor autophagy in plants. In contrast to yeast, where a single ATG8 gene is present, plants appear to contain a small gene family with several ATG8 isoforms, suggesting that autophagy is more complex in these photosynthetic organisms. For example, Arabidopsis (Arabidopsis thaliana) and maize (Zea mays) encode nine and five ATG8 genes, respectively (Doelling et al., 2002; Hanaoka et al., 2002; Ketelaar et al., 2004; Chung et al., 2009). However, despite the high complexity of the ATG8-conjugating system in plants, important findings have been recently reported on the molecular characterization of autophagy using ATG8 as an autophagy marker in these organisms. The use of specific markers for autophagy in plants has revealed that this process is active at a basal level under normal growth and is induced upon nitrogen- or carbon-limiting conditions as well as in response to oxidative stress (Yoshimoto et al., 2004; Thompson et al., 2005; Xiong et al., 2005, 2007; Chung et al., 2009). Reverse genetic approaches have also been applied to a number of Arabidopsis ATG genes using T-DNA insertional mutants or RNA interference in order to investigate the physiological roles of autophagy in plants. The initial characterization of autophagy-deficient plants demonstrated that the ATG system is not essential under nutrient-rich conditions. However, a detailed analysis of these mutants indicated that autophagy is required for the proper response of the plant to nutrient limitation or pathogen infection. Plants lacking the ATG4, ATG5, ATG7, ATG9, or ATG10 gene display premature leaf senescence and are hypersensitive to nitrogen or carbon limitation (Doelling et al., 2002; Hanaoka et al., 2002; Yoshimoto et al., 2004; Thompson et al., 2005; Phillips et al., 2008). Arabidopsis plants with reduced levels of ATG18, which is required for autophagosome formation, are more sensitive to methyl viologen treatment and accumulate high levels of oxidized proteins, demonstrating that autophagic processes participate in the response of the plant to oxidative stress (Xiong et al., 2005, 2007). Plants deficient in the autophagy genes ATG6/Beclin1, ATG3, ATG7, and ATG9 exhibit unrestricted hypersensitive response lesions in response to pathogen infection (Liu et al., 2005; Hofius et al., 2009). These findings implicate autophagy as a prosurvival mechanism to restrict programmed cell death associated with the pathogen-induced hypersensitive response in plants. Arabidopsis ATG6 has also been shown to mediate pollen germination in a manner independent of autophagy (Fujiki et al., 2007).As mentioned above, autophagy is triggered among other factors by a reduction in the availability of nutrients. This starvation signal is transmitted to the autophagic machinery by important regulatory factors, including the Ser/Thr kinases Target of Rapamycin (TOR), ATG1, and SNF1 and the phosphatidylinositol 3-kinase ATG6/Beclin1 (Diaz-Troya et al., 2008b; Bassham, 2009; Cebollero and Reggiori, 2009). TOR has been identified as a negative regulator of autophagy in yeast, mammals, and fruit flies (Diaz-Troya et al., 2008b). The pharmacological inhibition of TOR by rapamycin leads to autophagy activation through a mechanism that requires the activation of the ATG1 kinase (Kamada et al., 2000). It has been recently demonstrated in mammals and fruit flies that a rapamycin-sensitive TOR signaling complex termed TORC1 directly phosphorylates and inhibits the ATG1 kinase and its regulatory protein ATG13 (Chang and Neufeld, 2009; Hosokawa et al., 2009; Jung et al., 2009). These regulatory proteins are conserved in plants, although except for ATG6 (Liu et al., 2005), there is no direct evidence for regulation of autophagy by these signaling pathways.The unicellular green alga Chlamydomonas reinhardtii has been used as a model for the study of important cellular and metabolic processes in photosynthetic organisms (Harris, 2001). More recently, Chlamydomonas has also been proposed as a useful system for the characterization of the TOR signaling pathway in photosynthetic eukaryotes based on the finding that, unlike plants, Chlamydomonas cell growth is sensitive to rapamycin (Crespo et al., 2005; Diaz-Troya et al., 2008a). Treatment of Chlamydomonas cells with rapamycin results in a pronounced increase of vacuole size that resembles autophagy-like processes (Crespo et al., 2005). However, a role of TOR in autophagy regulation could not be demonstrated due to the absence of an autophagy marker in Chlamydomonas. Actually, no studies have been reported on any autophagy-related protein in green algae, despite the high conservation of ATG genes in Chlamydomonas (Diaz-Troya et al., 2008b).This study reports the molecular and cellular characterization of autophagy in the green alga Chlamydomonas. We demonstrate that the ATG8 protein from Chlamydomonas (CrATG8) may be used as a specific autophagy marker. Nutrient limitation and cell aging trigger an autophagic response that can be traced as an increase at the level of CrATG8, the detection of modified forms of CrATG8, and a change in the cellular localization of this protein. Furthermore, we demonstrate that autophagy is inhibited by a rapamycin-sensitive TOR cascade in Chlamydomonas.     

Spermidine induces autophagy by inhibiting the acetyltransferase EP300

Several natural compounds found in health-related food items can inhibit acetyltransferases as they induce autophagy. Here we show that this applies to anacardic acid, curcumin, garcinol and spermidine, all of which reduce the acetylation level of cultured human cells as they induce signs of increased autophagic flux (such as the formation of green fluorescent protein-microtubule-associated protein 1A/1B-light chain 3 (GFP-LC3) puncta and the depletion of sequestosome-1, p62/SQSTM1) coupled to the inhibition of the mammalian target of rapamycin complex 1 (mTORC1). We performed a screen to identify the acetyltransferases whose depletion would activate autophagy and simultaneously inhibit mTORC1. The knockdown of only two acetyltransferases (among 43 candidates) had such effects: EP300 (E1A-binding protein p300), which is a lysine acetyltranferase, and NAA20 (N(α)-acetyltransferase 20, also known as NAT5), which catalyzes the N-terminal acetylation of methionine residues. Subsequent studies validated the capacity of a pharmacological EP300 inhibitor, C646, to induce autophagy in both normal and enucleated cells (cytoplasts), underscoring the capacity of EP300 to repress autophagy by cytoplasmic (non-nuclear) effects. Notably, anacardic acid, curcumin, garcinol and spermidine all inhibited the acetyltransferase activity of recombinant EP300 protein in vitro. Altogether, these results support the idea that EP300 acts as an endogenous repressor of autophagy and that potent autophagy inducers including spermidine de facto act as EP300 inhibitors.Macroautophagy (herein referred to as ‘autophagy'') consist in the sequestration of cytoplasmic material in autophagosomes, followed by their fusion with lysosomes for the bulk degradation of autophagic cargo by lysosomal hydrolases.¹ This phenomenon can be measured by following the redistribution of green fluorescent protein-microtubule-associated protein 1A/1B-light chain 3 (GFP-LC3) fusion proteins from a diffuse location to autophagosomes (that results in the formation of the so-called GFP-LC3 ‘puncta''), the diminution of the overall abundance of autophagic substrates (such as sequestosome-1, p62/SQSTM1), and the stereotyped activation of proautophagic signals (such as the inhibition of the mammalian target of rapamycin complex 1, mTORC1).²There is growing consensus that the induction of autophagy by nutritional, pharmacological or genetic interventions can reduce age-related pathologies (such as neurodegenerative diseases or type 2 diabetes) and/or extend longevity.^{3, 4, 5, 6} This applies to caloric restriction or intermediate fasting,⁷ continuous or intermittent medication of rapamycin,^{8, 9, 10} administration of the sirtuin 1-activator resveratrol,^{11, 12} external supply of the polyamine spermidine,¹³ or genetic ablation of p53.¹⁴ In all these cases, inhibition of autophagy by deleting or silencing relevant genes abolishes the extension of health span and/or lifespan.^{13, 14, 15, 16, 17} Moreover, direct induction of autophagy by transgenic expression of autophagy-relevant genes such as ATG5 in mice is sufficient to increase lifespan.¹⁸Recently, acetyltransferases have emerged as a potential target for the pharmaceutical induction of autophagy. Thus, depletion of the sole donor of acetyl groups, acetyl-coenzyme A (acetyl-CoA), is sufficient to reduce the acetylation of cytoplasmic and nuclear proteins coupled to the induction of autophagy.^{19, 20, 21, 22} Culture of mammalian cells in nutrient-free (NF) conditions or starvation of mice for 24 h reduced the intracellular nucleocytosolic concentrations of acetyl-CoA at the same time as autophagy was induced, and replenishment of acetyl-CoA by external sources (for instance, by providing a membrane-permeant precursor of α-ketoglutarate for anaplerotic reactions or by microinjection of acetyl-CoA) was sufficient to inhibit starvation-induced autophagy.^{19, 20, 21, 22} Beyond the inhibition of acetyltransferases by acetyl-CoA depletion, direct pharmacological inhibition of acetyltransferases might also contribute to the induction of autophagy. A close correlation between autophagy induction and deacetylation of cytoplasmic proteins was observed in a screen conceived to identify autophagy-stimulating polyphenols²³ as well as in in vivo experiments designed to explore the health-improving effects of coffee.²⁴ Spermidine turned out to be an efficient inhibitor of histone acetyltransferases in vitro¹³ and reduced the global protein acetylation levels in cultured cells.^{25, 26}Driven by these premises, we investigated the hypothesis that several health-related compounds including anacardic acid, curcumin, garcinol and spermidine might induce autophagy by inhibition of acetyltranferases. Here we report results supporting this hypothesis. Moreover, we demonstrate that one particular acetyltransferase, EP300 (E1A-binding protein p300), negatively controls autophagy and that anacardic acid, curcumin, garcinol and spermidine may induce autophagy by directly inhibiting EP300.     

Shear stress regulates endothelial cell autophagy via redox regulation and Sirt1 expression

Disturbed cell autophagy is found in various cardiovascular disease conditions. Biomechanical stimuli induced by laminar blood flow have important protective actions against the development of various vascular diseases. However, the impacts and underlying mechanisms of shear stress on the autophagic process in vascular endothelial cells (ECs) are not entirely understood. Here we investigated the impacts of shear stress on autophagy in human vascular ECs. We found that shear stress induced by laminar flow, but not that by oscillatory or low-magnitude flow, promoted autophagy. Time-course analysis and flow cessation experiments confirmed that this effect was not a transient adaptive stress response but appeared to be a sustained physiological action. Flow had no effect on the mammalian target of rapamycin-ULK pathway, whereas it significantly upregulated Sirt1 expression. Inhibition of Sirt1 blunted shear stress-induced autophagy. Overexpression of wild-type Sirt1, but not the deacetylase-dead mutant, was sufficient to induce autophagy in ECs. Using both of gain- and loss-of-function experiments, we showed that Sirt1-dependent activation of FoxO1 was critical in mediating shear stress-induced autophagy. Shear stress also induced deacetylation of Atg5 and Atg7. Moreover, shear stress-induced Sirt1 expression and autophagy were redox dependent, whereas Sirt1 might act as a redox-sensitive transducer mediating reactive oxygen species-elicited autophagy. Functionally, we demonstrated that flow-conditioned cells are more resistant to oxidant-induced cell injury, and this cytoprotective effect was abolished after inhibition of autophagy. In summary, these results suggest that Sirt1-mediated autophagy in ECs may be a novel mechanism by which laminar flow produces its vascular-protective actions.Vascular endothelial cells (ECs) are fundamentally important in maintaining structural and functional homeostasis of blood vessels. Normal biological functions of ECs are highly sensitive to the biomechanical stimuli induced by blood flow, of which shear stress acting on the surface of EC has been recognized to be one of the most important vasoactive factors in EC.^{1, 2} A relatively high level of laminar shear stress is cytoprotective, whereas abnormal (low-magnitude or oscillatory) shear stress is a detrimental cellular stress to ECs.¹ Transduction of the mechanical signals involves multiple messenger molecules and signaling proteins, which collectively regulate important endothelial functions, such as gene expression, proliferation, migration, morphogenesis, permeability, thrombogenicity, and inflammation.²Autophagy (also known as macroautophagy) is an evolutionarily conserved cellular stress response.^{3, 4} Autophagy is a cellular self-digestion process, which is responsible for degradation of misfolded proteins and damaged organelles. Autophagic process is mainly mediated by the formation of autophagosome, a double-membrane vacuole structure containing engulfed cellular components. This process requires expression of a group of key genes involved in autophagy, including LC3A, beclin-1, Atg5, Atg7, and Atg12, for example.^{3, 5} Autophagosomes fuse with lysosomes, forming autolysosomes, where the cellular components are degraded by various hydrolases in an acidified environment.^{4, 5} In ECs, an autophagic response can be initiated by different stress stimuli.^{6, 7, 8} It is noted that the cellular outcome following autophagy induction in ECs varies depending on the nature of stimuli and specific experimental settings.^{6, 7, 9, 10} Moreover, there is evidence showing that autophagy may also be involved in modulating other EC functions such as angiogenesis and cellular senescence.^{11, 12} Therefore, understanding the regulatory mechanisms of autophagy in ECs will be important for discovery of strategies to protect normal endothelial functions. Recently, Guo et al. provided some evidence indicating that the autophagic process in EC might be affected by shear stress.¹³ This argument, however, was only based on observations of changed expression levels of LC3 and beclin-1; further experimental evidence is needed to confirm such an effect of shear stress on autophagy. More importantly, the mechanisms underlying this phenomenon are not understood. Different signaling pathways may be involved in modulating autophagy in ECs.^{14, 15, 16} For example, inhibition of the mTOR (mammalian target of rapamycin) pathway by rapamycin-induced endothelial autophagy and prevented energy stress-triggered cell damage.¹⁶ There is also evidence indicating a potential role of Sirt1.¹⁴ Moreover, accumulating evidence has suggested that reactive oxygen species (ROS) are closely implicated in modulating autophagic responses via complex interactions with other autophagy-related factors.¹⁵ Despite of these results, the signaling mechanisms of shear stress-regulated autophagy in EC remain to be defined. Hence, here we aim to delineate the impacts and underlying mechanisms of shear stress on autophagy in human vascular ECs.     

The generation of neutrophils in the bone marrow is controlled by autophagy

Autophagy has been demonstrated to have an essential function in several cellular hematopoietic differentiation processes, for example, the differentiation of reticulocytes. To investigate the role of autophagy in neutrophil granulopoiesis, we studied neutrophils lacking autophagy-related (Atg) 5, a gene encoding a protein essential for autophagosome formation. Using Cre-recombinase mediated gene deletion, Atg5-deficient neutrophils showed no evidence of abnormalities in morphology, granule protein content, apoptosis regulation, migration, or effector functions. In such mice, however, we observed an increased proliferation rate in the neutrophil precursor cells of the bone marrow as well as an accelerated process of neutrophil differentiation, resulting in an accumulation of mature neutrophils in the bone marrow, blood, spleen, and lymph nodes. To directly study the role of autophagy in neutrophils, we employed an in vitro model of differentiating neutrophils that allowed modulating the levels of ATG5 expression, or, alternatively, intervening pharmacologically with autophagy-regulating drugs. We could show that autophagic activity correlated inversely with the rate of neutrophil differentiation. Moreover, pharmacological inhibition of p38 MAPK or mTORC1 induced autophagy in neutrophilic precursor cells and blocked their differentiation, suggesting that autophagy is negatively controlled by the p38 MAPK–mTORC1 signaling pathway. On the other hand, we obtained no evidence for an involvement of the PI3K-AKT or ERK1/2 signaling pathways in the regulation of neutrophil differentiation. Taken together, these findings show that, in contrast to erythropoiesis, autophagy is not essential for neutrophil granulopoiesis, having instead a negative impact on the generation of neutrophils. Thus, autophagy and differentiation exhibit a reciprocal regulation by the p38–mTORC1 axis.Autophagy is an evolutionarily conserved mechanism, by which portions of cytoplasm are engulfed in a double-membrane structure, known as the autophagosome, and delivered to lysosomes for subsequent degradation. Autophagy is dependent on autophagy-related (ATG) proteins.¹ Autophagosome formation, elongation, and completion of enclosure require two ubiquitin-like conjugation systems: the first one generates the ATG5-ATG12 conjugate, which functions as a complex together with ATG16, and binds to the sequestering (pre-autophagosomal) phagophore. The second system conjugates an ATG8 homolog, LC3-I, with phosphatidylethanolamine to generate the lipidated LC3-II that associates with autophagosomes.^{2, 3, 4} The conversion of LC3-I into LC3-II represents a hallmark of autophagic activity and is widely used for the detection of autophagosome formation. Another frequently used marker for monitoring autophagic activity is p62, a protein, which is specifically degraded through autophagy.⁵The vital role of autophagy in cell growth, development, and homeostasis has long been appreciated. Recent data also indicate its involvement in the differentiation of hematopoietic cells. For instance, the importance of autophagy for efficient differentiation of leukocytes has been demonstrated with lymphocytes,^{6, 7, 8} monocytes,⁹ and dendritic cells,¹⁰ as well as reticulocytes.^11,12 Since granulopoiesis in the bone marrow is characterized by significant morphological changes and the acquisition of a range of effector functions, we hypothesized that autophagy might have a crucial role in the differentiation of neutrophils.ATG5 is an essential protein for the elongation of pre-autophagosomal structures and subsequent autophagosome formation. Therefore, it represents a suitable genetic target for blocking autophagy. Using this strategy, we demonstrate here that neutrophil differentiation is controlled by autophagy, which in turn is negatively regulated by the p38 signaling pathway. Surprisingly, and in contrast to differentiation in other non-granulocytic hematopoietic lineages, autophagy was downregulated during physiological neutrophil differentiation and its inappropriate induction delayed the differentiation process.     

Autophagy supports survival and phototransduction protein levels in rod photoreceptors

Damage and loss of the postmitotic photoreceptors is a leading cause of blindness in many diseases of the eye. Although the mechanisms of photoreceptor death have been extensively studied, few studies have addressed mechanisms that help sustain these non-replicating neurons for the life of an organism. Autophagy is an intracellular pathway where cytoplasmic constituents are delivered to the lysosomal pathway for degradation. It is not only a major pathway activated in response to cellular stress, but is also important for cytoplasmic turnover and to supply the structural and energy needs of cells. We examined the importance of autophagy in photoreceptors by deleting the essential autophagy gene Atg5 specifically in rods. Loss of autophagy led to progressive degeneration of rod photoreceptors beginning at 8 weeks of age such that by 44 weeks few rods remained. Cone photoreceptor numbers were only slightly diminished following rod degeneration but their function was significantly decreased. Rod cell death was apoptotic but was not dependent on daily light exposure or accelerated by intense light. Although the light-regulated translocation of the phototransduction proteins arrestin and transducin were unaffected in rods lacking autophagy, Atg5-deficient rods accumulated transducin-α as they degenerated suggesting autophagy might regulate the level of this protein. This was confirmed when the light-induced decrease in transducin was abolished in Atg5-deficient rods and the inhibition of autophagy in retinal explants cultures prevented its degradation. These results demonstrate that basal autophagy is essential to the long-term health of rod photoreceptors and a critical process for maintaining optimal levels of the phototransduction protein transducin-α. As the lack of autophagy is associated with retinal degeneration and altered phototransduction protein degradation in the absence of harmful gene products, this process may be a viable therapeutic target where rod cell loss is the primary pathologic event.Autophagy is an intracellular pathway where cytoplasmic constituents are delivered to the lysosomes for degradation. Defective autophagy can contribute to the age-dependent accumulation of damaged proteins and organelles leading to altered cellular homeostasis and loss of function.^{1, 2, 3, 4, 5} The metabolic roles of autophagy can be classified into two types, basal and induced. In nutrient-rich conditions, autophagy is suppressed but still occurs at low levels (basal autophagy); however, when cells are subjected to stress (starvation, injury, hypoxia), autophagy is activated immediately (induced autophagy).⁶ Induced autophagy maintains the amino acid pool inside cells to adapt to starvation while constitutive autophagy has been shown to function as a cell-repair mechanism that is important for long-lived postmitotic cells.^{7, 8, 9, 10, 11} Defects in autophagy have been associated with neurodegenerative diseases,^{12, 13, 14, 15} diabetes,^{16, 17} lysosomal storage disease¹⁸ and the loss of vision.¹⁹ In addition to macroautophagy, microautophagy and chaperone-mediated autophagy (CMA) have been described. Although little is known about microautophagy in mammalian cells, macroautophagy (hereafter autophagy) is a major pathway for bulk degradation of cytoplasmic components. CMA is a more selective pathway for degradation of cytosolic proteins that can compensate for the loss of macroautophagy.^{2, 20, 21, 22}Inherited retinal degenerative diseases such as retinitis pigmentosa or Leber''s congenital amaurosis are characterized by premature and progressive death of rod and cone photoreceptor cells.²³ These diseases are characterized by the loss of night vision due to the death of rods followed by the loss of cones leading to diminished visual acuity and a reduction in the quality of life for patients. Disease is typically associated with the production of harmful gene products that promote pathology by inhibiting critical pathways resulting in cell death.^{24, 25, 26} Strategies to prevent photoreceptor death during retinal degenerative disease such as gene replacement therapies or inhibition of cell death pathways have been undertaken with some success;^{27, 28, 29} however, effective treatments for these blinding disorders are lacking.Another strategy that could be used in conjunction with other therapies might be to enhance survival by stimulating autophagy. Augmenting autophagy would increase the supply of nutrients to stressed cells and accelerate removal of damaged proteins thereby prolonging cell survival beyond what would be possible by only preventing cell death. Although canonical^{22, 30, 31, 32, 33} and noncanonical autophagic mechanisms³⁴ have been detected in the eye, our knowledge of basic autophagy functions in this organ is still limited. In order to understand how autophagy maintains retinal homeostasis and function, we undertook studies to examine the consequences of deleting the essential autophagy gene Atg5 in rod photoreceptors.     

Oncogenic BRAF induces chronic ER stress condition resulting in increased basal autophagy and apoptotic resistance of cutaneous melanoma

The notorious unresponsiveness of metastatic cutaneous melanoma to current treatment strategies coupled with its increasing incidence constitutes a serious worldwide clinical problem. Moreover, despite recent advances in targeted therapies for patients with BRAF^V600E mutant melanomas, acquired resistance remains a limiting factor and hence emphasises the acute need for comprehensive pre-clinical studies to increase the biological understanding of such tumours in order to develop novel effective and longlasting therapeutic strategies. Autophagy and ER stress both have a role in melanoma development/progression and chemoresistance although their real impact is still unclear. Here, we show that BRAF^V600E induces a chronic ER stress status directly increasing basal cell autophagy. BRAF^V600E-mediated p38 activation stimulates both the IRE1/ASK1/JNK and TRB3 pathways. Bcl-XL/Bcl-2 phosphorylation by active JNK releases Beclin1 whereas TRB3 inhibits the Akt/mTor axes, together resulting in an increase in basal autophagy. Furthermore, we demonstrate chemical chaperones relieve the BRAF^V600E-mediated chronic ER stress status, consequently reducing basal autophagic activity and increasing the sensitivity of melanoma cells to apoptosis. Taken together, these results suggest enhanced basal autophagy, typically observed in BRAF^V600E melanomas, is a consequence of a chronic ER stress status, which ultimately results in the chemoresistance of such tumours. Targeted therapies that attenuate ER stress may therefore represent a novel and more effective therapeutic strategy for BRAF mutant melanoma.Cutaneous melanoma represents one of the most aggressive and difficult to treat forms of human cancer, with a worldwide incidence that has steadily increased over the past 40 years.^{1, 2}Notoriously unresponsive to conventional chemotherapy, metastatic disease is highly invasive and evolves with an extensive repertoire of molecular defences against immunological and cytotoxic attack.³Although linked to exposure to ultraviolet light, it is widely accepted that both genotypic and phenotypic changes in melanocytes predispose to melanocyte transformation and the onset of melanoma.^{4, 5}Surprisingly, p53 mutations are very rare in melanoma, but activity is, however, impaired through direct or indirect inactivation of key elements of this pathway, including through the suppression of APAF-1 expression,⁶ loss of PTEN function,⁷ dysregulation of Bcl-2 expression,⁸ upregulation of the anti-apoptotic protein Mcl-1 together with its altered slice variant expression ^{9, 10} and the ER chaperone GRP78.^{11, 12, 13} Oncogenic mutations, however, in the Ras/Raf pathway are the most well-described genetic mutations associated with melanoma development and progression.¹⁴ Indeed, up to 90% of all melanomas harbour activating NRAS or BRAF mutations, with BRAF^V600E representing more than 90% of BRAF mutations,^{15, 16} the consequence of which is the constitutive activation of RAF-extracellular signal-regulated kinase/ERK signalling promoting melanoma proliferation and resistance to apoptosis.¹⁷ Nevertheless, mutation of NRAS/BRAF alone is not sufficient to initiate melanomagenesis, because these common mutations are also present in benign nevi, thereby highlighting the requirement of other factors to drive melanocyte transformation and melanoma development.^{15, 16} Dysregulation of autophagy has accordingly been postulated as a secondary event contributing to melanoma progression and, importantly, also has a key role in chemoresistance.^{18, 19, 20}Autophagy is the principal catabolic process for the bulk degradation and recycling of aged/damaged cellular components, organelles and proteins through the formation of a double-membraned cytosolic vesicle able to wrap targeted material. The subsequent fusion with lysosomes and degradation of cargo provide nutrients in times of environmental stress, such as nutrient deprivation or hypoxia.²¹ Though essential for the maintenance of cellular homeostasis under conditions of nutrient deprivation, paradoxically, autophagy promotes both tumour suppression and tumour development.²² Although the accumulation of damaged organelles/cytosolic proteins may lead to cellular transformation, autophagy may also sustain tumour growth in a microenvironment which is commonly poor of oxygen and nutrients.²² Thus, not surprisingly, autophagy activation is frequently observed in late-stage malignancy although the molecular mechanisms mediating its activation/regain of function remain unclear.ER stress may also constitute a key secondary event in melanoma development.²³ Primarily a cytoprotective pro-survival process, ER stress is activated as a result of accumulated unfolded proteins, protein overload or depletion of ER calcium stores and mediated through the activity of the master ER chaperone Grp78 and three signalling pathways; PERK/eIF2α/ATF4, IRE-1/Xbp-1 and ATF6 which collectively maintain ER homeostasis through the instigation of an unfolded protein response (UPR)²⁴ or sustained ER stress may lead to the induction of apoptosis.^{25, 26} Increasing evidence indicates that nutrient deprivation and hypoxia lead to activation of the UPR in various solid tumours, frequently correlating with resistance to chemotherapy.²⁷ The accepted hypothesis is thus that activation of the UPR in cancer cells enables their adaption to such ER stress resulting in the resistance to apoptosis through the persistent expression of pro-survival instead of pro-apoptotic proteins.²⁸Although under stress conditions, autophagy and ER stress seem to act in parallel, indeed they are closely related, because one can regulate the other and vice versa. In fact, ER stress is able to promptly stimulate autophagy,²⁶ whereas autophagy selectively removes the membranes of the endoplasmic reticulum at the end of the UPR, although the molecular mechanisms are still largely unclear.²⁹In the present study, we investigated the link between oncogenic BRAF^V600E and increased basal autophagy in melanoma cells, highlighting the pivotal role played by ER stress, possibly responsible for tumour growth and chemoresistance.     

Atg5-dependent autophagy contributes to the development of acute myeloid leukemia in an MLL-AF9-driven mouse model

Acute myeloid leukemia (AML) is a hierarchical hematopoietic malignancy originating from leukemic stem cells (LSCs). Autophagy is a lysosomal degradation pathway that is hypothesized to be important for the maintenance of AML as well as contribute to chemotherapy response. Here we employ a mouse model of AML expressing the fusion oncogene MLL-AF9 and explore the effects of Atg5 deletion, a key autophagy protein, on the malignant transformation and progression of AML. Consistent with a transient decrease in colony-forming potential in vitro, the in vivo deletion of Atg5 in MLL-AF9-transduced bone marrow cells during primary transplantation prolonged the survival of recipient mice, suggesting that autophagy has a role in MLL-AF9-driven leukemia initiation. In contrast, deletion of Atg5 in malignant AML cells during secondary transplantation did not influence the survival or chemotherapeutic response of leukemic mice. Interestingly, autophagy was found to be involved in the survival of differentiated myeloid cells originating from MLL-AF9-driven LSCs. Taken together, our data suggest that Atg5-dependent autophagy may contribute to the development but not chemotherapy sensitivity of murine AML induced by MLL-AF9.Acute myeloid leukemia (AML) is a clonal hematopoietic malignancy characterized by the uncontrolled proliferation of immature myeloid cells within the bone marrow (BM), eventually suppressing normal hematopoiesis.¹ Recurrent chromosomal translocations frequently occur in AML, one of which involves the fusions of the KMT2A gene on chromosome 11 to a number of potential partners that are diagnosed as prognostically intermediate to poor.¹ Among these fusions, the MLL-AF9 fusion oncogene, resulting from the t(9;11)(p22;q23) translocation, is well studied owing to its robust phenotype in various mouse models of AML.^{2, 3, 4} It has been previously reported that BM transplantation of hematopoietic progenitors expressing exogenous MLL-AF9 leads to rapid in vivo transformation and progression of AML in a syngeneic, immunocompetent mouse model and recapitulates the poor chemotherapy response of t(9;11)(p22;q23) fusion human AML.^{2, 5}Autophagy is an evolutionarily conserved catabolic pathway by which cellular components are engulfed by double-membraned vesicles, called autophagosomes, and delivered to the lysosome for degradation and recycling. Autophagy is best characterized to be induced under stressful conditions, such as organelle damage or nutrient deprivation, and is followed by the elongation of the autophagosome membrane around its cargo. In Atg5-dependent autophagy, the conversion of LC3-I to LC3-II by lipidation is crucial for autophagosome membrane expansion, which is mediated by a series of ubiquitin-like conjugation systems.⁶ Within this pathway, the Atg5-Atg12-Atg16 complex acts as an E3-ubiquitin-ligase-like enzyme that specifically mediates the conjugation of LC3-I to phosphatidylethanolamine to form LC3-II, which inserts to the autophagosomal membrane. Autophagosome maturation is followed by fusion to lysosomes, at which time the inner compartment is degraded. The genetic ablation of Atg5 leads to a complete and highly selective inhibition of LC3-dependent autophagosome formation.^{6, 7}Autophagy is known to be implicated in cancer as both a tumor promoter and a tumor suppressor.⁸ The genetic ablation of autophagy in mouse hematopoietic stem cells (HSCs) has been shown to result in severe impairments to HSC maintenance.^{9, 10, 11, 12, 13} Autophagy dysregulation has also been implicated in AML,^{12, 13, 14} suggesting that targeting autophagy could be promising for AML treatment. As an expanding arsenal of pharmacological autophagy modulators are being developed,^{15, 16} it has become increasingly important to specifically determine whether autophagy has an important role in AML using a genetic mouse model. Therefore, we sought to dissect the role of autophagy through the in vivo homozygous deletion of Atg5 in MLL-AF9-driven murine AML. We discover in this study that Atg5 deletion during primary transplantation prolongs the survival of animals, whereas Atg5 deletion after secondary transplantation has no effect on animal survival, suggesting a role for autophagy in the initiation, but not maintenance, of AML in our model. We additionally assessed the effect of autophagy in chemotherapeutic response and found that Atg5 deletion in our MLL-AF9 model had no effect on the in vivo response to cytarabine and doxorubicin combination therapy, suggesting that autophagy does not significantly contribute to chemotherapy response in this model.     

The LRRK2 inhibitor GSK2578215A induces protective autophagy in SH-SY5Y cells: involvement of Drp-1-mediated mitochondrial fission and mitochondrial-derived ROS signaling

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been associated with Parkinson''s disease, and its inhibition opens potential new therapeutic options. Among the drug inhibitors of both wild-type and mutant LRRK2 forms is the 2-arylmethyloxy-5-subtitutent-N-arylbenzamide GSK257815A. Using the well-established dopaminergic cell culture model SH-SY5Y, we have investigated the effects of GSK2578215A on crucial neurodegenerative features such as mitochondrial dynamics and autophagy. GSK2578215A induces mitochondrial fragmentation of an early step preceding autophagy. This increase in autophagosome results from inhibition of fusion rather than increases in synthesis. The observed effects were shared with LRRK2-IN-1, a well-described, structurally distinct kinase inhibitor compound or when knocking down LRRK2 expression using siRNA. Studies using the drug mitochondrial division inhibitor 1 indicated that translocation of the dynamin-related protein-1 has a relevant role in this process. In addition, autophagic inhibitors revealed the participation of autophagy as a cytoprotective response by removing damaged mitochondria. GSK2578215A induced oxidative stress as evidenced by the accumulation of 4-hydroxy-2-nonenal in SH-SY5Y cells. The mitochondrial-targeted reactive oxygen species scavenger MitoQ positioned these species as second messengers between mitochondrial morphologic alterations and autophagy. Altogether, our results demonstrated the relevance of LRRK2 in mitochondrial-activated pathways mediating in autophagy and cell fate, crucial features in neurodegenerative diseases.Nowadays, Parkinson''s disease (PD) constitutes the main motor disorder and the second neurodegenerative disease after Alzheimer''s disease. Etiology of PD remains unknown, but both environmental and genetic factors have been implicated. Among the genes associated with PD is the leucine-rich repeat kinase 2 (LRRK2, PARK8, OMIM 607060) encoding gene encoded by PARK8. Indeed, LRRK2 mutations have been described in a substantial number of idiopathic late-onset PD patients without a known family history of the disease.^{1, 2, 3}The physiologic function remains unknown. It localizes in the cytosol as well as in specific membrane subdomains, including mitochondria, autophagosomes and autolysosomes,⁴ and interacts with a whole array of proteins, including both α- and β-tubulin,^{5, 6} tau,⁷ α-synuclein⁸ and F-actin.⁹ LRRK2 gene mutations, including the most common G2019S,³ are associated with increases in toxic putative kinase activity.^{1, 10} LRRK2 overexpression is toxic to cultured cells,^{11, 12} and LRRK2 loss did not cause neurodegenerative changes (for a review see Tong and Shen¹³). However, LRRK2 transgenic mice lack obvious PD-like behavioral phenotypes.¹⁴ LRRK2-associated PD patients show degeneration of dopaminergic neurons in the substantia nigra.¹⁵ Data from our own group and others have associated mitochondrial apoptotical pathways with PD,^{16, 17, 18} and, in this context, LRRK2 mutant-mediated toxicity could be due to mitochondria-dependent apoptosis.¹⁹ There is considerable evidence for impaired mitochondrial function and morphology in both early-onset, autosomal recessive inherited PD and late-onset sporadic PD.Mitochondrial dynamics include several mechanisms, such as fission, fusion and mitophagy.^{20, 21} Altered fission/fusion dynamics might be a common pathogenic pathway of neurodegenerative diseases. It is well documented that mitochondrial dynamics constitute a relevant issue in some experimental neurodegenerative models.^{20, 22, 23, 24, 25} Mitochondrial dynamics is tightly regulated by cellular pathways including those participated by the dynamin-related protein-1 (Drp1). Drp1 mostly locates in the cytoplasm, but is stimulated after fission stimuli to migrate to the mitochondria. Once there, Drp1 forms ring-like structures, which wrap around the scission site to constrict the mitochondrial membrane resulting in mitochondrial fission.^{26, 27} Interestingly, a functional interaction between PD-associated LRRK2 and members of the dynamin GTPase superfamily has been described.²⁸Macroautophagy (hereafter referred to as autophagy) is an active cellular response, which functions in the intracellular degradation system of cellular debris such as damaged organelles. Whether autophagy promotes cell death or enhances survival is still controversial.^{29, 30} It requires the formation of autophagosomes where cellular content is to be degraded by the action of lysosomal enzymatic content. Autophagosome formation is regulated by an orderly action of >30 autophagy-related (Atg) proteins. Among them is the microtubule-associated protein 1A/1B-light chain 3 (LC3), a homolog of Apg8p, which is essential for autophagy in yeast and is associated with autophagosome membranes.³¹ Interestingly, these vesicles are mostly highly mobile in the cytoplasm.³² Wild-type and mutant LRRK2 expression has been related to autophagy.^{4, 33, 34, 35, 36} Reactive oxygen species (ROS) function as relevant second messengers after several stimuli, including mitochondrial disruption. Exacerbated ROS increases might result in overactivation of antioxidant systems and yield harmful oxidative stress. Among oxidative stress hallmarks is the accumulation of α,β-unsaturated hydroxyalkenal 4-hydroxy-2-nonenal (4-HNE), whose accumulation has been reported in PD post-mortem patient brains,^{37, 38} thus giving a significant relevance to ROS in the pathogenesis of PD.All these results indicate LRRK2 as a promising pharmacologic target in PD treatment.³⁹ Several LRRK2 inhibitor drugs have been synthetized, such as the potent and highly selective 2-arylmethyloxy-5-substitutent-N-arylbenzamide (GSK2578215A). GSK2578215A exhibits biochemical IC₅₀s of 10.9 nM against wild-type LRRK2, and possesses a high ratio of brain to plasma distribution.⁴⁰ This study provides key insights into the mechanisms downstream of LRRK2 inhibition, and spreads light onto an underexplored, yet potentially tractable therapeutic target for treating LRRK2-associated PD. We demonstrate how inhibition of this kinase results in the activation of cellular death pathways such as the mitochondrial fission machinery, and how cells reply by activating a protective autophagic response. Our results show the presence of oxidative stress hallmarks, thus pointing to a key function for ROS, placed downstream of mitochondrial fission.     

Germ cell-specific Atg7 knockout results in primary ovarian insufficiency in female mice

Primary ovarian insufficiency (POI) is a common cause of infertility in around 1–2% of women aged <40 years. However, the mechanisms that cause POI are still poorly understood. Here we showed that germ cell-specific knockout of an essential autophagy induction gene Atg7 led to subfertility in female mice. The subfertility of Atg7 deletion females was caused by severe ovarian follicle loss, which is very similar to human POI patients. Further investigation revealed that germ cell-specific Atg7 knockout resulted in germ cell over-loss at the neonatal transition period. In addition, our in vitro studies also demonstrated that autophagy could protect oocytes from over-loss by apoptosis in neonatal ovaries under the starvation condition. Taken together, our results uncover a new role for autophagy in the regulation of ovarian primordial follicle reservation and hint that autophagy-related genes might be potential pathogenic genes to POI of women.Primary ovarian insufficiency (POI), also known as premature ovarian failure (POF), is an ovarian defect characterized by the premature depletion of ovarian follicles before the age of 40 years. POI is a common cause of infertility in women, affecting 1–2% of individuals aged <40 years and 0.1% of individuals aged <30 years.¹ Potential etiologies for POI are highly heterogeneous, which include iatrogenic, infectious, autoimmune, metabolic, chromosomal and genetic factors.² At present, about 25% of all forms of POF can be classified as iatrogenic and are related to cancer treatment, but >50% of the cases remain idiopathic. Though the pathogenic mechanism remains unexplained in the majority of the cases, several observations support a prevalent role of genetic mechanisms in the pathogenesis of idiopathic POI. It has been reported that mutations in FMR1, BMP-15, GDF-9, FOCL2, FSHR, LHR, INHA, GALT and AIRE are associated with POI.^{3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13} The genetic information of POI is very useful for family counseling, because it can predict the female relatives who may be at higher risk for POI and fertility loss in young age. The female carriers will be able to plan their conception before ovarian failure occurs. This requirement is becoming more and more important, because women nowadays tend to conceive ever more frequently in their thirties and forties,¹⁰ when the risk of POI in the general population is about 1–2%. However, still few genes could be identified that can explain a substantial proportion of the cases of POI.An important phenotype of POI is infertility, thus POI patients do not have large family histories, and therefore are difficult to study using traditional genetic methods, such as linkage analysis. Animal models of POI have been successfully used to identify candidate genes in this disease. The disruption of meiosis-specific genes, Bcl-2 family apoptotic-related genes, Pten-PI3K-Akt-Foxo3 pathway and Tsc1/2-mTOR signaling pathway result in POI-like phenotype in mice.^{14, 15, 16, 17} However, as a complex disorder, the genetic etiologies of POI still need to be further investigated to better understand the underlying molecular mechanisms.Macroautophagy (hereafter referred to as autophagy) is the primary intracellular catabolic mechanism for degrading and recycling long-lived proteins and organelles, which is evolutionarily conserved from yeast to mammals.¹⁸ During autophagy, isolation membrane enwraps parts of the cytoplasm and intracellular organelles, and fuse with each other forming a double membrane structure, known as the autophagosome. Then the outer membrane of the autophagosome fuses with the lysosome to form autolysosome, in which the cytoplasm-derived materials are degraded by resident hydrolases.¹⁹ The primary function of autophagy is to allow cells or organisms to survive nutrient starvation conditions by recycling either proteins or other cellular components. This process is important for cells to adapt their metabolism to starvation caused by decreased extracellular nutrients or by decreased intracellular metabolite concentrations. In addition to nutrient supply and adaptation to stress conditions, a number of observations have revealed that autophagy also functions in many physiological processes in mammalian systems, such as cell death, antiaging mechanisms, innate immunity, development and tumor suppression.^{20, 21, 22, 23, 24, 25}From the discovery of the molecular mechanism underlying autophagy, it was found that autophagy is required for the reproductive process in budding yeast.²⁶ In mammals, fertilization induces massive autophagy to degrade maternal proteins and messenger RNAs, and autophagy functions as a major nutrient-providing system for embryos before their implantation.²⁷ Our recent work indicates that autophagy is required for acrosome biogenesis during spermatogenesis in mice, thus essential to male fertility.²⁴ However, whether autophagy is involved in female gametogenesis or not is still unknown. Here, we showed that germ cell-specific knockout of an essential autophagy induction gene Atg7 led to POI in female mice, and the numbers of the oocytes and follicles were significantly declined in the adult mutant mice. Further investigation revealed that autophagy protected oocytes over-loss during the neonatal transition period. Our results suggest that autophagy-related genes might be pathogenic genes to POI.