Mammalian Autophagy: How Does It Work?

Autophagy is a conserved intracellular pathway that delivers cytoplasmic contents to lysosomes for degradation via double-membrane autophagosomes. Autophagy substrates include organelles such as mitochondria, aggregate-prone proteins that cause neurodegeneration and various pathogens. Thus, this pathway appears to be relevant to the pathogenesis of diverse diseases, and its modulation may have therapeutic value. Here, we focus on the cell and molecular biology of mammalian autophagy and review the key proteins that regulate the process by discussing their roles and how these may be modulated by posttranslational modifications. We consider the membrane-trafficking events that impact autophagy and the questions relating to the sources of autophagosome membrane(s). Finally, we discuss data from structural studies and some of the insights these have provided.

Diverse autophagosome membrane sources coalesce in recycling endosomes.

Autophagic protein degradation is mediated by autophagosomes that fuse with lysosomes, where their contents are degraded. The membrane origins of autophagosomes may involve multiple sources. However, it is unclear if and where distinct membrane sources fuse during autophagosome biogenesis. Vesicles containing mATG9, the only transmembrane autophagy protein, are seen in many sites, and fusions with other autophagic compartments have not been visualized in mammalian cells. We observed that mATG9 traffics from the plasma membrane to recycling endosomes in carriers that appear to be routed differently from ATG16L1-containing vesicles, another source of autophagosome membrane. mATG9- and ATG16L1-containing vesicles traffic to recycling endosomes, where VAMP3-dependent heterotypic fusions occur. These fusions correlate with autophagosome formation, and both processes are enhanced by perturbing membrane egress from recycling endosomes. Starvation, a primordial autophagy activator, reduces membrane recycling from recycling endosomes and enhances mATG9-ATG16L1 vesicle fusion. Thus, this mechanism may fine-tune physiological autophagic responses.

Autophagosome precursor maturation requires homotypic fusion.

Autophagy is a catabolic process in which lysosomes degrade intracytoplasmic contents transported in double-membraned autophagosomes. Autophagosomes are formed by the elongation and fusion of phagophores, which can be derived from preautophagosomal structures coming from the plasma membrane and other sites like the endoplasmic reticulum and mitochondria. The mechanisms by which preautophagosomal structures elongate their membranes and mature toward fully formed autophagosomes still remain unknown. Here, we show that the maturation of the early Atg16L1 precursors requires homotypic fusion, which is essential for subsequent autophagosome formation. Atg16L1 precursor homotypic fusion depends on the SNARE protein VAMP7 together with partner SNAREs. Atg16L1 precursor homotypic fusion is a critical event in the early phases of autophagy that couples membrane acquisition and autophagosome biogenesis, as this step regulates the size of the vesicles, which in turn appears to influence their subsequent maturation into LC3-positive autophagosomes.

IBtkα Activates the ß-Catenin-Dependent Transcription of MYC through Ubiquitylation and Proteasomal Degradation of GSK3ß in Cancerous B Cells.

The IBTK gene encodes the IBtkα protein that is a substrate receptor of E3 ubiquitin ligase, Cullin 3. We have previously reported the pro-tumorigenic activity of Ibtk in MYC-dependent B-lymphomagenesis observed in Eµ-myc transgenic mice. Here, we provide mechanistic evidence of the functional interplay between IBtkα and MYC. We show that IBtkα, albeit indirectly, activates the ß-catenin-dependent transcription of the MYC gene. Of course, IBtkα associates with GSK3ß and promotes its ubiquitylation, which is associated with proteasomal degradation. This event increases the protein level of ß-catenin, a substrate of GSK3ß, and results in the transcriptional activation of the MYC and CCND1 target genes of ß-catenin, which are involved in the control of cell division and apoptosis. In particular, we found that in Burkitt's lymphoma cells, IBtkα silencing triggered the downregulation of both MYC mRNA and protein expression, as well as a strong decrease of cell survival, mainly through the induction of apoptotic events, as assessed by using flow cytometry-based cell cycle and apoptosis analysis. Collectively, our results shed further light on the complex puzzle of IBtkα interactome and highlight IBtkα as a potential novel therapeutic target to be employed in the strategy for personalized therapy of B cell lymphoma.

Complex inhibitory effects of nitric oxide on autophagy.

Autophagy, a major degradation process for long-lived and aggregate-prone proteins, affects various human processes, such as development, immunity, cancer, and neurodegeneration. Several autophagy regulators have been identified in recent years. Here we show that nitric oxide (NO), a potent cellular messenger, inhibits autophagosome synthesis via a number of mechanisms. NO impairs autophagy by inhibiting the activity of S-nitrosylation substrates, JNK1 and IKKß. Inhibition of JNK1 by NO reduces Bcl-2 phosphorylation and increases the Bcl-2-Beclin 1 interaction, thereby disrupting hVps34/Beclin 1 complex formation. Additionally, NO inhibits IKKß and reduces AMPK phosphorylation, leading to mTORC1 activation via TSC2. Overexpression of nNOS, iNOS, or eNOS impairs autophagosome formation primarily via the JNK1-Bcl-2 pathway. Conversely, NOS inhibition enhances the clearance of autophagic substrates and reduces neurodegeneration in models of Huntington's disease. Our data suggest that nitrosative stress-mediated protein aggregation in neurodegenerative diseases may be, in part, due to autophagy inhibition.

Revisiting Bacterial Ubiquitin Ligase Effectors: Weapons for Host Exploitation.

Protein ubiquitylation plays a central role in eukaryotic cell physiology. It is involved in several regulatory processes, ranging from protein folding or degradation, subcellular localization of proteins, vesicular trafficking and endocytosis to DNA repair, cell cycle, innate immunity, autophagy, and apoptosis. As such, it is reasonable that pathogens have developed a way to exploit such a crucial system to enhance their virulence against the host. Hence, bacteria have evolved a wide range of effectors capable of mimicking the main players of the eukaryotic ubiquitin system, in particular ubiquitin ligases, by interfering with host physiology. Here, we give an overview of this topic and, in particular, we detail and discuss the mechanisms developed by pathogenic bacteria to hijack the host ubiquitination system for their own benefit.

Connections between SNAREs and autophagy.

Autophagy involves the sequestration of portions of cytoplasm by double-membraned autophagosomes, which are then trafficked to lysosomes. After autophagosome-lysosome fusion, the contents of the autophagosomes are degraded by lysosomal hydrolases. SNAREs [soluble N-ethylmaleimide-sensitive fusion (NSF) attachment protein receptors] are molecules that mediate vesicular fusion events. Here, we review recent data implicating SNAREs as having key roles both in the genesis of autophagosomes, as well as in autophagosome-lysosome fusion, and we discuss the implications of these findings in the context of a long-standing mystery: the origin of autophagosomes.

Regulation of mammalian autophagy in physiology and pathophysiology.

(Macro)autophagy is a bulk degradation process that mediates the clearance of long-lived proteins and organelles. Autophagy is initiated by double-membraned structures, which engulf portions of cytoplasm. The resulting autophagosomes ultimately fuse with lysosomes, where their contents are degraded. Although the term autophagy was first used in 1963, the field has witnessed dramatic growth in the last 5 years, partly as a consequence of the discovery of key components of its cellular machinery. In this review we focus on mammalian autophagy, and we give an overview of the understanding of its machinery and the signaling cascades that regulate it. As recent studies have also shown that autophagy is critical in a range of normal human physiological processes, and defective autophagy is associated with diverse diseases, including neurodegeneration, lysosomal storage diseases, cancers, and Crohn's disease, we discuss the roles of autophagy in health and disease, while trying to critically evaluate if the coincidence between autophagy and these conditions is causal or an epiphenomenon. Finally, we consider the possibility of autophagy upregulation as a therapeutic approach for various conditions.

XIAP and cIAP1 amplifications induce Beclin 1-dependent autophagy through NFκB activation.

Perturbations in autophagy and apoptosis are associated with cancer development. XIAP and cIAP1 are two members of the inhibitors of apoptosis protein family whose expression is elevated in different cancers. Here we report that XIAP and cIAP1 induce autophagy by upregulating the transcription of Beclin 1, an essential autophagy gene. The E3 ubiquitin ligase activity of both proteins activates NFκB signalling, leading to the direct binding of p65 to the promoter of Beclin 1 and to its transcriptional activation. This mechanism may be relevant in cancer cells, since we found increased levels of autophagy in different B-cell lymphoma-derived cell lines where XIAP is overexpressed and pharmacological inhibition of XIAP in these cell lines reduced autophagosome biogenesis. Thus, the chemotherapy resistance associated with XIAP and cIAP1 overexpression observed in several human cancers may be, at least in part, due to the Beclin 1-dependent autophagy activation by IAPs described in this study. In this context, the disruption of this increased autophagy might represent a valuable pharmacological tool to be included in combined anti-neoplastic therapies.

IGF-1 receptor antagonism inhibits autophagy.

Inhibition of the insulin/insulin-like growth factor signalling pathway increases lifespan and protects against neurodegeneration in model organisms, and has been considered as a potential therapeutic target. This pathway is upstream of mTORC1, a negative regulator of autophagy. Thus, we expected autophagy to be activated by insulin-like growth factor-1 (IGF-1) inhibition, which could account for many of its beneficial effects. Paradoxically, we found that IGF-1 inhibition attenuates autophagosome formation. The reduced amount of autophagosomes present in IGF-1R depleted cells can be, at least in part, explained by a reduced formation of autophagosomal precursors at the plasma membrane. In particular, IGF-1R depletion inhibits mTORC2, which, in turn, reduces the activity of protein kinase C (PKCα/ß). This perturbs the actin cytoskeleton dynamics and decreases the rate of clathrin-dependent endocytosis, which impacts autophagosome precursor formation. Finally, with important implications for human diseases, we demonstrate that pharmacological inhibition of the IGF-1R signalling cascade reduces autophagy also in zebrafish and mice models. The novel link we describe here has important consequences for the interpretation of genetic experiments in mammalian systems and for evaluating the potential of targeting the IGF-1R receptor or modulating its signalling through the downstream pathway for therapeutic purposes under clinically relevant conditions, such as neurodegenerative diseases, where autophagy stimulation is considered beneficial.

α-Synuclein levels modulate Huntington's disease in mice.

α-Synuclein and mutant huntingtin are the major constituents of the intracellular aggregates that characterize the pathology of Parkinson's disease (PD) and Huntington's disease (HD), respectively. α-Synuclein is likely to be a major contributor to PD, since overexpression of this protein resulting from genetic triplication is sufficient to cause human forms of PD. We have previously demonstrated that wild-type α-synuclein overexpression impairs macroautophagy in mammalian cells and in transgenic mice. Overexpression of human wild-type α-synuclein in cells and Drosophila models of HD worsens the disease phenotype. Here, we examined whether α-synuclein overexpression also worsens the HD phenotype in a mammalian system using two widely used N-terminal HD mouse models (R6/1 and N171-82Q). We also tested the effects of α-synuclein deletion in the same N-terminal HD mouse models, as well as assessed the effects of α-synuclein deletion on macroautophagy in mouse brains. We show that overexpression of wild-type α-synuclein in both mouse models of HD enhances the onset of tremors and has some influence on the rate of weight loss. On the other hand, α-synuclein deletion in both HD models increases autophagosome numbers and this is associated with a delayed onset of tremors and weight loss, two of the most prominent endophenotypes of the HD-like disease in mice. We have therefore established a functional link between these two aggregate-prone proteins in mammals and provide further support for the model that wild-type α-synuclein negatively regulates autophagy even at physiological levels.

Metabolic rewiring and autophagy inhibition correct lysosomal storage disease in mucopolysaccharidosis IIIB.

Mucopolysaccharidoses (MPSs) are lysosomal disorders with neurological involvement for which no cure exists. Here, we show that recombinant NK1 fragment of hepatocyte growth factor rescues substrate accumulation and lysosomal defects in MPS I, IIIA and IIIB patient fibroblasts. We investigated PI3K/Akt pathway, which is of crucial importance for neuronal function and survival, and demonstrate that PI3K inhibition abolishes NK1 therapeutic effects. We identified that autophagy inhibition, by Beclin1 silencing, reduces MPS IIIB phenotype and that NK1 downregulates autophagic-lysosome (ALP) gene expression, suggesting a possible contribution of autophagosome biogenesis in MPS. Indeed, metabolomic analyses revealed defects of mitochondrial activity accompanied by anaerobic metabolism and inhibition of AMP-activated protein kinase (AMPK), which acts on metabolism and autophagy, rescues lysosomal defects. These results provide insights into the molecular mechanisms of MPS IIIB physiopathology, supporting the development of new promising approaches based on autophagy inhibition and metabolic rewiring to correct lysosomal pathology in MPSs.

A method for the analysis of the oligomerization profile of the Huntington's disease-associated, aggregation-prone mutant huntingtin protein by isopycnic ultracentrifugation.

Conformational diseases, such as Alzheimer's, Parkinson's and Huntington's diseases as well as ataxias and fronto-temporal disorders, are part of common class of neurological disorders characterised by the aggregation and progressive accumulation of mutant proteins which display aberrant conformation. In particular, Huntington's disease (HD) is caused by mutations leading to an abnormal expansion in the polyglutamine (poly-Q) tract of the huntingtin protein (HTT), leading to the formation of inclusion bodies in neurons of affected patients. Furthermore, recent experimental evidence is challenging the conventional view of the disease by revealing the ability of mutant HTT to be transferred between cells by means of extracellular vesicles (EVs), allowing the mutant protein to seed oligomers involving both the mutant and wild type forms of the protein. There is still no successful strategy to treat HD. In addition, the current understanding of the biological processes leading to the oligomerization and aggregation of proteins bearing the poly-Q tract has been derived from studies conducted on isolated poly-Q monomers and oligomers, whose structural properties are still unclear and often inconsistent. Here we describe a standardised biochemical approach to analyse by isopycnic ultracentrifugation the oligomerization of the N-terminal fragment of mutant HTT. The dynamic range of our method allows one to detect large and heterogeneous HTT complexes. Hence, it could be harnessed for the identification of novel molecular determinants responsible for the aggregation and the prion-like spreading properties of HTT in the context of HD. Equally, it provides a tool to test novel small molecules or bioactive compounds designed to inhibit the aggregation of mutant HTT.

Autophagic substrate clearance requires activity of the syntaxin-5 SNARE complex.

Autophagy is a lysosome-dependent cellular catabolic mechanism that mediates the turnover of intracellular organelles and long-lived proteins. Reduced autophagic activity has been shown to lead to the accumulation of misfolded proteins in neurons and might be involved in chronic neurodegenerative diseases. Here, we uncover an essential role for the syntaxin-5 SNARE complex in autophagy. Using genetic knockdown, we show that the syntaxin-5 SNARE complex regulates the later stages of autophagy after the initial formation of autophagosomes. This SNARE complex acts on autophagy by regulating ER-to-Golgi transport through the secretory pathway, which is essential for the activity of lysosomal proteases such as cathepsins. Depletion of syntaxin-5 complex components results in the accumulation of autophagosomes as a result of lysosomal dysfunction, leading to decreased degradation of autophagic substrates. Our findings provide a novel link between a fundamental process such as intracellular trafficking and human diseases that might be affected by defective biogenesis and/or homeostasis of the autophagosome-lysosome degradation system.

Rilmenidine attenuates toxicity of polyglutamine expansions in a mouse model of Huntington's disease.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by a polyglutamine expansion in huntingtin. There are no treatments that are known to slow the neurodegeneration caused by this mutation. Mutant huntingtin causes disease via a toxic gain-of-function mechanism and has the propensity to aggregate and form intraneuronal inclusions. One therapeutic approach for HD is to enhance the degradation of the mutant protein. We have shown that this can be achieved by upregulating autophagy, using the drug rapamycin. In order to find safer ways of inducing autophagy for clinical purposes, we previously screened United States Food and Drug Administration-approved drugs for their autophagy-stimulating potential. This screen suggested that rilmenidine, a well tolerated, safe, centrally acting anti-hypertensive drug, could induce autophagy in cell culture via a pathway that was independent of the mammalian target of rapamycin. Here we have shown that rilmenidine induces autophagy in mice and in primary neuronal culture. Rilmenidine administration attenuated the signs of disease in a HD mouse model and reduced levels of the mutant huntingtin fragment. As rilmenidine has a long safety record and is designed for chronic use, our data suggests that it should be considered for the treatment of HD and related conditions.

Antioxidants can inhibit basal autophagy and enhance neurodegeneration in models of polyglutamine disease.

Many neurodegenerative diseases exhibit protein accumulation and increased oxidative stress. Therapeutic strategies include clearing aggregate-prone proteins by enhancing autophagy or decreasing oxidative stress with antioxidants. Many autophagy-inducing stimuli increase reactive oxygen species (ROS), raising concerns that the benefits of autophagy up-regulation may be counterbalanced by ROS toxicity. Here we show that not all autophagy inducers significantly increase ROS. However, many antioxidants inhibit both basal and induced autophagy. By blocking autophagy, antioxidant drugs can increase the levels of aggregate-prone proteins associated with neurodegenerative disease. In fly and zebrafish models of Huntington's disease, antioxidants exacerbate the disease phenotype and abrogate the rescue seen with autophagy-inducing agents. Thus, the potential benefits in neurodegenerative diseases of some classes of antioxidants may be compromised by their autophagy-blocking properties.

Activation of Non-Canonical Autophagic Pathway through Inhibition of Non-Integrin Laminin Receptor in Neuronal Cells.

To fight neurodegenerative diseases, several therapeutic strategies have been proposed that, to date, are either ineffective or at the early preclinical stages. Intracellular protein aggregates represent the cause of about 70% of neurodegenerative disorders, such as Alzheimer's disease. Thus, autophagy, i.e., lysosomal degradation of macromolecules, could be employed in this context as a therapeutic strategy. Searching for a compound that stimulates this process led us to the identification of a 37/67kDa laminin receptor inhibitor, NSC48478. We have analysed the effects of this small molecule on the autophagic process in mouse neuronal cells and found that NSC48478 induces the conversion of microtubule-associated protein 1A/1B-light chain 3 (LC3-I) into the LC3-phosphatidylethanolamine conjugate (LC3-II). Interestingly, upon NSC48478 treatment, the contribution of membranes to the autophagic process derived mainly from the non-canonical m-TOR-independent endocytic pathway, involving the Rab proteins that control endocytosis and vesicle recycling. Finally, qRT-PCR analysis suggests that, while the expression of key genes linked to canonical autophagy was unchanged, the main genes related to the positive regulation of endocytosis (pinocytosis and receptor mediated), along with genes regulating vesicle fusion and autolysosomal maturation, were upregulated under NSC48478 conditions. These results strongly suggest that 37/67 kDa inhibitor could be a useful tool for future studies in pathological conditions.

Chemical inducers of autophagy that enhance the clearance of mutant proteins in neurodegenerative diseases.

Many of the neurodegenerative diseases that afflict people are caused by intracytoplasmic aggregate-prone proteins. These include Parkinson disease, tauopathies, and polyglutamine expansion diseases such as Huntington disease. In Mendelian forms of these diseases, the mutations generally confer toxic novel functions on the relevant proteins. Thus, one potential strategy for dealing with these mutant proteins is to enhance their degradation. This can be achieved by up-regulating macroautophagy, which we will henceforth call autophagy. In this minireview, we will consider the reasons why autophagy up-regulation may be a powerful strategy for these diseases. In addition, we will consider some of the drugs and associated signaling pathways that can be used to induce autophagy with these therapeutic aims in mind.

Autophagy induction reduces mutant ataxin-3 levels and toxicity in a mouse model of spinocerebellar ataxia type 3.

Spinocerebellar ataxia type 3 is a neurodegenerative disorder caused by the expansion of the polyglutamine repeat region within the ataxin-3 protein. The mutant protein forms intracellular aggregates in the brain. However, the cellular mechanisms causing toxicity are still poorly understood and there are currently no effective treatments. In this study we show that administration of a rapamycin ester (cell cycle inhibitor-779, temsirolimus) improves motor performance in a transgenic mouse model of spinocerebellar ataxia type 3. Temsirolimus inhibits mammalian target of rapamycin and hence upregulates protein degradation by autophagy. Temsirolimus reduces the number of aggregates seen in the brains of transgenic mice and decreases levels of cytosolic soluble mutant ataxin-3, while endogenous wild-type protein levels remain unaffected. Temsirolimus is designed for long-term use in patients and therefore represents a possible therapeutic strategy for the treatment of spinocerebellar ataxia type 3. Using this disease model and treatment paradigm, we employed a microarray approach to investigate transcriptional changes that might be important in the pathogenesis of spinocerebellar ataxia type 3. This identified ubiquitin specific peptidase-15, which showed expression changes at both the messenger ribonucleic acid and protein level. Ubiquitin specific peptidase-15 levels were also changed in mice expressing another mutant polyglutamine protein, huntingtin. In total we identified 16 transcripts that were decreased in transgenic ataxin-3 mice that were normalized following temsirolimus treatment. In this mouse model with relatively mild disease progression, the number of transcripts changed was low and the magnitude of these changes was small. However, the importance of these transcriptional alterations in the pathogenesis of spinocerebellar ataxia type 3 remains unclear.

Protocol for labeling and fixation of intact lysosomes with esterified amino acid analogs to assess lysosomal expansion in living eukaryotic cells.

The lysosomal compartment is a key hub for cell metabolism, and morphological alterations have been described in several pathological conditions. Here, we describe the use of amino acid analogs modified by the presence of a methyl ester group that accumulates within lysosomes. This generates an intraluminal osmotic effect able to trigger a rapid and selective expansion of the lysosomal compartment within 2 h of treatment. We also present protocols to preserve lysosomal morphology, which yields a more accurate size measurement. For complete details on the use and execution of this protocol, please refer to Scerra et al. (2021).