Maria Jimenez-Sanchez

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 Crohns 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.

Mutation in VPS35 associated with Parkinson’s disease impairs WASH complex association and inhibits autophagy

Endosomal protein sorting controls the localization of many physiologically important proteins and is linked to several neurodegenerative diseases. VPS35 is a component of the retromer complex, which mediates endosome-to-Golgi retrieval of membrane proteins such as the cation-independent mannose 6-phosphate receptor. Furthermore, retromer is also required for the endosomal recruitment of the actin nucleation promoting WASH complex. The VPS35 D620N mutation causes a rare form of autosomal-dominant Parkinson’s disease (PD). Here we show that this mutant associates poorly with the WASH complex and impairs WASH recruitment to endosomes. Autophagy is impaired in cells expressing PD-mutant VPS35 or lacking WASH. The autophagy defects can be explained, at least in part, by abnormal trafficking of the autophagy protein ATG9A. Thus, the PD-causing D620N mutation in VPS35 restricts WASH complex recruitment to endosomes, and reveals a novel role for the WASH complex in autophagosome formation.

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.

Mammalian macroautophagy at a glance

Autophagy refers to a set of non-specific bulk degradation processes in which cells deliver cytoplasmic substrates for lysosomal degradation. Types of autophagy include macroautophagy, chaperone-mediated autophagy and microautophagy. Chaperone-mediated autophagy is selective for specific cytosolic

PICALM modulates autophagy activity and tau accumulation

Genome-wide association studies have identified several loci associated with Alzheimer’s disease (AD), including proteins involved in endocytic trafficking such as PICALM/CALM (phosphatidylinositol binding clathrin assembly protein). It is unclear how these loci may contribute to AD pathology. Here we show that CALM modulates autophagy and alters clearance of tau, a protein which is a known autophagy substrate and which is causatively linked to AD, both in vitro and in vivo. Furthermore, altered CALM expression exacerbates tau-mediated toxicity in zebrafish transgenic models. CALM influences autophagy by regulating the endocytosis of SNAREs, such as VAMP2, VAMP3 and VAMP8, which have diverse effects on different stages of the autophagy pathway, from autophagosome formation to autophagosome degradation. This study suggests that the AD genetic risk factor CALM modulates autophagy, and this may affect disease in a number of ways including modulation of tau turnover.

Autophagy and polyglutamine diseases

Highlights ► The ubiquitin–proteasome system and autophagy are two main degradative pathways. ► Autophagy upregulation may protect against polyglutamine-expanded protein neurotoxicity. ► Autophagy compromise may occur in certain neurodegenerative diseases.

The Hedgehog signalling pathway regulates autophagy

Autophagy is a highly conserved degradative process that removes damaged or unnecessary proteins and organelles, and recycles cytoplasmic contents during starvation. Autophagy is essential in physiological processes such as embryonic development but how autophagy is regulated by canonical developmental pathways is unclear. Here we show that the Hedgehog signalling pathway inhibits autophagosome synthesis, both in basal and in autophagy-induced conditions. This mechanism is conserved in mammalian cells and in Drosophila, and requires the orthologous transcription factors Gli2 and Ci, respectively. Furthermore, we identify that activation of the Hedgehog pathway reduces PERK levels, concomitant with a decrease in phosphorylation of the translation initiation factor eukaryotic initiation factor 2α, suggesting a novel target of this pathway and providing a possible link between Hedgehog signalling and autophagy.

The Parkinson's disease-associated genes ATP13A2 and SYT11 regulate autophagy via a common pathway

Forms of Parkinsons disease (PD) are associated with lysosomal and autophagic dysfunction. ATP13A2, which is mutated in some types of early-onset Parkinsonism, has been suggested as a regulator of the autophagy–lysosome pathway. However, little is known about the ATP13A2 effectors and how they regulate this pathway. Here we show that ATP13A2 depletion negatively regulates another PD-associated gene (SYT11) at both transcriptional and post-translational levels. Decreased SYT11 transcription is controlled by a mechanism dependent on MYCBP2-induced ubiquitination of TSC2, which leads to mTORC1 activation and decreased TFEB-mediated transcription of SYT11, while increased protein turnover is regulated by SYT11 ubiquitination and degradation. Both mechanisms account for a decrease in the levels of SYT11, which, in turn, induces lysosomal dysfunction and impaired degradation of autophagosomes. Thus, we propose that ATP13A2 and SYT11 form a new functional network in the regulation of the autophagy–lysosome pathway, which is likely to contribute to forms of PD-associated neurodegeneration.

siRNA screen identifies QPCT as a druggable target for Huntington's disease

Huntington’s disease (HD) is a currently incurable neurodegenerative condition caused by an abnormally expanded polyglutamine tract in huntingtin (HTT). We identified novel modifiers of mutant HTT toxicity by performing a large-scale “druggable genome” siRNA screen in human cultured cells, followed by hit validation in Drosophila. We focused on glutaminyl cyclase (QPCT), which had one of the strongest effects on mutant HTT-induced toxicity and aggregation in the cell-based siRNA screen, and which also rescued these phenotypes in Drosophila. We found that QPCT inhibition induced the levels of the molecular chaperone alpha B-crystallin and reduced the aggregation of diverse proteins. We generated novel QPCT inhibitors using in silico methods followed by in vitro screens, which rescued the HD-related phenotypes in cell, Drosophila and zebrafish HD models. Our data reveal a novel HD druggable target affecting mutant huntingtin aggregation, and provide proof-of-principle for a discovery pipeline from druggable genome screen to drug development.

Retrophosphorylation of Mkk1 and Mkk2 MAPKKs by the Slt2 MAPK in the Yeast Cell Integrity Pathway

In Saccharomyces cerevisiae, a variety of stresses and aggressions to the cell wall stimulate the activation of the cell wall integrity MAPK pathway, which triggers the expression of a series of genes important for the maintenance of cell wall homeostasis. This MAPK module lies downstream of the Rho1 small GTPase and protein kinase C Pkc1 and consists of MAPKKK Bck1, MAPKKs Mkk1 and Mkk2, and the Slt2 MAPK. In agreement with previous reports suggesting that Mkk1 and Mkk2 were functionally redundant, we show here that both Mkk1 and Mkk2 alone or even chimerical proteins constructed by interchanging their catalytic and regulatory domains are able to efficiently maintain signal transduction through the pathway. Both Mkk1 and Mkk2 are phosphorylated in vivo concomitant to activation of the cell integrity pathway. Interestingly, hyperphosphorylation of the MEKs required not only the upstream components of the pathway, but also a catalytically competent Slt2 MAPK downstream. Active Slt2 purified from yeast extracts was able to phosphorylate Mkk1 and Mkk2 in vitro. We have mapped Ser50 as a direct phosphorylation target for Slt2 in Mkk2. However, substitution of all (Ser/Thr)-Pro canonical MAPK target sites with alanine did not totally abrogate Slt2-dependent Mkk2 phosphorylation. Mutation or deletion of a conserved MAPK-docking site at the N-terminal extension of Mkk2 precluded its interaction with Slt2 and negatively affected retrophosphorylation. Our data show that the cell wall integrity MAPKKs are targets for their downstream MAPK, suggesting the existence of complex feedback regulatory mechanisms at this level.