Eloy Bejarano

Chaperone-Mediated Autophagy

Continuous renewal of intracellular components is required to preserve cellular functionality. In fact, failure to timely turnover proteins and organelles leads often to cell death and disease. Different pathways contribute to the degradation of intracellular components in lysosomes or autophagy. In this review, we focus on chaperone-mediated autophagy (CMA), a selective form of autophagy that modulates the turnover of a specific pool of soluble cytosolic proteins. Selectivity in CMA is conferred by the presence of a targeting motif in the cytosolic substrates that, upon recognition by a cytosolic chaperone, determines delivery to the lysosomal surface. Substrate proteins undergo unfolding and translocation across the lysosomal membrane before reaching the lumen, where they are rapidly degraded. Better molecular characterization of the different components of this pathway in recent years, along with the development of transgenic models with modified CMA activity and the identification of CMA dysfunction in different severe human pathologies and in aging, are all behind the recent regained interest in this catabolic pathway.

Autophagy modulates dynamics of connexins at the plasma membrane in a ubiquitin-dependent manner

Connexins modulate intercellular communication when assembled in gap junctions. Compromised macroautophagy increases cellular communication due to failure to degrade connexins at gap junctions. Nedd4-mediated ubiquitinylation of the connexin molecule is required to trigger its autophagy-dependent internalization and degradation.

Connexins modulate autophagosome biogenesis

The plasma membrane contributes to the formation of autophagosomes, the double-membrane vesicles that sequester cytosolic cargo and deliver it to lysosomes for degradation during autophagy. In this study, we have identified a regulatory role for connexins (Cx), the main components of plasma membrane gap junctions, in autophagosome formation. We have found that plasma-membrane-localized Cx proteins constitutively downregulate autophagy through a direct interaction with several autophagy-related proteins involved in the initial steps of autophagosome formation, such as Atg16 and components of the PI(3)K autophagy initiation complex (Vps34, Beclin-1 and Vps15). On nutrient starvation, this inhibitory effect is released by the arrival of Atg14 to the Cx–Atg complex. This promotes the internalization of Cx–Atg along with Atg9, which is also recruited to the plasma membrane in response to starvation. Maturation of the Cx-containing pre-autophagosomes into autophagosomes leads to degradation of these endogenous inhibitors, allowing for sustained activation of autophagy.

Molecular determinants of selective clearance of protein inclusions by autophagy

Protein quality control is essential for cellular survival. Failure to eliminate pathogenic proteins leads to their intracellular accumulation in the form of protein aggregates. Autophagy can recognize protein aggregates and degrade them in lysosomes. However, some aggregates escape the autophagic surveillance. Here we analyze the autophagic degradation of different types of aggregates of synphilin-1 (Sph1), a protein often found in pathogenic protein inclusions. We show that small Sph1 aggregates and large aggresomes are differentially targeted by constitutive and inducible autophagy. Furthermore, we identify a region in Sph1 necessary for its own basal and inducible aggrephagy, and sufficient for the degradation of other pro-aggregating proteins. Although the presence of this peptide is sufficient for basal aggrephagy, inducible aggrephagy requires its ubiquitination, which diminishes protein mobility on the surface of the aggregate and favors the recruitment and assembly of the protein complexes required for autophagosome formation. Our study reveals different mechanisms for cells to cope with aggregate proteins via autophagy and supports the idea that autophagic susceptibility of prone-to-aggregate proteins may not depend on the nature of the aggregating proteins per se but on their dynamic properties in the aggregate.

Proteasome failure promotes positioning of lysosomes around the aggresome via local block of microtubule-dependent transport

ABSTRACT Ubiquitinated proteins aggregate upon proteasome failure, and the aggregates are transported to the aggresome. In aggresomes, protein aggregates are actively degraded by the autophagy-lysosome pathway, but why targeting the aggresome promotes degradation of aggregated species is currently unknown. Here we report that the important factor in this process is clustering of lysosomes around the aggresome via a novel mechanism. Proteasome inhibition causes formation of a zone around the centrosome where microtubular transport of lysosomes is suppressed, resulting in their entrapment and accumulation. Microtubule-dependent transport of other organelles, including autophagosomes, mitochondria, and endosomes, is also blocked in this entrapment zone (E-zone), while movement of organelles at the cell periphery remains unaffected. Following the whole-genome small interfering RNA (siRNA) screen for proteins involved in aggresome formation, we defined the pathway that regulates formation of the E-zone, including the Stk11 protein kinase, the Usp9x deubiquitinating enzyme, and their substrate kinase MARK4. Therefore, upon proteasome failure, targeting of aggregated proteins of the aggresome is coordinated with lysosome positioning around this body to facilitate degradation of the abnormal species.

Autophagy and amino acid metabolism in the brain: implications for epilepsy

Autophagy is a catabolic pathway responsible for the maintenance of the tissue and organism homeostasis. Several amino acids regulate autophagic activity in different tissues, such as liver and muscle, but much less is known about this regulation in the brain. The lack of autophagy in neurons leads to a strong neurodegenerative phenotype and epileptic disorders. We summarize the current knowledge about the regulation of autophagy mediated by amino acids and how macroautophagy could serve as source of amino acids. We review the contribution of macroautophagy in the brain physiology and pathology emphasizing the relevancy of the proper control of amino acid levels such as glutamate and GABA in the brain due to its role as neurotransmitters and energy source. Furthermore, we discuss how malfunction in autophagy may result in pathological consequences, because many genetic epileptic disorders are related to signaling or metabolic pathways controlling both macroautophagy and amino acid metabolism in the brain.

Defective recruitment of motor proteins to autophagic compartments contributes to autophagic failure in aging

Inability to preserve proteostasis with age contributes to the gradual loss of function that characterizes old organisms. Defective autophagy, a component of the proteostasis network for delivery and degradation of intracellular materials in lysosomes, has been described in multiple old organisms, while a robust autophagy response has been linked to longevity. The molecular mechanisms responsible for defective autophagic function with age remain, for the most part, poorly characterized. In this work, we have identified differences between young and old cells in the intracellular trafficking of the vesicular compartments that participate in autophagy. Failure to reposition autophagosomes and lysosomes toward the perinuclear region with age reduces the efficiency of their fusion and the subsequent degradation of the sequestered cargo. Hepatocytes from old mice display lower association of two microtubule‐based minus‐end‐directed motor proteins, the well‐characterized dynein, and the less‐studied KIFC3, with autophagosomes and lysosomes, respectively. Using genetic approaches to mimic the lower levels of KIFC3 observed in old cells, we confirmed that reduced content of this motor protein in fibroblasts leads to failed lysosomal repositioning and diminished autophagic flux. Our study connects defects in intracellular trafficking with insufficient autophagy in old organisms and identifies motor proteins as a novel target for future interventions aiming at correcting autophagic activity with anti‐aging purposes.

Unique Features of Neuronal Autophagy: Considerations for Therapeutic Targeting

Autophagy is an essential catabolic pathway responsible for the maintenance of organismal homeostasis. Degradation of damaged organelles and proteinaceous aggregates predominantly takes place via autophagy and a proper function of autophagy is vital for cellular surveillance. Given their postmitotic nature, neurons are particularly vulnerable to stress and, consequently, robust housekeeping systems are required to guarantee the adequate functionality and viability of neurons. A vast literature links defective autophagic function to neurodegenerative diseases and dietary/pharmacological activation of autophagy has been proposed as potential strategy to fight these diseases. Here we summarize the recent progress on the research of neuronal autophagy highlighting the unique features of autophagy in neurons. In the last section, we discuss about therapeutic strategies modulating autophagy to preserve neuronal surveillance during aging.

Sa1689 Microtubule Based Motility of Autophagic and Lysosomal Compartments In Vitro: Vesicles With LC3 on Their Surface Show Greater Motility Than Those That Contain Lamp1

Background and aim: Angiotensin 2, an important byproduct of renin angiotensin system can lead to increased extracellular matrix formation and fibrosis via enhanced TGF β-1 production in many organ systems including liver. Angiotensin converting enzyme (ACE) coding gen polymorphisms can lead to different degree of ACE expressions and hence, various angiotensin 2 tissue levels. ACE gen polymorphisms have been reported as ACE I/ D. Especially, ACE gen D homozygotic patients produce more tissue levels of ACE and this may be a potential hazard for many organ systems. We aimed to investigate if there is any role of ACE I/D gen polymorphism in the degree of liver fibrosis due to various etiologies. Methods: We enrolled 411 patients with a histopathological diagnosis of liver fibrosis. There were 240 patients with the diagnosis of mild and moderate fibrosis (Ishaks stage; 1-3) and the rest, 171 patients had advanced liver fibrosis (Ishaks stage; 4-6). Polymerase chain reaction was used to determine the type of ACE I/D gen polymorphisms. We also studied serum ACE levels with enzymatic kinetic testing method on spectrometry. Results: There were 208 male and 203 female subjects with a mean age of 51.1±13.5 years, (range; 1880 years). Within the mild and moderate liver fibrosis group, the etiologies were chronic viral hepatitis B in 131, chronic viral hepatitis C in 59, non-alcoholic steatohepatitis in 46, autoimmune hepatitis in 1 and primary biliary cirrhosis in 3 patients. Within the advanced liver fibrosis group, the etiologies were chronic viral hepatitis B in 71, chronic viral hepatitis C in 61, non-alcoholic steatohepatitis in 24, alcoholic liver disease in 11 and autoimmune hepatitis in 4 patients. On statistical analysis, there was no difference between the two groups with regard to ACE genotypes. Moreover, we could not find any significant difference between the groups regarding serum levels of angiotensin converting enzyme. Conclusion: The ACE I/D gen polymorphism do not seem to contribute as a significant risk factor for advanced liver fibrosis.