Analysis of ATG4C function in vivo

Despite the great advances in macroautophagy/autophagy research in the last years, the in vivo role of the different members of the four mammalian orthologs of yeast Atg4 protease (ATG4A-D) remain unclear. To gain further insights into the functional relevance of Atg4 orthologs, we have generated mutant mice deficient in Atg4c. These mice are viable and fertile, and do not display any obvious abnormalities, indicating that they are able to develop the autophagic response required during the early neonatal period. However, they show tissue-specific autophagy alterations, including reduced autophagic flux in diaphragm and show decreased breathing and locomotor activity after fasting. In addition, atg4c-/- mice show reduced number of circulating T and B lymphocytes, which is associated with accumulation of apoptotic cells in the spleen and an increased susceptibility to develop chemically-induced fibrosarcomas. Moreover, through the analysis of cells and mice simultaneously deficient for ATG4C and ATG4D proteases we also reveal a role for ATG4C in mATG8 proteins delipidation.This work was supported by grants from Ministerio Ciencia eInnovación (Spain) (PID2021-127534OB-I00), the South-Eastern 1315 Norway Regional Health Authority (2021088 to N.E.) and Instituto de Salud Carlos III (RTICC Spain). Jesús Prieto-Lloret is funded by Programa Estrategico IBGM, Escalera de Excelencia, ref. CCVC8485, Consejería de Educación, Junta de Castilla y León (Spain). Funding for open Access Charge: Roche Farma”, as the aricle will be published via Open access and the OA costs will be funded by Roche Farma.Peer reviewe

Autophagy-linked plasma and lysosomal membrane protein PLAC8 is a key host factor for SARS-CoV-2 entry into human cells

Better understanding on interactions between SARS-CoV-2andhost cells should help to identify host factors that may be tar-getable to combat infection and COVID-19pathology. To this end,we have conducted a genome-wide CRISPR/Cas9-based loss-of-function screen in human lung cancer cells infected with SARS-CoV-2-pseudotyped lentiviruses. Our results recapitulate manyfindings from previous screens that used full SARS-CoV-2viruses,but also unveil two novel critical host factors: the lysosomal effluxtransporter SPNS1and the plasma and lysosomal membrane pro-tein PLAC8. Functional experiments with full SARS-CoV-2virusesconfirm that loss-of-function of these genes impairs viral entry.We find that PLAC8is a key limiting host factor, whose overexpres-sion boosts viral infection in eight different human lung cancer celllines. Using single-cell RNA-Seq data analyses, we demonstratethat PLAC8is highly expressed in ciliated and secretory cells of therespiratory tract, as well as in gut enterocytes, cell types that arehighly susceptible to SARS-CoV-2infection. Proteomics and cellbiology studies suggest that PLAC8and SPNS1regulate theautophagolysosomal compartment and affect the intracellular fateof endocytosed virions.This work was supported by Instituto de Salud Carlos III(COV20/00652, MS19/00100,  PI20/01267, COV20/00571 and PT17/0019/0003), Ministerio de Ciencia e Innovación (Spain) (PDI2020-118394RB-100, SAF2017-87655-R, PID2021-127534OB-100, and PGC2018-097019-B-I00), “laCaixa” Banking Foundation (HR17-00247) and Consejería de Ciencia, Innovación y Universidad del Gobierno del Principado de Asturias (AYUD/2021/57167). D.R.V and D.M are supported by PhD fellowships from Ministerio de Ciencia e Innovación(Spain).Peer reviewe

Giant tortoise genomes provide insights into longevity and age-related disease

© 2018, The Author(s), under exclusive licence to Springer Nature Limited. Giant tortoises are among the longest-lived vertebrate animals and, as such, provide an excellent model to study traits like longevity and age-related diseases. However, genomic and molecular evolutionary information on giant tortoises is scarce. Here, we describe a global analysis of the genomes of Lonesome George—the iconic last member of Chelonoidis abingdonii—and the Aldabra giant tortoise (Aldabrachelys gigantea). Comparison of these genomes with those of related species, using both unsupervised and supervised analyses, led us to detect lineage-specific variants affecting DNA repair genes, inflammatory mediators and genes related to cancer development. Our study also hints at specific evolutionary strategies linked to increased lifespan, and expands our understanding of the genomic determinants of ageing. These new genome sequences also provide important resources to help the efforts for restoration of giant tortoise populations

AMPK: Regulation of metabolic dynamics in the context of autophagy

Eukaryotic cells have developed mechanisms that allow them to link growth and proliferation to the availability of energy and biomolecules. AMPK (adenosine monophosphate-activated protein kinase) is one of the most important molecular energy sensors in eukaryotic cells. AMPK activity is able to control a wide variety of metabolic processes connecting cellular metabolism with energy availability. Autophagy is an evolutionarily conserved catabolic pathway whose activity provides energy and basic building blocks for the synthesis of new biomolecules. Given the importance of autophagic degradation for energy production in situations of nutrient scarcity, it seems logical that eukaryotic cells have developed multiple molecular links between AMPK signaling and autophagy regulation. In this review, we will discuss the importance of AMPK activity for diverse aspects of cellular metabolism, and how AMPK modulates autophagic degradation and adapts it to cellular energetic status. We will explain how AMPK-mediated signaling is mechanistically involved in autophagy regulation both through specific phosphorylation of autophagy-relevant proteins or by indirectly impacting in the activity of additional autophagy regulators

Pathogenic Single Nucleotide Polymorphisms on Autophagy-Related Genes

In recent years, the study of single nucleotide polymorphisms (SNPs) has gained increasing importance in biomedical research, as they can either be at the molecular origin of a determined disorder or directly affect the efficiency of a given treatment. In this regard, sequence variations in genes involved in pro-survival cellular pathways are commonly associated with pathologies, as the alteration of these routes compromises cellular homeostasis. This is the case of autophagy, an evolutionarily conserved pathway that counteracts extracellular and intracellular stressors by mediating the turnover of cytosolic components through lysosomal degradation. Accordingly, autophagy dysregulation has been extensively described in a wide range of human pathologies, including cancer, neurodegeneration, or inflammatory alterations. Thus, it is not surprising that pathogenic gene variants in genes encoding crucial effectors of the autophagosome/lysosome axis are increasingly being identified. In this review, we present a comprehensive list of clinically relevant SNPs in autophagy-related genes, highlighting the scope and relevance of autophagy alterations in human disease

Autophagy couteracts weight gain, lipotoxicity and pancreatic β-cell death upon hypercaloric pro-diabetic regimens

International audienceIn the last years, autophagy has been revealed as an essential pathway for multiple biological processes and physiological functions. As a catabolic route, autophagy regulation by nutrient availability has been evolutionarily conserved from yeast to mammals. On one hand, autophagy induction by starvation is associated with a significant loss in body weight in mice. Here, we demonstrate that both genetic and pharmacological inhibition of the autophagy process compromise weight loss induced by starvation. Moreover, autophagic potential also impacts on weight gain induced by distinct hypercaloric regimens. Atg4b-deficient mice, which show limited autophagic competence, exhibit a major increase in body weight in response to distinct obesity-associated metabolic challenges. This response is characterized by the presence of larger adipocytes in visceral fat tissue, increased hepatic steatosis, as well as reduced glucose tolerance and attenuated insulin responses. Similarly, autophagy-deficient mice are more vulnerable to experimentally induced type-I diabetes, showing an increased susceptibility to acute streptozotocin administration. Notably, pharmacological stimulation of autophagy in wild-type mice by spermidine reduced both weight gain and obesity-associated alterations upon hypercaloric regimens. Altogether, these results indicate that systemic autophagic activity influences the resilience of the organism to weight gain induced by high-calorie diets, as well as to the obesity-associated features of both type-1 and type-2 diabetes

Analysis of ATG4C function <i>in vivo</i>

Despite the great advances in macroautophagy/autophagy research in the last years, the in vivo role of the different members of the four mammalian orthologs of yeast Atg4 protease (ATG4A-D) remain unclear. To gain further insights into the functional relevance of Atg4 orthologs, we have generated mutant mice deficient in Atg4c. These mice are viable and fertile, and do not display any obvious abnormalities, indicating that they are able to develop the autophagic response required during the early neonatal period. However, they show tissue-specific autophagy alterations, including reduced autophagic flux in diaphragm and show decreased breathing and locomotor activity after fasting. In addition, atg4c-/- mice show reduced number of circulating T and B lymphocytes, which is associated with accumulation of apoptotic cells in the spleen and an increased susceptibility to develop chemically-induced fibrosarcomas. Moreover, through the analysis of cells and mice simultaneously deficient for ATG4C and ATG4D proteases we also reveal a role for ATG4C in mATG8 proteins delipidation. ATG4 (autophagy related 4 cysteine peptidase); ATG4A (autophagy related 4A cysteine peptidase); ATG4B (autophagy related 4B cysteine peptidase); ATG4C (autophagy related 4C cysteine peptidase); ATG4D (autophagy related 4D cysteine peptidase); Atg8 (autophagy related 8); GABARAP (GABA type A receptor-associated protein); GABARAPL1(GABA type A receptor-associated protein like 1); GABARAPL2 (GABA type A receptor-associated protein like 2); MAP1LC3A/LC3A (microtubule associated protein 1 light chain 3 alpha); MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta); mATG8 (mammalian Atg8); PE (phosphatidylethanolamine); PS (phosphatydylserine); SQSTM1/p62 (sequestosome 1).</p

Serum and Urinary Soluble alfa-Klotho as Markers of Kidney and Vascular Impairment

This study was designed to investigate the controversy on the potential role of sKlotho as an early biomarker in Chronic Kidney Disease-Mineral Bone Disorder (CKD-MBD), to assess whether sKlotho is a reliable marker of kidney alpha-Klotho, to deepen the effects of sKlotho on vascular smooth muscle cells (VSMCs) osteogenic differentiation and to evaluate the role of autophagy in this process. Experimental studies were conducted in CKD mice fed a normal phosphorus (CKD+NP) or high phosphorus (CKD+HP) diet for 14 weeks. The patients' study was performed in CKD stages 2-5 and in vitro studies which used VSMCs exposed to non-calcifying medium or calcifying medium with or without sKlotho. The CKD experimental model showed that the CKD+HP group reached the highest serum PTH, P and FGF23 levels, but the lowest serum and urinary sKlotho levels. In addition, a positive correlation between serum sKlotho and kidney alpha-Klotho was found. CKD mice showed aortic osteogenic differentiation, together with increased autophagy. The human CKD study showed that the decline in serum sKlotho is previous to the rise in FGF23. In addition, both serum sKlotho and FGF23 levels correlated with kidney function. Finally, in VSMCs, the addition of sKlotho prevented osteogenic differentiation and induced autophagy. It can be concluded that serum sKlotho was the earliest CKD-MBD biomarker, a reliable indicator of kidney alpha-Klotho and that might protect against osteogenic differentiation by increasing autophagy. Nevertheless, further studies are needed to investigate the mechanisms of this possible protective effect