TDP-43 regulates LC3ylation in neural tissue through ATG4B cryptic splicing inhibition.

Amyotrophic lateral sclerosis (ALS) is an adult-onset motor neuron disease with a mean survival time of three years. The 97% of the cases have TDP-43 nuclear depletion and cytoplasmic aggregation in motor neurons. TDP-43 prevents non-conserved cryptic exon splicing in certain genes, maintaining transcript stability, including ATG4B, which is crucial for autophagosome maturation and Microtubule-associated proteins 1A/1B light chain 3B (LC3B) homeostasis. In ALS mice (G93A), Atg4b depletion worsens survival rates and autophagy function. For the first time, we observed an elevation of LC3ylation in the CNS of both ALS patients and atg4b-/- mouse spinal cords. Furthermore, LC3ylation modulates the distribution of ATG3 across membrane compartments. Antisense oligonucleotides (ASOs) targeting cryptic exon restore ATG4B mRNA in TARDBP knockdown cells. We further developed multi-target ASOs targeting TDP-43 binding sequences for a broader effect. Importantly, our ASO based in peptide-PMO conjugates show brain distribution post-IV administration, offering a non-invasive ASO-based treatment avenue for neurodegenerative diseases.

Analysis of ATG4C function in vivo.

ABBREVIATIONS: 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).

Serum and Urinary Soluble α-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 α-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 α-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 α-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.

Microglial phagocytosis dysfunction in stroke is driven by energy depletion and induction of autophagy.

Microglial phagocytosis of apoptotic debris prevents buildup damage of neighbor neurons and inflammatory responses. Whereas microglia are very competent phagocytes under physiological conditions, we report their dysfunction in mouse and preclinical monkey models of stroke (macaques and marmosets) by transient occlusion of the medial cerebral artery (tMCAo). By analyzing recently published bulk and single cell RNA sequencing databases, we show that the phagocytosis dysfunction was not explained by transcriptional changes. In contrast, we demonstrate that the impairment of both engulfment and degradation was related to energy depletion triggered by oxygen and nutrient deprivation (OND), which led to reduced process motility, lysosomal exhaustion, and the induction of a protective macroautophagy/autophagy response in microglia. Basal autophagy, in charge of removing and recycling intracellular elements, was critical to maintain microglial physiology, including survival and phagocytosis, as we determined both in vivo and in vitro using pharmacological and transgenic approaches. Notably, the autophagy inducer rapamycin partially prevented the phagocytosis impairment induced by tMCAo in vivo but not by OND in vitro, where it even had a detrimental effect on microglia, suggesting that modulating microglial autophagy to optimal levels may be a hard to achieve goal. Nonetheless, our results show that pharmacological interventions, acting directly on microglia or indirectly on the brain environment, have the potential to recover phagocytosis efficiency in the diseased brain. We propose that phagocytosis is a therapeutic target yet to be explored in stroke and other brain disorders and provide evidence that it can be modulated in vivo using rapamycin.Abbreviations: AIF1/IBA1: allograft inflammatory factor 1; AMBRA1: autophagy/beclin 1 regulator 1; ATG4B: autophagy related 4B, cysteine peptidase; ATP: adenosine triphosphate; BECN1: beclin 1, autophagy related; CASP3: caspase 3; CBF: cerebral blood flow; CCA: common carotid artery; CCR2: chemokine (C-C motif) receptor 2; CIR: cranial irradiation; Csf1r/v-fms: colony stimulating factor 1 receptor; CX3CR1: chemokine (C-X3-C motif) receptor 1; DAPI: 4',6-diamidino-2-phenylindole; DG: dentate gyrus; GO: Gene Ontology; HBSS: Hanks' balanced salt solution; HI: hypoxia-ischemia; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MCA: medial cerebral artery; MTOR: mechanistic target of rapamycin kinase; OND: oxygen and nutrient deprivation; Ph/A coupling: phagocytosis-apoptosis coupling; Ph capacity: phagocytic capacity; Ph index: phagocytic index; SQSTM1: sequestosome 1; RNA-Seq: RNA sequencing; TEM: transmission electron microscopy; tMCAo: transient medial cerebral artery occlusion; ULK1: unc-51 like kinase 1.

Ambra1 haploinsufficiency in CD1 mice results in metabolic alterations and exacerbates age-associated retinal degeneration.

Macroautophagy/autophagy is a key process in the maintenance of cellular homeostasis. The age-dependent decline in retinal autophagy has been associated with photoreceptor degeneration. Retinal dysfunction can also result from damage to the retinal pigment epithelium (RPE), as the RPE-retina constitutes an important metabolic ecosystem that must be finely tuned to preserve visual function. While studies of mice lacking essential autophagy genes have revealed a predisposition to retinal degeneration, the consequences of a moderate reduction in autophagy, similar to that which occurs during physiological aging, remain unclear. Here, we described a retinal phenotype consistent with accelerated aging in mice carrying a haploinsufficiency for Ambra1, a pro-autophagic gene. These mice showed protein aggregation in the retina and RPE, metabolic underperformance, and premature vision loss. Moreover, Ambra1+/gt mice were more prone to retinal degeneration after RPE stress. These findings indicate that autophagy provides crucial support to RPE-retinal metabolism and protects the retina against stress and physiological aging.Abbreviations : 4-HNE: 4-hydroxynonenal; AMBRA1: autophagy and beclin 1 regulator 1, AMD: age-related macular degeneration;; GCL: ganglion cell layer; GFAP: glial fibrillary acidic protein; GLUL: glutamine synthetase/glutamate-ammonia ligase; HCL: hierarchical clustering; INL: inner nuclear layer; IPL: inner plexiform layer; LC/GC-MS: liquid chromatography/gas chromatography-mass spectrometry; MA: middle-aged; MTDR: MitoTracker Deep Red; MFI: mean fluorescence intensity; NL: NH4Cl and leupeptin; Nqo: NAD(P)H quinone dehydrogenase; ONL: outer nuclear layer; OPL: outer plexiform layer; OP: oscillatory potentials; OXPHOS: oxidative phosphorylation; PCR: polymerase chain reaction; PRKC/PKCα: protein kinase C; POS: photoreceptor outer segment; RGC: retinal ganglion cells; RPE: retinal pigment epithelium; SI: sodium iodate; TCA: tricarboxylic acid.

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-2 and host cells should help to identify host factors that may be targetable to combat infection and COVID-19 pathology. 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 many findings from previous screens that used full SARS-CoV-2 viruses, but also unveil two novel critical host factors: the lysosomal efflux transporter SPNS1 and the plasma and lysosomal membrane protein PLAC8. Functional experiments with full SARS-CoV-2 viruses confirm that loss-of-function of these genes impairs viral entry. We find that PLAC8 is a key limiting host factor, whose overexpression boosts viral infection in eight different human lung cancer cell lines. Using single-cell RNA-Seq data analyses, we demonstrate that PLAC8 is highly expressed in ciliated and secretory cells of the respiratory tract, as well as in gut enterocytes, cell types that are highly susceptible to SARS-CoV-2 infection. Proteomics and cell biology studies suggest that PLAC8 and SPNS1 regulate the autophagolysosomal compartment and affect the intracellular fate of endocytosed virions.

Comparative genomics of mortal and immortal cnidarians unveils novel keys behind rejuvenation.

Turritopsis dohrnii is the only metazoan able to rejuvenate repeatedly after its medusae reproduce, hinting at biological immortality and challenging our understanding of aging. We present and compare whole-genome assemblies of T. dohrnii and the nonimmortal Turritopsis rubra using automatic and manual annotations, together with the transcriptome of life cycle reversal (LCR) process of T. dohrnii. We have identified variants and expansions of genes associated with replication, DNA repair, telomere maintenance, redox environment, stem cell population, and intercellular communication. Moreover, we have found silencing of polycomb repressive complex 2 targets and activation of pluripotency targets during LCR, which points to these transcription factors as pluripotency inducers in T. dohrnii. Accordingly, we propose these factors as key elements in the ability of T. dohrnii to undergo rejuvenation.

Autophagy Deficiency by Atg4B Loss Leads to Metabolomic Alterations in Mice.

Autophagy is an essential protective mechanism that allows mammalian cells to cope with a variety of stressors and contributes to maintaining cellular and tissue homeostasis. Due to these crucial roles and also to the fact that autophagy malfunction has been described in a wide range of pathologies, an increasing number of in vivo studies involving animal models targeting autophagy genes have been developed. In mammals, total autophagy inactivation is lethal, and constitutive knockout models lacking effectors of this route are not viable, which has hindered so far the analysis of the consequences of a systemic autophagy decline. Here, we take advantage of atg4b-/- mice, an autophagy-deficient model with only partial disruption of the process, to assess the effects of systemic reduction of autophagy on the metabolome. We describe for the first time the metabolic footprint of systemic autophagy decline, showing that impaired autophagy results in highly tissue-dependent alterations that are more accentuated in the skeletal muscle and plasma. These changes, which include changes in the levels of amino-acids, lipids, or nucleosides, sometimes resemble those that are frequently described in conditions like aging, obesity, or cardiac damage. We also discuss different hypotheses on how impaired autophagy may affect the metabolism of several tissues in mammals.

ATG4D is the main ATG8 delipidating enzyme in mammalian cells and protects against cerebellar neurodegeneration.

Despite the great advances in autophagy research in the last years, the specific functions of the four mammalian Atg4 proteases (ATG4A-D) remain unclear. In yeast, Atg4 mediates both Atg8 proteolytic activation, and its delipidation. However, it is not clear how these two roles are distributed along the members of the ATG4 family of proteases. We show that these two functions are preferentially carried out by distinct ATG4 proteases, being ATG4D the main delipidating enzyme. In mammalian cells, ATG4D loss results in accumulation of membrane-bound forms of mATG8s, increased cellular autophagosome number and reduced autophagosome average size. In mice, ATG4D loss leads to cerebellar neurodegeneration and impaired motor coordination caused by alterations in trafficking/clustering of GABAA receptors. We also show that human gene variants of ATG4D associated with neurodegeneration are not able to fully restore ATG4D deficiency, highlighting the neuroprotective role of ATG4D in mammals.

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 role in environmental pollutants exposure.

During the last decades, the potential harmfulness derived from the exposure to environmental pollutants has been largely demonstrated, with associated damages ranging from geno- and cyto-toxicity to tissue malfunction and alterations in organism physiology. Autophagy is an evolutionarily-conserved cellular mechanism essential for cellular homeostasis, which contributes to protect cells from a wide variety of intracellular and extracellular stressors. Due to its pivotal importance, its correct functioning is directly linked to cell, tissue and organismal fitness. Environmental pollutants, particularly industrial compounds, are able to impact autophagic flux, either by increasing it as a protective response, by blocking it, or by switching its protective role toward a pro-cell death mechanism. Thus, the understanding of the effects of chemicals exposure on autophagy has become highly relevant, offering new potential approaches for risk assessment, protection and preventive measures to counteract the detrimental effects of environmental pollutants on human health.

Relationship between PMN-endothelium interactions, ROS production and Beclin-1 in type 2 diabetes.

Type 2 diabetes is closely related to oxidative stress and cardiovascular diseases. In this study, we hypothesized that polymorphonuclear leukocytes (PMN)-endothelium interactions and autophagy are associated. We evaluated PMN-endothelial interactions, ROS production and autophagy parameters in 47 type 2 diabetic patients and 57 control subjects. PMNs from type 2 diabetic patients exhibited slower rolling velocity (p < 0.001), higher rolling flux (p < 0.001) and adhesion (p < 0.001) in parallel to higher levels of total (p < 0.05) and mitochondrial ROS (p < 0.05). When the protein expression of autophagy markers was analysed, an increase of Beclin-1 (p < 0.05), LC3I (p < 0.05), LC3II (p < 0.01) and LC3II/LC3I ratio (p < 0.05) was observed. Several correlations between ROS and leukocyte-endothelium parameters were found. Interestingly, in control subjects, an increase of Beclin-1 levels was accompanied by a decrease in the number of rolling (r = 0.561) and adhering PMNs (r = 0.560) and a rise in the velocity of the rolling PMNs (r = 0.593). In contrast, in the type 2 diabetic population, a rise in Beclin-1 levels was related to an increase in the number of rolling (r = 0.437), and adhering PMNs (r = 0.467). These results support the hypothesis that PMN-endothelium interactions, ROS levels and formation of autophagosomes, especially Beclin-1 levels, are enhanced in type 2 diabetes.

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.

Methionine Restriction Extends Lifespan in Progeroid Mice and Alters Lipid and Bile Acid Metabolism.

Dietary intervention constitutes a feasible approach for modulating metabolism and improving the health span and lifespan. Methionine restriction (MR) delays the appearance of age-related diseases and increases longevity in normal mice. However, the effect of MR on premature aging remains to be elucidated. Here, we describe that MR extends lifespan in two different mouse models of Hutchinson-Gilford progeria syndrome (HGPS) by reversing the transcriptome alterations in inflammation and DNA-damage response genes present in this condition. Further, MR improves the lipid profile and changes bile acid levels and conjugation, both in wild-type and in progeroid mice. Notably, treatment with cholic acid improves the health span and lifespan in vivo. These results suggest the existence of a metabolic pathway involved in the longevity extension achieved by MR and support the possibility of dietary interventions for treating progeria.

Aspirin Recapitulates Features of Caloric Restriction.

The age-associated deterioration in cellular and organismal functions associates with dysregulation of nutrient-sensing pathways and disabled autophagy. The reactivation of autophagic flux may prevent or ameliorate age-related metabolic dysfunctions. Non-toxic compounds endowed with the capacity to reduce the overall levels of protein acetylation and to induce autophagy have been categorized as caloric restriction mimetics (CRMs). Here, we show that aspirin or its active metabolite salicylate induce autophagy by virtue of their capacity to inhibit the acetyltransferase activity of EP300. While salicylate readily stimulates autophagic flux in control cells, it fails to further increase autophagy levels in EP300-deficient cells, as well as in cells in which endogenous EP300 has been replaced by salicylate-resistant EP300 mutants. Accordingly, the pro-autophagic activity of aspirin and salicylate on the nematode Caenorhabditis elegans is lost when the expression of the EP300 ortholog cpb-1 is reduced. Altogether, these findings identify aspirin as an evolutionary conserved CRM.

Inhibitor of growth protein 4 interacts with Beclin 1 and represses autophagy.

Beclin 1 (BECN1) is a multifunctional protein that activates the pro-autophagic class III phosphatidylinositol 3-kinase (PIK3C3, best known as VPS34), yet also interacts with multiple negative regulators. Here we report that BECN1 interacts with inhibitor of growth family member 4 (ING4), a tumor suppressor protein that is best known for its capacity to interact with the tumor suppressor protein p53 (TP53) and the acetyltransferase E1A binding protein p300 (EP300). Removal of TP53 or EP300 did not affect the BECN1/ING4 interaction, which however was lost upon culture of cells in autophagy-inducing, nutrient free conditions. Depletion of ING4 stimulated the enzymatic activity of PIK3C3, as visualized by means of a red fluorescent protein-tagged short peptide (FYVE) that specifically binds to phosphatidylinositol-3-phosphate (PI3P)-containing subcellular vesicles and enhanced autophagy, as indicated by an enhanced lipidation of microtubule-associated proteins 1A/1B light chain 3 beta (LC3B) and the redistribution of a green-fluorescent protein (GFP)-LC3B fusion protein to cytoplasmic puncta. The generation of GFP-LC3B puncta stimulated by ING4 depletion was reduced by simultaneous depletion, or pharmacological inhibition, of PIK3C3/VPS34. In conclusion, ING4 acts as a negative regulator of the lipid kinase activity of the BECN1 complex, and starvation-induced autophagy is accompanied by the dissociation of the ING4/BECN1 interaction.