Autophagy is a process by which components of the cell are degraded to maintain essential activity and viability in response to nutrient limitation. Extensive genetic studies have shown that the yeast ATG1 kinase has an essential role in autophagy induction. Furthermore, autophagy is promoted by AMP activated protein kinase (AMPK), which is a key energy sensor and regulates cellular metabolism to maintain energy homeostasis. Conversely, autophagy is inhibited by the mammalian target of rapamycin (mTOR), a central cell-growth regulator that integrates growth factor and nutrient signals. Here we demonstrate a molecular mechanism for regulation of the mammalian autophagy-initiating kinase Ulk1, a homologue of yeast ATG1. Under glucose starvation, AMPK promotes autophagy by directly activating Ulk1 through phosphorylation of Ser 317 and Ser 777. Under nutrient sufficiency, high mTOR activity prevents Ulk1 activation by phosphorylating Ulk1 Ser 757 and disrupting the interaction between Ulk1 and AMPK. This coordinated phosphorylation is important for Ulk1 in autophagy induction. Our study has revealed a signalling mechanism for Ulk1 regulation and autophagy induction in response to nutrient signalling.

/pdf/ampk-and-mtor-regulate-autophagy-through-direct-14wqsz16ke.pdf

AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes.

For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy.

Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-ijf2t30pbq.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-28d1pi1jvo.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy

Methods in Mammalian Autophagy Research

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.

https://biblio.ugent.be/publication/8555786/file/8555788.pdf

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

To characterize the mechanisms by which the highly conserved exocyst trafficking complex regulates eye physiology in zebrafish and mice, we focused on Exoc5 (also known as sec10), a central exocyst component We analyzed both exoc5 zebrafish mutants and retinal pigmented epithelium (RPE)-specific Exoc5 knockout mice Exoc5 is present in both the non-pigmented epithelium of the ciliary body and in the RPE In this study, we set out to establish an animal model to study the mechanisms underlying the ocular phenotype and to establish if loss of visual function is induced by postnatal RPE Exoc5-deficiency Exoc5-/- zebrafish had smaller eyes, with decreased number of melanocytes in the RPE and shorter photoreceptor outer segments At 35 days post-fertilization, loss of rod and cone opsins were observed in zebrafish exoc5 mutants Mice with postnatal RPE-specific loss of Exoc5 showed retinal thinning associated with compromised visual function and loss of visual photoreceptor pigments Abnormal levels of RPE65 together with a reduced c-wave amplitude indicate a dysfunctional RPE The retinal phenotype in Exoc5-/- mice was present at 20 weeks, but was more pronounced at 27 weeks, indicating progressive disease phenotype We previously showed that the exocyst is necessary for photoreceptor ciliogenesis and retinal development Here, we report that exoc5 mutant zebrafish and mice with RPE-specific genetic ablation of Exoc5 develop abnormal RPE pigmentation, resulting in retinal cell dystrophy and loss of visual pigments associated with compromised vision Together, these data suggest that exocyst-mediated signaling in the RPE is required for RPE structure and function, indirectly leading to photoreceptor degeneration

Conditional Loss of the Exocyst Component Exoc5 in Retinal Pigment Epithelium (RPE) Results in RPE Dysfunction, Photoreceptor Cell Degeneration, and Decreased Visual Function

Purpose: In a previous study, several quantitative trait loci (QTL) that influence age-related degeneration (ageRD) were identified in a cross between the albino strains B6(Cg)-Tyr(c-2J)/J (B6a) and BALB/cByJ (C). The Chromosome (Chr) 6 and Chr 10 QTL were the strongest and most highly significant loci and both involved B6a protective alleles. The QTL were responsible for 21% and 9% of the variance in phenotypes, respectively. We focused on these two QTL to identify candidate genes. Methods: DNA microarrays were used for the two mouse strains at four and eight months of age to identify genes that are differentially regulated and map to either QTL. Gene Ontology (GO) analysis of the differentially expressed genes was performed to identify possible processes and pathways associated with ageRD. To identify additional candidates, database analyses (Positional Medline or PosMed) were used. Based on differential expression, PosMed, and the presence of reported polymorphisms, five genes per QTL were selected for further study by sequencing analysis and qRT–PCR. Tumor necrosis factor, alpha- induced protein 3 (Tnfaip3; on a C57BL/6J (B6) background) was phenotypically tested. Single nucleotide polymorphisms (SNPs) flanking this gene were correlated with outer nuclear layer thickness (ONL), and eight-month-old Tnfaip3 +/− mice were tested for ageRD. Results: Polymorphisms were found in the coding regions of eight genes. Changes in gene expression were identified by qRT–PCR for Hexokinase 2 (Hk2) and Docking protein 1 (Dok1) at four months and for Dok1 and Tnfaip3 at eight months. Tnfaip3 was selected for phenotypic testing due to differential expression and the presence of two nonsynonymous mutations. However, when ONL thickness was compared in eight-month-old congenic Tnfaip3 +/− and Tnfaip3 +/+ mice, no differences were found, suggesting that Tnfaip3 is not the quantitative trait gene (QTG) for the Chr 10 QTL. The GO analysis revealed that GO terms associated with stress and cell remodeling are overrepresented in the ageRD-sensitive C strain compared with the B6a strain with age (eight months). In the ageRD-resistant B6a strain, compared with the C strain, GO terms associated with antioxidant response and the regulation of blood vessel size are overrepresented with age. Conclusions: The analyses of differentially expressed genes and the PosMed database yielded candidate genes for the Chr 6 and Chr 10 QTL. HtrA serine peptidase 2 (Htra2), Dok1, and Tnfaip3 were deemed most promising because of their known roles in apoptosis and our finding of nonsynonymous substitutions between B6a and C strains. While Tnfaip3 was excluded as the QTG for the Chr 10 QTL, Dok1 and Htra2 remain good candidates for the Chr 6 QTL. Finally, the GO term analysis further supports the general hypothesis that oxidative stress is involved in ageRD.

/pdf/candidate-genes-for-chromosomes-6-and-10-quantitative-trait-fpqnixsek4.pdf

Bärbel Rohrer

Papers

Conditional Loss of the Exocyst Component Exoc5 in Retinal Pigment Epithelium (RPE) Results in RPE Dysfunction, Photoreceptor Cell Degeneration, and Decreased Visual Function

Candidate genes for chromosomes 6 and 10 quantitative trait loci for age-related retinal degeneration in mice.

Elastin turnover in ocular diseases: A special focus on age-related macular degeneration.

Amyloid Precursor Protein Metabolism in Retinal Degeneration

Lipoprotein-Associated Phospholipase A2 and Risk of Aging Macula Disorder: The Rotterdam Study