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

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.

In 2009, the Nomenclature Committee on Cell Death (NCCD) proposed a set of recommendations for the definition of distinct cell death morphologies and for the appropriate use of cell death-related terminology, including 'apoptosis', 'necrosis' and 'mitotic catastrophe'. In view of the substantial progress in the biochemical and genetic exploration of cell death, time has come to switch from morphological to molecular definitions of cell death modalities. Here we propose a functional classification of cell death subroutines that applies to both in vitro and in vivo settings and includes extrinsic apoptosis, caspase-dependent or -independent intrinsic apoptosis, regulated necrosis, autophagic cell death and mitotic catastrophe. Moreover, we discuss the utility of expressions indicating additional cell death modalities. On the basis of the new, revised NCCD classification, cell death subroutines are defined by a series of precise, measurable biochemical features.

/pdf/molecular-definitions-of-cell-death-subroutines-q5g428rlyi.pdf

Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012

Cells constantly adapt their metabolism to meet their energy needs and respond to nutrient availability. Eukaryotes have evolved a very sophisticated system to sense low cellular ATP levels via the serine/threonine kinase AMP-activated protein kinase (AMPK) complex. Under conditions of low energy, AMPK phosphorylates specific enzymes and growth control nodes to increase ATP generation and decrease ATP consumption. In the past decade, the discovery of numerous new AMPK substrates has led to a more complete understanding of the minimal number of steps required to reprogramme cellular metabolism from anabolism to catabolism. This energy switch controls cell growth and several other cellular processes, including lipid and glucose metabolism and autophagy. Recent studies have revealed that one ancestral function of AMPK is to promote mitochondrial health, and multiple newly discovered targets of AMPK are involved in various aspects of mitochondrial homeostasis, including mitophagy. This Review discusses how AMPK functions as a central mediator of the cellular response to energetic stress and mitochondrial insults and coordinates multiple features of autophagy and mitochondrial biology.

/pdf/ampk-guardian-of-metabolism-and-mitochondrial-homeostasis-2xckib51mn.pdf

AMPK: guardian of metabolism and mitochondrial homeostasis.

The intracellular location of organelles was studied in avian embryos immediately before and during the initial detachment of cells from the dorsoanterolateral wall of the otocyst, using light and electron microscopy. The Golgi apparatus was silver-impregnated, and its location within the otic epithelium was determined quantitatively. The present study demonstrates that a sub-population of cells of the dorsoanterolateral otic epithelium changes the intracellular location of its organelles, particularly the Golgi apparatus and the centrosome, from an apical to a basal position. Concomitantly, cells lose specializations characteristically present at apical (tight junctions, microvilli) and basal (basal lamina) surfaces. At basolateral cell surfaces, filopodia form ahead of the Golgi apparatus and centrosome and penetrate the previously continuous underlying basal lamina. Thereafter, cells detach from the otocyst and migrate medially toward the hind-brain. Thus, concomitant with changes in surface polarity, the cells that comprise the dorsoanterolateral wall of the otocyst undergo profound changes in the intracellular location of their organelles, especially the Golgi apparatus and the centrosome, so that by the time cells detach from the otic epithelium a reversal in their "normal" internal polarity has occurred. We suggest that the change in cell polarity may be related to the mechanisms that allow cells to leave the otocyst.

Cell polarity changes and migration during early development of the avian peripheral auditory system

Aging of long-lived post-mitotic cells is characterized as a progressive and irreversible reduction of functional activity. In such cells, mitochondria are organelles critical for bioenergetic supply, whose turnover is mediated by an autophagic-lysosomal pathway. In human teeth, odontoblasts are post-mitotic cells responsible for sensory function and dentin preservation. Here, human odontoblasts were processed for immunohistochemistry with antibodies against mitochondrial (MTCO2) and lysosomal (LAMP2) markers, and comparatively analyzed in two age groups (young-adult and adult) with light and electron microscopy. Selective engulfment of mitochondrial profiles into autophagic vacuoles is common in young-adult odontoblasts, suggesting a microautophagic pathway. With age, the odontoblast layer is reduced in width, and mitochondrial elements converge around large clusters of autofluorescent lipofuscin deposits. Age-related changes in odontoblasts are observed as a long-term process in which the progressive accumulation of intralysosomal debris limits the autophagic turnover of mitochondrial components, causing an eventual decline in physiological cell functions, which leads to increased vulnerability under stress conditions.

Mitochondrial Autophagy and Lipofuscin Accumulation in Aging Odontoblasts

The thickness of the predentin layer was studied at three different levels of developing human premolars. The results demonstrate that at the growing end next to the apex, where dentinogenesis is most

Changes in the Predentin Thickness and Mineralization Front Configuration in Developing Human Premolars

Mammalian teeth have evolved as dentin units that enclose a complex system of sensory innervation to protect and preserve their structure and function. In human dental pulp (DP), mechanosensory and nociceptive fibers form a dense meshwork of nerve endings at the coronal dentin-pulp interface, which arise from myelinated and non-myelinated axons of the Raschkow plexus (RP). Schwann cells (SCs) play a crucial role in the support, maintenance and regeneration after injury of these fibers. We have recently characterized two SC phenotypes hierarchically organized within the coronal and radicular DP in human teeth. Myelinating and non-myelinating SCs (nmSCs) display a high degree of plasticity associated with nociceptive C-fiber sprouting and axonal degeneration in response to DP injuries from dentin caries or physiological root resorption (PRR). By comparative immunolabeling, confocal and electron microscopy, we have characterized short-term adaptive responses of SC phenotypes to nerve injuries, and long-term changes related to aging. An increase of SCs characterizes the early responses to caries progression in association with axonal sprouting in affected DP domains. Moreover, during PRR, the formation of bands of Bungner is observed as part of SC repair tracks functions. On the other hand, myelinated axon density is significantly reduced with tooth age, as part of a gradual decrease in DP defense and repair capacities. The remarkable plasticity and capacity of SCs to preserve DP innervation in different dental scenarios constitutes a fundamental aspect to improve clinical treatments. This review article discusses the central role of myelinating and non-mSCs in long-term tooth preservation and homeostasis.

/pdf/schwann-cell-responses-and-plasticity-in-different-dental-2ji0i90olw.pdf

Schwann Cell Responses and Plasticity in Different Dental Pulp Scenarios.

The dental pulp in human primary teeth is densely innervated by a plethora of nerve endings at the coronal pulp-dentin interface. This study analyzed how the physiological root resorption (PRR) process affects dental pulp innervation before exfoliation of primary teeth. Forty-four primary canine teeth, classified into 3 defined PRR stages (early, middle, and advanced) were fixed and demineralized. Longitudinal cryosections of each tooth were stained for immunohistochemical and quantitative analysis of dental pulp nerve fibers and associated components with confocal and electron microscopy. During PRR, axonal degeneration was prominent and progressive in a Wallerian-like scheme, comprising nerve fiber bundles and nerve endings within the coronal and root pulp. Neurofilament fragmentation increased significantly during PRR progression and was accompanied by myelin degradation and a progressive loss of myelinated axons. Myelin sheath degradation involved activation of autophagic activity by Schwann cells to remove myelin debris. These cells expressed a sequence of responses comprising dedifferentiation, proliferative activity, GAP-43 overexpression, and Bungner band formation. During the advanced PRR stage, increased immune cell recruitment within the dental pulp and major histocompatibility complex (MHC) class II upregulation by Schwann cells characterized an inflammatory condition associated with the denervation process in preexfoliative primary teeth. The ensuing loss of dental pulp axons is likely to be responsible for the progressive reduction of sensory function of the dental pulp during preexfoliative stages.

Eduardo Couve

Papers

Cell polarity changes and migration during early development of the avian peripheral auditory system

Mitochondrial Autophagy and Lipofuscin Accumulation in Aging Odontoblasts

Changes in the Predentin Thickness and Mineralization Front Configuration in Developing Human Premolars

Schwann Cell Responses and Plasticity in Different Dental Pulp Scenarios.

Axonal Degeneration in Dental Pulp Precedes Human Primary Teeth Exfoliation