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.

Several risk factors for Crohn's disease have been identified in recent genome-wide association studies. To advance gene discovery further, we combined data from three studies on Crohn's disease (a total of 3,230 cases and 4,829 controls) and carried out replication in 3,664 independent cases with a mixture of population-based and family-based controls. The results strongly confirm 11 previously reported loci and provide genome-wide significant evidence for 21 additional loci, including the regions containing STAT3, JAK2, ICOSLG, CDKAL1 and ITLN1. The expanded molecular understanding of the basis of this disease offers promise for informed therapeutic development.

/pdf/genome-wide-association-defines-more-than-30-distinct-4d37zou84l.pdf

Genome-wide association defines more than 30 distinct susceptibility loci for Crohn's disease

Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms Recent reviews have described the range of assays that have been used for this purpose(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi) Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes This set of guidelines is 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 verify an autophagic response

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-4sx3t3he7d.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes

A 38-year-old woman was evaluated for progressive leg weakness that started 8 years earlier after delivering her third child. There was also right hip and buttock pain initially. Her current physical limitations included an inability to climb stairs or rise from a sitting position. Her gait was abnormal and considered “spastic.” She experienced multiple falls weekly reporting that her ankles and knees just gave out. Her right leg was restless at night; after a prolonged static position, it became numb with pain radiating to the foot. She did not complain of upper-extremity dysfunction or other problems with sensation. Bulbar, cognitive, and bowel and bladder functions were intact. Cramps, fasciculations, or weight loss were denied. Magnetic resonance imaging of the entire spine showed mild degenerative changes and disc abnormalities at C3-4 and T8-9 and T11-12. An outside electromyogram (EMG) was interpreted as showing only a decreased interference pattern. She was referred for a possible upper motor neuron disorder. Her birth history was significant for a possible left clubfoot according to her mother; however, she developed normally and was involved in softball and soccer. There were no cardiac or respiratory problems. There was no family history of neuromuscular disease. On examination, the patient was overweight and had hyperlordosis. Cranial nerves were essentially normal. A transverse smile was noted. There was Medical Research Council (MRC) 4+ power in neck flexors and extensors, right hamstrings, and bilateral deltoid, biceps brachii, triceps, hip flexors, first dorsal interosseous, and abductor pollicis brevis muscles. Wrist and finger extensors were normal. Finger flexors, tibialis anterior, and gastrocnemius muscles were MRC 3. Quadriceps were MRC 4 right and 42 left; hip adductors were 52; the left hamstrings were MRC 4. Muscle atrophy without fasciculations was noted below both knees anteriorly and posteriorly. No scapular winging was present. Deep tendon reflexes were absent in both legs, and the right brachioradialis and biceps reflexes were brisk. The other upper extremity reflexes were normal. Tone was normal. Reduced vibration and proprioception were present in both legs. She had bilateral pes cavus and a bilateral steppage and waddling gait. Laboratory studies revealed significantly elevated serum creatine kinase, 2321–4177 U/L (normal, 23–173 U/L). Electrodiagnostic testing revealed normal nerve conductions in the arms and legs. Needle EMG performed on proximal and distal arm and leg and thoracic paraspinal muscles demonstrated increased insertional activity with fibrillation potentials and positive sharp waves only in the triceps and biceps femoris long head. Short duration, low amplitude, and polyphasic motor unit potentials were present in proximal arm muscles to a greater degree than in thoracic paraspinal and proximal and distal leg muscles. A biopsy was performed on the biceps brachii muscle (Fig. 1A, 1B, 1C) and the lower extremities were photographed (Fig. 1D, 1E). Journal of CLINICAL NEUROMUSCULAR DISEASE

Neuromuscular pathology case.

Parkinson’s disease (PD) is the second most common neurodegenerative disease after Alzheimer’s disease (AD) and affects 1% of the population above 60 years old. Although PD commonly manifests with motor symptoms, a majority of patients with PD subsequently develop cognitive impairment which often progresses to dementia, a major cause of morbidity and disability. PD is characterized by α-synuclein accumulation that frequently associates with amyloid beta (Aβ) and tau fibrils, the hallmarks of AD neuropathologic changes; this co-occurrence suggests that onset of cognitive decline in PD may be associated with appearance of pathologic Aβ and/or tau. Recent studies have highlighted the appearance of the soluble form of the Triggering Receptor Expressed on Myeloid cells 2 (sTREM2) receptor in CSF during development of AD. Given the known association of microglial activation with advancing PD, we investigated whether CSF and/or plasma sTREM2 increased with progression to PD dementia. We examined 165 participants consisting of 17 cognitively normal elderly, 45 PD patients with no cognitive impairment, 86 with mild cognitive impairment, and 17 with dementia. Stratification of subjects by CSF Aβ and tau levels revealed that CSF sTREM2 concentrations were elevated in PD subgroups with abnormal tau, but not Aβ, CSF concentration. These findings indicate that CSF sTREM2 could serve as a surrogate immune biomarker of neuronal injury in PD that is associated with cognitive decline. One sentence summary CSF sTREM2 correlates with CSF tau in PD

/pdf/csf-strem2-correlates-with-csf-tau-in-advancing-parkinson-s-33beykeukp.pdf

CSF sTREM2 correlates with CSF tau in advancing Parkinson's disease

Importance Retinal hemorrhages are an important sequela of fatal head trauma The accurate pathologic diagnosis of retinal hemorrhages has critical implications for determination of the manner of death Observations We describe an autolytic postmortem histologic artifact of eosinophilic Muller cell foot process swelling that mimics a nerve fiber layer hemorrhage From April 24, 2012, through November 11, 2014, we conducted postmortem examination of the eyes of 23 infants and children who were referred to our institution for possible nonaccidental head trauma A focal artifact of Muller cell foot process swelling was identified in most patients (16 of 23) up to 4 years of age Three infants, all of whom were younger than 3 months, demonstrated diffusely swollen Muller cell foot processes with intensely eosinophilic cytoplasm that mimicked erythrocytes of nerve fiber layer hemorrhages The difference in the mean age between patients with diffuse eosinophilic artifacts (17 months) and patients with only a multifocal, focal, or absent artifact (133 months) was 116 months (95% CI, 65-167 months) Glycophorin C immunohistochemical analysis was useful to differentiate this artifact from nerve fiber layer hemorrhage Conclusions and Relevance Our case review demonstrates an artifact of eosinophilic Muller cell foot processes swelling in postmortem examination of young infant eyes, a potential pitfall in the diagnosis of retinal hemorrhages Our findings have important implications for the diagnosis of retinal hemorrhages in potential cases of nonaccidental head injury

https://jamanetwork.com/journals/jamaophthalmology/articlepdf/2206336/ebr150001.pdf

Histologic Artifacts of Autolytic Müller Cell Foot Process Swelling in Postmortem Examination of Infant Eyes: Potential Pitfall in the Evaluation of Traumatic Retinal Hemorrhages

Unbiased proteomics has been employed to interrogate central nervous system (CNS) tissues (brain, spinal cord) and fluid matrices (CSF, plasma) from amyotrophic lateral sclerosis (ALS) patients; yet, a limitation of conventional bulk tissue studies is that motor neuron (MN) proteome signals may be confounded by admixed non-MN proteins. Recent advances in trace sample proteomics have enabled quantitative protein abundance datasets from single human MNs (Cong et al., 2020b). In this study, we leveraged laser capture microdissection (LCM) and nanoPOTS (Zhu et al., 2018c) single-cell mass spectrometry (MS)-based proteomics to query changes in protein expression in single MNs from postmortem ALS and control donor spinal cord tissues, leading to the identification of 2515 proteins across MNs samples (>900 per single MN) and quantitative comparison of 1870 proteins between disease groups. Furthermore, we studied the impact of enriching/stratifying MN proteome samples based on the presence and extent of immunoreactive, cytoplasmic TDP-43 inclusions, allowing identification of 3368 proteins across MNs samples and profiling of 2238 proteins across TDP-43 strata. We found extensive overlap in differential protein abundance profiles between MNs with or without obvious TDP-43 cytoplasmic inclusions that together point to early and sustained dysregulation of oxidative phosphorylation, mRNA splicing and translation, and retromer-mediated vesicular transport in ALS. Our data are the first unbiased quantification of single MN protein abundance changes associated with TDP-43 proteinopathy and begin to demonstrate the utility of pathology-stratified trace sample proteomics for understanding single-cell protein abundance changes in human neurologic diseases.

/pdf/tdp-43-stratified-single-cell-proteomic-profiling-of-2a8y4riy.pdf

Edward D. Plowey

Papers

Neuromuscular pathology case.

CSF sTREM2 correlates with CSF tau in advancing Parkinson's disease

Histologic Artifacts of Autolytic Müller Cell Foot Process Swelling in Postmortem Examination of Infant Eyes: Potential Pitfall in the Evaluation of Traumatic Retinal Hemorrhages

TDP-43-stratified single-cell proteomic profiling of postmortem human spinal motor neurons reveals protein dynamics in amyotrophic lateral sclerosis