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.

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

Bcl-2 is an integral membrane protein located mainly on the outer membrane of mitochondria. Overexpression of Bcl-2 prevents cells from undergoing apoptosis in response to a variety of stimuli. Cytosolic cytochrome c is necessary for the initiation of the apoptotic program, suggesting a possible connection between Bcl-2 and cytochrome c, which is normally located in the mitochondrial intermembrane space. Cells undergoing apoptosis were found to have an elevation of cytochrome c in the cytosol and a corresponding decrease in the mitochondria. Overexpression of Bcl-2 prevented the efflux of cytochrome c from the mitochondria and the initiation of apoptosis. Thus, one possible role of Bcl-2 in prevention of apoptosis is to block cytochrome c release from mitochondria.

https://science.sciencemag.org/content/sci/275/5303/1129.full.pdf

Prevention of Apoptosis by Bcl-2: Release of Cytochrome c from Mitochondria Blocked

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.

We describe the landscape of somatic genomic alterations based on multidimensional and comprehensive characterization of more than 500 glioblastoma tumors (GBMs). We identify several novel mutated genes as well as complex rearrangements of signature receptors, including EGFR and PDGFRA. TERT promoter mutations are shown to correlate with elevated mRNA expression, supporting a role in telomerase reactivation. Correlative analyses confirm that the survival advantage of the proneural subtype is conferred by the G-CIMP phenotype, and MGMT DNA methylation may be a predictive biomarker for treatment response only in classical subtype GBM. Integrative analysis of genomic and proteomic profiles challenges the notion of therapeutic inhibition of a pathway as an alternative to inhibition of the target itself. These data will facilitate the discovery of therapeutic and diagnostic target candidates, the validation of research and clinical observations and the generation of unanticipated hypotheses that can advance our molecular understanding of this lethal cancer.

The somatic genomic landscape of glioblastoma

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

The treatment of spinal cord injury (SCI) is currently a major challenge, with a severe lack of effective therapies for yielding meaningful improvements in function. Therefore, there is a great opportunity for the development of novel treatment strategies for SCI. The modulation of autophagy, a process by which a cell degrades and recycles unnecessary or harmful components (protein aggregates, organelles, etc.) to maintain cellular homeostasis and respond to a changing microenvironment, is thought to have potential for treating many neurodegenerative conditions, including SCI. The discovery of microRNAs (miRNAs), which are short ribonucleotide transcripts for targeting of specific messenger RNAs (mRNAs) for silencing, shows prevention of the translation of mRNAs to the corresponding proteins affecting various cellular processes, including autophagy. The number of known miRNAs and their targets continues to grow rapidly. This review article aims to explore the relationship between autophagy and SCI, specifically with the intent of identifying specific miRNAs that can be useful to modulate autophagy for neuroprotection and the improvement of functional recovery in SCI.

/pdf/specific-micrornas-for-modulation-of-autophagy-in-spinal-sbmwzr93.pdf

Specific microRNAs for Modulation of Autophagy in Spinal Cord Injury

Currently, 17 transcription factors are known to form the Kruppel-like factor (KLF) family. All transcription factors of the KLF family resemble the members of the specificity protein (Sp) family; however, all members of the KLF family are distinctly characterized by three Cys2/His2 zinc finger motifs at their C-terminal ends. The Cys2/His2 zinc finger motifs of KLF family members are used for efficient binding to the GC/GT box or CACCC element of the enhancer or promoter of the target genes. For the regulation of tumor cell growth or death, KLF4 is an important player. The precise localization of the KLF4 gene is known to be chromosome 9q31. As a transcription regulator, KLF4 is capable of controlling tumor cell proliferation, differentiation, apoptosis, migration, angiogenesis, and metastasis emphasizing its significance in tumor diagnosis, treatment, and prognosis. Various studies suggest that KLF4 plays important roles in tumor progression as well as in tumor suppression depending on the tumor types and contexts. Malignant neuroblastoma is a deadly tumor that mostly occurs in children. Emerging evidence strongly suggests that KLF4 inhibits the cell cycle and activates cell differentiation and death pathways in human malignant neuroblastoma, thereby behaving as a tumor suppressor. Our increasing understanding of the molecular mechanism of expression and activity of KLF4 in human malignant neuroblastoma will be highly useful for better diagnosis, therapy, and prognosis of this deadly pediatric tumor in the near future.

Emerging Evidence for Krüppel-Like Factor 4 (KLF4) as a Tumor Suppressor in Neuroblastoma

As the most malignant primary brain tumor in adults, a diagnosis of glioblastoma multiforme (GBM) continues to carry a poor prognosis. GBM is characterized by cytoprotective homeostatic processes such as the activation of autophagy, capability to confer therapeutic resistance, evasion of apoptosis, and survival strategy even in the hypoxic and nutrient-deprived tumor microenvironment. The current gold standard of therapy, which involves radiotherapy and concomitant and adjuvant chemotherapy with temozolomide (TMZ), has been a game-changer for patients with GBM, relatively improving both overall survival (OS) and progression-free survival (PFS); however, TMZ is now well-known to upregulate undesirable cytoprotective autophagy, limiting its therapeutic efficacy for induction of apoptosis in GBM cells. The identification of targets utilizing bioinformatics-driven approaches, advancement of modern molecular biology technologies such as clustered regularly interspaced short palindromic repeats (CRISPR)—CRISPR-associated protein (Cas9) or CRISPR-Cas9 genome editing, and usage of microRNA (miRNA)-mediated regulation of gene expression led to the selection of many novel targets for new therapeutic development and the creation of promising combination therapies. This review explores the current state of advanced bioinformatics analysis and genetic technologies and their utilization for synergistic combination with TMZ in the context of inhibition of autophagy for controlling the growth of GBM.

/pdf/advanced-bioinformatics-analysis-and-genetic-technologies-16c3g8ma.pdf

Advanced Bioinformatics Analysis and Genetic Technologies for Targeting Autophagy in Glioblastoma Multiforme

Co-treatment with GM-CSF/IL3 fusion protein pIXY321 significantly increased apoptosis and the loss of clonogenic survival of HL-60 cells due to a treatment with high dose Ara-C (100 μM) for 4 hours or prolonged treatment with lose dose Ara-C (10 nM) for 5 days. In contrast, pIXY321 inhibited high dose Ara-C-in-duced apoptosis in CD34+ normal bone marrow cells. Combined treatment with staurosporine also enhanced Ara-C induced apoptosis in HL-60 cells. None of these modulations of Ara-C-induced apoptosis in the leukemic or normal cells was associated with appreciable alterations in intracellular p26BCL-2 levels.

Improved Antileukemic Activity of the Combination of Ara-C with GM-CSF and IL-3 Fusion Protein (PIXY321)

The small non-coding RNAs are called microRNAs or miRNAs that play key roles in mRNA silencing as well as in post-transcriptional regulation of gene expression in eukaryotic cells. The finding of miRNAs as negative regulators of gene expression at post-transcriptional level have added a new layer to the fine regulation of cell signaling mechanisms in human health and disease. Many miRNAs are known to play key roles in tumor biology. The actions of miRNAs may produce oncogenic or tumor suppressive effects. A specific miRNA may act as an oncogenic miRNA or a tumor suppressor miRNA depending on the tumor. The pathogenesis of human malignant neuroblastoma, which is a deadly childhood tumor, is now known to be associated with upregulation of oncogenic miRNAs and down regulation of tumor suppressor miRNAs. Upregulation of oncogenic miRNAs drives the growth of human malignant neuroblastoma by avoiding cell cycle arrest, differentiation, and apoptotic death. On the other hand, the down regulation of tumor suppressor miRNAs promotes cell proliferation, hypoxia, autophagy, multidrug resistance, migration, invasion, angiogenesis, and metastasis in human malignant neuroblastoma. So, inhibition of expression of specific oncogenic miRNAs and promotion of expression of tumor suppressor miRNAs may provide novel therapeutic opportunities to induce cell cycle arrest, differentiation, and apoptosis in human malignant neuroblastoma cells in vitro as well as in vivo. The success of modulation of expression of miRNAs in preclinical models of human malignant neuroblastoma may eventually be translated to the clinics for successful treatment of neuroblastoma patients in the future.

Swapan K. Ray

Papers

Specific microRNAs for Modulation of Autophagy in Spinal Cord Injury

Emerging Evidence for Krüppel-Like Factor 4 (KLF4) as a Tumor Suppressor in Neuroblastoma

Advanced Bioinformatics Analysis and Genetic Technologies for Targeting Autophagy in Glioblastoma Multiforme

Improved Antileukemic Activity of the Combination of Ara-C with GM-CSF and IL-3 Fusion Protein (PIXY321)

Modulation of Expression of miRNAs for Therapeutic Effects in Human Malignant Neuroblastoma