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)

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

Fenugreek (Trigonella foenum-graecum) seeds and roots of wild yam (Dioscorea villosa) possess nutritional and medicinal properties and have been used for centuries in traditional medicine to treat different diseases and inflammatory responses. Diosgenin is a natural steroidal sapogenin extracted from fenugreek and wild yam and it is one of the major bioactive compounds used in the treatment of diabetes, hypercholesterolemia, and inflammation. Recent studies have shown a promising effect of diosgenin as an anti-tumor agent for inhibition of cell proliferation and induction of apoptosis in many cancers such as colon cancer, leukemia, breast cancer, and liver cancer. We examined the effects of different concentrations (5, 10, 15, 20, and 25 µM) of diosgenin on proliferation of rat C6 and human T98G glioblastoma cell lines. We noticed that diosgenin had a high inhibitory effect on the growth of both C6 and T98G cell lines. Diosgenin induced the differentiation of glioblastoma cells, as determined by the increase in the expression of the differentiation marker glial fibrillary acidic protein (GFAP); and decreased the dedifferentiation of the cells, as shown by the decrease in the abundance of the dedifferentiation marker proteins Id2, N-Myc, telomerase reverse transcriptase (TERT), and Notch-1. It also induced apoptosis in C6 and T98G cell lines and the molecular mechanisms involved in the induction of apoptosis included increase in pro-apoptotic Bax protein and decrease in anti-apoptotic Bcl-2 protein. Further, the diosgenin-induced suppression of cell migration was correlated with the decrease in expression of matrix metalloproteinase 2 (MMP2) and MMP9; and the inhibition of angiogenesis, as determined by the tube formation assay, was correlated with a decrease in the protein levels of vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2). In conclusion, diosgenin showed anti-tumor effects in glioblastoma cells by induction of differentiation and apoptosis and inhibition of migration, invasion, and angiogenesis.

Diosgenin as a Novel Alternative Therapy for Inhibition of Growth, Invasion, and Angiogenesis Abilities of Different Glioblastoma Cell Lines

N-(4-Hydroxyphenyl) retinamide potentiated paclitaxel for cell cycle arrest and apoptosis in glioblastoma C6 and RG2 cells.

Brain Tumor Angiogenesis.- Adrenoleukodystrophy: Molecular, Metabolic, Pathologic And Therapeutic Aspects.- Hyperammonemia.- Glutamate And Cytokine-mediated Alterations Of Phospholipids In Head Injury And Spinal Cord Trauma.- Kynurenines In The Brain: Preclinical And Clinical Studies, Therapeutic Considerations.- Neurofibromatosis Type I: From Genetic Mutation To Tumor Formations.- Amyloid And Neurodegeneration: Alzheimer's Disease And Retinal Degeneration.- Diabetic Retinopathy.- Neurotransmitters And Electrophysiology In Traumatic Brain Injury.- Free Radicals And Neuroprotection In Traumatic Brain And Spinal Cord Injury.- Neuroimmunology Of Paraproteinemic Neuropathies.- Proteolytic Mechanisms Of Cell Death In The Central Nervous System.- Nitric Oxide In Experimental Allergic Encephalomyelitis.- The BBB: Biology, Development And Brain Injury.- Phospholipase A2 In CNS Disorders: Implication On Traumatic Spinal Cord And Brain Injuries.- Axonal Damage Due To Traumatic Brain Injury.- Blood-Central Nervous System Barriers: The Gateway To Neurodegeneration, Neuroprotection And Neuroregeneration.- Engineered Antibody Fragments As Potential Therapeutics Against Misfolded Proteins In Neurodegenerative Diseases.- Pathogenesis And Treatment Of HIV-Associated Dementia: Recent Studies In A SCID Mouse Model.- Stem Cell Transplantation Therapy For Neurological Diseases.- Ubiquitin/Proteasome And Autophagy/Lysosome Pathways: Comparison And Role In Neurodegeneration.- Experimental Autoimmune Encephalomyelitis In The Pathogenesis Of Optic Neuritis: Is Calpain Involved?- Protein Carbonylation In Neurodegenerative And Demyelinating CNS Diseases.- Clinical Considerations In Translational Research With Chronic Spinal Cord Injury: Intervention Readiness And Intervention Impact.- Estrogen As A Promising Multi-Active Agent For The Treatment Of Spinal Cord Injury.- Immunotherapy Strategies For Lewy Body And Parkinson's Disease.- Clinical Outcomes After Spinal Cord Injury.- Spinal Cord Injury ~ A Clinical Perspective.- Cubing The Brain: Mapping Expression Patterns Genome-Wide.

Brain and spinal cord trauma

https://josorge.com/publications/Citations/NCR/027.pdf

N-Myc knockdown and apigenin treatment controlled growth of malignant neuroblastoma cells having N-Myc amplification.

Calcium-activated neutral proteinase (calpain) is a ubiquitous, cytosolic endopeptidase which is believed to play a role in many neural functions. In the present study, we examined the transcriptional and translational expression of microcalpain (microcalpain) and millicalpain (mcalpain) isoforms and the endogenous inhibitor calpastatin in rat and bovine spinal cord, brain stem, cerebellum, and cerebral cortex tissues using reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. In rat central nervous system (CNS) samples, the microcalpain and mcalpain transcriptional expression was highest in white matter-enriched areas. Calpastatin mRNA expression demonstrated no significant differences among the CNS areas. Calpain and calpastatin translational expression levels were greatest in the spinal cord. In bovine CNS, microcalpain transcriptional expression was greatest in the spinal cord, while other CNS regions showed no significant differences. Bovine mcalpain transcriptional expression was similar among various CNS regions but marginally greater in the cortex. Translational expression of bovine calpain was greatest in the brain stem, while that of calpastatin was highest in the cerebral cortex. These results indicate that calpain expression varies among different CNS regions and is often highest in white matter-enriched areas.

Swapan K. Ray

Papers

Diosgenin as a Novel Alternative Therapy for Inhibition of Growth, Invasion, and Angiogenesis Abilities of Different Glioblastoma Cell Lines

N-(4-Hydroxyphenyl) retinamide potentiated paclitaxel for cell cycle arrest and apoptosis in glioblastoma C6 and RG2 cells.

Brain and spinal cord trauma

N-Myc knockdown and apigenin treatment controlled growth of malignant neuroblastoma cells having N-Myc amplification.

Calpain expression varies among different rat and bovine central nervous system regions