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- Klionsky, Daniel J., et al.
(author)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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In: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Research review (peer-reviewed)abstract
- 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.
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| 2. |
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| 3. |
- Klionsky, Daniel J., et al.
(author)
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Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes
- 2008
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In: Autophagy. - : Landes Bioscience. - 1554-8627 .- 1554-8635. ; 4:2, s. 151-175
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Research review (peer-reviewed)abstract
- 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.
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| 4. |
- Roberg, Karin, 1957-
(author)
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The role of cathepsin D in apoptosis induced by oxidative stress
- 2001
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Doctoral thesis (other academic/artistic)abstract
- The lysosomal protease cathepsin D is translocated from lysosomes to the cytosol during apoptosis induced by oxidative stress. In the present studies, the redox-cycling, xenobiotic compound naphthazarin (5,8-dihydroxy-1,4-naphthoquinone) was used to create oxidative stress in rat cardiomyocytes and human foreskin fibroblasts. In naphthazarin exposed cells, lysosomal release of cathepsin D preceded liberation of cytochrome c from mitochondria and a decrease in the mitochondrial transmembrane potential (ΔΨm).A pre-embedding immunocytochemical method was used for ultrastructural examination of cathepsin D and cytochrome c in cultured cells. Electron microscopic morphometry confirmed that a statistically significant amount of cathepsin D was transferred from lysosome-like structures to the cytosol before any biochemical or morphological signs of apoptosis were detected. Pretreatment of the cells with atocopherol succinate largely prevented translocation of cathepsin D and also significantly decreased apoptosis. Electron microscopy also revealed that, during exposure to naphthazarin, a minor release of cytochrome c has occured after one hour and a more extensive release after two hours, and these results were verified by Western blotting. After the translocation of cathepsin D and cytochrome c, a decrease in ΔΨm was detected using the ΔΨm-sensitive probe JC-1 and confocal microscopy or measured by flow cytornetry. Pretreatment with the cathepsin D inhibitor pepstatin A prevented release of cytochrome c from mitochondria, maintained the ΔΨm and inhibited apoptosis.In conclusion, these findings show that translocation of cathepsin D precedes important incidents in mitochondria, such as release of cytochrome c and loss of ΔΨm during apoptosis induced by oxidative stress. Moreover, inhibition of cathepsin D prevented the apoptosis and the mitochondrial changes, which indicates that cathepsin D is an inducer of apoptosis upstream of cytochrome c release.
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