| 2. |
- Klionsky, Daniel J., et al.
(författare)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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Ingår i: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Forskningsöversikt (refereegranskat)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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| 3. |
- Civitelli, Livia, et al.
(författare)
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Herpes simplex virus type 1 infection in neurons leads to production and nuclear localization of APP intracellular domain (AICD): implications for Alzheimers disease pathogenesis
- 2015
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Ingår i: Journal of Neurovirology. - : SPRINGER. - 1355-0284 .- 1538-2443. ; 21:5, s. 480-490
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Tidskriftsartikel (refereegranskat)abstract
- Several data indicate that neuronal infection with herpes simplex virus type 1 (HSV-1) causes biochemical alterations reminiscent of Alzheimers disease (AD) phenotype. They include accumulation of amyloid-beta (A beta), which originates from the cleavage of amyloid precursor protein (APP), and hyperphosphorylation of tau protein, which leads to neurofibrillary tangle deposition. HSV-1 infection triggers APP processing and drives the production of several fragments including APP intracellular domain (AICD) that exerts transactivating properties. Herein, we analyzed the production and intracellular localization of AICD following HSV-1 infection in neurons. We also checked whether AICD induced the transcription of two target genes, neprilysin (nep) and glycogen synthase kinase 3 beta (gsk3 beta), whose products play a role in A beta clearance and tau phosphorylation, respectively. Our data indicate that HSV-1 led to the accumulation and nuclear translocation of AICD in neurons. Moreover, results from chromatin immunoprecipitation assay showed that AICD binds the promoter region of both nep and gsk3 beta. Time course analysis of NEP and GSK3 beta expression at both mRNA and protein levels demonstrated that they are differently modulated during infection. NEP expression and enzymatic activity were initially stimulated but, with the progression of infection, they were down-regulated. In contrast, GSK3 beta expression remained nearly unchanged, but the analysis of its phosphorylation suggests that it was inactivated only at later stages of HSV-1 infection. Thus, our data demonstrate that HSV-1 infection induces early upstream events in the cell that may eventually lead to A beta deposition and tau hyperphosphorylation and further suggest HSV-1 as a possible risk factor for AD.
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