| 1. |
- 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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| 2. |
- Milosch, N, et al.
(författare)
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Holo-APP and G-protein-mediated signaling are required for sAPPa-induced activation of the Akt survival pathway
- 2014
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Ingår i: Cell Death and Disease. - : Springer Science and Business Media LLC. - 2041-4889. ; 5
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Tidskriftsartikel (refereegranskat)abstract
- Accumulating evidence indicates that loss of physiologic amyloid precursor protein (APP) function leads to reduced neuronal plasticity, diminished synaptic signaling and enhanced susceptibility of neurons to cellular stress during brain aging. Here we investigated the neuroprotective function of the soluble APP ectodomain sAPPα (soluble APPα), which is generated by cleavage of APP by α-secretase along the non-amyloidogenic pathway. Recombinant sAPPα protected primary hippocampal neurons and SH-SY5Y neuroblastoma cells from cell death induced by trophic factor deprivation. We show that this protective effect is abrogated in neurons from APP-knockout animals and APP-depleted SH-SY5Y cells, but not in APP-like protein 1- and 2- (APLP1 and APLP2) depleted cells, indicating that expression of membrane-bound holo-APP is required for sAPPα-dependent neuroprotection. Trophic factor deprivation diminished the activity of the Akt survival pathway. Strikingly, both recombinant sAPPα and the APP-E1 domain were able to stimulate Akt activity in wild-type (wt) fibroblasts, SH-SY5Y cells and neurons, but failed to rescue in APP-deficient neurons or fibroblasts. The ADAM10 (a disintegrin and metalloproteinase domain-containing protein 10) inhibitor GI254023X exacerbated neuron death in organotypic (hippocampal) slice cultures of wt mice subjected to trophic factor and glucose deprivation. This cell death-enhancing effect of GI254023X could be completely rescued by applying exogenous sAPPα. Interestingly, sAPPα-dependent Akt induction was unaffected in neurons of APP-ΔCT15 mice that lack the C-terminal YENPTY motif of the APP intracellular region. In contrast, sAPPα-dependent rescue of Akt activation was completely abolished in APP mutant cells lacking the G-protein interaction motif located in the APP C-terminus and by blocking G-protein-dependent signaling with pertussis toxin. Collectively, our data provide new mechanistic insights into the physiologic role of APP in antagonizing neurotoxic stress: they suggest that cell surface APP mediates sAPPα-induced neuroprotection via G-protein-coupled activation of the Akt pathway.
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| 3. |
- Siewert, F, et al.
(författare)
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On the characterization of ultra-precise X-ray optical components: advances and challenges in ex situ metrology.
- 2014
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Ingår i: Journal of Synchrotron Radiation. - 1600-5775. ; 21:Pt 5, s. 968-975
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Tidskriftsartikel (refereegranskat)abstract
- To fully exploit the ultimate source properties of the next-generation light sources, such as free-electron lasers (FELs) and diffraction-limited storage rings (DLSRs), the quality requirements for gratings and reflective synchrotron optics, especially mirrors, have significantly increased. These coherence-preserving optical components for high-brightness sources will feature nanoscopic shape accuracies over macroscopic length scales up to 1000 mm. To enable high efficiency in terms of photon flux, such optics will be coated with application-tailored single or multilayer coatings. Advanced thin-film fabrication of today enables the synthesis of layers on the sub-nanometre precision level over a deposition length of up to 1500 mm. Specifically dedicated metrology instrumentation of comparable accuracy has been developed to characterize such optical elements. Second-generation slope-measuring profilers like the nanometre optical component measuring machine (NOM) at the BESSY-II Optics laboratory allow the inspection of up to 1500 mm-long reflective optical components with an accuracy better than 50 nrad r.m.s. Besides measuring the shape on top of the coated mirror, it is of particular interest to characterize the internal material properties of the mirror coating, which is the domain of X-rays. Layer thickness, density and interface roughness of single and multilayer coatings are investigated by means of X-ray reflectometry. In this publication recent achievements in the field of slope measuring metrology are shown and the characterization of different types of mirror coating demonstrated. Furthermore, upcoming challenges to the inspection of ultra-precise optical components designed to be used in future FEL and DLSR beamlines are discussed.
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