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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 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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| 2. |
- Zheng, Wei, et al.
(author)
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Divalent metal transporter 1 is involved in amyloid precursor protein processing and A{beta} generation.
- 2009
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In: FASEB Journal. - : Wiley. - 1530-6860 .- 0892-6638. ; 23:12, s. 4207-4217
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Journal article (peer-reviewed)abstract
- The amyloid-beta precursor protein (APP) and its pathogenic byproduct beta-amyloid peptide (Abeta) play central roles in the pathogenesis of Alzheimer's disease (AD). Reduction in levels of the potentially toxic Abeta is one of the most important therapeutic goals in AD. Recent studies have shown that bivalent metals such as iron, copper, and zinc are involved in APP expression, Abeta deposition, and senile plaque formation in the AD brain. However, the underlying mechanisms involved in abnormal homeostasis of bivalent metals in AD brain remain unclear. In the present study, we found that two isoforms of the divalent metal transporter 1 (DMT1), DMT1-IRE, and DMT1-nonIRE, were colocalized with Abeta in the plaques of postmortem AD brain. Using the APP/PS1 transgenic mouse model, we found that the levels of both DMT1-IRE and DMT1-nonIRE were significantly increased in the cortex and hippocampus compared with wild type-control. We further verified the proposed mechanisms by which DMT1 might be involved in APP processing and Abeta secretion by using the SH-SY5Y cell line stably overexpressing human APP Swedish mutation (APPsw) as a cell model. We found that overexpression of APPsw resulted in increased expression levels of both DMT1-IRE and DMT1-nonIRE in SH-SY5Y cells. Interestingly, silencing of endogenous DMT1 by RNA interference, which reduced bivalent ion influx, led to reductions of APP expression and Abeta secretion. These findings suggest both that DMT1 plays a critical role in ion-mediated neuropathogenesis in AD and that pharmacological blockage of DMT1 may provide novel therapeutic strategies against AD.-Zheng, W., Xin, N., Chi, Z.-H., Zhao, B.-L., Zhang, J., Li, J.-Y., Wang, Z.-Y. Divalent metal transporter 1 (DMT1) is involved in amyloid precursor protein processing and Abeta generation.
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