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- Dehvari, Nodi
(author)
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Effects of Alzheimer's and Parkinson's disease gene mutation on cell signaling
- 2009
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Doctoral thesis (other academic/artistic)abstract
- Alzheimer's disease (AD) and Parkinson's disease (PD) are the major neurodegenerative disorders affecting the elderly. The discovery of genes causing familial forms of these disorders has contributed enormously towards our understanding of underlying pathogenic mechanisms. This thesis explores mechanisms by which Phospholipase C (PLC) mediated signaling is regulated by presenilin (PS) and parkin, these being genes in which mutations cause familial AD and PD respectively. In Paper I, mouse embryonic fibroblasts (MEFs) lacking PS1, PS2 or both, were used to evaluate the dependence of PS on PLC and Protein Kinase C (PKC). Results revealed a dependence of both PS1 and PS2 on m-3M3FBS stimulated PLC activities. Also, PLC stimulated PKCalpha and PKCgamma activities were dependent on the presence of both PSs. Protein levels of PKCalpha/gamma were decreased in PS double knockout MEFs, while PKCdelta levels were elevated in the same cells. These results were also verified by transfecting back PS and by using another cell line lacking PS. Furthermore PKCalpha levels were shown to be dependent on amyloid precursor protein (APP) and APP intracellular domain. It is concluded that PS modulate PLC and PKC activity and differently regulates PKC protein levels by both APP dependent and independent mechanisms. Paper II explored the dependence of PS on Extracellular regulated kinase 1/2 (ERK1/2) activities. ERK1/2 has previously been implicated in the pathogenesis of AD by different mechanisms. Using MEF cells lacking PSs (same as in paper I) it was found that ERK1/2 activities were increased in PS1 or PS2 knockout MEFs. PKCalpha inhibition could reverse these elevated ERK1/2 activities. Results also revealed a lower PLC or PKC stimulation in PS double knockout cells. The total levels of ERK were only downregulated after phorbol-12,13-dibutyrate (PdBu) treatment in PS double knockout. These findings show that PS regulates ERK1/2 activity via a PKCalpha dependent manner. Lack of both PSs disrupts PLC/PKC signaling and this is also reflected in the lower downstream activation of ERK1/2. In Paper III, PLCgamma1 was found to interact with parkin, in cells transfected with parkin WT, mutants G328E and R42P. This interaction was also detected in cortical, striatal and nigral human brain homogenates. PLCgamma1 protein levels were found to be higher in parkin knockout mice and lower in parkin WT cells. Also, parkin mutants cells showed a disrupted ubiquitination. In summary, PLCgamma1 was identified as a novel substrate for parkin by using interaction and ubiquitination studies of PLCgamma1 and parkin. Paper IV describes functional studies that revealed an increased PLC phosphorylation and activity in parkin mutant cells. Also, intracellular calcium levels were elevated in these cells, which could be reversed by the PLC inhibitor Neomycin and ryanodine receptor inhibitor dantrolene, suggesting a deregulated PLC activity. Parkin WT was shown to be protective against 6-OH-dopamine toxicity. The toxicity seen in parkin mutants after 6-OH-dopamine, could be reversed by dantrolene treatment, suggesting that ryanodine receptor calcium deregulation contributes to toxicity. In conclusion, these studies provide evidence that PS and parkin regulate PLC mediated signaling.
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| 2. |
- Zheng, Lin, et al.
(author)
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Oxidative stress induces macroautophagy of amyloid beta-protein and ensuing apoptosis
- 2009
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In: Free Radical Biology & Medicine. - : Elsevier BV. - 0891-5849 .- 1873-4596. ; 46:3, s. 422-429
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Journal article (peer-reviewed)abstract
- There is increasing evidence for the toxicity of intracellular amyloid beta-protein (A beta) to neurons and the involvement of lysosomes in this process in Alzheimer disease (AD). We have recently shown that oxidative stress, a recognized determinant of AD. enhances macroautophagy and leads to intralysosomal accumulation of A beta in Cultured neuroblastoma cells. We hypothesized that oxidative stress promotes AD by stimulating macroautophagy of A that further may induce cell death by destabilizing lysosomal membranes. To investigate such possibility, we compared the effects of hyperoxia (40% ambient oxygen) in cultured HEK293 cells that were transfected with an empty vector (Vector), wild-type APP (APPwt), or Swedish mutant APP (APPswe). Exposure to hyperoxia for 5 days increased the number of cells with A beta-containing lysosomes, as well as the number of apoptotic cells, compared to normoxic conditions. The rate of apoptosis in all three cell lines demonstrated dependence on intralysosomal A beta content (Vector
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