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An in vivo and in vitro study of stress-induced senescence in neurones and its role in neurodegeneration

Vazquez Villasenor, Irina (2018) An in vivo and in vitro study of stress-induced senescence in neurones and its role in neurodegeneration. PhD thesis, University of Sheffield.

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Abstract

Cellular senescence, characterised by the development of a toxic secretory phenotype in response to persistent DNA damage, has been studied in proliferating cells but is not well understood in post-mitotic neurones. Recent evidence showed a neuronal senescent-like state in response to persistent DNA damage in vivo, which could contribute to neuronal dysfunction in aging and neurodegeneration. The current study hypothesised that oxidative stress, a hallmark of neurodegeneration, activates a persistent DNA damage response and promotes neuronal senescence. To investigate this, activation of senescence in response to oxidative DNA damage was studied in human post-mitotic neurones in culture, as well as in the brains of control and ALS/MND donors. For the in vitro study, post-mitotic LUHMES were stressed with a double dose of 50 µM H2O2, which caused a persistent DNA damage in the form of double-strand breaks that was detectable 96 hours’ post-stress. Expression of the “classical” senescence marker SA β-gal and formation of senescence-associated heterochromatin foci (SAHF) were evaluated at the 96 hours-timepoint, using cytochemical methods. A co-culture system of double stressed LUHMES and healthy LUHMES was developed to study DNA damage propagation; gene expression profiling was used to investigate changes in known senescence pathways in 96 hours-double stressed LUHMES. Results from this study revealed a highly variable SA-β-gal activity in healthy and double stressed LUHMES; SAHF were not present in these cells and propagation of DNA damage was not seen in the co-culture system. Transcriptomic analysis of double stressed LUHMES revealed dysregulation of the APC/C:Cdh1 cell cycle regulatory pathway, ATR signalling and II mitochondrial complex I activity, which could be related to oxidative stress but not to senescence. The current work also investigated the relevance of neuronal senescence in neurodegeneration. “Classical” senescence (SA-β-gal, p16 and p21) and oxidative DNA damage markers (8-OHdG and γH2AX) were investigated in the motor cortex, spinal cord and frontal association cortex of ALS/MND and control donors using immunohistochemistry. Transcriptome analysis of LCM neurones obtained from the frontal cortex of control and ALS/MND donors was used to investigate early changes in gene expression that could be linked to a senescent-like state. Transcriptomic analysis suggested dysregulation of DDR, cell cycle and oxidative phosphorylation pathways as a consequence of the persistent oxidative DNA damage, but no of “classical” senescence was found in the in vitro neuronal model. In vivo, p21 expression was found in neurones and glia, whereas p16 was exclusively expressed in glial cells. A significantly higher percentage of p21+ neurones was detected in the frontal association cortex of ALS/MND donors. Transcriptome analysis showed alteration of DDR pathways and mitochondrial function in these neurones, but did not reveal dysregulation of “classical” senescence pathways. Neurones may activate a senescent-like state in response to persistent DNA damage but signalling pathways involved in this mechanism may differ from the ones described in mitotic cells. Thus, “classical” senescence markers should be used cautiously when studying neuronal senescence, as they could reflect induction of related but different mechanisms in these cells. In order to study neuronal senescence, it is necessary to understand first the cell cycle regulatory mechanisms that occur in neurones as part of the DDR.

Item Type: Thesis (PhD)
Academic Units: The University of Sheffield > Faculty of Medicine, Dentistry and Health (Sheffield) > Medicine (Sheffield)
Identification Number/EthosID: uk.bl.ethos.739891
Depositing User: Miss Irina Vazquez Villasenor
Date Deposited: 16 Apr 2018 09:42
Last Modified: 12 Oct 2018 09:54
URI: http://etheses.whiterose.ac.uk/id/eprint/20040

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