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Department of Pharmacology

 
Author(s): 
Du, Z
Abstract: 

The naked mole-rat (NMR) is a fascinating animal which has unique biological features including eusociality, strict subterranean inhabitation and poikilothermy. It is the longest-living rodent, showing negligible senescence over the majority of its lifespan and high resistance to diseases such as cancer and neurodegeneration. This animal is therefore a compelling system for understanding ageing and age-related diseases. Recent evidence suggests that protein homeostasis (proteostasis) mechanisms may play a vital role in mediating the resistance to multiple forms of stress and diseases and, subsequently, contribute to the exceptional longevity of the NMR. However, this view has been predominately based on protein-level and/or cell viability analyses, and our knowledge is still limited about the modulation of proteotoxic stress responses at the transcriptional level due to a lack of reliable and validated molecular tools.

This thesis sets out to develop, optimise and apply new methods to investigate two important and complex proteostatic mechanisms, namely the unfolded protein response (UPR) in the endoplasmic reticulum (ER) and macroautophagy (autophagy), in the NMR. Using these methodologies, the effects of pharmacologically induced in vitro stress and the effects of disease-related neurotoxic protein species on the UPR and autophagy in NMR fibroblasts were investigated. In Chapter 3, RNA-based methods, including an Xbp1 splicing assay and quantitative PCR (RT-qPCR) assays were successfully established to probe the activation and outputs of the UPR in a NMR kidney fibroblast cell line in response to tunicamycin (TU) and thapsigargin (TG)-induced ER stress. In Chapter 4, differences between the UPR of NMR kidney fibroblasts and mouse homologues were identified, where a notably higher threshold of pharmacologically induced UPR activation was observed in the NMR under conditions of mild ER stress. In Chapter 5, LC3B turnover and transcriptional changes of autophagy markers under rapamycin (RA) and chloroquine (CQ)-treated conditions were monitored in an NMR skin fibroblast cell line, where the sensitivity of the NMR skin fibroblasts to CQ, when compared to NMR kidney fibroblasts and mouse NIH3T3 embryonic cells, seemed to be partly attributed to the downregulation of TFEB, a master transcription factor of autophagy. In Chapter 6, the effects of amyloid-beta (Aβ) and α-synuclein oligomers, which are believed to be the major pathogenic species of Alzheimer’s and Parkinson’s diseases, respectively, on the UPR and/or autophagy were investigated in NMR and mouse cells using a combination of molecular and cellular tools. Although no significant changes of UPR markers were observed under Aβ oligomer-treated conditions, the chronic toxicity of wild-type α-synuclein oligomers seemed to be associated with downregulation of genes encoding ER chaperones and autophagy proteins. In Chapter 7, we demonstrate the utility of rational design to create a protein-specific binding probe for the NMR LC3B protein by introducing a peptide derived from a LC3-interacting region (LIR) motif into the inter-repeat loop of a consensus-designed tetratricopeptide repeat protein (CTPR). The results provide proof-of-concept validation of using CTPR-based probes to detect proteins in emerging animal models.

Having established a set of reliable methods to investigate the molecular details of the UPR and autophagy in the NMR, we have demonstrated unique features of the NMR, at the transcriptional level, when different forms of in vitro stress are employed. Exploiting these assays to measure the UPR and autophagy, as well as other proteostastic mechanisms, in the NMR under more disease-relevant conditions, may ultimately shed light on therapeutic developments to combat age-related neurodegenerative diseases.

Publication ID: 
1310254
Published date: 
2 August 2021 (No publication date)
Publication source: 
manual
Publication type: 
Theses / dissertations
Journal name: 
Publication volume: 
Publisher: 
University of Cambridge
Parent title: 
Edition: 
Publication number: 
Editor(s): 
Itzhaki, L, Kumita, J