Steps to creating a co-culture to investigate reactive species in neurodegeneration

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Howard, C., 2019. Steps to creating a co-culture to investigate reactive species in neurodegeneration. PhD, Nottingham Trent University.

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Abstract

Introduction: Neurodegenerative diseases such as Alzheimer's and Parkinson's disease are becoming more prevalent as the aging population increases. Multiple factors are implicated including impaired protein degradation, mitochondrial dysfunction and oxidative stress; whose mechanisms may be interconnected in the so-called 'Neurodegenerative Bermuda triangle'. Understanding these hallmarks is essential for developing new therapies to help ameliorate symptoms of neurodegenerative diseases.

Methods and Aims: Human neuronal (SH-SY5Y) and glial (U-87MG) cell lines were used to investigate oxidative stress mediated neurodegeneration. Reactive species (RS), specifically peroxy radicals and superoxide radicals were produced using 2,2′-Azobis(2-methylpropionamidine) dihydrochloride (AAPH; a ROS generating lipid peroxidation agent) and rotenone (a mitochondrial disruptor that inhibits complex I), respectively. Using fluorescence probes, cellular function assays, PCR analysis, protein expression and proteomics the effects of RS in neural cells both in mono and co-cultures were investigated.

Results: AAPH and rotenone were shown to have divergent effects on neural cells exposing the differences between these cells. Furthermore, AAPH was found to induce greater lipid peroxidation than rotenone, suggesting different toxins induce specific RS. In particular, the NRF2-antioxidant response element was highlighted as a key pathway activated by oxidative stress. Experiments with primary embryonic cortical neuronal cultures show the limitations of in vitro differentiation models. Thus, steps to create a co-culture were investigated, demonstrating the difficulties associated with co-cultures but showing how neuronal susceptibility to RS was altered by glial cells.

Conclusion: This body of work highlights AAPH for future investigations into lipid peroxidation. Furthermore, the results herein suggest differentiation methods should be considered when evaluating RS susceptibility. Finally, initial investigations have determined how transwell inserts can be used to create co-cultures between neural cells with materials needing to be considered for future studies. Conditioned media provides an alternative way to investigate secreted factors where potential pathways have been determined for future studies.

Item Type:	Thesis
Creators:	Howard, C.
Date:	September 2019
Rights:	This work is the intellectual property of the author and may also be owned by the research sponsor(s) and/or Nottingham Trent University. You may copy up to 5% of this work for private study, or personal, non-commercial research. Any re-use of the information contained within this document should be fully referenced, quoting the author, title, university, degree level and pagination. Queries or requests for any other use, or if a more substantial copy is required, should be directed in the owner(s) of the Intellectual Property Rights.
Divisions:	Schools > School of Science and Technology
Record created by:	Linda Sullivan
Date Added:	18 Aug 2020 14:44
Last Modified:	31 May 2021 15:17
URI:	https://irep.ntu.ac.uk/id/eprint/40472