Optimisation and validation of tools for in situ visualisation of heteroplasmic mtDNA variants.

Mitochondria is more than just a powerhouse in our cells; it holds its own genome (mtDNA). Eukaryotic cells often contain many hundreds to thousands of copies of mtDNA. When a mutation occurs in one or more of these copies, this results in a mixture of mutant and wildtype mtDNA, known as heteroplasmy. Developmental and ageing-related expansion of mutant mtDNA heteroplasmy is the leading cause of adult-onset mitochondrial disease, affecting 1 in 5,000 people in the United Kingdom. It plays a role in neurodegenerative disorders like Alzheimer’s and Parkinson’s disease. Despite their importance, the processes that cause this expansion of mutant mtDNA are poorly understood, primarily due to a lack of suitable in vivo models to measure and modulate mitochondrial mutations in somatic tissues. We develop and validate tools to visualise and measure mtDNA mutations in situ to address this gap. We have established a novel approach to visualising and distinguishing different mtDNA molecules in Drosophila cells and tissues based on single-molecule fluorescent in situ hybridisation (smFISH) combined with hybridisation chain reaction (HCR) for detection of mtDNA. Taking advantage of Drosophila heteroplasmic strains, we have applied this in vivo in the developing larval brain. Our findings revealed extensive cell-to-cell variability in heteroplasmy levels between individual neural stem cells (NSCs), and between NSCs and their postmitotic progeny, providing evidence for the existence of a bottleneck effect during neurogenesis in the developing brain. The bottleneck effect occurs during early larval stages when the NSCs undergo rapid differentiation and proliferation. Although the initial heteroplasmy level is set during embryonic development, a significant variance is seen between NSCs and their progeny in the heteroplasmy level during larval development. To improve the sensitivity and specificity of this technique, we further optimised the protocol using heteroplasmic Drosophila brains. Our mtDNA-smFISH method also demonstrated evidence of purifying selection is seen against mutant mtDNA during the development of oocytes. We further aim to understand the selection mechanism which influences the ratio of mutant to wildtype mtDNA transferring into the progeny from neurons. Our research will provide a powerful new approach to visualise and measure mtDNA heteroplasmy in vivo. By using this technique, we can understand the processes that drive the expansion of mutant mtDNA and help to develop new treatments aimed at reducing the mutational burden to prevent late-onset neurological disorders.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Higher Doctorate

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2024

Author dc:creator

• Chandrasegaram, Rajini

Advisor dc:contributor.advisor

• van den Ameele, Jelle

• Mitochondria
• mtDNA
• Heteroplasmy
• smFISH
• In situ hybridisation

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/64ad195c-69e7-427f-a977-f948169a0ac9/download
• http://purl.org/NET/rdflicense/allrightsreserved

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.118319

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/384264