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Fixing breaks in the brain: Exploring the protective role of DNA repair pathways in the central nervous system
Secondary Supervisor(s): Dr Mariaelena Repici
University of Registration: Aston University
BBSRC Research Themes:
Project Outline
The DNA in our cells is essential for life as it contains the complete set of genetic information for every cell to function. However, DNA is not inert and is vulnerable to being damaged or altered. It is constantly being damaged due to exposure to DNA damaging agents that are produced within our cells as a byproduct of metabolism or are present in the environment. If damage to DNA is not repaired, it will result in mutation, genome instability and ultimately human disease. Therefore, our cells employ a variety of essential DNA repair pathways that function to rapidly detect and repair DNA damage when it arises. Due to the important of these repair pathways, the inheritance of mutations in DNA repair genes is a significant cause of human disease.
One debilitating symptom that is common to diseases caused by mutations in DNA repair genes is neurodegeneration. Neurodegeneration is caused by the progressive loss of neurons in the central nervous system, ultimately leading to reduced brain function. However, despite decades of research it is still unknown why neurons in the brain seem to be particularly sensitive to the loss of DNA repair pathways, and a particular challenge is the inability to obtain disease affected tissue from patients. The recent development of techniques to generate physiologically relevant neuronal disease models via the use of patient-derived induced pluripotent stem cells (iPSCs) has therefore proved revolutionary to human neurodegenerative disease research.
This project aims to investigate the role that DNA double strand break repair plays in promoting neuronal health and function using a variety of human neuronal models.
Aim 1: Establish neuronal models using immortalised neuroblastoma SH-SY5Y cell lines. CRISPR-Cas9 will be used to generate SH-SY5Y cell lines in which disease-associated genes are knocked out, or in which patient mutations are introduced. These cell lines will be differentiated into a more mature neuron-like phenotype. The ability of these cells to repair physiologically relevant types of DNA damage will then be measured using a variety of assays, including immunofluorescence for markers of DNA damage, immunoblotting, metaphase spread analysis and comet assay.
Aim 2: Investigate the cellular consequences of elevated levels of unrepaired DNA damage persisting in differentiated SH-SY5Y cells. Following the induction of DNA damage in wild-type and mutant SH-SY5Y cells, we will investigate:
- Levels of apoptotic cell death
- RNA transcription of critical neuronal maintenance genes
- The formation and composition of stress granules
Aim 3: Generate central nervous system cell types from iPSCs carrying deletion/mutation of DNA repair genes associated with neurodegenerative disease. These different cells will be first characterised to ensure they possess the appropriate markers and cellular morphologies, and their ability to respond to and repair DNA damage will be measured.
In summary, this PhD project will use cutting edge cell models to gain a fuller understanding of why the repair of DNA damage is critical for the health of the human central nervous system, and attempt to answer some important questions, such as why are neuronal types affected differently in different neurodegenerative diseases?
References
Reynolds JJ, Stewart GS. (2018) "Chapter 12: DNA Replication and Inherited Human Disease" in "DNA Repair and Replication: Mechanisms and Clinical Significance". Garland Science p249-289. (Book Chapter)
Scheijen and Wilson III, (2022). Genome Integrity and Neurological Disease. International Journal of Molecular Sciences.
Provasek, et al, (2022). DNA Double-Strand Breaks as Pathogenic Lesions in Neurological Disorders. International Journal of Molecular Sciences.
