Investigating the role of Parp1 in Xrcc1-linked neuropathology

Badman, Jack (2022) Investigating the role of Parp1 in Xrcc1-linked neuropathology. Doctoral thesis (PhD), University of Sussex.

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Abstract

The DNA repair scaffold protein XRCC1 orchestrates the activity of the DNA single-strand break repair (SSBR) machinery. DNA single-strand breaks (SSBs) are primarily detected by poly(ADP-ribose) Polymerase 1 (PARP1), which synthesises poly(ADP-ribose) at sites of damage, promoting XRCC1 recruitment. Mutations in XRCC1 have recently been identified in several human patients, resulting in ataxia with ocular motor apraxia (AOA). In cells lacking XRCC1, SSBR is reduced, leading to the retention of PARP1 at sites of damage and the continuous synthesis of poly(ADP-ribose). In conditional knockout Xrcc1Nes-Cre mice, poly(ADP-ribose) accumulates in the brain, resulting in progressive ataxia and fatal seizures. Here, I aimed to establish a cell autonomous model of endogenous SSB induction and Parp1 activation in neuronal cells.

To do so, I developed primary cultures of hippocampal neurons from Xrcc1Nes-Cremouse pups. I show that Xrcc1Nes-Cre hippocampal neurons exhibit elevated Parp1/NAD+ dependent poly(ADP-ribose) accumulation, recapitulating what has been observed in vivo in mouse brain. I also show that this elevated activity underlies the spontaneous death of Xrcc1Nes-Cre hippocampal neurons in vitro. Both the accumulation of poly(ADP-ribose) and spontaneous cell death can be rescued via the additional deletion of one or both alleles of Parp1, as well as with treatment by commercially available PARP inhibitors, highlighting the potential therapeutic application of PARP inhibition in the treatment of XRCC1-mediated neurodegeneration. In investigating the source of the endogenous DNA SSBs characterising Xrcc1-deficient cells, I highlight the nitric oxide (•NO) dependent production of reactive nitrogen species (RNS) as a likely source of damage.

I further demonstrate Parp1 dependent deregulation of presynaptic calcium signalling in Xrcc1Nes-Cre neurons, providing a compelling potential explanation for the seizures observed in the mouse brain. This synaptic phenotype can be rescued by chronic PARP inhibition, further indicating the therapeutic potential of PARP inhibition.

Finally, I aimed to investigate whether Parp1 dependent alterations to transcription could be identified in the Xrcc1Nes-Cre mouse brain. To do so, RNA sequencing was performed, highlighting a number of differentially expressed genes. This phenotype was, however, comparatively mild, and targets were not effectively validated in our small sample size.

In summary, I present a cell autonomous model of endogenous DNA SSBs, and demonstrate elevated poly(ADP-ribose) accumulation in Xrcc1Nes-Cre hippocampal neurons, resulting in aberrant presynaptic calcium signalling and a mild deregulation of transcription. These data improve our knowledge of the pathological contribution of PARP1 to Xrcc1-defective phenotypes, and the therapeutic potential of PARP in treating XRCC1-linked disease.

Item Type: Thesis (Doctoral)
Schools and Departments: School of Life Sciences > Neuroscience
Subjects: Q Science > QH Natural history > QH0301 Biology > QH0426 Genetics > QH0460 Mutations > QH0467 Genetic repair mechanisms
R Medicine > RC Internal medicine > RC0321 Neurosciences. Biological psychiatry. Neuropsychiatry > RC0346 Neurology. Diseases of the nervous system Including speech disorders
Depositing User: Library Cataloguing
Date Deposited: 30 May 2022 13:04
Last Modified: 30 May 2022 13:04
URI: http://sro.sussex.ac.uk/id/eprint/106188

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