We made excellent progress in addressing possible involvement of ataxia/neurodegeneration genes in the DNA damage response, and in identifying a pathological mechanism by which rare neurological diseases associated with loss of single-strand break repair arise. Our advances are summarised, below.
During the first quarter of this project, we employed a library of small interfering RNA's to disrupt ataxia disease genes of interest to examine their impact on the DNA damage response, both at the level of endogenous DNA damage-induced responses and following exposure to exogenous genotoxins relevant to our environment. This screen enabled us to shortlist a number of disease-related human genes for further analysis, such as BRAT1. In addition, we identified a novel role for one DNA damage sensor protein (denoted PARP1) during normal cell growth and cell division that was unexpected.
During the second quarter, we conducted additional screens and assays to extend our analysis to a broader range of neurodegenerative diseases in which the response to DNA strand breaks may be a factor (Huntington’s disease, Alzheimer’s disease, Parkinson’s disease). We have systematically examined patient-derived fibroblasts and cultured cell lines in which proteins associated with neurodegeneration are mutated or depleted for defects in the DNA damage response. The project progressed well, but did not discover any obvious defects in the DNA damage response in these contexts. However, these experiments did yield exciting results in terms of identifying new connections between key RNA processing factors such as FUS and DNA metabolism. We also continued to explore the role of the DNA strand break sensor protein, PARP1, in the pathological response to DNA damage, using mouse in vivo models (following on from our work published in 2017; Hoch and Hanzlikova et al, Nature, 2017). This additional work resulted in one manuscript that we posted on BioRxiv, and which is now published in EMBO Reports (Komulainen et al EMBO Reports, 2021), and five additional primary research papers during this reporting period under the auspices of the ERC award, including one in Molecular Cell (Mahjoub A, et al Neurol Genet. 2019 Kalasova et al, Neurol Genet. 2019; Martinez-Macias MI, et al Life Sci Alliance. 2019;
Zagnoli-Vieira G, et al Neurol Genet. 2018; Hanzlikova H, et al Mol Cell. 2018). The latter paper was the remarkable and unexpected discovery that the primary source of activation of the SSB. sensor protein PARP1, in proliferating cells at least, are canonical unligated Okazaki fragment intermediates of DNA replication. The work has attracted more than 180 citations since its publication in 2018.
During the third and fourth quarter, we advanced rapidly with understanding the PARP1-dependent mechanism by which DNA breaks trigger neuropathology. In addition, we continued to focus and assess the possibility of pharmacological approaches for clinical intervention, based on this mechanism. This is because PARP inhibitors are already developed and in use in the clinic, for anti-cancer therapy, rising the possibility thatchy can be repurposed for use on SSB repair defective diseases. We published the work on integrator complex and human disease that was in progress and referred to in the last reporting period, and is now in press at Nature Communications (Cihlarova et al, Nat. Comm. 2022). In addition, we have published our exciting work showing that base excision repair (BER) intermediates are a potent threat to transcription, and that it is these lesions these that trigger excessive PARP1 hyperactivation and, as a consequence, neuronal pathology (Adamowicz et al Nature Cell Biology, 2021; Demin et al Molecuar Cell 2021). These two pieces of work are particularly exciting, because we have identified the mechanism by which PARP1 becomes trapped on unprepared SSB intermediates of BER, leading to excessive PARP1 activity and neuropathology. We found that PARP1 hyperactivation leads to excessive recruitment and activity of the ubiquitin protease USP3, which in turn results in excessive deubiquitination of H2A (K119) and H2B (K120), which in turn results in massive transcriptional disruption. This extends our model that PARP1 is a possible therapeutic target in neurological diseases arising from defects in BER to also include USP3. This work also further informs our recent work showing that SSBs generated during BER are enriched at sites of super-enhancers in neurons, which we showed are the result of active DNA demethylation triggered deliberately in neurons to drive expression of genes that determine neural cell identify (Wu et al Nature, 2021). Although sadly the EU project has now ended, it is my plan to now pursue these exciting avenues towards clinical intervention, working closely with industry to develop potential drugs that can alleviate this type of neurological disease.
