Alkylation induced cerebellar degeneration dependent on Aag and Parp1 does not occur via previously established cell death mechanisms

Abstract

Alkylating agents are ubiquitous in our internal and external environments, causing DNA damage that contributes to mutations and cell death that can result in aging, tissue degeneration and cancer. Repair of methylated DNA bases occurs primarily through the base excision repair (BER) pathway, a multi-enzyme pathway initiated by the alkyladenine DNA glycosylase (Aag, also known as Mpg). Previous work demonstrated that mice treated with the alkylating agent methyl methanesulfonate (MMS) undergo cerebellar degeneration in an Aag-dependent manner, whereby increased BER initiation by Aag causes increased tissue damage that is dependent on activation of poly (ADP-ribose) polymerase 1 (Parp1). Here, we dissect the molecular mechanism of cerebellar granule neuron (CGN) sensitivity to MMS using primary ex vivo neuronal cultures. We first established a high-throughput fluorescent imaging method to assess primary neuron sensitivity to treatment with DNA damaging agents. Next, we verified that the alkylation sensitivity of CGNs is an intrinsic phenotype that accurately recapitulates the in vivo dependency of alkylation-induced CGN cell death on Aag and Parp1 activity. Finally, we show that MMS-induced CGN toxicity is independent of all the cellular events that have previously been associated with Parp-mediated toxicity, including mitochondrial depolarization, AIF translocation, calcium fluxes, and NAD+ consumption. We therefore believe that further investigation is needed to adequately describe all varieties of Parp-mediated cell death.

Excision of mutagenic replication-blocking lesions suppresses cancer but promotes cytotoxicity and lethality in nitrosamine-exposed mice

“…The results of these experiments are consistent with previous studies showing reduced toxicity from 3MeA relative to BER intermediates (Allocca et al, 2019; Calvo et al, 2013; Ebrahimkhani et al, 2014; Kisby et al, 2009; Margulies et al, 2017; Meira et al, 2009; Sobol et al, 2003). In fact, knockout of Aag has been shown to protect against alkylation-induced toxicity and degeneration in the retina and cerebellum (Allocca et al, 2019; Calvo et al, 2013; Margulies et al, 2017; Meira et al, 2009), and overexpression of Aag sensitizes cells to alkylation-induced toxicity (Hendricks et al, 2002; Ibeanu et al, 1992). In terms of cancer, Aag −/− mice have been found to be more susceptible to exposure-induced mutations and tumors in the colon (Calvo et al, 2012; Fahrer et al, 2015; Meira et al, 2008; Wirtz et al, 2010).…”

“…PARP-1 stimulates repair of SSBs by polymerizing ADP-ribose from NAD+, so an excess of DNA damage causes NAD+ depletion and a form of programmed necrotic cell death termed parthanatos (Xu et al, 2006; Yu et al, 2002; Zhao et al, 2018). We have previously shown that AagTg mice experience elevated cytotoxicity and tissue degeneration from DNA alkylating treatments in the cerebellum, retina, spleen, thymus, bone marrow, and pancreas (Allocca et al, 2017; Allocca et al, 2019; Calvo et al, 2013; Margulies et al, 2017), and that this is often dependent on PARP-1. Indeed, histopathology of NDMA-treated AagTg livers showed significantly elevated necrosis over that of WT and Aag −/− livers.…”

“…Interestingly, the predicted effects of Aag overexpression are very different. High levels of AAG activity cause an increase in the levels of SSBs (Alhumaydhi et al, 2020; Allocca et al, 2019; Margulies et al, 2017; Parrish et al, 2018) that cannot be bypassed by polymerases. Indeed, SSBs promote replication fork breakdown, leading to one-ended double strand breaks (DSBs) that can both signal for cell death and trigger HR.…”

“…In contrast, mutations and cancer were significantly reduced in AagTg mice relative to WT mice. Prior studies have linked AAG-induced SSBs to cytotoxicity (Allocca et al, 2017; Allocca et al, 2019; Calvo et al, 2013; Margulies et al, 2017), and so cell death may prevent mutant cells from surviving and inducing cancer. While seemingly beneficial, the cost is that there is increased tissue damage and lethality.…”

Inflammation, necrosis, and the kinase RIP3 are key mediators of AAG-dependent alkylation-induced retinal degeneration

“…It is important to stress that AAG-dependent alkylation-induced cell death is cell-type specific (29,64). We observed alkylation-induced cell death only in the PR cells but not in other retinal cell types.…”