Chronic Exposure to Cadmium and Antioxidants Does Not Affect the Dynamics of Expanded CAG•CTG Trinucleotide Repeats in a Mouse Cell Culture System of Unstable DNA.

Mário Gomes-Pereira,Darren G Monckton

doi:10.3389/fncel.2020.606331

Abstract

More than 30 human disorders are caused by the expansion of simple sequence DNA repeats, among which triplet repeats remain the most frequent. Most trinucleotide repeat expansion disorders affect primarily the nervous system, through mechanisms of neurodysfunction and/or neurodegeneration. While trinucleotide repeat tracts are short and stably transmitted in unaffected individuals, disease-associated expansions are highly dynamic in the germline and in somatic cells, with a tendency toward further expansion. Since longer repeats are associated with increasing disease severity and earlier onset of symptoms, intergenerational repeat size gains account for the phenomenon of anticipation. In turn, higher levels of age-dependent somatic expansion have been linked with increased disease severity and earlier age of onset, implicating somatic instability in the onset and progression of disease symptoms. Hence, tackling the root cause of symptoms through the control of repeat dynamics may provide therapeutic modulation of clinical manifestations. DNA repair pathways have been firmly implicated in the molecular mechanism of repeat length mutation. The demonstration that repeat expansion depends on functional DNA mismatch repair (MMR) proteins, points to MMR as a potential therapeutic target. Similarly, a role of DNA base excision repair (BER) in repeat expansion has also been suggested, particularly during the removal of oxidative lesions. Using a well-characterized mouse cell model system of an unstable CAG•CTG trinucleotide repeat, we tested if expanded repeat tracts can be stabilized by small molecules with reported roles in both pathways: cadmium (an inhibitor of MMR activity) and a variety of antioxidants (capable of neutralizing oxidative species). We found that chronic exposure to sublethal doses of cadmium and antioxidants did not result in significant reduction of the rate of trinucleotide repeat expansion. Surprisingly, manganese yielded a significant stabilization of the triplet repeat tract. We conclude that treatment with cadmium and antioxidants, at doses that do not interfere with cell survival and cell culture dynamics, is not sufficient to modify trinucleotide repeat dynamics in cell culture.

Highlights

The expansion of simple sequence DNA repeats has been implicated in human disease (Hannan, 2018)
To further elucidate the correlation between mismatch repair (MMR) protein levels and repeat dynamics we derived single cell clones from the progenitor D2763Kc2 culture (Gomes-Pereira et al, 2001; Gomes-Pereira and Monckton, 2004) by limiting dilution, and identified four subclones that exhibited different degrees of somatic mosaicism, in spite of the similar proliferative capacity, as revealed by their population doubling time (PDT): while the CAGCTG repeat tract was relatively stable in clones 1 and 2, it exhibited more pronounced repeat size variability in clones 3 and 4 (Figure 1A, Table 1)
Our main aim was to determine if the exposure to chemicals reported to reduce MMR activity and oxidative stress could modify the dynamics of expanded trinucleotide repeats

Summary

Introduction

The expansion of simple sequence DNA repeats has been implicated in human disease (Hannan, 2018). The majority of trinucleotide repeat diseases are caused by the expansion of CAGCTG repeats, which can either encode a toxic polyglutamine tract in various proteins (Stoyas and La Spada, 2018), or be transcribed into a non-coding CUG RNA that interferes with the activity of key RNA-binding proteins and RNA processing (Sicot and Gomes-Pereira, 2013; Swinnen et al, 2020). All these conditions exhibit some element of neurological dysfunction, demonstrating the vulnerability of the central nervous system to triplet repeat expansion mutations (Orr and Zoghbi, 2007)

Methods

Results

Conclusion