Examining big tau's role in DNA protection with CRISPR-Cas9 induced tau knockouts
Tubulin associated unit (tau) is a protein that is hyperphosphorylated and misfolded into neurofibrillary tangles (NFTs) in many neurodegenerative diseases, most notably Alzheimer’s Disease (AD). NFT formation prevents tau from entering the nucleus and binding to DNA. When bound to DNA, tau has been shown to help protect DNA from double-strand breaks (DSBs) caused by oxidative stress. The neurodegeneration that occurs in AD pathology is likely partially caused by an increased number of DSBs found in diseased cells. In comparison to neurons in the central nervous system (CNS), neurons in the peripheral nervous system (PNS) are more exposed to toxic chemicals and oxidative stress due to their lack of protection by the blood brain barrier. PNS neurons express a high molecular weight isoform of tau called “big tau” that is rarely expressed in the CNS. Big tau is defined by the inclusion of an extra 753 bp exon in its proline rich region, a region that has been suggested to contribute to DNA binding. We hypothesize that big tau may be more effective than normal CNS tau isoforms in protecting DNA from DSBs. To test this hypothesis, SK-N-MC (neuroepithelioma) cell lines with and without tau and big tau expression were established with a CRISPR-Cas9 plasmid vector system. Neutral comet assays were used to quantitate DNA DSBs between WT and tau knockout cell lines. In DNA-damaging conditions, cells with all tau isoforms knocked out contained significantly higher numbers of DNA DSBs than wild type cells. Interestingly, cell lines with only a partial knockout of big tau were seen to have more or about the same levels of DSBs as total tau knockdown cells. Our results support our hypothesis that big tau plays an important role in DNA protection. Future studies might investigate big tau’s affinity for DNA, and whether the extra inserts in big tau allow it to exclusively bind to factors that the other tau isoforms cannot.