The Protective Role of Protein Disulphide Isomerase (PDI) against DNA damage in Amyotrophic Lateral Sclerosis (ALS)

Amyotrophic lateral sclerosis (ALS) is characterized by the degeneration and death of motor neurons in the brain, brainstem, and spinal cord. Previous studies from our group have established that a chaperone usually associated with the endoplasmic reticulum (ER), protein disulphide isomerase (PDI) is protective against dysfunction to proteostasis mechanisms in vitro in ALS. However, it remains unclear if PDI is also protective in vivo, or against other processes in ALS, particularly DNA damage, which is increasingly implicated as a disease mechanism. We have also recently established that Tar DNA binding protein 43 (TDP-43) is involved in DNA repair but ALS-associated mutant TDP-43 lacks this activity, resulting in DNA damage.

In the studies described in this thesis, it was examined whether PDI is protective in vivo and against DNA damage (both in vivo and in vitro). In neuronal cells, PDI was found to be protective against DNA damage induced by etoposide, H₂O₂ or ALS-associated mutant TDP-43. In addition, it was demonstrated that PDI translocates into the nucleus following induction of DNA damage. PDI displays two main activities; it is a general protein chaperone and it also functions as an oxidoreductase. The latter activity is mediated by redox active cysteine residues in its active site. Using a mutant in which all four redox active site cysteines were removed (‘quad’ mutant), it was also demonstrated that the oxidoreductase activity mediates its protective activity against DNA damage. Proteomics analysis using wildtype PDI and the quad mutant identified the Nrf2 pathway as a possible mediator of this protective function. The oxidoreductase activities of PDI and several PDI family members were also examined in vitro using three different methods.

This study also demonstrates a protective activity for PDI in vivo. PDI was also found to be protective in zebrafish against DNA damage induced by H₂O₂ and in two ALS-associated models, expressing either A4V mutant superoxide dismutase 1 (SOD1^A4V) or S621G mutant cyclin F (CCNF^S621G). Moreover, knockdown of PDI in C. elegans was found to shorten the lifespan and reduce the fecundity of these animals, implying that PDI has an important protective activity against ageing and viability in C. elegans. These results therefore demonstrate the protective role of PDI against DNA damage both in vivo and in vitro. They also reveal that PDI has a much broader protective role than previously recognised. This study therefore has implications for future therapeutic studies based on PDI.