Functional consequences of the homozygous mutation p.Gln121X of PRDX5 in primary human dermal fibroblasts

In the hospital La Pitié-Salpêtrière, two children were brought exhibiting symptoms including epilepsy, brain atrophy, deep mental retardation, and abnormal movements, suggesting a neurodegenerative-type disease. The children's genotyping identified one nonsense, PRDX5 p.Gln121X, and two missense homozygous mutations, MARK2 p.Arg445Trp and NPAS4 p.Thr587Met. The mutation that was mainly investigated in previous works in the lab is the homozygous nonsense p.Gln121X affecting PRDX5. These previous studies used heterologous expression systems, genetically engineered cell lines and KO mice. It was discovered that, when experimentally produced, the truncated protein is regulated by the proteasome, therefore explaining its absence. It was also discovered that PRDX5 knockout did not lead to significant behavioral differences or CNS affections in mice. For the work presented here, dermal fibroblasts from the two affected children were obtained and used as model. Results from qRT-PCR experiments suggest a regulation of the mutant transcripts by the Nonsense-Mediated mRNA Decay pathway. A 100-fold downregulation in PRDX5 transcriptional expression was measured between the wild type and the two mutant fibroblasts. Additionally, viability assays identified an increased vulnerability in the mutant cells towards H2O2-induced oxidative stress compared to the control. Moreover, transcriptional expression of MARK2, impacted by the p.Arg445Trp mutation, was assessed and determined to be reduced by a factor 2 in the mutants. However, the effects of the mutation on MARK2 functions were not studied. As far as NPAS4 is concerned, its expression and function could not be studied in the present work since it is not expressed in skin tissue. Finally, transcriptional expression of TRMT112 was measured to be downregulated by a factor 2 in the mutants compared to the WT fibroblasts, probably due to PRDX5 p.Gln121X mutation being located in its regulatory region. Experiments also suggested a reduction of TRMT112 at the protein level. In conclusion, the absence of PRDX5 increases cell vulnerability in the mutants upon hydrogen peroxide exposure. While it is probably not the sole responsible for the whole pathogeny, it is clearly an important factor of the neurodegeneration. MARK2 and NPAS4 functions require investigation, preferably in a more relevant model such as neural stem cells transdifferentiated from the fibroblasts. For reasons discussed in this work, TRMT112 is a good candidate for explaining the pathogeny, and should therefore be further investigated.