Generation and phenotypic characterization of a PRDX5-/- and PRDX5 Q121X human astrocyte cell line

The homozygous and nonsense mutation Q121X has been identified in three young sisters in the gene coding for peroxiredoxin-5 (PRDX5). The children suffer from severe motor and intellectual disabilities, suggesting an important role of antioxidant protein PRDX5 in the central nervous system (CNS). This mutation is localized in the third exon of PRDX5 gene generating a stop codon and therefore coding for a truncated protein. Previous studies have shown that there was no detection of truncated protein in sisters’ fibroblasts in Western blot screenings. As PRDX5 gene is expressed in several CNS cell types, our research group investigated how the nonsense mutation Q121X could affect oligodendrocytes, neurons and astrocytes in vitro. Thus, the main goals of this project were to study the functional consequences of PRDX5 gene inactivation and the functional consequences of Q121X nonsense mutation in the1321N1 human astrocyte cell line. Different strategies with the use of CRISPR-Cas9 were considered to introduce Q121X mutation into PRDX5 gene or to produce PRDX5 knockout (KO) 1321N1 cell lines. Both strategies for Q121X only differed by the pair of guides used and involved insertion of a single strand DNA template containing the desired sequence. Regarding KO generation, a template carrying three stop codons was used to be integrated into the first exon of PRDX5 gene. Although the DNA template could not be inserted into the latter, the production of PRDX5 KOs was successful. Q121X mutation could not be obtained in the 1321N1 cell line. However, several clones comprising a premature stop codon were selected. One of these mutants was chosen to analyze PRDX5 mRNA degradation induced by the nonsense mediated mRNA decay (NMD) pathway, since one of the hypotheses for the absence of truncated PRDX5 in the sister’s fibroblasts was that PRDX5 mRNA degradation would occur. Other hypotheses predicted protein insolubility and protein degradation by the proteasome. NMD mechanism analysis consisted in exposing wild-type (WT) and mutant cells to NMDI-14 as an inhibitor of NMD. Cells were harvested for mRNA extraction and RT-qPCR analysis. Our results show that PRDX5 mRNA is not degraded for the selected mutant. Furthermore, astrocyte vulnerability in response to different oxidative stresses was analyzed for PRDX5 KO clones. Different concentrations of several oxidative compounds, such as H2O2, TBHP, AAPH and rotenone were administered to the three obtained KOs and PRDX5 WT 1321N1 cells. A resazurin assay was performed to test cell viability. Results showed a significant difference between PRDX5 KO and PRDX5 WT cells when exposed to AAPH and rotenone, while no difference could be detected with H2O2 and TBHP. Unexpectedly, PRDX5 KO cells were indeed more resistant to oxidative stress induced by AAPH and rotenone than the WT cells. We hypothesized that PRDX5 KO cells have developed compensatory mechanisms for the loss of antioxidant PRDX5 protein, making cells more tolerant or more resistant to oxidative stresses.