Functional consequences of PRDX5 inactivation and redox compensatory mechanism in neuronal SH-SY5Y cells

Molecular oxygen is universally known to be essential for survival of aerobic organisms. However, aerobic metabolism can lead to the release of chemically reactive molecules, known as reactive oxygen species (ROS) and also reactive nitrogen species (RNS). In low/moderate level, these permit to address particular targets for redox signaling, essential for maintaining normal cell physiology. However, excessive ROS/RNS levels can cause damage to DNA, proteins, and lipids, leading to negative effects on cellular homeostasis and, ultimately, cell death. Consequently, ROS/RNS may be important factors in several pathological conditions and diseases. To block or control their harmful effect, aerobic organisms have developed a large body of antioxidant defenses, comprising powerful non-enzymatic and enzymatic antioxidants such as peroxiredoxins (PRDXs). Highly conserved throughout evolution, PRDXs represent a superfamily of thiol-dependent peroxidases playing among the other antioxidant systems, key roles in the regulation of ROS/RNS levels and redox homeostasis maintenance. In this master thesis, a particular interest has been given to PRDX5, the most divergent of the six mammalian PRDX isoforms. PRDX5 exhibits a wide subcellular localization and high expression in the central nervous system (CNS). Researchers from La Pitié-Salpêtrière and Cochin hospitals in Paris discovered a homozygous nonsense mutation in PRDX5 gene in three young sisters. These children suffer from cerebral atrophy, severe mental retardation, epilepsy and motor abnormalities, suggesting that PRDX5 could exhibit important function in the human CNS although mutations in other genes expressed in the CNS were identified. Preliminary studies conducted in our laboratory showed that dopaminergic neuronal SH-SY5Y cells exhibit a reduced vulnerability towards H2O2-induced oxidative stress following CRISPR-Cas9-mediated PRDX5 KO, suggesting the existence of a redox-based mechanism that compensates for PRDX5 deficiency. The objective of this work was therefore to study the functional consequences according to whether it is a permanent or transient PRDX5 inactivation, which would highlight the potential existence of a compensatory mechanism. To do so, several functional assays were performed in SH-SY5Y cell line, a cell model widely used in experimental neurological studies. Firstly, our data indicate that PRDX5 knockdown significantly sensitizes SH-SY5Y cells to high exposure to H2O2, supporting PRDX5 as a viable target for neuroprotection as reported in previous studies. Secondly, FACS analysis showed that PRDX5 inactivation would have anti-apoptotic effects on SH-SY5Y cells when exposed to 200 μM H2O2, suggesting the involvement of a compensatory response system that protect cells against oxidative damage and cell death. In addition, immunoblot analyses revealed the weak upregulation of SOD1 protein, which could involve the latter in H2O2-based signaling pathways. Thirdly, our data showed also that PRDX5 KO significantly reduced cell growth in both neuronal SH-SY5Y and astroglia 1321N1 cell lines, suggesting PRDX5 exhibits antiproliferative function including in the CNS. In conclusion, our results suggest that PRDX5 loss would have important impact on neuronal SH-SY5Y cells, leading to the development of a compensatory mechanism to adapt to a more oxidizing environment. Hence, the present work could also serve as a basis for further studies.