Abstracts

Rationale: Ferroptosis is an iron-dependent form of regulated cell death driven by iron accumulation and the excessive production of lipid reactive oxygen species (ROS). Ferroptosis has been recently implicated in various diseases involving neuronal injury, including epilepsy and cognitive neurodegenerative disorders. However, the role of ferroptosis in Tuberous Sclerosis Complex (TSC), a genetic disorder manifesting with early life epilepsy, cognitive dysfunction and autism has not been investigated. Among other factors, intracellular iron content is regulated by the transferrin receptor (TFRC), one of the primary mechanisms for iron uptake, as well as the ferritin heavy and light chain storage proteins (FHC and FLC), essential for limiting the intracellular free iron pool and for protecting cells from iron-induced damage. Lipid peroxidation is controlled by several endogenous antioxidant defense systems including the Solute Carrier Family 7 Member 11 (SLC7A11), a key protein in the glutathione synthesis pathway, and the glutathione peroxidase 4 (GPX4). Since previous studies have demonstrated increased levels of neuronal ROS and lipid peroxidation in TSC, we hypothesized that the ferroptosis cascade might be activated and therefore may contribute to disease pathology._x000D_
Methods: Control (n = 15) and Tsc1^GFAP-/- mice with conditional inactivation of the Tsc1 gene in astrocytes and neurons (Tsc1^flox/flox-GFAP-Cre knockout mice; n = 14) were sacrificed at postnatal day (P) 50, a time when spontaneous seizures are well established in this model. Frozen cortical samples were used to examine the relative levels of ferroptosis-related proteins using western blot. Membranes were probed with the following primary antibodies: beta-actin (1:5000, Sigma-Aldrich A5441), GPX4 (1:1000, Cell Signaling 2455), SLC7A11 (1:1000, Cell Signaling 12691), TFRC (1:500, ThermoFisher Scientific PA-5 27739), FHC (1:1000, Cell Signaling 4393), and FLC (1:1000, Abcam ab69090). Data were normalized to actin and expressed as % of control mean. Statistical significance was established by unpaired t-tests. _x000D_  
Results: We found that the TFRC expression was significantly increased in the TSC mice (p < 0.0001), while both FHC and FLC were significantly downregulated (p < 0.01), suggesting an excessive deposition of non-transferrin bound iron. In addition, GPX4 was significantly downregulated in the TSC mouse cortex (p < 0.001), consistent with diminished cellular antioxidant capacity in these mice. No significant differences in SLC7A11 levels were observed between genotypes (p = 0.302).