Study On The Mechanism Of Neuron Loss Of Friedreich Ataxia

Background:Friedreich ataxia (FRDA) is an inherited neurodegenerative disease caused by the mutations of FXN gene that encodes frataxin. Neuropathology and clinical features are progressive spinocerebellar and sensory ataxia. FRDA is also characterized hypertrophic cardiomyopathy and diabetes. Mitochondria frataxin is an iron chaperone for the assembly of iron-sulfur clusters, so its deficiency induce dysregulation of the cellular iron metabolism, the mitochondrial dysregulation, and/or oxidative stress. Which results in the neuron death of FRDA patients. Iron accumulation in mitochondria and oxidative stress are the two hallmarks of cellular frataxin deficiency. As O2 and Fe2+ are cosubstrates of PHDs (prolyl Hydroxylases) that target HIFα for proteasomal degradation. The hypoxia response mediated by Hypoxia inducible factor (HIF) is closely associated with iron metabolism. The change induced by frataxin deficiency is similar to the situation while ferroptosis occurs. Ferroptosis is characterized by the overwhelming, iron-dependent accumulation of lethal lipid ROS, distinct from other forms of apoptotic and non-apoptotic death. So we postulate neuron loss induced by frataxin deficiency might be a process of ferroptosis. α-LA is an antioxidants and antioxidant protectant. We evaluate the protective effect of α-LA can from ferroptosis.Objectives:(1) Determine if ferroptosis is the mechanism of neuron loss of FRDA patients; (2) Test if α-LA may provide the protective effect against erastin-induced ferroptosis; (3) Study on the interplay of FXN deficiency and hypoxia response.Methods:(1) Aconitase activity assays to judge the efficiency of iron-sulfur cluster biogenesis. (2) Western blotting to test the expression of IRP1, IRP2, Ft, HIF1α in FRDA patient cells and normal cells when treated with DFO, DFP. (3) Perl DAB stain to determine the iron levels of the cells derived from healthy control and FRDA patient when treated with FAC. (4) Optimize the concentration of erastin by visualizing the cell morphology for induction of ferroptosis. (5) CCK-8 test the cell viability. Test effect of erastin and a-LA on the SH-SY5Y cell viability by visualizing the cell morphology. (6) QPCR to detemine the expression of HIF-target genes.Results:(1) Decrease of the aconitase activity and ferritin expression and increase of IRP1 and IRP2 protein levels in the FRDA patient cells were confirmed. (2) Erastin induced ferroptosis in both the normal human cells and FRDA patient cells. Unexpectedly the normal human cells were more sensitive than the FRDA patient cells to erastin. Fer-1 and DFO protected the normal human cells from the erastin-induced ferroptosis, while FAC enhanced the cytotoxicity of erastin. Fer-1 protected the FRDA patient cells from the erastin-induced ferroptosis. However, both DFO and FAC worsened the cytotoxicity of erastin. (3) When treated the cells with 200 μM of FAC, Perl DAB stain revealed that much more iron was accumulated in the FRDA patient cells then in healthy control cells. (4) α-LA protected the SH-SY5Y cells from the cytotoxicity induced by addition of glutamate or erastin. (5) Frataxin deficiency increased the expression of HIF1α and the expression of HIF itself and HIF-targeted genes were not response to hypoxia anymore.Conclusions:Ferroptosis might not be the mechanism of neuron loss of FRDA patient; α-LA may protect the cells from erastin-induced ferroptosis; Frataxin deficiency makes cells chronically response to stress and lost the ability to response to hypoxia.