Mechanistic Investigations into Hydropersulfide (RSSH) Donor-Mediated Rescue of Deficient Iron-Sulfur Cluster Biosynthesis in Friedreich's Ataxia Fibroblasts

Iron-sulfur (Fe-S) clusters are essential protein cofactors for a wide range of cellular processes, including respiration, DNA repair, and metabolism. Deficiencies in Fe-S cluster biosynthesis underlie numerous human diseases, such as Friedreich's ataxia (FA), and contribute to mitochondrial dysfunction and cellular stress. Central to Fe-S cluster assembly is the transfer of a sulfane sulfur, typically in the form of a persulfide, from cysteine desulfurase enzymes (e.g., NFS1) to scaffold proteins (e.g., ISCU2), where cluster formation occurs. Recent structural and biochemical studies have elucidated the mechanism of persulfide transfer and highlighted the critical role of persulfide intermediates in cluster assembly and maturation. However, disruptions in this transfer pathway can impair Fe-S cluster formation, leading to cellular dysfunction. FA is a debilitating neurodegenerative disorder caused by a deficiency in frataxin (FXN), a protein that catalyzes a critical sulfane sulfur transfer for Fe-S cluster assembly. This FXN deficiency leads to impaired Fe-S cluster biosynthesis, mitochondrial dysfunction, and heightened oxidative stress. This proposal will investigate the protective and restorative effects of hydropersulfide (RSSH) donors in FA patient-derived skin fibroblasts and compare those effects in control cells. Aim 1 will investigate how RSSH donors rescue defective Fe-S cluster biosynthesis in FXN-deficient cells. We will analyze whether RSSH donors restore Fe-S cluster formation in FA patient-derived skin fibroblasts (which are FXN-deficient), examining their impact on FXN levels, mitochondrial aconitase activity, ISCU2 persulfidation, and ferredoxin levels, all critical to Fe-S cluster assembly. Aim 2 will elucidate the role of RSSH donors in improving mitochondrial biogenesis and function. Persulfidation has been linked to enhanced mitochondrial bioenergetics and biogenesis, with mitochondrial persulfides supporting electron transport chain activity and membrane potential. We will evaluate mitochondrial biogenesis and function focusing on PGC-1a and SIRT3 signaling, mitochondrial count, membrane potential, and respiratory function/ATP production following RSSH donor treatment. Aim 3 will assess whether RSSH donors restore intracellular RSSH levels and prevent iron-mediated oxidative damage/ferroptosis in FA cells. Low RSSH levels in FA cells leave them susceptible to oxidative damage and iron overload in FA contributes to this oxidative damage and triggers ferroptotic cell death, exacerbated by dysregulated ferritinophagy. We will assess whether RSSH donors mitigate these processes by restoring redox balance and iron homeostasis, thereby ultimately reducing cellular vulnerability to ferroptosis. Collectively, these studies will provide mechanistic insights into how RSSH donors combat defective Fe-S cluster biosynthesis and alleviate mitochondrial dysfunction, oxidative damage, and iron toxicity. Project Number: 1R01GM164444-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: John Toscano | Institution: JOHNS HOPKINS UNIVERSITY, BALTIMORE, MD | Award Amount: $325,665 | Activity Code: R01 | Study Section: Macromolecular Structure and Function A Study Section[MSFA] View on NIH RePORTER: https://reporter.nih.gov/project-details/11342528