CDK1 regulates NRF2 stability through phosphorylation of USP29 and inhibits neuronal ferroptosis after traumatic brain injury.
TL;DR
CDK1 overexpression in TBI rats improved neurological function, ameliorated histopathological damage to the brain, restored neuronal numbers, and concomitantly inhibited cortical ferroptosis through the modulation of the same suite of biochemical markers.
Abstract
Background
AND
Purpose
Neuronal ferroptosis is a critical driver of traumatic brain injury (TBI). While ubiquitin-specific protease 29 (USP29) is involved in neurological injury, its specific function in TBI and regulation of ferroptosis remain unknown. This study was designed to define the role of USP29 in TBI-induced ferroptosis and elucidate its molecular mechanism.
Methods
TBI models were established using the controlled cortical impact (CCI) method in rats and oxygenglucose deprivation (OGD) treatment in human SH-SY5Y cells. The expression levels of key genes and proteins were analyzed by RTqPCR, Western blotting, and immunofluorescence. Ferroptosis was assessed by measuring mitochondrial membrane potential with JC-1 staining, lipid peroxidation levels with BODIPY 581/591 staining, and Fe²⁺ concentration with commercial assay kits. Furthermore, neurological deficits and histopathological changes were evaluated through modified neurological severity score (mNSS), hematoxylin and eosin (H&E) staining, and Nissl staining.
Results
USP29 expression was downregulated in SH-SY5Y cells subjected to OGD. USP29 overexpression attenuated OGD/erastin-induced ferroptosis, as evidenced by reduced cell death; decreased levels of Fe²⁺, MDA, and lipid peroxidation; increased SOD activity; and restored mitochondrial membrane potential. Mechanistically, USP29 stabilizes the transcription factor NRF2 via deubiquitination. Furthermore, CDK1 interacts with and phosphorylates USP29. The overexpression of CDK1 also suppressed OGD-induced ferroptosis in SH-SY5Y cells, which was largely abolished by the knockdown of either USP29 or NRF2. Consistent with the in vitro findings, CDK1 overexpression in TBI rats improved neurological function (lower mNSS scores), ameliorated histopathological damage to the brain, restored neuronal numbers, and concomitantly inhibited cortical ferroptosis through the modulation of the same suite of biochemical markers.
Conclusion
CDK1 phosphorylates USP29 to increase its deubiquitinating activity toward NRF2, thereby stabilizing NRF2 and inhibiting neuronal ferroptosis after TBI.