The deubiquitinase OTUD3 plays a neuroprotective role by reducing ferroptosis induced by cerebral ischaemia reperfusion via stabilizing PLK1 via deubiquitination

Ischaemic stroke is one of the most common serious diseases observed in elderly people, which is caused by ischaemia-reperfusion (I/R) injury. Ovarian tumour domain-containing protein 3 (OTUD3) is a member of the ovarian tumour proteases (OTUs) family of deubiquitination enzymes located in the cytoplasm. We previously showed that the expression of OTUD3 in neurons was significantly reduced after cerebral I/R in mice. In addition, OTUD3 knockdown aggravated ferroptosis and brain damage following I/R in mice, and overexpression of OTUD3 reduced the mortality of cortical neurons in an oxygen glucose deprivation model (OGD/R). Co-immunoprecipitation-mass spectrometry analysis revealed that OTUD3 could bind to the amino acid sequence 35-305 of PLK1. Single-cell sequencing results suggested that PLK1 expression was significantly reduced in mouse neurons after I/R injury. Similarly, reduced PLK1 expression was found in the cortical brain tissues of I/R mice and in the OGD/R-stimulated primary cortical neurons of mice. In vitro experiments showed that OTUD3 overexpression led to the upregulation of PLK1 expression, and inhibition of PLK1 suppressed the inhibitory effect of OTUD3 overexpression on ferroptosis. Moreover, PLK1 positively regulated the PI3K/AKT signalling pathway in neurons after I/R injury, and inhibition of PI3K activity suppressed the inhibitory effect of PLK1 on ferroptosis. Ubiquitination experiments showed that OTUD3 modified PLK1 through deubiquitinating K48-linked ubiquitination, thereby reducing its degradation by ubiquitination and stabilizing PLK1 expression. These results indicated that OTUD3 could upregulate PLK1 through deubiquitination modification, thereby activating the PI3K/AKT signalling pathway and reducing ferroptosis after cerebral I/R. Animal behavioural experiments and cellular methyl thiazolyl tetrazolium and lactate dehydrogenase experiments revealed that inhibition of PLK1 exacerbated brain damage after I/R in mice. Inhibition of OTUD3 deubiquitination enzyme activity attenuated the neuroprotective effect of OTUD3. In conclusion, our findings provide evidence that OTUD3 reduces ferroptosis by upregulating PLK1 expression through deubiquitination modification and exerts neuroprotective effects in cerebral I/R injury. KEY POINTS: For the first time, it has been clarified that OTUD3 exerts neuroprotective effects in cerebral ischemia/reperfusion injury by deubiquitinating PLK1 to regulate the PI3K/AKT pathway and inhibit ferroptosis. The study first demonstrates that OTUD3 binds to the amino acid residues 35305 of PLK1 and deubiquitinates PLK1 (targeting K48-linked ubiquitination), thereby reducing its degradation and stabilizing PLK1 protein expression.