Dimethyl malonate preserves brain and neurobehavioral phenotype following neonatal hypoxia-ischemia by inhibiting FTH1-mediated ferritinophagy.

BACKGROUND: Hypoxic-ischemic brain damage (HIBD) is a predominant cause of neuronal injury and mortality in newborns. Current preventive and therapeutic interventions demonstrate limited clinical efficacy. Emerging evidence reveals ferroptosis as a critical mechanism within HIBD pathophysiology, positioning it as a promising therapeutic target. Dimethyl malonate (DMM), a competitive inhibitor of succinate dehydrogenase, has demonstrated neuroprotective properties across multiple models of neurological disorders. However, the impact of DMM on the neonatal HIBD has not been studied. AIM: To investigate the neuroprotective effects of DMM against neonatal HIBD and elucidate its mechanisms of action. METHODS: We created a model of HIBD in neonatal male C57BL/6J mice and administered various doses of DMM or vehicle control. Quantitative assessments included cerebral infarct volume measurement, Nissl staining for neurons, neurological behavior, ferrous ion (Fe(2+)), malondialdehyde (MDA) level, 4-hydroxynonenal (4-HNE) expression, and solute carrier family 7 member 11 (SLC7A11, system Xc(-))/glutathione peroxidase 4 (GPX4) antioxidant axis expression level. Parallel studies in vitro employed oxygen-glucose deprivation/reperfusion-treated HT22 cells to investigate the effects of DMM on ferroptosis and its underlying mechanisms. Moreover, key factors of ferritinophagy, including nuclear receptor coactivator 4 (NCOA4), SQSTM1/p62, ferritin heavy chain 1 (FTH1), and microtubule-associated protein light 3 II (LC3II) were analyzed by western blotting. Molecular interactions between NCOA4 and FTH1 in brain cortical tissues of DMM-treated HIBD mice were analyzed by coimmunoprecipitation (Co-IP). Ferroptosis regulation by DMM was further investigated via Fth1 knockdown in cellular models. Immunofluorescence staining was used to evaluate the capacity of DMM to suppress ferritin degradation and lysosomal Fe(2+) accumulation at the organelle level. RESULTS: DMM treatment demonstrated its neuroprotective efficacy in HIBD models, as evidenced by a reduction in cerebral infarct volume, an increase in the number of Nissl-positive neurons, and improved cognitive and motor functions in neonatal mice compared with controls. Additionally, the DMM intervention significantly modulated ferroptosis-related biomarkers in brain cortical tissues and HT22 cells, decreasing ferrous ion (Fe(2+)) accumulation, reducing lipid peroxidation products (MDA and 4-HNE), and enhancing SLC7A11/GPX4 antioxidant system activity. Importantly, DMM specifically regulated core ferritinophagy components: suppressing NCOA4 and LC3II expression while upregulating FTH1 and p62 levels. Co-IP revealed that mechanistically, DMM disrupted the protein interaction between NCOA4 and FTH1, effectively inhibiting ferritinophagy progression. The effects of antiferroptosis were FTH1-dependent, as demonstrated by reversal of the DMM protective effect following Fth1 knockdown in vitro. Immunofluorescence analysis showed that DMM decreased the colocalization of FTH1-lysosome-associated membrane protein 2, and FerroOrange/LysoTracker Green dual staining confirmed its inhibition of lysosomal iron accumulation, collectively indicating regulation of DMM at the organelle level. CONCLUSION: DMM suppressed ferroptosis-induced neuronal death by specifically targeting FTH1 and disrupting the NCOA4-FTH1 interaction, thereby mitigating HIBD. These findings position DMM as a promising therapeutic candidate for the clinical management of neonatal hypoxic-ischemic encephalopathy.