As a global public health concern, traumatic brain injury (TBI) is one of the leading causes of morbidity, mortality, and long-term disability worldwide. Annually, approximately 60 million persons worldwide suffer from TBI (Dewan et al., 2018). TBI could be classified into primary and secondary injury based on the timing and nature of the injury. While primary injury is irreversible since it results directly from external forces, secondary injury could be mitigated since it refers to a cascade of tissue damage triggered by biochemical changes such as glutamatergic excitotoxicity, neuroinflammation, mitochondrial dysfunction, and lipid peroxidation, etc. The pathophysiology of TBI is intricate, involving varying degrees of damage to the hippocampus and cortex, and neuronal death represents the primary pathogenic event in this process (Paterno et al., 2017). Therefore, reducing neuronal death represents a pivotal strategy in eliminating the secondary pathological defects of TBI and serves as a potential therapeutic target.

As a recently identified type of programmed cell death, ferroptosis primarily occurs due to mitochondrial changes, characterized by excessive accumulation of iron ions and intracellular lipid peroxides. Increasingly studies have shown that there is a potential association between ferroptosis and central nervous system conditions including TBI (Kenny et al., 2019; Tan et al., 2021). Hence, the modulation of the ferroptosis inhibition pathway may represent a significant approach to ameliorate cognitive dysfunction following TBI. Notably, recent research has highlighted dihydroorotate dehydrogenase (DHODH) as a novel parallel defense mechanism against ferroptosis (Mao et al., 2021a). The molecular localization and anti-ferroptosis effects of DHODH are particularly focused on mitochondria (Mao et al., 2021b; Vasan et al., 2020).

Since the early stages of ferroptosis research, mitochondrial morphological changes have been observed under electron microscopy (Dixon et al., 2012). Subsequent research has further demonstrated that ferroptosis is consistently initiated and amplified by mitochondrial dysfunction. Mitochondria serve as the metabolic hubs and iron pool for ferroptosis, regulating cellular ferroptosis through the release of iron and reactive oxygen species (ROS) via iron and redox metabolism, respectively (Fuhrmann et al., 2020; Liang et al., 2022). Within the past decade, greater attention to the effects of mitochondrial dysfunction on cell death following TBI has been gained (Zhang et al., 2022). Based on these insights, we hypothesized that enhancing mitochondrial function following TBI may prove advantageous in mitigating neuronal death.

As an iron‑sulfur [2Fe-2S] cluster protein, mitoNEET (gene: CISD1) is located on the outer surface of mitochondria, acting as a pH and redox sensor for mitochondria (Wiley et al., 2007). MitoNEET plays a key role in regulating cellular energy use, lipid metabolism, and mitochondrial iron content (Lipper et al., 2019). In mammals, mitoNEET has been reported to be involved in numerous types of CNS diseases or insults such as TBI (Hubbard et al., 2021; Yonutas et al., 2020) and stroke (Vijikumar et al., 2022). Deletion of mitoNEET in mice causes iron accumulation in the striatum and motor deficits in physiological conditions (Geldenhuys et al., 2017). However, the function of mitoNEET in TBI and the underlying mechanisms remain unexplored.

MitoNEET ligand or inhibitor NL-1, which selectively targets mitoNEET without PPARγ activity (Saralkar et al., 2020), has been shown to exert protective effects in cerebral ischemia/reperfusion injury using aged female rats (Vijikumar et al., 2022), and in experimental models of TBI using adult male mice, which was lost in mitoNEET knockout (−/−) mice (Yonutas et al., 2020). However, it is unclear whether the protective effects of NL-1 are related to ferroptosis. Although the molecular mechanism underlying the involvement of mitoNEET and its ligand NL-1 in TBI remains unclear, their significant roles in maintaining mitochondrial function and energy metabolism have been well-established. Previous studies have demonstrated that mitoNEET helps maintain mitochondrial membrane potential and cellular redox metabolism homeostasis by regulating iron content within the mitochondrial matrix. Additionally, it influences cellular lipid metabolism by modulating adiponectin levels and mitochondrial respiratory function (Kusminski et al., 2012). In a mouse stroke model, the administration of NL-1 enhanced the functionality of complex I in the electron transport chain while suppressing the oxidative stress response (Saralkar et al., 2021). Given the strong interconnection between ferroptosis and cellular iron metabolism, lipid metabolism, and energy metabolism, we hypothesize that targeting mitoNEET and NL-1 could serve as effective therapeutic strategies for regulating ferroptosis in the treatment of TBI.

In this study, we utilized C57BL/6 J mice to establish controlled cortical impact (CCI) models and employed mitoNEET's ligand NL-1 to assess the role of mitoNEET post-TBI. Taking advantage of the unique properties of the mitochondrial membrane protein mitoNEET, we were able to significantly modulate mitochondrial function, iron metabolism, and lipid homeostasis after TBI. These findings suggested a dual role of mitoNEET: (1) under physiological conditions, mitoNEET likely functions as a protective regulator against ferroptosis by maintaining cellular redox homeostasis; (2) In the TBI model, however, targeted suppression of mitoNEET attenuated ferroptosis and ameliorated trauma-induced cognitive deficits, possibly through stabilization of DHODH-mediated mitochondrial function. Additionally, our research revealed a potential link between the anti-ferroptotic effect of mitoNEET and DHODH, as evidenced by DHODH knockdown. These findings provided new insights into the role of ferroptosis in TBI and suggested that mitoNEET may be of therapeutic relevance in the prevention of TBI.