Manganese (Mn) is a vital trace element that plays a significant role in several critical physiological processes within the body. It is essential for maintaining redox homeostasis, facilitates the proper metabolism of nutrients, contributes to bone formation, and supports the synthesis of neurotransmitters. Mn is essential for these biological functions because it helps to ensure the proper functioning of enzymatic reactions and cellular processes that are fundamental to overall health and well-being (Aschner et al., 2022). However, excessive Mn exposure, typically occurring through mining work or contaminated drinking water, can lead to neurotoxicity (Aiken and Ying, 2023; Oulhote et al., 2014; Studer et al., 2022; Wang et al., 2010). Chronic exposure results in Mn accumulation in the brain, contributing to neurodegenerative conditions characterized by motor dysfunction and cognitive impairment (Budinger et al., 2021; Ghaisas et al., 2021; Kim et al., 2022; Murumulla et al., 2024), such as Parkinson's and Alzheimer's-like diseases (Chen et al., 2025). Exposure to Mn has also been linked to the development of various neurological disorders, including autism spectrum disorder (ASD) and attention deficit hyperactivity disorder (ADHD) (Aschner et al., 2024; Schullehner et al., 2020). These mechanistically distinct yet clinically convergent outcomes underscore the necessity for systematic exposure surveillance and preventive strategies targeting Mn-associated neurotoxicity.

The neurotoxicity of Mn arises from a multimodal pathogenic cascade involving interdependent molecular perturbations. Central to this process is oxidative damage mediated by ROS overproduction, compounded by endoplasmic reticulum stress-induced proteostatic dysfunction (Lu et al., 2023). Concurrently, Mn disrupts neuroimmune homeostasis by activating microglial-mediated neuroinflammation (Pajarillo et al., 2021), while impairing autophagic flux - particularly in hippocampal circuits critical for memory consolidation (Tinkov et al., 2021; Pajarillo et al., 2022). These cellular insults converge with dopaminergic and glutamatergic transmission abnormalities caused by Mn-induced neurotransmitter dysregulation (Soares et al., 2020). The pathophysiological synergy between these mechanisms (oxidative/ER stress, neuroinflammation, autophagy inhibition, and synaptic dysfunction) creates self-reinforcing toxicity loops that amplify neuronal vulnerability (Zheng et al., 2024; Das et al., 2025). This mechanistic complexity necessitates: 1) systems-level mapping of crosstalk between pathways; 2) identification of nodal regulators mediating inter-pathway interactions; 3) development of combinatorial therapeutic approaches targeting multiple axes of toxicity. Prioritizing these research directions will enable precision interventions against Mn-associated neurodegeneration. Notably, compared to other heavy metals such as lead (Pb) and mercury (Hg), manganese exhibits several unique neurotoxicological features. While Pb and Hg primarily exert neurotoxicity through interference with calcium signaling, disruption of synaptic plasticity, and direct DNA damage, Mn preferentially accumulates in the basal ganglia and hippocampus, where it impairs mitochondrial respiration, disrupts iron homeostasis, and triggers ferroptosis through intracellular iron mobilization (Budinger et al., 2021; Kim et al., 2022; Murumulla et al., 2024). Additionally, Mn's neurotoxicity shows a stronger association with motor dysfunctions and neurodevelopmental disorders such as ASD and ADHD, highlighting its distinctive clinical presentation and mechanistic profile. These distinguishing characteristics underscore the necessity of investigating Mn-induced neurotoxicity as a unique model of environmentally driven neurodegeneration.

Ferroptosis has emerged as a pathogenic nexus in Mn neurotoxicity, mediating neuronal degeneration through iron-driven lipid peroxidation cascades (Aschner et al., 2022; Chen et al., 2024a; Zhang et al., 2023). This cell death modality exhibits mechanistic parallels across neurodegenerative pathologies, with demonstrated involvement in Alzheimer's and Parkinson's disease models (Chu et al., 2023; Martins et al., 2019; Soni et al., 2024; Hong et al., 2025; Schreiner and Schreiner, 2023). Ferroptosis contributes to neuronal loss by promoting oxidative membrane damage, primarily through the iron-dependent accumulation of lipid peroxides. In recent years, ferroptosis has been implicated in a wide range of organ toxicities and environmental stress models beyond the nervous system, including zinc-mediated alleviation of arsenic-induced renal injury in carp (Lu et al., 2024), and microplastic-induced mitochondrial dysfunction in the chicken spleen (Guo et al., 2024). These studies emphasize the ubiquity of ferroptosis in toxicological responses across diverse biological contexts. While manganese has been widely reported to induce ferroptosis in tumor cells via multiple mechanisms—including suppression of GPX4, activation of the cGAS-STING pathway, inhibition of DHODH, and YAP/TAZ-driven lipid remodeling—such findings have been largely confined to malignant contexts (Zheng et al., 2024; Wang et al., 2018, 2024a; Zhang et al., 2022, 2025a; Zhao et al., 2023). However, the specific role of ferroptosis in Mn-induced neurotoxicity, especially through iron dysregulation in neural cells, remains poorly characterized, representing a critical mechanistic gap that this study seeks to address.

Building upon our foundational hypothesis proposing Mn-mediated neural ferroptosis (Aschner et al., 2022), subsequent experimental validation using PC12 cell models revealed HIF-1α/p53 pathway activation as a key regulatory mechanism (Chen et al., 2024a). Critical gaps remain in understanding Mn-induced ferroptotic mechanisms, while current therapies remain constrained by limited targets (e.g., GPX4/SLC7A11) and nonspecific inhibitors disrupting multiple cellular pathways. These dual challenges underscore the imperative to define neural-specific ferroptotic cascades and develop precision interventions - a core focus of our study advancing both mechanistic insight and targeted therapeutic strategies.

Nuclear receptor coactivator 4 (NCOA4) is a selective autophagy receptor that mediates ferritin degradation, a key process for maintaining intracellular iron homeostasis. By targeting ferritin for autophagic degradation, NCOA4 regulates iron release in a controlled manner, ensuring its availability for essential cellular functions. This highlights NCOA4's pivotal role in cellular iron metabolism (Le et al., 2024). By directly binding to ferritin, NCOA4 forms a complex that is recognized by the autophagosome, triggering the autophagic degradation of ferritin. NCOA4-mediated ferritinophagy plays a dual role. It can prevent iron overload and oxidative stress under controlled conditions, thereby protecting cells from damage (Santana-Codina et al., 2021). However, when NCOA4 is overactivated, excess Fe2+ is released from ferritin, boosting hydroxyl radical (·OH) generation through Fenton's reaction (Bailey et al., 2021), which causes oxidative stress and cellular damage, exacerbating neurotoxicity, and contributing to the pathological processes of cardiovascular diseases and other conditions (Wu et al., 2020; Fang et al., 2019). Recent reviews have highlighted the crucial role of NCOA4-mediated iron homeostasis in the toxic effects of heavy metals, suggesting its involvement in metal-induced cellular damage and disease progression (Xu et al., 2025a). Given the established role of iron dysregulation in ferroptosis and its connection to Mn toxicity (Liu et al., 2024; Ye et al., 2017), we hypothesized that dysregulated NCOA4-mediated ferritinophagy is a potential mechanism underlying Mn-induced ferroptosis and neurotoxicity.

To elucidate the mechanisms of Mn-induced ferroptosis, we selected HT22 cells as an in vitro model due to their hippocampal origin, as the hippocampus is a key target in Mn toxicity and neurodegenerative diseases. HT22 cells demonstrate high sensitivity to Mn-induced oxidative stress and ferroptosis, effectively modeling neurotoxic processes (Xuan et al., 2022; Goujon et al., 2024). Additionally, the lack of active ionotropic glutamate and cholinergic receptors in HT22 cells enables focused investigation of non-receptor-mediated pathways, such as oxidative stress and iron dysregulation (He et al., 2013; Zhang et al., 2019). This study was designed to clarify the role of NCOA4-mediated ferritinophagy in the context of Mn-induced ferroptosis and neurotoxicity, utilizing both in vivo and in vitro models. We anticipate that our findings will provide valuable insights into the molecular and cellular processes that drive Mn-related neurotoxicity, potentially guiding therapeutic strategies for future environmental toxin exposures.