Deficiency of TEX264-mediated reticulophagy is linked to ER stress-induced neuronal necroptosis: Implication of fluoride-associated hippocampal injury and cognitive dysfunction.

Introduction

Fluoride is ubiquitously distributed in the natural environment of the Earth and can be absorbed by the human body via multiple sources, including drinking water, air, dietary supplements, and industrial pollution. Moderate fluoride intake is beneficial for preventing dental caries and strengthening bones, however, excessive ingestion poses risks to various organs, contributing to skeletal fluorosis (Srivastava and Flora, 2020), kidney and liver damage (Malin et al., 2019), mental retardation, as well as endocrine and reproductive disorders (Elghareeb et al., 2024).

Notably, fluoride is capable of entering and bioaccumulating in the brain by crossing the blood-brain barrier (Zwierello et al., 2023), leading to neuronal damage and functional deficits. Epidemiological evidence have demonstrated a negative association between fluoride exposure and cognitive outcomes in both children and the elderly (Li et al., 2016). Concurrently, the rodent studies have verified that high fluoride ingestion induces neuronal cell death and neurobehavioral abnormalities. Previous research has indicated that fluoride-induced neurotoxicity is associated with neuronal autophagy, neurotransmitter dysfunction, reduction-oxidation (redox) imbalance (Wu et al., 2015), activation of neuroinflammation (Reddy et al., 2021), and apoptosis (Yildiz et al., 2022). Unfortunately, the specific systematic mechanisms underlying high fluoride exposure-induced brain damage remain unclear.

Necroptosis, a new type of necrotic programmed cell death, is mainly caused by the mixed lineage kinase domain-like (MLKL) pathway, receptor-interacting protein kinase 1 (RIPK1), and receptor-interacting protein kinase 3 (RIPK3) (Shi et al., 2023). In response to stress stimuli, autophosphorylated RIPK1 interacts with RIPK3 through their RIP-homology-interacting motifs (RHIM), initiating the phosphorylation and oligomerization of MLKL and ultimately leading to necroptotic cell death (Chen et al., 2017). Fluoride exposure has been indicated to effectively facilitate the induction of necroptosis. Specifically, necroptotic cell death, characterized by the organelles swelling, membrane rupture, and the disintegration of the cytoplasm and nucleus, was observed in sodium fluoride (NaF)-exposed renal tubular epithelial cells (Wang et al., 2023). Long-term exposure to NaF promoted the transcription of RIPK1/RIPK3/MLKL in the hearts of chickens (Hou et al., 2024). Additionally, rat kidney epithelial cells exposed to fluoride for 24 h showed a substantial uptick in RIPK1 and RIPK3 protein expression, suggesting the induction of necroptosis (Urut et al., 2021). However, whether chronic exposure to fluoride triggers the necroptosis in mice hippocampi and the role of this process in fluoride neurotoxicity remains elusive.

As a complex and dynamic membrane-structured organelle, the endoplasmic reticulum (ER) regulates essential cellular processes, including protein folding, calcium homeostasis, and lipid synthesis. Upon internal and external stimuli, such as nutrient deprivation, hypoxia, and environmental toxicant exposure, disruption of ER homeostasis leads to the formation of unfolded or misfolded proteins in the ER lumen, which in turn causes ER stress. Prior research has illustrated the contribution of ER stress to fluoride cytotoxicity (Liu et al., 2016). Specifically, upregulation of ER stress was observed in the hippocampus of fluoride-exposed mice, and the manipulation of hippocampal ER stress exerted powerful neuroprotection against fluoride neurotoxicity (Niu et al., 2018). Interestingly, recent research has highlighted the role of ER stress in regulating necroptosis (Chen et al., 2022). However, the regulatory effect of ER stress on hippocampal neuronal necroptosis under fluoride stress and its underlying mechanisms remains unclear.

The ER undergoes constant remodeling via a selective autophagic pathway known as ER-phagy (reticulophagy). This pathway acts as a critical compensatory mechanism for maintaining ER homeostasis, minimizing ER stress, and ensuring the continuous function of the ER (Ferro-Novick et al., 2021). During ER-phagy, ER-resident cargo receptors directly mediate the tethering of ER membranes to LC3/GABARAP proteins on phagophore membranes, thereby facilitating the engulfment of ER components and their delivery to lysosomes for degradation (Reggiori and Molinari, 2022). Multiple ER-phagy cargo receptors have been identified in mammals, including FAM134B, TEX264, RTN3L, SEC62, and CCPG1. Dysfunction of ER-phagy mediated by these receptors has been linked to numerous neurological and neurodegenerative disorders (Deng et al., 2017). However, the precise role of ER-phagy in fluoride-induced ER stress and the underlying mechanisms by which it contributes to this pathological process remain poorly understood. Therefore, the present study was designed to investigate the role of necroptosis in fluoride-induced hippocampal neuronal damage and associated cognitive impairment and delineate the interplay between ER-phagy, ER stress, and necroptosis in this pathological cascade.