Traumatic brain injury (TBI) remains a leading cause of morbidity and mortality in children (Cheng et al., 2020). Infants and toddlers (0–4 years of age) are at high risk of poor developmental outcomes (Keenan et al., 2019), and decreased quality of life (Wade et al., 2020), even after mild TBI (Gagner et al., 2018). The primary injury is inevitable, however, the secondary injuries, such as endoplasmic reticulum (ER) stress, oxidative stress, and neuroinflammation, are amenable to therapeutic interventions (Aghili-Mehrizi et al., 2022; Zhang et al., 2019; Zhang et al., 2015). Given the complex cytoarchitecture of injured brain regions, multiple cell types are affected by TBI (Arneson et al., 2018; Chen et al., 2019; Fan et al., 2023; Todd et al., 2021). However, cell type-specific and sex-specific activation of ER stress and neuroinflammatory pathways remain poorly understood. Accordingly, there is an urgent need to identify cell type specific responses to secondary injuries to address the unmet needs for developing targeted treatments after brain injuries.

ER stress has been linked to impairments in neuronal function and cognition (Hetz and Saxena, 2017). Upon ER stress, key quality-control pathways, including unfolded protein response (UPR) and ER-associated degradation (ERAD), are activated to restore ER homeostasis (Hwang and Qi, 2018; Meusser et al., 2005). UPR consists of three major signaling cascades: inositol requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6), and double-stranded RNA-activated protein kinase (PKR)-like endoplasmic reticulum kinase (PERK) (Hetz and Saxena, 2017). IRE1 has RNase activity, and functions as the most conserved sensor for misfolded and unfolded proteins in the ER lumen (Gardner et al., 2013). IRE1 activation induces the unconventional splicing of transcription factor Xbox binding protein-1 (XBP1) to XBP1s, which translocates to the nucleus and increases the transcription of genes that are involved in protein folding, ER biogenesis, lipogenesis and ERAD to restore ER homeostasis (Hwang and Qi, 2018; Metcalf et al., 2020). Activation of PERK leads to transient shutdown of protein synthesis and increased translation of specific mRNAs (Smith et al., 2020; Tabas and Ron, 2011; Wolzak et al., 2022). Upon activation, ATF6 translocates to the nucleus and binds promoter sequences, leading to increased gene expression of ER protein chaperones and UPR regulators to increase the folding capacity of ER (Metcalf et al., 2020). ERAD maintains the integrity of the ER proteome by recognizing, extracting, and ubiquitinating unfolded or unwanted ER proteins for cytosolic proteasomal degradation (Hegde and Ploegh, 2010). ERAD components are organized into distinct functional networks (Christianson et al., 2011). Hydroxymethylglutaryl-coenzyme A reductase degradation protein 1 (HRD1) and its adaptor protein sel-1 homolog 1 (SEL1L) are the most conserved ERAD machinery (Christianson and Ye, 2014; Olzmann et al., 2013; Qi et al., 2017), and are indispensable for ER homeostasis (Iida et al., 2011; Sha et al., 2014; Sun et al., 2014). Although UPR and ERAD play a beneficial role in restoring ER homeostasis, chronic and irreversible activation of UPR and ERAD can lead to cell death via IRE1-TRAF2 (TNFα receptor–associated factor 2)-ASK1 (apoptosis signal–regulating kinase 1) and PERK- CHOP (C/EBP-homologous protein) pathways (Metcalf et al., 2020).

Studies indicate that ER stress is transmissible among cells (e.g. neurons, microglia, and astrocytes) in the central nervous system (CNS) (Sprenkle et al., 2019), and plays important roles in programmed cell death, such as apoptosis, necroptosis, and pyroptosis (Chen et al., 2020; Huang et al., 2020; Yuan et al., 2019). For example, ER-stressed astrocytes can polarize microglia to an inflammatory phenotype that synergizes with ER stress in astrocytes (Meares et al., 2014). More importantly, different cell types exhibit different responses to ER stress. For example, astrocytes are largely resistant to aberrant ER stress-induced cell death (Sims et al., 2022), however, ER stress can induce necroptosis of microglia/macrophages after spinal cord injury (Fan et al., 2015). Paradoxically, neurons tolerate PERK dysfunction better than astrocytes, suggesting the existence of cell type-specific resilience (Wolzak et al., 2022).

Microglia, the resident immune cells of the CNS, play an important role in brain development and homeostasis (Li and Barres, 2018), and modulate inflammatory responses after TBI (Loane and Kumar, 2016; Shao et al., 2022; Witcher et al., 2021). Evidence indicates that there is reciprocal communication between neurons and microglia (Szepesi et al., 2018), and neuronal ER stress has been linked to microglia activation in TBI (Harvey et al., 2015). We have previously demonstrated that TBI induces ER stress pathway activation, abnormal protein accumulation and cell loss (Faulkner et al., 2023). However, the cell-specific responses to ER stress in neurons and microglia after TBI remain poorly understood. In addition, growing evidence emphasizes sex-dependent transcriptional and functional differences in microglia (Guneykaya et al., 2018; Villa et al., 2018; Villapol et al., 2017) and neurons (Mecklenburg et al., 2020; Williams et al., 2021), as well as sex-specific neuroinflammatory response after TBI (Faulkner et al., 2023; Hamood et al., 2022; Neale et al., 2023). Therefore, we hypothesized that TBI could induce sex-dependent differential regulation of ER stress and neuroinflammatory pathways in neurons and microglia. In the present study, we evaluated the activation of UPR and ERAD pathways, abnormal protein accumulation, and neuroinflammation in neurons and microglia at the acute phase after TBI in both male and female mice.