ASK1-K716R reduces neuroinflammation and white matter injury via preserving blood–brain barrier integrity after traumatic brain injury

Abstract

Background Traumatic brain injury (TBI) is a significant worldwide public health concern that necessitates attention. Apoptosis signal-regulating kinase 1 (ASK1), a key player in various central nervous system (CNS) diseases, has garnered interest for its potential neuroprotective effects against ischemic stroke and epilepsy when deleted. Nonetheless, the specific impact of ASK1 on TBI and its underlying mechanisms remain elusive. Notably, mutation of ATP-binding sites, such as lysine residues, can lead to catalytic inactivation of ASK1. To address these knowledge gaps, we generated transgenic mice harboring a site-specific mutant ASK1 Map3k5-e (K716R), enabling us to assess its effects and elucidate potential underlying mechanisms following TBI. Methods We employed the CRIPR/Cas9 system to generate a transgenic mouse model carrying the ASK1-K716R mutation, aming to investigate the functional implications of this specific mutant. The controlled cortical impact method was utilized to induce TBI. Expression and distribution of ASK1 were detected through Western blotting and immunofluorescence staining, respectively. The ASK1 kinase activity after TBI was detected by a specific ASK1 kinase activity kit. Cerebral microvessels were isolated by gradient centrifugation using dextran. Immunofluorescence staining was performed to evaluate blood–brain barrier (BBB) damage. BBB ultrastructure was visualized using transmission electron microscopy, while the expression levels of endothelial tight junction proteins and ASK1 signaling pathway proteins was detected by Western blotting. To investigate TBI-induced neuroinflammation, we conducted immunofluorescence staining, quantitative real-time polymerase chain reaction (qRT-PCR) and flow cytometry analyses. Additionally, immunofluorescence staining and electrophysiological compound action potentials were conducted to evaluate gray and white matter injury. Finally, sensorimotor function and cognitive function were assessed by a battery of behavioral tests. Results The activity of ASK1-K716R was significantly decreased following TBI. Western blotting confirmed that ASK1-K716R effectively inhibited the phosphorylation of ASK1, JNKs, and p38 in response to TBI. Additionally, ASK1-K716R demonstrated a protective function in maintaining BBB integrity by suppressing ASK1/JNKs activity in endothelial cells, thereby reducing the degradation of tight junction proteins following TBI. Besides, ASK1-K716R effectively suppressed the infiltration of peripheral immune cells into the brain parenchyma, decreased the number of proinflammatory-like microglia/macrophages, increased the number of anti-inflammatory-like microglia/macrophages, and downregulated expression of several proinflammatory factors. Furthermore, ASK1-K716R attenuated white matter injury and improved the nerve conduction function of both myelinated and unmyelinated fibers after TBI. Finally, our findings demonstrated that ASK1-K716R exhibited favorable long-term functional and histological outcomes in the aftermath of TBI. Conclusion ASK1-K716R preserves BBB integrity by inhibiting ASK1/JNKs pathway in endothelial cells, consequently reducing the degradation of tight junction proteins. Additionally, it alleviates early neuroinflammation by inhibiting the infiltration of peripheral immune cells into the brain parenchyma and modulating the polarization of microglia/macrophages. These beneficial effects of ASK1-K716R subsequently result in a reduction in white matter injury and promote the long-term recovery of neurological function following TBI.

TREM2 alleviates white matter injury after traumatic brain injury in mice might be mediated by regulation of DHCR24/LXR pathway in microglia

“…In addition, neuroprotective medications such as ethyl pyruvate improved white matter remodelling by promoting microglial state conversion in a rat TBI model 51 . Other studies have demonstrated that alleviating endoplasmic reticulum stress and blood–brain barrier damage reduces neuroinflammation and eventually improves white matter integrity 52,53 . Furthermore, other studies revealed that microglia alleviate WMI by interacting with oligodendrocytes 54,55 .…”

“… 51 Other studies have demonstrated that alleviating endoplasmic reticulum stress and blood–brain barrier damage reduces neuroinflammation and eventually improves white matter integrity. 52 , 53 Furthermore, other studies revealed that microglia alleviate WMI by interacting with oligodendrocytes. 54 , 55 However, the exact underlying mechanisms have yet to be elucidated.…”

The Interplay between Ferroptosis and Neuroinflammation in Central Neurological Disorders

“…At the core of this positive feedback loop is microglia, which can initiate inflammatory pathways following brain damage from diverse causes, impact the structure of BMECs, and disrupt the tight junctions of the blood-brain barrier through the release of inflammatory cytokines. Once the cell-cell connections of the blood-brain barrier are compromised, the infiltration of peripheral immune cells and exogenous neurotoxic substances further stimulate microglia [52,197]. This sequence of events encompasses several classic inflammatory manifestations, including the release of TNF-α, IL-6, and IL-1β, as well as the transition of microglia from the M2 to M1 phenotype [198,199].…”

“…Secondly, the infiltration of the peripheral immune cell barrier into the brain can lead to damage of the original neurovascular unit (NVU) [52]. Finally, the proinflammatory phenotypic changes in microglia are also directly or indirectly associated with the infiltration of peripheral cells [197].…”