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

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 i