1. Construction of ASK1-K716R.
Inhibition of ASK1 activity holds immense therapeutic potential for addressing nervous system diseases and promoting neurological function recovery following brain injury [14, 20–22]. To investigate the implications of ASK1 in the pathological progression after TBI, we generated a novel ASK1-K716R mouse model using the CRIPR/Cas9 system (Fig. 1A). DNA sequencing (Fig. S1A) confirmed the precise substitution at site 716, resulting in the translation change from lysine to arginine by altering the sequence from “AAGGAAATC” to “AGAGAAATA”. qPCR was performed to assess the impact of the ASK1-K716R mutation on ASK1 transcription. Notably, no significant difference in ASK1 mRNA expression was observed among the experimental groups (Fig. 1B), indicating that ASK1-K716R does not influence ASK1 transcription.
2. ASK1-K716R suppresses phosphorylation of ASK1 and activation of JNKs/p38 signaling pathway following TBI.
To investigate the impact of ASK1-K716R on ASK1 kinase activity after TBI, we measured ASK1 kinase activity using an ASK1 kinase activity detection kit. The results revealed a significant reduction in ASK1 kinase activity following TBI in ASK1-K716R TBI mice (Fig. 1C). Additionally, western blot analysis was performed 3 days post-TBI, showing no change in the overall expression of ASK1 with the mutation. However, the phosphorylation levels of ASK1 were significantly reduced in ASK1-K716R TBI mice, compared to controls (Fig. 1D-E). Furthermore, immunofluorescence staining was performed to evaluate the colocalization of p-ASK1 and (Iba1+) microglia, (GFAP+) astrocytes and (NeuN+) neurons, respectively (Fig. S1B). The results demonstrated that ASK1-K716R inhibited the expression of p-ASK1 in neurons, microglia, and astrocytes following TBI, supporting the effective suppression of ASK1 kinase activity by ASK1-K716Rafter TBI.
ASK1 is known to modulate apoptosis and inflammation by activating downstream JNKs and p38 signaling pathways [23–29]. In order to examine the impact of ASK1-K716R on the activation of JNKs/p38, western blotting was performed on day 3 post-TBI. No significant changes in the expression levels of ASK1, p38, or JNKs were observed in WT and ASK1-K716R mice, regardless of TBI (Fig. 1F, H). However, ASK1-716R significantly inhibited the elevated expression of p-ASK1, p-JNKs, and p-p38 induced by TBI (Fig. 1F, G). Additionally, TUNEL and NeuN immunofluorescence co-staining of brain slices on day 3 post-TBI revealed a significant decrease in neuronal apoptosis inASK1-K716R mice compared to controls (Fig. S2A-C). These results indicate the effective inhibition of the ASK1/JNKs pro-apoptotic pathway by ASK1-K716R after brain injury, consequently reducing neuronal apoptosis.
3. ASK1-K716R inhibits the activation of ASK1/JNKs signaling pathway in endothelial cells of cerebral microvessels.
Elevated phosphorylation of ASK1 has been associated with endothelial inflammation, apoptosis, and oxidative stress [30, 31]. Therefore, to explore the potential impact of ASK1 signaling pathway on BBB integrity following TBI, cerebral microvessels were isolated from the injured cerebral hemisphere of TBI mice. The protein expression of ASK1/p38/JNKs signaling pathway was quantitatively analyzed using Western blot analysis. The results showed no significant changes in the expression of ASK1, p38, or JNKs in cerebral microvessels from both WT and ASK1-K716R mice, regardless of TBI (Fig. 2A, 2C). However, ASK1-K716R significantly reduced the phosphorylation levels of ASK1 and JNKs in cerebral microvessels following TBI. Interestingly, unlike the findings in brain tissue samples, ASK1-K716R did not alter the phosphorylation level of p38 in cerebral microvessels after TBI (Fig. 2A, 2B). These results were further supported by double staining of CD31 with p-ASK1, p-JNKs, and p-p38 (Fig. 2D). Considering the involvement of ASK1/JNKs axis in apoptosis, we performed TUNEL staining to evaluate endothelial apoptosis following TBI. Our results demonstrated a reduction in the number of TUNEL+ CD31+ microvessels in the ASK1-K716R TBI group compared to the WT TBI group (Fig. 2E), further confirming the effective inhibition of ASK1 signaling by ASK1-K716R after brain injury and its subsequent reduction in endothelial apoptosis.
Inhibition of ASK1 has been shown to stabilize endothelial tight junctions, thus ameliorating endothelial barrier dysfunction [32–35]. Additionally, we evaluated the expression of tight junction proteins in cerebral microvessels using Western blotting and immunofluorescence staining. The data indicated that ASK1-K716R alleviated the compromised expression of Occludin and VE-Cadherin induced by TBI (Fig. 2F, 2G, S3 A-B).
4. ASK1-K716R attenuates BBB permeability and preserves BBB integrity.
To investigate the potential protective role of ASK1-K716R in preserving BBB function by modulating endothelial apoptosis, we assessed BBB integrity on day 3 post-TBI by evaluating by evaluating the central permeation of peripherally injected tracers, including fluorescent Alexa 555 cadaverine and EB. Analysis of cadaverine fluorescence intensity revealed a decrease in leakage volume in the ASK1-K716R TBI group compared to the WT TBI group (Fig. 3A, 3C upper panel). In the EB assay, extensive leakage of EB was observed in the ipsilateral hemispheres of WT TBI mice (Fig. 3B), while EB extravasation was significantly reduced in ASK1-K716R TBI mice, as evident both visually and quantitatively (Fig. 3B&3C). Concurrently, immunofluorescence staining for IgG was performed to examine endogenous peripheral IgG leakage into the brain parenchyma, and a notable reduction in IgG leakage was observed in the coronal brain sections of ASK1-K716R TBI mice compared to WT TBI mice (Fig. 3D). Furthermore, transmission electron microscopy of BBB ultrastructure revealed characteristics of BBB damage in WT mice following TBI, including swollen astrocyte endfeet, thickened basement membranes, and decreased expression of endothelial tight junction protein. Conversely, ASK1-K716R ameliorated these impairments (Fig. 3E). The restoration of BBB integrity was further confirmed by western blotting analysis, which showed that the downregulation of tight junction proteins, such as Occludin and VE-Cadherin, induced by TBI was significantly alleviated by ASK1-K716R (Fig. 3F, 3G). It has been reported that MMP-9, originating from various cell sources, contributes to the degradation of tight junction proteins in brain injury [36–38]. Consistent with this, Western blotting revealed an upregulation of MMP-9 in WT-TBI mice, whereas ASK1-K716R treatment prominently suppressed MMP-9 expression after TBI (Fig. 3H). Collectively, these findings demonstrate that ASK1-K716R reduces BBB permeability and safeguards its integrity following TBI.
5. ASK1-K716R inhibits the infiltration of peripheral immune cells and microglia-mediated neuroinflammation following TBI.
Circulating leukocytes that breach the compromised BBB play a pivotal role in the aftermath of TBI. The substantial influx of leukocytes is a primary contributor to the surge of MMPs during the early stage of brain injury [39]. We employed a flow cytometry-based approach to quantitatively assess the infiltration of peripheral immune cells in the brain on day 3 after TBI (Fig. 4A). The findings revealed no significant variation in cell numbers between the two Sham groups, suggesting that ASK1-K716R does not exert an influence on brain immune cells under physiological conditions (Fig. 4B). Notably, the numbers of peripheral infiltrating immune cells (CD45hiCD11b+), dendritic cells (CD11b+CD45+CD11c+Ly6G−), macrophages (CD45hiCD11b+CD11c−Ly6G−), T cells (CD11b−CD45+CD3+), and B cells (CD11b−CD45+CD3−CD19+) were observed to increase after TBI. However, ASK1-K716R significantly attenuated these increases, except for B cells (Fig. 4B).
To investigate the impact of ASK1-K716Ron peripheral immune cell levels following TBI, we examined the changes in various immune cell populations in peripheral blood and spleen using flow cytometry 3 days post-TBI. In both peripheral blood and spleen, the results revealed no significant differences in populations of macrophages, dendritic cells, Ly6Chigh inflammatory macrophages, T cells and B cells (Fig. S4A-C). These results further support the notion that ASK1-K716R does not alter the levels of immune cells in peripheral blood and spleen. However, it primarily inhibits the infiltration of various immune cells into the brain following TBI.
The ASK1-K716R variant’s protective effect on BBB dysfunction may reduce microglia-mediated neuroinflammation following TBI. To evaluate the heterogeneity of microglia/macrophages on day 3 after TBI, we employed triple immunofluorescence staining with Iba1 (a marker for microglia/macrophage), CD16 (a marker for pro-inflammatory phenotype), and CD206 (a marker for anti-inflammatory phenotype) to categorize the microglia/macrophages into four distinct phenotypes (Fig. 4D): pro-inflammatory (CD16+CD206−Iba1+, Pro), transient (CD16+CD206+Iba1+, Trans), anti-inflammatory (CD16−CD206+Iba1+, Anti), and resting (CD16−CD206−Iba1+, Rest) microglia/macrophages. The results demonstrated that TBI induced the activation of microglia/macrophages in the peri-lesion area, while ASK1-K716R didn’t alter the activation of microglia/macrophages, as indicated by the similar proportion of resting microglia/macrophages between ASK1-K716R TBI and WT TBI groups. Importantly, ASK1-K716R significantly decreased the proportion of the Pro phenotype and increased the proportion of Anti phenotype in the peri-lesion area located 0-400 µm and/or 400–800 µm from damaged edge of the cortex and striatum. However, there was no significant change observed in the Trans phenotype within the area located 0-400 µm and/or 400–800 µm from damaged edge of the cortex and striatum, 3 days after TBI (Fig. 4C-F). Overall, these results suggest the involvement of ASK1-K716R in the regulation of microglia/macrophage heterogenization following TBI.
The infiltration of peripheral immune cells and alterations in microglia heterogeneity have a substantial impact on the inflammatory environment within the brain. To further investigate the impact of ASK1-K716R on the expression of inflammatory mediators, we performed qPCR analysis 3 days after TBI. Our findings revealed a significant upregulation of pro-inflammatory factors (Cd11b, Cd16, iNOS, Tnf-α, Il-1β, and Ccl3) as well as anti-inflammatory factors (Ym1/2, Tgf-β) following TBI (Fig. 4G). ASK1-K716R inhibited the expression of several pro-inflammatory factors, specifically Cd11b, Cd16, iNOS, Tnf-α and Ccl3, while concurrently enhancing the expression of an anti-inflammatory factor Ym1/2 (Fig. 4G). As a result, ASK1-K716R primarily inhibits the pro-inflammatory response and stimulates the anti-inflammatory response, consequently ameliorating the inflammatory microenvironment within the brain.
6. ASK1-K716R attenuates white matter injury (WMI) following TBI.
It is of interest to determine whether ASK1-K716R can have a sustained protective effect on TBI by preserving BBB integrity and suppressing inflammatory responses in the brain during the acute phase. To assess long-term tissue loss, immunohistochemical staining of NeuN was used as a standard method for estimating TBI (Fig. 5A). Our findings revealed that, on day 35 following TBI, ASK1-K716R significantly mitigated the extent and volume of neuronal tissue loss compared to WTTBI mice (Fig. 5B-C).
WMI is a significant pathological consequence in the context of TBI, playing a crucial role in the pathophysiological processes [40–41]. WMI, characterized by demyelination and/or axonal damage, has been closely linked to neuroinflammation following acute or chronic brain injury. In this study, we employed immunofluorescence co-staining of MBP/SMI32 and NF200 to label myelin and neurofilaments, respectively, on day 3 and 35 post-TBI (Fig. 5D upper panel, Fig. S6A-C). Our findings demonstrated that traumatic injury resulted in a reduction in MBP fluorescence intensity and an increase in the relative fluorescence intensity of SMI32/MBP within the external capsule (EC) and striatum. Notably, ASK1-K716R treatment effectively ameliorated TBI-induced demyelination on both days 3 and 35 post-TBI (Fig. 5E-F, S5D-E). Furthermore, the neuroprotective effect of ASK1-K716R against axonal injury was evident through NF200 staining (Fig. 5D lower panel, 5G, S5C, S5F). Nodes of Ranvier (NOR) are essential for signal conduction and represent unmyelinated segments between two myelin sheaths in myelinated nerve fibers. [42]. Immunofluorescence staining for contactin-associated protein (Caspr) and sodium channel (Nav1.6) was performed to evaluate NOR in the corpus callosum (CC) 35 days after TBI (Fig. 5I). We observed a significant reduction in the number of NOR and paranode length following TBI compared to the contralateral region. Notably, ASK1-K716R remarkably alleviated these impairments in comparison to the WT TBI group (Fig. 5J-L). Additionally, we measured the fast potential of myelinated axon conduction (N1) and the slow potential of unmyelinated axon conduction (N2) in the CC to assess the conductive capacity of white matter fibers (Fig. 5M). No significant difference was observed between the two Sham groups, indicating that ASK1-K716R did not affect nerve conduction under physiological conditions. TBI caused a decrease in both N1 and N2 potential amplitudes in WT mice. In contrast, ASK1-K716R TBI mice exhibited a mild improvement in the N1 amplitude and a notable recovery of the N2 amplitude to levels comparable to the control group (Fig. 5N, 5O). These findings suggest that ASK1-K716R has a potent neuroprotective effect against brain tissue loss and white matter injury, holding promise as a potential therapeutic target for TBI management.
7. ASK1-K716R ameliorates long-term neurological deficits following TBI.
Previous studies have demonstrated a significant association between white matter injury and behavioral changes [43, 44]. To evaluate the impact of ASK1-K716R on sensorimotor dysfunction induced by TBI, we performed a battery of behavioral tests, including the modified Garcia score test, rotarod test, and adhesive-removal testfrom 3 to 35 days post-CCI (Fig. 6A). Remarkably, mice in the ASK1-K716R Sham group exhibited no neurological deficits in multiple behavioral tests compared to WT Sham mice (Fig. 6B-E). Particularly noteworthy, the ASK1-K716R TBI group displayed a significantly faster improvement in the modified Garcia score compared to the WT TBI group (Fig. 6B). Furthermore, the WT-TBI group mice exhibited a shorter latency to fall off in the rotarod test (Fig. 6D) and a prolonged latency to touch and remove sticky paper in the adhesive removal test, in comparison to the ASK1-K716R-TBI group mice (Fig. 6C). The results of these behavioral tests indicate that ASK1-K716R significantly improved TBI-induced sensorimotor deficits without affecting neurological function in the Sham groups.
To investigate the effect of ASK1-K716R on cognitive abilities after TBI, we performed three behavioral tests, including the new object recognition test, three-chamber social test, and MWM test within 25–35 days post-TBI. The new object recognition test aimed to evaluate recognition memory based on the mice’s preference for exploring novel objects. Remarkably, WT TBI mice showed no variation in their exploration tendencies toward familiar or new objects, while ASK1-K716R TBI mice displayed a restored preference for novel objects, indicating an improvement in recognition memory deficit (Fig. 6F). The cognitive improvements resulting from ASK1-K716R were further confirmed in the three-chamber social test, which measures social novelty recognition ability. During the adaptation period, both WT and ASK1-K716R mice exhibited normal sociability, interacting frequently with other mice rather than empty cages (Fig. 6G). However, the decline in preference for novel mice observed in the WT TBI group was mitigated in the ASK1-K716R-TBI group, highlighting the potential of ASK1-K716R to alleviate the impairment in social novelty recognition (Fig. 6H). Furthermore, we evaluated spatial cognitive function using the Morris Water Maze (MWM) test at 31–35 days after CCI. Both TBI groups showed prolonged escape latency during the training period, ASK1-K716R did not influence the spatial learning decline in TBI mice (Fig. S6A upper panel, 6B). However, during the probe test, ASK1-K716R attenuated the decrease in the number of platform crossings, with no difference in swimming velocity, induced by TBI (Fig. 1E, S6A lower panel). Collectively, these behavioral findings demonstrate the beneficial effects of ASK1-K716R on social novelty recognition and memory restoration after TBI.