Acute neuroinflammation induces AIS structural plasticity in a NOX2-dependent manner

Background: Chronic microgliamediated inflammation and oxidative stress are wellcharacterized underlying factors in neurodegenerative disease, whereby reactive inflammatory microglia enhance ROS production and impact neuronal integrity. Recently, it has been shown that during chronic inflammation, neuronal integrity is compromised through targeted disruption of the axon initial segment (AIS), the axonal domain critical for action potential initiation. AIS disruption was associated with contact by reactive inflammatory microglia which wrap around the AIS, increasing association with disease progression. While it is clear that chronic microglial inflammation and enhanced ROS production impact neuronal integrity, little is known about how acute microglial inflammation influences AIS stability. Here, we demonstrate that acute neuroinflammation induces AIS structural plasticity in a ROSmediated and calpaindependent manner. Methods: C57BL/6J and NOX2-/mice were given a single injection of lipopolysaccharide (LPS; 5 mg/kg) or vehicle (0.9% saline, 10 mL/kg) and analyzed at 6 h2 weeks postinjection. Antiinflammatory Didox (250 mg/kg) or vehicle (0.9% saline, 10 mL/kg) was administered beginning 24 h postLPS injection and continued for 5 days; animals were analyzed 1 week postinjection. Microglial inflammation was assessed using immunohistochemistry (IHC) and RTqPCR, and AIS integrity was quantitatively analyzed using ankyrinG immunolabeling. Data were statistically compared by oneway or twoway ANOVA where mean differences were significant as assessed using Tukey's post hoc analysis. Results: LPSinduced neuroinflammation, characterized by enhanced microglial inflammation and increased expression of ROSproducing enzymes, altered AIS protein clustering. Importantly, inflammationinduced AIS changes were reversed following resolution of microglial inflammation. Modulation of the inflammatory response using antiinflammatory Didox, even after significant AIS disruption occurred, increased the rate of AIS recovery. qPCR and IHC analysis revealed that expression of microglial NOX2, a ROSproducing enzyme, was significantly increased correlating with AIS disruption. Furthermore, ablation of NOX2 prevented inflammationinduced AIS plasticity, suggesting that ROS drive AIS structural plasticity. Conclusions: In the presence of acute microglial inflammation, the AIS undergoes an adaptive change that is capable of spontaneous recovery. Moreover, recovery can be therapeutically accelerated. Together, these findings underscore the dynamic capabilities of this domain in the presence of a pathological insult and provide evidence that the AIS is a viable therapeutic target.