Neuronal Toll-like Receptor-4 regulation of Matrix Metalloproteinase-9 Activity Mediates Dentate Circuit Dysfunction after Traumatic Brain Injury

Abstract Neuroinflammatory pathways activated by traumatic brain injury (TBI) are critical mediators of long-term neurological dysfunction and represent promising therapeutic targets. Toll-like receptor 4 (TLR4), an innate immune receptor, was previously shown to contribute to increased seizure susceptibility and cognitive deficits in rats after lateral fluid percussion injury (FPI). However, the cellular and molecular mechanisms underlying TLR4-mediated circuit dysfunction early after brain injury are not fully understood. In this study, we define a cell- and circuit-specific neuroimmune-enzyme effector signaling axis that mediates early post-TBI circuit dysfunction in the hippocampal Dentate Gyrus (DG). Using ex vivo electrophysiology in rat and mouse models one-week after brain injury, we demonstrate that neuronal TLR4 signaling regulates both excitatory and inhibitory synaptic inputs to dentate granule cells (DGC). Collectively, pharmacological inhibition of TLR4 in rats and cell-type-specific deletion of TLR4 in mice show that neuronal TLR4 mediates injury-driven increase in DGC excitatory input frequency and relies on downstream activation of Matrix Metalloproteinase-9 (MMP-9). In contrast, TLR4 signaling contributed to decrease in inhibitory current frequency after injury, but independent of MMP-9, revealing a mechanistic divergence. Systemic inhibition of either TLR4 signaling or MMP-9 activity in rats within 24 hours after injury reduced network hyperexcitability and improved long-term potentiation (LTP) in the DG measured in vivo one-week after injury. Either TLR4 or MMP-9 inhibition early after injury effectively attenuated spatial memory deficits in a Barnes maze task one-month post-injury. Paradoxically, in sham controls, inhibition of TLR4 increased frequency of both excitatory and inhibitory inputs to DGCs and augmented network excitability, without altering MMP-9 levels, identifying context-dependent roles for TLR4 signaling. Together, these results identify a novel TLR4-MMP-9 axis as a key driver of early post-TBI dentate gyrus circuit dysfunction and behavioral deficits. Competing Interest Statement The authors have declared no competing interest. Footnotes Supplementary figures are updated and now include additional validations. Supplementary tables with detailed descriptive statistics and p values are added. List of Abbreviations - aCSF - Artificial Cerebrospinal Fluid - AMPAR - α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid Receptor - ANOVA - Analysis of Variance - CA1 SR - CA1 Stratum Radiatum - CaMKIIa - Calcium/Calmodulin-Dependent Protein Kinase II Alpha - CreERT2 - Cre Recombinase Estrogen Receptor T2 - DG - Dentate Gyrus - DǪ Gelatinase - Dye Ǫuenched fluorogenic substrate for zymography - ECM - Extracellular Matrix - EPSC - Excitatory Postsynaptic Current - FPI - Fluid Percussion Injury - HMGB1 - High-Mobility Group Box 1 - IEI - Inter-Event Interval - IPSC - Inhibitory Postsynaptic Current - JSH-23 - NF-κB Inhibitor - K-S test - Kolmogorov-Smirnov Test - LPS - Lipopolysaccharide - LTP - Long Term Potentiation - MMP-9 - Matrix Metalloproteinase-9 - NF-κB - Nuclear Factor Kappa B - TAK-242 - TLR4 Antagonist - TBS - Theta Burst Stimulation - TBI - Traumatic Brain Injury - TTX - Tetrodotoxin