Defining TRPV4-mediated cytoskeletal changes that trigger pathological blood-neural barrier disruption - PROJECT SUMMARY/ABSTRACT TRPV4 (transient receptor potential vanilloid 4) is a calcium-permeable ion channel whose activation has been implicated in both hereditary and acquired neurological diseases. Gain-of-function mutations cause debilitating forms of hereditary motor neuron disease and neuropathy/neuronopathy for which no treatment exists. In addition, increased TRPV4 ion channel activity is implicated in multiple other neurological diseases. While the association of TRPV4 with disease and the availability of TRPV4 antagonist drugs make TRPV4 a promising therapeutic target, fundamental aspects of TRPV4 signaling and function remain poorly understood. To address this knowledge gap, we have generated cellular, fly, and mouse models of TRPV4-associated nerve disease. Unbiased studies in these models have identified the calcium-regulated actin remodeling proteins RhoA, CaMKII, and INF2 as critical downstream mediators of TRPV4 signaling. In addition, novel TRPV4 mutant knock-in mice, which develop severe neuromuscular disease that rapidly progresses to death, have unexpectedly demonstrated that increased TRPV4 activation within neural vascular endothelial cells (ECs) drives pathological disruption of blood-neural barriers (BNBs). Together, these results suggest that TRPV4 causes neurodegeneration through dysregulation of actin cytoskeletal remodeling in ECs, resulting in BNB breakdown. As BNB disruption is a common pathological event in a variety of degenerative and traumatic neurological diseases, effective therapeutic strategies to limit BNB breakdown would have far-reaching clinical value. Here, we propose a series of complementary studies utilizing in vitro models of neural vascular ECs and TRPV4 knock-in mice to further define the molecular mechanisms by which TRPV4 modulates the actin cytoskeleton and BNB integrity. In Specific Aim 1, we will use a combination of dynamic molecular biosensors, candidate-based biochemical assessments, and unbiased phospho-proteomics to define downstream TRPV4 signaling events and their temporal activation profiles. Specific Aim 2 will address the contribution of the calcium-sensitive actin remodeling proteins RhoA, CaMKII, and INF2 to TRPV4-mediated calcium influx and modulation of cellular morphology and BNB integrity. In Specific Aim 3, we will use TRPV4 knock-in mice to investigate the topographic and temporal patterns of actin modulating protein activation in the context of TRPV4-mediated BNB disruption. We will also determine the contribution of RhoA and INF2 activity to TRPV4 knock-in phenotypes, including motor behavior, BNB disruption, and survival. Together, these studies will delineate TRPV4-mediated signaling cascades that modulate cytoskeletal changes and barrier function in neural vascular ECs. These results will define specific molecular pathways that modulate BNB integrity and reveal therapeutic opportunities to limit pathological BNB disruption in neurological disease.
