Increased permeability of blood-neural barriers (BNBs) has been implicated in the pathogenesis of multiple acute and chronic neurological disorders, including neurodegenerative disease, but the specific contributions of BNB impairments to neuronal dysfunction and degeneration have been difficult to pinpoint. During characterization of patients with inherited forms of motor neuron disease, we and others previously discovered that autosomal dominant mutations of the cell surface-expressed cation channel transient receptor potential vanilloid 4 (TRPV4) cause subtypes of spinal muscular atrophy and Charcot-Marie-Tooth disease. While our studies in cultured cells suggest that TRPV4 mutations cause a gain of channel function, there is little evidence that TRPV4 is functionally expressed in motor neurons. In order to further dissect the cellular basis of TRPV4 channelopathy, we recently generated novel mutant TRPV4 knock-in mouse models that develop severe neurological phenotypes associated with focal breakdown of BNBs, particularly in the ventral horn of the cervical spinal cord and brainstem. Strikingly, cell type-specific genetic deletion of TRPV4 from endothelial cells (ECs) or treatment of symptomatic mice with a TRPV4 small molecule antagonist markedly reverses these phenotypes. Together, these studies suggest that TRPV4 activation plays a fundamental role in regulating BNB integrity and that TRPV4 antagonists could be a novel therapeutic promoting BNB function. Here, in Specific Aim 1, we will characterize the topographical and temporal expression patterns of TRPV4 in neural vascular ECs and determine the effects of TRPV4 mutations on TRPV4 channel activity in both cultured primary mouse neural vascular ECs and human iPSC-derived neural vascular ECs.
In Specific Aim 2, we will determine how TRPV4 activity alters BNB permeability and structure in vitro, including in both 2D confluent monolayers and in 3D engineered microvessels, as well as in mutant TRPV4 mouse models in vivo. Finally, in Specific Aim 3, using patch clamp electrophysiology in spinal cord slices, we will determine how BNB leak affects motor neuron function and structure, and determine whether TRPV4 small molecule antagonists can reverse disease manifestations in mutant TRPV4 mice. Together, these studies will define a previously uncharacterized role for TRPV4 in neural vascular ECs in regulating BNB integrity, determine effects of BNB breakdown on motor neuron function, and investigate whether a TRPV4 small molecule antagonist could be a novel treatment for patients with TRPV4 mutations, as well as for patients with other neurological diseases characterized by impaired BNBs.
Impairments of blood-neural barriers (BNBs) play key roles in many neurodegenerative disease contexts, but little is known about the molecular mechanisms that initiate BNB opening or therapeutic strategies to limit BNB leak. By studying gain-of-function mutations of the transient receptor potential vanilloid 4 (TRPV4) cation channel that cause forms of inherited motor neuron disease, we have unexpectedly uncovered a novel role for TRPV4 in regulating BNB integrity. Here we will characterize how TRPV4 may regulate BNB permeability and investigate whether TRPV4 small molecule antagonism could be a potential therapeutic strategy to improve BNB function.
