Ongoing research
Overall goals
The overall goals of the McCray laboratory are to further the understanding and treatment of peripheral neuropathy, particularly inherited forms of peripheral neuropathy such as Charcot-Marie-Tooth (CMT) disease. The laboratory is particularly focused on inherited neuromuscular disease due to mutations in TRPV4 (transient receptor potential vanilloid 4).
Model of TRPV4-RhoA interactions and disruption by disease mutations. Adapted from McCray et al., 2021.
MN-1 cells expressing neuromuscular disease mutant TRPV4 (R269C) show elevated baseline calcium levels and increased stimulus-induced calcium influx as compared to wild-type (WT) TRPV4. Adapted from Taga et al., 2022.
MDCK cells expressing mutant TRPV4 show normal localization to the plasma membrane and colocalization with cortical actin (unpublished).
Moving towards treatments for TRPV4 neuromuscular disease
Charcot-Marie-Tooth disease (CMT) comprises a group of inherited peripheral neuropathies that together represent the most common inherited neurological condition worldwide, but currently lack any disease-modifying treatments. Mutations in TRPV4 cause a spectrum of TRPV4 neuromuscular disease, including a subtype of CMT known as CMT2C. TRPV4 neuromuscular diseases cause motor or sensorimotor dysfunction as well as vocal cord weakness, which can be debilitating and life-threatening. Affected patients demonstrate marked heterogeneity in age of onset, symptoms, and disease severity. Our prior work has shown that disease mutations cause gain of ion channel function and that channel inhibition can rescue toxicity in cellular and animal models of disease. This suggests the exciting possibility that TRPV4 neuromuscular disease is a potentially treatable condition. However, moving towards a clinical trial will require a more complete understanding of the natural history of the disease and identification of biomarkers and outcome measures that could be used to demonstrate efficacy in a treatment trial. To address these needs, we have worked with the Inherited Neuropathies Consortium (INC) to establish a TRPV4 patient registry and a TRPV4 natural history study. The natural history study was initially established at Johns Hopkins University, but the goal is to add additional clinical sites over time to facilitate this important work. Work on this project includes identification and enrollment of patients in clinical studies, clinical assessment of affected patients, analysis of potential disease biomarkers, data analysis, and interface with pharmaceutical and biotech industry partners.
Any patients who are interested in participating or would like to learn more about our research can find out more here.
Ongoing clinical studies of TRPV4 patients includes specialized assessment of the brain, retina, skeleton, and vocal cords to identify potential disease biomarkers (unpublished).
Defining TRPV4 function in other neurological diseases
Mutations in TRPV4 cause forms of neuromuscular disease, but there is increasing evidence for activation of wild-type TRPV4 across a host of neurological disease states, including stroke, traumatic brain and spinal cord injury, and peripheral nerve injury. Understanding the cell- and tissue-specific expression patterns of TRPV4 in the nervous system and beyond has been hindered by the unsuitability of available antibodies for detection of endogenous TRPV4. In collaboration with Dr. Charlotte Sumner's lab at Johns Hopkins, we have been using TRPV4 reporter mice to interrogate expression patterns of TRPV4 under normal conditions and in response to injury. We have also begun to explore how gain or loss of function of TRPV4 affects neurological injury and recovery across different disease states. Work on this project involves performing various nerve injury paradigms including sciatic nerve crush, immunohistochemistry of mouse tissue, mouse behavior, and electrophysiology.
Assessment of TRPV4 expression patterns in mouse sciatic nerve (unpublished).
Background
Autosomal dominant missense mutations of TRPV4, a calcium-permeable ion channel, cause a spectrum of hereditary neuromuscular diseases, including Charcot Marie Tooth disease type 2C (CMT2C), scapuloperoneal spinal muscular atrophy (SPSMA), and congenital distal spinal muscular atrophy (CDSMA). We collectively refer to these conditions as “TRPV4 neuromuscular disease” for simplicity. These conditions result in muscle weakness of the upper and lower extremities, characteristic vocal cord weakness that can affect speech and breathing, and variable sensory loss. Many patients also develop a range of skeletal abnormalities affecting the trunk and extremities. In prior work, we have demonstrated that disease mutations cause a gain of channel function in cultured cells and in a fly model of TRPV4 neuromuscular disease. Furthermore, the toxicity of mutant TRPV4 can be robustly rescued with available TRPV4 antagonist drugs, suggesting that TRPV4 neuromuscular disease may be readily treatable.
TRPV4 encodes a plasma membrane-expressed, non-selective ion channel that is preferentially permeable to calcium. TRPV4 ion channels are homotetramers with each subunit consisting of a transmembrane domain as well as large N- and C-terminal cytoplasmic domains. Notably, most neuromuscular disease-causing mutations occur within the ankyrin repeat domain (ARD) in the N terminus, suggesting that this domain is likely critical for disease pathogenesis.
Defining TRPV4 regulation of actin cytoskeletal dynamics
The normal function of TRPV4 within the various organs and tissues where it is expressed remains enigmatic, but likely relates to functions as an extracellular signal transducer. TRPV4 responds to a broad range of environmental stimuli, including mechanical stimuli related to shear stress, stretch, cell swelling, and heat. Work from our group and others has demonstrated that TRPV4 can strongly influence cellular morphogenesis and actin cytoskeletal changes, particularly through interactions with the actin remodeling small GTPase RhoA. Ongoing work is focused on understanding the specific pathways that are regulated by TRPV4 signaling in both health and in disease. Based on our prior work demonstrating direct binding and reciprocal regulation of TRPV4 and RhoA, we are particularly interested in how TRPV4 modulates RhoA-dependent signaling in distinct cell types and tissues. We are also engaged in studies to identify and characterize additional TRPV4-regulated cytoskeletal machinery. These projects utilize a combination of approaches in immortalized cell lines, iPS-derived cells, and rodent models of TRPV4 neuromuscular disease. Techniques include live-cell imaging, functional studies of cell-cell junction integrity, calcium imaging using both radiometric and single-channel calcium indicators, cellular and molecular biology, biochemistry, mouse histology, and mouse behavior.
Defining the genetic landscape of TRPV4 channelopathies
Many missense mutations in TRPV4 have been linked to TRPV4 neuromuscular disease, but there are also other mutations in TRPV4 that cause distinct diseases of connective tissue and bone, including various forms of skeletal dysplasia and hereditary arthropathy. These conditions together constitute a group of "TRPV4 channelopathies," which share features of dominant inheritance due to gain-of-function mutations in TRPV4. In addition, increased utilization of genetic testing in patients with suspected hereditary neuromuscular disease has led to identification of many TRPV4 variants of unclear pathogenicity, termed variants of unknown significance (VUSs). We have developed multiple in vitro assays to assess the functional effects of TRPV4 variants in order to help determine their pathogenicity and elucidate mechanisms of disease pathogenesis. Together, these studies also help to clarify the genetic landscape of TRPV4 channelopathies and improve understanding of the functional and structural underpinnings of pathogenic TRPV4 mutations. Work in this project area typifies the bench-to-bedside approach of our lab, with studies involving analysis of human genetic data, collection and review of relevant clinical records, structural modeling, and biochemistry and cellular biology approaches.
