Charlotte Sumner - US grants

[unreadable] DESCRIPTION (provided by applicant): Spinal muscular atrophy (SMA) is the most common inherited cause of infant mortality. The disease is caused by mutations in the survival motor neuron 1 (SMN1) gene. All patients retain a second copy of the gene (SMN2) that produces insufficient quantities of SMN protein to fully compensate for the loss of SMN1. One promising therapeutic strategy for SMA is to augment SMN protein levels by increasing SMN2 gene expression. Preliminary work done in our laboratory and by other groups indicates that histone deacetylase inhibitors can increase SMN levels in vitro. However, the specific mechanism of action of these compounds and the fundamental mechanisms that regulate SMN2 gene expression are currently unknown. Our goal is to understand the mechanisms that regulate SMN2 gene expression in order to advance therapy for SMA. We hypothesize that the SMN2 gene is regulated, in part, by the acetylation state of histones and the methylation state of DNA acting in concert with cis- and trans-acting factors within the SMN2 promoter and that manipulation of these determinants can lead to increased SMN levels in human cells. We plan to test our hypothesis by pursuing the following three Specific Aims: Specific Aim 1: To characterize the role of histone acetylation and DNA methylation in SMN gene expression regulation. Specific Aim 2: To identify transcription factors and SMN promoter elements that act in concert with epigenetic determinants to regulate SMN gene expression. Specific Aim 3: To characterize baseline SMN protein levels in peripheral blood cells of SMA patients and to test the ability of pharmacological compounds that modulate SMN gene expression to increase SMN protein levels in these cells in vitro. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Peripheral neuropathy results in axonal degeneration of motor and sensory nerve fibers causing muscle weakness and sensory loss. Development of effective therapies has been hampered by incomplete understanding of the underlying molecular and cellular triggers and a lack of druggable therapeutic targets. The discovery that mutations of transient receptor potential vanilloid 4 (TRPV4) cause Charcot- Marie-Tooth disease type 2C marks the first example of an ion channel causing inherited sensorimotor neuropathy. TRPV4 is expressed at the cell surface membrane and several small molecule antagonists have already been developed. In order to further investigate mechanisms of TRPV4-induced neuropathy, we recently developed a novel knock-in mouse model containing the R269C mutation in the endogenous mouse TrpV4 gene. Our preliminary studies of young knock-in mice indicate abnormalities of their peripheral nerve physiology. Using this model, we propose 1) to characterize the spatial and temporal evolution of neuronal dysfunction and degeneration in TRPV4R269C knock-in mice and 2) to test our hypothesis that the R269C mutation causes of gain-of-TRPV4 channel activity in neurons that can be suppressed by TRPV4 antagonists. Together these studies will determine how neuropathy-associated TRPV4 mutations alter TRPV4 function in peripheral neurons, assess the extent to which TRPV4 antagonists may represent a potential therapeutic strategy for patients, and define outcome measures of TRPV4-induced neurodegeneration in mice that can be utilized during future mechanistic and treatment studies.

PROJECT SUMMARY The motor neuron disease spinal muscular atrophy (SMA) is the leading inherited cause of death in infancy and childhood. It is caused by recessive mutations of the survival motor neuron 1 gene (SMN1). All patients retain one or more copies of the homologous SMN2 gene, but it produces inadequate levels of SMN protein due to an alternative splice event. Novel therapeutics aiming to modulate SMN2 splicing including antisense oligonucleotides and small molecules are recently FDA-approved or currently in clinical trials in SMA patients. While this is a success, it remains unknown why many patients have inadequate therapeutic responses. Defining the optimal timing and tissue targeting of SMN induction has been limited by poor understanding of early disease pathology in patients. To address this knowledge gap, in preliminary studies we examined ventral root axons in severe SMA patients and model mice and discovered marked impairments of motor axon sorting and radial growth, which begin prenatally and are followed by degeneration of immature axons perinatally. This project aims to determine if these pathologies may underlie the early disease onset, stereotypical pattern of weakness, and precipitous decline of severe SMA patients. In Specific Aim 1, we will characterize the temporal and topographic patterns of this pathology in both a severe and milder SMA mouse model and in human samples. In Specific Aim 2, we will define the cellular contributors to this pathology utilizing a series of conditional SMA mouse lines expressing increased SMN specifically in either motor neurons, Schwann cells, or muscle. We will also evaluate whether neuregulin 1 type III (NRG1-III), a key regulator of peripheral axon development, is dysregulated in SMA and explore whether overexpression of NRG1-III can ameliorate SMA axonal pathologies. Finally, in Specific Aim 3, we will establish when SMN- inducing drugs, including SMN2 splice-switching antisense oligonucleotides and the small molecule SMN-C3, must be delivered to restore axonal maturation, prevent motor unit degeneration, and provide optimal phenotypic rescue. Together, these studies will characterize a newly recognized and prominent pathology of severe SMA patients and define the optimal timing of therapeutics. The results of these investigations will provide important insights regarding the outcomes of patients currently enrolled in clinical trials, influence the design of future trials, and potentially uncover novel SMA therapeutic targets.

PROJECT SUMMARY Spinal muscular atrophies (SMAs) are monogenetic motor neuron (MN) diseases that cause debilitating muscle weakness and often early mortality. My research program focuses on advancing therapeutics for two forms of SMA: proximal SMA caused by recessive, loss-of-function mutations of the survival motor neuron 1 gene (SMN1) and distal SMA (dSMA) caused by dominant mutations of the transient receptor potential vanilloid 4 gene (TRPV4). Our overarching approach is to integrate findings from human patients with experimentation in animal and iPSC-derived models to elucidate pathomechanistic pathways relevant to human disease and identify promising therapeutic opportunities. Here, we will leverage unique resources and state-of-the-art technologies to define factors limiting efficacy of current SMA therapeutics, characterize cellular and molecular mechanisms driving SMA pathology, and identify and validate novel therapeutic strategies. Proximal SMA is at the forefront of rapidly evolving gene-targeting therapeutics, with two recently approved SMN-inducing treatments and a third under FDA review. While a transformative success, the clinical efficacy of these treatments is highly variable, ranging from normal attainment of early motor milestones to no improvement in motor function. In the last 5 years, our studies have revealed that proximal SMA pathology begins in utero, before treatments are currently initiated in patients. In both humans and mice, SMA MNs exhibit impaired maturation during gestation and precipitous neonatal degeneration, paralleled by a marked decline in SMN expression. Here, we will build on these observations to 1) dissect the specific mechanisms regulating SMN expression during development and treatment, 2) identify the molecular mechanisms causing impaired maturation and degeneration of SMA MNs, and 3) use these insights to develop novel and in utero SMA therapeutic strategies. In parallel studies on dSMA, we have recently demonstrated that neuropathogenic mutations in TRPV4, a cell surface cation channel, disrupt regulatory protein-protein interactions and cause a gain of channel function. Existing TRPV4 antagonists have good tolerability in humans, making the channel a promising therapeutic target. Strikingly, mutant TRPV4 knock-in mouse models develop severe neurological phenotypes due to focal breakdown of blood-neural barriers (BNBs), which are rescued by selective genetic deletion of TRPV4 from endothelial cells (ECs) or treatment of symptomatic mice with TRPV4 antagonists. These studies suggest that TRPV4 activation can drive neuropathology in a non-cell autonomous manner by regulating BNBs. Here, we will 1) characterize protein interactions regulating TRPV4 channel activity, 2) evaluate the role of TRPV4 in modulating EC barrier function, and 3) assess TRPV4 antagonists as a therapeutic strategy in dSMA mice and ultimately other disorders characterized by BNB disruption. Together, our studies will further our mechanistic understanding of SMA pathology, delineate novel therapeutic targets and strategies, and advance care of patients with SMAs and related neuromuscular diseases.