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): Spinal muscular atrophy (SMA) is a currently untreatable, autosomal recessive motor neuron disease that is the leading inherited cause of infant mortality. SMA is caused by deficiency of the survival motor neuron (SMN) protein. Our long term research goal is to understand the underlying pathogenesis of SMA in order to develop effective treatment strategies for this disease. Recent studies suggest that SMA begins because of intrinsic abnormalities of both muscle and motor nerve terminals;however the nature of these defects remains unknown. Histone deacetylase (HDAC) inhibitors have been shown to increase survival of SMA mice;but it is unclear how these drugs improve muscle and/or motor neuron function. In preliminary studies in SMA mice, we have shown that at a time when the mouse is profoundly weak, there is little structural denervation. However there are widespread immature and hypotrophic myofibers as well as simplified neuromuscular junctions (NMJs). Mice treated with a pan- HDAC inhibitor show a substantial extension of survival, increase in motor function, and improvement in the size and maturity of myofibers, without a change in motor neuron number. Based on these preliminary data, we hypothesize that SMN deficiency causes an arrest of muscle maturation and/or a failure of NMJ transmission that can be overcome with HDAC inhibitors, which accelerate the development of the SMA motor unit. We further hypothesize that HDAC isoform-specific drugs will have distinct biological effects on the SMA motor unit that will provide crucial insights into the therapeutic mechanism of these compounds. We will test these hypotheses by: 1) establishing whether or not a defect of muscle development contributes to SMA by studying cultured SMA muscle cells and by rescuing SMN expression specifically in muscle tissue in conditional SMA mice, 2) determining whether or not weakness in SMA is due to an impairment of neuromuscular transmission by examining the electrophysiology and morphology of the NMJs in SMA mice, and 3) characterizing the ability of HDAC inhibitors to facilitate muscle and NMJ maturation and ameliorate SMA in mice by treating SMA muscle cells and SMA mice with broadly active and HDAC-isoform specific HDAC inhibitors. PUBLIC HEALTH RELEVANCE: This work is important for public health because spinal muscular atrophy is the leading inherited cause of infant mortality and is currently untreatable. In this project, we plan to define the roles of muscle and motor neuron terminals in the pathogenesis of SMA and to explore the therapeutic mechanism of histone deacetylase inhibitors. These studies will provide important insights about what tissue and molecules are necessary to target therapeutically in SMA and will therefore guide future efforts to develop treatment for this disease.

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.

? DESCRIPTION (provided by applicant): 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), but all patients retain one or more copies of the homologous SMN2 gene that produce inadequate levels of SMN protein due to an alternative splice event. Novel therapeutics including splice-switching oligonucleotides (SSOs) can modulate this splice event thus increasing SMN expression. SSOs are currently being tested in phase III clinical trials in SMA patients, but they have a ceiling effect imposed by existing levels of SMN2 pre-mRNA. The efficacy of SMA splice-modulating treatments could be substantially accentuated by combining such treatments with specific SMN2 promoter activation. In preliminary data, we have identified a novel SMN2- associated long non-coding RNA (SMN-NAT), which is enriched in the CNS and represses SMN2 gene expression via recruitment of the epigenetic modifier Polycomb repressive complex 2 (PRC2). We have further shown that SMN-NAT-targeting antisense oligonucleotides (ASOs) can suppress SMN-NAT resulting in enhanced expression of SMN in cultured cells. In order to further explore whether SMN-NAT regulates SMN2 in human SMA patient cells and in neurons, in Specific Aim 1 we will examine the effects of SMN-NAT ASOs in several fibroblast cell lines derived from SMA patients and controls, in primary neurons derived from severe SMA mice, and in motor neurons derived from SMA induced pluripotent stem cells (iPSCs). In Specific Aim 2, we will further define the role of the PRC2 complex in the epigenetic control of SMN2 gene expression in SMA patient cells and neurons. Finally, in Specific Aim 3, we will examine the therapeutic potential of targeting SMN-NAT by treating severe SMA mice with SMN-NAT ASOs alone or in combination with SSOs and examining effects on SMN expression, behavioral outcomes, and motor unit histology. Together, these studies will characterize novel mechanisms of SMN2 gene control in neurons and determine whether targeting a SMN2-associated lncRNA could represent a new therapeutic strategy for SMA, which has the promise of working additively with splice modulating treatments.

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 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.

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.