Umrao Monani, Phd - US grants

[unreadable] DESCRIPTION (provided by applicant): Proximal spinal muscular atrophy (SMA) is a common neuromuscular disorder caused by mutations in the Survival of Motor Neuron 1 (SMN1) gene and insufficient levels of its translated product, the SMN protein. SMA is the most common genetic cause of childhood mortality. Hallmarks of the disease in SMA mice and human patients include spinal motor neuron loss and skeletal muscle atrophy. Based on these characteristics it is widely believed that motor neurons are selectively vulnerable to reduced SMN and that muscle atrophy is a secondary consequence of neurodegeneration. These long-held beliefs notwithstanding, there continues to be a vigorous debate about whether motor neurons are indeed uniquely susceptible to reduced levels of SMN acting cell autonomously within them. Alternatively, neurodegeneration could be triggered by primary effects on some other cell type closely associated with motor neurons. If SMN does function within motor neurons to ensure their health and survival, it is not clear why they and not other cells are so sensitive to reduced levels of the protein. To better understand the molecular and cellular causes of SMA, mouse models that genetically mimic the human condition have been generated. In this application for funding to the NIH, we have outlined experiments described in three related aims to determine if SMA is a disease dictated exclusively by the health of the motor neurons and whether restoring normal levels of the SMN protein to this cell type is sufficient to completely ameliorate the disease phenotype. We propose to answer this question in two ways. Firstly, we will restore SMN selectively to the motor neurons of mice with SMA and ask if this results in complete phenotypic correction. Secondly, we will selectively deplete the SMN protein in the motor neurons and two associated tissues, muscle and glia, of healthy mice and ask to what extent such manipulations create neuromuscular pathology. In a second set of experiments, we will determine why insufficient SMN protein causes a selective degeneration of the neuromuscular system. To answer this question, we will look at the effects of reduced SMN on the development of the nerve-muscle synapse of SMA mice. If reduced SMN disrupts the development of this synapse and its constituent proteins which are crucial in ensuring proper nerve-muscle function, it will explain the neuromuscular pathology so characteristic of the human disease. Given the high frequency of SMA among humans, the lack of an effective treatment and the consequent burden it places on society, it is imperative that questions such as those posed here be answered in as timely a manner as possible. PUBLIC HEALTH RELEVANCE: Spinal muscular atrophy is a devastating neurodegenerative disease and the leading genetic killer of infants and toddlers. SMA is not presently treatable. Understanding why SMA results in neuromuscular failure and death is important to designing an appropriate treatment. In this proposal, we will use mouse models of the human condition to determine which cell types contribute to neuromuscular failure and why they degenerate. We believe our results will profoundly impact the design of successful therapies for SMA. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Spinal muscular atrophy (SMA) is a common, frequently fatal, autosomal recessive disorder caused by homozygous mutations in the Survival of Motor Neuron 1 (SMN1) gene that lead to a deficiency of the SMN protein. Residual protein is expressed from SMN2, a partially functional homologue of the SMN1 gene. There is presently no cure for SMA. Currently available treatments are palliative at best. Although much has been learned about the pathology and natural history of the human disease and notwithstanding proof-of-concept studies demonstrating rescue of an SMA phenotype by restoring SMN to mouse models of the disease, the biochemical pathway(s) linking low levels of the protein to neurodegeneration remain(s) obscure. The single established function of SMN in orchestrating snRNP biogenesis has failed to shed adequate light on the motor neuron phenotype observed in SMA, prompting the search for additional functions of the protein and/or genes linking SMN paucity and disrupted snRNP biogenesis to neuromuscular disease. Increasing SMN2 copy number leads to higher levels of the SMN protein in patients and mutant mice and results in milder phenotypes. However, in rare instances the correlation between SMN2 copies and disease severity no longer holds, implying the existence of additional genetic modifiers of the SMA phenotype. Identifying such modifiers is one way to uncover new, disease-relevant functions of the SMN protein or reveal effector genes through which a disruption in snRNP biogenesis causes the SMA phenotype. In this application for funding to the NIH, we have outlined experiments in two related aims to exploit a modification of the disease phenotype in mouse models of SMA to map and identify modifying loci. In aim 1 congenic strains of SMA mice will be created to precisely define how different genetic backgrounds affect the mutant phenotype. Additionally, mutants from defined inter-strain crosses between the congenic SMA carriers will be generated and characterized by molecular, cellular and phenotypic means. In aim 2, mutants with the most distinct disease phenotypes will be used in linkage studies to map and eventually identify modifier loci. To confirm the disease modifying effects of the identified loci we will re-introduce them into SMA mice exhibiting a typical disease phenotype. Our studies will have two important outcomes. First, they will uncover novel, disease-relevant biochemical pathways and thus inform the underlying biology of spinal muscular atrophy. Second, they will identify genes that could serve as new molecular targets for future SMA therapies. The results of our experiments will constitute an important step toward the design of safe and effective treatments for SMA patients. PUBLIC HEALTH RELEVANCE: SMA is a debilitating, frequently fatal, incurable human neuromuscular disorder caused by reduced SMN protein. We wish to define pathways that lead from reduced SMN to dysfunction and disease. To do so we have made mouse models that allows us to identify such pathways and novel associated genes. Defining the pathways and genes will not only lead to a better understanding of SMA but also serve to identify potential targets for safe and effective treatments for the disease.

Spinal muscular atrophy is a common, recessively inherited, pediatric neuromuscular disorder caused by mutations in the Survival of Motor Neuron 1 (SMN1) gene and a deficiency of the SMN protein. SMN is ubiquitously expressed and reported to play a critical role in RNA processing, by orchestrating the biogenesis of spliceosomal small nuclear ribonucleoprotein (snRNP) particles. The assembly of these particles is severely compromised in SMA model mice. Restoring SMN to the mutants not only corrects this defect but also fully rescues the SMA phenotype. Nevertheless, SMN?s role in snRNP assembly, which is a requirement of all cells, has been difficult to reconcile with the selective neuromuscular disease phenotype characteristic of SMA. One way to explain this conundrum is to suggest that transcripts selectively expressed in one or more cells of the neuromuscular system fail to be properly processed owing to defects in SMN?s housekeeping function. Alternatively, the selective SMA phenotype could stem from novel SMN functions in the motor unit. In this project we wish to address each possibility. In aim 1 of the project we will determine if neuronal agrin, which was found to be mis-spliced in SMA motor neurons, presumably as a consequence of defects in snRNP biogenesis, is a true mediator of the SMA phenotype. Neuronal agrin is known to be important for the development of neuromuscular synapses, structures that are profoundly affected in SMA. To test possible links between agrin and the SMA phenotype, we will transgenically restore the protein selectively to the motor neurons of SMA model mice. We will then assess the consequences of agrin repletion in the mice at the molecular, cellular and phenotypic levels. In aim 2 of the project we will identify transcriptional/splice alterations in SMA motor neurons during a critical window of time that defines neuromuscular synapse maturation. This experiment takes advantage of a novel line of tamoxifen-induced SMN knockdown mice that we have developed, and exploits new findings suggesting that the requirements for the SMN protein are greatest when neuromuscular synapses mature. Following acute depletion of SMN prior to or immediately after neuromuscular synapses mature, we will catalogue motor neuronal gene expression changes in mutants and controls. This approach which complements Aim 1, but is unbiased with respect to any one gene, will uncover molecules that are important in the maturation of the neuromuscular synapses, a process that is disrupted in SMA. Some of these molecular alterations may eventually point to novel, disease-relevant and phenotype- specific functions of the protein. The collective results of the project will lead to new insights into a disease for which an optimal treatment has yet to be developed, and whose phenotype continues to puzzle scientists in light of what is currently known about the SMN protein.

Project Summary Spinal muscular atrophy (SMA) is a common, frequently fatal, neuromuscular disorder caused by mutations in the Survival of Motor Neuron 1 (SMN1) gene and, consequently, a paucity of the SMN protein. In humans, an almost identical copy gene, SMN2, is unable to fully compensate for loss of SMN1 owing to a splicing defect and thus an inability to express sufficient protein to stave off disease. In the two decades that we have researched SMA much progress has been made, from the identification of the disease gene and the description of its protein to the generation of pre-clinical models and, most recently, the approval of Spinraza, a promising drug that raises SMN levels and thus thwarts the inevitable paralysis and frequent death associated with SMA. While Spinraza, in particular, raises considerable optimism for SMA patients, significant challenges remain and, in our minds, stem from two crucial deficiencies. First, despite the milestones achieved, how low SMN protein evolves into the SMA phenotype, selectively triggering motor neuron death and preferentially disabling the neuromuscular system remains a singular mystery. This is especially perplexing considering SMN?s most widely-cited function of orchestrating the splicing cascade. Identifying mediators that provide a logical explanation for why splicing defects cause SMA or, uncovering additional, more disease-relevant SMN functions is therefore not only mechanistically but also therapeutically relevant. Second, while it is clear that administering Spinraza provides immediate benefit to patients, it is premature to make a determination of the long-term outcome of such treatment; the drug is selectively delivered to the CNS, raising questions about the effects of chronic low SMN in the periphery. Besides, the strategy of raising SMN appears inadequate in the symptomatic patient. Here we describe 3 related sets of experiments that address the deficiencies identified above. Aim 1 proposes to define disease-relevant mechanisms by exploiting a novel line of SMA mice in which early mortality, motor neuron loss and a severe phenotype are replaced by prolonged survival, intact motor neurons and a decidedly mild phenotype. We hypothesize that a spontaneous mutation in a chaperone protein that the mice express suppresses the SMA phenotype. We will confirm and extend this finding to determine how the chaperone modulates the effects of low SMN. In aim 2, we will examine the potential long- term adverse effects of persistently low levels of SMN in muscles of model mice expressing normal protein in the CNS. Such rodents represent a pre-clinical model of SMA patients administered Spinraza. We propose that chronic low SMN in skeletal muscle has profoundly serious consequences for the health of the tissue and contributes to the overall SMA phenotype. In aim 3, we will determine if the disease-causing effects of low SMN in muscle can nevertheless be mitigated upon restoring protein post-symptomatically. Reversing such defects will inform the manner in which current treatments may have to be modified to prove more potent. Our study thus addresses important mechanistic as well as clinical aspects of SMA.

Project Summary Spinal muscular atrophy (SMA) is a common, frequently fatal, neuromuscular disorder caused by mutations in the Survival of Motor Neuron 1 (SMN1) gene and, consequently, a paucity of the SMN protein. In humans, an almost identical copy gene, SMN2, is unable to fully compensate for loss of SMN1 owing to a splicing defect and thus an inability to express sufficient protein to stave off disease. In the two decades that we have researched SMA much progress has been made, from the identification of the disease gene and the description of its protein to the generation of pre-clinical models and, most recently, the approval of Spinraza, a promising drug that raises SMN levels and thus thwarts the inevitable paralysis and frequent death associated with SMA. While Spinraza, in particular, raises considerable optimism for SMA patients, significant challenges remain and, in our minds, stem from two crucial deficiencies. First, despite the milestones achieved, how low SMN protein evolves into the SMA phenotype, selectively triggering motor neuron death and preferentially disabling the neuromuscular system remains a singular mystery. This is especially perplexing considering SMN's most widely-cited function of orchestrating the splicing cascade. Identifying mediators that provide a logical explanation for why splicing defects cause SMA or, uncovering additional, more disease-relevant SMN functions is therefore not only mechanistically but also therapeutically relevant. Second, while it is clear that administering Spinraza provides immediate benefit to patients, it is premature to make a determination of the long-term outcome of such treatment; the drug is selectively delivered to the CNS, raising questions about the effects of chronic low SMN in the periphery. Besides, the strategy of raising SMN appears inadequate in the symptomatic patient. Here we describe 3 related sets of experiments that address the deficiencies identified above. Aim 1 proposes to define disease-relevant mechanisms by exploiting a novel line of SMA mice in which early mortality, motor neuron loss and a severe phenotype are replaced by prolonged survival, intact motor neurons and a decidedly mild phenotype. We hypothesize that a spontaneous mutation in a chaperone protein that the mice express suppresses the SMA phenotype. We will confirm and extend this finding to determine how the chaperone modulates the effects of low SMN. In aim 2, we will examine the potential long- term adverse effects of persistently low levels of SMN in muscles of model mice expressing normal protein in the CNS. Such rodents represent a pre-clinical model of SMA patients administered Spinraza. We propose that chronic low SMN in skeletal muscle has a profoundly negative impact on the health of the tissue and contributes to the overall SMA phenotype. In aim 3, we will determine if the disease-causing effects of low SMN in muscle can nevertheless be mitigated upon restoring protein post-symptomatically. Reversing such defects will inform the manner in which current treatments may have to be modified to prove more potent. Our study thus addresses important mechanistic as well as clinical aspects of SMA.