Spinal muscular atrophy (SMA) is the leading genetic cause of death in infants world-wide. SMA is caused by reduced levels of functional survival of motor neuron (SMN) protein, leading to cell autonomous defects at the neuromuscular junctions, axon degeneration, and loss of motor neurons in the spinal cord. The ubiquitously expressed SMN protein has a well characterized essential function in the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs) in all tissues, but it is still unclear to what extent pre- mRNA splicing defects contribute to SMA. It is a central question in the field why spinal motor neurons are more severely affected by low SMN protein levels than other cell types. We and others have discovered that axons of cultured SMN-deficient motor neurons have impaired axonal localization of specific mRNA binding proteins (mRBPs) and mRNAs that are known to play roles in axon growth and maintenance. These findings have led us to hypothesize that SMN plays a critical role in the assembly and trafficking of messenger ribonucleoproteins (mRNPs) in neuronal processes that serve axonal growth and maintenance. In SMA, defects in the maturation of NMJs followed by their progressive impairment and loss are among the earliest events in the disease, and lead to a decline in basic motor functions essential for survival. NMJ vulnerability represents a critical, yet poorly understood, component of the disease. Despite having a well- characterized understanding of the protein deficiency that causes SMA, the contributions of this protein to the function, maintenance, and maturation of the NMJ remains unknown. While recently developed therapeutics are expected to increase the life expectancy and quality of life for affected SMA patients, a better understanding of disease mechanisms may be required for a complete rescue. With the goal to reveal axonal mRNA processing defects in SMA and their contribution to the earliest manifestations of the disease phenotype, we propose two specific aims: In Aim 1, we will use motor neuron- specific tagging of ribosomes with the RiboTag system to thoroughly characterize differences in the ribosome- associated transcriptome in axons and NMJs of spinal cord motor neurons of SMA mouse models. In Aim 2, we will use motor neuron-specific metabolic labeling of proteins with mutant MetRS* to uncover disease- specific molecular axonal defects by a detailed and comprehensive analysis of differences in the motor neuron proteome in the spinal cord and muscle tissue of SMA mouse models. Our pilot data demonstrate the feasibility of these novel approaches for remote cellular compartments such as distal axons and NMJs. The results of this study will offer a unique and thorough examination of the mRNA and protein composition and processing at the NMJ, revealing novel aspects of NMJ biology, disease progression, and targets for therapeutic interventions.
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