Spinal Muscular Atrophy (SMA) is a neurological disorder characterized by loss of lower motor neurons. This degeneration is caused by genetic mutations that lead to decreased levels of Survival of Motor Neuron (SMN) protein. It is currently unknown how SMN reduction results in neuronal death, however recent evidence suggests that miRNA disruption may play a role. miRNAs are small non-coding RNAs predicted to regulate protein expression of a vast number of mRNAs. The RNA helicase Gemin3 pulls down with both SMN and numerous miRNAs in cultured mouse motor neurons. I hypothesize that Gemin3-associated miRNAs are misregulated in smn-1 loss-of-function(lf) animals leading to NMJ defects. smn-1(ok355) animals are defective on aldicarb, have reduced pumping and altered levels of synaptic proteins, suggesting neuromuscular junction (NMJ) defects. My preliminary data shows that loss of the C. elegans Gemin3 ortholog, mel-46, results in similar NMJ defects. Using tissue-specific rescue analysis and genetic epistasis, I will show that mel-46 is downstream of smn-1 in a pathway influencing NMJ signaling. Additionally, I have identified C. elegans orthologs of Gemin3-associated miRNAs that are necessary for proper NMJ synaptic transmission similar to smn-1 and mel-46. I will compile lists of potential mRNA targets for these candidate miRNAs using online bioinformatics tools; assigning priority to conserved targets with known synaptic function. For each potential mRNA target, I will confirm miRNA regulation and investigate whether miRNA function is altered in smn-1 lf and mel-46 lf. Furthermore, I have designed experiments to show loss of miRNA function in smn-1 lf animals is caused by mel-46 reduction. Using motor neurons from a SMA mouse model, I will show that the relationship between SMN, Gemin3 and miRNA function is conserved across species. These experiments will advance our knowledge of how SMN protein contributes to essential neuronal functions and expand our understanding of how miRNA misregulation may contribute to neurodegeneration.
The proposed work is significant to public health since Spinal Muscular Atrophy is the leading genetic cause of infantile death in the United States with an estimated 7.5 million American carriers for the mutation that causes the disorder. miRNAs have been implicated in a range of neurodegenerative disorders, including ALS and SMA, and have previously been used for therapies in a wide range of diseases and disorders. Utilizing the powerful screening methods of the model organism, C. elegans, this project aims to identify the specific miRNAs misregulated in an SMA model which will provide a better understanding of how miRNAs contribute to neurodegenerative disease.
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