Investigating the Role of the Helicase, IGHMBP2, and the RIG-I-Like Receptor Pathway in Spinal Muscular Atrophy with Respiratory Distress Type 1 and Neuromuscular Development (SMARD1)

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PROJECT ABSTRACT Deleterious recessive mutations in the immunoglobulin h mu-binding protein 2 gene (IGHMBP2) create a spectrum of motor neuron diseases (MNDs) ranging from the less severe young adult onset motor and sensory neuropathic disease, Charcot-Marie-Tooth disease type 2S (CMT2S), to the severe and often fatal young childhood disease, Spinal Muscular Atrophy with Respiratory Distress type 1 (SMARD1). IGHMBP2 mutations have also been associated with Sudden Infant Death Syndrome (SIDS). Due to the rarity of these diseases, not much research has been invested into possible therapeutics, let alone the role IGHMBP2 plays in causing the phenotype. IGHMBP2 is thought to encode a DNA/RNA helicase with low processivity. It is ubiquitously expressed at low levels throughout the body, with gonadal and nervous tissue having the highest expression. It is also believed to have a role in translation because of its association with ribosomes. Through the creation of several mouse models representing the spectrum of disease found in human patients, we have made strides in understanding that IGHMBP2 plays a role not only in these diseases, but in neuromuscular development/maintenance. We recently performed RNA sequencing on our severe SMARD1 and CMT2S models and found that the immune system RIG-I-like receptor (RLR) pathway is highly upregulated in the spinal cords of these mice. This pathway is associated with the detection of typically viral double-stranded RNA and causes eventual death of the infected cell via RNase L. We have also conducted studies on MND mouse models with deleterious recessive mutations in the nuclear export mediator factor-encoding gene Nemf. In these mice, we see a similar spectrum of disease severity. The NEMF protein is better known for its role in ribosome quality control. In our RNAseq analysis of the spinal cords of our mutated Nemf mice, we saw a similar upregulation of the RLR pathway. We also bred mice to be heterozygous for mutations in both Nemf and Ighmbp2. While a heterozygous phenotype is not typically seen for each mutated gene alone, these double heterozygous mutants showed a severe MND phenotype. This suggests that these mutations impact the same pathway. I hypothesize that dysfunctional IGHMBP2 and/or a decrease in IGHMBP2 causes certain RNA products associated with neural development/maintenance to build up, triggering the RLR pathway and causing death of the motor neurons. This study will determine if the RLR pathway has a reactive, ameliorative, or detrimental effect on neuromuscular degeneration. This study opens a novel category of MNDs associated with the RLR pathway. I propose a series of genetic crosses associated with knock-outs of RLR in addition to MND mutations. I also propose using single nuclear RNAseq and RNAscope, a single cell fluorescent in-situ hybridization assay, on the spinal cords of Ighmbp2 and Nemf mutant mice to determine the origin of the RLR signal.