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)

项目摘要 免疫球蛋白hMU结合蛋白2基因(IGHMBP2)的有害隐性突变会导致 运动神经元病(MND)的范围从不太严重的年轻人起病的运动和感觉 神经性疾病，Charcot-Marie-Tooth病2S型(CMT2S)，对严重且往往致命的年轻人 儿童疾病，脊髓性肌萎缩症合并呼吸窘迫1型(SMARD1)。IGHMBP2突变 也与婴儿猝死综合症(SID)有关。由于这些疾病的罕见，不是 许多研究都被投入到可能的治疗方法上，更不用说IGHMBP2在导致 表型。IGHMBP2被认为编码一种低加工性的DNA/RNA解旋酶。它无处不在 在全身低水平表达，性腺和神经组织表达最高。它 也被认为在翻译中起作用，因为它与核糖体有关。通过创建 几个代表在人类患者中发现的疾病谱的老鼠模型，我们在 了解IGHMBP2不仅在这些疾病中发挥作用，而且在神经肌肉中也发挥作用 开发/维护。我们最近对严重的SMARD1和CMT2S进行了RNA测序 模型，并发现免疫系统Rig-I样受体(RLR)途径在脊髓中高度上调 这些老鼠的绳索。这一途径与检测典型的病毒双链RNA和 通过核糖核酸酶L导致感染细胞的最终死亡。我们还在MND小鼠模型上进行了研究 核出口调节因子编码基因Nemf存在有害的隐性突变。在这些老鼠身上， 我们看到了类似的疾病严重程度。NEMF蛋白因其在核糖体质量中的作用而广为人知 控制力。在我们对突变的Nemf小鼠脊髓的RNAseq分析中，我们看到了类似的上调 RLR途径。我们还培育了Nemf和Ighmbp2基因突变的杂合子小鼠。当一个 杂合表型通常不是每个突变基因都单独出现的，这些双重杂合突变体 表现为严重的MND表型。这表明这些突变影响的是相同的途径。我假设 IGHMBP2功能失调和/或IGHMBP2减少会导致某些与 神经发育/维持建立，触发RLR通路并导致马达死亡 神经元。这项研究将确定RLR途径是否具有反应、改善或有害的影响 神经肌肉变性。这项研究开启了与RLR途径相关的一类新的MND。我 除了MND突变外，还提出了一系列与RLR基因敲除相关的基因杂交。我也是 建议使用单核RNAseq和RNAScope，一种单细胞荧光原位杂交分析方法 Ighmbp2和Nemf突变小鼠的脊髓以确定RLR信号的来源。

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.