Analysis of affected cellular targets causing cell-autonomous and non-cell-autonomous disease mechanisms in a mouse model for spinal muscular atrophy with respiratory distress type 1 (SMARD1).

肌肉萎缩和膈肌麻痹是脊髓性肌萎缩伴呼吸窘迫 1 型 (SMARD1) 的临床特征，在神经肌肉变性 (Nmd2J) 小鼠中得到很好的体现。在人类和小鼠中，IGHMBP2 基因的突变都会导致运动神经元变性。 IGHMBP2 是一种核糖体相关 ATP 酶/解旋酶，被认为参与核糖体和翻译事件/过程。然而，导致 SMARD1 的细胞水平疾病机制仍远未被了解。对 Nmd2J 小鼠胚胎的原代 Ighmbp2 缺陷运动神经元的分析仅显示出微小的形态变化，例如轴突分支的轻微增加。 Ighmbp2 缺陷运动神经元的 RNA 测序仅揭示了一些转录组改变（例如 Sparcl1 下调和 FGFR1 上调）。同样，我们没有检测到蛋白质合成的任何全局变化。然而，我们观察到 Ighmbp2 缺陷运动神经元的生长锥中 β-肌动蛋白水平降低。这伴随着体细胞和生长锥中 IMP1/ZBP1 水平的降低，对应于总 IMP1 的减少，但 IMP1 mRNA 量一致。基于这些数据，我们的项目将重点关注以下目标：（1）IMP1过度表达是否可以补偿Ighmbp-2缺陷运动神经元的功能改变？ (2) 非细胞自主疾病机制在多大程度上导致 SMARD1 运动神经元变性？为了发现 IMP1 在多大程度上导致 SMARD1 患者的 Nmd2J 运动神经元和神经元前体细胞 (NPC) 的细胞功能障碍，慢病毒基因转移将导致 IMP1 过表达。形态学和功能分析将揭示 IMP1 过表达是否可以挽救受影响的轴突分支、远端轴突中的 β-肌动蛋白缺陷并重新平衡 mRNA 谱。 IMP1 对 Ighmbp2 缺陷运动神经元功能失调的影响将通过 IMP1 过表达和敲低方法（包括高分辨率显微镜、质谱分析）以及 RiboTag/ChAT-Cre 小鼠实验的帮助来研究。此外，我们将扩展对 IMP1 新发现的相互作用伙伴 FGFR1 mRNA 的研究。关于 FGFR1 信号传导失调如何影响 Ighmbp2 缺陷下运动神经元存活的体外/体内研究将使我们首次了解 Nmd2J 小鼠的非细胞自主疾病机制。目标 (2) 将专门关注主要基于星形胶质细胞/运动神经元共培养的 SMARD1 小鼠模型中的非细胞自主疾病机制。共培养的结果、转录组数据、脊髓“三方”突触和突触输入的分析将增加我们对 SMARD1 疾病机制和进展的了解。

Muscle atrophy, and diaphragmatic palsy are the clinical characteristics of spinal muscular atrophy with respiratory distress type 1 (SMARD1), well represented in the neuromuscular degeneration (Nmd2J) mouse. Both in humans and mice mutations in the IGHMBP2 gene lead to motoneuron degeneration. IGHMBP2 is a ribosome-associated ATPase/helicase supposed to be involved in ribosomal and translational events/processes. However, disease mechanisms on cellular level leading to SMARD1 are still far from being understood.An analysis of primary Ighmbp2-deficient motoneurons from Nmd2J mouse embryos exhibit only minor morphological changes such as a slight increase of axonal branches. RNA sequencing of Ighmbp2-deficient motoneurons revealed only a few transcriptome alterations (e. g. Sparcl1 down-regulation and FGFR1 upregulation). Likewise, we did not detect any global changes in protein synthesis. However, we observed reduced β-actin protein levels at the growth cone of Ighmbp2-deficient motoneurons. This is accompanied by reduced levels of IMP1/ZBP1 in soma and growth cone, corresponding to a decrease of total IMP1 but consistent IMP1 mRNA amount. Based on these data, our project will focus on the following objectives:(1) Does IMP1 overexpression compensates for functional alterations in Ighmbp-2deficient motoneurons? (2) To what extend non-cell-autonomous disease mechanisms contribute to motoneuron degeneration in SMARD1?In order to discover, to what extend IMP1 contributes to cellular dysfunctions in Nmd2J motoneurons and in neuronal precursor cells (NPCs) from SMARD1 patients, IMP1 will be overexpressed by lentiviral gene transfer. Morphological and functional analyses will reveal whether IMP1 overexpression rescues affected axonal branching, β-actin deficit in the distal axon and re-balances mRNA profiles. The contribution of IMP1 to the functional dysregulations in Ighmbp2-deficient motoneurons will be investigated by IMP1 overexpression and knockdown approaches including high resolution microscopy, mass spectrometry analysis, and with the aid of RiboTag/ChAT-Cre mouse experiments. In addition, we will extend our studies on a newly identified interaction partner of IMP1, the FGFR1 mRNA. In vitro/vivo studies on how dysregulation of FGFR1 signaling influences motoneuron survival under Ighmbp2 defciency will give us first insights into non-cell-autonomous disease mechanisms in Nmd2J mice. Objective (2) will focus exclusively on non-cell autonomous disease mechanisms in the SMARD1 mouse model primarily based on astrocyte/motoneuron co-cultures. Results from the co-cultures, the transcriptome data, the analysis of the spinal "tripartite" synapse and the synaptic input will increase our knowledge about disease mechanisms and progression in SMARD1.