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）小鼠中有很好的体现。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是一种核糖体相关的atp酶/解旋酶，被认为参与核糖体和翻译事件/过程。然而，在细胞水平上导致SMARD1的疾病机制仍远未被了解。对来自Nmd2J小鼠胚胎的原发性ighmbp2缺陷运动神经元的分析显示，只有轻微的形态学变化，如轴突分支的轻微增加。对缺乏ighmbp2的运动神经元的RNA测序显示只有少数转录组改变（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缺陷运动神经元生长锥的β-肌动蛋白水平降低。这伴随着胞体和生长锥中IMP1/ZBP1水平的降低，对应于IMP1总量的减少，但与IMP1 mRNA数量一致。基于这些数据，我们的项目将集中在以下目标上：(1)IMP1过表达是否补偿ighmbp -2缺陷运动神经元的功能改变？(2)在何种程度上非细胞自主疾病机制导致了SMARD1的运动神经元退化？为了发现IMP1在多大程度上导致了来自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，IMP1将通过慢病毒基因转移过表达。形态学和功能分析将揭示IMP1过表达是否能挽救受影响的轴突分支、远端轴突β-肌动蛋白缺陷和重新平衡mRNA谱。IMP1对ighmbp2缺陷运动神经元功能失调的贡献将通过IMP1过表达和敲低的方法进行研究，包括高分辨率显微镜、质谱分析和RiboTag/ChAT-Cre小鼠实验。此外，我们将扩展我们对新发现的IMP1相互作用伙伴FGFR1 mRNA的研究。在体外/体内研究FGFR1信号失调如何影响Ighmbp2缺乏症下的运动神经元存活，将使我们首次了解Nmd2J小鼠的非细胞自主疾病机制。目的(2)将专注于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.