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).

在患有 1 型呼吸窘迫的脊髓性肌萎缩症 (SMARD1) 小鼠模型中，分析导致细胞自主和非细胞自主疾病机制的受影响细胞靶点。

• 批准号: 467428013
• 负责人: Privatdozentin Dr. Sibylle Jablonka
• 金额: --
• 依托单位: Institut für Klinische Neurobiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/467428013?language=en
• 关键词: Analysis; affected; cellular; targets; causing

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.

肌肉萎缩和痉挛性麻痹是脊髓性肌萎缩伴呼吸窘迫1型（SMARD 1）的临床特征，在神经肌肉变性（Nmd 2 J）小鼠中有很好的代表性。在人类和小鼠中，IGHMBP 2基因的突变均导致运动神经元变性。IGHMBP 2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，导致SMARD 1的细胞水平上的疾病机制仍远未被理解。来自Nmd 2 J小鼠胚胎的原代Ighmbp 2缺陷运动神经元的分析仅表现出轻微的形态学变化，例如轴突分支的轻微增加。Ighmbp 2缺陷型运动神经元的RNA测序仅显示少数转录组改变（例如，G. Sparcl 1下调和FGFR 1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Ighmbp 2缺陷运动神经元的生长锥中β-肌动蛋白蛋白水平降低。这伴随着索马和生长锥中IMP 1/ZBP 1水平的降低，对应于总IMP 1的降低，但IMP 1 mRNA量一致。基于这些数据，我们的项目将集中在以下目标：（1）IMP 1过表达是否补偿IgHMBP-2缺陷运动神经元的功能改变？(2)非细胞自主性疾病机制在何种程度上导致SMARD 1中运动神经元变性？为了发现IMP 1在何种程度上导致来自SMARD 1患者的Nmd 2 J运动神经元和神经元前体细胞（NPC）中的细胞功能障碍，将通过慢病毒基因转移过表达IMP 1。形态学和功能分析将揭示IMP 1过表达是否挽救了受影响的轴突分支、远端轴突中的β-肌动蛋白缺陷以及重新平衡mRNA谱。IMP 1对Ighmbp 2缺陷型运动神经元功能失调的贡献将通过IMP 1过表达和敲低方法进行研究，包括高分辨率显微镜、质谱分析以及RiboTag/ChAT-Cre小鼠实验。此外，我们将扩展我们的研究对一个新确定的相互作用的合作伙伴IMP 1，FGFR 1 mRNA。在Ighmbp 2缺陷下，FGFR 1信号失调如何影响运动神经元存活的体外/体内研究将使我们首次深入了解Nmd 2 J小鼠的非细胞自主性疾病机制。目的（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SMARD 1小鼠模型中的非细胞自主疾病机制。来自共培养、转录组数据、脊髓“三联体”突触和突触输入的分析的结果将增加我们对SMARD 1的疾病机制和进展的认识。