Identification of affected cellular targets, mechanisms and signalling pathways in mouse and cell models for spinal muscular atrophy with respiratory distress type 1 (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，并且在神经肌肉变性（Nmd 2 J）小鼠中得到了很好的表现，模拟了SMARD 1的幼年形式。在人类和小鼠中，IGHMBP 2基因的突变均导致运动神经元变性。我们先前可以证明，用IGF 1的聚乙二醇偶联变体（PEG-IGF 1）治疗可以改善运动功能，同时减少Nmd 2 J小鼠腓肠肌和膈肌的纤维变性，但对运动神经元的存活没有有益的影响。IGHMBP 2/Ighmbp 2是一种核糖体相关的ATP酶/解旋酶，被认为参与核糖体事件/编排和/或翻译事件。这些数据提出了一个问题，即影响细胞自主性疾病的机制和信号通路有助于Ighmbp 2缺陷运动神经元和肌纤维的功能障碍和损失。来自Nmd 2 J小鼠的原发性Ighmbp 2缺陷运动神经元和脊髓组织的分析显示出分化缺陷，兴奋性失败对应于N型特异性电压门控钙通道（Cav2.2）积累减少和瞬时受体电位通道（TRPC）表达失调。为了发现受影响的细胞机制和信号通路，我们将开始一个详细的形态和功能分析的主要IgHmbp 2缺陷运动神经元。将在特定培养条件下对原代Ighmbp 2缺陷型小鼠运动神经元进行光漂白后恢复（FRAP）和钙成像研究。最后，我们想找出哪些特定的信号通路受到影响IgHMBP 2缺陷运动神经元导致功能障碍。在体外研究的同时，我们将详细分析TRPC 5和Nav1.9敲除背景下的Nmd 2 J小鼠，以发现这些离子通道是否是Nmd 2 J表型的修饰剂。细胞靶标的这种间接鉴定将通过微阵列研究完成，以在RNA和蛋白质水平上鉴定其他受影响的细胞靶标。此外，计划用来自SMARD 1患者的皮肤成纤维细胞进行再分化。将再分化的人运动神经元与原代小鼠运动神经元进行功能和形态学比较，以验证其作为进一步分析Ighmbp 2突变运动神经元中功能缺陷的可靠工具的效力。在最后一个工作页中，我们想详细分析导致SMARD 1小鼠膈肌和腓肠肌肌病变化的细胞自主机制。通过我们的项目，我们想了解Ighmbp 2缺陷运动神经元和肌肉中受影响的细胞机制，以确定可以被刺激的信号通路，从而绕过受影响的细胞机制。

Muscle atrophy, and diaphragmatic palsy are the clinical characteristics of spinal muscular atrophy with respiratory distress type 1 (SMARD1), and are well represented in the neuromuscular degeneration (Nmd2J) mouse, modeling the juvenile form of SMARD1. Both in humans and mice mutations in the IGHMBP2 gene lead to motoneuron degeneration. We could previously demonstrate that treatment with a polyethylene glycol-coupled variant of IGF1 (PEG-IGF1) improves motor functions accompanied by reduced fiber degeneration in the gastrocnemius muscle and the diaphragm of the Nmd2J mouse, but has no beneficial effect on motoneuron survival. IGHMBP2/Ighmbp2 is a ribosome associated ATPase/helicase supposed to be involved in ribosomal events/orchestration and/or translational events. These data raised the question which affected cell autonomous disease mechanisms and signalling pathways contribute to dysfunction and loss of Ighmbp2 deficient motoneurons and muscle fibers. An analysis of primary Ighmbp2 deficient motoneurons and spinal cord tissue from Nmd2J mice exhibited differentiation deficits with excitability failures corresponding to reduced N-type specific voltage-gated calcium channel (Cav2.2) accumulation and a dysregulation in the expression of transient receptor potential channels (TRPCs). In order to discover affected cellular mechanisms and signalling pathways, we will start a detailed morphological and functional analysis of primary Ighmbp2 deficient motoneurons. Primary Ighmbp2 deficient mouse motoneurons under specific culture conditions will be subjected to a Fluorescence-Recovery after Photobleaching (FRAP) and a Calcium Imaging study. We finally want to find out which specific signalling pathways are affected in Ighmbp2 deficient motoneurons leading to functional failures. Simultaneously to the in vitro study, we will analysed in detail Nmd2J mice on a TRPC5 and Nav1.9 knock-out background to discover whether these ion channels are modifiers of the Nmd2J phenotype. This indirect identification of a cellular target will be completed by microarray studies in order to identify other affected cellular targets on RNA and protein levels. Furthermore, a re-differentiation approach with skin fibroblast from SMARD1 patients is planned. The re-differentiated human motoneurons will be functional and morphological compared with primary mouse motoneurons to verify their potency as a reliable tool for the further analysis of functional deficits in Ighmbp2 mutated motoneurons. In a last workpage we want to analyse in detail the cell autonomous mechanisms which lead to myopathic changes of diaphragm and gastrocnemius muscle in the SMARD1 mouse. By means of our project we would like to understand affected cellular mechanisms in Ighmbp2 deficient motoneurons and muscles in order to identify signalling pathways which could be stimulated and thus bypass the affected cellular mechanisms.