Identification of affected cellular targets, mechanisms and signalling pathways in mouse and cell models for spinal muscular atrophy with respiratory distress type 1 (SMARD1).

肌肉萎缩和隔膜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)小鼠中得到很好的表现，模拟了SMARD1的幼年型。在人类和小鼠中，IGHMBP2基因的突变都会导致运动神经元退化。我们以前可以证明，使用IGF1的聚乙二醇偶联体(PEG-IGF1)治疗可以改善Nmd2J小鼠的腓肠肌和横隔肌的运动功能，并减少纤维变性，但对运动神经元的存活没有任何有利影响。IGHMBP2/Ighmbp2是一个核糖体相关的ATPase/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，即影响细胞自主疾病的机制和信号通路导致Ighmbp2缺陷的运动神经元和肌肉纤维功能障碍和丢失。对Nmd2J小鼠原代Ighmbp2缺陷运动神经元和脊髓组织的分析显示，分化缺陷与兴奋性障碍对应的是N型特异性电压门控钙通道(Cav2.2)积聚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，我们将开始对初级Ighmbp2缺陷运动神经元进行详细的形态和功能分析。在特定培养条件下，原代Ighmbp2缺陷小鼠运动神经元将接受光漂白后荧光恢复(FRAP)和钙成像研究。我们最终想要找出在Ighmbp2缺陷运动神经元中哪些特定的信号通路受到影响，从而导致功能故障。在体外研究的同时，我们将详细分析TRPC5和Nav1.9基因敲除背景下的Nmd2J小鼠，以发现这些离子通道是否是Nmd2J表型的修饰物。这种细胞靶标的间接鉴定将通过微阵列研究来完成，以便在RNA和蛋白质水平上识别其他受影响的细胞靶标。此外，还计划使用SMARD1患者的皮肤成纤维细胞进行再分化。重新分化的人运动神经元将与原代小鼠运动神经元进行功能和形态的比较，以验证它们作为进一步分析Ighmbp2突变运动神经元功能缺陷的可靠工具的有效性。在最后一个工作页面中，我们想要详细分析导致SMARD1小鼠横隔肌和腓肠肌发生肌病变化的细胞自主机制。通过我们的项目，我们希望了解Ighmbp2缺陷运动神经元和肌肉中受影响的细胞机制，以便识别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.