Spinal muscular atrophy: Analysis of pathomechanistic impact of protective genetic modifiers in mouse models

脊髓性肌萎缩症(SMA)是一种常见的神经肌肉疾病，在约50%的患者中会导致儿童早期死亡。SMN1基因突变导致脊髓运动神经元(MN)功能丧失，主要影响神经肌肉接头的发育和成熟。突触传递受损会导致近端随意肌无力和萎缩。疾病的严重程度主要受复制基因SMN2的影响，该基因在90%的转录本中缺少外显子7而异常剪接，很少受到额外的遗传修饰物的影响。在过去的几年里，我们确定了人类遗传修饰物PLS3(PLS3)的功能特征，而在上一次资助期间，我们确定了第二个修饰物，在这里命名为MOD2。最重要的是，最近只有这两个修饰物的发现才将我们指向SMA的主要病理细胞紊乱。我们分析了一个大型SMA家族，该家族有5个SMN1缺失个体，尽管只携带4个SMN1拷贝，但他们完全没有症状，通常导致II型或III型SMA。转录组和连锁分析揭示了MOD2作为一种新的SMA保护性修饰物。所有5名无症状个体的MOD2表达均低于家族中的患者和其他具有4个SMN2拷贝的独立SMA患者。MOD2是一种神经元性钙感受器蛋白，在大脑和NMJ水平强烈表达。重要的是，抑制mod2可以恢复SMA在细胞培养中引起的表型和各种SMA模型中的MN功能，包括斑马鱼、蠕虫，根据初步数据，在小鼠中也是如此。在下一个资助期内，我们的目标是利用一种新的小鼠模型来揭示mod2基因敲除的影响，以及它对两种不同的SMA模型的修改效果：一种是类似于I型SMA患者的重度SMA模型，另一种是类似于II型患者的较轻SMA模型。将进行详细的形态、组织学和功能分析。为了揭示mod2基因敲除对神经元发育和突触传递的影响，我们将广泛分析培养的MNS和海马神经元，并使用pH敏感的GFP报告结构和活细胞成像来研究突触神经传递。为抑制MOD2提供所需的科学证据可能会使其在未来的SMA临床试验中使用。

Spinal muscular atrophy (SMA) is a common neuromuscular disorder leading to early childhood lethality in about 50% of patients. Mutations in the SMN1 gene cause functional loss of the ¿-motor neurons (MNs) in the spinal cord mainly affecting development and maturation of neuromuscular junctions. Impaired synaptic transmission causes muscle weakness and atrophy of proximal voluntary muscles. The disease severity is mainly influenced by a copy gene, SMN2, which is aberrantly spliced lacking exon 7 in 90% of transcripts and, rarely, by additional genetic modifiers. In the past years we identified and functionally characterized the human genetic modifier plastin 3 (PLS3), while during the last funding period we identified a second modifier, denominated here as MOD2. Most importantly, only the discovery of both modifiers pointed us recently towards the main pathocellular disturbance in SMA.We analyzed a large SMA family with five SMN1-deleted individuals, who were fully asymptomatic despite carrying only four SMN2 copies, usually causing type II or III SMA. Transcriptome and linkage analysis unraveled MOD2 as a novel SMA protective modifier. All five asymptomatic individuals showed low MOD2 expression in comparison to the affected persons in the family and other independent SMA patients with four SMN2 copies. MOD2 is a neuronal calcium sensor protein, strongly expressed in brain and at NMJ level. Importantly, suppression of Mod2 restores SMA caused phenotype in cell culture and MN function across various SMA models, including zebrafish, worm, and, according to preliminary data, in mice. Within the next funding period we aim to unravel the impact of knock-down of Mod2 using a novel mouse model and its modifying effect on two different SMA models: a severe SMA model resembling a type I SMA patient and a milder SMA model resembling a type II patient. Detailed morphological, histological and functional analysis will be carried out. To unravel the impact of Mod2 knock-down on neuronal development and synaptic transmission, we will extensively analyze cultured MNs and hippocampal neurons and investigate synaptic neurotransmission using pH-sensitive GFP-reporter constructs and live cell imaging. Providing the required scientific proof for MOD2 suppression may allow its use in future SMA clinical trials.