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，约50%的患者在儿童早期死亡。SMN1基因突变导致脊髓中运动神经元（MNs）的功能丧失，主要影响神经肌肉连接的发育和成熟。突触传递受损导致肌无力和近端随意肌萎缩。该疾病的严重程度主要受拷贝基因SMN2的影响，SMN2在90%的转录本中异常剪接，缺乏外显子7，很少受其他遗传修饰因子的影响。在过去的几年里，我们确定了人类遗传修饰物plastin 3 （PLS3）并对其进行了功能表征，而在上一个资助期间，我们确定了第二个修饰物，命名为MOD2。最重要的是，只有这两种修饰因子的发现才使我们最近发现了SMA的主要病理细胞紊乱。我们分析了一个大的SMA家族，其中有5个smn1缺失的个体，尽管只携带4个SMN2拷贝，但完全无症状，通常导致II型或III型SMA。转录组和连锁分析揭示了MOD2是一种新的SMA保护修饰子。与家族中受影响的人和其他具有4个SMN2拷贝的独立SMA患者相比，所有5名无症状个体的MOD2表达均较低。MOD2是一种神经元钙传感器蛋白，在脑内和NMJ水平上强烈表达。重要的是，抑制Mod2可以恢复细胞培养中SMA引起的表型和各种SMA模型中的MN功能，包括斑马鱼，蠕虫，以及根据初步数据，小鼠。在下一个资助期内，我们的目标是利用一种新型小鼠模型揭示Mod2敲除的影响及其对两种不同SMA模型的修饰作用：一种类似I型SMA患者的严重SMA模型和一种类似II型SMA患者的轻度SMA模型。将进行详细的形态学、组织学和功能分析。为了揭示Mod2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报告构建物和活细胞成像研究突触神经传递。为抑制MOD2提供所需的科学证据，可能允许其在未来的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.