Arakawa Reiko, Arakawa Masayuki, Kaneko Kaori, Otsuki Noriko, Aoki Ryoko, Saito Kayoko
Institute of Medical Genetics, Tokyo Women's Medical University, Tokyo, Japan.
Laboratory of Virology, Institute of Microbial Chemistry, Microbial Chemistry Research Foundation, Tokyo, Japan.
Pediatr Neurol. 2016 Aug;61:70-5. doi: 10.1016/j.pediatrneurol.2016.05.009. Epub 2016 May 26.
Spinal muscular atrophy is a neurodegenerative disorder caused by the deficient expression of survival motor neuron protein in motor neurons. A major goal of disease-modifying therapy is to increase survival motor neuron expression. Changes in survival motor neuron protein expression can be monitored via peripheral blood cells in patients; therefore we tested the sensitivity and utility of imaging flow cytometry for this purpose.
After the immortalization of peripheral blood lymphocytes from a human healthy control subject and two patients with spinal muscular atrophy type 1 with two and three copies of SMN2 gene, respectively, we used imaging flow cytometry analysis to identify significant differences in survival motor neuron expression. A bright detail intensity analysis was used to investigate differences in the cellular localization of survival motor neuron protein.
Survival motor neuron expression was significantly decreased in cells derived from patients with spinal muscular atrophy relative to those derived from a healthy control subject. Moreover, survival motor neuron expression correlated with the clinical severity of spinal muscular atrophy according to SMN2 copy number. The cellular accumulation of survival motor neuron protein was also significantly decreased in cells derived from patients with spinal muscular atrophy relative to those derived from a healthy control subject.
The benefits of imaging flow cytometry for peripheral blood analysis include its capacities for analyzing heterogeneous cell populations; visualizing cell morphology; and evaluating the accumulation, localization, and expression of a target protein. Imaging flow cytometry analysis should be implemented in future studies to optimize its application as a tool for spinal muscular atrophy clinical trials.
脊髓性肌萎缩症是一种神经退行性疾病，由运动神经元中生存运动神经元蛋白表达不足引起。疾病修饰疗法的一个主要目标是提高生存运动神经元的表达。患者外周血细胞可监测生存运动神经元蛋白表达的变化；因此，我们为此测试了成像流式细胞术的敏感性和实用性。
分别从一名健康对照者和两名1型脊髓性肌萎缩症患者（分别携带2份和3份SMN2基因）的外周血淋巴细胞永生化后，我们使用成像流式细胞术分析来确定生存运动神经元表达的显著差异。采用明亮细节强度分析来研究生存运动神经元蛋白细胞定位的差异。
与健康对照者来源的细胞相比，脊髓性肌萎缩症患者来源的细胞中生存运动神经元表达显著降低。此外，根据SMN2拷贝数，生存运动神经元表达与脊髓性肌萎缩症的临床严重程度相关。与健康对照者来源的细胞相比，脊髓性肌萎缩症患者来源的细胞中生存运动神经元蛋白的细胞积累也显著降低。
成像流式细胞术用于外周血分析的优点包括能够分析异质细胞群体、可视化细胞形态以及评估靶蛋白的积累、定位和表达。未来的研究应采用成像流式细胞术分析，以优化其作为脊髓性肌萎缩症临床试验工具的应用。
