T3S, INSERM UMR1124, Faculté des Sciences Fondamentales et Biomédicales, Université Paris Cité, F-75006 Paris, France.
INSERM UMR1163, Institut Imagine, Université Paris Cité, F-75015 Paris, France.
Int J Mol Sci. 2024 Sep 18;25(18):10039. doi: 10.3390/ijms251810039.
Dysregulated RNA metabolism caused by SMN deficiency leads to motor neuron disease spinal muscular atrophy (SMA). Current therapies improve patient outcomes but achieve no definite cure, prompting renewed efforts to better understand disease mechanisms. The calcium channel blocker flunarizine improves motor function in -deficient mice and can help uncover neuroprotective pathways. Murine motor neuron-like NSC34 cells were used to study the molecular cell-autonomous mechanism. Following RNA and protein extraction, RT-qPCR and immunodetection experiments were performed. The relationship between flunarizine mRNA targets and RNA-binding protein GEMIN5 was explored by RNA-immunoprecipitation. Flunarizine increases demethylase transcripts across cell cultures and mouse models. It causes, in NSC34 cells, a temporal expression of GEMIN5 and KDM6B. GEMIN5 binds to flunarizine-modulated mRNAs, including transcripts. depletion reduces mRNA and protein levels and hampers responses to flunarizine, including neurite extension in NSC34 cells. Moreover, flunarizine increases the axonal extension of motor neurons derived from SMA patient-induced pluripotent stem cells. Finally, immunofluorescence studies of spinal cord motor neurons in -deficient mice reveal that flunarizine modulates the expression of KDM6B and its target, the motor neuron-specific transcription factor HB9, driving motor neuron maturation. Our study reveals GEMIN5 regulates expression with implications for motor neuron diseases and therapy.
SMN 缺乏导致的 RNA 代谢失调会引发运动神经元疾病脊髓性肌萎缩症（SMA）。目前的治疗方法可以改善患者的预后，但无法根治疾病，因此人们再次努力深入了解疾病的发病机制。钙通道阻滞剂氟桂利嗪可改善 -/- 小鼠的运动功能，有助于揭示神经保护途径。本研究使用鼠运动神经元样 NSC34 细胞来研究分子细胞自主机制。提取 RNA 和蛋白质后，进行 RT-qPCR 和免疫检测实验。通过 RNA 免疫沉淀实验，研究了氟桂利嗪 mRNA 靶标与 RNA 结合蛋白 GEMIN5 之间的关系。氟桂利嗪可增加细胞培养物和小鼠模型中的去甲基化酶 转录本。它可在 NSC34 细胞中引起 GEMIN5 和 KDM6B 的时间表达。GEMIN5 可与氟桂利嗪调节的 mRNAs（包括 转录本）结合。 耗竭会降低 mRNA 和蛋白水平，并阻碍对氟桂利嗪的反应，包括 NSC34 细胞中的神经突延伸。此外，氟桂利嗪可增加源自 SMA 患者诱导多能干细胞的运动神经元的轴突延伸。最后，对 -/- 小鼠脊髓运动神经元的免疫荧光研究表明，氟桂利嗪可调节 KDM6B 及其靶基因，即运动神经元特异性转录因子 HB9 的表达，从而驱动运动神经元成熟。本研究揭示了 GEMIN5 可调节 的表达，这与运动神经元疾病和治疗有关。
