The expansion of GGC repeats (typically exceeding 60 repeats) in the 5’ untranslated region (UTR) of the NOTCH2NLC gene (N2C) is linked to N2C-related repeat expansion disorders (NREDs), such as neuronal intranuclear inclusion disease (NIID), frontotemporal dementia (FTD), essential tremor (ET), and Parkinson’s disease (PD). These disorders share common clinical manifestations, including parkinsonism, dementia, seizures, and muscle weakness. Intermediate repeat sizes ranging from 40 to 60 GGC repeats, particularly those with AGC-encoded serine insertions, have been reported to be associated with PD; however, the functional implications of these intermediate repeats with serine insertion remain unexplored. Here, we utilized cellular models harbouring different sizes of N2C variant 2 (N2C2) GGC repeat expansion and CRISPR-Cas9 engineered transgenic mouse models carrying N2C2 GGC intermediate repeats with and without serine insertion to elucidate the underlying pathophysiology associated with N2C intermediate repeat with serine insertion in NREDs. Our findings revealed that the N2C2 GGC intermediate repeat with serine insertion (32G13S) led to mitochondrial dysfunction and cell death in vitro. The neurotoxicity was influenced by the length of the repeat and was exacerbated by the presence of the serine insertion. In 12-month-old transgenic mice, 32G13S intensified intranuclear aggregation and exhibited early PD-like characteristics, including the formation of α-synuclein fibers in the midbrain and the loss of tyrosine hydroxylase (TH)-positive neurons in both the cortex and striatum. Additionally, 32G13S induced neuronal hyperexcitability and caused locomotor behavioural impairments. Transcriptomic analysis of the mouse cortex indicated dysregulation in calcium signaling and MAPK signaling pathways, both of which are critical for mitochondrial function. Notably, genes associated with myelin sheath components, including MBP and MOG, were dysregulated in the 32G13S mouse. Further investigations using immunostaining and transmission electron microscopy revealed that the N2C intermediate repeat with serine induced mitochondrial dysfunction-related hypermyelination in the cortex. Our in vitro and in vivo investigations provide the first evidence that the N2C-GGC intermediate repeat with serine promotes intranuclear aggregation of N2C, leading to mitochondrial dysfunction-associated hypermyelination and neuronal hyperexcitability. These changes contribute to motor deficits in early PD-like neurodegeneration in NREDs.

中文翻译：

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NOTCH2NLC 丝氨酸的 GGC 中间重复序列诱导小鼠髓鞘形成过多和早期帕金森病样表型


GGC 重复序列在 NOTCH2NLC 基因 （N2C） 的 5' 非翻译区 （UTR） 中的扩增（通常超过 60 个重复序列）与 N2C 相关重复扩增障碍 （NRED） 有关，例如神经元核内包涵体病 （NIID）、额颞叶痴呆 （FTD）、特发性震颤 （ET） 和帕金森病 （PD）。这些疾病具有共同的临床表现，包括帕金森综合征、痴呆、癫痫发作和肌无力。据报道，40 至 60 个 GGC 重复序列的中间重复大小，尤其是那些具有 AGC 编码的丝氨酸插入的重复序列，与 PD 相关;然而，这些带有丝氨酸插入的中间重复序列的功能意义仍未得到探索。在这里，我们利用了携带不同大小的 N2C 变体 2 （N2C2） GGC 重复扩增的细胞模型和 CRISPR-Cas9 工程转基因小鼠模型，携带 N2C2 GGC 中间重复序列，有和没有丝氨酸插入，以阐明与 NRED 中丝氨酸插入的 N2C 中间重复相关的潜在病理生理学。我们的研究结果显示，丝氨酸插入的 N2C2 GGC 中间重复序列 （32G13S） 在体外导致线粒体功能障碍和细胞死亡。神经毒性受重复长度的影响，并因丝氨酸插入的存在而加剧。在 12 月龄的转基因小鼠中，32G13S 加强了核内聚集并表现出早期 PD 样特征，包括在中脑中形成 α-突触核蛋白纤维以及皮层和纹状体中酪氨酸羟化酶 （TH） 阳性神经元的丢失。此外，32G13S 诱导神经元过度兴奋并导致运动行为障碍。 小鼠皮层的转录组学分析表明钙信号传导和 MAPK 信号通路失调，这两者都对线粒体功能至关重要。值得注意的是，与髓鞘成分相关的基因，包括 MBP 和 MOG，在 32G13S 小鼠中失调。使用免疫染色和透射电子显微镜的进一步研究表明，丝氨酸的 N2C 中间重复诱导了皮层中线粒体功能障碍相关的髓鞘形成过多。我们的体外和体内研究提供了第一个证据，证明丝氨酸的 N2C-GGC 中间重复序列促进 N2C 的核内聚集，导致线粒体功能障碍相关的髓鞘形成过多和神经元过度兴奋。这些变化导致 NRED 早期 PD 样神经变性的运动缺陷。