Neural Stem Cell Research Lab, Research Department, National Neuroscience Institute, Singapore, 308433, Singapore.
Department of Psychological Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 119228, Singapore.
Mol Neurodegener. 2024 Nov 28;19(1):91. doi: 10.1186/s13024-024-00780-2.
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.
NOTCH2NLC 基因（N2C）5'非翻译区（UTR）中 GGC 重复扩增（通常超过 60 个重复）与 N2C 相关重复扩增障碍（NREDs）有关，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，包括帕金森病、痴呆、癫痫发作和肌肉无力。已经报道中间重复大小在 40 到 60 GGC 重复之间，特别是那些具有 AGC 编码丝氨酸插入的，与 PD 有关；然而，具有丝氨酸插入的这些中间重复的功能意义仍未被探索。
在这里，我们利用携带不同大小的 N2C 变体 2（N2C2）GGC 重复扩增的细胞模型和携带 N2C2 GGC 中间重复且无丝氨酸插入的 CRISPR-Cas9 工程转基因小鼠模型，阐明了与 NREDs 中 N2C 中间重复带丝氨酸插入相关的潜在病理生理学。
我们的研究结果表明，N2C2 GGC 中间重复带丝氨酸插入（32G13S）导致体外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 个月大的转基因小鼠中，32G13S 加剧了核内聚集，并表现出早期 PD 样特征，包括中脑α-突触核蛋白纤维的形成和皮质和纹状体中酪氨酸羟化酶（TH）阳性神经元的丧失。此外，32G13S 诱导神经元过度兴奋并导致运动行为障碍。对小鼠皮质的转录组分析表明，钙信号和 MAPK 信号通路的调节失常，这两者对线粒体功能都至关重要。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进一步研究表明，N2C 中间重复带丝氨酸诱导皮质中线粒体功能障碍相关的过度髓鞘形成。
我们的体外和体内研究提供了第一个证据，表明 N2C-GGC 中间重复带丝氨酸促进 N2C 的核内聚集，导致与线粒体功能障碍相关的过度髓鞘形成和神经元过度兴奋。这些变化导致 NREDs 中早期 PD 样神经退行性变中的运动缺陷。
