Department of Biochemistry, Duke University School of Medicine, Durham, NC, 27710, USA.
Department of Cell Biology, Duke University School of Medicine, Durham, NC, 27710, USA.
Nat Commun. 2023 Oct 17;14(1):6558. doi: 10.1038/s41467-023-42227-0.
The neurofilament (NF) cytoskeleton is critical for neuronal morphology and function. In particular, the neurofilament-light (NF-L) subunit is required for NF assembly in vivo and is mutated in subtypes of Charcot-Marie-Tooth (CMT) disease. NFs are highly dynamic, and the regulation of NF assembly state is incompletely understood. Here, we demonstrate that human NF-L is modified in a nutrient-sensitive manner by O-linked-β-N-acetylglucosamine (O-GlcNAc), a ubiquitous form of intracellular glycosylation. We identify five NF-L O-GlcNAc sites and show that they regulate NF assembly state. NF-L engages in O-GlcNAc-mediated protein-protein interactions with itself and with the NF component α-internexin, implying that O-GlcNAc may be a general regulator of NF architecture. We further show that NF-L O-GlcNAcylation is required for normal organelle trafficking in primary neurons. Finally, several CMT-causative NF-L mutants exhibit perturbed O-GlcNAc levels and resist the effects of O-GlcNAcylation on NF assembly state, suggesting a potential link between dysregulated O-GlcNAcylation and pathological NF aggregation. Our results demonstrate that site-specific glycosylation regulates NF-L assembly and function, and aberrant NF O-GlcNAcylation may contribute to CMT and other neurodegenerative disorders.
神经丝（NF）细胞骨架对于神经元的形态和功能至关重要。特别是，神经丝轻链（NF-L）亚基对于体内 NF 的组装是必需的，并且在几种遗传性神经病 Charcot-Marie-Tooth（CMT）疾病中发生突变。NFs 具有高度的动态性，NF 组装状态的调控尚不完全清楚。在这里，我们证明人类 NF-L 可通过 O-连接-β-N-乙酰氨基葡萄糖（O-GlcNAc）进行营养敏感修饰，O-GlcNAc 是一种普遍存在的细胞内糖基化形式。我们鉴定了五个 NF-L O-GlcNAc 位点，并表明它们调节 NF 的组装状态。NF-L 与自身和 NF 成分α-中间丝蛋白（internexin）进行 O-GlcNAc 介导的蛋白-蛋白相互作用，这表明 O-GlcNAc 可能是 NF 结构的普遍调节剂。我们进一步表明，NF-L 的 O-GlcNAc 糖基化对于原代神经元中的正常细胞器运输是必需的。最后，几种导致 CMT 的 NF-L 突变体表现出 O-GlcNAc 水平的紊乱，并抵抗 O-GlcNAc 化对 NF 组装状态的影响，这表明失调的 O-GlcNAc 化与病理性 NF 聚集之间可能存在潜在联系。我们的研究结果表明，特定部位的糖基化调节 NF-L 的组装和功能，并且异常的 NF O-GlcNAc 化可能导致 CMT 和其他神经退行性疾病。
