功能失调的线粒体的积累是阿尔茨海默病 (AD) 的早期特征。受损线粒体的周转受损会增加活性氧的产生并降低 ATP 的产生，从而导致细胞毒性和神经退行性变。有趣的是，AD 表现出整体翻译后修饰 β-N-乙酰氨基葡萄糖 (O-GlcNAc) 的破坏。 O-GlcNAc 是一种普遍存在于核蛋白、细胞质蛋白和线粒体蛋白中的单糖修饰。细胞分别通过 O-GlcNAc 转移酶 (OGT) 或 O-GlcNAcase (OGA) 循环添加和去除糖来维持 O-GlcNAc 的稳态水平。我们使用患者来源的诱导多能干细胞，这是一种转基因小鼠模型AD、SH-SY5Y 神经母细胞瘤细胞系，利用生化分析检查 Thiamet-G (TMG) 或 OGT 缺乏对 O-GlcNAcase 持续抑制对线粒体自噬的影响。在这里，我们确定了 O-GlcNAc 在调节线粒体自噬中的重要作用。线粒体选择性自噬）。使用尿石素 A (UA) 刺激线粒体自噬会降低细胞 O-GlcNAc 并升高线粒体 O-GlcNAc。通过使用 Thiamet-G (TMG) 药理学抑制 OGA，O-GlcNAcNAc 持续升高，从而增加线粒体自噬蛋白 PTEN 诱导激酶 1 (PINK1) 和自噬相关蛋白轻链 3 (LC3) 的线粒体水平。此外，我们在 PINK1 上检测到 O-GlcNAc，并且 TMG 增加了其 O-GlcNAc 化水平。相反，通过敲低 OGT 来减少细胞 O-GlcNAc 化会降低 PINK1 蛋白表达和 LC3 蛋白表达。从 CAMKII-OGT-KO 小鼠中分离的线粒体也减少了 PINK1 和 LC3。此外，与对照组相比，经 TMG 处理的人脑类器官的 LC3 显着升高。然而，TMG 处理的 AD 类器官显示 LC3 表达没有变化。总的来说，这些数据表明 O-GlcNAc 在线粒体自噬的激活和进展中起着至关重要的作用，并且这种激活在 AD 中被破坏。版权所有 © 2024 Alghusen, Carman,威尔金斯、斯特罗普、吉莫尔、费多修克、沙瓦、埃弗莱姆、丹森、王、斯沃德洛和斯劳森。

The accumulation of dysfunctional mitochondria is an early feature of Alzheimer's disease (AD). The impaired turnover of damaged mitochondria increases reactive oxygen species production and lowers ATP generation, leading to cellular toxicity and neurodegeneration. Interestingly, AD exhibits a disruption in the global post-translational modification β-N-acetylglucosamine (O-GlcNAc). O-GlcNAc is a ubiquitous single sugar modification found in the nuclear, cytoplasmic, and mitochondrial proteins. Cells maintain a homeostatic level of O-GlcNAc by cycling the addition and removal of the sugar by O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA), respectively.We used patient-derived induced pluripotent stem cells, a transgenic mouse model of AD, SH-SY5Y neuroblastoma cell lines to examine the effect of sustained O-GlcNAcase inhibition by Thiamet-G (TMG) or OGT deficiency on mitophagy using biochemical analyses.Here, we established an essential role for O-GlcNAc in regulating mitophagy (mitochondria-selective autophagy). Stimulating mitophagy using urolithin A (UA) decreases cellular O-GlcNAc and elevates mitochondrial O-GlcNAc. Sustained elevation in O-GlcNAcylation via pharmacologically inhibiting OGA using Thiamet-G (TMG) increases the mitochondrial level of mitophagy protein PTEN-induced kinase 1 (PINK1) and autophagy-related protein light chain 3 (LC3). Moreover, we detected O-GlcNAc on PINK1 and TMG increases its O-GlcNAcylation level. Conversely, decreasing cellular O-GlcNAcylation by knocking down OGT decreases both PINK1 protein expression and LC3 protein expression. Mitochondria isolated from CAMKII-OGT-KO mice also had decreased PINK1 and LC3. Moreover, human brain organoids treated with TMG showed significant elevation in LC3 compared to control. However, TMG-treated AD organoids showed no changes in LC3 expression.Collectively, these data demonstrate that O-GlcNAc plays a crucial role in the activation and progression of mitophagy, and this activation is disrupted in AD.Copyright © 2024 Alghusen, Carman, Wilkins, Strope, Gimore, Fedosyuk, Shawa, Ephrame, Denson, Wang, Swerdlow and Slawson.