Li Qiang, Yuan Yiyuan, Huang Shi, Di Guangfu, Chen Haoyuan, Zhuang Yani, Fang Wanzhen, Huang Yanjiao, Tao Yinan, Jiang Jing, Xu Zhiliang
Human Anatomy Experimental Training Center, School of Basic Medical Sciences, Wannan Medical College, 241002 Wuhu, Anhui, China.
School of Clinical Medicine, Wannan Medical College, 241002 Wuhu, Anhui, China.
J Integr Neurosci. 2024 Dec 24;23(12):223. doi: 10.31083/j.jin2312223.
K48-linked ubiquitin chain (Ub-K48) is a crucial ubiquitin chain implicated in protein degradation within the ubiquitin-proteasome system. However, the precise function and molecular mechanism underlying the role of Ub-K48 in the pathogenesis of Alzheimer's disease (AD) and neuronal cell abnormalities remain unclear. The objective of this study was to examine the function of K48 ubiquitination in the etiology of AD, and its associated mechanism of neuronal apoptosis.
A mouse model of AD was constructed, and behavioral phenotypic changes were detected using an open field test (OFT). The expression of glial fibrillary acidic protein (GFAP), an early marker of AD, was detected by western blotting (WB). Neuronal apoptosis in the hippocampal region was assessed by hematoxylin and eosin (HE) and Nissl staining. Immunohistochemistry and immunofluorescence were performed to observe the changes in Phosphorylated tubulin associated unit (p-Tau) and Ub-K48 colocalization in neurons of the hippocampal region of AD mice. WB was further applied to detect the degree of ubiquitylation of K48 and the expression of Tau, p-Tau, B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X (Bax) proteins in neuronal cells of the hippocampus and cortical regions of mice.
Mice with AD exhibited significantly longer resting times ( < 0.05) and shorter average speeds ( < 0.01), total distances travelled ( < 0.01), and distances travelled ( < 0.01) in the central region than those in the control group. This indicated cognitive impairment, which occurred concurrent with an increased expression of the AD marker GFAP protein ( < 0.001). The hippocampal region of AD mice showed abnormalities with sparsely and irregularly arranged cells, large gaps between cells, lighter staining, unclear boundaries of the cell membranes and nuclei, and agglutinated and condensed nuclei ( < 0.01). The neuronal cells of AD mice exhibited significantly elevated levels of p-Tau ( < 0.01) and Ub-K48 ( < 0.01), as well as a notable degree of co-localization within the cells. The intracellular pro-inflammatory protein Bax was significantly upregulated ( < 0.05), while the Bcl-2/Bax ratio was significantly lower than that in the control group ( < 0.05), thus inducing apoptosis in AD neuronal cells.
Ub-K48 is strongly linked to the development of AD. p-Tau aggregate in neuronal cells in the hippocampal region of the AD brain and colocalize with Ub-K48, which in turn leads to cellular inflammation and the induction of apoptosis in neuronal cells.
K48连接的泛素链（Ub-K48）是泛素-蛋白酶体系统中参与蛋白质降解的关键泛素链。然而，Ub-K48在阿尔茨海默病（AD）发病机制和神经元细胞异常中的确切功能及分子机制仍不清楚。本研究的目的是探讨K48泛素化在AD病因中的作用及其相关的神经元凋亡机制。
构建AD小鼠模型，采用旷场试验（OFT）检测行为表型变化。通过蛋白质免疫印迹法（WB）检测AD早期标志物胶质纤维酸性蛋白（GFAP）的表达。采用苏木精-伊红（HE）染色和尼氏染色评估海马区神经元凋亡。进行免疫组织化学和免疫荧光检测，观察AD小鼠海马区神经元中磷酸化微管相关单位（p-Tau）与Ub-K48的共定位变化。进一步应用WB检测小鼠海马和皮质区神经元细胞中K48的泛素化程度以及Tau、p-Tau、B细胞淋巴瘤-2（Bcl-2）和Bcl-2相关X蛋白（Bax）的表达。
与对照组相比，AD小鼠的静息时间显著延长（<0.05），平均速度、总移动距离和中央区域移动距离显著缩短（<0.01）。这表明存在认知障碍，同时AD标志物GFAP蛋白表达增加（<0.001）。AD小鼠海马区细胞排列稀疏且不规则，细胞间隙大，染色浅，细胞膜和细胞核边界不清，细胞核凝集浓缩，呈现异常（<0.01）。AD小鼠神经元细胞中p-Tau（<0.01）和Ub-K48（<0.01）水平显著升高，且在细胞内有明显的共定位。细胞内促炎蛋白Bax显著上调（<0.05），而Bcl-2/Bax比值显著低于对照组（<0.05），从而诱导AD神经元细胞凋亡。
Ub-K48与AD的发生密切相关。AD脑海马区神经元细胞中p-Tau聚集并与Ub-K48共定位，进而导致细胞炎症并诱导神经元细胞凋亡。
