Supplementary Material for: Frontal Cortex and Hippocampal γ-Secretase Activating Protein Levels in Prodromal Alzheimer Disease

<b><i>Background:</i></b> β-Amyloid (Aβ) is the product of concerted cleavage of the amyloid precursor protein (APP) by β- and γ-secretases. However, the molecular mechanisms that regulate this process are not well understood. Recently, evidence was reported that γ-secretase activating protein (GSAP, 16 kDa), derived from a larger precursor protein (98 kDa), plays a role in Aβ metabolism through a mechanism involving its interaction with both γ-secretase and APP. However, a detailed evaluation of GSAP protein levels and their association with clinical and neuropathological variables are lacking during the clinical progression of Alzheimer disease (AD). <b><i>Methods:</i></b> We quantified levels of the GSAP precursor (98 kDa) and its active form (16 kDa) in the frontal cortex and hippocampus, areas displaying extensive Aβ and neurofibrillary tangle (NFT) pathology, in subjects who came to autopsy with a premortem clinical diagnosis of noncognitive impairment, mild cognitive impairment, mild to moderate AD, and severe AD using Western blotting. <b><i>Results:</i></b> Analysis found that 98-kDa GSAP levels were increased, while those of 16 kDa were reduced in the frontal cortex of severe-AD subjects. By contrast, GSAP levels remained stable in the hippocampus. Frontal cortex and hippocampal GSAP 98- and 16-kDa levels were not associated with Aβ, NFT, and neuropathological criteria across clinical groups. Interestingly, only neocortical 98-kDa GSAP values showed a significant correlation with the Mini-Mental State Examination and episodic memory scores. <b><i>Conclusions:</i></b> These data demonstrate that GSAP proteins are differentially dysregulated in severe AD, but only the full-length form was associated with cognitive test scores in AD.

**背景**：β-淀粉样蛋白（β-Amyloid, Aβ）是淀粉样前体蛋白（amyloid precursor protein, APP）经β分泌酶与γ分泌酶协同切割所产生的产物。然而，调控该过程的分子机制尚未得到充分阐明。近期有研究报道，由分子量98 kDa的前体蛋白剪切而来的γ分泌酶激活蛋白（γ-secretase activating protein, GSAP，分子量16 kDa），可通过同时结合γ分泌酶与APP的机制参与Aβ的代谢过程。但在阿尔茨海默病（Alzheimer disease, AD）的临床进展过程中，尚未有研究对GSAP蛋白水平及其与临床、神经病理变量的关联进行详细评估。

**方法**：本研究采用蛋白质免疫印迹（Western blotting）技术，对尸检受试者的额叶皮层与海马体这两个存在广泛Aβ与神经原纤维缠结（neurofibrillary tangle, NFT）病理改变的脑区中，GSAP前体（分子量98 kDa）及其活性形式（分子量16 kDa）的蛋白水平进行了定量分析。所有受试者生前的临床诊断分别为非认知损害、轻度认知损害、轻中度阿尔茨海默病以及重度阿尔茨海默病。

**结果**：分析结果显示，重度阿尔茨海默病受试者的额叶皮层中，98 kDa GSAP的蛋白水平显著升高，而16 kDa GSAP的蛋白水平则明显降低；与之形成对比的是，海马体中的GSAP蛋白水平未出现显著变化。在所有临床分组中，额叶皮层与海马体的98 kDa和16 kDa GSAP蛋白水平均与Aβ负荷、NFT病理以及神经病理诊断标准无显著关联。值得注意的是，仅新皮层中的98 kDa GSAP蛋白水平与简易精神状态检查（Mini-Mental State Examination, MMSE）评分以及情景记忆得分存在显著相关性。

**结论**：本研究数据表明，GSAP蛋白在重度阿尔茨海默病中存在差异性失调，但仅全长形式的GSAP与阿尔茨海默病患者的认知测试得分存在关联。