Mutations in APP (amyloid precursor protein), PSEN1 (presenilin 1) or PSEN2 (presenilin 2) are the main cause of early-onset familial forms of Alzheimer's disease (autosomal dominant AD or ADAD). These genes affect γ-secretase-dependent generation of Amyloid β (Aβ) peptides, the main constituent of amyloid plaques and one of the pathological hallmarks of AD. Evaluation of patients with ADAD includes assessment of family history, clinical presentation, biomarkers, neuropathology when available and DNA sequencing data. These analyses frequently uncover novel variants of unknown significance in ADAD genes. This presents a barrier to recruitment of such individuals into clinical trials, unless a biochemical test can demonstrate that a novel mutation results in altered APP processing in a manner consistent with pathogenicity. Here we describe generation and characterization of a novel presenilin 1 and 2 double knock-out in N2A mouse neuroblastoma cells using CRISPR/Cas9, which results in complete ablation of Aβ production, decreased Pen-2 expression and Nicastrin glycosylation. Because of the absence of background Aβ secretion from endogenous γ-secretases, these cells can be used for validation of PSEN1 and PSEN2 variant effects on production of Aβ or other γ-secretase substrates and for biochemical studies of γ-secretase function using novel variants. We examined several PSEN1 and PSEN2 mutations of known and unknown pathogenicity. Known mutants increased Aβ42/Aβ40 ratio with varying effect on Aβ40, Aβ42, total Aβ levels and Pen-2 expression, which aligns with previous work on these mutants. Our data on novel PSEN1 V142F, G206V and G206D mutations suggest that these mutations underlie the reported clinical observations in ADAD patients. We believe our novel cell line will be valuable for the scientific community for reliable validation of presenilin mutations and helpful in defining their pathogenicity to improve and facilitate evaluation of ADAD patients, particularly in the context of enrollment in clinical trials.

中文翻译：

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用于体外验证 PSEN1 和 PSEN2 突变的新型早老素 1 和 2 双敲除细胞系。


APP（淀粉样前体蛋白）、PSEN1（早老素 1）或 PSEN2（早老素 2）突变是早发家族性阿尔茨海默病（常染色体显性 AD 或 ADAD）的主要原因。这些基因影响 β 淀粉样蛋白 (Aβ) 肽的 γ 分泌酶依赖性生成，β 淀粉样蛋白斑的主要成分，也是 AD 的病理标志之一。 ADAD 患者的评估包括评估家族史、临床表现、生物标志物、可用的神经病理学和 DNA 测序数据。这些分析经常发现 ADAD 基因中未知意义的新变体。这对招募此类个体进入临床试验构成了障碍，除非生化测试能够证明新的突变以与致病性一致的方式导致 APP 处理的改变。在这里，我们描述了使用 CRISPR/Cas9 在 N2A 小鼠神经母细胞瘤细胞中产生和表征新型早老素 1 和 2 双敲除，从而完全消除 Aβ 产生，减少 Pen-2 表达和 Nicastrin 糖基化。由于不存在内源性 γ 分泌酶的背景 Aβ 分泌，这些细胞可用于验证 PSEN1 和 PSEN2 变体对 Aβ 或其他 γ 分泌酶底物产生的影响，并可用于使用新变体对 γ 分泌酶功能进行生化研究。我们检查了几种已知和未知致病性的 PSEN1 和 PSEN2 突变。已知的突变体增加了 Aβ42/Aβ40 比率，对 Aβ40、Aβ42、总 Aβ 水平和 Pen-2 表达产生不同的影响，这与之前对这些突变体的研究结果一致。我们关于新型 PSEN1 V142F、G206V 和 G206D 突变的数据表明，这些突变是 ADAD 患者临床观察报告的基础。 我们相信，我们的新型细胞系对于科学界可靠验证早老素突变具有重要价值，并有助于确定其致病性，以改善和促进 ADAD 患者的评估，特别是在临床试验入组的情况下。