Lysosomal dysfunction in Down syndrome is APP-dependent and mediated by APP-ßCTF (C99)

Lysosomal failure underlies pathogenesis of numerous congenital neurodegenerative disorders and is an early and progressive feature of Alzheimer's disease (AD) pathogenesis. Here, we report that lysosomal dysfunction in Down Syndrome (Trisomy 21) requires the extra gene copy of amyloid precursor protein (APP) and is mediated by the beta cleaved carboxy terminal fragment of APP (APP-ßCTF, C99). In primary fibroblasts from individuals with Down Syndrome (DS), lysosomal degradation of autophagic and endocytic substrates is selectively impaired causing them to accumulate in enlarged autolysosomes/lysosomes. Direct measurements of lysosomal pH uncovered a significant elevation (0.6 units) associated with slowed LC3 turnover and the inactivation of cathepsin D (CTSD) and other lysosomal hydrolases known to be unstable or less active when lysosomal pH is persistently elevated. RNA sequencing analysis excluded a transcriptional contribution to hydrolase declines. Normalizing lysosome pH by delivering acidic nanoparticles to lysosomes ameliorated lysosomal deficits, implicating pH elevation as their primary basis. Cortical neurons cultured from the Ts2 mouse model of DS exhibited lysosomal deficits similar to those in DS cells. Lowering APP expression with siRNA or BACE1 inhibition reversed cathepsin deficits in both fibroblasts and neurons. Deleting one BACE1 allele from adult Ts2 mice had similar rescue effects in vivo. The modest elevation of endogenous APP-ßCTF needed to disrupt lysosomal function in DS is relevant to sporadic AD where APP-ßCTF, but not APP, is also elevated. Our results extend evidence that impaired lysosomal acidification drives progressive lysosomal failure in multiple forms of AD. Overall design: (1) mRNA-Seq profiling of six Trisomic and six Disomic human fibroblasts samples (3 replicates from 2 individuals in each group) from 5 months (3 replicates) and 2 years (3 replicates) old unrelated inviduals treated with either siRNA against human APP (siAPP) or a negative control DsiRNA (siNC). (2) A separate Differential Gene Expression (DGE) analysis was also carried out to compare transcriptomic profile of human Disomic and Trisomic fibroblasts using Disomic samples treated with siNC (3 replicates each of 5 months and 2 years individuals; Total = 6) in conjunction with age matched untreated human Disomic fibroblasts samples already deposited by Letourneau et al. (GSM1338333, GSM1338336) and Sullivan et al. (GSM2105075, GSM2105077); and Trisomic samples treated with siNC (3 replicates each of 5 months and 2 years individuals; Total = 6) in conjunction with age matched untreated human Trisomic fibroblasts samples from Letourneau et al. (GSM1338325, GSM1338326, GSM1338327) and Sullivan et al. (GSM2105044, GSM2105047). The final count of samples for the Disomic and Trisomic group is 11 and 10 respectively.

溶酶体功能衰竭（lysosomal failure）是众多先天性神经退行性疾病（congenital neurodegenerative disorders）的发病基础，同时也是阿尔茨海默病（Alzheimer's disease, AD）发病进程中早期且进行性的核心特征。本研究发现，唐氏综合征（Down Syndrome, Trisomy 21, DS）中的溶酶体功能障碍依赖于额外的淀粉样前体蛋白（amyloid precursor protein, APP）基因拷贝，并由APP的β剪切羧基末端片段（APP-βCTF, C99）介导。在来自唐氏综合征患者的原代成纤维细胞（primary fibroblasts）中，自噬与内吞底物的溶酶体降解过程受到选择性损伤，导致底物在膨大的自噬溶酶体/溶酶体（autolysosomes/lysosomes）中异常蓄积。对溶酶体pH（lysosomal pH）的直接检测发现，pH显著升高（0.6个单位），这与LC3周转（LC3 turnover）减慢、组织蛋白酶D（cathepsin D, CTSD）及其他已知在溶酶体pH持续升高时不稳定或活性降低的溶酶体水解酶（lysosomal hydrolases）失活密切相关。RNA测序（RNA sequencing）分析排除了水解酶表达下调的转录调控因素。通过向溶酶体递送酸性纳米颗粒（acidic nanoparticles）以校正溶酶体pH，可显著改善溶酶体功能缺陷，提示pH升高是溶酶体缺陷的核心致病基础。从唐氏综合征Ts2小鼠模型中分离培养的皮层神经元（Cortical neurons），表现出与DS患者细胞高度相似的溶酶体功能缺陷。通过小干扰RNA（small interfering RNA, siRNA）下调APP表达，或抑制β位淀粉样前体蛋白裂解酶1（BACE1），均可逆转成纤维细胞与神经元中的组织蛋白酶功能缺陷。在成年Ts2小鼠中敲除单个BACE1等位基因，在体内同样可产生类似的挽救效应。在唐氏综合征中破坏溶酶体功能所需的内源性APP-βCTF轻度升高，与散发性阿尔茨海默病（sporadic AD）的病理特征高度相关——后者中APP-βCTF的水平显著升高，而APP本身的水平并无明显变化。本研究结果进一步证实，溶酶体酸化受损会驱动多种阿尔茨海默病亚型的进行性溶酶体功能衰竭。整体实验设计如下：(1) 对6份三体组与6份二体组人类成纤维细胞样本开展mRNA测序分析（mRNA-Seq profiling）：每组均来自2名无关个体，各设3次生物学重复，样本分别取自5月龄（3次重复）与2岁龄（3次重复）的受试者，且均用针对人APP的小干扰RNA（siAPP）或阴性对照双链RNA（siNC）处理。(2) 另一项独立的差异基因表达分析（Differential Gene Expression, DGE），通过整合多组样本比较人类二体与三体成纤维细胞的转录组谱（transcriptomic profile）：① 以siNC处理的二体成纤维细胞样本（5月龄与2岁龄各3次重复，共6份），搭配Letourneau等人已上传的年龄匹配的未处理人类二体成纤维细胞样本（GSM1338333、GSM1338336）与Sullivan等人的对应样本（GSM2105075、GSM2105077）；② 以siNC处理的三体成纤维细胞样本（5月龄与2岁龄各3次重复，共6份），搭配Letourneau等人的年龄匹配的未处理人类三体成纤维细胞样本（GSM1338325、GSM1338326、GSM1338327）与Sullivan等人的对应样本（GSM2105044、GSM2105047）。最终二体组与三体组的有效样本数分别为11份与10份。