Characterization and application of a disease-cell model for a neurodegenerative lysosomal disease
Characterization and application of a disease-cell model for a neurodegenerative lysosomal disease
复制标题
DOI：
10.1016/j.ymgme.2013.09.011
复制
发表时间：
2014-02-01
影响因子：
3.8
通讯作者：
Maegawa, Gustavo H. B.
中科院分区：
文献类型：
--
作者：
Ribbens, Jameson. J.;Moser, Ann B.;Maegawa, Gustavo H. B.
概括特定分子表型的疾病细胞模型对于研究具有主要神经系统表现的神经退行性疾病（包括溶酶体贮积病（LSD））的分子发病机制是必不可少的。在此，我们报告了一种快速神经退行性LSD，球状细胞脑白质营养不良（GLD），主要被称为克拉伯病的细胞模型的开发和表征。GLD是由E-半乳糖苷酶（GALC）缺乏引起的，GALC是一种溶酶体酶，可水解两种鞘糖脂，即精神病肽和半乳糖神经酰胺。不幸的是，来自GLD患者的可用培养成纤维细胞由有限的研究工具组成，因为这些细胞不能积累精神病碱，精神病碱是导致严重脱髓鞘的LSD中的中心致病性鞘糖脂。首先，我们从Twitch（Twi）小鼠（GALC（twi/twt））获得脑样品，GALC（twi/twt）是GALC缺陷的天然小鼠模型。我们用SV 40大T抗原使原代神经胶质细胞培养细胞永生化，分别从Twi和对照小鼠产生145 M-Twi和145 C-Wt细胞系。两种细胞系均表达特异性少突胶质细胞标记物，包括A2 B5和GalC。145 M-Twi细胞显示与GLD神经发病机制相关的生化和细胞紊乱，包括显著的半胱天冬酶-3活化、细胞色素C释放到胞质溶胶中和溶酶体隔室扩张。在鞘糖脂处理下，145 M-Twi细胞显示出增加的LOB水平，这是自噬的标志物。使用我们开发的LC-MS/MS方法，145 M-Twi细胞显示出显著更高水平的精神碱。145 M-Twi和145 C-Wt细胞系允许开发稳健的通量LC-MS/MS测定法来测量细胞的精神病肽水平。在该通量测定中，L-环丝氨酸显示出显著降低了145 M-Twi细胞中的精神病肽水平。已建立的145 M-Twi细胞是研究神经学相关致病途径以及开发用于鉴定GLD和潜在其他鞘糖脂疾病治疗剂的初步筛选测定的强大研究工具。(C)2013 Elsevier Inc. All rights reserved.
Disease-cell models that recapitulate specific molecular phenotypes are essential for the investigation of molecular pathogenesis of neurodegenerative diseases including lysosomal storage diseases (LSDs) with predominant neurological manifestations. Herein we report the development and characterization of a cell model for a rapid neurodegenerative LSDs, globoid-cell leukodystrophy (GLD), mostly known as Krabbe disease. GLD is caused by the deficiency of E-galactocerebrosidase (GALC), a lysosomal enzyme that hydrolyzes two glycosphingolipids, psychosine and galactosylceramide. Unfortunately, the available culture fibroblasts from GLD patients consist of a limited research tool as these cells fail to accumulate psychosine, the central pathogenic glycosphingolipid in this LSD that results in severe demyelination. Firstly, we obtained brain samples from the Twitcher (Twi) mice (GALC(twi/twt)), the natural mouse model with GALC deficiency. We immortalized the primary neuroglial cultured cells with SV40 large T antigen, generating the 145M-Twi and the 145C-Wt cell lines from the Twi and control mice, respectively. Both cell lines expressed specific oligodendrocyte markers including A2B5 and GalC The 145M-Twi cells showed biochemical and cellular disturbances related to GLD neuropathogenesis including remarkable caspase-3 activation, release of cytochrome C into the cytosol and expansion of the lysosomal compartment. Under treatment with glycosphingolipids, 145M-Twi cells showed increased LOB levels, a marker of autophagy. Using the LC-MS/MS method that we developed, the 145M-Twi cells showed significantly higher levels of psychosine. The 145M-Twi and 145C-Wt lines allowed the development of a robust throughput LC-MS/MS assay to measure cellular psychosine levels. In this throughput assay, L-cycloserine showed to significantly reduce the 145M-Twi cellular levels of psychosine. The established 145M-Twi cells are powerful research tools to investigate the neurologically relevant pathogenic pathways as well as to develop primary screening assays for the identification of therapeutic agents for GLD and potentially other glycosphingolipid disorders. (C) 2013 Elsevier Inc. All rights reserved.