Characterization and application of a disease-cell model for a neurodegenerative lysosomal disease

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• 作者：Jameson J. Ribbens ; Ann B. Moser ; Walter C. Hubbard ; Ernesto R. Bongarzone ; Gustavo H.B. Maegawa
• 关键词：Ac-DEDV-AMC ; N-acetyl-DEVD 7-amido-4-methylcoumarin ; BEH ; Bridged Ethyl Hybrid ; GALC ; 尾-galactocerebrosidase ; GalC ; galactosylceramide ; GFAP ; glial fibrillary acidic protein ; GLD ; globoid-cell leukodystrophy ; HMU尾Gal ; 6-hexadecanoylamino-4-methylumbelliferyl-尾-d-galactoside ; ISD ; internal standard ; LSD ; lysosomal storage disease ; MAP-2 ; microtubule-associated protein 2 ; MPA ; mobile phase A ; MPB ; mobile phase B ; MS ; mass spectrometry ; RT ; retention time ; SPE ; solid-phase extraction ; SV40T ; SV4
• 刊名：Molecular Genetics and Metabolism
• 出版年：February, 2014
• 年：2014
• 卷：111
• 期：2
• 页码：172-183
• 全文大小：2171 K

文摘

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 尾-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/twi), 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 LC3B 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.