Shyng Charles, Macauley Shannon L, Dearborn Joshua T, Sands Mark S
Department of Internal Medicine, Washington University School of Medicine, Campus Box 8007, 660 S. Euclid Ave, St. Louis, MO, 63110, USA.
Department of Neurology, Washington University School of Medicine, St. Louis, MO, 63110, USA.
JIMD Rep. 2017;36:85-92. doi: 10.1007/8904_2017_1. Epub 2017 Feb 18.
"Cross-correction," the transfer of soluble lysosomal enzymes between neighboring cells, forms the foundation for therapeutics of lysosomal storage disorders (LSDs). However, "cross-correction" poses a significant barrier to studying the role of specific cell types in LSD pathogenesis. By expressing the native enzyme in only one cell type, neighboring cell types are invariably corrected. In this study, we present a strategy to limit "cross-correction" of palmitoyl-protein thioesterase-1(PPT1), a lysosomal hydrolase deficient in Infantile Neuronal Ceroid Lipofuscinosis (INCL, Infantile Batten disease) to the lysosomal membrane via the C-terminus of lysosomal associated membrane protein-1 (LAMP1). Tethering PPT1 to the lysosomal membrane prevented "cross-correction" while allowing PPT1 to retain its enzymatic function and localization in vitro. A transgenic line harboring the lysosomal membrane-tethered PPT1 was then generated. We show that expression of lysosome-restricted PPT1 in vivo largely rescues the INCL biochemical, histological, and functional phenotype. These data suggest that lysosomal tethering of PPT1 via the C-terminus of LAMP1 is a viable strategy and that this general approach can be used to study the role of specific cell types in INCL pathogenesis, as well as other LSDs. Ultimately, understanding the role of specific cell types in the disease progression of LSDs will help guide the development of more targeted therapeutics. One Sentence Synopsis: Tethering PPT1 to the lysosomal membrane is a viable strategy to prevent "cross-correction" and will allow for the study of specific cellular contributions in INCL pathogenesis.
“交叉校正”，即可溶性溶酶体酶在相邻细胞间的转移，构成了溶酶体贮积症（LSDs）治疗的基础。然而，“交叉校正”对研究特定细胞类型在LSDs发病机制中的作用构成了重大障碍。通过仅在一种细胞类型中表达天然酶，相邻细胞类型总会得到校正。在本研究中，我们提出了一种策略，即将棕榈酰蛋白硫酯酶-1（PPT1，一种在婴儿神经元蜡样脂褐质沉积症（INCL，婴儿型巴滕病）中缺乏的溶酶体水解酶）通过溶酶体相关膜蛋白-1（LAMP1）的C末端限制在溶酶体膜上，以限制其“交叉校正”。将PPT1拴系到溶酶体膜上可防止“交叉校正”，同时使PPT1在体外保留其酶功能和定位。然后生成了一个携带溶酶体膜拴系PPT1的转基因品系。我们表明，体内溶酶体限制的PPT1表达在很大程度上挽救了INCL的生化、组织学和功能表型。这些数据表明，通过LAMP1的C末端将PPT1拴系到溶酶体膜上是一种可行的策略，并且这种通用方法可用于研究特定细胞类型在INCL发病机制以及其他LSDs中的作用。最终，了解特定细胞类型在LSDs疾病进展中的作用将有助于指导更具针对性的治疗方法的开发。一句话总结：将PPT1拴系到溶酶体膜上是防止“交叉校正”的可行策略，并将有助于研究INCL发病机制中特定细胞的作用。
