Ciccotosto Giuseppe D, Jana Metta, Cappai Roberto
Department of Pathology, The University of Melbourne, Parkville, VIC, 3010, Australia.
Methods Mol Biol. 2017;1658:147-165. doi: 10.1007/978-1-4939-7244-9_12.
Prion neurotoxicity has been modeled in vitro using synthetic peptides derived from the PrP sequence. The major region of neurotoxicity has been localized to the hydrophobic domain located in the middle of the PrP protein. Neurotoxicity assays are typically performed on cultured mouse cerebellar neurons derived from neonatal pups, and cell viability can be monitored by assays including MTT or MTS, cell death by LDH release, or apoptosis by caspase cleavage assays. These neurotoxicity studies have been useful in identifying cofactors, such as PrP and metals, as modulators of PrP peptide-mediated neurotoxicity. Given the biosafety issues associated with handling and purifying infectious prions, the use of synthetic peptides, which display a dependence upon PrP expression for toxicity, as per the PrP agent for infectivity, supports the relevance of using these synthetic peptides for understanding PrP-mediated neurotoxicity.
朊病毒神经毒性已在体外通过源自朊蛋白（PrP）序列的合成肽进行模拟。神经毒性的主要区域已定位到位于PrP蛋白中部的疏水结构域。神经毒性测定通常在源自新生幼崽的培养小鼠小脑神经元上进行，细胞活力可通过包括MTT或MTS的测定来监测，细胞死亡通过乳酸脱氢酶（LDH）释放来监测，或细胞凋亡通过半胱天冬酶切割测定来监测。这些神经毒性研究有助于确定辅因子，如PrP和金属，作为PrP肽介导的神经毒性的调节剂。鉴于与处理和纯化传染性朊病毒相关的生物安全问题，合成肽的使用（其毒性表现出对PrP表达的依赖性，如同感染性的PrP病原体）支持了使用这些合成肽来理解PrP介导的神经毒性的相关性。
