Lim Youngshin, Cho Il-Taeg, Schoel Leah J, Cho Ginam, Golden Jeffrey A
Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA.
Ann Neurol. 2015 Nov;78(5):679-96. doi: 10.1002/ana.24488. Epub 2015 Sep 16.
Mutations in receptor expression enhancing protein 1 (REEP1) are associated with hereditary spastic paraplegias (HSPs). Although axonal degeneration is thought to be a predominant feature in HSP, the role of REEP1 mutations in degeneration is largely unknown. Previous studies have implicated a role for REEP1 in the endoplasmic reticulum (ER), whereas others localized REEP1 with mitochondria. We sought to resolve the cellular localization of REEP1 and further elucidate the pathobiology underlying REEP1 mutations in patients.
A combination of cellular imaging and biochemical approaches was used to refine the cellular localization of REEP1. Next, Reep1 mutations associated with HSP were functionally tested in neuritic growth and degeneration assays using mouse cortical culture. Finally, a novel assay was developed and used with wild-type and mutant Reep1s to measure the interactions between the ER and mitochondria.
We found that REEP1 is present at the ER-mitochondria interface, and it contains subdomains for mitochondrial as well as ER localization. Knockdown of Reep1 and expression of pathological Reep1 mutations resulted in neuritic growth defects and degeneration. Finally, using our novel split-RLuc8 assay, we show that REEP1 facilitates ER-mitochondria interactions, a function diminished by disease-associated mutations.
Our data potentially reconcile the current conflicting reports regarding REEP1 being either an ER or a mitochondrial protein. Furthermore, our results connect, for the first time, the disrupted ER-mitochondria interactions to a failure in maintaining health of long axons in HSPs. Finally, the split-RLuc8 assay offers a new tool to identify potential drugs for multiple neurodegenerative diseases with ER-mitochondria interaction defects.
受体表达增强蛋白1（REEP1）突变与遗传性痉挛性截瘫（HSP）相关。尽管轴突变性被认为是HSP的主要特征，但REEP1突变在变性中的作用很大程度上未知。先前的研究表明REEP1在内质网（ER）中发挥作用，而其他研究则将REEP1定位于线粒体。我们试图确定REEP1的细胞定位，并进一步阐明患者中REEP1突变的病理生物学机制。
结合细胞成像和生化方法来优化REEP1的细胞定位。接下来，在使用小鼠皮质培养的神经突生长和变性试验中对与HSP相关的Reep1突变进行功能测试。最后，开发了一种新的试验，并用于野生型和突变型Reep1，以测量ER和线粒体之间的相互作用。
我们发现REEP1存在于ER-线粒体界面，并且它包含线粒体以及ER定位的亚结构域。Reep1的敲低和病理性Reep1突变的表达导致神经突生长缺陷和变性。最后，使用我们新的分裂型Renilla荧光素酶8（split-RLuc8）试验，我们表明REEP1促进ER-线粒体相互作用，这种功能因疾病相关突变而减弱。
我们的数据可能调和了目前关于REEP1是ER蛋白还是线粒体蛋白的相互矛盾的报道。此外，我们的结果首次将ER-线粒体相互作用的破坏与HSP中长轴突健康维持的失败联系起来。最后，split-RLuc8试验提供了一种新工具，用于识别具有ER-线粒体相互作用缺陷的多种神经退行性疾病的潜在药物。
