Soshana Svendsen1, Erin McMillan2, Chad Zimprich1, Allison Ebert2, Craig Tork2, Clive N. Svendsen2, Georgi V. Los1
1Research and Development, Promega Corporation, Madison, WI
2Waisman Center, University of Wisconsin, Madison, WI
Over expression of the α-synuclein protein can lead to familial Parkinson’s disease. However, the cause of cell loss and dysfunction in human neurons is still unknown. In addition, the relevance of synuclein subcellular localization within the cytoplasm versus the nucleus remains unclear.
The HaloTag® technology, based on the formation of a specific covalent bond between a HaloTag reporter protein and fluorescent ligands, was used to explore synuclein over production in human neural progenitor cells. Our previous studies have shown that synuclein and its mutations can be expressed in human neural stem and progenitor cells (Schneider et al, 2007). To establish whether human synuclein can be tagged and specifically directed to either the cytoplasm or the nucleus, we fused synuclein to either HaloTag or HaloTag and a nuclear localization sequence (NLS). Lentiviral vectors encoding these synuclein fusions were generated and used for stable expression in human neural progenitor cells. Image analysis showed proper localization in progenitors and in differentiated human neurons and astrocytes. Stable expression permits the study of old and new protein pools overtime in live cells.
Finally, cells stably expressing synuclein-HaloTag fusions were transplanted to assess survival, expression and protein localization in vivo. Transplants, visualized with the HaloTag® technology,