Abstract
α-Synuclein is a synaptic protein that accumulates primarily in synucleinopathies and secondarily in certain lysosomal storage disorders. However, its physiological roles in health and disease are not fully understood. In part, this has been hampered by the inability to visualize α-synuclein and its cellular localization, due to the lack of specific antibodies and faithful reporters. Here, we used CRISPR/Cas9-based genome editing to generate human induced pluripotent stem cell (iPSC) lines in which the α-synuclein (SNCA) gene has been tagged with the short HA peptide either at the N-terminus or C-terminus, or with the fluorescent protein mCherry at the C-terminus of the protein. These diverse strategies revealed the C-terminus HA-tag as the best option. C-terminus HA-tagged α-synuclein had unchanged protein expression and did not generate degradation by-products. Importantly, we show that following differentiation to neurons the C-terminus HA-tagged iPSC line had unaffected electrophysiological properties and could be used to visualize accumulation of α-synuclein upon inhibition of lysosomal function and under physiological protein levels. It is our expectation that this line and tagging approach will be very useful in further studies examining α-synuclein aggregation and its role in cellular dysfunction and neurodegeneration.
Significance Statement We present an optimal genome editing strategy for incorporating the short peptide HA at the C-terminus of α-synuclein in human induced pluripotent stem cells. We also show that this newly generated C-terminus tagged line can be differentiated towards functional neurons to facilitate visualization of the protein and its accumulation upon inhibition of lysosomal function, which will be useful for studying aggregation in models of neurodegenerative diseases.
Footnotes
We thank the Strategic Research Area's MultiPark (Multidisciplinary Research on Parkinson’s Disease) and the Lund Stem Cell Center for infrastructure support; the Electrophisiology Core Facility at Lund University for the neuronal recordings and analysis of data; and the StemTherapy Imaging Platform at Lund University and the Center for Microscopy and Image Analysis at University of Zurich for technical assistance. Some illustrations where created in BioRender: Fig 1A, Fig 1B, Fig 1-5A, Fig 1-6A, Fig 2A and Fig 3A.
The authors declare no competing financial interests.
This study was supported by Swedish Society of Medical Research (grant no. S20-003 to I.C.),
Swedish Research Council (grant no. 2023-02409 to H.A.), Hjärnfonden (grant no. FO2023-0391 to I.C.), Åhlén foundation (grant no. 233003 to I.C.), Magnus Bergvall foundation (grant no. 2023-471 to I.C.), National MPS Society (grant no. 1749 to I.C.), Olga Mayenfisch foundation (grant to I.C.), Swiss National Science Foundation (grant no. 320030E 219127 and 320030-231175 to D.S.F.) and the UZH Postdoc Grant (grant no. FK-24-044 to J.C.). M.A.G., D.S.F., A.G. and I.C. are supported by the University of Zurich Research Priority Program ITINERARE – Innovative therapies in rare diseases.
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