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Generation of iPSC Lines with Tagged α-Synuclein for Visualization of Endogenous Protein in Human Cellular Models of Neurodegenerative Disorders

Zetterdahl, Oskar G LU ; Crowe, James A LU orcid ; Reyhani, Samira LU ; Güra, Miriam A ; Labastida-Botey, Ot ; Girard, Aline S LU ; Froese, D Sean ; Ahlenius, Henrik LU and Canals, Isaac LU (2025) In eNeuro 12(6).
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... (More)

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.

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author
; ; ; ; ; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Humans, alpha-Synuclein/metabolism, Induced Pluripotent Stem Cells/metabolism, Neurodegenerative Diseases/metabolism, Neurons/metabolism, Cell Line, CRISPR-Cas Systems, Gene Editing, Cell Differentiation
in
eNeuro
volume
12
issue
6
publisher
Society for Neuroscience
external identifiers
  • pmid:40456614
  • scopus:105009380397
ISSN
2373-2822
DOI
10.1523/ENEURO.0093-25.2025
language
English
LU publication?
yes
additional info
Copyright © 2025 Zetterdahl et al.
id
dcb8a87b-a966-4397-bf1a-fb623a8a15c7
date added to LUP
2025-08-26 11:03:13
date last changed
2025-08-27 09:01:21
@article{dcb8a87b-a966-4397-bf1a-fb623a8a15c7,
  abstract     = {{<p>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 (<br>
 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.<br>
 </p>}},
  author       = {{Zetterdahl, Oskar G and Crowe, James A and Reyhani, Samira and Güra, Miriam A and Labastida-Botey, Ot and Girard, Aline S and Froese, D Sean and Ahlenius, Henrik and Canals, Isaac}},
  issn         = {{2373-2822}},
  keywords     = {{Humans; alpha-Synuclein/metabolism; Induced Pluripotent Stem Cells/metabolism; Neurodegenerative Diseases/metabolism; Neurons/metabolism; Cell Line; CRISPR-Cas Systems; Gene Editing; Cell Differentiation}},
  language     = {{eng}},
  number       = {{6}},
  publisher    = {{Society for Neuroscience}},
  series       = {{eNeuro}},
  title        = {{Generation of iPSC Lines with Tagged α-Synuclein for Visualization of Endogenous Protein in Human Cellular Models of Neurodegenerative Disorders}},
  url          = {{http://dx.doi.org/10.1523/ENEURO.0093-25.2025}},
  doi          = {{10.1523/ENEURO.0093-25.2025}},
  volume       = {{12}},
  year         = {{2025}},
}