Method for measuring cell-to-cell transmission of α-synuclein aggregates using bimolecular fluorescence complementation system and method for screening a substance for preventing or treating neurodegenerative disease using the same
Inventors
Lee, Seung-jae • LEE, He-Jin • Bae, Eun-Jin • Kim, Dong Kyu
Assignees
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Publication Number
US-9757402-B2
Publication Date
2017-09-12
Expiration Date
Abstract
The present disclosure relates to dual-cell model and Caenorhabditis elegans model systems for measuring neuron-to-neuron transmission of protein aggregates, and more particularly to transgenic cell and animal model systems expressing fusion proteins of N-terminus or C-terminus of fluorescent proteins with α-synuclein proteins, methods for measuring continuous cell-to-cell transmission of α-synuclein aggregates using the same, and methods for screening substances for preventing or treating neurodegenerative diseases.
Core Innovation
The invention relates to measuring continuous cell-to-cell transmission of α-synuclein aggregates and analyzing co-aggregation of α-synuclein proteins derived from adjacent cells by using a cell system split across two cells expressing complementary BiFC fusion proteins. A first cell expresses an N-terminal fragment of a fluorescent protein fused to α-synuclein, and a second cell expresses a C-terminal fragment of the fluorescent protein fused to α-synuclein. Through co-culture of the first cell and the second cell, α-synuclein co-aggregation and cell-to-cell transfer are detected by quantitative detection of a bimolecular fluorescence complementation (BiFC) fluorescent signal generated by linking the N-terminal and C-terminal fragments.
The invention addresses the need to measure cell-to-cell transmission and co-aggregation in a way that enables analysis of transfer between adjacent cells using a fluorescence complementation readout. By expressing complementary split fluorescent protein–α-synuclein fusion proteins in donor and recipient cells and detecting BiFC-positive signals in each cell type, the approach provides a quantitative way to analyze cell-to-cell transfer and co-aggregation of α-synuclein proteins derived from adjacent cells. The document reports that co-culture and serial subculturing increase BiFC-positive populations and secondary secretion, supporting perpetual transmission.
The described cell system includes transgenic neuroblastoma cell lines having Accession No. KCLRF-BP-00322 as the first cell and Accession No. KCLRF-BP-00323 as the second cell. The disclosed models connect BiFC signal outcomes to lysosomal dysfunction and aging-related pathways, including GBA1 (GCase) loss in recipient cells and GBA1 rescue with wild-type. In Caenorhabditis elegans, aging-rate modulation (daf-2/daf-16), anti-aging treatment GlcNAc, and lysosomal biogenesis regulator hlh-30/TFEB are reported to modulate BiFC signals, inclusion bodies, nerve degeneration, pumping behavior, and lifespan.
Claims Coverage
The partial content includes one independent claim describing a cell-based method to measure cell-to-cell transmission of α-synuclein aggregates using split-fluorescent BiFC fusion proteins expressed in co-cultured donor/recipient cells, with quantitative detection of BiFC signals and with specified transgenic neuroblastoma cell line accessions. The independent claim includes three main inventive aspects corresponding to the donor/recipient split BiFC design, co-culture-based measurement and analysis of transfer/co-aggregation, and the specified transgenic neuroblastoma cell lines for the first and second cells.
Split fluorescent protein BiFC fusion in donor and recipient cells
A cell system comprising a first cell expressing a first fusion protein where an N-terminal fragment of a fluorescent protein and α-synuclein are fused, and a second cell expressing a second fusion protein where a C-terminal fragment of the fluorescent protein and α-synuclein are fused.
Co-culture to generate BiFC for analyzing transfer and co-aggregation
Co-expressing the first fusion protein and the second fusion protein in the first cell and the second cell, respectively, through co-culture of the first cell and the second cell; and quantitatively detecting, in the first cell and the second cell, a bimolecular fluorescence complementation (BiFC) fluorescent signal generated by linking the N-terminal fragment and the C-terminal fragment of the fluorescent protein to analyze cell-to-cell transfer and co-aggregation of α-synuclein proteins derived from adjacent cells.
Transgenic neuroblastoma cell line pair for donor/recipient
Wherein the cell model system includes a transgenic neuroblastoma cell line having Accession No. KCLRF-BP-00322 as the first cell and a transgenic neuroblastoma cell line having Accession No. KCLRF-BP-00323 as the second cell.
Across the independent claim, the inventive coverage focuses on a split-fluorescent BiFC system fused to α-synuclein in two co-cultured cells, quantitative detection of the BiFC signal to analyze cell-to-cell transfer and co-aggregation, and use of specified transgenic neuroblastoma cell line accessions as the first and second cells.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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