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In vivo Validation of Bimolecular Fluorescence Complementation (BiFC) to Investigate Aggregate Formation in Amyotrophic Lateral Sclerosis (ALS)
Amyotrophic lateral sclerosis (ALS) is a form of motor neuron disease (MND) that is characterized by the progressive loss of motor neurons within the spinal cord, brainstem, and motor cortex. Although ALS clinically manifests as a heterogeneous disease, with varying disease onset and survival, a uni...
Autores principales: | , , , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Springer US
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8018926/ https://www.ncbi.nlm.nih.gov/pubmed/33415684 http://dx.doi.org/10.1007/s12035-020-02238-0 |
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author | Don, Emily K. Maschirow, Alina Radford, Rowan A. W. Scherer, Natalie M. Vidal-Itriago, Andrés Hogan, Alison Maurel, Cindy Formella, Isabel Stoddart, Jack J. Hall, Thomas E. Lee, Albert Shi, Bingyang Cole, Nicholas J. Laird, Angela S. Badrock, Andrew P. Chung, Roger S. Morsch, Marco |
author_facet | Don, Emily K. Maschirow, Alina Radford, Rowan A. W. Scherer, Natalie M. Vidal-Itriago, Andrés Hogan, Alison Maurel, Cindy Formella, Isabel Stoddart, Jack J. Hall, Thomas E. Lee, Albert Shi, Bingyang Cole, Nicholas J. Laird, Angela S. Badrock, Andrew P. Chung, Roger S. Morsch, Marco |
author_sort | Don, Emily K. |
collection | PubMed |
description | Amyotrophic lateral sclerosis (ALS) is a form of motor neuron disease (MND) that is characterized by the progressive loss of motor neurons within the spinal cord, brainstem, and motor cortex. Although ALS clinically manifests as a heterogeneous disease, with varying disease onset and survival, a unifying feature is the presence of ubiquitinated cytoplasmic protein inclusion aggregates containing TDP-43. However, the precise mechanisms linking protein inclusions and aggregation to neuronal loss are currently poorly understood. Bimolecular fluorescence complementation (BiFC) takes advantage of the association of fluorophore fragments (non-fluorescent on their own) that are attached to an aggregation-prone protein of interest. Interaction of the proteins of interest allows for the fluorescent reporter protein to fold into its native state and emit a fluorescent signal. Here, we combined the power of BiFC with the advantages of the zebrafish system to validate, optimize, and visualize the formation of ALS-linked aggregates in real time in a vertebrate model. We further provide in vivo validation of the selectivity of this technique and demonstrate reduced spontaneous self-assembly of the non-fluorescent fragments in vivo by introducing a fluorophore mutation. Additionally, we report preliminary findings on the dynamic aggregation of the ALS-linked hallmark proteins Fus and TDP-43 in their corresponding nuclear and cytoplasmic compartments using BiFC. Overall, our data demonstrates the suitability of this BiFC approach to study and characterize ALS-linked aggregate formation in vivo. Importantly, the same principle can be applied in the context of other neurodegenerative diseases and has therefore critical implications to advance our understanding of pathologies that underlie aberrant protein aggregation. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12035-020-02238-0. |
format | Online Article Text |
id | pubmed-8018926 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Springer US |
record_format | MEDLINE/PubMed |
spelling | pubmed-80189262021-04-16 In vivo Validation of Bimolecular Fluorescence Complementation (BiFC) to Investigate Aggregate Formation in Amyotrophic Lateral Sclerosis (ALS) Don, Emily K. Maschirow, Alina Radford, Rowan A. W. Scherer, Natalie M. Vidal-Itriago, Andrés Hogan, Alison Maurel, Cindy Formella, Isabel Stoddart, Jack J. Hall, Thomas E. Lee, Albert Shi, Bingyang Cole, Nicholas J. Laird, Angela S. Badrock, Andrew P. Chung, Roger S. Morsch, Marco Mol Neurobiol Article Amyotrophic lateral sclerosis (ALS) is a form of motor neuron disease (MND) that is characterized by the progressive loss of motor neurons within the spinal cord, brainstem, and motor cortex. Although ALS clinically manifests as a heterogeneous disease, with varying disease onset and survival, a unifying feature is the presence of ubiquitinated cytoplasmic protein inclusion aggregates containing TDP-43. However, the precise mechanisms linking protein inclusions and aggregation to neuronal loss are currently poorly understood. Bimolecular fluorescence complementation (BiFC) takes advantage of the association of fluorophore fragments (non-fluorescent on their own) that are attached to an aggregation-prone protein of interest. Interaction of the proteins of interest allows for the fluorescent reporter protein to fold into its native state and emit a fluorescent signal. Here, we combined the power of BiFC with the advantages of the zebrafish system to validate, optimize, and visualize the formation of ALS-linked aggregates in real time in a vertebrate model. We further provide in vivo validation of the selectivity of this technique and demonstrate reduced spontaneous self-assembly of the non-fluorescent fragments in vivo by introducing a fluorophore mutation. Additionally, we report preliminary findings on the dynamic aggregation of the ALS-linked hallmark proteins Fus and TDP-43 in their corresponding nuclear and cytoplasmic compartments using BiFC. Overall, our data demonstrates the suitability of this BiFC approach to study and characterize ALS-linked aggregate formation in vivo. Importantly, the same principle can be applied in the context of other neurodegenerative diseases and has therefore critical implications to advance our understanding of pathologies that underlie aberrant protein aggregation. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12035-020-02238-0. Springer US 2021-01-07 2021 /pmc/articles/PMC8018926/ /pubmed/33415684 http://dx.doi.org/10.1007/s12035-020-02238-0 Text en © The Author(s) 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. |
spellingShingle | Article Don, Emily K. Maschirow, Alina Radford, Rowan A. W. Scherer, Natalie M. Vidal-Itriago, Andrés Hogan, Alison Maurel, Cindy Formella, Isabel Stoddart, Jack J. Hall, Thomas E. Lee, Albert Shi, Bingyang Cole, Nicholas J. Laird, Angela S. Badrock, Andrew P. Chung, Roger S. Morsch, Marco In vivo Validation of Bimolecular Fluorescence Complementation (BiFC) to Investigate Aggregate Formation in Amyotrophic Lateral Sclerosis (ALS) |
title | In vivo Validation of Bimolecular Fluorescence Complementation (BiFC) to Investigate Aggregate Formation in Amyotrophic Lateral Sclerosis (ALS) |
title_full | In vivo Validation of Bimolecular Fluorescence Complementation (BiFC) to Investigate Aggregate Formation in Amyotrophic Lateral Sclerosis (ALS) |
title_fullStr | In vivo Validation of Bimolecular Fluorescence Complementation (BiFC) to Investigate Aggregate Formation in Amyotrophic Lateral Sclerosis (ALS) |
title_full_unstemmed | In vivo Validation of Bimolecular Fluorescence Complementation (BiFC) to Investigate Aggregate Formation in Amyotrophic Lateral Sclerosis (ALS) |
title_short | In vivo Validation of Bimolecular Fluorescence Complementation (BiFC) to Investigate Aggregate Formation in Amyotrophic Lateral Sclerosis (ALS) |
title_sort | in vivo validation of bimolecular fluorescence complementation (bifc) to investigate aggregate formation in amyotrophic lateral sclerosis (als) |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8018926/ https://www.ncbi.nlm.nih.gov/pubmed/33415684 http://dx.doi.org/10.1007/s12035-020-02238-0 |
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