Rapid in vitro quantification of TDP-43 and FUS mislocalisation for screening of gene variants implicated in frontotemporal dementia and amyotrophic lateral sclerosis

Abstract Identified genetic mutations cause 20% of frontotemporal dementia (FTD) and 5-10% of amyotrophic lateral sclerosis (ALS) cases: however, for the remainder of patients the origin of disease is uncertain. The overlap in genetic, clinical and pathological presentation of FTD and ALS suggests t...

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Autores principales:	Lisa J. Oyston, Stephanie Ubiparipovic, Lauren Fitzpatrick, Marianne Hallupp, Lauren M. Boccanfuso, John B. Kwok, Carol Dobson-Stone
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/d6b114cd448a4c3994d1dd8d4af96bb4
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spelling	oai:doaj.org-article:d6b114cd448a4c3994d1dd8d4af96bb42021-12-02T16:17:28ZRapid in vitro quantification of TDP-43 and FUS mislocalisation for screening of gene variants implicated in frontotemporal dementia and amyotrophic lateral sclerosis10.1038/s41598-021-94225-12045-2322https://doaj.org/article/d6b114cd448a4c3994d1dd8d4af96bb42021-07-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-94225-1https://doaj.org/toc/2045-2322Abstract Identified genetic mutations cause 20% of frontotemporal dementia (FTD) and 5-10% of amyotrophic lateral sclerosis (ALS) cases: however, for the remainder of patients the origin of disease is uncertain. The overlap in genetic, clinical and pathological presentation of FTD and ALS suggests these two diseases are related. Post-mortem, ~ 95% of ALS and ~ 50% of FTD patients show redistribution of the nuclear protein TDP-43 to the cytoplasm within affected neurons, while ~ 5% ALS and ~ 10% FTD show mislocalisation of FUS protein. We exploited these neuropathological features to develop an unbiased method for the in vitro quantification of cytoplasmic TDP-43 and FUS. Utilising fluorescently-tagged cDNA constructs and immunocytochemistry, the fluorescence intensity of TDP-43 or FUS was measured in the nucleus and cytoplasm of cells, using the freely available software CellProfiler. Significant increases in the amount of cytoplasmic TDP-43 and FUS were detectable in cells expressing known FTD/ALS-causative TARDBP and FUS gene mutations. Pharmacological intervention with the apoptosis inducer staurosporine and mutation in a secondary gene (CYLD) also induced measurable cytoplasmic mislocalisation of endogenous FUS and TDP-43, respectively. These findings validate this methodology as a novel in vitro technique for the quantification of TDP-43 or FUS mislocalisation that can be used for initial prioritisation of predicted FTD/ALS-causative mutations.Lisa J. OystonStephanie UbiparipovicLauren FitzpatrickMarianne HalluppLauren M. BoccanfusoJohn B. KwokCarol Dobson-StoneNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-10 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Lisa J. Oyston Stephanie Ubiparipovic Lauren Fitzpatrick Marianne Hallupp Lauren M. Boccanfuso John B. Kwok Carol Dobson-Stone Rapid in vitro quantification of TDP-43 and FUS mislocalisation for screening of gene variants implicated in frontotemporal dementia and amyotrophic lateral sclerosis
description	Abstract Identified genetic mutations cause 20% of frontotemporal dementia (FTD) and 5-10% of amyotrophic lateral sclerosis (ALS) cases: however, for the remainder of patients the origin of disease is uncertain. The overlap in genetic, clinical and pathological presentation of FTD and ALS suggests these two diseases are related. Post-mortem, ~ 95% of ALS and ~ 50% of FTD patients show redistribution of the nuclear protein TDP-43 to the cytoplasm within affected neurons, while ~ 5% ALS and ~ 10% FTD show mislocalisation of FUS protein. We exploited these neuropathological features to develop an unbiased method for the in vitro quantification of cytoplasmic TDP-43 and FUS. Utilising fluorescently-tagged cDNA constructs and immunocytochemistry, the fluorescence intensity of TDP-43 or FUS was measured in the nucleus and cytoplasm of cells, using the freely available software CellProfiler. Significant increases in the amount of cytoplasmic TDP-43 and FUS were detectable in cells expressing known FTD/ALS-causative TARDBP and FUS gene mutations. Pharmacological intervention with the apoptosis inducer staurosporine and mutation in a secondary gene (CYLD) also induced measurable cytoplasmic mislocalisation of endogenous FUS and TDP-43, respectively. These findings validate this methodology as a novel in vitro technique for the quantification of TDP-43 or FUS mislocalisation that can be used for initial prioritisation of predicted FTD/ALS-causative mutations.
format	article
author	Lisa J. Oyston Stephanie Ubiparipovic Lauren Fitzpatrick Marianne Hallupp Lauren M. Boccanfuso John B. Kwok Carol Dobson-Stone
author_facet	Lisa J. Oyston Stephanie Ubiparipovic Lauren Fitzpatrick Marianne Hallupp Lauren M. Boccanfuso John B. Kwok Carol Dobson-Stone
author_sort	Lisa J. Oyston
title	Rapid in vitro quantification of TDP-43 and FUS mislocalisation for screening of gene variants implicated in frontotemporal dementia and amyotrophic lateral sclerosis
title_short	Rapid in vitro quantification of TDP-43 and FUS mislocalisation for screening of gene variants implicated in frontotemporal dementia and amyotrophic lateral sclerosis
title_full	Rapid in vitro quantification of TDP-43 and FUS mislocalisation for screening of gene variants implicated in frontotemporal dementia and amyotrophic lateral sclerosis
title_fullStr	Rapid in vitro quantification of TDP-43 and FUS mislocalisation for screening of gene variants implicated in frontotemporal dementia and amyotrophic lateral sclerosis
title_full_unstemmed	Rapid in vitro quantification of TDP-43 and FUS mislocalisation for screening of gene variants implicated in frontotemporal dementia and amyotrophic lateral sclerosis
title_sort	rapid in vitro quantification of tdp-43 and fus mislocalisation for screening of gene variants implicated in frontotemporal dementia and amyotrophic lateral sclerosis
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/d6b114cd448a4c3994d1dd8d4af96bb4
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Rapid in vitro quantification of TDP-43 and FUS mislocalisation for screening of gene variants implicated in frontotemporal dementia and amyotrophic lateral sclerosis

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