Cargando…

Live Neuron High-Content Screening Reveals Synaptotoxic Activity in Alzheimer Mouse Model Homogenates

Accurate quantification of synaptic changes is essential for understanding the molecular mechanisms of synaptogenesis, synaptic plasticity, and synaptic toxicity. Here we demonstrate a robust high-content imaging method for the assessment of synaptic changes and apply the method to brain homogenates...

Descripción completa

Detalles Bibliográficos
Autores principales: Jiang, Hao, Esparza, Thomas J., Kummer, Terrance T., Zhong, Haining, Rettig, Jens, Brody, David L.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7042280/
https://www.ncbi.nlm.nih.gov/pubmed/32098978
http://dx.doi.org/10.1038/s41598-020-60118-y
_version_ 1783501278313185280
author Jiang, Hao
Esparza, Thomas J.
Kummer, Terrance T.
Zhong, Haining
Rettig, Jens
Brody, David L.
author_facet Jiang, Hao
Esparza, Thomas J.
Kummer, Terrance T.
Zhong, Haining
Rettig, Jens
Brody, David L.
author_sort Jiang, Hao
collection PubMed
description Accurate quantification of synaptic changes is essential for understanding the molecular mechanisms of synaptogenesis, synaptic plasticity, and synaptic toxicity. Here we demonstrate a robust high-content imaging method for the assessment of synaptic changes and apply the method to brain homogenates from an Alzheimer’s disease mouse model. Our method uses serial imaging of endogenous fluorescent labeled presynaptic VAMP2 and postsynaptic PSD95 in long-term cultured live primary neurons in 96 well microplates, and uses automatic image analysis to quantify the number of colocalized mature synaptic puncta for the assessment of synaptic changes in live neurons. As a control, we demonstrated that our synaptic puncta assay is at least 10-fold more sensitive to the toxic effects of glutamate than the MTT assay. Using our assay, we have compared synaptotoxic activities in size-exclusion chromatography fractioned protein samples from 3xTg-AD mouse model brain homogenates. Multiple synaptotoxic activities were found in high and low molecular weight fractions. Amyloid-beta immunodepletion alleviated some but not all of the synaptotoxic activities. Although the biochemical entities responsible for the synaptotoxic activities have yet to be determined, these proof-of-concept results demonstrate that this novel assay may have many potential mechanistic and therapeutic applications.
format Online
Article
Text
id pubmed-7042280
institution National Center for Biotechnology Information
language English
publishDate 2020
publisher Nature Publishing Group UK
record_format MEDLINE/PubMed
spelling pubmed-70422802020-03-03 Live Neuron High-Content Screening Reveals Synaptotoxic Activity in Alzheimer Mouse Model Homogenates Jiang, Hao Esparza, Thomas J. Kummer, Terrance T. Zhong, Haining Rettig, Jens Brody, David L. Sci Rep Article Accurate quantification of synaptic changes is essential for understanding the molecular mechanisms of synaptogenesis, synaptic plasticity, and synaptic toxicity. Here we demonstrate a robust high-content imaging method for the assessment of synaptic changes and apply the method to brain homogenates from an Alzheimer’s disease mouse model. Our method uses serial imaging of endogenous fluorescent labeled presynaptic VAMP2 and postsynaptic PSD95 in long-term cultured live primary neurons in 96 well microplates, and uses automatic image analysis to quantify the number of colocalized mature synaptic puncta for the assessment of synaptic changes in live neurons. As a control, we demonstrated that our synaptic puncta assay is at least 10-fold more sensitive to the toxic effects of glutamate than the MTT assay. Using our assay, we have compared synaptotoxic activities in size-exclusion chromatography fractioned protein samples from 3xTg-AD mouse model brain homogenates. Multiple synaptotoxic activities were found in high and low molecular weight fractions. Amyloid-beta immunodepletion alleviated some but not all of the synaptotoxic activities. Although the biochemical entities responsible for the synaptotoxic activities have yet to be determined, these proof-of-concept results demonstrate that this novel assay may have many potential mechanistic and therapeutic applications. Nature Publishing Group UK 2020-02-25 /pmc/articles/PMC7042280/ /pubmed/32098978 http://dx.doi.org/10.1038/s41598-020-60118-y Text en © The Author(s) 2020 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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Jiang, Hao
Esparza, Thomas J.
Kummer, Terrance T.
Zhong, Haining
Rettig, Jens
Brody, David L.
Live Neuron High-Content Screening Reveals Synaptotoxic Activity in Alzheimer Mouse Model Homogenates
title Live Neuron High-Content Screening Reveals Synaptotoxic Activity in Alzheimer Mouse Model Homogenates
title_full Live Neuron High-Content Screening Reveals Synaptotoxic Activity in Alzheimer Mouse Model Homogenates
title_fullStr Live Neuron High-Content Screening Reveals Synaptotoxic Activity in Alzheimer Mouse Model Homogenates
title_full_unstemmed Live Neuron High-Content Screening Reveals Synaptotoxic Activity in Alzheimer Mouse Model Homogenates
title_short Live Neuron High-Content Screening Reveals Synaptotoxic Activity in Alzheimer Mouse Model Homogenates
title_sort live neuron high-content screening reveals synaptotoxic activity in alzheimer mouse model homogenates
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7042280/
https://www.ncbi.nlm.nih.gov/pubmed/32098978
http://dx.doi.org/10.1038/s41598-020-60118-y
work_keys_str_mv AT jianghao liveneuronhighcontentscreeningrevealssynaptotoxicactivityinalzheimermousemodelhomogenates
AT esparzathomasj liveneuronhighcontentscreeningrevealssynaptotoxicactivityinalzheimermousemodelhomogenates
AT kummerterrancet liveneuronhighcontentscreeningrevealssynaptotoxicactivityinalzheimermousemodelhomogenates
AT zhonghaining liveneuronhighcontentscreeningrevealssynaptotoxicactivityinalzheimermousemodelhomogenates
AT rettigjens liveneuronhighcontentscreeningrevealssynaptotoxicactivityinalzheimermousemodelhomogenates
AT brodydavidl liveneuronhighcontentscreeningrevealssynaptotoxicactivityinalzheimermousemodelhomogenates