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PU.1 regulates Alzheimer’s disease-associated genes in primary human microglia

BACKGROUND: Microglia play critical roles in the brain during homeostasis and pathological conditions. Understanding the molecular events underpinning microglial functions and activation states will further enable us to target these cells for the treatment of neurological disorders. The transcriptio...

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Autores principales: Rustenhoven, Justin, Smith, Amy M., Smyth, Leon C., Jansson, Deidre, Scotter, Emma L., Swanson, Molly E. V., Aalderink, Miranda, Coppieters, Natacha, Narayan, Pritika, Handley, Renee, Overall, Chris, Park, Thomas I. H., Schweder, Patrick, Heppner, Peter, Curtis, Maurice A., Faull, Richard L. M., Dragunow, Mike
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6102813/
https://www.ncbi.nlm.nih.gov/pubmed/30124174
http://dx.doi.org/10.1186/s13024-018-0277-1
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author Rustenhoven, Justin
Smith, Amy M.
Smyth, Leon C.
Jansson, Deidre
Scotter, Emma L.
Swanson, Molly E. V.
Aalderink, Miranda
Coppieters, Natacha
Narayan, Pritika
Handley, Renee
Overall, Chris
Park, Thomas I. H.
Schweder, Patrick
Heppner, Peter
Curtis, Maurice A.
Faull, Richard L. M.
Dragunow, Mike
author_facet Rustenhoven, Justin
Smith, Amy M.
Smyth, Leon C.
Jansson, Deidre
Scotter, Emma L.
Swanson, Molly E. V.
Aalderink, Miranda
Coppieters, Natacha
Narayan, Pritika
Handley, Renee
Overall, Chris
Park, Thomas I. H.
Schweder, Patrick
Heppner, Peter
Curtis, Maurice A.
Faull, Richard L. M.
Dragunow, Mike
author_sort Rustenhoven, Justin
collection PubMed
description BACKGROUND: Microglia play critical roles in the brain during homeostasis and pathological conditions. Understanding the molecular events underpinning microglial functions and activation states will further enable us to target these cells for the treatment of neurological disorders. The transcription factor PU.1 is critical in the development of myeloid cells and a major regulator of microglial gene expression. In the brain, PU.1 is specifically expressed in microglia and recent evidence from genome-wide association studies suggests that reductions in PU.1 contribute to a delayed onset of Alzheimer’s disease (AD), possibly through limiting neuroinflammatory responses. METHODS: To investigate how PU.1 contributes to immune activation in human microglia, microarray analysis was performed on primary human mixed glial cultures subjected to siRNA-mediated knockdown of PU.1. Microarray hits were confirmed by qRT-PCR and immunocytochemistry in both mixed glial cultures and isolated microglia following PU.1 knockdown. To identify attenuators of PU.1 expression in microglia, high throughput drug screening was undertaken using a compound library containing FDA-approved drugs. NanoString and immunohistochemistry was utilised to investigate the expression of PU.1 itself and PU.1-regulated mediators in primary human brain tissue derived from neurologically normal and clinically and pathologically confirmed cases of AD. RESULTS: Bioinformatic analysis of gene expression upon PU.1 silencing in mixed glial cultures revealed a network of modified AD-associated microglial genes involved in the innate and adaptive immune systems, particularly those involved in antigen presentation and phagocytosis. These gene changes were confirmed using isolated microglial cultures. Utilising high throughput screening of FDA-approved compounds in mixed glial cultures we identified the histone deacetylase inhibitor vorinostat as an effective attenuator of PU.1 expression in human microglia. Further characterisation of vorinostat in isolated microglial cultures revealed gene and protein changes partially recapitulating those seen following siRNA-mediated PU.1 knockdown. Lastly, we demonstrate that several of these PU.1-regulated genes are expressed by microglia in the human AD brain in situ. CONCLUSIONS: Collectively, these results suggest that attenuating PU.1 may be a valid therapeutic approach to limit microglial-mediated inflammatory responses in AD and demonstrate utility of vorinostat for this purpose. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s13024-018-0277-1) contains supplementary material, which is available to authorized users.
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spelling pubmed-61028132018-08-27 PU.1 regulates Alzheimer’s disease-associated genes in primary human microglia Rustenhoven, Justin Smith, Amy M. Smyth, Leon C. Jansson, Deidre Scotter, Emma L. Swanson, Molly E. V. Aalderink, Miranda Coppieters, Natacha Narayan, Pritika Handley, Renee Overall, Chris Park, Thomas I. H. Schweder, Patrick Heppner, Peter Curtis, Maurice A. Faull, Richard L. M. Dragunow, Mike Mol Neurodegener Research Article BACKGROUND: Microglia play critical roles in the brain during homeostasis and pathological conditions. Understanding the molecular events underpinning microglial functions and activation states will further enable us to target these cells for the treatment of neurological disorders. The transcription factor PU.1 is critical in the development of myeloid cells and a major regulator of microglial gene expression. In the brain, PU.1 is specifically expressed in microglia and recent evidence from genome-wide association studies suggests that reductions in PU.1 contribute to a delayed onset of Alzheimer’s disease (AD), possibly through limiting neuroinflammatory responses. METHODS: To investigate how PU.1 contributes to immune activation in human microglia, microarray analysis was performed on primary human mixed glial cultures subjected to siRNA-mediated knockdown of PU.1. Microarray hits were confirmed by qRT-PCR and immunocytochemistry in both mixed glial cultures and isolated microglia following PU.1 knockdown. To identify attenuators of PU.1 expression in microglia, high throughput drug screening was undertaken using a compound library containing FDA-approved drugs. NanoString and immunohistochemistry was utilised to investigate the expression of PU.1 itself and PU.1-regulated mediators in primary human brain tissue derived from neurologically normal and clinically and pathologically confirmed cases of AD. RESULTS: Bioinformatic analysis of gene expression upon PU.1 silencing in mixed glial cultures revealed a network of modified AD-associated microglial genes involved in the innate and adaptive immune systems, particularly those involved in antigen presentation and phagocytosis. These gene changes were confirmed using isolated microglial cultures. Utilising high throughput screening of FDA-approved compounds in mixed glial cultures we identified the histone deacetylase inhibitor vorinostat as an effective attenuator of PU.1 expression in human microglia. Further characterisation of vorinostat in isolated microglial cultures revealed gene and protein changes partially recapitulating those seen following siRNA-mediated PU.1 knockdown. Lastly, we demonstrate that several of these PU.1-regulated genes are expressed by microglia in the human AD brain in situ. CONCLUSIONS: Collectively, these results suggest that attenuating PU.1 may be a valid therapeutic approach to limit microglial-mediated inflammatory responses in AD and demonstrate utility of vorinostat for this purpose. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s13024-018-0277-1) contains supplementary material, which is available to authorized users. BioMed Central 2018-08-20 /pmc/articles/PMC6102813/ /pubmed/30124174 http://dx.doi.org/10.1186/s13024-018-0277-1 Text en © The Author(s). 2018 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided 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 Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research Article
Rustenhoven, Justin
Smith, Amy M.
Smyth, Leon C.
Jansson, Deidre
Scotter, Emma L.
Swanson, Molly E. V.
Aalderink, Miranda
Coppieters, Natacha
Narayan, Pritika
Handley, Renee
Overall, Chris
Park, Thomas I. H.
Schweder, Patrick
Heppner, Peter
Curtis, Maurice A.
Faull, Richard L. M.
Dragunow, Mike
PU.1 regulates Alzheimer’s disease-associated genes in primary human microglia
title PU.1 regulates Alzheimer’s disease-associated genes in primary human microglia
title_full PU.1 regulates Alzheimer’s disease-associated genes in primary human microglia
title_fullStr PU.1 regulates Alzheimer’s disease-associated genes in primary human microglia
title_full_unstemmed PU.1 regulates Alzheimer’s disease-associated genes in primary human microglia
title_short PU.1 regulates Alzheimer’s disease-associated genes in primary human microglia
title_sort pu.1 regulates alzheimer’s disease-associated genes in primary human microglia
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6102813/
https://www.ncbi.nlm.nih.gov/pubmed/30124174
http://dx.doi.org/10.1186/s13024-018-0277-1
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