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CRISPRi screens in human astrocytes elucidate regulators of distinct inflammatory reactive states

Kun Leng, Brendan Rooney, Hyosung Kim, Wenlong Xia, Mark Koontz, Mitchell Krawczyk, Ye Zhang, Erik M. Ullian, Stephen P.J. Fancy, Matthew S. Schrag, Ethan S. Lippmann, View ORCID ProfileMartin Kampmann
doi: https://doi.org/10.1101/2021.08.23.457400
Kun Leng
1Institute for Neurodegenerative Diseases, University of California, San Francisco, San Francisco, CA, USA
2Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA, USA
3Medical Scientist Training Program, University of California, San Francisco, San Francisco, CA, USA
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  • For correspondence: kun.leng@ucsf.edu martin.kampmann@ucsf.edu
Brendan Rooney
1Institute for Neurodegenerative Diseases, University of California, San Francisco, San Francisco, CA, USA
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Hyosung Kim
4Department of Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN, USA. Department of Neurology, Vanderbilt University, Nashville, TN, United States
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Wenlong Xia
5Departments of Neurology and Pediatrics, School of Medicine, University of California, San Francisco
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Mark Koontz
6Department of Ophthalmology, School of Medicine, University of California, San Francisco
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Mitchell Krawczyk
7Neuroscience Graduate Program, University of California, Los Angeles, Los Angeles, CA, USA
8Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA
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Ye Zhang
8Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA
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Erik M. Ullian
6Department of Ophthalmology, School of Medicine, University of California, San Francisco
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Stephen P.J. Fancy
5Departments of Neurology and Pediatrics, School of Medicine, University of California, San Francisco
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Matthew S. Schrag
9Department of Neurology, Vanderbilt University Medical Center, Nashville, TN, USA
10Vanderbilt Memory and Alzheimer’s Center, Vanderbilt University Medical Center, Nashville, TN, USA
11Vanderbilt Brain Institute, Vanderbilt University, Nashville, TN, USA
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Ethan S. Lippmann
4Department of Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN, USA. Department of Neurology, Vanderbilt University, Nashville, TN, United States
9Department of Neurology, Vanderbilt University Medical Center, Nashville, TN, USA
10Vanderbilt Memory and Alzheimer’s Center, Vanderbilt University Medical Center, Nashville, TN, USA
11Vanderbilt Brain Institute, Vanderbilt University, Nashville, TN, USA
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Martin Kampmann
1Institute for Neurodegenerative Diseases, University of California, San Francisco, San Francisco, CA, USA
12Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, CA, USA
13Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA, USA
14Chan Zuckerberg Biohub, San Francisco, CA, USA
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  • ORCID record for Martin Kampmann
  • For correspondence: kun.leng@ucsf.edu martin.kampmann@ucsf.edu
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ABSTRACT

In response to central nervous system injury or disease, astrocytes become reactive, adopting context-dependent states with altered functions. Certain inflammatory insults induce reactive astrocyte states that lose homeostatic functions and gain harmful outputs, and likely contribute to neuroinflammatory and neurodegenerative diseases. However, the cellular pathways controlling these states are not fully understood. Here, we combined single-cell transcriptomics with CRISPRi screening in human iPSC-derived astrocytes to systematically interrogate inflammatory reactivity. We found that autocrine-paracrine IL-6 and interferon signaling downstream of canonical NF-κB activation drove two distinct inflammatory reactive states promoted by and inhibited by STAT3, respectively. Furthermore, these states corresponded with those observed in other experimental contexts, including in vivo, and their markers were upregulated in the human brain in Alzheimer’s disease and hypoxic ischemic encephalopathy. These results and the platform we established have the potential to guide the development of therapeutics to selectively modulate different aspects of inflammatory astrocyte reactivity.

Competing Interest Statement

M. Kampmann has filed a patent application related to CRISPRi and CRISPRa screening (PCT/US15/40449) and serves on the Scientific Advisory Board of Engine Biosciences, Casma Therapeutics, and Cajal Neuroscience, and is an advisor to Modulo Bio and Recursion Therapeutics. None of the other authors declare competing interests.

Footnotes

  • https://www.crisprbrain.org/

  • https://kampmannlab.ucsf.edu/inflammatory-reactive-astrocyte-analysis

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted August 24, 2021.
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CRISPRi screens in human astrocytes elucidate regulators of distinct inflammatory reactive states
Kun Leng, Brendan Rooney, Hyosung Kim, Wenlong Xia, Mark Koontz, Mitchell Krawczyk, Ye Zhang, Erik M. Ullian, Stephen P.J. Fancy, Matthew S. Schrag, Ethan S. Lippmann, Martin Kampmann
bioRxiv 2021.08.23.457400; doi: https://doi.org/10.1101/2021.08.23.457400
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CRISPRi screens in human astrocytes elucidate regulators of distinct inflammatory reactive states
Kun Leng, Brendan Rooney, Hyosung Kim, Wenlong Xia, Mark Koontz, Mitchell Krawczyk, Ye Zhang, Erik M. Ullian, Stephen P.J. Fancy, Matthew S. Schrag, Ethan S. Lippmann, Martin Kampmann
bioRxiv 2021.08.23.457400; doi: https://doi.org/10.1101/2021.08.23.457400

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