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Using Single-Molecule Fluorescence Microscopy to Uncover Neuronal Vulnerability to Protein Damage

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Neuronal Cell Death

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2515))

Abstract

Neurodegenerative disorders (NDs) are diverse age-related conditions also described as “conformational diseases.” The hallmark of NDs is the accumulation of disease-specific proteins as toxic misfolded aggregates in some areas of the brain. They lead to the loss of protein homeostasis (proteostasis) that causes neuronal dysfunction and death. A potential therapeutic strategy for NDs is to prevent the accumulation of misfolded proteins by activating the heat shock response (HSR). The HSR maintains proteostasis through the upregulation of heat shock proteins (HSPs), molecular chaperones that recognize misfolded proteins, and either refold them to their functional conformations and/or target them for degradation. However, how to manipulate the expression of HSPs to obtain a therapeutic effect in neurons remains unclear. Furthermore, the regulation of the HSR in neurons is more complex than what we have learned from culturing somatic nonneuronal cells. This chapter describes a method to investigate the induction of HSP70 in primary hippocampal neurons using single-molecule fluorescence in situ hybridization (smFISH). Quantification of smFISH provides the means to analyze neuron-to-neuron variability in the activation of the HSR and enables us to study the transcriptional induction and localization of HSP70 mRNA in primary neurons. This information might be critical to find the druggable steps for developing effective therapies to treat age-related NDs.

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Acknowledgments

This work was supported by NIH grant AG05583 and NSERC grant RGPIN-2019-04767 to M.V.

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Author notes

  1. Suleima Jacob-Tomas and Lokha R. Alagar Boopathy contributed equally with all other contributors.

Authors and Affiliations

  1. Department of Biochemistry, Center de Recherche en Biologie Structurale, McGill University, Montreal, QC, Canada

    Suleima Jacob-Tomas, Lokha R. Alagar Boopathy & Maria Vera

Authors
  1. Suleima Jacob-Tomas
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  2. Lokha R. Alagar Boopathy
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  3. Maria Vera
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Corresponding author

Correspondence to Maria Vera .

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Editors and Affiliations

  1. Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada

    Arezu Jahani-Asl

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© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Jacob-Tomas, S., Alagar Boopathy, L.R., Vera, M. (2022). Using Single-Molecule Fluorescence Microscopy to Uncover Neuronal Vulnerability to Protein Damage. In: Jahani-Asl, A. (eds) Neuronal Cell Death. Methods in Molecular Biology, vol 2515. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2409-8_15

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  • DOI: https://doi.org/10.1007/978-1-0716-2409-8_15

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2408-1

  • Online ISBN: 978-1-0716-2409-8

  • eBook Packages: Springer Protocols

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