Abstract
Autophagy is a highly conserved, cytoprotective, catabolic process induced in response to conditions of cellular stress and nutrient deprivation. It is responsible for the degradation of large intracellular substrates such as misfolded or aggregated proteins and organelles. This self-degradative mechanism is crucial for proteostasis in post-mitotic neurons, requiring its careful regulation. Due to its homeostatic role and the implications, it has for certain disease pathologies, autophagy has become a growing area of research. We describe here two assays that can be used as part of a tool kit for measuring autophagy-lysosomal flux in human iPSC-derived neurons.
One way to measure autophagic flux is through a western blotting assay, which can be used to analyze two important autophagy proteins: microtubule-associated protein 1 light chain 3 (LC3) and p62. In this chapter, we describe a western blotting assay for use in human iPSC neurons that can be used to quantify these two proteins of interest to measure autophagic flux.
In addition to conventional western blotting techniques, more sophisticated tools have come available to readout autophagic flux in a sensitive and high-throughput manner. In the latter portion of this chapter, we describe a flow cytometry assay which utilizes a pH-sensitive fluorescent reporter which can also be used to measure autophagic flux.
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Acknowledgments
This work was supported by grants from National Institutes of Health (R21AG077697 and P20GM119943 [Subaward 7137296] to Y.A.H), and the Brown UTRA SPRINT Summer Award (to G.A.E.). Y.A.H. is a GFL Translational Professor from Center for Translational Neuroscience at the Brown Institute for Translational Sciences.
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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
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O’Rourke, R., Erdemir, G.A., Huang, YW.A. (2023). Assays of Monitoring and Measuring Autophagic Flux for iPSC-Derived Human Neurons and Other Brain Cell Types. In: Huang, YW.A., Pak, C. (eds) Stem Cell-Based Neural Model Systems for Brain Disorders. Methods in Molecular Biology, vol 2683. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3287-1_18
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DOI: https://doi.org/10.1007/978-1-0716-3287-1_18
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Publisher Name: Humana, New York, NY
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Online ISBN: 978-1-0716-3287-1
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