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Imaging Neuropeptide Release and Localization with Genetically Engineered Reporters

Citation

Ding, Ke (2022) Imaging Neuropeptide Release and Localization with Genetically Engineered Reporters. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/3ey5-9p35. https://resolver.caltech.edu/CaltechTHESIS:06012022-062105760

Abstract

Neuropeptides are a class of neural signaling molecules that play a pivotal role in brain function and human health through neuromodulatory influences. There are over 100 types of neuropeptides identified and characterized, yet genomic analysis suggests that it is only the tip of the iceberg, with extra hundreds of putative neuropeptides awaiting further investigation. Neuropeptides collectively regulate a variety of developmental, physiological, and behavioral functions. While each neuropeptide is idiosyncratic in regard to its molecular structure, chemical properties, and anatomical distribution, they impinge on the nervous system in a similar fashion.

Surprisingly, despite their fundamental importance, techniques for measuring the localization, expression and release of neuropeptides, at large scale and with high spatio­temporal resolution, have lagged far behind. Microdialysis and fast-scanning cyclic voltammetry are useful primarily for measuring "volume transmission," but are invasive, and have poor spatial resolution and limited general applicability. FP-tagged vesicle reporters are mainly tested and used in limited cell types. Little is characterized about their functional universality and specificity. GPCR-based sensors are designed to visualize the binding, instead of expression and release, of a neuropeptide.

Therefore, I aim to develop new methods for visualizing, detecting, and inhibiting NP expression and release in vivo. The long-term goal is to apply these methods to understanding the dynamics of neuromodulation of specific, behaviorally relevant neural circuits, and to providing a dynamic, high-resolution view of chemical modulation of circuit function.

In Chapter 2, I will describe the design, screening, and proof-of-concept validation of novel genetically engineered neuropeptide release reporters (NPRR) in Drosophila. I further demonstrated the idiosyncrasy of neuropeptide release dynamics, as well as cell-type specific release properties of a neuropeptide. In Chapter 3, I conceived and constructed a neuropeptide imaging platform that exploits the discoveries and strategies from Drosophila NPRRs. Besides a series of redesign of mammalian NPRRs, a collection of sister reporters to visualize localization and expression (Neuropeptide Localization and Expression Reporter, NPLER) were built in parallel. I also established a prototypical pipeline to systematically screen for appropriate cell lines for the purpose of NPRR/NPLER applications.

Malfunctioning of neuropeptide pathways can potentially result in a variety of mental illnesses triggered by stress, and metabolic disorders including obesity. Drugs targeting neuropeptide signaling have received heavy investment, but most have failed in the clinical trials. We therefore propose alternative strategies to target the processing/release of the neuropeptide from neurons, rather than blocking its receptor. In Chapter 4, I describe the ongoing process of adapting modern biotechnologies to the imaging platform to explore novel therapeutic strategies for neuropeptide- relevant disorders and abnormalities.

The Appendix includes a serendipitous finding from our attempt to generalize NPRR to Caenorhabditis elegans.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Neuropeptide, Neuromodulation, Drosophila
Degree Grantor:California Institute of Technology
Division:Biology and Biological Engineering
Major Option:Neurobiology
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Anderson, David J.
Thesis Committee:
  • Prober, David A. (chair)
  • Chan, David C.
  • Sternberg, Paul W.
  • Anderson, David J.
Defense Date:25 May 2022
Record Number:CaltechTHESIS:06012022-062105760
Persistent URL:https://resolver.caltech.edu/CaltechTHESIS:06012022-062105760
DOI:10.7907/3ey5-9p35
Related URLs:
URLURL TypeDescription
https://doi.org/10.7554/eLife.46421UNSPECIFIEDArticle adapted for Chapter 2.
ORCID:
AuthorORCID
Ding, Ke0000-0002-5261-4843
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:14918
Collection:CaltechTHESIS
Deposited By: Ke Ding
Deposited On:08 Jun 2022 15:17
Last Modified:23 Nov 2022 17:24

Thesis Files

[img] PDF (Thesis with Chapters 3 and 4 redacted.) - Final Version
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[img] PDF (Chapter 1) - Final Version
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[img] PDF (Chapter 2) - Final Version
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[img] PDF (Chapter 5) - Final Version
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[img] PDF (Appendix) - Final Version
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