Dissertation
Characterization of Calcium binding protein 1 (CaBP1/Caldendrin) expression and function in dorsal root ganglion neurons
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Summer 2021
DOI: 10.17077/etd.005957
Abstract
The dorsal root ganglion (DRG) is a cluster of heterogenous neurons that are responsible for transmitting sensory input signals such as touch, pain, and temperature from the periphery to the spinal cord up to the brain. During development, DRG neuron (DRGN) axons navigate three-dimensional space following attractive and repulsive cues for proper innervation to achieve proper function. After nerve injury or neuropathy, DRGNs have the capacity to regenerate; however, there are multiple factors that could hinder functional axon regeneration. In culture, DRGNs have robust regeneration ability compare to neurons from the central nervous system (CNS). Therefore, it is important to advance our understanding of the molecular mechanisms involved in DRGNs axon regeneration, not only to develop approaches for new nerve injury and neuropathy therapies, but to uncover molecular mechanisms that can be exploited in CNS neurons. Although the mechanisms involved in DRG axon growth and regeneration have been widely studied, the full understanding of how this heterogenous population of neurons achieves this feat remain fragmented. One common pathway has emerged that influences axon growth and regeneration, which involves Ca2+ signaling. CaBP1 is a member of the Ca2+ binding proteins family. CaBP1 splice variant Caldendrin is highly expressed in neuronal cells and has multiple binding partners, which regulate Ca2+ pathways that are involved in neurite growth and neuronal morphology. Our lab has previously shown that transcription dependent regulation of neurite growth is altered in spiral ganglion neurons which lack CaBP1 compared to WT, resulting in increased neurite initiation. It remains unknown if CaBP1 regulates similar functions in other sensory neurons, such as DRGNs. In this thesis I characterize the role of CaBP1 in DRGNs. I demonstrated that Caldendrin Short and Long are the major splice variants expressed in myelinated medium and large diameter DRGNs. In vitro experiments with primary DRGNs cultures indicate Caldendrin is a negative regulator of axonal regeneration. Caldendrin represses axonal growth by coupling L-Type voltage-gated Ca2+ channel to transcriptional mechanisms in a sex dependent manner. Further understanding the transcription mechanisms by which CaBP1 regulates DRGN neurite regrowth will provide insight to the molecular mechanisms involved in DRGN axonal regeneration after nerve injury.
To aid in sorting DRGN images based on morphological characteristics, we developed a novel in silico tool (NeuriteNet) in a multi-lab collaboration. NeuriteNet is a machine learning based program that was able to differentiate DRGN images based in morphology, sex, and genotype. This tool will be a useful resource for future image analyses that remain difficult and time-consuming due to the complexity of DRGNs. NeuriteNet will be available to researchers to utilize for their research.
As part of this work, Caldendrin expression was observed to be enriched in mechanosensory DRGNs population. Stimulus-evoked behavioral assays further implicated Caldendrin with mechanosensation. We discovered that CaBP1 KO mice have increased mechanical sensitivity, but no deficiency in thermal sensation. Further, in vitro experiments showed that Caldendrin interacts with a major mechanically-activated cation channel Piezo2. In collaboration with Dr. Vasquez’s lab, experts in measuring whole-cell patch-clamp recordings evoked by mechanical stimulation provided preliminary data further demonstrating that Piezo2 mechanical currents are modulated by Caldendrin.
The findings from this thesis provide an outlook on future approaches and limitations of CaBP1 research based on the tools currently available. We provide the groundwork for future research to better understand the molecular pathways that Caldendrin is involved in regulating DRG axonal regeneration and mechanosensory function, which can help to understand the basic mechanism involved in nerve injury, neuropathy and mechanical allodynia.
Details
- Title: Subtitle
- Characterization of Calcium binding protein 1 (CaBP1/Caldendrin) expression and function in dorsal root ganglion neurons
- Creators
- Josue Alan Lopez
- Contributors
- Amy Lee (Advisor)Mark Stamnes (Committee Member)Robert Piper (Committee Member)Steven Green (Committee Member)Peter Snyder (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Molecular Physiology and Biophysics
- Date degree season
- Summer 2021
- Publisher
- University of Iowa
- DOI
- 10.17077/etd.005957
- Number of pages
- xiii, 110 pages
- Copyright
- Copyright 2021 Josue Alan Lopez
- Comment
- This thesis has been optimized for improved web viewing. If you require the original version, contact the University Archives at the University of Iowa: https://www.lib.uiowa.edu/sc/contact/
- Language
- English
- Description illustrations
- color illustrations
- Description bibliographic
- Includes bibliographical references (pages 99-110).
- Public Abstract (ETD)
Humans sense their external environment using their senses: touch, sight, hearing, smell and taste. Touch consist of several sensations including light touch, pressure, vibration, temperature, pain and the awareness self-movement and body position. During human embryo development, axons elongate from a cluster of sensory neurons termed the dorsal root ganglion neurons (DRGNs), connecting to periphery of the skin and central axon which makes contact with the central nervous system. DRG nerves act as wires relaying information from the skin to the brain which is then perceived as touch sensation. The longest nerves connect to the extremities such as the feet and hands; these nerves are prone to damage by physical injury and neuropathies which can result in loss of sensation, pain, or increased sensitivity resulting in a painful sensation. Although there are strategies to alleviate the symptoms, unfortunately there are no therapies to regenerate these nerves and regain function. My thesis work focuses on a calcium binding protein called Caldendrin, which is highly expressed in DRGNs involved in touch sensation. Utilizing DRGNs cultures from mice, I have determined that Caldendrin is a negative regulator of axonal regeneration. Independently, with the use of behavioral assays, a potential role for Caldendrin was revealed in modulating a mechanosensitive ion channel (Piezo2) that has a key role in sensing touch. Therefore, my work serves as a foundation for the role of Caldendrin in DRGNs which was previously unknown and may lead to future studies to identify therapies for people suffering from nerve injury.
- Academic Unit
- Molecular Physiology and Biophysics
- Record Identifier
- 9984124470302771
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