Date of Award

3-2023

Document Type

Dissertation

Degree Name

Ph.D.

Organizational Unit

College of Natural Science and Mathematics, Biological Sciences

First Advisor

Yan Qin

Second Advisor

Michelle Knowles

Third Advisor

Joe Angleson

Fourth Advisor

Ann-Charlotte Granholm-Bentley

Keywords

Calcium, Hippocampal neurons, Metallothionein 3, pH, Zinc

Abstract

Zn2+ has been suggested to act as an intracellular signaling molecule due to its regulatory effects on numerous protein targets including enzymes, transcription factors, ion channels, neurotrophic factors, and postsynaptic scaffolding proteins. However, intracellular Zn2+ concentration is tightly maintained at steady levels under natural physiological conditions. Dynamic changes in intracellular Zn2+ concentration have only been detected in cells exposed to pathologic stimuli or upon receptor ligand binding. In the first project of my dissertation, for the first time, we revealed that developing neurons fire spontaneous and synchronous Zn2+ spikes at a specific time window (from 14 to 25 days in vitro (DIV)) without external stimuli. By blocking Ca2+ influx from voltage-gated Ca2+ channels (VGCCs) and glutamate receptors, we found such Zn2+ spikes are Ca2+ spikes dependent and are driven by glutamate-mediated spontaneous neural excitability. Cellular acidification is the bridge that connects Zn2+ spikes with Ca2+ spikes. My second project aims to identify the mechanism by which Zn2+ spikes are generated via Ca2+ influx. Interestingly, such Ca2+-induced Zn2+ increases can only be detected in brain cells including neurons and astrocytes, but not in HeLa cells. When brain-specific metallothionein (MT3) was overexpressed in HeLa cells, Ca2+ influx can induce Zn2+ spikes, while silencing of MT3 expression in neurons depleted the Ca2+-induced Zn2+ spikes. Our results revealed that Ca2+ influx induces cellular acidification, which liberates Zn2+ from MT3, causing Zn2+ spikes in neurons. Further, we found evidence that such Zn2+ spikes may protect neurons against Ca2+ dysregulations and glutamate excitotoxicity. My third project focused on characterizing the in situ responses of different genetically encoded Ca2+ sensors. Our results showed that GCaMP6f is a great choice to detect cytosolic Ca2+ signals due to its high sensitivity. In summary, my dissertation provides paramount evidence supporting the signaling roles of Zn2+ by uncovering Zn2+ and Ca2+ crosstalk in primary hippocampal neurons. We established the neuronal protection roles of MT3 and Zn2+ spikes during glutamate excitotoxicity, indicating that Zn2+ is essential to maintain neuronal health during development of functional neuronal network, where large synchronous neuronal assembly activities occur.

Publication Statement

Copyright held by the author. User is responsible for all copyright compliance.

Rights Holder

Chen Zhang

Provenance

Received from ProQuest

File Format

application/pdf

Language

en

File Size

171 pgs

Discipline

Biology, Neurosciences, Molecular biology

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Available for download on Friday, April 11, 2025



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