Uncovering Zn2+ and Ca2+ Homeostasis and Crosstalk in Primary Hippocampal Neurons

<p>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<em> in situ</em> 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.</p>

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Year dc:date.available

2023

Author dc:creator

• Zhang, Chen

Contributors dc:contributor

• Yan Qin
• Michelle Knowles
• Joe Angleson
• Ann-Charlotte Granholm-Bentley

• Calcium
• Hippocampal neurons
• Metallothionein 3
• pH
• Zinc
• Biochemistry, Biophysics, and Structural Biology
• Life Sciences
• Molecular Biology

Statement dc:rights

• <p>Copyright held by the author. User is responsible for all copyright compliance.</p>

Language dc:language

en

Repository record dc:identifier

https://digitalcommons.du.edu/etd/2179

OAI identifier oai:identifier

oai:digitalcommons.du.edu:etd-3175