Characterization of Intracellular Zn2+ Dynamics and Zn2+- Dependent Gene Expression In Primary Dissociated Hippocampal Neurons

Sanford, Lynn

Graduate Thesis Or Dissertation

Characterization of Intracellular Zn2+ Dynamics and Zn2+- Dependent Gene Expression In Primary Dissociated Hippocampal Neurons Público Deposited

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Citeable URL: https://scholar.colorado.edu/concern/graduate_thesis_or_dissertations/qj72p7983

Abstract

Ionic zinc (Zn²⁺) is an essential cofactor in many proteins, but labile (non-protein bound) Zn²⁺ also functions as a signaling ion in many cell types. In specific neurons of the brain and nervous system, Zn²⁺ is highly enriched and operates as a neurotransmitter that modulates neuronal signaling. Zn²⁺ has crucial physiological roles in memory consolidation and sensory processing, yet abnormal levels of Zn²⁺ have been observed to be pathological in neurological disorders ranging from Alzheimer’s Disease to depression. Both extracellular and intracellular Zn²⁺ are necessary for neuronal signaling, but little is known about the dynamics of intracellular Zn²⁺ during neuronal stimulation and how intracellular Zn²⁺ influences signaling in physiological or pathological processes. We employed fluorescence imaging with genetically encoded and small molecule Zn²⁺ sensors to quantify intracellular Zn²⁺ dynamics during different stimulations in primary dissociated hippocampal neurons. We found that Zn²⁺ rose transiently during both chemical and electrical stimulation, although there were differences in peak Zn²⁺ concentrations depending on the stimulus used. Furthermore, we perturbed intracellular Zn²⁺ signaling to explore Zn²⁺-dependent changes in gene expression through global mRNA sequencing of hippocampal neurons. Our RNA-Seq results indicated that mild stimulation with potassium chloride induced Zn²⁺-dependent expression of genes related to synaptic growth and signaling, while more intensive stimulation with glutamate promoted Zn²⁺-dependent expression of genes involved in endoplasmic reticulum stress. Our results therefore show distinct differences in the role of Zn²⁺ signaling during potassium chloride and glutamate stimulation of neurons, which may reflect differences between physiological and pathological Zn²⁺ signaling. In future studies we aim to elucidate the underlying signaling mechanisms behind these differences.

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