Metallothionein III Mediates Ca ²⁺ -Dependent Zn ²⁺ Spikes to Inhibit Dendritic Arborization

Abstract

Zinc is crucial for neuron function, but whether and how labile zinc ion (Zn 2+ ) acts as an intracellular signaling molecule remains unclear. In this work, we investigate the relationship between Ca 2+ and Zn 2+ dynamics using fluorescence imaging. Our findings reveal that manipulating Ca 2+ influx through various pathways induces intracellular acidification, which subsequently elicits Zn 2+ spikes that reflect transient increases in cytosolic Zn 2+ levels. These Ca 2+ -dependent Zn 2+ spikes have been recorded in both rat ( Rattus norvegicus ) primary neuron cultures and organotypic mouse ( Mus musculus ) hippocampal slice cultures prepared from both males and females. They are specific to neurons and astrocytes but are absent in other cell types we tested including HeLa cells, COS-7 cells, and fibroblasts. We further identify Metallothionein III (MT3), a Zn 2+ buffering protein specifically expressed in brain cells, as the source of these Zn 2+ spikes. Reduction in MT3 expression by knockdown with shRNAmiR techniques significantly decreases the amplitude of Zn 2+ spikes, while overexpression of MT3 in HeLa and COS-7 cells is sufficient to induce Ca 2+ -dependent Zn 2+ spikes, demonstrating the crucial roles of MT3 in Zn 2+ release. Lastly, we explore the biological roles of MT3-mediated Zn 2+ spikes in neurons. Suppressing Zn 2+ spikes with either MT3 knockdown or mild Zn 2+ chelation results in increased dendritic branching in primary rat hippocampal neurons. These results suggest that Zn 2+ release from endogenous MT3 acts as a regulatory signal to inhibit dendrite branching and growth, establishing a critical role for Zn 2+ spikes in neurite outgrowth and neuronal development.