Metallothionein III mediates Ca²⁺-dependent Zn²⁺ spikes to inhibit dendritic arborization

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. Significance statement Zinc is essential for brain development, primarily known for its role in supporting protein structure and enzymatic activity. However, its function as an intracellular signaling molecule has been debated because labile zinc (Zn 2+ ) concentrations inside cells are typically stable. In this study, we discovered a unique pathway where Ca 2+ influx triggers cellular acidification, which subsequently releases Zn 2+ from Metallothionein III (MT3), a Zn 2+ -binding protein highly expressed in the brain. More importantly, we found that depletion of these Zn 2+ spikes via MT3 knockdown or chelation increases dendritic arborization, a critical step in forming neural connections. Our findings reveal that Ca 2+ influx activates MT3-mediated Zn 2+ signaling, which fine-tunes the neuronal network maturation, highlighting previously unrecognized signaling roles of Zn 2+ in brain development.