Cellular Zn²⁺ chelators cause "dying-back" neurite degeneration associated with energy impairment

Most cellular zinc is tightly associated with metalloproteins and other Zn²⁺-dependent proteins, which along with cellular Zn²⁺ compartments may coordinately regulate cytoplasmic free Zn²⁺ levels in the picomolar range. Moreover, Zn²⁺-containing endosomes or protein complexes appear to move along axons or dendrites, suggesting a dynamic mechanism for trafficking, exchanging, or scavenging Zn²⁺ and/or Zn²⁺ protein complexes in neurons. It is therefore interesting to examine whether cellular Zn²⁺ levels might alter neurite integrity and dynamics. Here we show that membrane-permeable zinc chelators, including 1,10-phenanthroline, N,N,N′,N′-tetrakis-(2-pyridylmethyl)- ethylenediamine (TPEN), and zinquin, selectively elicit axon and dendrite degeneration but leave the cell body intact in sympathetic neurons. The process begins distally and then moves retrogradely, with a distinct "dying-back" pattern. An inactive isomer of 1,10-phenanthroline failed to cause neuite degeneration, and these chelators mediated their effects by selectively chelating Zn²⁺, but not other metals. Moreover, neurite degeneration was associated with a decrease in neuritic ATP levels and was caused by energy failure, because an exogenous supply of nicotinamide adenine dinucleotide (NAD) or its precursor nicotinamide suppressed the degeneration by delaying axonal ATP reduction caused by Zn²⁺ depletion. Blockage of autophagy by 3-methyladenine provided partial protection against degeneration of terminal axons or dendrites; there was, however, no obvious alteration in that of medial portions. Collectively, our results show that cellular Zn²⁺ depletion induces a "dying-back" degeneration characterized by an NAD- and autophagy-dependent process, independently of neurite elongation dynamics.