Subcellular Localization Determines the Stability and Axon Protective Capacity of Axon Survival Factor Nmnat2

Abstract

Modulation of the subcellular localization of the endogenous axon survival factor Nmnat2 boosts its axon protective capacity, suggesting a novel approach to delaying axon degeneration in neurodegenerative disease.

Abstract

Axons require a constant supply of the labile axon survival factor Nmnat2 from their cell bodies to avoid spontaneous axon degeneration. Here we investigate the mechanism of fast axonal transport of Nmnat2 and its site of action for axon maintenance. Using dual-colour live-cell imaging of axonal transport in SCG primary culture neurons, we find that Nmnat2 is bidirectionally trafficked in axons together with markers of the trans-Golgi network and synaptic vesicles. In contrast, there is little co-migration with mitochondria, lysosomes, and active zone precursor vesicles. Residues encoded by the small, centrally located exon 6 are necessary and sufficient for stable membrane association and vesicular axonal transport of Nmnat2. Within this sequence, a double cysteine palmitoylation motif shared with GAP43 and surrounding basic residues are all required for efficient palmitoylation and stable association with axonal transport vesicles. Interestingly, however, disrupting this membrane association increases the ability of axonally localized Nmnat2 to preserve transected neurites in primary culture, while re-targeting the strongly protective cytosolic mutants back to membranes abolishes this increase. Larger deletions within the central domain including exon 6 further enhance Nmnat2 axon protective capacity to levels that exceed that of the slow Wallerian degeneration protein, Wld ^S. The mechanism underlying the increase in axon protection appears to involve an increased half-life of the cytosolic forms, suggesting a role for palmitoylation and membrane attachment in Nmnat2 turnover. We conclude that Nmnat2 activity supports axon survival through a site of action distinct from Nmnat2 transport vesicles and that protein stability, a key determinant of axon protection, is enhanced by mutations that disrupt palmitoylation and dissociate Nmnat2 from these vesicles.

Author Summary

Neurons are polarized cells that rely on bidirectional transport to deliver thousands of cargos between the cell body and the most distal ends of their axons. One cargo that is of particular importance is the NAD-synthesising enzyme Nmnat2. This surprisingly unstable protein is produced in the cell body and its constant supply into axons is required to keep them alive. If this supply is interrupted, Nmnat2 levels in the distal axon drop below a critical threshold, leading to axon degeneration. The rapid turnover of Nmnat2 contributes critically to the time course of axon degeneration. If its half-life could be extended, axons may be able to survive transient interruptions of its supply. In this study, we find that disruption of Nmnat2 localization to axonal transport vesicles increases both its half-life and its capacity to protect injured neurites. Specifically, association of Nmnat2 with transport vesicles reduces it stability by making it vulnerable to ubiquitination and proteasome-mediated degradation. These findings suggest that modulation of the subcellular localization of Nmnat2 on transport vesicles could serve as a potential avenue for therapeutic treatment of axon degeneration.