The Highwire Ubiquitin Ligase Promotes Axonal Degeneration by Tuning Levels of Nmnat Protein

Abstract

Axonal degeneration is a hallmark of many neuropathies, neurodegenerative diseases, and injuries. Here, using a Drosophila injury model, we have identified a highly conserved E3 ubiquitin ligase, Highwire (Hiw), as an important regulator of axonal and synaptic degeneration. Mutations in hiw strongly inhibit Wallerian degeneration in multiple neuron types and developmental stages. This new phenotype is mediated by a new downstream target of Hiw: the NAD+ biosynthetic enzyme nicotinamide mononucleotide adenyltransferase (Nmnat), which acts in parallel to a previously known target of Hiw, the Wallenda dileucine zipper kinase (Wnd/DLK) MAPKKK. Hiw promotes a rapid disappearance of Nmnat protein in the distal stump after injury. An increased level of Nmnat protein in hiw mutants is both required and sufficient to inhibit degeneration. Ectopically expressed mouse Nmnat2 is also subject to regulation by Hiw in distal axons and synapses. These findings implicate an important role for endogenous Nmnat and its regulation, via a conserved mechanism, in the initiation of axonal degeneration. Through independent regulation of Wnd/DLK, whose function is required for proximal axons to regenerate, Hiw plays a central role in coordinating both regenerative and degenerative responses to axonal injury. Axons degenerate after injury and during neurodegenerative diseases, but we are still searching for the cellular mechanism responsible for this degeneration. Here, using a nerve crush injury assay in the fruit fly Drosophila, we have identified a role for a conserved molecule named Highwire (Hiw) in the initiation of axonal degeneration. Hiw is an E3 ubiquitin ligase thought to regulate the levels of specific downstream proteins by targeting their destruction. We show that Hiw promotes axonal degeneration by regulating two independent downstream targets: the Wallenda (Wnd) kinase, and the NAD+ biosynthetic enzyme nicotinamide mononucleotide adenyltransferase (Nmnat). Interestingly, Nmnat has previously been implicated in a protective role in neurons. Our findings indicate that Nmnat protein is down-regulated in axons by Hiw and that this regulation plays a critical role in the degeneration of axons and synapses. The other target, the Wnd kinase, was previously known for its role in promoting new axonal growth after injury. We propose that Hiw coordinates multiple responses to regenerate damaged neuronal circuits after injury: degeneration of the distal axon via Nmnat, and new growth of the proximal axon via Wnd.