Local axonal protection by WldS as revealed by conditional regulation of protein stability

Abstract

The expression of the mutant Wallerian degeneration slow (WldS) protein significantly delays axonal degeneration from various nerve injuries and in multiple species; however, the mechanism for its axonal protective property remains unclear. Although WldS is localized predominantly in the nucleus, it also is present in a smaller axonal pool, leading to conflicting models to account for the WldS fraction necessary for axonal protection. To identify where WldS activity is required to delay axonal degeneration, we adopted a method to alter the temporal expression of WldS protein in neurons by chemically regulating its protein stability. We demonstrate that continuous WldS activity in the axonal compartment is both necessary and sufficient to delay axonal degeneration. Furthermore, by specifically increasing axonal WldS expression postaxotomy, we reveal a critical period of 4-5 h postinjury during which the course of Wallerian axonal degeneration can be halted. Finally, we show that NAD + , the metabolite of WldS/nicotinamide mononucleotide adenylyltransferase enzymatic activity, is sufficient and specific to confer WldS-like axon protection and is a likely molecular mediator of WldS axon protection. The results delineate a therapeutic window in which the course of Wallerian degeneration can be delayed even after injures have occurred and help narrow the molecular targets of WldS activity to events within the axonal compartment.xon degeneration is a characteristic event in many neurodegenerative conditions including stroke, glaucoma, and motor neuropathies. Remarkably, expression of the Wallerian degeneration slow (WldS) transgene delays nerve degeneration in these events, and the protection is conserved across many species, including rats (1), Drosophila (2, 3), and even in human neurons (4). Thus, identifying the molecular components of the degeneration pathway with which WldS interferes provides a window of opportunity to understand how axons are normally lost after injury.The WldS mutation results in the formation of a chimeric gene product consisting of the N-terminal 70 amino acids of ubiquitination factor 4B (Ube4B), which contains no enzymatic activity, and the full functional sequence of a NAD + synthetic enzyme, nicotinamide mononucleotide adenylyltransferase (Nmnat1) (5). The Ube4B portion in WldS contains a binding site for valosin-containing protein (VCP) (6), a cytoplasmic protein with diverse cellular functions (7). Both this VCP-binding domain and the enzymatic activity of Nmnat1 are required for WldS-mediated axon protection (8, 9). Although the WldS protein is localized predominantly in the nucleus because of the endogenous nuclear localization of Nmnat1, trace amounts of WldS protein also have been identified in extranuclear compartments in the axoplasm and in axonal organelles including the mitochondria and phagosomes (10, 11), suggesting that the N-terminal Ube4B region of WldS partially redistributes the nuclear Nmnat1 to the axon.Despite the remarkable phenotype, little is known regarding the m...

Acute Axonal Degeneration Drives Development of Cognitive, Motor, and Visual Deficits after Blast-Mediated Traumatic Brain Injury in Mice

“…Although axonal degeneration is a major aspect of many forms of neurodegeneration (Warner et al, 2010; Wang et al, 2012; Johnson et al, 2013; Smith et al, 2013), its contribution to the pathological and neurobehavioral deficits of blast-mediated TBI, the most common form of TBI sustained by soldiers in Iraq and Afghanistan, has not previously been investigated. Here, we show for the first time that early axonal degeneration is a critical driver of the development of neurologic deficits after blast-mediated TBI.…”

Axon Self-Destruction: New Links among SARM1, MAPKs, and NAD+ Metabolism

“…Direct transduction of NMNAT1 protein into severed axons in vitro within four hours after axon transection (see Figure 1) is sufficient to prevent later fragmentation of the axons (Sasaki and Milbrandt, 2010), definitively demonstrating that NMNAT1 exerts its protective effect locally within the axonal compartment. Moreover, Wlds-expressing axons rapidly degenerate when Wlds is depleted after injury by protein destabilization, demonstrating a continuous local requirement for NMNAT activity in isolated axons (Wang et al, 2015). …”

“…Other findings are also difficult to reconcile with the NMN hypothesis. First, NMN is postulated to trigger axon degeneration, yet exogenous addition of NMN protects axons after injury (Wang et al, 2015). Second, overexpression of NAMPT, which synthesizes NMN, leads to short-lived axonal protection following injury (Sasaki et al, 2006), although the NMN hypothesis would predict this to accelerate or induce axon degeneration.…”

Metabolic Vulnerability in the Neurodegenerative Disease Glaucoma

“…Mutations that extend the half-life of NMNAT2 increase its ability to delay axon degeneration, beyond that observed with the Wld s mutation (Milde et al, 2013). The Wld s protein can protect a cut axon for up to 4 h in vitro , while NMNAT2 depletion within 5 h commits the axon to degeneration (Gilley and Coleman, 2010; Wang et al, 2015). The longer half-life of Wld s over NMNAT2 may explain its ability to radically extend axon survival.…”