Targeting NMNAT1 to Axons and Synapses Transforms Its Neuroprotective PotencyIn Vivo

Abstract

Axon and synapse degeneration are common components of many neurodegenerative diseases, and their rescue is essential for effective neuroprotection. The chimeric Wallerian degeneration slow protein (Wld S ) protects axons dose dependently, but its mechanism is still elusive. We recently showed that Wld S acts at a non-nuclear location and is present in axons. This and other recent reports support a model in which Wld S protects by extranuclear redistribution of its nuclear NMNAT1 portion. However, it remains unclear whether cytoplasmic NMNAT1 acts locally in axons and synapses or at a non-nuclear site within cell bodies. The potency of axon protection by non-nuclear NMNAT1 relative to Wld S also needs to be established in vivo. Because the N-terminal portion of Wld S (N70) localized to axons, we hypothesized that it mediates the trafficking of the NMNAT1 portion. To test this, we substituted N70 with an axonal targeting peptide derived from amyloid precursor protein, and fused this to NMNAT1 with disrupted nuclear targeting. In transgenic mice, this transformed NMNAT1 from a molecule unable to inhibit Wallerian degeneration, even at high expression levels, into a protein more potent than Wld S , able to preserve injured axons for several weeks at undetectable expression levels. Preventing NMNAT1 axonal delivery abolished its protective effect. Axonally targeted NMNAT1 localized to vesicular structures, colocalizing with extranuclear Wld S , and was cotransported at least partially with mitochondria. We conclude that axonal targeting of NMNAT activity is both necessary and sufficient to delay Wallerian degeneration, and that promoting axonal and synaptic delivery greatly enhances the effectiveness.

Generation of genetically-modified human differentiated cells for toxicological tests and the study of neurodegenerative diseases

“…This fusion protein has powerful neurite-protective properties in vivo and in vitro (Conforti et al, 2007;Beirowski et al, 2009;Babetto et al, 2010). expression of wld(s) in lUHMeS may help to answer the question whether such neurite protection may also be observed in human cells.…”

“…On the N-terminus, a 15 aa sequence from amyloid precursor protein (APP) was introduced, as this peptide sequence is known for its role as axonal targeting motif. To reduce the intrinsic nuclear targeting of wld(s), two point mutations in the NMNAT-1 region (R213A; R215A) were introduced (Beirowski et al, 2009;Babetto et al, 2010). Localization of this mutated wld(s) protein was assessed both by eGFP detection and by wld(s) antibody staining (Fig.…”

Molecular chaperones protect against JNK- and Nmnat-regulated axon degeneration in Drosophila

“…However, Wld S function remains unclear. For example, despite its predominant nuclear localisation, it is axonal localisation that appears to be key to neuroprotection, even though Wld S and different Nmnat isoforms have subtle and distinct subcellular locations (Berger et al, 2005;Conforti et al, 2007;Beirowski et al, 2009;Babetto et al, 2010;Sasaki and Milbrandt, 2010;Yahata et al, 2009) (supplementary material Fig. S2).…”

Differential protection of neuromuscular sensory and motor axons and their endings in Wld mutant mice

“…A nuclear localisation signal (NLS) embedded in Nmnat-1 results, as expected, in strong intranuclear transport of Wld S protein (Mack et al, 2001;Wilbrey et al, 2008). However, recent studies show that either inhibition of nuclear localisation of Wld S or tar-geting low levels of cytoplasmic Nmnat activity to an intra-axonal compartment, result in strengthening of both axonal and synaptic protection and counteract its agedepen-dence (Gillingwater et al, 2002;Beirowski et al, 2009;Babetto et al, 2010;Coleman and Freeman, 2010;Gilley and Coleman, 2010). Thus, at least two variables determine the amount and extent of protection by Wld S protein: its expression level and the proportion localised to intracellular components of the axon.…”

“…The age-dependence of the protective phenotype is also much less in transgenic mice in which Wld S protein (or Nmnat-1 activity) was targeted to axons, even at very low expression levels Babetto et al, 2010). Expression level of the mutant protein is another important factor in determining the strength of the Wld S phenotype (Mack et al, 2001; Gilling-water et al, 2002).…”