Non-Nuclear Wld^SDetermines Its Neuroprotective Efficacy for Axons and SynapsesIn Vivo

Abstract

Axon degeneration contributes widely to neurodegenerative disease but its regulation is poorly understood. The Wallerian degeneration slow (Wld S ) protein protects axons dose-dependently in many circumstances but is paradoxically abundant in nuclei. To test the hypothesis that Wld S acts within nuclei in vivo, we redistributed it from nucleus to cytoplasm in transgenic mice. Surprisingly, instead of weakening the phenotype as expected, extranuclear Wld S significantly enhanced structural and functional preservation of transected distal axons and their synapses. In contrast to native Wld S mutants, distal axon stumps remained continuous and ultrastructurally intact up to 7 weeks after injury and motor nerve terminals were robustly preserved even in older mice, remaining functional for 6 d. Moreover, we detect extranuclear Wld S for the first time in vivo, and higher axoplasmic levels in transgenic mice with Wld S redistribution. Cytoplasmic Wld S fractionated predominantly with mitochondria and microsomes. We conclude that Wld S can act in one or more nonnuclear compartments to protect axons and synapses, and that molecular changes can enhance its therapeutic potential.

A recoverable state of axon injury persists for hours after spinal cord contusion in vivo

“…Thy1-TNXXL and DNLS R213AR215A WLD S (ref. 35) mice were maintained in a C57BL/6 background. C57BL/6 mice were used for tracing experiments.…”

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.…”

Targeting NMNAT1 to Axons and Synapses Transforms Its Neuroprotective PotencyIn Vivo

“…Mice were anesthetized with isoflurane (Abbot Animal Health) and subcutaneous injection of 2.5 g of buprenorphine hydrochloride (Vetergesic, Alstoe Lt. Animal Heath), and unilateral sciatic nerve lesion was performed as described by Beirowski et al (2009); the contralateral side served as control. A 5 mm segment of sciatic nerve was removed to prevent regeneration complicating the analysis of the distal stump at longer lesion durations (14 -35 d).…”

“…Brain nuclear and postnuclear fractions (see Fig. 5) were obtained as described by Beirowski et al (2009). More extensive subcellular fractionation (see Fig.…”

“…4C). Because enzymatic NMNAT activity is essential for Wld S neuroprotection (Araki et al, 2004;Jia et al, 2007;Conforti et al, 2009;Sasaki et al, 2009a) and measurements of NMNAT enzyme activity also allow quantitative assessment of transgenic protein levels (Conforti et al, 2007;Beirowski et al, 2009), we assayed total NMNAT activity in brains from Ax-NMNAT1 mice. Line 1 samples showed activity similar to wild-type (WT) littermates, further indicating that transgene expression in this line is very low.…”