Synaptic Protection in the Brain of WldS Mice Occurs Independently of Age but Is Sensitive to Gene-Dose

Disruption of synaptic connectivity is a significant early event in many neurodegenerative conditions affecting the aging CNS, including Alzheimer's disease and Parkinson's disease. Therapeutic approaches that protect synapses from degeneration in the aging brain offer the potential to slow or halt the progression of such conditions. A range of animal models expressing the slow Wallerian Degeneration (Wld(S)) gene show robust neuroprotection of synapses and axons from a wide variety of traumatic and genetic neurodegenerative stimuli in both the central and peripheral nervous systems, raising that possibility that Wld(S) may be useful as a neuroprotective agent in diseases with synaptic pathology. However, previous studies of neuromuscular junctions revealed significant negative effects of increasing age and positive effects of gene-dose on Wld(S)-mediated synaptic protection in the peripheral nervous system, raising doubts as to whether Wld(S) is capable of directly conferring synapse protection in the aging brain.We examined the influence of age and gene-dose on synaptic protection in the brain of mice expressing the Wld(S) gene using an established cortical lesion model to induce synaptic degeneration in the striatum. Synaptic protection was found to be sensitive to Wld(S) gene-dose, with heterozygous Wld(S) mice showing approximately half the level of protection observed in homozygous Wld(S) mice. Increasing age had no influence on levels of synaptic protection. In contrast to previous findings in the periphery, synapses in the brain of old Wld(S) mice were just as strongly protected as those in young mice.Our study demonstrates that Wld(S)-mediated synaptic protection in the CNS occurs independently of age, but is sensitive to gene dose. This suggests that the Wld(S) gene, and in particular its downstream endogenous effector pathways, may be potentially useful therapeutic agents for conferring synaptic protection in the aging brain

Quantitative analysis of synaptic degeneration confirmed a dose-dependent protection of striatal synapses in <i>Wld^S</i> mice 3 days after cortical lesion.

<p>A – Bar chart (mean±SEM) showing the number of degenerating synapses in the striatum of wild-type (WT), heterozygous <i>Wld^S</i> (Het) and homozygous <i>Wld^S</i> (Hom) mice 3 days after cortical lesion (***P<0.001, *P<0.05, ANOVA with Tukey's post-hoc test; N = 4 wild-type mice, 3 heterozygous <i>Wld^S</i>, 3 homozygous <i>Wld^S</i>). B – Bar chart showing the total number of synapses remaining in the striatum of wild-type (WT), heterozygous <i>Wld^S</i> (Het) and homozygous <i>Wld^S</i> (Hom) mice 3 days after cortical lesion (**P<0.01, *P<0.05, nsP>0.05, ANOVA with Tukey's post-hoc test; N = 4 wild-type mice, 3 heterozygous <i>Wld^S</i>, 3 homozygous <i>Wld^S</i>).</p

Cortical lesion model for initiating synaptic degeneration in the striatum of wild-type, heterozygous <i>Wld^S</i> and homozygous <i>Wld^S</i> mice.

<p>A/B – Schematic diagram of the mouse brain viewed from above (A), showing the extent of cortical lesion produced (grey area). The dotted line in panel A represents the level of brain shown in coronal section in panel B (note the lesion to the left cortex). The box in panel B shows the region of striatum selected for ultrastructural experiments. C – Quantitative fluorescent western blotting of protein extracted from tail tips was used to confirm the genotype of experimental mice generated from heterozygous <i>Wld^S</i> (+/−) breeding colonies. Example blots show Wld^S protein levels (red; labelled with the Wld-18 antibody specific for Wld^S protein) and levels of actin loading control (green) in tail tips from 18 mice. 2 membranes are shown side by side with randomly arranged samples from individual mice numbered 1–18. A molecular weight marker is also shown (L). Lanes numbered 2,5,9,12 and 17 show wild-type mice (no Wld^S protein present), lanes 6,7,8,14,16 and 18 show heterozygous <i>Wld^S</i> mice (intermediate levels of Wld^S protein present), and lanes 1,3,4,10,11,13 and 15 show homozygous <i>Wld^S</i> mice (high levels of Wld^S protein present). D/E – Bar charts (mean±SEM) showing quantification of fluorescent western blots (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015108#s4" target="_blank">methods</a>) shown in panel C (pooled to give a mean value for each genotype), confirming that heterozygous <i>Wld^S</i> mice had approximately half the expression levels of Wld^S protein observed in homozygous <i>Wld^S</i> mice (D), whilst levels of actin loading control remained constant (E) across mice of all genotypes. N = 5 wild-type mice, 6 heterozygous <i>Wld^S</i> mice & 7 homozygous <i>Wld^S</i> mice.</p

Quantitative western blotting for synaptic proteins confirmed dose-dependent protection of striatal synapses in <i>Wld^S</i> mice 3 days after cortical lesion.

<p>A – Representative bands from western blots showing expression levels of two major synaptic proteins (SNAP and synaptophysin) as well as two loading controls (actin and tubulin) in the striatum of wild-type, heterozygous <i>Wld^S</i> and homozygous <i>Wld^S</i> mice 3 days after cortical lesion. Note lower levels of synaptic markers in heterozygous <i>Wld^S</i> mice compared to homozygous <i>Wld^S</i> mice and lower still levels in wild-type mice, indicative of a loss of synapses. B/C – Bar charts (mean±SEM) showing relative expression levels of SNAP (B) and synaptophysin (C) in the striatum of wild-type, heterozygous <i>Wld^S</i> and homozygous <i>Wld^S</i> mice 3 days after cortical lesion (ns = not significant, *P<0.05, ***P<0.001; ANOVA with Tukey's post-hoc test; N = 3 mice per genotype). Levels of actin and tubulin remained constant between samples (data not shown but see Panel A).</p