Synaptic protection in the brain of WldS mice occurs independently of age but is sensitive to gene-dose

Abstract

Background: 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.

Methodology/principal findings: 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.

Conclusions/significance: 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.

Figure 1. Cortical lesion model for initiating synaptic degeneration in the striatum of wild-type, heterozygous Wld^S and homozygous Wld^S mice.

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 Wld^S (+/−) 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 Wld^S mice (intermediate levels of Wld^S protein present), and lanes 1,3,4,10,11,13 and 15 show homozygous Wld^S mice (high levels of Wld^S protein present). D/E – Bar charts (mean±SEM) showing quantification of fluorescent western blots (see methods) shown in panel C (pooled to give a mean value for each genotype), confirming that heterozygous Wld^S mice had approximately half the expression levels of Wld^S protein observed in homozygous Wld^S mice (D), whilst levels of actin loading control remained constant (E) across mice of all genotypes. N = 5 wild-type mice, 6 heterozygous Wld^S mice & 7 homozygous Wld^S mice.

Figure 2. Widespread synaptic degeneration in the striatum of young (2 month old) wild-type, but not heterozygous Wld^S or homozygous Wld^S mice, 3 days after cortical lesion.

Representative electron micrographs of striatal synapses at low power (A) and higher power (B) from a young wild-type mouse (top panel), heterozygous Wld^S mouse (middle panel) and homozygous Wld^S mouse (bottom panel). Asterisks indicate degenerating synaptic profiles (identified principally by their electron dense cytoplasm) and arrows indicate healthy (i.e. non-degenerating) synaptic profiles. Degenerating synapses were readily identified in wild-type mice, occasionally observed in heterozygous Wld^S mice and rarely observed in homozygous Wld^S mice. However, the morphological appearance of degenerating synapses was indistinguishable between the different genotypes, suggesting that synapses in mice expressing the Wld^S gene ultimately degenerate by the same mechanism as in wild-type mice, albeit after a delay. Scale bars; A = 1 µm, B = 0.5 µm.

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

A – Bar chart (mean±SEM) showing the number of degenerating synapses in the striatum of wild-type (WT), heterozygous Wld^S (Het) and homozygous Wld^S (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 Wld^S, 3 homozygous Wld^S). B – Bar chart showing the total number of synapses remaining in the striatum of wild-type (WT), heterozygous Wld^S (Het) and homozygous Wld^S (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 Wld^S, 3 homozygous Wld^S).

Figure 4. Quantitative western blotting for synaptic proteins confirmed dose-dependent protection of striatal synapses in Wld^S mice 3 days after cortical lesion.

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 Wld^S and homozygous Wld^S mice 3 days after cortical lesion. Note lower levels of synaptic markers in heterozygous Wld^S mice compared to homozygous Wld^S 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 Wld^S and homozygous Wld^S 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).

Figure 5. Synaptic protection in the striatum remains robust in old (∼12 month old) homozygous Wld^S mice after cortical lesion.

Representative electron micrographs of striatal synapses at low power (A) and higher power (B) from an old wild-type mouse (top panel) and old homozygous Wld^S mouse (bottom panel) 3 days after cortical lesion. Asterisks indicate degenerating synaptic profiles (identified principally by their electron dense cytoplasm) and arrows indicate healthy (i.e. non-degenerating) synaptic profiles. Degenerating synapses were readily identified in wild-type mice but rarely observed in homozygous Wld^S mice. As in young mice (see Figure 2), the morphological appearance of degenerating synapses was indistinguishable between the different genotypes. Scale bars; A = 1 µm, B = 0.5 µm.

Figure 6. Quantitative analysis of synaptic degeneration confirmed robust protection of striatal synapses in old (∼12 mth) Wld^S mice 3 and 5 days after cortical lesion.

A – Bar chart (mean±SEM) showing the number of degenerating synapses in the striatum of wild-type (WT) and homozygous Wld^S (Wlds) mice at 3 and 5 days after cortical lesion (**P<0.01, ANOVA with Tukey's post-hoc test; N = 4 wild-type mice per time-point, 3 homozygous Wld^S mice per time-point). B – Bar chart showing the total number of synapses remaining in the striatum of wild-type and homozygous Wld^S mice at 3 and 5 days after cortical lesion (*P<0.05, nsP>0.05, ANOVA with Tukey's post-hoc test; N = 4 wild-type mice per time-point, 3 homozygous Wld^S mice per time-point).