Dysmyelinated axons in shiverer mice are highly vulnerable to alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor-mediated toxicity

Abstract

Glutamate excitotoxicity plays a role in white matter injury in many neurological diseases. Oligodendrocytes in particular are highly vulnerable to excitotoxicity, mediated through activation of AMPA/kainate receptors. Myelin may also be injured independently via NMDA (N-methyl-D-aspartic acid) receptors located on peripheral oligodendroglial processes. Central axons are susceptible to glutamate receptor activation in vivo, but it is unclear whether this is mediated directly by activation of receptors expressed on axons, or indirectly through glutamate toxicity of myelin or neighboring glial cells. We examined axonal vulnerability in mice deficient in myelin basic protein (shiverer), also expressing yellow fluorescent protein (YFP) in a subset of axons. YFP fluorescence, EM, and mouse behavior were assessed 24 h after microstereotactical injections of S-AMPA or NMDA into lumbar dorsal columns. S-AMPA injection led to impaired rotarod performance and widespread axonal degeneration and was more pronounced in shiverer mice than controls. In contrast, NMDA injection did not cause axonal injury or behavioral changes in either group. These results indicate that spinal cord axons in vivo are vulnerable to toxicity mediated by AMPA but not NMDA receptors. The presence of compact myelin is not required for excitotoxic axon damage, and its absence may increase vulnerability. Further understanding of AMPA receptor-mediated axonal toxicity may provide new targets for neuroprotective therapy in WM diseases.

Figure 1.

AMPA-induced axon injury in shiverer mice. (a–c) Electron micrographs of dorsal column white matter in untreated mice. Axons are unmyelinated or severely hypomyelinated in homozygous shiverer (shi/shi) mice (a). In contrast, myelin in heterozygous +/shi mice is compact (b) and appears similar to wild type mice (c). (d, e) Dorsal column axons after injection with S-AMPA 20 mM in shiverer mice (d) and heterozygous controls (e) displaying axonal edema and disintegration of organelles and cytoskeletal filaments (calibration bar, 1μm).

Figure 2.

AMPA induced injury of YFP+ axons in shiverer mice and controls. (a, b) Longitudinal sections of dorsal columns from uninjected thy1-YFP2.2 control (a) and thy1-YFP2.2 shiverer mice (b). Axonal distentions (torpedos) are seen in shiverer mice (inset). (c, d) Axonal injury 24 hours after S-AMPA injection (20 mM) characterized by beading and fragmentation in control (c) and shiverer mice (d).

Figure 3.

Dose-dependence of S-AMPA induced axonal injury. (a) YFP+ axon morphology was scored in longitudinal sections 24 hours after AMPA injection in shiverer or control animals at various rostral-caudal distances from the injection site. Injection with vehicle resulted in minimal damage at the injection site. Increasing concentrations of S-AMPA resulted in increasing and widespread axonal damage (n=5 or 6 per concentration and group). (b) Axonal damage differed significantly between shiverers and control at S-AMPA concentrations of 10 mM and 20 mM but not 30 mM or vehicle. All S-AMPA concentrations produced significant damage compared to vehicle injections in both groups. Linear repeated measures mixed models were used to describe effects of study group, concentration and distance from injection site on axon counts per unit area. (Data are mean ± SEM. * p<0.002, ** p<0.0001)

Figure 4.

Rotarod performance of shiverer and control mice after S-AMPA injection. Performance (time on rotarod) is expressed as percentage of performance prior to injection. S-AMPA injection resulted in both groups in significant drop of performance at all concentrations compared to vehicle. Rotarod performance differed significantly between groups only at a S-AMPA concentration of 20 mM. Values shown are the mean ± SEM from two independent experiments with a total of 6 or 7 mice per data point. Asterisk indicates p<0.01, two-way ANOVA with Bonferroni post-tests.

Figure 5.

Injection of NMDA does not damage axons. (b, c) Injection of dorsal columns with NMDA (80 mM) did not induce axonal damage in thy1-YFP2.2 shiverer (b) or control mice (c). Injection sites are labeled with microruby (red) that was co-injected with NMDA. (a) No behavioral deficits were noted on rotarod testing in either group. Data are mean ± SEM, n = 5 per group.

Figure 6.

Upregulation of GluR1 in shiverer white matter. (a) Fluorescence micrographs show anti-glutamate receptor 1–4 antibodies (green) and nuclear stain (DAPI, blue) in spinal cord dorsal columns from shiverer and control mice. (a) Labeling for GluR1 but not GluR2/3 or GluR4 was markedly more intense in shiverer. Bright signal was apparent on cell bodies and elongated processes running in parallel with axons. (b) GluR1 (red) is expressed in close proximity to axons labeled by the pan-axonal marker SMI 312 (green, top) and colocalizes with the astrocytic marker GFAP (blue, bottom).