Disruption of glial glutamate transport by reactive oxygen species produced in motor neurons

Abstract

Observations of elevated CSF glutamate in amyotrophic lateral sclerosis (ALS), together with findings that motor neurons are selectively vulnerable to glutamate receptor-mediated ("excitotoxic") injury, support an excitotoxic contribution to the motor neuron loss in the disease. However, the basis of the apparent loss of astrocytic glutamate transport capacity in affected areas of motor cortex and spinal cord, which probably underlies the extracellular glutamate elevations, is unexplained. Here, we find that glutamate induces far greater reactive oxygen species (ROS) generation in cultured motor neurons than in other spinal neurons. In addition, we found that the ROS seem to be able to leave the motor neurons and induce oxidation and disruption of glutamate uptake in neighboring astrocytes. Correspondingly, in a transgenic mouse model of ALS, protein oxidation was increased in regions immediately surrounding motor neurons. These results provide a mechanism that can account for the localized loss of glial glutamate transport seen in the disease. Furthermore, the observations lend support for a feedforward model involving reciprocal interactions between motor neurons and glia, which may prove useful in understanding ALS pathogenesis.

Fig. 1.

Characterization of cultures. A, Multifluorescence microscopy shows neurons (fluorescent Nissl stain,red), MNs (SMI-32, blue), and astrocytes (anti-GFAP, green). B, High-magnification image shows MNs (SMI-32,red), astrocytes (anti-GFAP,green), and nuclei (primarily of astrocytes; Hoechst 33258, blue). C, Under confocal microscopy, the close spatial relationship between the MN (SMI-32,red) and glial glutamate transporters (anti-GLT-1, green) is apparent. Scale bars, 50 μm.

Fig. 2.

Glutamate exposure causes preferential ROS generation in cultured MNs. Spinal cultures were loaded with the oxidant-sensitive fluorophores HEt (left column) or DHR (right column) and exposed to glutamate (250 μm). Pseudocolor images depict fluorescence intensity before (A, E) and 20 min after (B, F) addition of glutamate. The pseudocolor scale depicts fluorescence ratios to baseline for HEt and raw fluorescence for DHR. Scale bars, 50 μm. Motor neuron identity was confirmed subsequently by SMI-32 immunoreactivity (C, G) and morphological criteria (arrows mark representative nonmotor neurons).Traces show time course of fluorescence changes in cultures loaded with HEt (D) or DHR (H), in MNs (circles) and other spinal neurons (squares), before and after addition of glutamate (indicated by bars). Eachtrace represents mean ±SEM of 8–10 MNs or >80 other spinal neurons.

Fig. 3.

Mechanisms of MN ROS generation. Spinal cultures were loaded with the oxidant-sensitive fluorophore HEt and exposed to glutamate (250 μm). A, Ca²⁺ dependence of MN ROS generation (circles) was examined by exposing cultures to glutamate (bar) in Ca²⁺-free buffer before the addition of Ca²⁺ (1.8 mm) (arrow). B, The role of mitochondria in MN ROS generation was tested by addition of the electron transport blocker rotenone (10 μm) to cultures before and during the glutamate exposure (black circles). Fluorescence changes in MNs from matched cultures exposed to glutamate alone are shown for comparison (gray circles). Eachtrace represents mean ± SEM of 8–10 MNs.

Fig. 4.

MN ROS generation induces oxidation in neighboring astrocytes. A, Pseudocolor images depict HEt fluorescence, as ratios to baseline, 20 min after addition of kainate (100 μm plus 10 μm MK-801) alone (left) or with addition of the antioxidant SOD (100 U/ml) to the bath (+AO, right).Arrows indicate HEt fluorescence signal from representative astrocyte nuclei (pseudocolor scaling to visualize astrocytic signals renders MN signals off-scale), andinsets show imaged MNs after SMI-32 labeling. Scale bar, 50 μm. B, Time course. In HEt-loaded cultures, fluorescence changes were measured in neurons (top) and astrocytes (bottom) before and after addition of kainate (indicated by bar). Note strong fluorescence increases in MNs (circles) in both the absence (black) or presence (white) of SOD, with minimal response in other neurons (squares). Astrocytic responses (triangles) were measured in nuclei at various distances from the MN soma (<50 μm, red; 50–100 μm, yellow; 100–150 μm, green) during exposure in the absence (bottom left) or presence (bottom right) of extracellular SOD (+AO). Each trace represents mean ± SEM of 80–200 astrocytes, 11 MNs, or >70 other spinal neurons. At the end point, fluorescence increases in nearby (<50 μm) astrocytes during exposure to kainate alone were greater than astrocytic responses in all other conditions (p < 0.001, by ANOVA with Student–Newman–Keuls post hoc test).

Fig. 5.

MN ROS can disrupt glutamate uptake in surrounding astrocytes. A, Spinal cultures were exposed to sham wash or to kainate (KA; 100 μm plus 10 μm MK-801) alone or with addition of the antioxidants SOD (100 U/ml) and catalase (400 U/ml) to the bath (KA+AO) before [³H]glutamate uptake assays as described. A control condition examined [³H]glutamate uptake in Na⁺-free buffer (0 Na⁺). Fluorescent images (left) show SMI-32-labeled MNs and some faintly labeled surrounding neurons. Transmitted light images (right) show autoradiographic granules corresponding to glutamate uptake.Colored lines indicate zones of increasing distance from the MN (<50 μm, red; 50–100 μm,yellow; 100–150 μm, green). Scale bar, 50 μm. B, Quantitative assessment of uptake. Relative uptake was calculated by normalizing optical density values in the two closer zones (<50, red; 50–100 μm,yellow) to that in the distal zone (100–150 μm) for each cell (mean ± SEM from zones surrounding 60–80 individual MNs from 13–14 cultures). * indicates difference from same zone in blank condition; ‡ indicates difference from same zone after kainate exposure; p < 0.001 by two-tailedt test.

Fig. 6.

Oxidative changes in regions surrounding MNs in transgenic mouse spinal cord. Lumbar spinal cord sections from 3-month-old SOD1 transgenic mice (G93A) and nontransgenic controls (non-TG) (n = 4 of each) were examined for 3-nitrotyrosine immunoreactivity.A, Spinal cord hemisection from G93A mouse.B, Representative ventral horn details from G93A and control mice. C, Fluorescent 3-nitrotyrosine labeling of region surrounding ventral horn motor neurons from G93A and control mice displayed (top) on a pseudocolor intensity scale (in arbitrary fluorescence units). Bottom images show labeling with SMI-32 (red) and Hoechst 33258 (blue) to identify MNs and nuclei, respectively.Lines demarcate confluent regions of increased labeling around ventral horn MNs seen in G93A mice. Scale bars:A, B, 100 μm; C, 50 μm.