Glutamate potentiates the toxicity of mutant Cu/Zn-superoxide dismutase in motor neurons by postsynaptic calcium-dependent mechanisms

Abstract

Mutations in the Cu/Zn-superoxide dismutase (SOD-1) gene are responsible for a subset of familial cases of amyotrophic lateral sclerosis. Using a primary culture model, we have demonstrated that normally nontoxic glutamatergic input, particularly via calcium-permeable AMPA/kainate receptors, is a major factor in the vulnerability of motor neurons to the toxicity of SOD-1 mutants. Wild-type and mutant (G41R, G93A, or N139K) human SOD-1 were expressed in motor neurons of dissociated cultures of murine spinal cord by intranuclear microinjection of plasmid expression vector. Both a general antagonist of AMPA/kainate receptors (CNQX) and a specific antagonist of calcium-permeable AMPA receptors (joro spider toxin) reduced formation of SOD-1 proteinaceous aggregates and prevented death of motor neurons expressing SOD-1 mutants. Partial protection was obtained by treatment with nifedipine, implicating Ca2+ entry through voltage-gated calcium channels as well as glutamate receptors in potentiating the toxicity of mutant SOD-1 in motor neurons. Dramatic neuroprotection was obtained by coexpressing the calcium-binding protein calbindin-D28k but not by increasing intracellular glutathione levels or treatment with the free radical spin trap agent, N-tert-butyl-alpha-phenylnitrone. Thus, generalized oxidative stress could have contributed in only a minor way to death of motor neurons expressing the mutant SOD-1. These studies demonstrated that the toxicity of these mutants is calcium-dependent and provide direct evidence that calcium entry during neurotransmission, coupled with deficiency of cytosolic calcium-binding proteins, is a major factor in the preferential vulnerability of motor neurons to disease.

Fig. 1.

Phase-contrast micrographs of a motor neuron (A) and DRG sensory neurons (B) in living spinal cord–DRG cultures.C, Lower magnification view of spinal cord–DRG culture 4 weeks in vitro labeled with antibody SMI32 against neurofilament proteins. Arrowhead points to cell identified as a motor neuron (see Materials and Methods). Also visible are small spinal neurons and larger DRG neurons. Scale bars, 20 μm.D–F, Distribution of wild-type human SOD-1 (D) and G93A mutant SOD-1 (E, F) in motor neurons after intranuclear microinjection of plasmid expression vector (200 μg/ml). Three days after microinjection, cells were immunolabeled with antibody specific to human SOD-1 (Sigma) followed by anti-mouse IgG-Texas red. Two general patterns of mSOD-1 distribution are observed: diffuse distribution of mSOD-1 throughout the motor neuron similar to that observed with wild-type human SOD-1 (E) and localization in punctate aggregates (F). Scale bar, 20 μm.

Fig. 2.

A, Motor neuron death induced by mSOD-1 expression was prevented by blockade of non-NMDA ionotropic glutamate receptors but not NMDA receptors. Motor neurons were microinjected with pwtSOD-1 or pG93A (200 μg/ml) plus the fluorescent marker, 70 kDa dextran-FITC. Survival of injected neurons was evaluated at days 1, 3, 6, 9, and 12 after injection by counting cells containing the marker. Experiments were conducted in the presence and absence of kynurenic acid (antagonist of both NMDA and non-NMDA ionotropic receptors; 1 mm), CNQX (non-NMDA ionotropic receptor blocker; 5 μm), or APV (NMDA receptor antagonist; 100 μm). For additional details, see Materials and Methods. B, Blockers of ionotropic glutamate receptors were neither generally neuroprotective nor toxic to motor neurons in culture. Motor neurons were microinjected with pCEP4 vector or pwtSOD-1 alone plus dextran-FITC. The usual attrition of cells normally observed in cultures of this age was not affected by kynurenic acid (1 mm) or by a combination of CNQX and APV. C, D, CNQX also protected motor neurons from death induced by different human SOD-1 mutants (200 μg/ml) pN139K (C) or pG41R (D). Shown are means ± SD of results from four to seven different cultures; *significant reduction of cell death; Student’s t test (unpaired, two-tailed; p < 0.05).

Fig. 3.

JSTX-3, an inhibitor of Ca²⁺-permeable AMPA receptors, prevented motor neuron death induced by both AMPA and the G93A SOD-1 mutant.A, The day after microinjection of motor neurons with 15 mg/ml dextran-FITC, marked neurons were counted, and the various glutamate receptor blockers were added to the culture medium (0.1 mm APV ± 5 μm CNQX or 0.5 μm JSTX-3). Five hours later, cultures were challenged with 5 μm AMPA. Viability was assessed by counting surviving marked neurons after an additional 24 hr. B, pG93A (200 μg/ml) was injected into motor neuronal nuclei along with dextran-FITC, and viability was assessed daily. We added 0.5 μm JSTX-3 to the culture medium 5 hr after microinjection and replenished it every 3 d.C, JSTX-3 reduced formation of aggregates in motor neurons expressing G93A SOD-1. On day 3, cultures were immunolabeled with antibody specific to human SOD-1, and the percentage of cells in which most of the immunoreactive mutant SOD-1 was localized in punctate aggregates was determined. Shown are means ± SD for results obtained from three or four cultures per treatment group; *significant difference in the absence and presence of JSTX-3, unpaired, two-tailed Student’s t test; p < 0.05.

Fig. 4.

Blockade of voltage-gated calcium channels by nifedipine (1 μm) delayed loss of viability of motor neurons expressing mutant SOD-1. pG93A (200 μg/ml) was injected into motor neuronal nuclei along with dextran-FITC, and viability was assessed daily. Shown are means ± SD for results obtained from four cultures per treatment group; *significant difference between G93A SOD-1 alone and G93A SOD-1 plus nifedipine, unpaired, two-tailed Student’st test; p < 0.05.

Fig. 5.

Calbindin-D28k protected motor neurons from the toxicity of G93A SOD-1. A, Top panel, Increased level of calbindin-D28k was demonstrated 3 d after microinjection of pcalbindin-D28k (30 μg/ml) by immunolabeling with antibody specific to calbindin-D28k. Middle panel, Motor neuron injected with control vector pCEP4 was not labeled (phase micrograph of this neuron is presented in bottom panel). Scale bar, 20 μm. B,C, Motor neuronal nuclei were injected with pG93A or pwtSOD-1 ± pcalbindin-D28k along with dextran-FITC.B, Coexpression of calbindin-D28k dramatically preserved the viability of motor neurons expressing mutant SOD-1.C, Coexpression of calbindin-D28k reduced the percentage of motor neurons with aggregated SOD-1 on day 3 after microinjection by 56%. D, Calbindin-D28k protected motor neurons from death induced by both glutamate and paraquat. See Materials and Methods for details. Shown are means ± SD for results obtained from three to six cultures per treatment group; *significant difference in the absence and presence of pcalbindin-D28k, unpaired, two-tailed Student’s t test; p < 0.05.

Fig. 6.

Increase in GSH levels in cultured motor neurons by treatment with glutathione ethyl ester. GSH was visualized using the fluorescent probe, Cell Tracker Green. A, Untreated cultures. Note minimal fluorescence (i.e., reduced GSH) in the two motor neurons indicated by arrows and inB by phase contrast. C, Culture treated with 1 mm glutathione ethyl ester in the medium for 3 d showing marked increase in Cell Tracker Green fluorescence relative to control. Scale bar, 20 μm.

Fig. 7.

Glutathione ethyl ester failed to protect motor neurons from toxicity of G93A SOD-1. A, 1 mmglutathione ethyl ester (GSH EE) completely protected motor neurons from death induced by paraquat and provided partial protection against glutamate-induced death. See Materials and Methods for details.B, pCEP4 or pG93A (200 μg/ml) was injected into motor neuronal nuclei along with dextran-FITC (15 mg/ml). Addition of 1 mm GSH EE, beginning 5 hr after microinjection, failed to preserve the viability of motor neurons expressing G93A SOD-1 or (C) to alter the percentage of motor neurons in which most of the immunoreactive mutant SOD-1 was localized in punctate aggregates. D, The free radical spin trap agent, 50 μm PBN failed to protect motor neurons from death induced by expression of G93A mSOD-1. Shown are means ± SD for results obtained from four cultures per treatment group; *significant difference in the presence or absence of GSH EE, unpaired, two-tailed Student’s t test;p < 0.05.