Cu/Zn-superoxide dismutase (GLY93→ALA) mutation alters AMPA receptor subunit expression and function and potentiates kainate-mediated toxicity in motor neurons in culture
Introduction
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder, characterised by the progressive loss of motor neurons (MN) in brainstem, spinal cord and motor cortex. ALS occurs in two forms: sporadic and familial ALS (FALS) that are clinically indistinguishable. FALS accounts for approximately 10% of all ALS cases, with 20% of FALS cases associated with dominantly inherited mutations in the Cu2+/Zn2+ superoxide dismutase (SOD1) gene Cudkowicz et al., 1997, Rosen et al., 1993. This genetic linkage revealed the first causal factor related to the disease Bruijn et al., 1997, Ripps et al., 1995, Wong et al., 1995. The development of transgenic mice overexpressing this mutant form of SOD1 has provided a valuable animal model of the disease.
Transgenic G1H mice overexpress a mutant human SOD1 gene containing a glycine93→alanine (G93A) substitution. These mice have been extensively validated as an animal model of FALS due to the development of symptoms and a pathology that mimic those found in ALS patients (Gurney et al., 1994).
Although ALS is now regarded as a multisystem degenerative disease, the earliest and most severe degenerative changes tend to affect MN. It is recognised that MN may be particularly susceptible to excitotoxicity because of their size and the consequent high-energy requirements of such a large cell. Among several theories of ALS, a substantial body of literature suggests that MN degeneration may be a consequence of a disturbance in glutamatergic neurotransmission Kong and Xu, 1998, Plaitakis, 1991, Rothstein et al., 1992, Rothstein et al., 1995, Wong et al., 1995, involving excessive activation of glutamate receptors and the disruption of intracellular Ca2+ homeostasis Choi, 1994, Dugan and Choi, 1994. A pathogenic role for AMPA receptor (AMPAR)-mediated neurotoxicity has been identified in the selective loss of MN seen in ALS Bar-Peled et al., 1999, Carriedo et al., 1996, Rothstein et al., 1993, Roy et al., 1998.
AMPAR are cation-permeable heteromeric complexes composed of various combinations of four subunits, GluR1 to GluR4 (Hollmann and Heinemann, 1994). The presence of the GluR2 subunit in the assembled AMPAR determines its Ca2+ permeability Burnashev et al., 1992, Geiger et al., 1995, Jonas et al., 1994. Numerous groups have shown that spinal MN express substantial amounts of GluR2 mRNA and protein and that GluR2 is virtually completely edited at the Q/R site Morrison et al., 1998, Tölle et al., 1993, Vandenberghe et al., 2000a. Vandenberghe et al. (2001) proposed a model in which GluR2-containing and GluR2-lacking AMPAR are coexpressed and they cluster at synapses in the same MN. Other groups have measured, by in situ hybridization and immunohistochemistry, a lower level of the GluR2 subunit in human MN compared with other neuronal populations Petralia et al., 1997, Shaw and Ince, 1997, Williams et al., 1997. Recently, Heath et al. (2002) have quantitatively assessed the expression of the GluR2 subunit in human MN compared with dorsal horn neurons, they have found a lower expression in MN, they conclude that this finding may have important pathophysiological implications for the selective MN vulnerability. Thus, AMPAR expressed by MN may conduct more Ca2+ than they do in other neurons.
Alternative splicing generates further diversity, giving either Flip (Fi) or Flop (Fo) variants of all subunits Bettler and Mulle, 1995, Monyer et al., 1991, affecting the kinetic properties of AMPAR Lambolez et al., 1996, Mosbacher et al., 1994. The AMPAR desensitisation to prolonged agonist stimulation is a property that might affect vulnerability to excitotoxicity. AMPAR desensitisation protects neurons against the excitotoxic effects of their activation, as suggested by the pharmacological block of AMPAR desensitisation that enhances excitotoxicity in neurons, including spinal motor neurons Ballerini et al., 1995, Brorson et al., 1995, Carriedo et al., 2000.
Thus, we examined if (i) MN from G93A mice are, in fact, more susceptible to toxicity following prolonged AMPAR stimulation compared with control and SOD1 MN; (ii) the extent of AMPAR desensitisation of G93A MN AMPAR differed from those of control and SOD1; (iii) the relative expression of the GluR2 was elevated in the G93A MN, and/or if the Fi/Fo isoform expression was different in these neurons compared with control and SOD1 MN.
Section snippets
Transgenic mice
Transgenic mice carrying the Gly93→Ala mutation B6SJL-TgN(SOD1-G93A) 1Gur and B6SJL-TgN(SOD-1)2Gur carrying the wild-type human SOD1 constructed by Gurney et al. (1994) were obtained from Jackson Laboratories (Bar Harbor, ME, USA). Mice heterozygous for the wild-type SOD1, the mutated SOD1 (G93A) and control littermates were housed individually in small cages in a controlled environment. Transgenic progeny was identified by screening for the presence of the human transgene in the embryos after
Results
We identified MN in our mixed spinal cord culture by their positive SMI-32 immunoreactivity (Carriedo et al., 1996), and their characteristic morphology of large and multipolar cell bodies and long axon-like processes. The cell body size correlated with the probability of staining with the motor neurons markers (Fig. 1). For electrophysiological recording and single-cell experiments, MN were identified by morphological criteria only.
Discussion
Several lines of circumstantial evidence suggest that an imbalance in the glutamatergic system may contribute to the selective MN death seen in ALS (Heath and Shaw, 2002). In particular, the AMPAR-mediated excitotoxicity seems to exert a pivotal role. Indeed, numerous scientific reports have focused on the characteristics of AMPAR in motor neurons compared with other spinal neurons that do not degenerate in ALS Fryer et al., 1999, Heath et al., 2002, Takuma et al., 1999, Tomiyama et al., 1996,
Acknowledgements
We wish to thank B. Liss and J. Roeper for kindly introducing us to the single-cell RT-PCR techniques. C. P. Bengtson for reading the manuscript, and R. Sorge for support in the statistical analysis. The work was supported by a grant of Telethon number 1185.
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