Cu/Zn-superoxide dismutase (GLY93 !ALA) mutation alters AMPA receptor subunit expression and function and potentiates kainate- mediated toxicity in motor neurons in culture Alida Spalloni, a Federica Albo, a Francesca Ferrari, a Nicola Mercuri, a,b Giorgio Bernardi, a,b Cristina Zona, a,b and Patrizia Longone a, * a Fondazione Santa Lucia IRCCS, 00179 Rome, Italy b Department of Neuroscience, University of Tor Vergata, 00133 Rome, Italy Received 6 February 2003; revised 26 October 2003; accepted 10 November 2003 The cause of the selective degeneration of motor neurons in amyotrophic lateral sclerosis (ALS) remains a mystery. One potential pathogenic mechanism is excitotoxicity due to disturbances of glutamatergic neurotransmission, particularly via AMPA-sensitive glutamate receptors. We report here that motor neurons from a familial ALS-linked superoxide dismutase (SOD1) mutant G93A mouse show an higher susceptibility to kainate-induced excitotoxicity. Moreover, they ex- pressed GluR 3 and GluR 4 mRNA at detectable levels more frequently, with a modified electrophysiology when compared with control and wild-type SOD1 motor neurons. Thus, the SOD1 G93A mutation causes changes in the AMPA-receptor expression and function, as well as a susceptibility to kainate-mediated excitotoxicity, which may promote the motor neuron degeneration seen in ALS. D 2004 Elsevier Inc. All rights reserved. Keywords: Amyotrophic lateral sclerosis; ALS; Superoxide dismutase-1; Motor neuron; Excitotoxicity; Glutamate receptors; Selective vulnerability; scRT-PCR Introduction Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenera- tive 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 degenera- tion may be a consequence of a disturbance in glutamatergic neurotransmission (Kong and Xu, 1998; Plaitakis, 1991; Rothstein et al., 1992, 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 neurotox- icity 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 com- posed of various combinations of four subunits, GluR 1 to GluR 4 (Hollmann and Heinemann, 1994). The presence of the GluR 2 subunit in the assembled AMPAR determines its Ca2+ permeabil- ity (Burnashev et al., 1992; Geiger et al., 1995; Jonas et al., 1994). Numerous groups have shown that spinal MN express substantial amounts of GluR 2 mRNA and protein and that GluR 2 is virtually completely edited at the Q/R site (Morrison et al., 1998; To ¨lle et al., 1993; Vandenberghe et al., 2000a). Vandenberghe et al. (2001) proposed a model in which GluR 2 -containing and GluR 2 -lacking AMPAR are coexpressed and they cluster at synapses in the same MN. Other groups have measured, by in situ hybridization and 0969-9961/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2003.11.012 Abbreviations: ALS, amyotrophic lateral sclerosis; G93A, mutated (Gly93!Ala) form of SOD1; AMPAR, a-amino-3-hydroxy-5-methyl-4- isoxazole propionic acid receptors; CTZ, cyclothiazide; NMDA, N-methyl- D-aspartic acid; SOD1, superoxide dismutase; scRT-PCR, single-cell reverse transcriptase polymerase chain reaction. * Corresponding author. Laboratory of Molecular Neurobiology, Santa Lucia Foundation, IRCCS, Via Ardeatina 306, 00179 Rome, Italy. Fax: +39-0651501384. E-mail address: p.longone@hsantalucia.it (P. Longone). Available online on ScienceDirect (www.sciencedirect.com.) www.elsevier.com/locate/ynbdi Neurobiology of Disease 15 (2004) 340 – 350