Protective Effect of Parvalbumin on Excitotoxic Motor Neuron Death L. Van Den Bosch,* B. Schwaller,† V. Vleminckx,* B. Meijers,* S. Stork,‡ T. Ruehlicke,† E. Van Houtte,* H. Klaassen,* M. R. Celio,† L. Missiaen,§ W. Robberecht,* ,1 and M. W. Berchtold‡ *Neurobiology and §Laboratory of Physiology, Campus Gasthuisberg, Herestraat 49, Leuven, Belgium; †Institute of Histology and General Embryology, University of Fribourg, Fribourg, Switzerland; and ‡Institute of Molecular Biology, University of Copenhagen, Copenhagen, Denmark Received May 14, 2001; accepted October 31, 2001 The mechanism responsible for the selective vulnera- bility of motor neurons in amyotrophic lateral sclerosis (ALS) is poorly understood. Several lines of evidence indicate that susceptibility of motor neurons to Ca 2 overload induced by excitotoxic stimuli is involved. In this study, we investigated whether the high density of Ca 2 -permeable AMPA receptors on motor neurons gives rise to higher Ca 2 transients in motor neurons com- pared to dorsal horn neurons. Dorsal horn neurons were chosen as controls as these cells do not degenerate in ALS. In cultured spinal motor neurons, the rise of the cytosolic Ca 2 concentration induced by kainic acid (KA) and mediated by the AMPA receptor was almost twice as high as in spinal neurons from the dorsal horn. Further- more, we investigated whether increasing the motor neuron’s cytosolic Ca 2 -buffering capacity protects them from excitotoxic death. To obtain motor neurons with increased Ca 2 buffering capacity, we generated trans- genic mice overexpressing parvalbumin (PV). These mice have no apparent phenotype. PV overexpression was present in the central nervous system, kidney, thy- mus, and spleen. Motor neurons from these transgenic mice expressed PV in culture and were partially pro- tected from KA-induced death as compared to those iso- lated from nontransgenic littermates. PV overexpres- sion also attenuated KA-induced Ca 2 transients, but not those induced by depolarization. We conclude that the high density of Ca 2 -permeable AMPA receptors on the motor neuron’s surface results in high Ca 2 transients upon stimulation and that the low cytosolic Ca 2 -buffer- ing capacity of motor neurons may contribute to the selective vulnerability of these cells in ALS. Overexpres- sion of a high-affinity Ca 2 buffer such as PV protects the motor neuron from excitotoxicity and this protective effect depends upon the mode of Ca 2 entry into the cell. © 2002 Elsevier Science (USA) Key Words: amyotrophic lateral sclerosis; ALS; AMPA receptor; calcium-binding proteins; calcium buffering; excitotoxicity; kainic acid; motor neuron; parvalbumin. INTRODUCTION Amyotrophic lateral sclerosis (ALS) is a neurodegen- erative disorder, characterized by loss of cortical, bul- bar, and spinal motor neurons. This neuronal death results in progressive muscle weakness, ultimately leading to death due to bulbar and respiratory involve- ment. In about 1 to 2% of cases, mutations in the superoxide dismutase 1 (SOD1) gene have been iden- tified to underlie the disorder, but in the remainder 98 to 99% of patients, the pathogenesis remains unknown (reviews: 11, 34). A number of studies suggest that vulnerability of motor neurons to excitotoxicity is involved in establish- ing the selectivity of ALS for motor neurons. This cell type is particularly more vulnerable to AMPA receptor agonists than other spinal neurons both in vivo (20, 21) and in vitro (3, 6, 36, 42). At least two factors are thought to contribute to this selective vulnerability: the high density of Ca 2+ -permeable -amino-3-hy- droxy-5-methyl-4-isoxazole propionic acid (AMPA)- type of glutamate receptors and the low Ca 2+ -buffering capacity of motor neurons. The density of Ca 2+ -permeable AMPA receptors on the motor neuron’s surface is high as compared to that on dorsal horn neurons, as was shown before (43). It was demonstrated that the AMPA receptors on these cultured motor neurons have normal Ca 2+ permeabil- ity at the single cell level and contain normal amounts of GluR2 mRNA (17, 44). Others have suggested that the selective vulnerability of motor neurons might re- sult from the predominant expression of Ca 2+ -perme- able AMPA receptors that lack the GluR2 subunit (47). Whatever the underlying mechanism is, it remains unknown whether the high density of Ca 2+ -permeable AMPA receptors results in higher intracellular Ca 2+ concentrations ([Ca 2+ ] i ) upon stimulation. Motor neurons affected during ALS are also thought to have a limited capacity to buffer Ca 2+ that enters the cell upon stimulation. This has been demonstrated in several electrophysiological studies (26, 27, 30, 46). 1 Fax: 32 16 34 42 85. E-mail: wim.robberecht@uz.kuleuven.ac.be. Experimental Neurology 174, 150 –161 (2002) doi:10.1006/exnr.2001.7858, available online at http://www.idealibrary.com on 150 0014-4886/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.