Cytoprotective effects of NR3A and NR3B upon cell death mediated by NR1/NR2B receptors expessed in HEK 293 cells

H. E. Chaffey1 , M.J. Gunthorpe2 and P. L. Chazot1, Centre for Integrative Neuroscience (CINS)1, School of Biological and Biomedical Sciences, Durham University, UK. Neurology CEDD2, GSK, Harlow, Essex, UK

NMDA receptors are fast-acting ligand-gated ion channels constituting a major subclass of excitatory glutamate receptors. They play an essential role in normal neurological development and function, and are critically involved in neuropathological processes. Functional NMDA receptors are hetero-oligomers comprising the obligatory NR1 subunit, and at least one NR2A-D subunit type. NR3A and NR3B subunit types have been shown to associate and reduce the Ca2+ permeability of recombinant NMDA receptors but to date the native assemblies containing the NR3 subunits and potential role in neuronal loss have yet to be fully elucidated (Chazot, 2004). Motor neuron disease (MND)/ Amyotrophic lateral sclerosis (ALS) is a devastating set of neurodegenerative diseases, characterized by selective loss of spinal motor neurons (MN). No treatment is currently available to halt the loss of MN and, consequently, the disease process. NR2A, NR2B and NR3B subunits are all co-expressed in rodent and human motor neurons in the spinal cord; interestingly the NR2A appears to be more prominent than NR2B in the rat, the reverse being the case in the human spinal cord (Chaffey & Chazot, unpublished). In this initial study, we have investigated the potential cytoprotective effects of the rNR3A and rNR3B subunits when co-expressed with rNR1/NR2B receptor subtypes in HEK 293 cells, using an optimised lipofectamine protocol (50% mean transfection efficiency). Expression of subunits was confirmed using immunoblotting with our panel of selective NMDA receptor subunit antibodies (Chazot et al., 2002), including a new anti-rNR3B antibody recently developed in our laboratory. Levels of cell death were assayed by release of lactate dehydrogenase (LDH) activity in the growth medium as described previously (Cik et al., 1994).

Co-expression of NR3B with NR1/2B receptor subtype resulted in a 52 + 18% (mean + SD, n = 6) reduction in cell death in comparison to NR1/NR2B alone. Preliminary data from the co-expression of NR3A with NR1/2B receptor type also revealed cytoprotection (23 + 10%, n = 6). This study shows that incorporation of either NR3B or NR3A significantly reduces the cell death known to be due, in part, to calcium influx through recombinant NMDA receptors (Cik et al., 1994). We showed that the reduced levels of calcium influx, previously determined electrophysiologically, are sufficient to provide profound cytoprotective effects in this useful in vitro model. These findings have implications for the treatment of neurological disorders such as MND in which NMDA mediated neuronal death has been suggested. Further studies with the other likely major motor neuron NR1/NR2A receptor subtype are currently underway, to assess whether or not this latter subtype is similarly influenced by NR3A or NR3B.

Chazot P.L 2004, Current Medicinal Chemistry, 11, 389-396.
Chazot P.L. et al., 2002, Neuropharmacology, 42 , 319-24.
Cik, M., et al., 1994 Eur J Pharmacol, 266, R1-3.

We thank Prof Anne Stephenson (SOP, London) for the NR1 and NR2 subunit clones.