NMDA RECEPTORS IN NEURODEGENERATION

NMDA receptors are glutamate-gated ion channels that are permeable to Ca. NMDA receptors are tetramers formed by a combination of subunits. In the present thesis, we describe the novel NR3B subunit that we cloned in mouse and human. NR3B was found to be expressed in the adult rat and human central nervous system (CNS). Also, human NR3A was cloned and it's expression in the adult human CNS was investigated. In addition, we identified, cloned, and characterized tissue expression of a previously unknown gene, which we named membralin because it contains several transmembrane regions. Notably, the genes that encode membralin and NR3B were found to be oriented in an anti-parallel fashion, both in the mouse and the human genomes. Excessive Ca influx through NMDA receptors is a major mechanism for neurodegeneration following stroke and brain trauma. To better understand the mechanisms behind NMDA-dependent neurodegeneration, we used, developed, and characterized three injury models in the rat: crush injury in entorhinal hippocampal slices, global ischemia, and subarachnoid hemorrhage. Temporal and regional patterns of cell death were evaluated in these models with focus on delayed cell death. Crush-injury in slices induced two phases of cell death, including an early phase that peaked within a day after injury and a delayed phase that appeared at 4 DAI. Treatment with the NMDA receptor antagonist MK-801 (30 μM) for 2 h at the time of the injury prevented the delayed but not the early phase of cell death. Also, we analyzed the inflammatory response at various time-points after crush-injury. Two complement components: C1q and C5b-9 and the complement inhibitor clusterin were dramatically up-regulated after injury. These studies indicate that delayed cell death in this model is dependent on NMDA receptor activation and that the complement cascade can be activated in vitro, without the influence of exogenous blood products and cells. A new reproducible global ischemia model was developed. Eleven min of global ischemia induced delayed cell death of CA1 neurons in the hippocampus, as shown by Fluoro-Jade, cresyl-violet and NeuN staining. The death of CA1 neurons was correlated to deficiencies in learning and memory, as analyzed using the Morris water maze and a novel water T-maze. At 90 days after ischemia, new CA1 neurons had formed, associated with a marked recovery in learning and memory. At 250 days after ischemia, the ischemia-induced new neurons were found to degenerate in association with the appearance of Ca deposits, but without significant attenuation of learning and memory performance. In a model of subarachoidal hemorrhage, we demonstrated delayed nerve cell death in CA1 and CA3, and found the extent of cell death to relate to the severity of the transient ischemia. We found that the delayed nerve cell death in both the global ischemia model and in the subarachnoid hemorrhage model was preceded by down regulation of specific NMDA receptor subunits, with somewhat different profiles in the two models. Also the total number of NMDA receptors was found to be decreased using [H]MK-801 autoradiography. These studies may give a better understanding of the role of NMDA receptors in delayed neurodegeneration, and provide a basis for the development of new neuroprotective treatments. LIST OF PUBLICATIONS I. Andersson, O. , Stenqvist, A. , Attersand, A. and von Euler, G. , Nucleotide sequence, genomic organization, and chromosomal localization of genes encoding the human NMDA receptor subunits NR3A and NR3B. Genomics. 2001 Dec; 78(3):178-84 II. Bendel, O. , Meijer, B. , Hurd, Y. and von Euler, G. , Cloning and expression of the human NMDA receptor subunit NR3B in the adult human hippocampus. Neurosci Lett. 2005 Mar 22; 377(1): 31-6 III. Andersson, O. and von Euler, G. , Characterization and expression of the gene encoding membralin, an evolutionary conserved protein expressed in the central nervous system. Gene Expr Patterns. 2002 Oct; 1(3-4): 205-12 IV. Bendel, O. , Langmoen, I. A. , and von Euler, G. , Crush injury induces NMDAreceptor-dependent delayed nerve cell death in rat entorhinal-hippocampal slice cultures. Brain Res. 2004 Oct 29; 1025(1-2): 35-42 V. Bellander, B. M. , Bendel, O. , von Euler, G. , Ohlsson, M. , and Svensson, M. , Activation of microglial cells and complement following traumatic injury in rat entorhinal-hippocampal slice cultures. J Neurotrauma. 2004 May; 21(5): 605-15 VI. Bendel, O. , Alkass, K. , Bueters, T. , von Euler, M. and von Euler, G. , Reproducible loss of CA1 neurons following carotid artery occlusion combined with halothane-induced hypotension. Brain Res. 2005 Feb 08; 1033(2): 135-42 VII. Bendel, O. , Bueters, T. , von Euler, M. , Ögren S-O. , Sandin, J. and von Euler, G. , Reappearance of hippocampal CA1 neurons after ischemia is associated with recovery of learning and memory. J Cereb Blood Flow Metab. 2005 Dec; 25(12): 1586-95 VIII. von Euler, M. , Bendel, O. , Bueters, T. , Sandin, J. and von Euler, G. , Profound but transient deficits in learning and memory after global ischemia using a novel water maze test. Behav Brain Res. 2006 Jan 30; 166(2): 204-10 IX. Bueters, T. , von Euler, M. , Bendel, O. and von Euler, G. Degeneration of newly formed CA1 neurons following global ischemia in the rat. Experimental Neurology in press 2007 X. Bueters, T. , Changes in NMDA receptor subunit expression precedes degeneration of CA1 neuron following transient global ischemia in the rat. Manuscript 2007 XI. Bendel, O. , Prunell, G. , Stenqvist, A. , Mathiesen, T. , Holmin, S. , Svendgaard, N-A. von Euler, G. Experimental subarachnoid hemorrhage induces changes in the levels of hippocampal NMDA receptor subunit mRNA. Brain Res Mol Brain Res. 2005 Jun 13; 137(1-2): 119-25