NMDA receptors in neurodegeneration : studies on NMDA receptor subunit expression preceding ischemia-induced delayed nerve cell death

Abstract

NMDA receptors are glutamate-gated ion channels that are permeable to Ca2+. 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 Ca2+ 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 ìM) 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 Ca2+ 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 [3H]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.