Characterization of recombinant NMDA receptor-mediated cell toxicity.

Format:

Book

Thesis/Dissertation

Author/Creator:

Anegawa, Norifusa John.

Contributor:

Lindstrom, Jon M., advisor.

University of Pennsylvania.

Language:

English

Subjects (All):

Pharmacology.

Neurosciences.

Molecular biology.

0307.

0317.

0419.

Penn dissertations--Pharmacology.

Pharmacology--Penn dissertations.

Local Subjects:

Penn dissertations--Pharmacology.

Pharmacology--Penn dissertations.

0307.

0317.

0419.

Physical Description:

166 pages

Contained In:

Dissertation Abstracts International 58-11B.

System Details:

Mode of access: World Wide Web.

text file

Summary:

The N-Methyl-D-Aspartate (NMDA) receptor is a subclass of glutamate ionotropic receptors which mediates fast neuronal excitation in the CNS. NMDA receptor (NR) dysfunction has been implicated in a variety of acute as well as chronic neuropathological processes such as ischemia-induced neuronal death. Huntington's disease, and HIV dementia. The receptor is highly permeable to $\rm Ca\sp{2+}$ and excessive and prolonged activation of NMDA receptors results in massive influx of Ca$\sp{2+}$ which leads to neuronal cell death through various Ca$\sp{2+}$ activated pathways. The specific mechanisms however are not well understood. The process of neuronal death induced by excessive activation of NMDA receptors has been termed 'excitotoxicity' because neurons literally excite themselves to death. At least five genes (NR 1 and NR 2A-2D) encode NMDA receptor subunits. In the brain, NMDA receptor subunits are thought to form heteromeric pentamers consisting of NR 1 with at least one of the NR 2 subunits. To study the molecular aspects of excitotoxicity, we have used heterologous expression systems to express specific combinations of NMDA receptor subunits.

We have expressed NMDA receptors in human embryonic kidney (HEK) 293 cells by transiently transfecting various combinations of NR subunit cDNAs. Through binding studies with known agonists and antagonists of NMDA receptors, we show that recombinant receptors reconstitute many of the characteristics of native NMDA receptors in the brain. We have also discovered that expression of NMDA receptors in this non-neuronal HEK 293 cell line causes the cells to die. This cell death appears to be mediated by activation of the NMDA receptors because incubating the cells with drugs which block NMDA receptor function protects cells from cell death. Even though several active forms of NMDA receptors can be synthesized by transfecting the NR 1 subunit with various NR 2 subunits, not all receptor subunits were equally toxic: NR 2A subunits were consistently more toxic than 2B or 2C. The technique we developed to quantitate this phenomenon, GFP-gated FACS (Green Fluorescent Protein-gated Fluorescence Activated Cell Sorting), could more specifically and sensitively detect cell death than hand-counting cells. The observed cell death is at least partially mediated by Ca$\sp{2+}$-stimulated processes such as activation of calpains and generation of reactive oxygen species. Some of the cells appear to die apoptotically as well.

Additionally, transfected cells expressing a lower-Ca$\sp{2+}$-permeable mutant NR receptors are less vulnerable to cell death, suggesting that Ca$\sp{2+}$ flux through the NMDA receptor/ion channel complex may be important in mediating exicitotoxic cell death. This observation led us to construct various deletional mutations in the C-terminus of the NR 2A subunit. When a truncated NR 2A subunit was co-expressed with a NR 1, decreased levels of toxicity was observed. The C-terminal deleted constructs had equivalent levels of expression as the wild-type constructs and generated equivalent currents in the cells. These results suggest that the expression of NMDA receptors may be the major neuron-specific component of excitotoxicity and that there is a region in the C-terminus of NMDAR 2A which may at least partially contribute to the generation of a cell toxic signal.

Notes:

Thesis (Ph.D. in Pharmacology) -- University of Pennsylvania, 1997.

Source: Dissertation Abstracts International, Volume: 58-11, Section: B, page: 5899.

Supervisor: Jon M. Lindstrom.

Local Notes:

School code: 0175.

ISBN:

9780591659498

Access Restriction:

Restricted for use by site license.