Novel Application of Stem Cell-Derived Neurons to Evaluate the Time-and Dose-Dependent Progression of Excitotoxic Injury
Abstract
Glutamate receptor (GluR)-mediated neurotoxicity is implicated in a variety of disorders ranging from ischemia to neural degeneration. Under conditions of elevated glutamate, the excessive activation of GluRs causes internalization of pathologic levels of Ca2+, culminating in bioenergetic failure, organelle degradation, and cell death. Efforts to characterize cellular and molecular aspects of excitotoxicity and conduct therapeutic screening for pharmacologic inhibitors of excitogenic progression have been hindered by limitations associated with primary neuron culture. To address this, we evaluated glutamate-induced neurotoxicity in highly enriched glutamatergic neurons (ESNs) derived from murine embryonic stem cells. As of 18 days in vitro (DIV 18), ESNs were synaptically coupled, exhibited spontaneous network activity with neurotypic mEPSCs and expressed NMDARs and AMPARs with physiological current:voltage behaviors. Addition of 0.78 200 mM glutamate evoked reproducible time- and dose-dependent metabolic failure in 6 h, with a calculated EC50 value of 0.44 mM at 24 h. Using a combination of cell viability assays and electrophysiology, we determined that glutamate-induced toxicity was specifically mediated by NMDARs and could be inhibited by addition of NMDAR antagonists, increased extracellular Mg2+ or substitution of Ba2+ for Ca2+. Glutamate treatment evoked neurite fragmentation and focal swelling by both immunocytochemistry and scanning electron microscopy. Presentation of morphological markers of cell death was dose-dependent, with 0.78 200 mM glutamate resulting in apoptosis and 3000 mM glutamate generating a mixture of necrosis and apoptosis. Addition of neuroprotective small molecules reduced glutamate-induced neurotoxicity in a dosedependent fashion.
Document Details
- Document Type
- Technical Report
- Publication Date
- May 14, 2013
- Accession Number
- ADA590112
Entities
People
- Ian M. Gut
- Kyle S. Hubbard
- Megan E. Lyman
- Patrick M. McNutt
- Phillip H. Beske
- Tracey A. Hamilton
Organizations
- United States Army Medical Research Institute of Chemical Defense