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Updated: May 23, 2026
Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons
Published on: November 6, 2017
Possible involvement of mitochondrial uncoupling protein-2 in cytotoxicity mediated by acquired N-methyl-D-aspartate
Ryo Fukumori1, Takeshi Takarada, Yuki Kambe
1Laboratory of Molecular Pharmacology, Division of Pharmaceutical Sciences, Kanazawa University Graduate School of Natural Science and Technology, Kanazawa, Ishikawa 920-1192, Japan.
Abstract:
We have previously shown the possible involvement of mitochondrial membrane potential disruption in the mechanisms underlying the neurotoxicity seen after activation of N-methyl-d-aspartate (NMDA) receptors (NMDAR) in primary cultured rat hippocampal neurons. In this study, we attempted to demonstrate a pivotal role of mitochondrial uncoupling protein-2 (UCP2) as a determinant of the NMDA neurotoxicity by using acquired NMDAR channels artificially orchestrated in HEK293 cells. In cells with overexpression of UCP2, immunoreactive UCP2 was exclusively detected at intracellular locations stained with the mitochondrial marker MitoTracker. In cells with acquired NMDAR channels, exposure to either NMDA or the calcium ionophore A23187 similarly led to a significant increase in cytosolic Ca(2+) levels determined by Fluo-3 imaging irrespective of the overexpression of UCP2. By contrast, NMDA, but not A23187, was significantly more effective in increasing mitochondrial Ca(2+) levels determined by Rhod-2 fluorescence imaging in cells transfected with NMDAR subunit and UCP2 expression vectors than in those without UCP2 overexpression. Overexpression of UCP2 significantly increased the number of cells stained with propidium iodide in cultures with acquired NMDAR channels, but failed to significantly affect that in cells exposed to A23187. Immunocytochemical and immunoprecipitation analyses similarly revealed the possible interaction between GluN1 subunit and UCP2 in HEK293 cells with acquired NMDAR channels and UCP2 overexpression. These results suggest that UCP2 could play a role as a determinant of the neurotoxicity mediated by NMDAR through a mechanism related to the unidentified interaction with the essential GluN1 subunit toward modulation of mitochondrial Ca(2+) levels in neurons.
Insights
Mitochondrial uncoupling protein-2 (UCP2) may determine N-methyl-d-aspartate receptor (NMDAR) neurotoxicity. UCP2 influences mitochondrial calcium levels and cell death, potentially interacting with the GluN1 subunit.
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- N-methyl-d-aspartate receptors (NMDARs) activation can cause neurotoxicity, potentially involving mitochondrial dysfunction.
- Mitochondrial uncoupling protein-2 (UCP2) is implicated in cellular energy regulation and stress responses.
Purpose of the Study:
- To investigate the role of UCP2 in NMDAR-mediated neurotoxicity.
- To determine if UCP2 modulates mitochondrial calcium levels and cell death following NMDAR activation.
Main Methods:
- Utilized HEK293 cells with artificially expressed NMDAR channels and UCP2.
- Measured cytosolic and mitochondrial calcium levels using Fluo-3 and Rhod-2 imaging, respectively.
- Assessed cell viability using propidium iodide staining and analyzed protein interactions via immunocytochemistry and immunoprecipitation.
Main Results:
- Overexpression of UCP2 enhanced NMDA-induced increases in mitochondrial calcium, but not cytosolic calcium.
- UCP2 overexpression increased cell death in response to NMDA, but not to a calcium ionophore.
- Evidence suggests an interaction between the NMDAR GluN1 subunit and UCP2.
Conclusions:
- UCP2 plays a role in determining NMDAR-mediated neurotoxicity.
- UCP2 may influence neurotoxicity by modulating mitochondrial calcium levels, potentially through interaction with the GluN1 subunit.
