Biochemical and Biophysical Research Communications
A deficiency of the GluN2C subunit of the N-methyl-D-aspartate receptor is neuroprotective in a mouse model of ischemic stroke
Graphical abstract
Absence of the GluN2C subunit from the N-methyl-D-aspartate pool of ion channels diminished neuroedema in a murine model of ischemic stroke. The penumbral neurons of the core infarct of the GluN2C-/- mice showed diminished cytoskeletal disruption, decreased tauopathy, and increased phosphorylation of the pro-survival CREB molecule compared to WT mice. Thus, the GluN2C subunit impairs stroke-mediated neurological deficits, thereby, making the GluN2C subunit an attractive therapeutic target.
Introduction
The N-methyl-D-aspartate receptor (NMDAR) is an ionotropic receptor that plays a critical role in the mammalian central nervous system. Physiologically, this receptor is co-agonized by Glutamate (Glu) and Glycine (Gly), and its activation regulates synaptic activity and pro-survival pathways important for the long-term potentiation required for learning and memory [1]. However, hyperactivation of the NMDAR is centrally involved in several neurological disorders, including ischemic stroke. During ischemic stroke, release of excess glutamate causes preferential hyperactivation of the extrasynaptic NMDAR with influx of excessive levels of intracellular Ca2+, that causing inactivation of the cAMP response element-binding protein (CREB) and neuronal cell death [2].
The functional heterotetrameric NMDAR is composed of two Gly-binding (GluN1) and two Glu-binding (GluN2) subunits. The GluN1 subunits (GluN1a-h) are splice variants of a single gene, and the GluN2 subunits (GluN2A, 2 B, 2C, 2D) are separate gene products [3]. A third subunit class, GluN3 (A/B), binds to Gly and can co-assemble with the GluN1/GluN2 subunits to form a functional triheteromeric complex [4]. Expression of these subunits is temporally, developmentally, and regionally regulated. Since, ion channels formed by each of the GluN2 subunits in the NMDAR display disparate biochemical, pharmacological, and electrophysiological properties, the type of GluN2 subunit present in NMDAR ion channels dictates its functional properties, and, consequently, its involvement in different neurological diseases [5].
Previous studies have reported that ischemia was attenuated in GluN2A-/- and GluN2A-/-/GluN2B+/- mice compared to WT mice [6]. Additionally, selective pharmacological blocking of the GluN2B subunit resulted in decreased ischemic infarcts [7]. Apart from the role of GluN2C in psychogenetic disorders, such as fear acquisition, working memory [8], schizophrenia [9], and seizure generation [10], few studies have investigated the mechanistic role of GluN2C subunit during ischemia. One brief report demonstrated a 50–60% reduction in cerebral infarct volume 24 h after ischemia in GluN2C-/- mice compared to WT mice [11]. However, no mechanistic insights regarding attenuation of stroke in the GluN2C-/- mice was provided. Conversely, another study determined that the GluN2C subunit played a neuroprotective role in ischemic excitotoxicity [12]. Thus, published data regarding the role of GluN2C in ischemic stroke is conflicting.
In this study we sought to determine the contribution of GluN2C subunit in stroke, which is expressed in the retrospinal cortex, hippocampus, striatum, thalamus, olfactory bulb, and cerebral cortex [13]. The current study summarizes the results of an investigation of the effects of ablation of the GluN2C subunit in the brain using an ischemic/reperfusion stroke model in GluN2C-/- mice. We show that ischemic neuroedema, neurological and physiological recovery, and cytoarchitecture, are influenced by the presence of the GluN2C subunit.
Section snippets
Mice
Male (to minimize experimental variance due to gender) wild-type (WT) and GluN2C knockout/nβ-galactosidase knockin mice (GluN2C-/-) (NIH) in a C57BL/6 background and >F7, were used to induce stroke [13]. The mice were housed on a 12 h light/dark cycle with ad libitum access to food and water. All animal procedures were performed in accordance with the University of Notre Dame animal care committee's regulations, Protocol number 14–086.
Ischemic stroke induction
For induction of MCAO, the mice (23–27 g) were anesthetized
GluN2C-/- mice show attenuated neurological deficits, cerebral edema, and weight loss
An average of neurological scores showed that deficits between WT and GluN2C-/- mice at 24 h post-MCAO were similar (p = 0.79). However, at 72 h post-MCAO, GluN2C-/- mice showed significantly lower neurological scores compared to WT mice, which was indicative of recovery from ischemia compared to WT mice (Fig. 1A). Similarly, the average weight loss of GluN2C-/- mice was substantially lower than that of WT mice at 72 h post-MCAO (Fig. 1B). No significant differences in infarct size between WT
Discussion
The role of GluN2C subunit in ischemic stroke was investigated, as the available literature on this subunit is sparse and the data are conflicting. We demonstrated that the GluN2C subunit plays a differential role in the pathology of ischemic stroke. The absence of GluN2C did not significantly decrease infarct size, but the extent of neuroedema was reduced by 45% compared to stroked WT mice. Additionally, GluN2C-/- mice showed fewer perturbations in neuronal integrity and increased pro-survival
Disclosure
None declared.
Conflicts of interest
This manuscript, in part or in entirety is not under consideration by any other journal. The animal study has been reviewed and approved by the ethics committee of the University of Notre Dame. All authors have approved the manuscript and have agreed to the submission. Authors report no conflict of interest.
Acknowledgements
We acknowledge Ms. Juan Fu for genotyping the animals.
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