NMDA receptor-dependent glutamate excitotoxicity in human embryonic stem cell-derived neurons

Highlights • Human embryonic stem cell derived neurons develop NMDA and AMPA receptors in culture.• Enriched HESC-derived neurons develop functional glutamate responses in vitro.• MK 801 confers a protective effect on glutamate-induced excitotoxic neuronal death.• Studies using human pluripotent stem cells may aid the discovery of novel therapeutic targets.


Introduction
Glutamate excitotoxicity, both acute and chronic, has been implicated widely in the pathogenesis of a range of neurological diseases, including Alzheimer's disease, Huntington's disease and traumatic brain injury [25,28]. Knowledge of the role and mechanisms of glutamate excitotoxicity can potentially enhance our understanding of key downstream signalling processes that mediate neuronal death and identify novel therapeutic targets. A major cause of glutamate excitotoxicity is activation of the NMDA subtype of glutamate receptor (NMDAR), which mediates Ca 2+dependent cell death [26]. Most NMDARs contain two obligate GluN1 subunits plus two GluN2 subunits [11], of which there are four subtypes, GluN2A-D, with GluN2A and GluN2B predominant in the forebrain [30,40]. While physiological patterns of synaptic NMDAR activity have the potential to be neuroprotective [15,19,39], excessive or inappropriate activity is harmful. The work of many laboratories has advanced our understanding of how loss of glutamate homeostasis, ionic imbalance and inappropriate NMDAR activity can contribute to degenerative disorders [8,16,22,25,27]. Antagonism of pathological glutamate-signalling in rodent models of neurological diseases has yielded promising results [6,10], though unsuccessful clinical trials highlight the need for further studies [1,31]. Currently, excitotoxic neuronal cell death has primarily been characterised in rodent systems. Advances in human pluripotent stem cell (HPSC) technology permit the generation of enriched neurons of differing positional identities as well as astrocytes from human embryonic stem cells (HESC) and allow investigation of cell injury and rescue paradigms in a human in vitro system [12,13,23,24,35,38]. In light of the inter-species differences between rodent and human [34], studies utilising human in vitro systems may prove informative in interpreting the relevance of findings in rodent studies to the human pathological condition [3,13,18].
Here, we describe the functional maturation of HESC-derived neurons and the development of glutamate-responsiveness, in order to study neuronal responses to pathologically relevant glutamate concentrations [5].
Calcium-imaging: The day before imaging, HESC-derived neurons were removed from their trophic differentiation medium into a trophically deprived, glutamate-free minimum medium as described above. HESC-neurons, identified by morphology and confirmed by TuJ1 immunostaining, were imaged at various timepoints in artificial cerebrospinal fluid (aCSF) [39] on a 37 • C-heated stage. Cells were loaded with 11 M Fluo-3 for 30 min and washed in aCSF. Coverslips were mounted in a perfusion chamber. Fluo-3 fluorescence images (excitation 488 nm/emission 520 nm) were taken at baseline and during saturating glutamate (200 M) and NMDA (150 M) stimulation. Glycine (100 M) was applied as a co-agonist. D-APV (50 M) was used as an NMDAR antagonist. For calibration, ionomycin was used to saturate the Fluo-3 signal (50 M), followed by MnCl 2 (10 mM) to quench. The quenched Fluo-3 signal corresponds to ∼100 nM Ca ++ . The K d of Fluo-3 was taken as 315 nM and free Ca ++ concentration was calculated by the formula Ca ++ = K d (F − F min )/(F max -F), where F is fluorescence. Images were taken every second for the duration of each experiment (∼10 min) [17].
Neuronal injury and Cell viability assays: The day before application of the excitotoxic insult, HESC-derived neurons were removed from their trophic differentiation medium into a trophically deprived, glutamate-free minimal medium. Neurons were treated with glutamate with and without MK801 (10 M) [29]. 24 h after excitotoxic challenge, cell viability was quantified using CellTiter-Glo Luminescent Cell Viability Assay (Promega), following manufacturer's instructions. Neuronal injury experiments using high-dose NMDA (150 M) [29], were performed in magnesiumfree SGG medium [2]; cell viability was quantified after 24 h by the same protocol.
Data analysis: All experiments were performed with at least 3 biological repeats; within each, 3 technical repeats were generated. qPCR data were normalised to GAPDH. Data are presented as mean ± standard error of the mean; statistical analyses were performed in Microsoft Excel and GraphPad Prism 5 (GraphPad Software, Inc., San Diego) by univariate and two-way ANOVA with Newman-Keuls post-test and two-tailed Students' t-test. P < 0.05 was considered statistically significant.

Temporal profile of in vitro acquisition of HESC-derived neuronal glutamate responses
In order to study excitotoxic responses in a human platform, enriched cultures of HESC-derived neurons were examined for functional glutamate-mediated responses. Neurons were stimulated with saturating glutamate (200 M) and functional glutamate receptor activity was determined by calcium-imaging over increasing duration in culture. While initially unresponsive to glutamate (including doses of 1 mM glutamate: data not shown), HESCderived neurons developed calcium currents in response to bath-extracellular glutamate application with increasing duration in culture. 2-week old HESC-derived neurons demonstrated no significant calcium influx, while later time points demonstrated significant calcium influx with glutamate treatment, with peak calcium currents increasing over time to maximal at 6 and 8-weeks duration in culture (p < 0.001, Fig. 1a). The proportion of HESC-derived neurons that exhibited glutamate-evoked calcium currents similarly showed a time-dependent increase from 13 ± 3% at 2-weeks and 15 ± 1% at 3-weeks, 54.4 ± 5% at 4-weeks, to 77.7 ± 4% and 73.0 ± 3% at 6 and 8-weeks respectively (p < 0.001, Fig. 1b). Thus, HESC-derived neurons acquired functional glutamate responsiveness over time in culture.

Development of functional glutamate responses coincides with expression of NMDA and AMPA receptor subunits
In order to determine if the temporal emergence of glutamateresponsiveness was due to increasing expression of glutamate receptors, we quantified the expression of NMDAR and AMPAR subunits in glutamate-unresponsive neurons at 2-weeks and in glutamate-responsive neurons at 6 and 8-weeks by qRT-PCR.
The NMDAR and AMPAR profiles were dynamic over the course of the period studied, with increasing expression of AMPAR and NMDAR subunits GluN1 and GluN2B coinciding with the development of functional glutamate responses in vitro. Given that during development rodent neurons undergo a transition from GluN2B subunit-containing NMDARs to GluN2A predominance [41], the NMDAR subunit profile in HESC-derived neurons may change further over time.

HESC-derived neurons express functional NMDA receptors
In order to determine whether the glutamate-induced calcium influx was dependent on the expression of functional NMDARs, receptor-mediated calcium currents were quantified in 8-week HESC-derived neuronal cultures using NMDA-glycine co-treatment. Data demonstrated that HESC-derived neurons exhibited calcium currents upon NMDA application, which were blocked with application of D-APV, a selective NMDAR inhibitor (p < 0.05, Fig. 2a). NMDAR currents were further confirmed by patch-clamp electrophysiology (Fig. 2b).

HESC-derived neurons demonstrate excitotoxic cell death at pathophysiological glutamate concentrations, and are rescued by MK801-mediated NMDA receptor blockade
The susceptibility of 7-week-old HESC-derived neurons to glutamate-induced cell death was determined over a range of concentrations. With increasing glutamate concentrations, increasing cell death was observed: at 20 M, 58% ± 5.98%; at 40 M, 72 ± 6.45%; and at 80 M, 71 ± 6.64% cell death was noted (Fig. 2c). 100% cell death was induced with application of 50 M staurosporine. As 20 M glutamate is in the pathophysiological range for extracellular glutamate concentration in human traumatic brain injury patients [5], MK801 was assayed against this concentration.
In vitro studies on rodent neurons demonstrated the efficacy of NMDAR antagonists in preventing excitotoxic neuronal death [6]. To investigate the role of NMDARs in glutamate excitotoxicity in our system, enriched HESC-derived neuronal cultures were exposed to 20 M glutamate in the presence and absence of MK801 (10 M) for 24 h. MK801 conferred a significant neuroprotective effect against glutamate-induced excitotoxic injury (33.2 ± 8.4% cell death compared to control: 57.5 ± 3.4%; p < 0.05, Fig. 2d). When challenged with 150 M NMDA, neuronal death was also observed (27.7 ± 3.6%; p < 0.001, Fig. 2e), however at lower levels than those induced by glutamate. Further work is required to elucidate the downstream signalling pathways that mediate glutamate and NMDA-induced excitotoxic neuronal dysfunction and death. Establishment of a robust platform for functional glutamate transmission and excitotoxicity in human neurons permits examination of these downstream pathways and could potentially aid the discovery of novel therapeutic targets.

Discussion
Pathological stimulation of NMDAR by glutamate has been implicated in excitotoxic injury and channel blockers have been trialled in human conditions to poor effect [20], suggesting that our understanding of human NMDAR-signalling is incomplete and reliable human-based experimental models could be informative, alongside animal studies. However, the development of glutamatedependent currents was previously uncharacterised in enriched HESC-derived neurons in vitro.
In these studies, we demonstrate that HESC-derived neurons, within the context of the current experimental platform, acquired maturation-dependent glutamate-responsiveness over time in culture; knowledge of these characteristics is critical for the use of HESC-neural derivatives in models of neurological injury. By mRNA expression studies, there were large increases in transcription of GRIN1 encoding the constitutive NMDAR1 subunit, GRIA1 encoding the AMPAR1 subunit, and modest increases in further NMDAR and AMPAR subunit genes. These modest transcriptional increases translated to the expression of functional and properly assembled NMDARs as determined by functional studies herein, utilising NMDA as a specific agonist and the selective inhibitor D-APV. Furthermore, quantitative studies of current amplitude and the proportion of glutamate-responsive cells demonstrated gradual increase of receptor function over duration in culture, thus suggesting neuronal-intrinsic modulation of glutamate-receptor expression and functional downstream-signalling over time. Direct confirmation of NMDAR and AMPAR subunit profiles could be elucidated further by electrophysiological studies with appropriate subunit-specific inhibitors. The mRNA expression data also suggested that NMDAR subunit expression was dynamic over the in vitro time course, with GluN2B predominance. It remained to be seen whether the subunit profile would develop over time to recapitulate the conversion from GluN2B to GluN2A predominance in experimental mammalian systems [41].
In this study, glutamate was investigated as a physiologically relevant stressor due to its direct pathophysiological role in disease states. Dose-survival curves demonstrated that HESC-derived neurons underwent glutamate-induced cell death over the range of concentrations seen in human brain injury patients in vivo [5], and could be rescued with MK801 co-administration. Similar concentrations of glutamate and MK801 have also been demonstrated to mediate excitotoxic cell death and protection in rodent in vitro studies [7,37], and in human neurons derived from the human NT2 teratocarcinoma cell-line [14,32].
Interestingly, MK801 did not completely block glutamatemediated neuronal death and, furthermore, NMDA alone induced a lower level of neuronal death than that mediated by glutamate. These findings suggest other mechanisms of glutamate-induced neuronal death in addition to NMDAR-mediated excitotoxicity. Glutamate in high concentrations has been demonstrated to cause oxidative-stress mediated neuronal death by competitively inhibiting cystine uptake via the Xct cystine/glutamate antiporter and impairing glutathione production [33,37]. While the concentrations used to experimentally induce glutamateinduced oxidative cell death are typically in the millimolar range [36], excitotoxic neuronal death at lower glutamate concentrations, mediated by NMDAR activation, has been shown to result in further glutamate release and accumulation in medium to higher concentrations capable of mediating oxidative neuronal death [37]. Calcium-permeable AMPARs, lacking the GluR2 subunit that prevents calcium permeability, have also been demonstrated to mediate hippocampal neuronal death in rodent slice cultures. Oxygen-glucose-deprivation increases synaptic-trafficking of GluR2-lacking AMPARs in CA1 hippocampal neurons, causing AMPAR mediated excitotoxic cell death [9]. The pre-exposure of HESC-neurons to trophically-deprived medium in the current experimental model may have affected the trafficking of AMPAR and partially accounted for the MK801-insensitive excitotoxic neuronal death observed.

Conclusions
Human pluripotent stem cell technology, including HESCs and iPSCs, has tremendous potential for modelling neurological disease processes. Technological advances have allowed the generation of enriched human neurons and glia under defined conditions, permitting study of human neural cells in vitro [13,24,35]. Here, we characterised a population of enriched HESC-derived neurons in terms of development of glutamatergic responses and investigated their utility in modelling glutamate-induced neuronal injury. We established that after 2 months in culture, HESC-derived neurons underwent excitotoxic cell death at concentrations of glutamate found in the brains of human patients suffering acquired brain injury, and thus have the potential to recapitulate aspects of human pathophysiology. Furthermore, insights from the development and refinement of HESC-based platforms for in vitro modelling of neuronal injury and rescue will be critical for the development of other HPSC-based complementary systems, including iPSCs.