3.1. Functional expression of P2X7Rs in hiPSC-derived neurons
In order to assess the functional expression of P2X7Rs and if targeting of these receptors reduces hyperexcitability in a human brain-relevant cellular model, we differentiated neurons from two hiPSC lines (Kesavan et al., 2023).
As a first approach, immunocytochemistry was performed on cells at 18-22 days after plating NSCs using two different P2X7R antibodies and the neuronal marker β-III tubulin. Of note, while the β-III tubulin antibody produced a more diffuse staining, the P2X7R antibodies showed the expected punctate staining in the neuronal soma and along the processes, demonstrating the presence of P2X7Rs on hiPSC-derived neuronal soma and processes (Figure 1a-c ).
To complement the immunocytochemistry findings, we next sought to assess whether these human neurons respond to the application of P2X7R-stimulating agonists. Previous studies have shown that the activation of the P2X7R requires high (mM range) concentrations of ATP. The P2X7R is, however, 10-30 times more sensitive to the ATP analog and non-selective P2X7R agonist, 2′,3′-O-(4-benzoylbenzoyl)-ATP (BzATP) (Donnelly-Roberts et al., 2009). To assess the functional expression of P2X7Rs, hiPSC line 1-derived neurons grown on coverslips without glial monolayer were loaded with 2 μM Cal-520 AM calcium-sensitive fluorescent dye to eliminate unspecific fluorescence from glia. The application of the P2X7R agonist BzATP (300 μM) resulted in a discernable increase in calcium fluorescence in hiPSc line 1-derived neurons (Figure 2a ). Fluorescence rose quickly in response to a pulse of 300 μM BzATP for 5 s signifying a persistent rise in [Ca2+]i during constant agonist exposure. In contrast, when the cells were pre-incubated with the P2X7R antagonist AFC-5128 (Fischer et al., 2016; Kesavan et al., 2023) (30 nM) pulse ejection of 300 μM BzATP and 30 nM AFC-5128, changes in [Ca2+]i were strongly reduced (BzATP 0.0819 ± 0.0060, BzATP and AFC-5128 0.0127 ± 0.0050; p < 0.0001). The peak amplitude of the individual ΔF/F0 traces upon BzATP application was reduced when stimulated in the presence of AFC-5128 (BzATP 0.2086 ± 0.0362, BzATP and AFC-5128 0.0975 ± 0.024;p < 0.0001) (Figure 2b ). Similar results were evident when the area under ΔF/F0 curve (AUC) of individual ΔF/F0 traces were compared (BzATP 2.170 ± 0.2276, BzATP and AFC-5128 0.5178 ± 0.1307;p < 0.0001) (Figure 2c ), suggesting BzATP-evoked responses are mediated mainly via the P2X7R.
To further confirm the functional expression of P2X7Rs, we repeated the BzATP application in the presence of another highly-specific P2X7R antagonist, JNJ-47965567. Similar to AFC-5128, BzATP-evoked [Ca2+]i signals were also reduced in the presence of a second P2X7R antagonist JNJ-47965567 (BzATP 0.1089 ± 0.0134, BzATP and JNJ-47965567 0.0209 ± 0.0004; p < 0.000) (Figure 2d ). The peak amplitude of the individual ΔF/F0 traces upon BzATP application was reduced when stimulated in the presence of JNJ-47965567 (BzATP 0.6528 ± 0.0428, BzATP and JNJ-47965567 0.0460 ± 0.0064, mean ± SEM; p < 0.0001) (Figure 2e ). As before, similar results were evident when the area under ΔF/F0 curve of individual ΔF/F0 traces were compared (BzATP 2.808 ± 0.1811, BzATP and JNJ-47965567 0.0777 ± 0.0114;p < 0.0001) (Figure 2f ).
We further confirmed the expression of P2X7Rs using hiPSC line 2-derived neurons. Bz-ATP evoked Ca2+ influx was also blocked by AFC-5128 (BzATP 0.0592 ± 0.0038, BzATP and AFC-5128 0.0035 ± 0.0011) (Figure 2g ). The peak amplitude of the individual ΔF/F0 traces upon BzATP application was reduced when stimulated in the presence of AFC-5128 (BzATP 0.1359 ± 0.0233, BzATP and AFC-5128 0.0674 ± 0.0086; p < 0.0111) (Figure 2h) . Similar results were obtained when the area under ΔF/F0 curve of individual ΔF/F0 traces were compared (BzATP 1.367 ± 0.2385, BzATP and JNJ-47965567 0.4862 ± 0.0849; p = 0.0007) (Figure 2i ). BzATP-evoked Ca2+ influx was also blocked by JNJ-47965567 (BzATP 0.0241 ± 0.0035, BzATP and JNJ-47965567 0.0018 ± 0.0008; p < 0.0001) (Figure 2j ). The peak amplitude of the individual ΔF/F0 traces upon BzATP application was reduced when stimulated in the presence of JNJ-47965567 (BzATP 0.2110 ± 0.0299, BzATP and JNJ-47965567 0.0631 ± 0.0115; p = 0.0002) (Figure 2k ). A comparison of area under ΔF/F0 curve of individual ΔF/F0 traces reveals a significant reduction in the presence of JNJ-47965567 (BzATP 1.584 ± 0.2567, BzATP and JNJ-47965567 0.5119 ± 0.1265; p = 0.0002) (Figure 2l ). These results demonstrate the functional expression of P2X7Rs in two lines of hiPSC-derived neurons.
3.2. Antagonism of P2X7Rs alleviates PTX-induced chronic epileptiform-like events under inflammatory conditions
Pharmacological targeting of P2X7Rs have repeatedly been proven effective in rodent models of epilepsy (Engel et al., 2012; Jimenez-Pacheco et al., 2016; Jimenez-Pacheco et al., 2013; Mamad et al., 2023). The effects of P2X7R antagonism on hyperexcitability in a human neuronal network has, however, not been reported to date. For this, we first developed an in vitro model consisting of hiPSC-derived neurons differentiated on human primary astrocytes. When co-cultured with astrocytes, functional maturation of hiPSC-derived neurons has been shown to be improved (Hedegaard et al., 2020; Johnson et al., 2007).Here, network perturbations leading to epileptiform-like bursts were induced via the application of PTX (100 µM), a non-competitive antagonist of inhibitory γ-aminobutyric acid (GABAA) receptors (Figure 3a, b). Loose patch-clamp experiments were performed to assess network excitability. As depicted by exemplary traces and quantifications, loose patch-clamp experiments showed that treatment with PTX for 10 min increased the burst frequency from 0.0558 ± 0.0146 Hz at baseline to 0.0925 ± 0.0149 Hz; p = 0.0358) post-treatment (Figure 3c). Similarly, calcium imaging experiments using Cal-520 loaded neurons also revealed PTX induced an increase in network activity and synchronisation, hallmarks of seizures (Figure 3d, e ).
We next assessed the effect of P2X7R antagonism in this model (Figure 4a). Comparison of burst parameters during PTX application vs. co-application of PTX and AFC-5128 revealed no significant difference in burst frequency ([F(2,11) = 1.79; p = 0.2053)]: baseline 0.0800 ± 0.0277, PTX 0.1421 ± 0.044, PTX and AFC-5128 0.0995 ± 0.0227 Hz), number of spikes in burst (([F(2,11) = 1.79; p = 0.2053)]: baseline 19.65 ± 2.62, PTX 19.17 ± 3.551, PTX and AFC-5128 24.94 ± 4.708; burst duration ([F(2,11) = 1.327; p = 0.2857)]: baseline 1025 ± 202.9, PTX 1209 ± 220.7, PTX and AFC-5128 1367 ± 299.5 ms) and interburst interval ([F(2,11) = 1.365; p = 0.2725)]: baseline 20701 ± 5737, PTX 12334 ± 2778, PTX and AFC-5128 17696 ± 4797 ms) (Figure 4b-e). Similar results were observed with the P2X7R antagonist JNJ-47965567. Comparison of burst parameters during PTX application vs. co-application of PTX and JNJ-47965567 revealed no significant difference in burst frequency ([F(2,6) = 0.1479; p=0.4297)]: baseline 0.0477 ± 0.01258, PTX 0.0692 ± 0.01705, PTX and JNJ-47965567 0.06347 ± 0.02547 Hz), number of spikes in burst ([F(2,6) = 1.696; p = 0.2322)]: baseline 31.56 ± 8.213, PTX 59.16 ± 22.14, PTX and JNJ-47965567 26.93.3 ± 9.223), burst duration (baseline 1179 ± 272.6, PTX 1562 ± 281.1, PTX and JNJ-47965567 1230 ± 526.2 ms) and interburst interval ([F(2,6) = 1.606; p = 0.1916)]: baseline 15.03± 4.294, PTX 10.19 ± 2.126, PTX and JNJ-47965567 19.90± 7.103 s) (Figure 4f-i).
The above findings indicate that P2X7R antagonism is not sufficient to prevent acute epileptiform-like activity evoked by PTX in this human model. We hypothesized this may because of the absence of any background inflammatory tone. To test this idea, we adapted the model to more closely reflect chronic inflammatory conditions by co-treating the PTX-exposed hiPSC-derived neuronal networks with a cocktail of pro-inflammatory agents (TNF-α, IL-1α, IL-1β and C1q) for 7 to 12 days (Figure 4j). Previous data have shown that treatment with these agents leads to a neuroinflammatory phenotype in hiPSC-derived neuronal networks (Hyvarinen et al., 2019; Stoberl et al., 2023). Loose patch-clamp recordings were performed in the presence of PTX alone or PTX and AFC-5128 from the neuronal cultures treated with PTX and pro-inflammatory agents. In contrast to results from our acute settings, burst frequency was significantly reduced in the presence of AFC-5128 (PTX 0.1012 ± 0.0156, PTX and AFC-5128 0.0662 ± 0.0159 Hz;p = 0.0342) (Figure 4k) illustrating that P2X7R antagonism alleviates hyperexcitability in a chronic model of epileptiform-like events under inflammatory conditions. Moreover, the mean number of spikes in burst (PTX 19.20 ± 2.771, PTX and AFC-5128 14.95 ± 2.542 Hz;p = 0.1688) and burst duration (PTX 1404 ± 274.4, PTX and AFC-5128 1264 ± 209.5 ms; p = 0.1688) were slightly reduced in the presence of AFC-5128, however, this did not reach statistical significance (Figure 4l, m). Finally, mean inter-burst-intervals (IBI) were also increased following AFC-5128 application (PTX 10.61 ± 2.370, PTX and AFC-5128 40.87 ± 16.28 s; p = 0.0479) (Figure 4n).
Next, we used histograms and kernel density plots to delineate IBI subpopulations Figure 4o-q). After the addition of AFC-5128, a distinct peak appeared (indicated by arrow in Figure 4p) at longer IBIs indicating AFC-5128 increases successive intervals between bursts. Furthermore, the shape and scale parameters of the distribution of the IBIs were measured by fitting a gamma distribution to the histogram (Figure 4r, s and Table 1). We found that the mean shape parameter of the gamma distribution of IBI was 0.794 in PTX-treated group and 0.482 in PTX and AFC-5128 co-treated group. Similarly, the mean scale parameter from the gamma distribution was 8.722 in PTX-treated group but increased to 31.048 in PTX and AFC-5128 co-treated group demonstrating increase in longer intervals between bursts when AFC-5128 was co-applied. Comparison of the cumulative frequency distributions of PTX or PTX and AFC-5128 treated groups also revealed increase in IBI after AFC-5128 application (Figure 4t; p = 0.0198). These results provide additional confirmation that AFC-5128 increases the IBI in chronic inflammatory model of epileptiform-like events.