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Targeting Purinergic Pathway in drug-resistant epilepsy using human Neurons and Glia.

Periodic Reporting for period 1 - TaPPiNG-EPI (Targeting Purinergic Pathway in drug-resistant epilepsy using human Neurons and Glia.)

Reporting period: 2019-04-01 to 2021-03-31

Issue: Epilepsy is a neurological disorder characterized by recurrent seizures affecting affects around 65 million people worldwide and antiseizure drugs (ASDs) are routinely used to control seizures as a first line of treatment. Despite the development of many new ASDs in recent years, approximately one-third of all epilepsy cases are refractory to the existing anticonvulsants, even in combination therapy involving ASDs with different mechanisms of action.

Social Impact: Given that approximately 30% of patients do not respond to available anti-seizure drugs, identification of a new pharmacological target will help to address such a significant clinical problem and therefore would have important impacts with respect to tailoring patient treatments to their needs and enhancing quality of life. The validation of a novel potential pharmacological target may prove efficient in a group of patients for whom invasive surgery is currently the only treatment choice.

Objective: P2X7Rs have emerged as a potential pharmacological target to treat hyperexcitabily in animal models of brain diseases. To advance findings from animal models towards the clinical use of drugs targeting P2X7Rs for epilepsy, this project aims at using human induced pluripotent stem cell (hiPSC)-derived neurons and glia as human brain-relevant cellular models to identify a new potential pharmacological target.

Conclusion: The results of this project demonstrates that i) human iPSC-derived cortical neurons and glia express functional P2X7Rs ii) establishment of an in vitro seizure model suitable for pharmacological targeting of P2X7 receptors.
1) Differentiation of forebrain cortical neurons and astrocytes from hiPSC-derived primitive neural stem cells:
We differentiated neural stem cells (NSCs) from hiPSCs. For neuronal differentiation, NSCs were plated in neuronal differentiation medium. At 14 days in vitro, cells exhibited neuronal morphology and were positive for the neuron-specific cytoskeletal marker β-III-tubulin and forebrain marker FOXG1. For the differentiation of astrocytes, dissociated NSCs were plated in an astrocyte differentiation medium and immunocytochemical analysis at passage 7 confirmed the expression of the astrocyte marker S100 β in cells maintained in astrocyte differentiation medium (Figure 1).

2) In vitro differentiated hiPSC-derived cortical neurons exhibit fundamental electrophysiological properties.
The functional assessment of the differentiated neurons were performed using patch-clamp recordings from individual neurons. The recordings demonstrated the presence of fast activating and inactivating inward currents and slow and sustained outward currents resembling voltage-gated Na+ (Nav) and K+ (Kv) channels. Whole-cell voltage clamp recordings revealed spontaneous postsynaptic currents (sPSCs) at 21-28 days in vitro differentiation (Figure 2).

3. Expression of P2X7Rs in hiPSC-derived neurons and astrocytes
To investigate if P2X7Rs are expressed in hiPSC-derived neurons, immunohistochemistry was performed on neurons and astrocytes. In double labelling experiments using the neuronal marker β-III tubulin and P2X7-specific affinity purified antibody, expression of P2X7Rs was evident by a punctate staining pattern. Likewise, to assess the expression of P2X7Rs on hiPSC-derived astrocytes, double immunostaining was performed on astrocytes at 4 weeks in vitro using antibodies against the astrocyte marker GFAP and P2X7 (Figure 3).

4. BzATP evokes AFC-5128-sensitive Ca2+ transients in hiPSC-derived neurons
To confirm the results obtained using immunocytochemistry, we next sought to assess whether P2X7Rs also respond to the application of P2X7-stimulating agonists. During appication of 300 μM BzATP, hiPSc-derived neurons responded to application by a discernable increase in fluorescence. In contrast, when the cells were pre-incubated with the P2X7R antagonist AFC-5128 (30 nM) pulse ejection of 300 μM BzATP and 30 nM AFC-5128 the change in [Ca2+]i was significantly reduced. Taken together, these results show that the BzATP-gated change in [Ca2+]i in hiPSC-derived neurons is mediated via the P2X7R (Figure 4).

5. BzATP evokes AFC-5128-sensitive Ca2+ transients in iPSC-derived astrocytes
Similar to neurons, we then assessed the functional expression of P2X7Rs in hiPSC-derived astrocytes by monitoring changes in [Ca2+]i upon BzATP application. To assess the functional expression of P2X7Rs, hiPSC-derived astrocytes grown on coverslips were loaded with 2 μM Cal-520 AM. Pulse ejection of BzATP (300 μM) for 5 s evoked an increase in [Ca2+]i in of hiPSC-derived astrocytes.
In support of the role for the P2X7R in mediating this response, when astrocytes were pre-incubated with the P2X7R antagonist AFC-5128, co-application of BzATP with AFC-5128 (30 nM) significantly reduced the [Ca2+]i response.

Plans for exploitation and dissemination of results: To explore the potential of the research findings, and the use of in vitro seizure model using human iPSC-derived neuron and glia as preclinical cellular models neurological drug discovery industry will be further exploited with support from the RCSI Office of Research and Innovation and Enterprise Ireland –funded activities. The market survey/feasibility study is already underway with funding from Enterprise Ireland to explore such a possibility. If a potential market is identified in this feasibility study, efforts to establish an RCSI spin-off focused on a platform for drug screening and toxicity testing using human brain-relevant cellular models of disease will be initiated.

The research findings have been submitted for publication and is under revision in an open access journal (bioRxiv doi: 10.1101/2021.03.28.437391
https://biorxiv.org/cgi/content/short/2021.03.28.437391v1)
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Despite the potential benefits of pharmacologically targeting P2X7Rs in brain diseases, and accumulated data on mRNA expression in human brain tissue, if functional P2X7Rs are expressed in human neurons and glia remains elusive. TaPPiNG-EPI demonstrates the functional expression of P2X7Rs in the hiPSC-derived neurons and glia.

Economy: Developing & investigating hiPSC-derived cellular model of epilepsy has both academic value and commercial potential. The global market for iPSCs is projected to reach US$2.4 billion by the year 2027, trailing a post COVID-19 CAGR of 6.6%, over the analysis period 2020 through 2027. (Source: Global Industry Analysts, Inc., Report ID: 4805485, April 2021). The in vitro seizure model using human iPSC-derived neuron and glia used in this project as preclinical cellular models is of high demand in neurological drug discovery industry and can be further exploited with support from the RCSI Office of Research and Innovation and Enterprise Ireland–funded activities.

Wider societal implications: Targeting of the P2X7Rs has been suggested as possible treatment avenue not only fin epilepsy but also for numerous pathological diseases of the central nervous system including the most common comorbidities associated with epilepsy (e.g. depression, schizophrenia). Furthermore, proof of concept for the functional expression of P2X7Rs in human brain-relevant pre-clinical cellular models will promote the development of drugs at a faster pace, which in the long term will improve treatment options for patients with neurological diseases.
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