Modeling mTORopathy-related epilepsy in cultured murine hippocampal neurons using the multi-electrode array

Abstract

The mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway is a ubiquitous cellular pathway. mTORopathies, a group of disorders characterized by hyperactivity of the mTORC1 pathway, illustrate the prominent role of the mTOR pathway in disease pathology, often profoundly affecting the central nervous system. One of the most debilitating symptoms of mTORopathies is drug-resistant epilepsy, emphasizing the urgent need for a deeper understanding of disease mechanisms to develop novel anti-epileptic drugs. In this study, we explored the multiwell Multi-electrode array (MEA) system as a tool to identify robust network activity parameters in an approach to model mTORopathy-related epilepsy in vitro. To this extent, we cultured mouse primary hippocampal neurons on the multiwell MEA to identify robust network activity phenotypes in mTORC1-hyperactive neuronal networks. mTOR-hyperactivity was induced either through deletion of Tsc1 or overexpression of a constitutively active RHEB variant identified in patients, RHEBp.P37L. mTORC1 dependency of the phenotypes was assessed using rapamycin, and vigabatrin was applied to treat epilepsy-like phenotypes. We show that hyperactivity of the mTORC1 pathway leads to aberrant network activity. In both the Tsc1-KO and RHEB-p.P37L models, we identified changes in network synchronicity, rhythmicity, and burst characteristics. The presence of these phenotypes is prevented upon early treatment with the mTORC1-inhibitor rapamycin. Application of rapamycin in mature neuronal cultures could only partially rescue the network activity phenotypes. Additionally, treatment with the anti-epileptic drug vigabatrin reduced network activity and restored burst characteristics. Taken together, we showed that mTORC1-hyperactive neuronal cultures on the multiwell MEA system present reliable network activity phenotypes that can be used as an assay to explore the potency of new drug treatments targeting epilepsy in mTORopathy patients and may give more insights into the pathophysiological mechanisms underlying epilepsy in these patients. ABBREVIATIONS AED, anti-epileptic drug, CoVNIBI, coefficient of variance of NIBI, CTR, control transduced cultures, DIV, days in vitro, DMEM, Dulbeccos modified Eagle medium, DMSO, dimethyl sulfoxide, GABA, gamma-aminobutyric acidergic, GAPDH, Glyceraldehyde-3-Phosphate Dehydrogenase, iPSC, induced pluripotent stem cell, KO, knock-out, LV, lentivirus, MEA, multi-electrode array, MFR, mean firing rate, mTORC1, mechanistic target of rapamycin complex 1, NB, network burst, NBC, network burst composition, NBD, network burst duration, NBM, neurobasal medium, NBR, network burst rate, NIBI, network interburst interval, NT, non-transduced, RHEB, Ras-homolog enriched in brain, %RS, percentage of random spikes, TBS, tris buffered saline, TSC, Tuberous sclerosis complex, WT, wildtype