Abstracts

Rationale: Low-level somatic mutation in neurons in the brain causes intractable focal epilepsy, including focal cortical dysplasia type 2 (FCD II) (Lim et al. Nat Med 2015;21:395-400) and ganglioglioma (Koh et al. Nat Med 2018;24:1662-1668). Neurons carrying somatic mutations in ~1-2% of MTOR, TSC1, or TSC2 were found to induce epileptogenesis with spontaneous behavioral seizures. However, how only a few mutated cells in focal brain regions are able to elicit synchronized electrical discharges leading to epilepsy through disrupting the local network remains poorly understood.

Methods: We generated FCD II model mice having a somatic mutation in MTOR L2427P presenting seizures by in utero electroporation and ascertained the number of mutated neurons (MTOR^L2427P) throughout the whole brain along with patients-derived tissue. To probe the origin of epileptogenesis, we measured the neuronal excitability in MTOR^L2427Pand nearby non-mutated neurons (L2427P^nearby) by current injection and multi-electrode recording comparing the topographic distribution of cells having the mutation. Computational connectivity using the leaky-integrate-and-fire model recapitulates network changes based on measured properties. To examine the underlying mechanism, we measured excitatory and inhibitory (E-I) synaptic inputs in MTOR^L2427P and L2427P^nearby by electrophysiological and immunofluorescence studies. To explain non-cell autonomous hyperexcitability, an inhibitor of adenosine kinase (ADK), which translation was increased by MTOR^L2427P in ribosome-sequencing, was injected into mice in vivo.

Results: Only 1.85±0.80% of neurons carried the MTOR somatic mutation in the whole epileptic-brain (Fig. 1A). Interestingly, the seizure-triggering hyperexcitability was originated from L2427P^nearby, but MTOR^L2427P was less excitable than L2427P^nearby. This suggests that neurons carrying MTOR^L2427P may not be the source of the hyperactivity observed. Our comprehensive data from computational simulations and immunofluorescence and electrophysiological experiments implicated non-cell autonomous effects in the hyperexcitability of nearby non-mutated neurons (Fig. 1B and C), rather than cell-autonomous or intrinsic properties in mutated neurons, such as an increased cell size and an alternation of synaptic connectivity. E-I synaptic inputs onto MTOR^L2427P remained unchanged in mice and FCD II patients’ tissues, implying that intrinsic synaptic changes driven by MTOR mutation are less likely to be explanatory. Instead, we found that inhibition of ADK, an extrinsic factor induced by neurons carrying MTOR^L2427P, reduced the hyperexcitability of L2427P^nearby affecting adenosine metabolism in the local network (Fig. 2).

Conclusions: Taken together, providing an example of how low-level brain somatic mutation can change the entire brain activity, this study showed that neurons carrying somatic mutations in MTOR lead to focal epileptogenesis via non-cell autonomous hyperexcitability of nearby non-mutated neurons.

Funding: Please list any funding that was received in support of this abstract.: Suh Kyungbae Foundation and the Korean Health Technology Research and Development Project (H15C3143) and National Research Foundation (2019R1A2C2005161, 2019R1A2C4069863 and 2019R1A3B2066619).