Abstracts

Rationale:
Low-frequency somatic mutations in key genes within the mechanistic target of rapamycin (mTOR) pathway have been frequently identified in patients with focal cortical dysplasia type II (FCDII), the most common underlying pathologies in children with severe refractory epilepsies. The treatment failure of these neuron and mTOR centric strategies highlights our knowledge gaps in conceptual and mechanistic understanding of epileptogenesis in the malformed cortex. For example, previous studies have only focused on low-frequency, at times ultra-low-level, mutant neurons but largely ignored the surrounding wild-type cell populations, e.g., interneurons, astrocytes, and microglial cells. In addition, patients with mTORopathies develop more severe focal imaging abnormalities and seizures over time, suggesting a progressive epileptogenesis. However, current experimental paradigms have not investigated the pathophysiology to guide disease-modifying therapies, one of the most clinically critical questions.

Methods:
We generated two highly clinically relevant animal models of mTORopathies respectively arising from the disturbed amino-acid sensing pathway (DEPDC5-related epilepsy) and the overactivated growth factor signaling branch (RHEB-related epilepsy). To provide a defined spatiotemporal frame to interrogate the logical cellular and molecular networks, we used in vivo and in vitro electrophysiology to determine its latent period of epileptogenesis and intrinsic epileptogenicity. We next investigated the cellular and molecular landscape in experimental and human mTORopathies to identify targetable pathways. Last, we took advantage of novel C3 knockout animals to investigate whether C3 inhibition could improve seizure outcomes and reverse disease progression.

Results:
FCDII animal models recapitulate human disease and provide opportunities to investigate converged mechanisms underlying mTORopathies. Our data further show that animals develop more severe seizures over time, correlating progressive astrogliosis, microglia activation, and inhibitory synapse loss that are restricted to the malformed cortex. In addition, although those low-frequency mutant excitatory neurons have severely disrupted homeostasis (e.g., prominent cell overgrowth, autophagy blockage, and oxidative stress), their intrinsic membrane properties are not hyperexcitable. These preliminary data suggest mTORopathies/FCDII is a progressive disease and its progressive epileptogenicity is beyond a few low-frequency mutant cells. Molecular profiling showed complement overactivation in the dysplastic cortex during latent period of epileptogenesis and late disease stage mediating preferential inhibitory synapse loss.

Conclusions:
Aberrant complement overexpression from activated microglia results in a sustained local pro-epileptogenic microenvironment in the dysplastic cortex that progressively sculpts cellular architectures, disrupts inhibitory circuits, and enhances cortical excitability. Complement inhibition could restore the disturbed cellular and molecular architectures, modify disease progression, and improve seizure outcomes.

Funding: R01NS113824