DEPDC5, the key gene within the mechanistic target of rapamycin (mTOR) pathway, is one of the most common causative genes in patients with epilepsy and malformation of cortical development (MCD). Although somatic mutations in the dorsal cortical progenitors generate the malformed cortex, its pathogenesis of hyperexcitability is complex and remains unclear. We specifically deleted Depdc5 in the mouse forebrain dorsal progenitors to model DEPDC5-related epilepsy, and investigated whether and how parvalbumin interneurons were non-cell autonomously affected in the malformed cortex. We showed that long before seizures, coincident with microglia inflammation, proteolytic enzymes degraded perineuronal nets (PNN) in the malformed cortex, resulting in parvalbumin (PV+) interneuron loss and presynaptic inhibition impairment. Our studies therefore uncovered the hitherto unknown role of PNN in mTOR-related MCD, providing a new framework for mechanistic-based therapeutic development.

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Efficient genetic manipulation in the developing central nervous system is crucial for investigating mechanisms of neurodevelopmental disorders and the development of promising therapeutics. Common approaches including transgenic mice and in utero electroporation, although powerful in many aspects, have their own limitations. In this study, we delivered vectors based on the AAV9.PHP.eB pseudo-type to the fetal mouse brain, and achieved widespread and extensive transduction of neural cells. When AAV9.PHP.eB-coding gRNA targeting PogZ or Depdc5 was delivered to Cas9 transgenic mice, widespread gene knockout was also achieved at the whole brain level. Our studies provide a useful platform for studying brain development and devising genetic intervention for severe developmental diseases.

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Objective: Low-level somatic mosaicism in the brain has been shown to be a major genetic cause of intractable focal epilepsy. However, how a relatively few mutation-carrying neurons are able to induce epileptogenesis at the local network level remains poorly understood. Methods: To probe the origin of epileptogenesis, we measured the excitability of neurons with MTOR mutation and nearby nonmutated neurons recorded by whole-cell patch-clamp and array-based electrodes comparing the topographic distribution of mutation. Computational simulation is used to understand neural network-level changes based on electrophysiological properties. To examine the underlying mechanism, we measured inhibitory and excitatory synaptic inputs in mutated neurons and nearby neurons by electrophysiological and histological methods using the mouse model and postoperative human brain tissue for cortical dysplasia. To explain non-cell-autonomous hyperexcitability, an inhibitor of adenosine kinase was injected into mice to enhance adenosine signaling and to mitigate hyperactivity of nearby nonmutated neurons. Results: We generated mice with a low-level somatic mutation in MTOR presenting spontaneous seizures. The seizure-triggering hyperexcitability originated from nonmutated neurons near mutation-carrying neurons, which proved to be less excitable than nonmutated neurons. Interestingly, the net balance between excitatory and inhibitory synaptic inputs onto mutated neurons remained unchanged. Additionally, we found that inhibition of adenosine kinase, which affects adenosine metabolism and neuronal excitability, reduced the hyperexcitability of nonmutated neurons. Interpretation: This study shows that neurons carrying somatic mutations in MTOR lead to focal epileptogenesis via non-cell-autonomous hyperexcitability of nearby nonmutated neurons.