Rationale:
Somatic mutations impairing mTOR pathway regulators, such as Pten or Tsc1/2, lead to the development of focal lesions, autism, and epilepsy in affected children. Disease causing mutations are believed to primarily involve excitatory neuron lineages, leading to neuronal hypertrophy, excess synaptogenesis, and hyperexcitability. Abnormal excitatory neurons still receive inhibitory input, however, which may substantially restrain the epileptogenic potential of these cells. Interneuron loss, as is evident in animal models, may promote disease progression by “releasing” dysmorphic excitatory cells. To assess the role of interneurons in mTORopathies, we developed a dual-targeted cre recombinase/flip (FlpO) recombinase mouse model system to combine variable loads of dysmorphic mTOR hyperactive Pten knockout hippocampal granule cells (DGC-Pten KOs) with selective ablation of hippocampal interneuron subtypes. The ability to combine Pten loss from excitatory neurons with interneuron ablation will make it possible to determine whether—and if so, which—populations of interneurons play the greatest role in restraining mTOR hyperactive (Pten KO) neurons.
Methods:
On postnatal day 21, triple transgenic Gli1-CreER
T2, Pten
fl/fl, SST-FlpO
+/- mice received tamoxifen to activate CreER
T2 and delete Pten from a subset of DGCs. At eight to ten weeks of age, mice received EEG implants and underwent stereotaxic hippocampal injection of AAV9-CAG-frt-DTr to express diphtheria toxin receptor (DTr) in somatostatin (SST) interneurons, allowing for their selective ablation with diphtheria toxin (DT). After one week of baseline recording, animals were randomly divided into treatment groups to receive five days of either DT or saline. Video-EEG recording continued for a minimum of two weeks post-treatment. Experimental groups included: DGC-Pten KO alone, SST ablation alone, and combined DGC-Pten KO and SST ablation.
Results:
Histological studies confirmed the efficacy of Pten deletion from granule cells and DTr-mediated ablation of SST interneurons under both individual and combined conditions. Ablation of SST interneurons alone consistently resulted in seizures and protracted epileptiform activity (n=5 mice, cumulative seizure duration 124.6 ± 46.4 minutes/week [mean ± SEM]). Animals with SST ablation and low rates of Pten KO DGC exhibited a similar phenotype with no significant difference in time spent in seizures (n=6 mice, 243.3 ± 48.1 minutes/week; unpaired t-test, p=0.113). Mortality, although occurring, was limited in ablation groups. Seizures were absent from controls.
Conclusions:
The intersectional approach utilizing FLP-FRT recombination was effective in ablating SST interneurons, producing a consistent epilepsy phenotype. Results indicate that this approach can be used in combination with Cre-Lox methods to investigate the importance of different interneuron populations in inhibiting mTOR hyperactive excitatory neurons. Ongoing work will combine interneuron ablation with Pten deletion from higher proportions of excitatory neurons to assess its impact on epilepsy severity.
Funding:
R01-NS-065020 and R01-NS-121042