Abstracts

Rationale: Hyperactivation of mechanistic target of rapamycin (mTOR) signaling, due to genetic mutations in components of the mTOR pathway, is associated with intractable epilepsy. We previously reported that expression of an mTOR pathway gene, which harbors a mutation leading to constitutive activation of mTOR signaling, Rheb^S16H, results in aberrant hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) expression and altered excitability in cortical pyramidal neurons. To assess whether this finding is conserved in other epilepsy-associated mTOR pathway genes, we examined the impact of two other mTOR pathway activating variants, Rheb^Y35L and MTOR^S2215Y, on the resting membrane potential (RMP) and HCN current amplitude of layer 2/3 pyramidal neurons in the mouse medial prefrontal cortex (mPFC). We also assessed electrophysiological changes following CRISPR/Cas9-mediated knockout of the mTOR negative regulators Pten, Tsc1, and Depdc5.

Methods: Plasmids encoding Rheb^Y35L or mTOR^S2215Y, or Pten, Tsc1, or Depdc5 CRISPR guide RNAs, were expressed in developing mouse embryos via in utero electroporation at embryonic day 15. Whole-cell patch-clamp recordings were obtained from layer 2/3 mPFC pyramidal neurons in acute slices from mice at postnatal day 26-51. A series of hyperpolarizing voltage steps from −130 to −40 mV was applied to evoke HCN currents. HCN current amplitudes were measured as the difference between the instantaneous current and the steady-state current at -90 mV (when there is no current contamination from inwardly-rectifying potassium channels). Statistical tests were performed using one-way ANOVA with Holm-Šídák's multiple comparisons test. Shown p-values are from multiple comparison tests following significant ANOVAs.

Results: Expression of Rheb^Y35L and mTOR^S2215Y in pyramidal neurons both led to significantly more depolarized RMPs compared to control (p< 0.0001). Knockout of Pten and Tsc1 also led to significantly decreased RMPs (p= 0.0233 and p< 0.0001, respectively), while knockout of Depdc5 was not significantly different from the respective control (p= 0.4452). HCN currents were detected in all evaluated variants to variable degrees. HCN current amplitudes were largest in the mTOR^S2215Y condition, though both mTOR^S2215Y and Rheb^Y35L expression led to significantly larger HCN current amplitudes compared to control (p< 0.0001 and p= 0.0461, respectively). Increased HCN current amplitudes were also found following Pten and Tsc1 knockout (p= 0.0232 and p< 0.0001, respectively), with the largest amplitudes in the Tsc1 knockout condition. Some individual neurons in the Depdc5 knockout condition displayed prominent HCN current amplitudes, but as a group, the mean difference did not reach statistical difference from control (p= 0.2371).