Abstracts

Rationale: Hyperactivation of the mechanistic target of rapamycin (mTOR) signaling pathway is implicated in epileptogenesis. The pathway contains two central mTOR complexes, mTORC1 and mTORC2, which are regulated by the Regulatory-associated protein of mTOR (Raptor) and the Rapamycin-insensitive companion of mTOR (Rictor), respectively.

Hippocampal dentate granule cells are a known target of mTOR hyperactivation in temporal lobe epilepsy (TLE). However, the exact roles played by mTORC1 and mTORC2 in regulating granule cell pathology in TLE are largely unexplored. Because granule cell pathology has been hypothesized to mediate TLE, understanding whether and how mTOR regulates granule cell dysmorphogenesis may have therapeutic implications.

Methods: We developed a viral cre-recombinase driven strategy to produce sparse deletions of Raptor or Rictor from hippocampal dentate granule cells in mice during epileptogenesis. Sparse deletions were titrated to produce knockout (KO) cells in each mouse without impacting epileptogenesis. One week after pilocarpine-induced status epilepticus (SE), Raptor^fl/fl;tdTomato^+/- or Rictor^fl/fl;tdTomato^+/- mice received hippocampal injections of AAV9.CamKII.HI.eGFP and AAV9.CamKII.Cre (4 x 10⁹ vg/mL), producing a mixture of eGFP+ wildtype and tdTomato+ Raptor or Rictor knockout cells in each mouse. Animals were video monitored for seizures during week seven, and brains were collected at the end of the eighth week for histology and cell morphometry. The study consisted of 6 groups (Control, n=10 mice; Control + SE, n=6; Raptor KO, n=8; Raptor KO + SE, n=7; Rictor KO, n=9; Rictor KO + SE, n=17).

Results: The low titer viral cocktail approach was effective in producing a mosaic of GFP+ wildtype and tdTomato+ knockout granule cells in each animal. The sparse deletion approach was titrated to avoid disrupting whole-animal epileptogenic changes, and this was confirmed by characterizing mice for hilar cell loss and mossy fiber sprouting. Hippocampal pathology was absent from control (no SE) mice and present in mice that went through SE (z test, p=0.004). Within animal analyses of soma area revealed that Raptor KO cells were consistently smaller than adjacent wildtype cells, while Rictor KO cells were statistically identical to adjacent wildtype cells (values are % control (wildtype) cells: Raptor KO; n=6, 92.4±3.8%; Raptor KO + SE, n=6, 87.1±3.8%; Rictor KO, n=7, 99.6±3.5%; Rictor KO + SE, n=14, 97.8±2.4%; two-way ANOVA, p=0.015 for genotype [Raptor]).

Conclusions: We describe a novel AAV/transgenic approach in which GFP+ wildtype and tdTomato+ Raptor/Rictor KO cells can be generated side-by-side in an epileptic animal, allowing us to assess the cell-intrinsic effects of blocking mTORC1 or mTORC2 signaling. Initial studies confirm the known effect of mTORC1 on regulating soma size. Raptor KO cells were consistently smaller than wildtype cells. Studies are ongoing to reveal cell intrinsic roles of mTORC1 and mTORC2 signaling in mediating granule cell dysmorphogenesis in epilepsy.

Funding: Please list any funding that was received in support of this abstract.: This work was supported by the National Institute of Neurological Disorders and Stroke (R01-NS-062806, F31-NS11552501).