Abstracts

Rationale: The mechanistic target of rapamycin (mTOR) signaling pathway is involved in organismal growth and homeostasis. Inhibition of the mTOR pathway with the mTORC1 antagonist rapamycin has been shown to be anti-epileptogenic in rodent TLE models. However, the exact cellular and molecular targets by which rapamycin produces its positive effects remain uncertain. The systemically applied drug could act anywhere in the brain, and prolonged treatment with rapamycin can inhibit mTORC2. To determine where and how enhanced mTOR signaling might contribute to epileptogenesis, we developed an AAV-mediated gene deletion strategy to selectively delete Raptor from hippocampal dentate granule cells. Raptor is necessary for mTORC1 signaling, and mTOR-mediated changes in granule cells are implicated in epileptogenesis. Methods: To delete Raptor from granule cells, AAV9.CamKII.HI.Cre-eGFP was injected into the hippocampus of Raptor fl/fl mice at a titer of 1.25 x 10^11 vg/mL. To control for any off-target effects of cre-recombinase, Raptor wt/wt mice underwent the same paradigm. Additionally, Raptor fl/fl and Raptor wt/wt l animals were injected with a control virus, AAV9.CamKII.HI.eGFP. AAV was injected into the hippocampus and examined across serial sections between Bregma levels 0.94 – 3.40. To confirm the efficacy of cre-recombinase, AAV was also injected into double transgenic animals containing a floxed reporter construct (Raptorfl/fl TdTomato+/- mice). Brain sections were imaged to assess viral spread, and immunostained with pS6, a marker of mTOR signaling. Results: Combined dorsal and ventral AAV injections into each dentate produced a pattern of viral spread throughout the rostral-caudal extent of the dentate gyrus. Analysis of GFP-labeling in the dentate revealed that 55-100% (n=5 mice) of granule cells were infected. Injection into Raptorfl/fl TdTomato+/- mice produced similar patterns of labeling, with cells expressing both GFP and tdTomato. Moderate to strong labeling of mossy cells were also found in the polymorphic layer of the dentate gyrus. pS6 immunostaining confirmed effective Raptor deletion in infected cells. In animals injected with control virus, 100% of GFP-expressing granule cells were immunoreactive for Raptor, while in animals injected with AAV-Cre virus, only 1.3% of GFP-expressing cells were pS6 immunoreactive (control, n=137 cells; AAV-Cre, n=147; p<0.001, z-test). GFP-expressing raptor knockout DGCs appeared grossly normal, exhibiting spine covered dendrites projecting to the hippocampal fissure. Obvious degenerative changes were absent. Conclusions: The AAV9.CamKII.HI.Cre-eGFP/Raptorflox/flox strategy was effective at labeling mature granule cells throughout the rostral-caudal extent of the dentate gyrus and deleting Raptor. At optimized titers, cre toxicity was absent. Raptor knockout cells had decreased pS6 expression, indicative of decreased mTORC1 signaling. Gross degenerative changes were absent following raptor deletion, suggesting that the protein is not required for cell survival. This approach will be useful for assessing the role of mTORC1 signaling in epileptogenesis. Funding: This work was supported by the National Institute of Neurological Disorders and Stroke (R01-NS-062806).