Excessive activation of mTOR in postnatally generated granule cells is sufficient to cause epilepsy

Abstract

The dentate gyrus is hypothesized to function as a "gate," limiting the flow of excitation through the hippocampus. During epileptogenesis, adult-generated granule cells (DGCs) form aberrant neuronal connections with neighboring DGCs, disrupting the dentate gate. Hyperactivation of the mTOR signaling pathway is implicated in driving this aberrant circuit formation. While the presence of abnormal DGCs in epilepsy has been known for decades, direct evidence linking abnormal DGCs to seizures has been lacking. Here, we isolate the effects of abnormal DGCs using a transgenic mouse model to selectively delete PTEN from postnatally generated DGCs. PTEN deletion led to hyperactivation of the mTOR pathway, producing abnormal DGCs morphologically similar to those in epilepsy. Strikingly, animals in which PTEN was deleted from ≥ 9% of the DGC population developed spontaneous seizures in about 4 weeks, confirming that abnormal DGCs, which are present in both animals and humans with epilepsy, are capable of causing the disease.

Figure 1

Confocal optical sections of NeuN staining (green) reveal the dentate granule cell layer (boxed region in inset, top left panel) in cre control and PTEN KO mice treated with tamoxifen on P14. Sections were double-immunostained for PTEN (red), and in control animals, 100% of NeuN labeled granule cells also co-localize PTEN. In double transgenic mice, however, recombined granule cells appear as gaps in the PTEN staining. Note that these gaps correspond exactly with NeuN stained granule cells, and these PTEN-negative cells appear in black and white in the merged image. As expected, recombined granule cells were localized primarily to the inner region of the granule cell layer, where postnatally-generated granule cells are typically present. Scale bars = 25 μm. See also figures S1 and S2.

Figure 2

A–D: Disrupted development of PTEN-immunonegative granule cells in Gli1-CreER^T2 X PTEN^flox/flox X Thy1-GFP mice. In these animals, the Thy1 promoter drives GFP expression in a subset of wildtype and PTEN KO granule cells. A: PTEN immunostaining of two GFP-expressing dentate granule cells. The upper cell (arrow) is PTEN-immunonegative, while the lower cell is immunoreactive for PTEN protein (arrowhead). The asterisk denotes a PTEN-negative, GFP-negative granule cell. B: Confocal maximum projection of a GFP-expressing, PTEN-negative granule cell (arrow) with GFP-expressing, PTEN-positive neighbors. Note how the PTEN-negative cell dwarfs its neighbors. C,D: Confocal maximum projections of GFP-labeled dendritic segments from PTEN-negative (C) and PTEN-positive (D) cells. The dendrite of the PTEN-negative cells exhibits dramatically increased thickness and spine density. E,F: GFP-expressing granule cells from cre control (E) and PTEN KO (F) mice. The white line in both images marks the dentate granule cell layer – hilar border. Note the presence of hilar ectopic granule cells in the KO. Scale bars = 10 μm (A), 30 μm (B), 25 μm (C), 20 μm (E,F).

Figure 3

A: Confocal maximum projection showing aberrant hilar basal dendrites on a PTEN KO granule cell. Single optical sections of these basal dendrites reveal dendritic spines (A.1) apposed to ZnT-3 immunoreactive puncta (arrowheads, A.2 and merged image in A.3) indicative of recurrent mossy fiber input. Higher resolution confocal optical sections of a subset of the spines shown in A are shown in B.1, with apposed ZnT-3 immunoreactive puncta in B.2, and merged images in B.3. White arrows denote spines and green arrows denote apposed puncta. Additional examples of basal dendrite spines and apposed ZnT-3 immunoreactive puncta are shown in C.1-C.3, and D.1-D.3. E: Confocal optical section of a basal dendrite spine in the hilus (E.1, white arrow) apposed to ZnT-3 immunoreactive puncta (E.2, green arrows). The spine is immunoreactive for PSD-95 (E.3, white arrow), providing further evidence of a functional synapse. Merged images are shown in E.4. Scale bars = 5 μm (A), 3 μm (B, C, D), 5 μm (E).

Figure 4

PTEN KO mice treated with tamoxifen on P14 develop epileptic seizures. For all panels, cortical traces (ctx) are shown in black and hippocampal traces (hp) in blue. A: Typical electrographic seizure recorded using hippocampal depth electrodes. B: Dual trace EEG recording showing a long (≈3.5 minute) hippocampal seizure (hp, blue) preceded by two shorter seizures. Regions denoted by red lines are shown at higher resolution below. Note the absence of cortical involvement despite extensive hippocampal seizure activity. C: Example of a typical seizure recorded in cortex. D: Dual trace EEG showing seizure activity in cortex which gradually increases in amplitude over the course of the ≈30 minute event. In this trace, high amplitude intermittent burst activity is seen in both the hippocampal and cortical traces. E: Example of cortical burst suppression activity, which could persist for 30 minutes or longer and was typically observed as animal health declined. F: Example of epileptiform activity in hippocampus. See also figures S3 and S4.

Figure 5

Confocal maximum projections showing GFP-expressing olfactory granule neurons from control (A) and PTEN KO (B, C, D) mice. Olfactory granule cells from a PTEN KO animal immunostained for GFP and PTEN are shown in C and D. The cell in C (arrow) was immunoreactive for GFP (C.1) and PTEN (C.2), indicative of incomplete cre-mediated recombination in the cell (the GFP reporter was activated but at least one allele of PTEN was left intact). The cell in D (arrow) expressed GFP but was immunonegative for PTEN protein, indicative of complete recombination at all three lox-p splice sites. PTEN KO olfactory cells exhibited subtle, but statistically significant increases in soma area. Scale bars = 10 μm. E: Simultaneous EEG recording from hippocampus and olfactory bulb revealed epileptiform activity and seizures in the hippocampal trace (blue). Olfactory EEG (black) was unchanged during these events, and no seizures originating from, or isolated to, olfactory bulb were observed.

Figure 6

Confocal maximum projections of NeuN (green) and PTEN (red) double-immunostaining reveal PTEN KO granule cells in animals treated with vehicle (V) or rapamycin (RAP). PTEN KO cells are shown in gray in the merged image. Confocal maximum projections showing pS6 staining (magenta) reveal robust staining in vehicle-treated PTEN KOs, and virtually absent staining in rapamycin treated animals. Confocal maximum projections showing ZnT-3 staining (cyan) demonstrate that rapamycin treatment was able to block the sprouting of mossy fiber axons into the dentate granule cell layer (DGC-L) and inner molecular layer (IML). Both groups exhibited the normal pattern of ZnT-3 staining in the dentate hilus (H). Scale bars = 25 μm.

Figure 7

Confocal maximum projections of hippocampi from tamoxifen-treated control and PTEN KO mice immunostained for GFP (red) and ZnT3 (cyan) are shown. ZnT3-labeling reveals the normal mossy fiber axon terminal field (hilus and stratum lucidum) in the control animal, while mossy fiber sprouting into the dentate granule cell layer (DGC-L) and inner molecular layer (IML) is evident in the knockout animal (green boxes in the top row correspond to high resolution images shown in the bottom row). Scale bars = 200 μm (top row) and 30 μm (bottom row). Top graph: Correlation between the degree of mossy fiber sprouting (assessed by ZnT3 immunoreactivity) in the IML and the percentage of PTEN KO granule cells. Control animals exhibited neither recombined cells nor mossy fiber sprouting (n=9, black diamonds), while mossy fiber sprouting in PTEN KO animals (blue circles) was significantly correlated with the percentage of recombined granule cells in the dentate gyrus (P<0.01). Bottom graph: PTEN KO granule cells exhibited somatic hypertrophy in all KO animals (n=8, blue dots) relative to GFP-expressing cells from cre-control animals (n=4, black diamonds), regardless of the degree of recombination or whether seizures were observed.

Figure 8

Confocal optical sections of GFP-expressing mossy fiber axons in the dentate inner molecular layer from PTEN KO cells are shown in red. Double-immunostaining for the presynaptic granule cell terminal marker ZnT-3 is shown in cyan. White arrows denote ZnT-3 immunoreactive, GFP-expressing axon varicosities (presumptive PTEN KO cell axon terminals), while blue arrowheads denote ZnT-3 immunoreactive, GFP negative puncta (presumptive wildtype granule cell terminals). Scale bars= 5 μm.