Ablation of peri-insult generated granule cells after epilepsy onset halts disease progression.

Aberrant integration of newborn hippocampal granule cells is hypothesized to contribute to the development of temporal lobe epilepsy. To test this hypothesis, we used a diphtheria toxin receptor expression system to selectively ablate these cells from the epileptic mouse brain. Epileptogenesis was initiated using the pilocarpine status epilepticus model in male and female mice. Continuous EEG monitoring was begun 2-3 months after pilocarpine treatment. Four weeks into the EEG recording period, at a time when spontaneous seizures were frequent, mice were treated with diphtheria toxin to ablate peri-insult generated newborn granule cells, which were born in the weeks just before and after pilocarpine treatment. EEG monitoring continued for another month after cell ablation. Ablation halted epilepsy progression relative to untreated epileptic mice; the latter showing a significant and dramatic 300% increase in seizure frequency. This increase was prevented in treated mice. Ablation did not, however, cause an immediate reduction in seizures, suggesting that peri-insult generated cells mediate epileptogenesis, but that seizures per se are initiated elsewhere in the circuit. These findings demonstrate that targeted ablation of newborn granule cells can produce a striking improvement in disease course, and that the treatment can be effective when applied months after disease onset.

Ablation of Newly Generated Hippocampal Granule Cells Has Disease-Modifying Effects in Epilepsy.

Hippocampal granule cells generated in the weeks before and after an epileptogenic brain injury can integrate abnormally into the dentate gyrus, potentially mediating temporal lobe epileptogenesis. Previous studies have demonstrated that inhibiting granule cell production before an epileptogenic brain insult can mitigate epileptogenesis. Here, we extend upon these findings by ablating newly generated cells after the epileptogenic insult using a conditional, inducible diphtheria-toxin receptor expression strategy in mice. Diphtheria-toxin receptor expression was induced among granule cells born up to 5 weeks before pilocarpine-induced status epilepticus and these cells were then eliminated beginning 3 d after the epileptogenic injury. This treatment produced a 50% reduction in seizure frequency, but also a 20% increase in seizure duration, when the animals were examined 2 months later. These findings provide the first proof-of-concept data demonstrating that granule cell ablation therapy applied at a clinically relevant time point after injury can have disease-modifying effects in epilepsy. SIGNIFICANCE STATEMENT: These findings support the long-standing hypothesis that newly generated dentate granule cells are pro-epileptogenic and contribute to the occurrence of seizures. This work also provides the first evidence that ablation of newly generated granule cells can be an effective therapy when begun at a clinically relevant time point after an epileptogenic insult. The present study also demonstrates that granule cell ablation, while reducing seizure frequency, paradoxically increases seizure duration. This paradoxical effect may reflect a disruption of homeostatic mechanisms that normally act to reduce seizure duration, but only when seizures occur frequently.

Impact of rapamycin on status epilepticus induced hippocampal pathology and weight gain.

Growing evidence implicates the dentate gyrus in temporal lobe epilepsy (TLE). Dentate granule cells limit the amount of excitatory signaling through the hippocampus and exhibit striking neuroplastic changes that may impair this function during epileptogenesis. Furthermore, aberrant integration of newly-generated granule cells underlies the majority of dentate restructuring. Recently, attention has focused on the mammalian target of rapamycin (mTOR) signaling pathway as a potential mediator of epileptogenic change. Systemic administration of the mTOR inhibitor rapamycin has promising therapeutic potential, as it has been shown to reduce seizure frequency and seizure severity in rodent models. Here, we tested whether mTOR signaling facilitates abnormal development of granule cells during epileptogenesis. We also examined dentate inflammation and mossy cell death in the dentate hilus. To determine if mTOR activation is necessary for abnormal granule cell development, transgenic mice that harbored fluorescently-labeled adult-born granule cells were treated with rapamycin following pilocarpine-induced status epilepticus. Systemic rapamycin effectively blocked phosphorylation of S6 protein (a readout of mTOR activity) and reduced granule cell mossy fiber axon sprouting. However, the accumulation of ectopic granule cells and granule cells with aberrant basal dendrites was not significantly reduced. Mossy cell death and reactive astrocytosis were also unaffected. These data suggest that anti-epileptogenic effects of mTOR inhibition may be mediated by mechanisms other than inhibition of these common dentate pathologies. Consistent with this conclusion, rapamycin prevented pathological weight gain in epileptic mice, suggesting that rapamycin might act on central circuits or even peripheral tissues controlling weight gain in epilepsy.

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

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.