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.
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.
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.
► Direct evidence that selective disruption of the dentate gyrus causes epilepsy ► PTEN deletion from as few as 9% of granule cells is sufficient to cause epilepsy ► Findings suggest a plausible mechanism of epileptogenesis
Abnormal hippocampal granule cells are hypothesized to be critical for temporal lobe epileptogenesis, but direct supporting evidence has been limited. Here, Pun and colleagues demonstrate that selective disruption of granule cells by PTEN deletion is sufficient to cause the disease.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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.
•Rapamycin prevents pathological weight gain in epileptic rodents.•Rapamycin reduces mossy fiber sprouting in epilepsy.•Rapamycin does not block ectopic granule cell accumulation or somatic hypertrophy.•Rapamycin does not block hippocampal inflammatory changes.•Rapamycin does not prevent hilar mossy cell death.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Granule cells are one of the few neuronal populations that continue to be born throughout life and into old age. The protracted development of this cell population appears to make it uniquely ...vulnerable to anatomical and physiological abnormalities following epileptogenic insults. Under normal conditions, this cell population typically is located densely in the granule cell body layer of the hippocampus, projects its dendritic trees outwardly through the molecular layer to the edges of the hippocampal fissure, and sends its mossy fiber axons inwardly to the hilus and CA3. However, following an epileptogenic insult, the defined arrangement of dentate granule cells becomes unstructured. Interestingly, it is the newly generated granule cells that are more prone to improper migration and cellular development. Furthermore, newly generated dentate granule cells exhibit striking physiological changes including depolarized membrane potentials, prolonged action potentials, spontaneous bursting, and increased firing. At the same time, the dentate gyrus becomes less capable at limiting the amount of synaptic flow through the hippocampus. These data underlie the hypothesis that newly generated dentate granule cells are pro-epileptogenic. To test this hypothesis I have completed studies which aim at decreasing granule cell anatomic and physiologic abnormalities. In the first study (chapter 2), I used the mTOR inhibitor rapamycin to try to prevent the improper development of dentate granule cells following pilocarpine-induced status epilepticus. The mTOR pathway is involved in cell growth and division, making it a promising candidate target to inhibit aberrant cell development. Rapamycin treatment prevented aberrant axonal sprouting, but had no effect on other hippocampal pathologies. Interestingly, treatment also inhibited epilepsy induced weight gain. In the second study (chapter 3), I used a novel transgenic approach to target newly generated dentate granule cells prior to pilocarpine-induced status epilepticus. These cells were then ablated shortly following the epileptogenic injury. Results of this study demonstrated that removing the cells prophylactically could reduce the number of ectopically located granule cells and also reduce seizure frequency several weeks later. As a follow up to this study, the project described in the final study ( chapter 4) used the same transgenic approach to target newly generated granule cells; however, cell ablation occurred months after the injury. In this study, treatment prevented further increases in seizure frequency, and also resulted in a robust reduction of ectopically located granule cells. The studies conducted as a part of this dissertation research demonstrate that newly generated dentate granule cells are contributing to the formation of seizures in the pilocarpine model of temporal lobe epilepsy. The data supports the hypothesis that these effects are driven in part by ectopically located granule cells generated after the epileptogenic insult. Finally, this data provides the first line of evidence that targeting newly born dentate granule cells could lead to new therapeutic approaches for patients with epilepsy, especially since treatment was effective at inhibiting seizure progression when applied months after spontaneous seizure onset.
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