Targeting Newly Generated Granule Cells: A Double-Edged Sword

Commentary

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

Hosford BE, Liska JP, Danzer SC., J Neurosci 2016;3643:11013–11137.

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.

Until the turn of the century, it was believed that neurogenesis did not occur in adult brains. Now, it is well established that adult neurogenesis does occur, albeit in specific brain areas. In the hippocampus, newly generated granule cells are produced by neural progenitor cells in the subgranular zone. In the healthy brain, these cells then migrate to the granule cell layer of the dentate gyrus, where they project dendrites into the molecular layer and axons into the hilus and CA3. It is believed that these adult-born granule cells play an important role in learning and memory. Specifically, scientists have hypothesized that new-born neurons are engaged in new episodes of learning in order to reduce memory interference, thereby aiding in pattern separation and encoding of temporal contexts (1). There is little doubt that adult neurogenesis serves an important and beneficial purpose in the healthy brain. However, in an epileptic brain, adult neurogenesis can stop being merely beneficial and may actually contribute to the epileptogenic process (2).

A critical time window exists where immature and newborn granule cells are vulnerable to aberrant seizure-induced plasticity. An epileptogenic insult can induce newly generated granule cells to develop aberrant dendritic projections (hilar basal dendrites) and axonal projections (mossy fiber sprouting) (3). In addition, cells born after the insult can ectopically migrate and integrate improperly into the hilus (Figure 1). Together, these abnormalities are hypothesized to contribute to a breakdown of the dentate gate, the formation of recurrent excitatory loops in the hippocampus, and temporal lobe epileptogenesis (4). As such, blocking neurogenesis may actually be beneficial for treating epilepsy. Previously, Jung et al. (5) blocked neurogenesis through infusion of an antimitotic agent from 1 day before to 14 days after the epileptogenic insult and observed decreases in seizure frequency and duration. In a different study, Cho et al. (6) demonstrated that ablating granule cell progenitors 4 weeks before an epileptogenic insult resulted in a long-term reduction in seizure frequency but had no effect on seizure duration (Figure 1). To achieve a more complete ablation, Cho et al. also experimented with actively ablating granule cell progenitors 4 weeks before and again 4 weeks after the epileptogenic insult. This double-hit ablation strategy was expected to improve seizure attenuation, but instead, no significant seizure reduction was observed. The authors suggested that this may be a result of the second hit ablating both newly generated granule cells as well as reactive astrocytes activated after the insult. Although the role of reactive astrocytes in epileptogenesis is unclear, they could potentially play a protective role. Their ablation may then offset the benefits of ablating newly generated granule cells. An additional caveat to these prior studies is that the intervention was started before the epileptogenic insult—making the intervention strategy less clinically translatable and potentially altering the severity of the insult.

FIGURE 1.

Anti-neurogenesis treatments and granule cell abnormalities with respect to the timing of an epileptogenic insult. The timeline illustrates the morphologic abnormalities (below the timeline) associated with granule cells generated at various time points relative to an epileptogenic insult (pilocarpine-induced status epilepticus; illustrated as a lightning bolt) and when previous studies (above the timeline) have intervened with anti-neurogenesis treatments. Granule cells born before and after the epileptogenic insult have been shown to develop hilar basal dendrites and mossy fiber sprouting. More specifically, hilar basal dendrites have been observed in granule cells born 4 weeks before (3) to 3 weeks after (8) the epileptogenic insult. Mossy fiber sprouting has also been observed in newly generated granule cells born 4 weeks before the insult to 4 days after the insult, and it is possible that neurons generated later will continue to contribute to mossy fiber sprouting. Granule cells generated before the insult do not undergo ectopic migration into the hilus, but improper migration has been seen with cells born 4 days (3) to 3 weeks after the insult (8). Ablation of granule cell progenitors 4 weeks before the insult decreased seizure frequency but had no effect on seizure duration (6). Application of an antimitotic agent beginning 1 day before the insult and continuing for 2 weeks after the insult reduced both seizure frequency and duration (5). In their recent study, Hosford and colleagues targeted progenitor cells and granule cells that were generated up to 5 weeks before the insult. The ablation started 3 days after the insult. With this approach, they reduced seizure frequency but simultaneously increased individual seizure duration.

Hosford and colleagues therefore cleverly designed experiments that would target new granule cells generated before and after the insult but would only actually ablate the cells after the insult. To achieve this, they expressed simian diphtheria toxin receptor in progenitor cells (and thereafter newly generated granule cells) beginning 5 weeks before the epileptogenic insult. However, the diphtheria toxin itself was not given until 3 days after the insult. This strategy effectively ablated and reduced the generation of granule cells born within the critical time window for aberrant integration (Figure 1). Interestingly, with this ablation strategy, seizure control was still incomplete; seizure frequency decreased by 50% when examined 2 months after the insult. It is plausible that this may still be due to an incomplete ablation. Because of limitations with the Crerecombinase technology, hilar ectopic granule cells were only reduced by 50%. With a more effective ablation, the effect on seizure reduction may be more pronounced. Surprisingly, alongside the observed reduction in seizure frequency, the treatment simultaneously increased seizure duration. Therefore, the approach of ablating newborn granule cells may be a double-edged sword. One potential reason for the increase in seizure duration is that the ablation strategy did not exclusively target pathological granule cells but also those that had or would have migrated and integrated properly into the granule cell body layer. Indeed, it has been suggested that newly generated granule cells in a healthy brain may play a role in inhibiting synchronized network activity, perhaps by preferentially activating inhibitory neurons (7). Thus, this ablation strategy may cut both ways: diminishing pathological, hyperexcitable granule cells while simultaneously decreasing beneficial granule cells that could otherwise be contributing to limiting excitability. The net effect of ablating these cells is antiepileptic, but a more targeted method for ablating only pathological granule cells while preserving properly integrated cells would be ideal and may result in greater seizure control.

Preserving properly integrated granule cells may also have benefits regarding cognitive outcomes. As mentioned earlier, in a healthy system, newly generated granule cells contribute to learning and memory. As such, an additional concern with broad targeting of newly generated granule cells as an epilepsy therapy is that it may contribute to or exacerbate cognitive deficits. However, ongoing seizures may also result in cognitive impairment, and the benefits of stopping seizures may outweigh the negative consequences for cognition of reduced neurogenesis. Supporting this possibility, Cho et al. (6) previously examined the effect of disrupting neurogenesis in epileptic animals on cognitive outcomes and found that epilepsy-associated cognitive deficits actually improved when neurogenesis was blocked. Therefore, the net effect of ablating newly generated granule cells in the epileptic brain could be beneficial for cognition. Nevertheless, a more selective ablation could further limit any cognitive deficiencies that may arise from this treatment strategy.

Broadly, the findings of Hosford and colleagues support the hypothesis that abnormally integrated newly generated granule cells are detrimental to the dentate gate and are pro-epileptogenic. Their approach of targeting immature granule cells and ablating the cells after the epileptogenic insult is a novel method that successfully reduces seizure incidence. Yet, this strategy is not without shortcomings, as individual seizure duration is increased. Furthermore, properly integrated granule cells are collateral damage in this ablation strategy. Moving forward, more selective targeting of aberrant granule cells would be valuable, as this may improve seizure attenuation while also minimizing potential cognitive deficits. Currently, no clinical approach exists for the targeted ablation of adult-generated cells. Should someone develop the technology necessary for selective ablation of improperly integrated granule cells in epilepsy, it would be a promising novel treatment for epilepsy.