Targeting Seizure-Induced Neurogenesis in a Clinically Relevant Time Period Leads to Transient But Not Persistent Seizure Reduction.

Mesial temporal lobe epilepsy (mTLE), the most common form of medically refractory epilepsy in adults, is usually associated with hippocampal pathophysiology. Using rodent models of mTLE, many studies including work from our laboratory have shown that new neurons born around the onset of severe acute seizures known as status epilepticus (SE) are crucial for the process of epileptogenesis and targeting seizure-induced neurogenesis either genetically or pharmacologically can impact the frequency of chronic seizures. However, these studies are limited in their clinical relevance as none of them determines the potential of blocking new neurons generated after the epileptogenic insult to alleviate the development of chronic seizures. Therefore, using a pilocarpine-induced SE model of mTLE in mice of either sex, we show that >4 weeks of continuous and concurrent ablation of seizure-induced neurogenesis after SE can reduce the formation of spontaneous recurrent seizures by 65%. We also found that blocking post-SE neurogenesis does not lead to long-term seizure reduction as the effect was observed only transiently for 10 d with >4 weeks of continuous and concurrent ablation of seizure-induced neurogenesis. Thus, these findings provide evidence that seizure-induced neurogenesis when adequately reduced in a clinically relevant time period has the potential to transiently suppress recurrent seizures, but additional mechanisms need to be targeted to permanently prevent epilepsy development.SIGNIFICANCE STATEMENT Consistent with morphological and electrophysiological studies suggesting aberrant adult-generated neurons contribute to epilepsy development, ablation of seizure-induced new neurons at the time of the initial insult reduces the frequency of recurrent seizures. In this study, we show that continuous targeting of post-insult new neurons in a therapeutically relevant time period reduces chronic seizures; however, this effect does not persist suggesting possible additional mechanisms.

Neural stem cells and epilepsy: functional roles and disease-in-a-dish models.

Epilepsy is a disorder of the central nervous system characterized by spontaneous recurrent seizures. Although current therapies exist to control the number and severity of clinical seizures, there are no pharmacological cures or disease-modifying treatments available. Use of transgenic mouse models has allowed an understanding of neural stem cells in their relation to epileptogenesis in mesial temporal lobe epilepsy. Further, with the significant discovery of factors necessary to reprogram adult somatic cell types into pluripotent stem cells, it has become possible to study monogenic epilepsy-in-a-dish using patient-derived neurons. This discovery along with some of the newest technological advances in recapitulating brain development in a dish has brought us closer than ever to a platform in which to study and understand the mechanisms of this disease. These technologies will be critical in understanding the mechanism of epileptogenesis and ultimately lead to improved therapies and precision medicine for patients with epilepsy.

Mice with conditional NeuroD1 knockout display reduced aberrant hippocampal neurogenesis but no change in epileptic seizures.

Adult neurogenesis is significantly increased in the hippocampus of rodent models of temporal lobe epilepsy (TLE). These adult-generated neurons have recently been shown to play a contributing role in the development of spontaneous recurrent seizures (SRS). In order to eventually target pro-epileptic adult neurogenesis in the clinical setting, it will be important to identify molecular players involved in the control of aberrant neurogenesis after seizures. Here, we focused on NeuroD1 (ND1), a member of the bHLH family of transcription factors previously shown to play an essential role in the differentiation and maturation of adult-generated neurons in the hippocampus. Wild-type mice treated with pilocarpine to induce status epilepticus (SE) showed a significant up-regulation of NeuroD1+ immature neuroblasts located in both the granule cell layer (GCL), and ectopically localized to the hilar region of the hippocampus. As expected, conditional knockout (cKO) of NeuroD1 in Nestin-expressing stem/progenitors and their progeny led to a reduction in the number of NeuroD1+ adult-generated neurons after pilocarpine treatment compared to WT littermates. Surprisingly, there was no change in SRS in NeuroD1 cKO mice, suggesting that NeuroD1 cKO fails to reduce aberrant neurogenesis below the threshold needed to impact SRS. Consistent with this conclusion, the total number of adult-generated neurons in the pilocarpine model, especially the total number of Prox1+ hilar ectopic granule cells were unchanged after NeuroD1 cKO, suggesting strategies to reduce SRS will need to achieve a greater removal of aberrant adult-generated neurons.

NEUROD1 Instructs Neuronal Conversion in Non-Reactive Astrocytes.

Currently, all methods for converting non-neuronal cells into neurons involve injury to the brain; however, whether neuronal transdifferentiation can occur long after the period of insult remains largely unknown. Here, we use the transcription factor NEUROD1, previously shown to convert reactive glial cells to neurons in the cortex, to determine whether astrocyte-to-neuron transdifferentiation can occur under physiological conditions. We utilized adeno-associated virus 9 (AAV9), which crosses the blood-brain barrier without injury, to deliver NEUROD1 to astrocytes through an intravascular route. Interestingly, we found that a small, but significant number of non-reactive astrocytes converted to neurons in the striatum, but not the cortex. Moreover, astrocytes cultured to minimize their proliferative potential also exhibited limited neuronal transdifferentiation with NEUROD1 expression. Our results show that a single transcription factor can induce astrocyte-to-neuron conversion under physiological conditions, potentially facilitating future clinical approaches long after the acute injury phase.

REST regulation of gene networks in adult neural stem cells.

Adult hippocampal neural stem cells generate newborn neurons throughout life due to their ability to self-renew and exist as quiescent neural progenitors (QNPs) before differentiating into transit-amplifying progenitors (TAPs) and newborn neurons. The mechanisms that control adult neural stem cell self-renewal are still largely unknown. Conditional knockout of REST (repressor element 1-silencing transcription factor) results in precocious activation of QNPs and reduced neurogenesis over time. To gain insight into the molecular mechanisms by which REST regulates adult neural stem cells, we perform chromatin immunoprecipitation sequencing and RNA-sequencing to identify direct REST target genes. We find REST regulates both QNPs and TAPs, and importantly, ribosome biogenesis, cell cycle and neuronal genes in the process. Furthermore, overexpression of individual REST target ribosome biogenesis or cell cycle genes is sufficient to induce activation of QNPs. Our data define novel REST targets to maintain the quiescent neural stem cell state.

Aberrant hippocampal neurogenesis contributes to epilepsy and associated cognitive decline.

Acute seizures after a severe brain insult can often lead to epilepsy and cognitive impairment. Aberrant hippocampal neurogenesis follows the insult but the role of adult-generated neurons in the development of chronic seizures or associated cognitive deficits remains to be determined. Here we show that the ablation of adult neurogenesis before pilocarpine-induced acute seizures in mice leads to a reduction in chronic seizure frequency. We also show that ablation of neurogenesis normalizes epilepsy-associated cognitive deficits. Remarkably, the effect of ablating adult neurogenesis before acute seizures is long lasting as it suppresses chronic seizure frequency for nearly 1 year. These findings establish a key role of neurogenesis in chronic seizure development and associated memory impairment and suggest that targeting aberrant hippocampal neurogenesis may reduce recurrent seizures and restore cognitive function following a pro-epileptic brain insult.