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.

Delivery of a protein transduction domain-mediated Prdx6 protein ameliorates oxidative stress-induced injury in human and mouse neuronal cells.

Oxidative stress or reduced expression of naturally occurring antioxidants during aging has been identified as a major culprit in neuronal cell/tissue degeneration. Peroxiredoxin (Prdx) 6, a protective protein with GSH peroxidase and acidic calcium-independent phospholipase A2 activities, acts as a rheostat in regulating cellular physiology by clearing reactive oxygen species (ROS) and thereby optimizing gene regulation. We found that under stress, the neuronal cells displayed reduced expression of Prdx6 protein and mRNA with increased levels of ROS, and the cells subsequently underwent apoptosis. Using Prdx6 fused to TAT transduction domain, we showed evidence that Prdx6 was internalized in human brain cortical neuronal cells, HCN-2, and mouse hippocampal cells, HT22. The cells transduced with Prdx6 conferred resistance against the oxidative stress inducers paraquat, H2O2, and glutamate. Furthermore, Prdx6 delivery ameliorated damage to neuronal cells by optimizing ROS levels and overstimulation of NF-κB. Intriguingly, transduction of Prdx6 increased the expression of endogenous Prdx6, suggesting that protection against oxidative stress was mediated by both extrinsic and intrinsic Prdx6. The results demonstrate that Prdx6 expression is critical to protecting oxidative stress-evoked neuronal cell death. We propose that local or systemic application of Prdx6 can be an effective means of delaying/postponing neuronal degeneration.

Clonal Analysis of Newborn Hippocampal Dentate Granule Cell Proliferation and Development in Temporal Lobe Epilepsy.

Hippocampal dentate granule cells are among the few neuronal cell types generated throughout adult life in mammals. In the normal brain, new granule cells are generated from progenitors in the subgranular zone and integrate in a typical fashion. During the development of epilepsy, granule cell integration is profoundly altered. The new cells migrate to ectopic locations and develop misoriented "basal" dendrites. Although it has been established that these abnormal cells are newly generated, it is not known whether they arise ubiquitously throughout the progenitor cell pool or are derived from a smaller number of "bad actor" progenitors. To explore this question, we conducted a clonal analysis study in mice expressing the Brainbow fluorescent protein reporter construct in dentate granule cell progenitors. Mice were examined 2 months after pilocarpine-induced status epilepticus, a treatment that leads to the development of epilepsy. Brain sections were rendered translucent so that entire hippocampi could be reconstructed and all fluorescently labeled cells identified. Our findings reveal that a small number of progenitors produce the majority of ectopic cells following status epilepticus, indicating that either the affected progenitors or their local microenvironments have become pathological. By contrast, granule cells with "basal" dendrites were equally distributed among clonal groups. This indicates that these progenitors can produce normal cells and suggests that global factors sporadically disrupt the dendritic development of some new cells. Together, these findings strongly predict that distinct mechanisms regulate different aspects of granule cell pathology in epilepsy.

Morphological changes among hippocampal dentate granule cells exposed to early kindling-epileptogenesis.

Temporal lobe epilepsy is associated with changes in the morphology of hippocampal dentate granule cells. These changes are evident in numerous models that are associated with substantial neuron loss and spontaneous recurrent seizures. By contrast, previous studies have shown that in the kindling model, it is possible to administer a limited number of stimulations sufficient to produce a lifelong enhanced sensitivity to stimulus evoked seizures without associated spontaneous seizures and minimal neuronal loss. Here we examined whether stimulation of the amygdala sufficient to evoke five convulsive seizures (class IV or greater on Racine's scale) produce morphological changes similar to those observed in models of epilepsy associated with substantial cell loss. The morphology of GFP-expressing granule cells from Thy-1 GFP mice was examined either 1 day or 1 month after the last evoked seizure. Interestingly, significant reductions in dendritic spine density were evident 1 day after the last seizure, the magnitude of which had diminished by 1 month. Further, there was an increase in the thickness of the granule cell layer 1 day after the last evoked seizure, which was absent a month later. We also observed an increase in the area of the proximal axon, which again returned to control levels a month later. No differences in the number of basal dendrites were detected at either time point. These findings demonstrate that the early stages of kindling epileptogenesis produce transient changes in the granule cell body layer thickness, molecular layer spine density, and axon proximal area, but do not produce striking rearrangements of granule cell structure.