Epilepsy is a common neurologic disorder yet no treatments aimed at preventing epilepsy have been developed. Several molecules including genes containing cAMP response elements (CREs) in their promoters have been identified that contribute to the development of epilepsy, a process called epileptogenesis. When phosphorylated cAMP response element binding protein (CREB) increases transcription from CRE regulated promoters. CREB phosphorylation is increased in rodent epilepsy models, and in the seizure onset region of humans with medically intractable epilepsy (Rakhade, et al., 2005, Lee, et al., 2007, Lund, et al., 2008). Here we show that mice with decreased CREB levels (CREB αΔ mutants) have a ~50% reduction in spontaneous seizures following pilocarpine induced status epilepticus (SE) and require more stimulation to electrically kindle. Following SE, brain derived neurotrophic factor (BDNF) and inducible cAMP early repressor (ICER) mRNAs are differentially up-regulated in the hippocampus and cortex of the CREB αΔ mutants compared to wild-type mice, which may be contributing to differences in the severity of epilepsy. In contrast, we found no difference in KCC2 mRNA levels between the CREB αΔ and wild-type mice after SE. The mechanism by which BDNF and ICER mRNAs increase specifically in the CREB αΔ compared to wild-type mice following SE is not known. We did, however, find an increase in specific cAMP response element modulator (CREM) mRNA transcripts in the CREB αΔ mutants that might be responsible for the differential regulation of BDNF and ICER after SE. Altering CREB activity following a neurologic insult provides a therapeutic strategy for modifying epileptogenesis.
epilepsy; cyclic amp response element binding protein (CREB); cAMP response element modulator (CREM); inducible cAMP early repressor (ICER); pilocarpine; kindling
In the rodent model of temporal lobe epilepsy, there is extensive synaptic reorganization within the hippocampus following a single prolonged seizure event, after which animals eventually develop epilepsy. The perineuronal net (PN), a component of the neural extracellular matrix, primarily surrounds inhibitory interneurons and under normal conditions restricts synaptic reorganization. The objective of the current study was to explore the effects of status epilepticus (SE) on PNs in the adult hippocampus. The aggrecan component of the PN was studied, acutely (48 hours post-SE), sub-acutely (1 week post-SE), and during the chronic period (2 months post-SE). Aggrecan expressing PNs decreased by one week, likely contributing to a permissive environment for neuronal reorganization and remained attenuated at 2 months. The SE exposed hippocampus showed many PNs with poor structural integrity, a condition rarely seen in controls. Additionally, the decrease in the aggrecan component of the PN was preceded by a decrease in hyaluronan and proteoglycan link protein 1 (HAPLN1) and hyaluronan synthase 3 (HAS3), which are components of the PN known to stabilize the connection between aggrecan and hyaluronan, a major constituent of the extracellular matrix. These results were replicated in vitro with the addition of excess KCl to hippocampal cultures. Enhanced neuronal activity caused a decrease in aggrecan, HAPLN1, and HAS3 around hippocampal cells in vivo and in vitro leaving inhibitory interneurons susceptible to increased synaptic reorganization. These studies are the foundation for future experiments to explore how loss of the PN following SE contributes to the development of epilepsy.
Aggrecan; perineuronal net; seizure; HAPLN1; HAS3
Genetics and environment likely contribute to the development of medically intractable epilepsy, however, in most patients the specific combination of etiologies remains unknown. Here we undertook a multicenter retrospective cohort study of sex distribution in pediatric patients undergoing epilepsy surgery, and carried out a secondary analysis of the same population subdivided by histopathologic diagnosis. In the multicenter cohort of intractable epilepsy patients undergoing surgery regardless of etiology (n=206), 63% were boys, which is significantly more boys than expected for the general population (Fisher exact two-tailed p=0.017). Subgroup analysis found that of the 90 patients with a histopathologic diagnosis of focal cortical dysplasia, 72% were boys, giving an odds ratio (OR) of 2.5 (95% CI, 1.34 to 4.62) for male sex. None of the other etiologies had a male sex predominance. Future studies could examine the biological relevance and potential genetic and pathophysiological mechanisms of this observation.
focal cortical dysplasia; pediatric epilepsy; epilepsy surgery; gender; sex
Identify seizure onset electrodes that need to be resected for seizure freedom in children undergoing intracranial electroencephalography recording for treatment of medically refractory epilepsy. All children undergoing intracranial electroencephalography subdural grid electrode placement at the Children’s Hospital of Philadelphia from 2002-2008 were asked to enroll. We utilized intraoperative pictures to determine the location of the electrodes and define the resection cavity. A total of 15 patients had surgical fields that allowed for complete identification of the electrodes over the area of resection. Eight of 15 patients were seizure free after a follow up of 1.7 to 8 yr. Only one seizure-free patient had complete resection of all seizure onset associated tissue. Seizure free patients had resection of 64.1% of the seizure onset electrode associated tissue, compared to 35.2% in the not seizure free patients (p=0.05). Resection of tissue associated with infrequent seizure onsets did not appear to be important for seizure freedom. Resecting ≥ 90% of the electrodes from the predominant seizure contacts predicted post-operative seizure freedom (p=0.007). The best predictor of seizure freedom was resecting ≥ 90% of tissue involved in majority of a patient’s seizures. Resection of tissue under infrequent seizure onset electrodes was not necessary for seizure freedom.
Epilepsy; epilepsy surgery; cortical dysplasia; neocortical epilepsy; intracranial electroencephalography
A prolonged seizure, status epileptics (SE), is a potent stimulus for increased neurogenesis in the dentate gyrus of the hippocampus. Molecular mechanisms that regulate normal and pathologic cell birth in the dentate gyrus are poorly understood.
Lithium-pilocarpine was used to induce SE in immature post-natal day 20 rats. Newborn cells in the dentate were labeled with bromo-deoxyuridine to determine a time course of cell proliferation, and measure cell cycle length. In addition, we studied expression by western blot and immunohistochemistry of two known inhibitors of G1-S cell-cycle progression P27/Kip1 and P15/Ink4b following SE.
Cell proliferation in the dentate gyrus increases starting 2 hours after SE and is sustained for 40 days. Increased cell proliferation following SE is associated with a shortened dentate gyrus progenitor’s cell cycle, 15 hours in control to 12 hours in the SE animals. To identify molecules responsible for the shortened progenitor cell cycle we studied inhibitors of cell cycle progression P27/Kip1, and P15/Ink4b. We find decreased phosphorylation at P27/Kip1 Serine 10 and Threonine 187 following SE. While total P27/Kip1 and P15/Ink4b levels were not altered after SE. P27/Kip1 immunoreactivity was minimal in newborn but increased with maturation of the dentate granule neurons.
The sustained increase in dentate gyrus cell proliferation following SE provides a large pool of immature dentate granule cells prior to development of spontaneous seizures. A decrease in cell cycle length of dentate gyrus progenitors is at least partially responsible for increased numbers of newborn cells following SE.
Neurogenesis; Rats; Epilepsy; P27/Kip1
Historically, adrenal cortico-tropic hormone (ACTH) was used as first line treatment for infantile spasms, however there has been increasing use of topiramate as initial therapy. Here we report a retrospective study of ACTH and topiramate as initial treatment of infantile spasms. The neurology patient database at the Children's Hospital of Philadelphia was searched using the ICD-9 code for infantile spasms, and 50 patients were randomly chosen for chart review. We identified thirty-one patients receiving either ACTH or topiramate monotherapy (ACTH, n = 12, topiramate n = 19) as a first line treatment for infantile spasms. Twenty-six patients were symptomatic and five cryptogenic. Six patients treated with ACTH had resolution of clinical spasms and hypsarrhithmia within a month, but three relapsed. Four of the nineteen patients treated with topiramate eventually, though over a period of 0,1,8 or 69 months, had resolution of spasms and hypsarrhythmia.
Infantile spasms; ACTH; topiramate
Alterations in the brain that contribute to the development of epilepsy, also called epileptogensis, are not well understood, which makes it difficult to develop strategies for preventing epilepsy. Here we have studied the role of the CRE binding transcription factors, CREM and ICER, in the development of epilepsy following pilocarpine induced status epilepticus (SE) in mice. Following SE, ICER mRNA and protein is increased in neurons. The increase in ICER, however, is not necessary for neuronal injury following SE as pilocarpine treatment induces equivalent neuronal injury in pyramidal neurons of wild type and CREM/ICER null mice. Following SE, the CREM/ICER null mice develop a more severe epileptic phenotype experiencing ~3-fold more frequent spontaneous seizures. Together these data suggest that the increase in ICER mRNA following SE may have a role in suppressing the severity of epilepsy.
Seizures; Apoptosis; Epileptogenesis; Cyclic-AMP response element binding protein; CREB
Rates of regrowth after resection of subependymal giant cell astrocytoma (SEGA) are low, making surgical resection a successful and permanent therapeutic strategy. In addition to surgical resection of SEGAs, other treatment options now include medications and Gamma Knife™ therapy. Advising patients on medical versus surgical management of SEGAs is currently not easy. SEGAs have been reported to regrow if mTOR inhibitor therapy is stopped, raising the possibility that long-term medication may be required to prevent tumor growth and hydrocephalus. The question of regrowth following medication withdrawal will need to be addressed in more patients to help establish the optimal duration of therapy. The risks of surgery include acute morbidity and the permanent need for ventriculoperitoneal shunting, which must be balanced against the adverse effects of mTOR inhibitors, including immunosuppression (infections, mouth sores), hypercholesterolemia, and the need for chronic drug monitoring. Some additional benefits of mTOR inhibition in patients with tuberous sclerosis complex, however, may include shrinkage of angiofibromas and angiomyolipomas as well as a possible decrease in seizure burden. Recent reports of successful nonsurgical treatment of SEGAs are promising, and it is hoped that further specifics on dosing, duration, and long-term outcome will help patients and physicians to make informed therapeutic choices.
Present treatment recommendations for SEGAs include routine surveillance neuroimaging and close clinical follow-up, paying particular attention to signs and symptoms of acute hydrocephalus. If symptoms arise, or if serial neuroimaging demonstrates tumor growth, neurosurgical intervention is recommended. When gross total resection is impossible, rapamycin and everolimus should be considered, but may not offer a durable response.
Recently, we demonstrated that mRNA for the neuronal glutamate transporter, excitatory amino acid carrier 1 (EAAC1), is found in dendrites of hippocampal neurons in culture and in dendrites of hippocampal pyramidal cells after pilocarpine-induced status epilepticus (SE). We also showed that SE increased the levels of EAAC1 mRNA ~15-fold in synaptoneurosomes. In the present study, the effects of SE on the distribution EAAC1 protein in hippocampus were examined. In addition, the effects of Group 1 mGluR receptor activation on the levels of EAAC1 protein were examined in synaptoneurosomes prepared from sham control animals and from animals that experience pilocarpine-induced SE. We find that EAAC1 immunoreactivity increases in pyramidal cells of the hippocampus after 3 h of SE. In addition, the group I mGluR agonist, (S)-3,5-dihydroxyphenylglycine (DHPG), caused an increase in EAAC1 protein levels in hippocampal synaptoneurosomes; this effect of DHPG was much larger (~3- to 5-fold) after 3 h of SE. The DHPG-induced increases in EAAC1 protein were blocked by two different inhibitors of translation but not by inhibitors of transcription. mGluR1 or mGluR5 antagonists completely blocked the DHPG-induced increases in EAAC1 protein. DHPG also increased the levels of GluR2/3 protein, but this effect was not altered by SE. The DHPG-induced increase in EAAC1 protein was blocked by an inhibitor of the mammalian target of rapamycin (mTOR) or an inhibitor of extracellular signal-regulated kinase (ERK). These studies provide the first evidence EAAC1 translation can be regulated, and they show that regulated translation of EAAC1 is up-regulated after SE.
glutamate transport; EAAC1; epilepsy; pilocarpine; seizure; mGluR; dendritic targeting
The neuronal Na+-dependent glutamate transporter, excitatory amino acid carrier 1 (EAAC1, also called EAAT3), has been implicated in the control of synaptic spillover of glutamate, synaptic plasticity, and the import of cysteine for neuronal synthesis of glutathione. EAAC1 protein is observed in both perisynaptic regions of the synapse and in neuronal cell bodies. Although amino acid residues in the carboxyl terminal tail have been implicated in the dendritic targeting of EAAC1 protein, it is not known if mRNA for EAAC1 may also be targeted to dendrites. Sorting of mRNA to specific cellular domains provides a mechanism by which signals can rapidly increase translation in a local environment; this form of regulated translation has been linked to diverse biological phenomena ranging from establishment of polarity during embryogenesis to synapse development and synaptic plasticity. In the present study, EAAC1 mRNA sequences were amplified from dendritic samples that were mechanically harvested from low-density hippocampal neuronal cultures. In parallel analyses, mRNA for histone deacetylase 2 (HDAC-2) and glial fibrillary acidic protein (GFAP) was not detected, suggesting that these samples are not contaminated with cell body or glial mRNAs. EAAC1 mRNA also co-localized with Map2a (a marker of dendrites) but not Tau1 (a marker of axons) in hippocampal neuronal cultures by in situ hybridization. In control rats, EAAC1 mRNA was observed in soma and proximal dendrites of hippocampal pyramidal neurons. Following pilocarpine- or kainate-induced seizures, EAAC1 mRNA was present in CA1 pyramidal cell dendrites up to 200 μm from the soma. These studies provide the first evidence that EAAC1 mRNA localizes to dendrites and suggest that dendritic targeting of EAAC1 mRNA is increased by seizure activity and may be regulated by neuronal activity/depolarization.
glutamate transport; EAAC1; EAAT3; epilepsy; pilocarpine; seizure; mRNA targeting
The perineuronal net (PN), a component of the neural extracellular matrix (ECM), is a dynamic structure whose expression decreases following diminished physiological activity. Here, we analyzed the effects of increased neuronal activity on the development of aggrecan, a component of the PN, in the hippocampus. We show aggrecan expression to be prominent around parvalbumin (PV) interneurons in the postnatal hippocampus. Moreover, after seizure induction in early life there was a significant increase in aggrecan expression in a region specific manner during the course of development. We conclude that increased neuronal activity leads to accelerated expression of PNs in the hippocampus that attenuates in the adult hippocampus. This study shows the dynamic nature of the PN component of the ECM and the role neuronal activity has in molding the extracellular milieu of inhibitory interneurons.
Aggrecan; perineuronal net; extracellular matrix; hippocampus; kainic acid; neonatal seizure; interneurons; parvalbumin
The role of sharps and spikes, interictal epileptiform discharges (IEDs), in guiding epilepsy surgery in children remains controversial, particularly with intracranial EEG (IEEG). While ictal recording is the mainstay of localizing epileptic networks for surgical resection, current practice dictates removing regions generating frequent IEDs if they are near the ictal onset zone. Indeed, past studies suggest an inconsistent relationship between IED and seizure onset location, though these studies were based upon relatively short EEG epochs.
We employ a previously validated, computerized spike detector, to measure and localize IED activity over prolonged, representative segments of IEEG recorded from 19 children with intractable, mostly extra temporal lobe epilepsy. Approximately 8 hours of IEEG, randomly selected thirty-minute segments of continuous interictal IEEG per patient were analyzed over all intracranial electrode contacts.
When spike frequency was averaged over the 16-time segments, electrodes with the highest mean spike frequency were found to be within the seizure onset region in 11 of 19 patients. There was significant variability between individual 30-minute segments in these patients, indicating that large statistical samples of interictal activity were required for improved localization. Low voltage fast EEG at seizure onset was the only clinical factor predicting IED localization to the seizure onset region.
Our data suggest that automated IED detection over multiple representative samples of IEEG may be of utility in planning epilepsy surgery for children with intractable epilepsy. Further research is required to better determine which patients may benefit from this technique a priori.
Spike density; intracranial EEG; Seizure onset; Pediatric Epilepsy
Multiple studies have highlighted how seizures induce different molecular, cellular and physiologic consequences in an immature as compared to a mature brain. In keeping with these studies, seizures early in life alter dentate granule cell birth in different and even opposing fashion to adult seizure models (see Table1). During the first week of rodent post-natal life, seizures decrease cell birth in the post-ictal period but do not alter maturation of newborn cells. Seizures during the second week of life have varied effects on dentate granule cell birth, either causing no change or increasing birth, and may promote a mild increase in neuronal survival. During the third and fourth weeks of life, seizures begin to increase cell birth similar to that seen in adult seizure models. Interestingly, animals that experienced seizure during the first month of life have an increase in cell birth during adulthood opposite to the reported decrease in chronic animals experiencing a prolonged seizure as an adult. Children have more ongoing cell birth in the dentate gyrus than adults and markers of cell division are further increased in children with refractory temporal lobe epilepsy. There are clear age dependent differences in how seizures alter cell birth in the dentate gyrus both acutely and chronically. Future studies need to focus on how these changes in neurogenesis influence dentate gyrus function, and what they imply for epileptogenesis and the learning and memory impairments so commonly found in children with temporal lobe epilepsy.
MEF2C haploinsufficiency syndrome is an emerging neurodevelopmental disorder associated with intellectual disability, autistic features, epilepsy, and abnormal movements. We report 16 new patients with MEF2C haploinsufficiency, including the oldest reported patient with MEF2C deletion at 5q14.3. We detail the neurobehavioral phenotype, epilepsy, and abnormal movements, and compare our subjects with those previously reported in the literature. We also investigate Mef2c expression in the developing mouse forebrain. A spectrum of neurofunctional deficits emerges, with hyperkinesis a consistent finding. Epilepsy varied from absent to severe, and included intractable myoclonic seizures and infantile spasms. Subjects with partial MEF2C deletion were statistically less likely to have epilepsy. Finally, we confirm that Mef2c is present both in dorsal primary neuroblasts and ventral gamma-aminobutyric acid(GABA)ergic interneurons in the forebrain of the developing mouse. Given interactions with several key neurodevelopmental genes such as ARX, FMR1, MECP2, and TBR1, it appears that MEF2C plays a role in several developmental stages of both dorsal and ventral neuronal cell types.
MEF2C haploinsufficiency; Intellectual disability; Autism; Infant-onset myoclonic epilepsy; Infantile spasms; Hyperkinesis; Deletion 5q14.3
MicroRNAs regulate protein synthesis by binding non-translated regions of mRNAs and suppressing translation and/or increasing mRNA degradation. MicroRNAs play an important role in the nervous system including controlling synaptic plasticity. Their expression is altered in disease states including stroke, head injury and epilepsy. To better understand microRNA expression changes that might contribute to the development of epilepsy, microRNA arrays were performed on rat hippocampus 4 hours, 48 hours and 3 weeks following an episode of pilocarpine induced status epilepticus. Eighty microRNAs increased at one or more of the time points. No microRNAs decreased at 4 hours, and only a few decreased at 3 weeks, but 188 decreased 48 hours after status epilepticus. The large number of microRNAs with altered expression following status epilepticus suggests that microRNA regulation of translation has the potential to contribute to changes in protein expression during epileptogenesis. We carried out a second set of array’s comparing microRNA expression at 48 hours in synaptoneurosome and nuclear fractions of the hippocampus. In control rat hippocampi multiple microRNAs were enriched in the synaptoneurosomal fraction as compared to the nuclear fraction. In contrast, 48 hours after status epilepticus only one microRNA was enriched in the synaptoneurosome fraction. The loss of microRNAs enriched in the synaptoneurosomal fraction implies a dramatic change in translational regulation in synapses 48 hours after status epilepticus.