Abstract
Selective Suppression of Excessive GluN2C Expression Rescues Early Epilepsy in a Tuberous Sclerosis Murine Model.
Lozovaya N, Gataullina S, Tsintsadze T, Tsintsadze V, Pallesi-Pocachard E, Minlevaeuv M, Goriounova NA, Buhler E, Watrin F, Shityakov S, Becker AJ, Bordey A, Milh M, Scavarda D, Bulteau C, Dorfmuller G, Delalande O, Represa A, Cardoso C, Dulac O, Ben-Ari Y, Burnashev N. Nat Commun 2014;5:4563.
Tuberous sclerosis complex (TSC), caused by dominant mutations in either TSC1 or TSC2 tumour suppressor genes is characterized by the presence of brain malformations, the cortical tubers that are thought to contribute to the generation of pharmacoresistant epilepsy. Here we report that tuberless heterozygote Tsc1+/− mice show functional upregulation of cortical GluN2C-containing N-methyl-D-aspartate receptors (NMDARs) in an mTOR-dependent manner and exhibit recurrent, unprovoked seizures during early postnatal life (<P19). Seizures are generated intracortically in the granular layer of the neocortex. Slow kinetics of aberrant GluN2C-mediated currents in spiny stellate cells promotes excessive temporal integration of persistent NMDAR-mediated recurrent excitation and seizure generation. Accordingly, specific GluN2C/D antagonists block seizures in Tsc1+/− mice in vivo and in vitro. Likewise, GluN2C expression is upregulated in TSC human surgical resections, and a GluN2C/D antagonist reduces paroxysmal hyperexcitability. Thus, GluN2C receptor constitutes a promising molecular target to treat epilepsy in TSC patients.
Commentary
The role of tubers in causing seizures in the genetic epilepsy, tuberous sclerosis complex (TSC), is often debated. TSC is an autosomal dominant disorder due to mutation of the TSC1 or TSC2 gene, involving hamartoma or tumor development in multiple organs, and associated with abnormal mechanistic target of rapamycin (mTOR) signaling.1 Neurological symptoms—including epilepsy, intellectual disability, and autism—usually represent the most disabling symptoms of the disease, with drug-resistant epilepsy being especially common. Traditionally, seizures in TSC have been attributed to cortical tubers, which are focal cortical malformations consisting of loss of normal cortical lamination and a variety of abnormal, enlarged, or dysmorphic cell types. Surgical removal of tubers can sometimes eliminate seizures in some TSC patients, supporting the idea that tubers may drive seizures. However, it remains controversial as to whether seizures typically start within the tubers directly or originate from perituberal regions surrounding tubers or, in rare cases, completely remote from tubers.2 Potentially triggered by excessive mTOR pathway signaling, a number of cellular and molecular abnormalities have been reported as a result of TSC gene mutations that may occur independently of, or outside of, gross pathological lesions, such as tubers. Thus, recently a nontuber hypothesis has emerged to challenge, or at least complement, the tuber dogma as to the pathophysiology of neurological manifestations of TSC, including epilepsy.
Another important question about the pathogenesis of TSC relates to the molecular genetic mechanisms required to generate the disease phenotype. As most TSC patients are heterozygous for either a TSC1 or TSC2 mutation, the phenotype could emerge simply due to haploinsufficiency from an approximately 50% reduction in the TSC1 or TSC2 gene products, hamartin or tuberin. However, in some autosomal dominant disorders, an alternative mechanism is that, in addition to the single germline mutation that the patient carries, a second, somatic mutation of the other gene copy is required to produce a phenotype, leaving no remaining functional gene (“loss of heterozygosity” or Knudsen's “two hit hypothesis”). There is strong evidence that a second mutation occurs in many tumors in TSC. However, the reported rate of finding loss of heterozygosity in tuber specimens is relatively low,3–4 suggesting that the genetic mechanisms causing neurological manifestations of TSC may differ from tumor mechanisms.
Animal models of TSC have shed some light on these important questions about the pathophysiology of neurological symptoms of TSC. Rodents that are heterozygous for either a Tsc1 or Tsc2 mutation generally show minimal pathological brain abnormalities, including the naturally occurring Eker rat and genetically engineered heterozygous knock-out mice. Interestingly, however, these heterozygous TSC models do show evidence of cognitive and behavioral deficits,5–7 indicating that gross pathological lesions and loss of heterozygosity are not necessary to cause these neurological symptoms. On the other hand, epilepsy has not been previously demonstrated in these heterozygous models. By comparison, severe epilepsy phenotypes have been produced in multiple knock-out mouse models of TSC, involving homozygous inactivation of either Tsc1 or Tsc2 in subsets of neurons and glia.8–9 All these homozygous models show significant pathological abnormalities, such as dysmorphic, cytomegalic neurons and astrocytosis, usually leading to megalencephaly, although focal, tuber-like abnormalities are not present in most of these cases.
The recent study by Lozovaya and colleagues provides some intriguing new insights into the pathophysiology of epilepsy in TSC. First, with detailed intracranial EEG recordings, they document for the first time that heterozygous Tsc1+/− mice do, in fact, have epilepsy. However, the seizures appear to be restricted to a limited developmental time window of less than 19 days of age (roughly equivalent to a young human child), which may explain why previous studies of heterozygous mice did not detect any seizures. As Tsc1+/− mice have minimal pathological brain abnormalities, the demonstration of seizures suggests that, similar to other cognitive and behavioral deficits that occur in these mice, gross pathological lesions and loss of heterozygosity are not necessary to cause epilepsy in TSC.
From a mechanistic standpoint, the major finding of this study is that Tsc1+/− mice exhibit excessive N-methyl-D-aspartate (NMDA) receptor-mediated glutamate synaptic activity, which appears to originate in layer 4 of the neocortex—particularly in a type of neuron called the spiny stellate cell—and then spreads to other cortical layers. This increased NMDA-mediated excitatory activity is due to upregulation of a specific NMDA receptor subunit, GluN2C, which is mTOR-dependent, as the mTOR inhibitor rapamycin reverses this abnormality. The dependence on mTOR signaling provides a direct mechanistic link between Tsc1 gene inactivation and the dysregulated expression of the glutamate receptor subunit. Interestingly, using another genetic modeling technique, they induced and compared heterozygyous and homozygous deletions of Tsc1 and found similar degrees of increased NMDA activity in both cases, indicating that the heterozygous state was maximally effective in inducing this change.
To assess the causal relationship of the NMDA receptor abnormality to epilepsy, a specific GluN2C/D antagonist was tested and found to have significant inhibitory effects on seizures in the Tsc1+/− mice. Furthermore, human brain samples (resected from TSC patients with intractable epilepsy) similarly showed excessive NMDA receptor activity, which was also blocked by the GluN2C/D antagonist, demonstrating the clinical relevance of the mouse findings and suggesting the potential utility of specific NMDA antagonists for treating epilepsy in TSC patients.
Overall, this study is significant in demonstrating a specific molecular abnormality in an ion channel, which can potentially cause seizures in TSC, independent of tubers and homozygous loss of Tsc1. This finding brings epilepsy in TSC full circle from a mechanistic standpoint. While channelopathies have been linked to several types of nonlesional epilepsy, the emphasis of epileptogenesis in TSC has traditionally been focused on the structural lesions of TSC. This study indicates that TSC may be a primary defect in neurotransmission.
There are, of course, unanswered questions and future directions from this work. The natural history of epilepsy in Tsc1+/− mice is atypical for human TSC in spontaneously resolving at an early developmental stage. The reasons for this epilepsy remission were not investigated, in particular whether this was related to a reversal in the NMDA receptor abnormality in the mice. Other studies with human TSC tissue and mouse models have found abnormalities in different glutamate receptor subunits and channels,10 indicating that there could be a multiplicity of molecular abnormalities that affect brain excitability in TSC. On the other hand, some currently used drugs have glutamate receptor antagonistic properties but are only moderately effective for TSC. While tubers may not be the entire story, epilepsy in TSC likely results from a combination of structural, cellular, and molecular mechanisms.