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Tuberous Sclerosis Complex (TSC) is an autosomal dominant, multi-system disorder, typically involving severe neurological symptoms, such as epilepsy, cognitive deficits and autism. Two genes, TSC1 and TSC2, encoding the proteins hamartin and tuberin, respectively, have been identified as causing TSC. Although there is a substantial overlap in the clinical phenotype produced by TSC1 and TSC2 mutations, accumulating evidence indicates that TSC2 mutations cause more severe neurological manifestations than TSC1 mutations. In this study, the neurological phenotype of a novel mouse model involving conditional inactivation of the Tsc2 gene in glial-fibrillary acidic protein (GFAP)-positive cells (Tsc2(GFAP1)CKO mice) was characterized and compared with previously generated Tsc1(GFAP1)CKO mice. Similar to Tsc1(GFAP1)CKO mice, Tsc2(GFAP1)CKO mice exhibited epilepsy, premature death, progressive megencephaly, diffuse glial proliferation, dispersion of hippocampal pyramidal cells and decreased astrocyte glutamate transporter expression. However, Tsc2(GFAP1)CKO mice had an earlier onset and higher frequency of seizures, as well as significantly more severe histological abnormalities, compared with Tsc1(GFAP1)CKO mice. The differences between Tsc1(GFAP1)CKO and Tsc2(GFAP1)CKO mice were correlated with higher levels of mammalian target of rapamycin (mTOR) activation in Tsc2(GFAP1)CKO mice and were reversed by the mTOR inhibitor, rapamycin. These findings provide novel evidence in mouse models that Tsc2 mutations intrinsically cause a more severe neurological phenotype than Tsc1 mutations and suggest that the difference in phenotype may be related to the degree to which Tsc1 and Tsc2 inactivation causes abnormal mTOR activation.

Identification of cell signaling mechanisms mediating seizure-related neuronal death and epileptogenesis is important for developing more effective therapies for epilepsy. The mammalian target of rapamycin (mTOR) pathway has recently been implicated in regulating neuronal death and epileptogenesis in rodent models of epilepsy. In particular, kainate-induced status epilepticus causes abnormal activation of the mTOR pathway, and the mTOR inhibitor, rapamycin, can decrease the development of neuronal death and chronic seizures in the kainate model. Here, we discuss the significance of these findings and extend them further by identifying upstream signaling pathways through which kainate status epilepticus activates the mTOR pathway and by demonstrating limited situations where rapamycin may paradoxically increase mTOR activation and worsen neuronal death in the kainate model. Thus, the regulation of seizure-induced neuronal death and epileptogenesis by mTOR is complex and may have dual, opposing effects depending on the physiological and pathological context. Overall, these findings have important implications for designing potential neuroprotective and antiepileptogenic therapies that modulate the mTOR pathway.

Astrocyte dysfunction may contribute to epileptogenesis and other neurological deficits in Tuberous Sclerosis Complex (TSC). In particular, decreased expression and function of astrocyte glutamate transporters have been implicated in causing elevated extracellular glutamate levels, neuronal death, and epilepsy in a mouse model of TSC (Tsc1(GFAP)CKO mice), involving inactivation of the Tsc1 gene primarily in astrocytes. Here, we tested whether pharmacological induction of astrocyte glutamate transporter expression can prevent the neurological phenotype of Tsc1(GFAP)CKO mice. Early treatment with ceftriaxone prior to the onset of epilepsy increased expression of astrocyte glutamate transporters, decreased extracellular glutamate levels, neuronal death, and seizure frequency, and improved survival in Tsc1(GFAP)CKO mice. In contrast, late treatment with ceftriaxone after onset of epilepsy increased glutamate transporter expression, but had no effect on seizures. These results indicate that astrocyte glutamate transporters contribute to epileptogenesis in Tsc1(GFAP)CKO mice and suggest novel therapeutic strategies for epilepsy in TSC directed at astrocytes., (2009 Elsevier Inc. All rights reserved.)

Abnormalities in astrocytes occur in the brains of patients with Tuberous Sclerosis Complex (TSC) and may contribute to the pathogenesis of neurological dysfunction in this disease. Here, we report that knock-out mice with Tsc1 gene inactivation in glia (Tsc1(GFAP)CKO mice) exhibit decreased expression of the astrocytic connexin protein, Cx43, and an associated impairment in gap junction coupling between astrocytes. Correspondingly, hippocampal slices from Tsc1(GFAP)CKO mice have increased extracellular potassium concentration in response to stimulation. This impaired potassium buffering can be attributed to abnormal gap junction coupling, as a gap junction inhibitor elicits an additional increase in potassium concentration in control, but not Tsc1(GFAP)CKO slices. Furthermore, treatment with a mammalian target of rapamycin inhibitor reverses the deficient Cx43 expression and impaired potassium buffering. These findings suggest that Tsc1 inactivation in astrocytes causes defects in astrocytic gap junction coupling and potassium clearance, which may contribute to epilepsy in Tsc1(GFAP)CKO mice.

Objective: Tuberous sclerosis complex (TSC) represents one of the most common genetic causes of epilepsy. TSC gene inactivation leads to hyperactivation of the mammalian target of rapamycin signaling pathway, raising the intriguing possibility that mammalian target of rapamycin inhibitors might be effective in preventing or treating epilepsy in patients with TSC. Mice with conditional inactivation of the Tsc1 gene primarily in glia (Tsc1(GFAP)CKO mice) develop glial proliferation, progressive epilepsy, and premature death. Here, we tested whether rapamycin could prevent or reverse epilepsy, as well as other cellular and molecular brain abnormalities in Tsc1(GFAP)CKO mice., Methods: Tsc1(GFAP)CKO mice and littermate control animals were treated with rapamycin or vehicle starting at postnatal day 14 (early treatment) or 6 weeks of age (late treatment), corresponding to times before and after onset of neurological abnormalities in Tsc1(GFAP)CKO mice. Mice were monitored for seizures by serial video-electroencephalogram and for long-term survival. Brains were examined histologically for astrogliosis and neuronal organization. Expression of phospho-S6 and other molecular markers correlating with epileptogenesis was measured by Western blotting., Results: Early treatment with rapamycin prevented the development of epilepsy and premature death observed in vehicle-treated Tsc1(GFAP)CKO mice. Late treatment with rapamycin suppressed seizures and prolonged survival in Tsc1(GFAP)CKO mice that had already developed epilepsy. Correspondingly, rapamycin inhibited the abnormal activation of the mammalian target of rapamycin pathway, astrogliosis, and neuronal disorganization, and increased brain size in Tsc1(GFAP)CKO mice., Interpretation: Rapamycin has strong efficacy for preventing seizures and prolonging survival in Tsc1(GFAP)CKO mice.

Purpose: Patients with tuberous sclerosis complex (TSC) often have severe epilepsy that is intractable to available therapies. The development of novel treatments for epilepsy in TSC would benefit greatly from a suitable animal model, but most animal models of TSC to date have few reported neurological abnormalities, such as epilepsy. We previously described a novel model of TSC, due to conditional inactivation of the Tsc1 gene in glia (Tsc1(GFAP)CKO mice), in which mice develop epilepsy and premature death. Here, we characterize the natural history of the epilepsy in Tsc1(GFAP)CKO mice in more detail and report acute effects of treatment with standard antiepileptic drugs on seizures in these mice., Methods: Video-EEG recordings were obtained from Tsc1(GFAP)CKO mice on a weekly basis, starting at 4 weeks of age until death, to monitor progression of interictal EEG abnormalities and seizures. In separate experiments, Tsc1(GFAP)CKO mice were monitored for interictal EEG abnormalities and seizures before and during treatment with phenobarbital, phenytoin, or saline., Results: Tsc1(GFAP)CKO mice developed seizures around 4-6 weeks of age and subsequently had progressive worsening of the interictal EEG background and seizure frequency over a month, culminating in death. Treatment with phenobarbital or phenytoin caused a reduction in seizure frequency, but did not improve EEG background or prevent death., Conclusions: Tsc1(GFAP)CKO mice develop progressive epilepsy. Acute treatment with standard antiepileptic drugs suppresses seizures in these mice, but does not affect long-term prognosis. Tsc1(GFAP)CKO mice represent a good model to test other drugs that may have true antiepileptogenic actions in TSC.

Aims Previous study suggests that mTOR signaling pathway may play an important role in epileptogenesis. The present work was designed to explore the contribution of raptor protein to the development of epilepsy and comorbidities. Methods Mice with conditional knockout of raptor protein were generated by cross-bred Rptorflox/flox mice with nestin- CRE mice. The expression of raptor protein was analyzed by Western blotting in brain tissue samples. Neuronal death and mossy fiber sprouting were detected by FJB staining and Timm staining, respectively. Spontaneous seizures were recorded by EEG-video system. Morris water maze, open field test, and excitability test were used to study the behaviors of Rptor CKO mice. Results As the consequence of deleting Rptor, downstream proteins of raptor in mTORC1 signaling were partly blocked. Rptor CKO mice exhibited decrease in body and brain weight under 7 weeks old and accordingly, cortical layer thickness. After kainic acid ( KA)-induced status epilepticus, overactivation of mTORC1 signaling was markedly reversed in Rptor CKO mice. Although low frequency of spontaneous seizure and seldom neuronal cell death were observed in both Rptor CKO and control littermates, KA seizure-induced mossy fiber spouting were attenuated in Rptor CKO mice. Additionally, cognitive-deficit and anxiety-like behavior after KA-induced seizures were partly reversed in Rptor CKO mice. Conclusion Loss of the Rptor gene in mice neural progenitor cells affects normal development in young age and may contribute to alleviate KA seizure-induced behavioral abnormalities, suggesting that raptor protein plays an important role in seizure comorbidities. [ABSTRACT FROM AUTHOR]

Abstract: Mice with inactivation of the Tuberous sclerosis complex-1 (Tsc1) gene in glia (Tsc1 GFAPCKO mice) have deficient astrocyte glutamate transporters and develop seizures, suggesting that abnormal glutamate homeostasis contributes to neurological abnormalities in these mice. We examined the hypothesis that Tsc1 GFAPCKO mice have elevated extracellular brain glutamate levels that may cause neuronal death, abnormal glutamatergic synaptic function, and associated impairments in behavioral learning. In vivo microdialysis documented elevated glutamate levels in hippocampi of Tsc1 GFAPCKO mice and several cell death assays demonstrated neuronal death in hippocampus and neocortex. Impairment of long-term potentiation (LTP) with tetanic stimulation was observed in hippocampal slices from Tsc1 GFAPCKO mice and was reversed by low concentrations of NMDA antagonist, indicating that excessive synaptic glutamate directly inhibited LTP. Finally, Tsc1 GFAPCKO mice exhibited deficits in two hippocampal-dependent learning paradigms. These results suggest that abnormal glutamate homeostasis predisposes to excitotoxic cell death, impaired synaptic plasticity and learning deficits in Tsc1 GFAPCKO mice. [Copyright &y& Elsevier]

Abstract: Seizures may exert pathophysiological effects on dendritic spines, but the molecular mechanisms mediating these effects are poorly understood. Actin represents a major structural protein of dendritic spines, and actin filaments (F-actin) can be depolymerized by the regulatory molecule, cofilin, leading to structural or functional changes in spines in response to normal physiological activity. To investigate mechanisms by which pathophysiological stimuli may affect dendritic spine structure and function, we examined changes in F-actin and cofilin in hippocampus due to 4-aminopyridine (4-AP)-induced seizures/epileptiform activity in vivo and in vitro and investigated possible structural correlates of these changes in actin dynamics. Within an hour of induction, seizure activity caused both a significant decrease in F-actin labeling, indicating depolymerization of F-actin, and a corresponding decrease in phosphorylated cofilin, signifying an increase in cofilin activity. However, 4-AP seizures had no overt short-term structural effects on dendritic spine density. By comparison, high potassium caused a more dramatic decrease in cofilin and an immediate dendritic beading and loss of dendritic spines. These findings indicate that activation of cofilin and depolymerization of F-actin represent mechanisms by which seizures may exert pathophysiological modulation of dendritic spines. In addition to affecting non-structural functions of spines, the degree to which overt structural changes occur with actin depolymerization is dependent on the severity and type of the pathophysiological stimulus. [Copyright &y& Elsevier]