Ling-Hui Zeng

Rapamycin prevents epilepsy in a mouse model of tuberous sclerosis complex

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 (Tsc1GFAPCKO 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 Tsc1GFAPCKO mice.

The Mammalian Target of Rapamycin Signaling Pathway Mediates Epileptogenesis in a Model of Temporal Lobe Epilepsy

Understanding molecular mechanisms mediating epileptogenesis is critical for developing more effective therapies for epilepsy. We recently found that the mammalian target of rapamycin (mTOR) signaling pathway is involved in epileptogenesis, and mTOR inhibitors prevent epilepsy in a mouse model of tuberous sclerosis complex. Here, we investigated the potential role of mTOR in a rat model of temporal lobe epilepsy initiated by status epilepticus. Acute kainate-induced seizures resulted in biphasic activation of the mTOR pathway, as evident by an increase in phospho-S6 (P-S6) expression. An initial rise in P-S6 expression started ∼1 h after seizure onset, peaked at 3–6 h, and returned to baseline by 24 h in both hippocampus and neocortex, reflecting widespread stimulation of mTOR signaling by acute seizure activity. After resolution of status epilepticus, a second increase in P-S6 was observed in hippocampus only, which started at 3 d, peaked 5–10 d, and persisted for several weeks after kainate injection, correlating with the development of chronic epileptogenesis within hippocampus. The mTOR inhibitor rapamycin, administered before kainate, blocked both the acute and chronic phases of seizure-induced mTOR activation and decreased kainate-induced neuronal cell death, neurogenesis, mossy fiber sprouting, and the development of spontaneous epilepsy. Late rapamycin treatment, after termination of status epilepticus, blocked the chronic phase of mTOR activation and reduced mossy fiber sprouting and epilepsy but not neurogenesis or neuronal death. These findings indicate that mTOR signaling mediates mechanisms of epileptogenesis in the kainate rat model and that mTOR inhibitors have potential antiepileptogenic effects in this model.

Tsc2 gene inactivation causes a more severe epilepsy phenotype than Tsc1 inactivation in a mouse model of Tuberous Sclerosis Complex

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.

Kainate Seizures Cause Acute Dendritic Injury and Actin Depolymerization In Vivo

Seizures may cause brain injury via a variety of mechanisms, potentially contributing to cognitive deficits in epilepsy patients. Although seizures induce neuronal death in some situations, they may also have “nonlethal” pathophysiological effects on neuronal structure and function, such as modifying dendritic morphology. Previous studies involving conventional fixed tissue analysis have demonstrated a chronic loss of dendritic spines after seizures in animal models and human tissue. More recently, in vivo time-lapse imaging methods have been used to monitor acute changes in spines directly during seizures, but documented spine loss only under severe conditions. Here, we examined effects of secondary generalized seizures induced by kainate, on dendritic structure of neocortical neurons using multiphoton imaging in live mice in vivo and investigated molecular mechanisms mediating these structural changes. Higher-stage kainate-induced seizures caused dramatic dendritic beading and loss of spines within minutes, in the absence of neuronal death or changes in systemic oxygenation. Although the dendritic beading improved rapidly after the seizures, the spine loss recovered only partially over a 24 h period. Kainate seizures also resulted in activation of the actin-depolymerizing factor, cofilin, and a corresponding decrease in filamentous actin, indicating that depolymerization of actin may mediate the morphological dendritic changes. Finally, an inhibitor of the calcium-dependent phosphatase, calcineurin, antagonized the effects of seizures on cofilin activation and spine morphology. These dramatic in vivo findings demonstrate that seizures produce acute dendritic injury in neocortical neurons via calcineurin-dependent regulation of the actin cytoskeleton, suggesting novel therapeutic targets for preventing seizure-induced brain injury.

Rapamycin Attenuates the Development of Posttraumatic Epilepsy in a Mouse Model of Traumatic Brain Injury

Posttraumatic epilepsy is a major source of disability following traumatic brain injury (TBI) and a common cause of medically-intractable epilepsy. Previous attempts to prevent the development of posttraumatic epilepsy with treatments administered immediately following TBI have failed. Recently, the mammalian target of rapamycin complex 1 (mTORC1) pathway has been implicated in mechanisms of epileptogenesis and the mTORC1 inhibitor, rapamycin, has been proposed to have antiepileptogenic effects in preventing some types of epilepsy. In this study, we have tested the hypothesis that rapamycin has antiepileptogenic actions in preventing the development of posttraumatic epilepsy in an animal model of TBI. A detailed characterization of posttraumatic epilepsy in the mouse controlled cortical impact model was first performed using continuous video-EEG monitoring for 16 weeks following TBI. Controlled cortical impact injury caused immediate hyperactivation of the mTORC1 pathway lasting at least one week, which was reversed by rapamycin treatment. Rapamycin decreased neuronal degeneration and mossy fiber sprouting, although the effect on mossy fiber sprouting was reversible after stopping rapamycin and did not directly correlate with inhibition of epileptogenesis. Most posttraumatic seizures occurred greater than 10 weeks after TBI, and rapamycin treatment for one month after TBI decreased the seizure frequency and rate of developing posttraumatic epilepsy during the entire 16 week monitoring session. These results suggest that rapamycin may represent a rational treatment for preventing posttraumatic epilepsy in patients with TBI.

Impaired Astrocytic Gap Junction Coupling and Potassium Buffering in a Mouse Model of Tuberous Sclerosis Complex

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.

Modulation of astrocyte glutamate transporters decreases seizures in a mouse model of Tuberous Sclerosis Complex

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.

The Natural History and Treatment of Epilepsy in a Murine Model of Tuberous Sclerosis

Summary:  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 (Tsc1GFAPCKO mice), in which mice develop epilepsy and premature death. Here, we characterize the natural history of the epilepsy in Tsc1GFAPCKO mice in more detail and report acute effects of treatment with standard antiepileptic drugs on seizures in these mice.

Regulation of Cell Death and Epileptogenesis by the Mammalian Target of Rapamycin (mTOR): A Double-Edged Sword?

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.

Hippocampal seizures cause depolymerization of filamentous actin in neurons independent of acute morphological changes.

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.