Progressive multifocal cystlike cortical tubers in tuberous sclerosis complex: Clinical and neuropathologic findings

Tuberous sclerosis complex (TSC) is a genetic disease characterized by the presence of hamartomatous lesions in multiple organs and cortical tubers in the brain. The majority of patients with TSC have epilepsy, although the mechanisms underlying epileptogenesis remain unknown. Tubers are traditionally thought to be stable lesions that result from abnormal corticogenesis in early fetal development. Recently, cystlike tubers have been identified in nearly half of patients with TSC, although the spectrum and natural history of these lesions remains unknown. Herein we report eight children with a high burden of cystlike tubers and present detailed clinical information on two children with documented progression. We also report neuropathologic findings of one of the cystlike cortical tubers resected in epilepsy surgery. These cases support the notion that cystlike tubers in TSC are not static lesions and can exhibit evolving characteristics over time. Further work evaluating how these lesions relate to epileptogenesis needs to be done.     

Impaired glial glutamate transport in a mouse tuberous sclerosis epilepsy model

Excessive astrocytosis in cortical tubers in tuberous sclerosis complex (TSC) suggests that astrocytes may be important for epileptogenesis in TSC. We previously demonstrated that astrocyte-specific Tsc1 gene inactivation in mice (Tsc1 cKO mice) results in progressive epilepsy. Here, we report that glutamate transporter expression and function is impaired in Tsc1 cKO astrocytes. Tsc1 cKO mice exhibit decreased GLT-1 and GLAST protein expression. Electrophysiological assays demonstrate a functional decrease in glutamate transport currents of Tsc1 cKO astrocytes in hippocampal slices and astrocyte cultures. These findings suggest that Tsc1 inactivation in astrocytes causes dysfunctional glutamate homeostasis, leading to seizure development in TSC.     

Mammalian target of rapamycin (mTOR) inhibition as a potential antiepileptogenic therapy: From tuberous sclerosis to common acquired epilepsies

Most current treatments for epilepsy are symptomatic therapies that suppress seizures but do not affect the underlying course or prognosis of epilepsy. The need for disease-modifying or "antiepileptogenic" treatments for epilepsy is widely recognized, but no such preventive therapies have yet been established for clinical use. A rational strategy for preventing epilepsy is to target primary signaling pathways that initially trigger the numerous downstream mechanisms mediating epileptogenesis. The mammalian target of rapamycin (mTOR) pathway represents a logical candidate, because mTOR regulates multiple cellular functions that may contribute to epileptogenesis, including protein synthesis, cell growth and proliferation, and synaptic plasticity. The importance of the mTOR pathway in epileptogenesis is best illustrated by tuberous sclerosis complex (TSC), one of the most common genetic causes of epilepsy. In mouse models of TSC, mTOR inhibitors prevent the development of epilepsy and underlying brain abnormalities associated with epileptogenesis. Accumulating evidence suggests that mTOR also participates in epileptogenesis due to a variety of other causes, including focal cortical dysplasia and acquired brain injuries, such as in animal models following status epilepticus or traumatic brain injury. Therefore, mTOR inhibition may represent a potential antiepileptogenic therapy for diverse types of epilepsy, including both genetic and acquired epilepsies.     

Epileptogenesis and reduced inward rectifier potassium current in tuberous sclerosis complex-1-deficient astrocytes

PURPOSE: Individuals with tuberous sclerosis complex (TSC) frequently have intractable epilepsy. To gain insights into mechanisms of epileptogenesis in TSC, we previously developed a mouse model of TSC with conditional inactivation of the Tsc1 gene in glia (Tsc1(GFAP)CKO mice). These mice develop progressive seizures, suggesting that glial dysfunction may be involved in epileptogenesis in TSC. Here, we investigated the hypothesis that impairment of potassium uptake through astrocyte inward rectifier potassium (Kir) channels may contribute to epileptogenesis in Tsc1(GFAP)CKO mice. METHODS: Kir channel function and expression were examined in cultured Tsc1-deficient astrocytes. Kir mRNA expression was analyzed in astrocytes microdissected from neocortical sections of Tsc1(GFAP)CKO mice. Physiological assays of astrocyte Kir currents and susceptibility to epileptiform activity induced by increased extracellular potassium were further studied in situ in hippocampal slices. RESULTS: Cultured Tsc1-deficient astrocytes exhibited reduced Kir currents and decreased expression of specific Kir channel protein subunits, Kir2.1 and Kir6.1. mRNA expression of the same Kir subunits also was reduced in astrocytes from neocortex of Tsc1(GFAP)CKO mice. By using pharmacologic modulators of signalling pathways implicated in TSC, we showed that the impairment in Kir channel function was not affected by rapamycin inhibition of the mTOR/S6K pathway, but was reversed by decreasing CDK2 activity with roscovitine or retinoic acid. Last, hippocampal slices from Tsc1(GFAP)CKO mice exhibited decreased astrocytic Kir currents, as well as increased susceptibility to potassium-induced epileptiform activity. CONCLUSIONS: Impaired extracellular potassium uptake by astrocytes through Kir channels may contribute to neuronal hyperexcitability and epileptogenesis in a mouse model of TSC.     

Basic Science

Laura A. Jansen , Erik J. Uhlmann , Peter B. Crino , David H. Gutmann , and Michael Wong
Individuals with Tuberous Sclerosis Complex (TSC) frequently suffer from intractable epilepsy. To gain insight into the causes of epilepsy in TSC, we previously developed a mouse model of TSC with inactivation of the Tsc1 gene selectively in brain astrocytes (Tsc1GFAPCKO mice). Astrocytes, the main category of glial cells in brain, are important supporting cells in the brain, and also have direct effects on brain physiology, development, and repair. These mice develop progressive seizures, suggesting that astrocyte dysfunction may be involved in the development of epilepsy in TSC. Since one of the important functions of astrocytes is to limit sudden elevations of extracellular potassium in the brain, which would lead to neuronal excitability, in this study we investigated the hypothesis that impairment of potassium uptake may contribute to the development of seizures in Tsc1GFAPCKO mice. Astrocytes take up potassium from the extracellular space via a membrane channel, called the Kir channel. Cultured astrocytes from Tsc1GFAPCKO mice exhibited reduced Kir potassium currents and decreased expression of specific Kir channel protein subunits. mRNA expression of the same Kir subunits was also reduced in astrocytes from Tsc1GFAPCKO mice. Furthermore, we showed that the impairment in Kir channel function was reversed with drugs (roscovitine and retinoic acid) that modulate cell signaling pathways implicated in TSC. Lastly, hippocampal slices from Tsc1GFAPCKO mice exhibited decreased astrocytic Kir currents, as well as increased susceptibility to potassium-induced seizure-like activity. In conclusion, impaired extracellular potassium uptake by astrocytes through Kir channels may contribute to increased neuronal excitability and the development of epilepsy in a mouse model of TSC.     

Mouse models of tuberous sclerosis complex

The most devastating complications of tuberous sclerosis complex affect the central nervous system and include epilepsy, mental retardation, autism, and glial tumors. Mutations in one of two genes, TSC1 and TSC2, result in a similar disease phenotype by disrupting the normal interaction of their protein products, hamartin and tuberin, which form a functional signaling complex. Disruption of these genes in the brain results in abnormal cellular differentiation, migration, and proliferation, giving rise to characteristic brain lesions called cortical tubers. Relevant animal models, including conventional and conditional knockout mice, are valuable tools for studying the normal functions of tuberin and hamartin and how disruption of their expression gives rise to the variety of clinical features that characterize tuberous sclerosis complex. In the future, these animals will be invaluable preclinical models for the development of highly specific and efficacious treatments for children affected with tuberous sclerosis complex.     

Expression of microRNAs miR21, miR146a,and miR155 in tuberous sclerosis complex cortical tubers and their regulation in human astrocytes and SEGA‐derived cell cultures

Tuberous sclerosis complex (TSC) is a genetic disease presenting with multiple neurological symptoms including epilepsy, mental retardation, and autism. Abnormal activation of various inflammatory pathways has been observed in astrocytes in brain lesions associated with TSC. Increasing evidence supports the involvement of microRNAs in the regulation of astrocyte‐mediated inflammatory response. To study the role of inflammation‐related microRNAs in TSC, we employed real‐time PCR and in situ hybridization to characterize the expression of miR21, miR146a, and miR155 in TSC lesions (cortical tubers and subependymal giant cell astrocytomas, SEGAs). We observed an increased expression of miR21, miR146a, and miR155 in TSC tubers compared with control and perituberal brain tissue. Expression was localized in dysmorphic neurons, giant cells, and reactive astrocytes and positively correlated with IL‐1β expression. In addition, cultured human astrocytes and SEGA‐derived cell cultures were used to study the regulation of the expression of these miRNAs in response to the proinflammatory cytokine IL‐1β and to evaluate the effects of overexpression or knockdown of miR21, miR146a, and miR155 on inflammatory signaling. IL‐1β stimulation of cultured glial cells strongly induced intracellular miR21, miR146a, and miR155 expression, as well as miR146a extracellular release. IL‐1β signaling was differentially modulated by overexpression of miR155 or miR146a, which resulted in pro‐ or anti‐inflammatory effects, respectively. This study provides supportive evidence that inflammation‐related microRNAs play a role in TSC. In particular, miR146a and miR155 appear to be key players in the regulation of astrocyte‐mediated inflammatory response, with miR146a as most interesting anti‐inflammatory therapeutic candidate. GLIA 2016;64:1066–1082     

Neuroimaging in tuberous sclerosis complex

PURPOSE OF REVIEW: In this review we discuss recent advances in the neuroimaging of patients with tuberous sclerosis complex (TSC), highlighting its application in improving clinical management, particularly in the case of intractable epilepsy. RECENT FINDINGS: Progress in structural and functional imaging has led to further characterization of the brain lesions in TSC. New magnetic resonance imaging techniques that can delineate the extent of structural brain abnormalities in TSC have been developed. Diffusion tensor imaging unveils the microstructural abnormalities of the brain lesions and of the morphologically normal appearing white matter in TSC. It can potentially identify the epileptogenic zone. Positron emission tomography scanning with 2-deoxy-2-[18F]fluoro-D-glucose can assess the full extent of functional brain abnormalities in TSC. The use of alpha [11C] methyl-L-tryptophan positron emission tomography scanning has proven to be a useful tool in the identification of epileptogenic tubers and has improved the outcome of surgery for epilepsy in TSC. SUMMARY: Major advances of neuroimaging in TSC have shown evidence of widespread structural and functional brain abnormalities. In TSC patients with intractable epilepsy, new neuroimaging modalities can now provide an accurate assessment of the epileptogenic zone, thereby permitting improved identification of patients who can have good seizure outcome following surgery for epilepsy.     

microRNAs在癫痫发生中的作用（英文）

Patients who have sustained brain injury or had developmental brain lesions present a non-negligible risk for developing delayed epilepsy. Finding therapeutic strategies to prevent development of epilepsy in at-risk patients represents a crucial medical challenge. Noncoding microRNA molecules( miRNAs) are promising candidates in this area. Indeed,deregulation of diverse brainspecific miRNAs has been observed in animal models of epilepsy as well as in patients with epilepsy,mostly in temporal lobe epilepsy( TLE). Herein we review deregulated miRNAs reported in epilepsy with potential roles in key molecular and cellular processes underlying epileptogenesis,namely neuroinflammation,cell proliferation and differentiation,migration,apoptosis,and synaptic remodeling. We provide an up-to-date listing of miRNAs altered in epileptogenesis and assess recent functional studies that have interrogated their role in epilepsy. Last,we discuss potential applications of these findings for the future development of disease-modifying therapeutic strategies for antiepileptogenesis.     

Mechanisms of epileptogenesis in tuberous sclerosis complex and related malformations of cortical development with abnormal glioneuronal proliferation

Malformations of cortical development (MCDs) are increasingly recognized as causes of medically intractable epilepsy. In order to develop more effective, rational therapies for refractory epilepsy related to MCDs, it is important to achieve a better understanding of the underlying mechanisms of epileptogenesis, but this is complicated by the wide variety of different radiographic, histopathological, and molecular features of these disorders. A subset of MCDs share a number of characteristic cellular and molecular abnormalities due to early defects in neuronal and glial proliferation and differentiation and have a particularly high incidence of epilepsy, suggesting that this category of MCDs with abnormal glioneuronal proliferation may also share a common set of primary mechanisms of epileptogenesis. This review critically analyzes both clinical and basic science evidence for overlapping mechanisms of epileptogenesis in this group of disorders, focusing on tuberous sclerosis complex, focal cortical dysplasia with balloon cells, and gangliogliomas. Specifically, the role of lesional versus perilesional regions, circuit versus cellular/molecular defects, and nonneuronal factors, such as astrocytes, in contributing to epileptogenesis in these MCDs is examined. An improved understanding of these various factors involved in epileptogenesis has direct clinical implications for optimizing current treatments or developing novel therapeutic approaches for epilepsy in these disorders.     

Involvement of microRNAs in epileptogenesis

Patients who have sustained brain injury or had developmental brain lesions present a non‐negligible risk for developing delayed epilepsy. Finding therapeutic strategies to prevent development of epilepsy in at‐risk patients represents a crucial medical challenge. Noncoding microRNA molecules (miRNAs) are promising candidates in this area. Indeed, deregulation of diverse brain‐specific miRNAs has been observed in animal models of epilepsy as well as in patients with epilepsy, mostly in temporal lobe epilepsy (TLE). Herein we review deregulated miRNAs reported in epilepsy with potential roles in key molecular and cellular processes underlying epileptogenesis, namely neuroinflammation, cell proliferation and differentiation, migration, apoptosis, and synaptic remodeling. We provide an up‐to‐date listing of miRNAs altered in epileptogenesis and assess recent functional studies that have interrogated their role in epilepsy. Last, we discuss potential applications of these findings for the future development of disease‐modifying therapeutic strategies for antiepileptogenesis.     

The neurobiology of the tuberous sclerosis complex

Tuberous sclerosis complex (TSC) is a multisystem disorder that affects numerous organ systems. Brain lesions that form during development, known as tubers, are highly associated with epilepsy, cognitive disability, and autism. Following the identification of two genes and their encoded proteins, TSC1 (hamartin) and TSC2 (tuberin), responsible for TSC, identification of several downstream protein cascades that might be affected in TSC have been discovered. Of primary importance is the mammalian target of rapamycin pathway that controls cell growth and protein synthesis. The mechanisms governing brain lesion growth have not been fully identified but likely altered regulation of the mammalian target of rapamycin cascade by hamartin and tuberin during development leads to aberrant cell growth. Secondary effects of TSC gene mutations might disrupt normal neuronal migration and cerebral cortical lamination. Numerous studies have identified changes in gene and protein expression in animal models of TSC and in human TSC brain specimens that contribute to altered brain cytoarchitecture. This review will provide an overview of the neurobiological aspects of TSC. Author to whom all correspondence and reprint requests should be addressed.     

Immunohistochemical expression of fibroblast growth factor (FGF)‐2 in epilepsy‐associated malformations of cortical development (MCDs)

To elucidate the biological significance of dysplastic cells in malformations of cortical development, an immunohistochemical study was performed to investigate fibroblast growth factor‐2 (FGF‐2) expression in corticectomy specimens from epilepsy patients, including focal cortical dysplasia (FCD) with balloon cells (BCs) (n = 4; age/sex = 2M, 14F, 24M, 45M), tubers of tuberous sclerosis complex (TSC‐tubers) (n = 2; 1F, 3F), FCD without BCs (n = 3; 23F, 23M, 25M), and gliotic lesions (n = 3; 12M, 25M, 29M). The nucleus and/or cytoplasm of astrocytes in all cases examined were positive for FGF‐2; however, FGF‐2 immunoreactivity was not detected in oligodendroglial cells. In all dysplastic lesions, FGF‐2 was detected in the astrocytic nuclei, and cytoplasm and/or nuclei of BCs. Dysplastic neurons (DNs) in FCD with BCs and TSC‐tubers were variably positive for FGF‐2 in the cytoplasm, but FGF‐2 was not detected in the neurons of FCD without BCs. The number of FGF‐2 immunoreactive cells (FGF‐2‐IR%) in FCD with BCs (46.0 ± 4.1%) was higher than that in FCD without BCs (19.8 ± 3.1%) and gliotic lesions (19.5 ± 3.3%) with statistical significance (P < 0.001). These results, together with previous reports showing FGF‐2 expression in neuroblasts and glioblasts in human fetal brain, and mainly in astrocytes in adult brain, suggest that FGF‐2 expression in MCDs reflects incomplete differentiation and maturation of dysplastic cells, and that FGF‐2‐IR% is associated with histological subtypes of MCD, reflecting the timing of insults underlying the pathogenesis of each disorder.     

Clinicopathological and immunohistochemical findings in an autopsy case of tuberous sclerosis complex

Tuberous sclerosis complex (TSC) is an autosomal dominant, multisystem disorder caused by mutations in either the TSC1 or TSC2 genes and characterized by developmental brain abnormalities. In the present study we discuss the neuropathological findings of a 32‐year‐old patient with a germ‐line mutation in the TSC2 gene. Post mortem MRI combined with histology and immunocytochemical analysis was applied to demonstrate widespread anatomical abnormalities of gray and white matter structure. TSC brain lesions were analyzed for loss of heterozygosity (LOH) on chromosome 16p13. The neuropathological supratentorial abnormalities were represented by multiple subependymal nodules (SENs) and cortical tubers. In addition to cerebral cortical lesions, cerebellar lesions and hippocampal sclerosis were also observed. LOH was not found in the cortical tubers and SENs of this patient. Immunocytochemical analysis of the TSC brain lesions confirmed the cell‐specific activation of the mTOR pathway in cortical tubers, SENs and cerebellum, as well as differential cellular localization of hamartin and tuberin, the TSC1 and TSC2 gene products. Examination of the pathological brain regions revealed activated microglial cells and disruption of blood‐brain barrier permeability. Predominant intralesional cell‐specific distribution was also detected for the multidrug transporter protein P‐gp, possibly explaining the mechanisms underlying the pharmacoresistance to antiepileptic drugs. Autopsy findings confirm the complexity of the brain abnormalities encountered in TSC patients and proved useful in clarifying certain aspects of the pathogenesis, epileptogenesis and pharmacoresistance of TSC lesions.     

Inflammatory processes in cortical tubers and subependymal giant cell tumors of tuberous sclerosis complex

Cortical tubers and subependymal giant cell tumors (SGCT) are two major cerebral lesions associated with tuberous sclerosis complex (TSC). In the present study, we investigated immunocytochemically the inflammatory cell components and the induction of two major pro-inflammatory pathways (the interleukin (IL)-1beta and complement pathways) in tubers and SGCT resected from TSC patients. All lesions were characterized by the prominent presence of microglial cells expressing class II-antigens (HLA-DR) and, to a lesser extent, the presence of CD68-positive macrophages. We also observed perivascular and parenchymal T lymphocytes (CD3(+)) with a predominance of CD8(+) T-cytotoxic/suppressor lymphoid cells. Activated microglia and reactive astrocytes expressed IL-1beta and its signaling receptor IL-1RI, as well as components of the complement cascade, such as C1q, C3c and C3d. Albumin extravasation, with uptake in astrocytes, was observed in both tubers and SGCT, suggesting that alterations in blood brain barrier permeability are associated with inflammation in TSC-associated lesions. Our findings demonstrate a persistent and complex activation of inflammatory pathways in cortical tubers and SGCT.     

Tuberous sclerosis complex and epilepsy: recent developments and future challenges

Tuberous sclerosis complex (TSC) is a congenital syndrome characterized by the widespread development of benign tumors in multiple organs, caused by mutations in one of the tumor suppressor genes, TSC1 or TSC2. About 80% of affected patients have a new mutation, and the remaining 20% have inherited a TSC gene mutation from a parent. The disorder affects approximately 1 in 6000 individuals. Cortical tubers are the neuropathological hallmark of TSC. The most common neurological manifestations of TSC are epilepsy, mental retardation, and autistic behavior. Epilepsy occurs in up to 80-90% of patients and is often intractable, with a poor response to anticonvulsant medications. While the molecular basis of TSC is well established, far less is known about the mechanisms of epilepsy in this disorder. In this article, we first summarize known clinical aspects of TSC with emphasis on its neurological features. Then, based on the molecular, pathological, immunohistochemical, neurochemical, and physiological properties of tubers in patients with TSC and in animal models, we discuss possible mechanisms of seizures and epileptogenesis in TSC. Finally, we provide an updated literature review and a consensus statement from the Tuberous Sclerosis Complex Working Group for future research into the mechanisms of epilepsy in TSC.     

Proliferative potential of human astrocytes

Although a number of studies have demonstrated proliferation of nonneoplastic astrocytes in experimental animal models, the proliferative potential of human astrocytes has not been well defined. Using double-label immunohistochemistry, we identified proliferating cells with the proliferation marker MIB-1 and astrocytes with glial fibrillary acidic protein staining in human biopsy and autopsy tissue. MIB-1 labeling of astrocytes was monitored in a variety of conditions containing significant numbers of reactive astrocytes, including infections, arteriovenous malformations, demyelinating lesions, metastatic tumors, and long-standing gliosis. Twenty-nine of a total of 54 cases showed no evidence of astrocyte-specific MIB-1 labeling despite prominent reactive changes. An average proliferation rate of 0.9% was present in the remaining 25 cases. Labeling indices were highest in infectious conditions and acute demyelinating lesions. We also examined astrocyte proliferation in 5 cases of progressive multifocal leukoencephalopathy. Astrocytic labeling indices were notably elevated in these cases, with an average labeling index of 5.8%. We conclude that low, but appreciable, astrocytic proliferation may occur in nonneoplastic human astrocytes. These findings have implications for astrocyte function in the normal and disease states and for the diagnostic distinction between reactive lesions and low-grade astrocytic neoplasms.