Tuberous Sclerosis Complex: Molecular Pathogenesis and Animal Models

Neurosurg Focus 20 (1):E4, 2006

Tuberous sclerosis complex: molecular pathogenesis and animal models

LEANDRO R. PIEDIMONTE, M.D., IAN K. WAILES, M.D., AND HOWARD L. WEINER, M.D. Division of Pediatric Neurosurgery, Department of Neurosurgery, New York University School of Medicine, New York, New York

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 the characteristic brain lesions of tuberous sclerosis complex (TSC) called cortical tubers. The most devastating complications of TSC affect the central nervous system and include epilepsy, mental retardation, autism, and glial tumors. Relevant animal models, including conventional and conditional knockout mice, are valuable tools for studying the normal functions of tuberin and hamartin and the way in which disruption of their expression gives rise to the variety of clinical features that characterize TSC. In the future, these animals will be invaluable preclinical models for the development of highly specific and efficacious treatments for children affected with TSC.

KEY WORDS • tuberous sclerosis • pathogenesis • animal model

Tuberous sclerosis complex is an autosomal-dominant zures, mental retardation, and autism.1,51 The number and tumor predisposition syndrome that affects approximately location of cortical tubers and the patient’s age at seizure 1 in 7500 individuals worldwide.19 The TSC is character- onset are highly correlated with neurological outcomes.1,30 ized by benign hamartomatous growths in multiple organs, Epilepsy occurs in approximately 80% of affected indi- including the kidney, skin, retina, lung, and brain. Rarely, viduals. Infants affected with TSC commonly present with malignant tumors, including renal cell carcinoma, can partial motor seizures or infantile spasms within the 1st develop. Although it has been nearly a decade since link- year of life.9 An increase in seizure frequency and severity age analyses first revealed the two genetic loci associated is common during early childhood. With maturity, TSC- with TSC, the mechanism by which disruption of these associated infantile spasms frequently evolve into other seizure types, including partial motor, complex partial, and genes produces associated abnormalities remains poorly 8 understood. The current efforts aimed at developing pre- secondarily generalized ones. Epileptogenic foci identified by electroencephalography frequently correlate with corti- clinical animal models have provided important insights 9,20 into the pathogenesis of TSC and will continue to provide cal tubers visualized on magnetic resonance imaging. In useful tools for studying potential clinical therapies. addition, the progression from infantile spasms to other seizure types might reflect the formation of tubers at different times during cortical development. Thus, tubers in CLINICAL ASPECTS areas of the brain that become functionally mature earlier Neurological Manifestations and Brain Lesions can become epileptogenic before tubers located in brain regions that mature more slowly. Finally, due to the prema- Clinically, cortical tubers (pathognomonic lesions) cause turity of the patient, some as young as 20 weeks of gesta- extremely disabling CNS complications, which character- tion, physicians cannot accurately visualize each of these ize the TSC. Tubers are detected as high-intensity signals lesions, thus making the progression of epilepsy in patients that are often located in the junctions between the gray and with TSC extremely difficult to predict. white matter on T2-weighted magnetic resonance images. Tuberous sclerosis complex–associated seizures are The size and location of cortical tubers have been suggest- often refractory to conventional pharmacological therapies.␥ ed to correlate with the major CNS manifestations: sei- Improved seizure control has been achieved using the - aminobutyric acid agonist vigabatrin in some patients with 23 Abbreviations used in this paper: CNS = central nervous system; TSC. Pathological evidence suggests that cortical tubers GDP = guanosine diphosphate; GTP = guanosine triphosphate; represent areas of abnormal differentiation and migration. GTPase = guanosine triphosphatase; mTOR = mammalian target of Nevertheless, because surrounding areas of cortex are not rapamycin; Rheb = Ras homolog enriched in brain; TSC = tuberous disrupted and because tubers often disrupt normal cortical sclerosis complex. lamination, it has been hypothesized that these lesions

Neurosurg. Focus / Volume 20 / January, 2006 1 Unauthenticated | Downloaded 09/26/21 03:25 PM UTC L. R. Piedimonte, I. K. Wailes, and H. L. Weiner result from the defective differentiation, migration, and high frequency of TSC2 mutation might reflect a higher proliferation of a subpopulation of precursor cells (Fig. 1). intrinsic mutation rate for the TSC2 gene. Tubers contain dysmorphic neurons and an increased Individuals who are born with a mutated copy of the number of astrocytes; thus they are characterized as “giant TSC1 or TSC2 gene in all cells of their bodies are predis- cells.” The cellular origin of these giant cells is unknown; posed to the development of tumors in numerous organs; however, they may arise from a neuroglial precursor cell, this suggests that these genes act as tumor suppressors. Tu- because giant cells often express both mature neuronal and mors develop in these individuals when one or more somat- astrocytic proteins. Many giant cells also express proteins ic cells undergo a “second hit” and the remaining wild-type found in immature CNS cells, including nestin and TSC gene is inactivated. This loss of TSC1 or TSC2 expres- vimentin.6,35 Similarly, the morphological features of giant sion results in abnormal cell growth and proliferation. cells are variable.40 Recent evidence supports the proposal Indeed, hamartomas from patients with both familial and that cells of cortical tubers can undergo active proliferation sporadic forms of the disease have displayed a loss of the and that, despite their early formation, these lesions can be normal copy of TSC1 or TSC2, known as a loss of hetero- 35 zygosity, which indicates that these genes act as classic tu- more dynamic than previously assumed. 24,41,59 In addition to cortical tubers, low-grade astrocytic neo- mor suppressors. In vitro studies have also supported plasms develop in individuals affected with TSC. Subepen- the role of these genes as tumor suppressors. When either tuberin or hamartin is overexpressed in cultured cells, the dymal nodules are benign proliferative lesions that line the 2,37 surface of the lateral ventricles. Unlike the cortical tubers, cells undergo growth arrest. Similarly, downregulation of TSC2 expression in culture fibroblasts by antisense inhi- these lesions are often asymptomatic, but can develop into 54 subependymal giant cell astrocytomas. Although not frank- bition has been shown to increase cell proliferation. Tuberin and hamartin are highly expressed in the CNS of ly malignant, subependymal giant cell astrocytomas can 21,39 cause ventricular obstruction and hydrocephalus, which re- humans and mice in both neurons and glial cells. Both quire neurosurgical intervention.52 In addition to glial cells, of these proteins appear to be required for CNS development, and tuberin may be essential for neuronal differen- subependymal nodules and subependymal giant cell astro- 53 cytomas can also contain giant cells. These lesions, like cor- tiation. The effect these proteins have in the CNS is prob- tical tubers, are thought to develop early in life and have ably the same as they have where they act as tumor 38 suppressors. This is hypothesized because tuberin expres- been identified as early as 27 weeks of gestation. Like cor- 61 tical tubers, these lesions are thought to arise from abnormal sion is reduced or absent in 30% of sporadic astrocytomas. development of precursor cells during brain formation. There is conflicting evidence whether somatic mutations of the wild-type TSC1 or TSC2 allele in a precursor cell are required for cortical tuber formation in patients who have Genetics and Molecular Pathogenesis 24,41,49 only one normal copy of one of the two TSC genes. Linkage studies have identified two distinct loci that 48 Because these lesions are composed of many different cell undergo mutational inactivation in individuals with TSC. types, it can be difficult to identify the subset of cells that The phenotypic expression of TSC is demonstrated when has undergone a second mutational event. In support of the mutations occur in one of the two genes, TSC1 or TSC2. hypothesis that two genetic “hits” are required for TSC- The TSC1 gene is located on 9q43; this gene contains 23 59 associated tuber formation, recent evidence suggests that, exons and encodes the 130-kD hamartin protein. Hamar- in mice, neuroepithelial cell progenitors that lack TSC2 tin has little sequence homology to other known proteins. expression have many features of the giant cells found in The TSC2 gene on 16p13 encodes the 180- to 200-kD cortical tubers.42 tuberin protein. Tuberin contains a small region with The two abnormal cellular phenotypes in TSC involve sequence similarity to proteins with GTPase–activating an increase in both cellular proliferation and cell size. In- protein function. The GTPase–activating proteins are mo- sights into how the tuberin–hamartin complex might regu- lecules that negatively regulate small GTPase roteins relat- late this process originated from experiments with Droso- ed to the Ras oncogene. These Ras-like molecules are phila. When the homologs of TSC1 or TSC2 were mutated active when bound to GTP and are inactivated by its con- in flies, organogenesis proceeded normally, but dramatic version to GDP by GTPase. The GTPase proteins acceler- increases in organ size were observed.28,46 These size de- ate the conversion of the GTP–bound active form to the in- fects were attributed to both an increase in cell prolifera- active GDP–bound form by triggering this intrinsic tion, determined by measuring the number of mitotic cells, GTPase activity of Ras and related proteins. and an increase in individual cell size when TSC1 was Both hamartin and tuberin contain predicted coil–coil mutated.46 Overexpression of both TSC1 and TSC2 in these domains that mediate their interaction to form the flies reversed these defects. Because the insulin–signaling tuberin–hamartin protein complex.45,60 The formation of pathway has been linked to the regulation of cell growth this complex is critical for tuberin and hamartin functions, and proliferation,56 the possibility that the tuberin–hamartin and TSC-associated mutations frequently disrupt this inter- complex might regulate this signaling pathway seemed action and render the tuberin–hamartin complex function- plausible. In support of this hypothesis, overexpression of ally inactive. Identified TSC gene mutations include mis- TSC1 or TSC2 reinstated lethality in Drosophila mutants in sense and nonsense mutations, in-frame deletions, and which the insulin receptor was nonfunctional; it also re- large deletions.25,34 Whereas familial cases of TSC have an versed the cell proliferation defects in flies that overex- approximately equal distribution in families with TSC1 and pressed the insulin receptor.15,26,46 TSC2 mutations, TSC2 mutations are approximately four Additional studies, first in Drosophila and then in mam- times more common in sporadic cases.4,34 Because two malian cells, indicated that the tuberin–hamartin complex thirds of all patients with TSC have sporadic mutations, the functions in the insulin-like growth factor receptor pathway

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Fig. 1. Schematic showing how cellular differentiation in the CNS is altered by inactivation of the tuberin–hamartin complex. During development, neurons and astrocytes arise from neuroglial precursors. Giant cells that express a variety of cellular markers, including those characteristic of immature nervous system cells, are found in CNS lesions associated with TSC. This suggests that tuberin–hamartin complex inactivation interferes with the normal maturation of precursor cells and impairs their differentiation into more mature neurons and astrocytes. The black stars symbolize tuberin– hamartin complex inactivation. downstream of phosphatidylinositol 3–kinase and Akt (Fig. binding protein–1, whereas overexpression of TSC1 or 2). This pathway is known to regulate both cell prolifera- TSC2 inhibits S6 kinase pathway hyperactivation.27,33,42,57 tion and cell size. When the insulin receptor is activated, Phosphorylation of S6 kinase also increases in cells that phosphatidylinositol 3–kinase is recruited to the membrane express a TSC2 gene containing a human TSC gene muta- and triggers the production and release of the second mes- tion.17 senger phosphatidylinositol 3,4,5-trisphosphate. Activation The mechanism by which the tuberin–hamartin complex of Akt, a serine/threonine kinase, is effected by phospha- regulates mTOR, however, remained unclear until Rheb, tidylinositol 3,4,5-trisphosphate, and Akt has been shown a Ras-like GTPase, was identified as a target of tuberin to phosphorylate tuberin, resulting in its dissociation from GTPase-activating protein activity in Drosophila.17,64 The hamartin and inactivation of the tuberin–hamartin com- 11,47 Rheb molecule is required for cell-cycle progression and plex. cell growth in Drosophila.44 In the presence of the tuberin– The functional tuberin–hamartin complex normally in- hamartin complex, the GTP bound to Rheb is hydrolyzed hibits the activity of the mTor.16,27,42,57 Nevertheless, when to GDP, resulting in Rheb inactivation. When the tuber- the tuberin–hamartin complex is inactivated by Akt-medi- in–hamartin complex is inactivated, either by Akt-mediat- ated phosphorylation of tuberin, mTOR inhibition is ed tuberin phosphorylation or genetic mutation, Rheb is relieved. Activation of mTOR has been shown to trigger constitutively active, resulting in upregulation of mTOR- the phosphorylation of ribosomal S6 kinase and factor 4E dependent pathways, thereby increasing cell size. In this re- binding protein–1.14,18 The activation of these proteins regard, increased S6 kinase phosphorylation in Rheb-overex- pressing cells can be blocked by treatment with rapamycin, sults in an increase in protein synthesis and, ultimately, cell 3 growth. In cells in which the tuberin–hamartin complex is a drug that inhibits the mTOR pathway. permanently inactivated due to a genetic mutation, in- Although all functions of Rheb in mammalian cells are creased levels of phosphorylated S6 kinase and factor 4E not understood, it is known that posttranslational farnesyla- binding protein–1 are constitutively present, resulting in tion of Rheb is required for its activity and cell-cycle pro- unregulated cell growth. Several research groups have gression.5 In this regard, farnesyltransferase inhibitors that demonstrated that TSC1 and TSC2 mutant cells have ele- block the posttranslational activation of Rheb have been vated levels of phosphorylated S6 kinase and factor 4E shown to inhibit mTOR dependent S6 kinase activity.3 The

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Fig. 2. Schematic showing mechanisms for cell growth and proliferation. To relieve the tuberin–hamartin complex- mediated inhibition of cell size and proliferation, phosphatidylinositol 3–kinase (PI3k) and Akt are activated after stimu- lation of the insulin-like growth factor (IGF) receptor. The Akt phosphorylates tuberin, inactivating the complex and resulting in increased cell size and proliferation. The tuberin–hamartin complex negatively regulates Rheb, a small GTPase molecule that activates the mTOR/S6 kinase (S6K)/factor 4E binding protein (4E-BP)–1 signaling cascade, which is important for the regulation of cell size and proliferation. In addition, the tuberin–hamarin complex may regulate the expression of the cyclin-dependent kinase inhibitor p27Kip1, which is involved in both cell growth and size control. mechanism by which Rheb regulates mTOR is not under- animal models were made using Eker rats; these animals stood and is presently an area of active investigation. carry a spontaneous germline mutation in the rat homolog In addition to its effects on cell size, Rheb overexpres- of the human TSC2 gene and have been used as a model of Dro- hereditary renal cell carcinoma.63 These rats develop bilat- sion resulted in an increase in cell proliferation in 62 sophila.50,55 Nevertheless, it is not known if this effect of eral renal tumors and subependymal nodules. Unfortu- Rheb is mTOR dependent. In mammalian cells, cyclin-de- nately, genetic manipulations of rats have been hampered by a lack of suitable methods for generating targeted muta- pendent kinases are required for the transition from a quies- 29 cent state to one of active proliferation. Inhibitory mole- tions. In contrast, methods for generating mice with dis- cules, such as p27kip1, regulate these kinases. The expression ease-associated genetic changes are well established and kip1 have been widely used to study tumor suppressor gene of the cyclin-dependent kinase inhibitor p27 is reduced in 36 cells in which TSC1 or TSC2 expression is decreased.53,58 function. Not only does the use of transgenic mice allow Tuberin can regulate nuclear localization of p27kip1 because analysis of disruption or overexpression of single genes, the molecule is mislocalized to the cytoplasm in tuberin- but these mice can be interbred to assess the effects of mul- deficient fibroblasts.53 Moreover, inactivation of p27kip1 in tiple genetic changes. In this way, these genetically engi- mice is associated with increases in both cell size and pro- neered mice can serve as useful preclinical models for the liferation.13 Still, it is not clear whether Rheb or mTOR is study of disease pathophysiology and potential therapies. kip1 required for regulation of p27 expression. Initial attempts to recapitulate the human TSC phenotype in mice were performed using conventional knockout mice ANIMAL MODELS in which germline expression of TSC1 or TSC2 was inactivated. Mice that are homozygous for loss of TSC1 or TSC2 Despite the advances that Drosophila genetic and in die in midembryogenesis of apparent cardiac malformations 31–33 vitro systems brought to the understanding of the signaling and liver hypoplasia, whereas heterozygous animals areϩրϪ activities involving these proteins, a more desirable viable but develop renal and liver tumors. The TSC2ϩ Ϫ approach is now used. This technique is used to analyze the mice developed tumors at younger ages than TSC1 / ani- signaling and developmental roles of tuberin and hamartin mals. This finding is interesting in light of the observation by using in vivo animal models, with the molecules studied that patients who harbor TSC2 mutations often present with both individually and as a protein complex. Initially, the more severe diseases than those with TSC1 mutations.10 In

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ϩրϪ ϩրϪ the CNS of TSC2 and TSC1 mice, the numbers of as- lography in 34 children with tuberous sclerosis. Epilepsia 31: trocytes were increased, a finding that suggests that ha- 747–755, 1990 10. Dabora SL, Joswiak S, Franz DN, et al: Mutational analysis in martin and tuberin are important astrocyte growthϩրϪ regula- 58 TSC2 a cohort of 224 tuberous sclerosis patients indicates increased tors. When grown in vitro, however, astrocytes severity of TSC2, compared with TSC1, disease in multiple or- did not demonstrate a cell autonomous growth advantage. kip1 gans. Am J Hum Genet 68:64–80, 2001 Expression of the ϩրϪcell cycle–associated protein p27 was 11. Dan HC, Sun M, Yang L, et al: Phosphatidylinositol-3-kinase/ reduced in TSC2 astrocytes compared with wild-type Akt pathway regulates tuberous sclerosis tumor suppressor com- ones, which has led to the suggestion that tuberin might reg- J Biol Chem 4: kip1 58 plex by phosphorylation of tuberin. 35364–35370, ulate cell growth via regulation of p27 expression. In 2002 addition, compound heterozygotes that lacked one copy of 12. European Chromosome 16 Tuberous Sclerosis Consortium: Iden- both TSC1 and TSC2 exhibited further increases in the num- tification and characterization of the tuberous sclerosis gene on ber of astrocytes. Together, these observations support the chromosome 16. Cell 75:1305–1315,1993 role of tuberin and hamartin as regulators of cell growth and 13. Fero ML, Rivkin M, Tasch M, et al: A syndrome of multiorgan hyperplasia with features of gigantism, tumorigenesis and fe- proliferation for the CNS. male sterility in p27(Kip1)-deficient mice. Cell 85:733–744, 1996 14. Fingar DC, Salama S, Tsou C, et al: Mammalian cell size is con- CONCLUSIONS trolled by mTOR and its downstream targets S6K1 and 4EBP1/ eIF4E. Genes Dev 16:1472–1487, 2002 In the last several years, much has been learned about the 15. Gao X, Pan D: TSC1 and TSC2 tumor suppressors antagonize cellular functions of the TSC1 and TSC2 genes and the pro- insulin signaling in cell growth. Genes Dev 15:1383–1392, teins they encode. An improved understanding of the mol- 2001 ecular pathogenesis of TSC is critical to the development of 16. Gao X, Zhang Y, Arrazola P, et al: Tsc tumor suppressor pro- effective, highly specific therapies. Rapamycin, a specific teins antagonize aminoacid-TOR signaling. Nat Cell Biol 4: inhibitor of mTOR, is currently being tested in clinical can- 699–704, 2002 cer trials and might prove to be useful in some TSC-relat- 17. Garami A, Zwartkruis FJT, Nobukuni T, et al: Insulin activation ed tumors, including those that affect the CNS. 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