Genetics and Molecular Biology of Tuberous Sclerosis Complex

Tuberous Sclerosis Complex is a multisystem disorder exhibiting a wide range of manifestations characterized by tumour-like lesions called hamartomas in the brain, skin, eyes, heart, lungs and kidneys. Tuberous Sclerosis Complex is genetically determined with an autosomal dominant inheritance and is caused by inactivating mutations in either the TSC1 or TSC2 genes. TSC1/2 genes play a fundamental role in the regulation of phosphoinositide 3-kinase (PI3K) signalling pathway, inhibiting the mammalian target of rapamycin (mTOR) through activation of the GTPase activity of Rheb. Mutations in TSC1/2 genes impair the inhibitory function of the hamartin/tuberin complex, leading to phosphorylation of the downstream effectors of mTOR, p70 S6 kinase (S6K), ribosomal protein S6 and the elongation factor binding protein 4E-BP1, resulting in uncontrolled cell growth and tumourigenesis.     

Phosphorylation and binding partner analysis of the TSC1-TSC2 complex

Tuberous sclerosis complex (TSC) is an autosomal dominant benign tumour syndrome caused by mutations to either the TSC1 or TSC2 tumour suppressor gene. The TSC1 and TSC2 gene products, TSC1 and TSC2, form a protein complex that integrates inputs from multiple signalling cascades to inactivate the small GTPase rheb, and thereby inhibit mTOR-dependent cell growth. We have used matrix-assisted laser desorption/ionisation time-of-flight and Fourier transform mass spectrometry to identify TSC1 and TSC2 phosphorylation sites and candidate TSC1 and TSC2 interacting proteins. We identified three sites of TSC2 phosphorylation and a novel site of TSC1 phosphorylation, and investigated the roles of these sites in regulating the activity of the TSC1-TSC2 complex. In addition, we identified three TSC1-TSC2 interacting proteins, including DOCK7 a putative rhebGEF.     

The tuberous sclerosis genes and regulation of the cyclin-dependent kinase inhibitor p27

Tuberous sclerosis complex (TSC) is an autosomal dominant tumor syndrome that affects approximately 1 in 6000 individuals. It is characterized by the development of tumors, named hamartomas, in the kidneys, heart, skin and brain. The latter often cause seizures, mental retardation, and a variety of developmental disorders, including autism. This disease is caused by mutations within the tumor suppressor gene TSC1 on chromosome 9q34 encoding hamartin or within TSC2 on chromosome 16p13.3 encoding tuberin. TSC patients carry a mutant TSC1 or TSC2 gene in each of their somatic cells, and loss of heterozygosity has been documented in a wide variety of TSC tumors. Recent data suggest that functional inactivation of TSC proteins might also be involved in the development of other diseases not associated with TSC, such as sporadic bladder cancer, breast cancer, ovarian carcinoma, gall bladder carcinoma, non-small-cell carcinoma of the lung, and Alzheimer's disease. Tuberin and hamartin form a heterodimer, suggesting they might affect the same processes. Tuberin is assumed to be the functional component of the complex and has been implicated in the regulation of different cellular functions. The TSC proteins regulate cell size control due to their involvement in the insulin signalling pathway. Furthermore, they are potent positive regulators of the cyclin-dependent kinase inhibitor p27, a major regulator of the mammalian cell cycle. Here we review the current knowledge on how mutations within the TSC genes could trigger deregulation of stability and localization of the tumor suppressor p27.     

Tumour suppressors hamartin and tuberin: intracellular signalling

Tumour suppressors hamartin and tuberin, encoded by tuberous sclerosis complex 1(TSC1) and TSC2 genes, respectively, are critical regulators of cell growth and proliferation. Mutations in TSC1 and TSC2 genes are the cause of an autosomal dominant disorder known as tuberous sclerosis complex (TSC). Another genetic disorder, lymphangioleiomyomatosis (LAM), is also associated with mutations in the TSC2 gene. Hamartin and tuberin control cell growth by negatively regulating S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1), potentially through their upstream modulator mammalian target of rapamycin (mTOR). Growth factors and insulin promote Akt/PKB-dependent phosphorylation of tuberin, which in turn, releases S6K1 from negative regulation by tuberin and results in the activation of S6K1. Although much has been written regarding the molecular genetics of TSC and LAM, which is associated with either the loss of or mutation in the TSC1 and TSC2 genes, few reviews have addressed the intracellular signalling pathways regulated by hamartin and tuberin. The current review will fill the gap in our understanding of their role in cellular signalling networks, and by improving this understanding, an integrated picture regarding the normal function of tuberin and hamartin is beginning to emerge.     

Mechanisms of TSC-mediated control of synapse assembly and axon guidance

Tuberous sclerosis complex is a dominant genetic disorder produced by mutations in either of two tumor suppressor genes, TSC1 and TSC2; it is characterized by hamartomatous tumors, and is associated with severe neurological and behavioral disturbances. Mutations in TSC1 or TSC2 deregulate a conserved growth control pathway that includes Ras homolog enriched in brain (Rheb) and Target of Rapamycin (TOR). To understand the function of this pathway in neural development, we have examined the contributions of multiple components of this pathway in both neuromuscular junction assembly and photoreceptor axon guidance in Drosophila. Expression of Rheb in the motoneuron, but not the muscle of the larval neuromuscular junction produced synaptic overgrowth and enhanced synaptic function, while reductions in Rheb function compromised synapse development. Synapse growth produced by Rheb is insensitive to rapamycin, an inhibitor of Tor complex 1, and requires wishful thinking, a bone morphogenetic protein receptor critical for functional synapse expansion. In the visual system, loss of Tsc1 in the developing retina disrupted axon guidance independently of cellular growth. Inhibiting Tor complex 1 with rapamycin or eliminating the Tor complex 1 effector, S6 kinase (S6k), did not rescue axon guidance abnormalities of Tsc1 mosaics, while reductions in Tor function suppressed those phenotypes. These findings show that Tsc-mediated control of axon guidance and synapse assembly occurs via growth-independent signaling mechanisms, and suggest that Tor complex 2, a regulator of actin organization, is critical in these aspects of neuronal development.     

Rheb is a direct target of the tuberous sclerosis tumour suppressor proteins

Mutations in the TSC1 or TSC2 genes cause tuberous sclerosis, a benign tumour syndrome in humans. Tsc2 possesses a domain that shares homology with the GTPase-activating protein (GAP) domain of Rap1-GAP, suggesting that a GTPase might be the physiological target of Tsc2. Here we show that the small GTPase Rheb (Ras homologue enriched in brain) is a direct target of Tsc2 GAP activity both in vivo and in vitro. Point mutations in the GAP domain of Tsc2 disrupted its ability to regulate Rheb without affecting the ability of Tsc2 to form a complex with Tsc1. Our studies identify Rheb as a molecular target of the TSC tumour suppressors.     

Generation of a conditional disruption of the Tsc2 gene

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations in the TSC1 or TSC2 gene. Patients afflicted with TSC develop tumors in various organ systems, but cerebral pathology is particularly severe. Conventional gene disruption of the Tsc1 or Tsc2 gene in mice cause limited central nervous system pathology. Homozygous deletion of either gene causes midgestation lethality. To circumvent the homozygous lethality of the conventional Tsc2 knockout we have generated a conditional allele of the Tsc2 gene by homologous recombination in mouse ES cells. The homozygous Tsc2(flox/flox) mice are identical to wildtype in many organs typically affected by TSC, especially the brain. Using this Tsc2(flox) allele we have generated a null allele using Cre recombination. This allele will be useful in investigating TSC pathology with appropriate cell and organ specific Cre-transgenic mice.     

Evidence that lymphangiomyomatosis is caused by TSC2 mutations: chromosome 16p13 loss of heterozygosity in angiomyolipomas and lymph nodes from women with lymphangiomyomatosis.

Lymphangiomyomatosis (LAM) is a rare disease, of unknown etiology, affecting women almost exclusively. Lung transplantation is the only consistently effective therapy for LAM. Microscopically, LAM consists of a diffuse proliferation of smooth muscle cells. LAM can occur without evidence of other disease (referred to as "sporadic LAM") or in association with tuberous sclerosis complex (TSC). TSC is an autosomal dominant tumor suppressor gene syndrome characterized by seizures, mental retardation, and tumors in the brain, heart, skin, and kidney. Renal angiomyolipomas occur in approximately 50% of sporadic LAM patients and in 70% of TSC patients. Loss of heterozygosity (LOH) in the chromosomal region for the TSC2 gene occurs in 60% of TSC-associated angiomyolipomas. Because of the similar pulmonary and renal manifestations of TSC and sporadic LAM, we hypothesized that LAM and TSC have a common genetic basis. We analyzed renal angiomyolipomas, from 13 women with sporadic LAM, for LOH in the regions of the TSC1 (chromosome 9q34) and TSC2 (chromosome 16p13) genes. TSC2 LOH was detected in seven (54%) of the angiomyolipomas. We also found TSC2 LOH in four lymph nodes from a woman with retroperitoneal LAM. No TSC1 LOH was found. Our findings indicate that the TSC2 gene may be involved in the pathogenesis of sporadic LAM. However, genetic transmission of LAM has not been reported. Women with LAM may have low-penetrance germ-line TSC2 mutations, or they may be mosaic, with TSC2 mutations in the lung and the kidney but not in other organs.     

The tuberous sclerosis complex: balancing proliferation and survival

Mutations in genes encoding either hamartin [TSC1 (tuberous sclerosis complex 1)] or tuberin (TSC2) result in a multisystem disorder characterized by the development of benign tumours and hamartomas in several organs. The TSC1 and TSC2 proteins form a complex that lies at the crossroad of many signalling pathways integrating the energy status of the cell with signals induced by nutrients and growth factors. The TSC1/2 complex is a critical negative regulator of mTORC1 [mTOR (mammalian target of rapamycin) complex 1], and by that controls anabolic processes to promote cell growth, proliferation and survival. In the present paper, we review recent evidence highlighting the notion that the TSC1/2 complex simultaneously controls mTOR-dependent and mTOR-independent signals critical for the balancing of cell proliferation and cell death.     

Enhanced episodic-like memory and kindling epilepsy in a rat model of tuberous sclerosis

Tuberous sclerosis complex (TSC) is a common neurological autosomal-dominant syndrome caused by mutations in the TSC1 or TSC2 genes. TSC starts in early childhood and is characterized by cerebral hamartomas (benign tumours), severe epilepsy and cognitive deficits such as mental retardation and autism. The hamartomas are characterized by loss of the remaining wild-type TSC allele, and clinical data implicate cerebral hamartomas in the generation of epileptic seizures, which may play a significant role in the development of mental retardation. The TSC2 mutation predicts alterations in mitogen-associated protein kinase (MAPK) and, together with the TSC1 mutation, in mammalian target of rapamycin (mTOR) signalling pathways. Both pathways are involved in neuronal plasticity. We therefore hypothesized that the heterozygous mutation itself, besides cerebral hamartomas, contributes to the pathogenesis of cognitive deficits and possibly also epilepsy. Here, we show that young adult TSC2+/- rats, which are virtually free of cerebral hamartomas, exhibit enhanced episodic-like memory and enhanced responses to chemically-induced kindling. The activation of cyclic adenosine monophosphate (cAMP) in the hippocampus results in stronger induction of phospho-p42-MAPK in TSC2+/- rats than in wild-type animals. Thus, the cognitive phenotype and, possibly, epilepsy in TSC patients may result not only from the focal hamartomatous lesions but also, from altered neuronal plasticity in the heterozygous tissue.     

Identification of Regions Critical for the Integrity of the TSC1-TSC2-TBC1D7 Complex

The TSC1-TSC2-TBC1D7 complex is an important negative regulator of the mechanistic target of rapamycin complex 1 that controls cell growth in response to environmental cues. Inactivating TSC1 and TSC2 mutations cause tuberous sclerosis complex (TSC), an autosomal dominant disorder characterised by the occurrence of benign tumours in various organs and tissues, notably the brain, skin and kidneys. TBC1D7 mutations have not been reported in TSC patients but homozygous inactivation of TBC1D7 causes megaencephaly and intellectual disability. Here, using an exon-specific deletion strategy, we demonstrate that some regions of TSC1 are not necessary for the core function of the TSC1-TSC2 complex. Furthermore, we show that the TBC1D7 binding site is encoded by TSC1 exon 22 and identify amino acid residues involved in the TSC1-TBC1D7 interaction.     

TSC1 stabilizes TSC2 by inhibiting the interaction between TSC2 and the HERC1 ubiquitin ligase

Tuberous sclerosis complex (TSC) is an autosomal dominant disease characterized by hamartoma formation in various organs. Two genes responsible for the disease, TSC1 and TSC2, have been identified. The TSC1 and TSC2 proteins, also called hamartin and tuberin, respectively, have been shown to regulate cell growth through inhibition of the mammalian target of rapamycin pathway. TSC1 is known to stabilize TSC2 by forming a complex with TSC2, which is a GTPase-activating protein for the Rheb small GTPase. We have identified HERC1 as a TSC2-interacting protein. HERC1 is a 532-kDa protein with an E3 ubiquitin ligase homology to E6AP carboxyl terminus (HECT) domain. We observed that the interaction of TSC1 with TSC2 appears to exclude TSC2 from interacting with HERC1. Disease mutations in TSC2, which result in its destabilization, allow binding to HERC1 in the presence of TSC1. Our study reveals a potential molecular mechanism of how TSC1 stabilizes TSC2 by excluding the HERC1 ubiquitin ligase from the TSC2 complex. Furthermore, these data reveal a possible biochemical basis of how certain disease mutations inactivate TSC2.     

Angiomyolipoma have common mutations in TSC2 but no other common genetic events

Renal angiomyolipoma are part of the PEComa family of neoplasms, and occur both in association with Tuberous Sclerosis Complex (TSC) and independent of that disorder. Previous studies on the molecular genetic alterations that occur in angiomyolipoma are very limited. We evaluated 9 angiomyolipoma for which frozen tissue was available from a consecutive surgical series. Seven of 8 samples subjected to RT-PCR-cDNA sequencing showed mutations in TSC2; none showed mutations in TSC1 or RHEB. Six of the seven mutations were deletions. We searched for 983 activating and inactivating mutations in 115 genes, and found none in these tumors. Similarly analysis for genomic regions of loss or gain, assessed by Affymetrix SNP6.0 analysis, showed no abnormalities. Loss of heterozygosity in the TSC2 region was commonly seen, except in patients with low frequency TSC2 mutations. We conclude that sporadic renal angiomyolipoma usually have mutations in TSC2, but not TSC1 or RHEB, and have no other common genomic events, among those we searched for. However, chromosomal translocations and gene fusion events were not assessed here. TSC2 inactivation by mutation is a consistent and likely necessary genetic event in the pathogenesis of most angiomyolipoma.     

Insulin activation of Rheb,a mediator of mTOR/S6K/4E-BP signaling,is inhibited by TSC1 and 2

Tumor suppressor genes evolved as negative effectors of mitogen and nutrient signaling pathways, such that mutations in these genes can lead to pathological states of growth. Tuberous sclerosis (TSC) is a potentially devastating disease associated with mutations in two tumor suppressor genes, TSC1 and 2, that function as a complex to suppress signaling in the mTOR/S6K/4E-BP pathway. However, the inhibitory target of TSC1/2 and the mechanism by which it acts are unknown. Here we provide evidence that TSC1/2 is a GAP for the small GTPase Rheb and that insulin-mediated Rheb activation is PI3K dependent. Moreover, Rheb overexpression induces S6K1 phosphorylation and inhibits PKB phosphorylation, as do loss-of-function mutations in TSC1/2, but contrary to earlier reports Rheb has no effect on MAPK phosphorylation. Finally, coexpression of a human TSC2 cDNA harboring a disease-associated point mutation in the GAP domain, failed to stimulate Rheb GTPase activity or block Rheb activation of S6K1.     

Conditional and domain‐specific inactivation of the Tsc2 gene in neural progenitor cells

Tuberous sclerosis complex (TSC) is a genetic disease characterized by multiorgan benign tumors as well as neurological manifestations. Epilepsy and autism are two of the more prevalent neurological complications and are usually severe. TSC is caused by mutations in either the TSC1 (encodes hamartin) or the TSC2 (encodes tuberin) genes with TSC2 mutations being associated with worse outcomes. Tuberin contains a highly conserved GTPase‐activating protein (GAP) domain that indirectly inhibits mammalian target of rapamycin complex 1 (mTORC1). mTORC1 dysregulation is currently thought to cause much of the pathogenesis in TSC but mTORC1‐independent mechanisms may also contribute. We generated a novel conditional allele of Tsc2 by flanking exons 36 and 37 with loxP sites. Mice homozygous for this knock‐in Tsc2 allele are viable and fertile with normal appearing growth and development. Exposure to Cre recombinase then creates an in‐frame deletion involving critical residues of the GAP domain. Homozygous conditional mutant mice generated using Emx1^Cre have increased cortical mTORC1 signaling, severe developmental brain anomalies, seizures, and die within 3 weeks. We found that the normal levels of the mutant Tsc2 mRNA, though GAP‐deficient tuberin protein, appear unstable and rapidly degraded. This novel animal model will allow further study of tuberin function including the requirement of the GAP domain for protein stability. genesis 51:284–292. © 2013 Wiley Periodicals, Inc.     

TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling

Tuberous sclerosis (TSC) is an autosomal dominant disorder characterized by the formation of hamartomas in a wide range of human tissues. Mutation in either the TSC1 or TSC2 tumour suppressor gene is responsible for both the familial and sporadic forms of this disease. TSC1 and TSC2 proteins form a physical and functional complex in vivo. Here, we show that TSC1-TSC2 inhibits the p70 ribosomal protein S6 kinase 1 (an activator of translation) and activates the eukaryotic initiation factor 4E binding protein 1 (4E-BP1, an inhibitor of translational initiation). These functions of TSC1-TSC2 are mediated by inhibition of the mammalian target of rapamycin (mTOR). Furthermore, TSC2 is directly phosphorylated by Akt, which is involved in stimulating cell growth and is activated by growth stimulating signals, such as insulin. TSC2 is inactivated by Akt-dependent phosphorylation, which destabilizes TSC2 and disrupts its interaction with TSC1. Our data indicate a molecular mechanism for TSC2 in insulin signalling, tumour suppressor functions and in the inhibition of cell growth.     

Pam and its ortholog highwire interact with and may negatively regulate the TSC1.TSC2 complex

Tuberous Sclerosis Complex (TSC) is an autosomal dominant disorder associated with mutations in TSC1, which codes for hamartin, or TSC2, which codes for tuberin. The brain is one of the most severely affected organs, and CNS lesions include cortical tubers and subependymal giant cell astrocytomas, resulting in mental retardation and seizures. Tuberin and hamartin function together as a complex in mammals and Drosophila. We report here the association of Pam, a protein identified as an interactor of Myc, with the tuberin-hamartin complex in the brain. The C terminus of Pam containing the RING zinc finger motif binds to tuberin. Pam is expressed in embryonic and adult brain as well as in cultured neurons. Pam has two forms in the rat CNS, an approximately 450-kDa form expressed in early embryonic stages and an approximately 350-kDa form observed in the postnatal period. In cortical neurons, Pam co-localizes with tuberin and hamartin in neurites and growth cones. Although Pam function(s) are yet to be defined, the highly conserved Pam homologs, HIW (Drosophila) and RPM-1 (Caenorhabditis elegans), are neuron-specific proteins that regulate synaptic growth. Here we show that HIW can genetically interact with the Tsc1.Tsc2 complex in Drosophila and could negatively regulate Tsc1.Tsc2 activity. Based on genetic studies, HIW has been implicated in ubiquitination, possibly functioning as an E3 ubiquitin ligase through the RING zinc finger domain. Therefore, we hypothesize that Pam, through its interaction with tuberin, could regulate the ubiquitination and proteasomal degradation of the tuberin-hamartin complex particularly in the CNS.     

Analysis of TSC1 truncations defines regions involved in TSC1 stability,aggregation and interaction

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterised by the development of hamartomas in a variety of organs and tissues. The disease is caused by mutations in either the TSC1 gene on chromosome 9q34, or the TSC2 gene on chromosome 16p13.3. The TSC1 and TSC2 gene products, TSC1 and TSC2, interact to form a protein complex that inhibits signal transduction to the downstream effectors of the target of rapamycin complex 1 (TORC1). Here we investigate TSC1 structure and function by analysing a series of truncated TSC1 proteins. We identify specific regions of the protein that are important for TSC1 stability, localisation, interactions and function.     

A novel TSC1 mutation (c.1964delA) in a Chinese patient with tuberous sclerosis complex

Tuberous sclerosis complex is an autosomal-dominant heritable disease caused by mutations in the TSC1 and TSC2 genes. We studied a Chinese patient with sporadic tuberous sclerosis complex. The clinical features of this patient included epilepsy, hypomelanotic macules and angiofibromas on his back; a cranial CT scan showed subependymal nodules along the lateral walls of the lateral ventricles. The TSC1 and TSC2 genes were studied by PCR and direct sequencing of the entire coding region and exon-intron boundaries of these genes. A novel deletion mutation (c.1964delA) in the TSC1 gene exon 15 was identified, which was not present in his parents or 100 unrelated normal controls. This is the first report of this c.1964delA mutation of the TSC1 gene, associated with tuberous sclerosis complex, expanding the spectrum of TSC1 mutations that cause this disease.