- Using a Drosophila Model to Screen for Molecules to Reverse the Effects of Tsc Gene Mutations
- Role of Altered mGluR Activity in Cognitive Impairments in TSC: Implications for a Novel Method of Treatment
- LAMS Clinical Research Network
- Understanding the Etiology of Tuberous Sclerosis Complex
- TSC-FoxO Signaling Network in Kidney Cancer Development
- The Role of Plk1 in Tuberous Sclerosis Complex
- Regulation of Neuronal Differentiation in TSC
- Protein Synthesis-Dependent Synaptic Changes in Tuberous Sclerosis Complex
- Angiogenesis and Lymphangiogenesis as Chemotherapeutic Targets in Tuberous Sclerosis Complex
- TSC1 and TSC2 Variants Identified in Patients with Tuberous Sclerosis Complex
- The Rags Necessary to Mediate Amino Acid Signaling to mTORC1
- Mechanism of TSC Regulation of Neuronal Differentiation
- The Importance of Rheb in Tuberous Sclerosis
- Update to the Tuberous Sclerosis Complex (TSC) Natural History Database Studies
- New Software for Cell Image Analysis
- TSC2 Regulates Cell Motility and Invasiveness
- TSC1 and TSC2 Regulate Neuronal Morphology and Function
- Tuberin Regulates mTOR Function in Response to Hypoxia
- Elucidating the Functions of the TSC1 and TSC2 Genes
- Understanding the Causes of TSC-Related Epilepsy
Tuberous Sclerosis Complex (TSC) is a genetic disorder resulting from loss of function of the TSC1 or TSC2 genes and characterized by the development of nonmalignant tumors in many different organs, primarily in the brain where they are called "tubers", heart, kidney, skin, and lungs. While some pharmacological targets for the treatment of TSC have been identified, available therapies are largely ineffective in TSC patients with intractable epilepsy, autism, and developmental delays. Additional tools to facilitate the understanding of TSC gene function and to catalyze the discovery of novel therapeutic strategies are needed. Mouse models of TSC are limited because deletion of the Tsc1 or Tsc2 genes is lethal, and mice that are heterozygous for Tsc1 or Tsc2 mutations have a very mild disease phenotype. Dr. Kevin Ess at the Vanderbilt University Medical Center has developed a zebrafish model of TSC by inactivating the tsc2 gene. Zebrafish are an attractive research model because they produce a large number of embryos, are transparent, simplifying study of their internal development, and can be easily modified genetically, which aids in the study of gene interactions.
Dr. Ess received an Idea Development Award from the TSC Research Program to characterize brain abnormalities, hamartoma formation, and gene interactions in the mTOR signaling pathway in tsc2-deficient zebrafish. He has found alterations in brain development in tsc2-deficient zebrafish, including increased cell size in the brain and poor organization of forebrain neurons, leading to extensive disorganization of gray and white matter and ectopically positioned cells. Tsc2-deficient fish also have increased mTOR Complex 1 (TORC1) activity, which was reversed by treatment with rapamycin or expression of zebrafish or human TSC2 mRNA. Further, transplantation of fluorescently labeled tsc2-deficient cells into wild-type zebrafish embryos resulted in wild-type neurons in the host brain to become mispositioned. Eight months following transplantation, tsc2-deficient cells were seen in multiple organs and displayed increased TORC1 signaling. One year after cell transplantation, apparent hamartomas of tsc2-deficient cells were seen in the brain. These findings lead Dr. Ess to conclude that the tsc2 mutant cells have cell autonomous and possibly non-cell autonomous mechanisms that lead to aberrant brain development. Additional experiments to better understand the role of tsc2 in zebrafish development are ongoing. The knowledge gained from these studies will not only impact the TSC community but will provide insight into the role of mTOR signaling in development and disease pathogenesis.
Brain of wild type zebrafish 8 months following the transplantation of tsc2 homozygous mutant cells labeled with green fluorescent protein (GFP). Upper left: GFP (green); upper right: phospho-S6, a marker of mTORC1 signaling (red); lower left: DAPI to identify cell nuclei (blue); lower right: merged image. Scale bar equals 200 μm.
Tsc1 and Tsc2 normally function to suppress cellular growth. Tuberous sclerosis complex (TSC) results from mutation of the Tsc1 or Tsc2 genes, causing excessive cell growth and tumor formation. Dr. Tin Tin Su, with funding from a Fiscal Year 2005 (FY05) Tuberous Sclerosis Complex Research Program (TSCRP) Concept Award, previously developed a Drosophila melanogaster (fruit fly) model to screen for small molecules that can reverse the effects of Tsc1/2 mutations. Through funding from an FY09 TSCRP Exploration - Hypothesis Development Award, Dr. Su's research group utilized this Drosophila model to screen approximately 6,000 small molecules. This work led to the identification of two small molecules that reliably inhibit the effects of mutant Tsc1 in Drosophila. The next steps in this work will involve validating the activity of these candidate small molecules using the Drosophila model and characterizing their mechanism of action.
Schematic of Drosophila melanogaster (fruit fly) model to screen for small molecules that can reverse the effects of Tsc1/2 mutations.
Patients with Tuberous Sclerosis Complex (TSC) develop benign tumors and suffer from cognitive deficiencies including mental retardation, epilepsy, autism, anxiety, and mental disorders. TSC is a genetic disorder resulting from the mutation of either the TSC1 or TSC2 gene. In healthy cells, the TSC proteins function to modulate protein synthesis by suppressing mTOR (mammalian target of rapamycin) signaling. Interestingly, maintenance of synaptic plasticity, critical for learning, memory, and cognition, requires precise regulation of protein synthesis. Based on these observations, Dr. Mark Bear hypothesized that dysregulation of synaptic protein synthesis may contribute to TSC-associated learning and cognitive deficiencies. Dr. Bear's research team, with funding from an FY10 TSCRP Idea Development Award, demonstrated that loss of TSC2 resulted in unregulated mTOR activity and suppressed hippocampal protein synthesis. In addition, the team also determined that treatment with an mGluR5 positive allosteric modulator (PAM) restored hippocampal protein synthesis. Additionally, treatment with mGluR5 PAM also reversed hippocampal-dependent behavioral deficits in Tsc2+/- mice. These exciting results suggest that mGluR5 PAM may be a novel therapeutic intervention for TSC-associated cognitive deficiencies.
Tuberous sclerosis complex (TSC) is a rare genetic disorder, affecting approximately one million people worldwide, that may result in seizures, cognitive impairment, and benign tumors of the skin, eyes, brain, and other organs. Lymphangioleiomyomatosis (LAM), a life-threatening progressive lung manifestation of TSC, affects approximately 40 percent of women with TSC. Although a significant number of molecular targets have been identified as potential therapeutics for LAM, the design and implementation of clinical trials has been limited due to the rare occurrence of the disease. Dr. Francis McCormack at the University of Cincinnati received a fiscal year 2009 Clinical and Translational Research Award from the TSC Research Program to develop a novel paradigm for conducting clinical trials in rare diseases more quickly and more conveniently to patients by bringing clinical trials to local LAM treatment sites. To this end, Dr. McCormack established the LAM Clinical Research Network (LCRN), a collaboration of 24 LAM clinics throughout the United States. LAM clinic directors are in the process of developing a longitudinal dataset that would be collected from all consenting participants to build a natural history database and maintain a list of prospective subjects for future clinical trials. The LCRN will allow patients to enroll in studies at their local LAM clinics with their physician, rather than traveling, sometimes long distances, to gain entry into cutting-edge clinical trials.
One of the initial LCRN aims is to develop and validate a biomarker for diagnosis of LAM using vascular endothelial growth factor-D (VEGF-D). Preliminary results suggest that serum VEGF-D levels can be utilized to diagnosis LAM in women with TSC. A second project of the LCRN is a Phase I/II trial of Letrozole, an oral non-steroidal aromatase inhibitor, in postmenopausal women with LAM (Trial of Aromatase Inhibition in LAM, or TRAIL), which is expected to begin enrollment at six LCRN clinics in 2012.
Dr. McCormack and the LCRN are in a unique position to increase access to clinical trials for individuals with LAM and, in turn, facilitate the delivery of novel LAM treatment options to patients.
Tuberous sclerosis complex (TSC) patients have mutations in the Tsc1 or Tsc2 genes and suffer from the formation of lesions on multiple organs. Patients also develop neurological symptoms including seizures, mental retardation, and autism. Cortical tuber lesions that form during embryonic development are associated with the development of seizures in TSC patients. The mechanisms involved in TSC lesion formation and seizure generation are not well understood and validated animal models to investigate the etiology or cause of TSC lesions and subsequent development of neurological disorders are currently unavailable. Through funding from a fiscal year 2009 Idea Development Award, Dr. Angelique Bordey, at Yale University, has developed and validated a mouse model to bridge this gap in knowledge. Dr. Bordey and her research team have determined that deletion of Tsc1 in this mouse model leads to formation of lesions that contain many of the hallmark characteristics associated with human cortical tubers in TSC. This animal model will now allow TSC researchers to study the development of cortical tuber lesions at specific time points during embryonic development. This work was recently published in the Journal of Clinical Investigation (Feliciano 2011).
Tuberous Sclerosis Complex (TSC) Syndrome is an autosomal dominant genetic disease characterized by the development of benign tumors, called hamartomas, in multiple organs. Renal complications are a frequent cause of death in patients with TSC. The renal manifestations in TSC patients include the development of renal angiomyolipomas (AMLs), renal cell carcinomas (RCCs), and polycystic kidney disease (PKD). Although great progress has been made in the last decade in the field of TSC, little is known about TSC’s role in renal cancer development, and the cooperative events in TSC-mediated renal tumorigenesis. With funding from an FY09 Career Transition Award, Dr. Boyi Gan aims to elucidate the molecular pathogenesis of TSC-related renal tumorigenesis and to provide novel insights of targeted therapies against renal complications in TSC patients. Dr. Gan’s central hypothesis is that FoxO transcriptional factors play a key role in the molecular pathogenesis of TSC-related renal tumorigenesis. Dr. Gan proposes to (1) determine the mechanism and role of FoxO activation in TSC-deficient cells and renal tumors, (2) investigate the underlying mechanisms by which FoxOs cooperate with the TSC1-TSC2 complex to inhibit mTORC1 signaling, and (3) identify direct FoxO transcriptional targets that contribute to TSC-mediated renal tumorigenesis. Dr. Gan’s research has the potential to advance the understanding of TSC-mediated renal tumorigenesis and to expand drug development for those TSC patients with renal complications.
Tuberous Sclerosis Complex (TSC) is an inherited disorder characterized by seizures, mental retardation, and benign tumors in multiple organs including the brain, kidney, heart, and skin. Worldwide, there are approximately 900,000 patients with TSC, 40,000 of whom are in the United States. TSC is caused by mutations in two genes: TSC1 and TSC2. The rare disorder Lymphangioleiomyomatosis (LAM), which manifests primarily in the lungs and affects exclusively women, is another disease caused by TSC1 and TSC2 mutations.
Loss of TSC1 or TSC2 function leads to uncontrolled cell growth via activation of mTOR, a protein that is sensitive to the antibiotic rapamycin. Clinical trials are underway to determine rapamycin�s potential for the treatment of TSC and LAM. Targeting multiple proteins in the TSC/mTOR pathway may provide additional TSC and LAM treatment options. Plk1, a protein regulating several aspects of cell division, including cytokinesis (the final stage of cell division) may serve as another protein target in TSC and LAM. Recent investigations in the laboratory of Dr. Aristotelis Astrinidis have identified Plk1 to be a new interacting partner of TSC1. His research team has also found that mTOR is activated by Plk1, that cells lacking TSC1 have abnormal cytokinesis, and that Plk1 expression is increased in cells without TSC1 or TSC2 and in samples from LAM patients. With funding from an FY09 Tuberous Sclerosis Research Program Idea Development Award, Dr. Astrinidis proposes to (1) define the pathway leading to mTOR activation by Plk1, (2) investigate the consequences of TSC1 or TSC2 loss in cell division, more specifically cytokinesis, and (3) determine whether targeting Plk1 by specific inhibitors causes death in cells without TSC1 or TSC2. This project will potentially lead to a new preclinical model for TSC treatment.
Children with tuberous sclerosis complex (TSC) characteristically suffer from autism, mental retardation, epilepsy, and psychiatric disorders. This inherited disease is caused by mutations on the protein complex TSC1 and TSC2. Dr. Helen McNeill, recipient of a Fiscal Year 2005 Tuberous Sclerosis Complex Research Program Idea Development Award, began looking at TSC's role in controlling neuronal cell fate and how these growth pathways lead to neurological defects. Dr. McNeill's focus is on the insulin receptor/target of rapamycin kinase (InR/TOR) pathway of the Drosophila retina, where she found that TOR regulates growth by controlling the activity of the S6 kinase (S6K) and eIF4E proteins. S6K controls differentiation and acts downstream or parallel to TOR signaling, but eIF4E does not affect the timing of differentiation, only growth. Dr. McNeill also found that activation of the InR/Tor pathway regulates expression at the transcriptional level of the EGFR pathway components Argos, rhomboid, and pointedP2. Reduction of the EGFR signaling shows similar behavior to inhibition of the InR/TOR pathway in regulating differentiation, thus suggesting that transcriptional crosstalk between InR/TOR and EGFR pathways control developing neuron timing. Understanding how loss of TSC leads to premature cell fate decisions in Drosophila could lead to significant findings in humans and could lead to treatment for many of the neurological disorders that affect TSC patients.
McNeill H, Craig G, and Bateman JM. 2008. Regulation of neurogenesis and EGFR signaling by the insulin receptor/TOR pathway in Drosophila. Genetics 179:843-853.
Tuberous sclerosis complex (TSC) is a genetic disorder characterized by neurological symptoms such as seizures, autistic behavior, and mental retardation. Patients with TSC also have non-malignant brain tumors called cortical hamartomas. A mutation in two genes, TSC1 and TSC2, causes TSC. TSC1 and TSC2 both play important roles in protein synthesis and cell growth. TSC proteins are negative regulators of mTOR, an enhancer of protein synthesis. In theory, mutations causing hypofunction of TSC1 or TSC2 can cause increased protein production, but it is unknown which neuronal proteins mTOR regulates and how malfunction in the TSC/mTOR pathway results in the neurological symptoms of TSC.
Cortical hamartomas often become the focus of seizures in TSC patients, leaving some aspects of TSC's neurological features unexplained. Dr. Akira Yoshii (recipient of a Fiscal Year 2008 Tuberous Sclerosis Complex Research Program Career Transition Award), of the Massachusetts Institute of Technology, is studying whether it is neurons themselves that are malfunctioning in TSC. Neurons communicate with each other at junctions called synapses. At a synapse, one neuron releases neurotransmitters and another neuron receives the signal via its receptors. There are excitatory and inhibitory synapses, which normally exist in equilibrium; however, in seizures excitation overwhelms neural circuitry. This type of imbalance is also thought to cause autistic behavior. Dr. Yoshii plans to test the hypothesis that the balance between excitation and inhibition is skewed in TSC and that this synaptic dysregulation is caused by altered protein synthesis. To test this hypothesis, Dr. Yoshii will use biochemical assays to determine the levels of both excitatory and inhibitory synapse-associated proteins in brains of TSC mutant mice compared to wild type (normal) mice. He will look for evidence of upregulation of excitatory synaptic proteins and/or downregulation of inhibitory synaptic proteins. Rapamycin, an mTOR inhibitor, has been reported to be beneficial for seizure control and the overall health of TSC mutant mice. Dr. Yoshii plans to examine whether rapamycin will affect surface expression of excitatory and inhibitory receptors. Results of these experiments will potentially advance understanding of the neurobiological basis of TSC and facilitate the identification of a therapeutic target for neurological symptoms of this challenging disorder.
Angiogenesis and Lymphangiogenesis as Chemotherapeutic Targets in Tuberous Sclerosis Complex
Posted June 29, 2009
Thomas Darling, M.D., Ph.D., Uniformed Services University of the Health Sciences, Bethesda, Maryland
Patients with tuberous sclerosis complex (TSC) develop skin tumors that bleed with minor trauma and can be disfiguring. There are no effective oral or topical treatments for these TSC skin tumors. As a result, patients must undergo several surgical procedures that can ultimately leave scarring. These tumors are highly vascularized with both blood and lymphatic vessels. Angiogenesis (the formation of blood vessels) and lymphangiogenesis (the formation of lymphatic vessels) are important for the growth and spread of cancerous tumors, but little is known about angiogenesis and lymphangiogenesis in TSC. In experimental models, drugs that inhibit angiogenesis and lymphangiogenesis have decreased the size and spread of cancers. Dr. Thomas Darling of the Uniformed Services University of the Health Sciences, recipient of a Department of Defense Fiscal Year 2008 Tuberous Sclerosis Complex Research Program Idea Development Award, hypothesizes that similar treatment could inhibit skin tumors in TSC patients. Dr. Darling's laboratory has found that TSC skin tumors produce higher-than-normal levels of proteins that stimulate angiogenesis and lymphangiogenesis. Rapamycin and tranilast have been used for the treatment of TSC-related tumors with varying success. The mechanisms by which rapamycin and tranilast exert their effects on the processes of angiogenesis and lymphangiogenesis are not completely understood. Dr. Darling plans to test the effects of rapamycin on the production of proteins involved in angiogenesis and lymphangiogenesis in TSC skin tumors. He also plans to test the effectiveness of tranilast in blocking angiogenesis and lymphangiogenesis in TSC skin tumors. To test the effects of rapamycin and tranilast on TSC skin tumors, Dr. Darling will use early-passage primary TSC2-null cells grown from human TSC skin tumors in his novel xenograft mouse model. This model system will allow for quantification of the tumor microenvironment in response to experimental manipulations. Dr. Darling will use more than one human cell type in these grafts (TSC tumor cells, normal human keratinocytes, and melanocytes), making these more representative of the human tumor. If successful, Dr. Darling's work would have three potential clinical applications: (1) a better understanding of how rapamycin and tranilast inhibit tumor growth in TSC, (2) a determination as to whether rapamycin and tranilast have a synergistic effect when combined, and (3) identification of proteins that would be useful in blood tests to determine if tumors are responding to treatment.
Tuberous sclerosis complex (TSC) is an inherited disease characterized by seizures, mental retardation, and the development of hamartomas in several organs and tissues. TSC is caused by mutations in the TSC1 and TSC2 genes. Mutation analysis of the TSC1 and TSC2 genes is a valuable diagnostic tool for TSC, and in most cases a definite disease causing TSC1 or TSC2 mutation is identified. However, there are some unclassified variants in which it is difficult to determine whether or not sequence changes identified in the TSC1 or TSC2 genes are pathogenic. These variants present significant diagnostic and genetic counseling challenges. Dr. Mark Nellist (recipient of a Fiscal Year 2006 Tuberous Sclerosis Complex Research Program Idea Development Award) of the Erasmus MC-Daniel den Hoed Cancer Center is developing and applying assays to determine whether or not specific unclassified TSC1 and TSC2 variants are pathogenic. If successful, individuals and families carrying these variant mutations will be able to obtain clearer information about the condition and the associated risks. Additionally, these studies could provide insight into genotype-phenotype correlations and identify regions of these proteins that are important for TSC1 and TSC2 function. Using an immunoblot assay, Dr. Nellist has identified three regions essential for TSC1 or TSC2 function: (1) amino acid substitutions within the N-terminal region (amino acids 1-200) of TSC1 that destabilize TSC1, (2) substitutions to a central region of TSC2 (amino acids 600-900) that disrupt TSC1-TSC2 binding, and (3) substitutions outside the predicted TSC2 GAP domain that inactivate the complex. He has also identified a region of TSC1 (amino acids 50-224) required for maintaining TSC1 at sufficient levels in the cell to form a stable TSC1-TSC2 complex and inhibit mTOR.
Nellist M, Sancak O, Goedbloed M, Adriaans, Wessels M, Maat-Kievit A, Baars M, Dommering C, van den Ouweland A, and Halley D. 2008. Functional characterisation of the TSC1-TSC2 complex to assess multiple TSC2 variants identified in single families affected by tuberous sclerosis complex. BMC Med Genet 9:10.
Nellist M, van den Heuvel D, Schluep D, Exalto C, Goedbloed M, Maat-Kievit A, van Essen T, van Spaendonck-Zwarts, Jansen F, Helderman P, Bartalini G, Vierimaa O, Penttinen M, van den Ende J, van den Ouweland A, and Halley D. 2009. Missense mutations to the TSC1 gene cause tuberous sclerosis complex. Eur J Hum Genet 17(3):319-328.
Coevoets R, Arican S, Hoogeveen-Westerveld M, Simons E, van den Ouweland A, Halley D and Nellist M. 2009. A reliable cell-based assay for testing unclassified TSC2 gene variants. Eur J Hum Genet 17(3):301-310.
Tuberous sclerosis complex (TSC) is caused by inactivating mutations of the TSC1 and TSC2 tumor suppressor genes, but how TSC1/2 loss of function leads to tumor formation is not well understood. The protein kinase mammalian target of rapamycin (mTOR) plays a central role in regulating cell growth, proliferation, and survival. The deregulation of the mTOR pathway has been implicated in many human diseases. In TSC, the mTOR pathway is hyperactive. mTOR participates in two distinct multi?protein complexes, one of which is the mammalian target of rapamycin complex 1 (mTORC1). The mTORC1 protein complex has recently emerged as a key downstream regulator of TSC1/2, which negatively regulates the small GTPase Rheb, an activator of the mTORC1 pathway. Although mTORC1 is a major therapeutic target, little is known about its structure and the molecular mechanisms through which TSC1/2 regulates the mTOR kinase.
Raptor (regulatory associated protein of mTOR) is one of the mTOR-interacting proteins being studied by Dr. David Sabatini, of the Whitehead Institute for Biomedical Research, recipient of a Fiscal Year 2006 Tuberous Sclerosis Complex Research Program Idea Development Award. While analyzing the Rheb?mediated phosphorylation of Raptor in the regulation of mTORC1, Dr. Sabatini found a new Raptor-interacting protein, RagC. The Rag proteins are a unique family of small GTPases that have been shown to interact with each other in mammalian cells and in yeast. In mammals, there are four Rag genes, Rag A, B, C, and D. Dr. Sabatini found that binding of the Rag GTPase to Raptor is necessary to mediate amino acid signaling to mTORC1 and that binding also mediates the amino acid-induced relocalization of mTOR within the endomembrane system of the cell. Given the prevalence of cancer-linked mutations in the pathways that control mTORC1, Dr. Sabatini suggests that it is possible that Rag function is also deregulated in human tumors. Dr. Sabatini is currently assessing the details of amino acid-induced mTORC1 activation while trying to identify other Rag-interacting proteins.
Role of Rag GTPases in signaling amino acid availability to mTORC1
Sancak Y, Peterson TR, Shaul YD, Lindquist RA, Thoreen CC, Bar-Peled L, and Sabatini DM. 2008. The rag GTPases bind raptor and mediate amino acid signaling to mTORC1. Science 302(5882):1496-1501.
While the symptoms of tuberous sclerosis complex (TSC) are well understood, many of the basic questions regarding the etiology of TSC still remain unanswered. Through her Department of Defense Fiscal Year 2005 Idea Development Award, Dr. Helen McNeill has studied how TSC loss affects the timing of neuronal differentiation in Drosophila. As Drosophila-based insect models are well-established for discovering the molecular and cellular mechanisms underlying TSC, these findings are regularly translated into the clinic. Using the latest techniques in high-throughput genome analysis technology, combined with in vivo analysis, Dr. McNeill has identified genes that cooperate with TSC to regulate neuronal differentiation in Drosophila. To date, two critical findings have resulted from these studies. The novel transcription factor, Pointed-P2 (PntP2), was identified as playing a role in mediating precocious neuronal differentiation. Interestingly, activation of PntP2 was induced by either loss of TSC or activation by growth factors such as insulin. Dr. McNeill also characterized another gene, S6K, which influenced neuronal development. Loss of S6K blocked the process of precocious neuronal differentiation in Drosophila, which lacked TSC. The role of S6K in the development of TSC symptoms has recently become a significant area of research. Findings such as these may lead to the development of novel, early clinical interventions for individuals with TSC to prevent or ameliorate the nervous system defects associated with this disease.
McNeill H, Gavin C, and Bateman J. 2008. Regulation of neurogenesis and EGFR signaling by the insulin receptor/TOR pathway in Drosophila. Genetics 179:1-11.
Bateman JM and McNeill H. 2006. Insulin/IGF signaling in neurogenesis. Cellular and Molecular Life Sciences 63(15):1701-1705.
Tuberous sclerosis is caused by mutations in either the Tsc1 or the Tsc2 gene. Products of these genes form a complex that acts as a negative regulator of Rheb GTPase, an activator of the molecular target of rapamycin (mTOR). Thus, one of the major problems with tuberous sclerosis is that the TSC/Rheb/mTOR signaling pathway is over-activated. Dr. Fuyuhiko Tamanoi, of the University of California, Los Angeles, received a Fiscal Year 2004 Idea Development Award through the Department of Defense Tuberous Sclerosis Research Program to explore how the TSC/Rheb/mTOR signaling pathway is regulated and what the consequences of alteration of this signaling pathway are. Dr. Tamanoi found that, unlike Rheb1, Rheb2 protein expression was not ubiquitous. Activation of the TSC/Rheb/mTOR signaling pathway led to constitutive activation of Cdk2, a cell cycle protein that functions at the G1/S phase boundary. In addition, activation of the TSC/Rheb/mTOR signaling pathway blocked the translocation to the nucleus of p27, a Cdk inhibitor protein. Using constitutively active mTOR mutants, Dr. Tamanoi found that these mutations affect mTORC1, but not mTORC2, and did not affect the overall structure of the mTOR complex. Dr. Tamanoi found that mTOR forms a heterodimer in which heterozygous mutations could result in constitutive activation of mTOR. The constitutively active mTOR mutants were still inhibited by rapamycin. Cells stably transformed with constitutively active mTOR exhibited resistance to hydrogen peroxide, although they retained sensitivity to both sorbitol and rapamycin. Research such as this lays the foundation for a better understanding of tuberous sclerosis.
Urano J, Sato T, Matsuo T, Otsubo Y, Yamamoto M, and Tamanoi F. 2007. Point mutations in TOR confer Rheb-independent growth in fission yeast and nutrient-independent mTOR signaling in mammalian cells. Proceedings of the National Academy of Sciences of the United States of America 104:3514-3519.
Update to the Tuberous Sclerosis Complex (TSC) Natural History Database Studies
Posted December 13, 2007
Steven P. Sparagana, M.D., Texas Scottish Rite Hospital for Children, Dallas, Texas and Vicky Whittemore, Ph.D., Tuberous Sclerosis Alliance, Silver Spring, Maryland
Dr. Steven Sparagana of Texas Scottish Rite Hospital for Children and the University of Texas Southwestern Medical Center at Dallas began development of the TSC Natural History Database using funds from a Fiscal Year 2004 (FY04) Natural History Development Award. The TS Alliance and a consortium of TSC clinics completed development of this database (see http://cdmrp.army.mil/highlights/default.shtml), and the TS Alliance is currently leading this project (see http://tsalliance.org/pages.aspx?content=558). This comprehensive clinical database of TSC cases documents the natural history and variability of TSC over the lifespan of individuals with the disease. Patient information collected for the database includes demographics, enrollment in the database, initial TSC diagnosis, genotype, participation in investigational studies, mortality, family history, prenatal history, vital signs, TSC-related diagnoses, diagnostic tests, and treatments. Understanding the clinical aspects of TSC could lead to more accurate disease prognosis, development of new targeted therapies, and prediction of patient response to treatments.
Phase I of the project was completed at the first two clinics (Minnesota Epilepsy Group, P.A.®, and Texas Scottish Rite Hospital for Children) in April 2007. As of June 2007, these two clinics enrolled more than 80 individuals with TSC, 80% of whom were children. The database was optimized during Phase I; Phase II of the project was launched in 2007. In this phase, the following six clinics are being added: The Carol and James Herscot Center for Children and Adults with Tuberous Sclerosis Complex at Massachusetts General Hospital; the Tuberous Sclerosis Center at New York University Medical Center; Washington Metro Area Tuberous Sclerosis Research Clinic; Chicago Corner Children's Hospital Neurogenetic Clinic; The Jack and Julia Center for TSC at Children's Hospital and Research Center at Oakland; and the TSC Clinic at UCLA. Additional TSC clinics are expected to be added in 2008.
Dr. David Sabatini and Dr. Anne Carpenter of the Whitehead Institute for Biomedical Research have developed new image analysis software for identifying and quantifying cell phenotypes using funds from a Fiscal Year 2004 Tuberous Sclerosis Complex (TSC) Research Program Concept Award. Dr. Sabatini's team created this software to overcome limitations found in existing software packages, such as price, availability of applications, challenges in using algorithms and macros, and flexibility. Their program, CellProfiler, is the first free, open-source system designed for flexible, high-throughput cell image analysis. CellProfiler can be used for assaying cell count, size, per-cell protein levels, cell/organelle shape, and subcellular localization of DNA or protein.
Dr. Sabatini's group is using CellProfiler as part of a high throughput screen to identify new drug targets for treating TSC. The team is using cultured Drosophila cells as living cell microarrays, and they are identifying all the genes in the genome whose RNAi-mediated reduction in expression (1) prevents growth and proliferation of TSC1- or TSC2-deficient cells without affecting normal cells, (2) induces apoptosis and cell death in TSC1- or TSC2-deficient cells without killing normal cells, or (3) reverts TSC1- or TSC2-deficient cells to a normal phenotype.
In addition to TSC research, CellProfiler can be utilized for diverse types of biological research, and it has been used by several researchers for a variety of applications, including analysis of yeast, Drosophila, worm, and mammalian cells. As CellProfiler is already being employed to count cells, identify tumors, and quantify wound healing (see www.cellprofiler.org for more information), it clearly has the potential to speed TSC research, as well as other types of cell-based biological research.
Lamprecht M, Sabatini DM, and Carpenter AE. 2007. CellProfilerTM: Free, versatile software for automated biological image analysis. BioTechniques 42(1):71-75.
Jones TR, Carpenter AE, Golland P, and Sabatini DM. 2006. Methods for high-content, high-throughput image-based cell screening. MIABB 2006 Workshop Proceedings.
Carpenter AE, Jones TR, Wheeler DB, Lamprecht M, Clarke C, Friman O, Guertin DA, Kang IH, Lindquist R, Chang JH, Moffat J, Golland P, and Sabatini DM. 2006. CellProfiler: Image analysis software for identifying and quantifying cell phenotypes. Genome Biology 7:R100.
Tuberous sclerosis complex (TSC) is a genetic disorder arising from inactivating mutations in the tumor suppressor genes TSC1 or TSC2. The complex formed by TSC1 and TSC2 proteins has long been associated with the regulation of protein synthesis and cell growth, achieved through inhibition of the mammalian target of rapamycin (mTOR) signaling pathway. However, neurologic complications of TSC, such as epilepsy, autism, and cognitive impairments, are considered to result from perturbations in neuronal migration. Similarly, lymphangioleiomyomatosis (LAM), a progressive lung disease that is another common complication of TSC, is also associated with loss of TSC1 and TSC2 function due to cell migration and metastasis. Dr. Vera P. Krymskaya of the University of Pennsylvania therefore hypothesized that complex formation between TSC1 and TSC2 regulates cell adhesion and motility and that dysregulation of the complex formation may contribute to the pathogenesis of TSC. With funding from a Department of Defense Tuberous Sclerosis Complex Research Program Fiscal Year 2003 Idea Development Award, Dr. Krymskaya and her colleagues found that TSC2 plays a critical role in regulating actin cytoskeleton, focal adhesion, cell motility, and invasiveness. TSC2 is required for Rac1 activation. Binding of TSC2 to TSC1 inhibits the Rho-activating function of TSC1, preventing abnormal cell migration and invasiveness. When TSC2 is mutated, as seen in the disease state, TSC1 inhibits Rac1, activates Rho, and promotes cell migration and invasiveness. These results indicate that loss of function of TSC2 results in downregulation of Rac1 and upregulation of Rho activities, leading to abnormal cell motility and invasiveness associated with the pathology of both TSC and LAM.
Goncharova E, Goncharov D, Noonan D, and Krymskaya VP. 2004. TSC2 modulates actin cytoskeleton and focal adhesion through TSC1-binding domain and the Rac1 GTPase. The Journal of Cell Biology 167(6):1171-1182.
Goncharova EA, Goncharov DA, Lim PN, Noonan D, and Krymskaya VP. 2006. Modulation of cell migration and invasiveness by tumor suppressor TSC2 in lymphangioleiomyomatosis. American Journal of Respiratory Cell and Molecular Biology 34:473-480.
Tuberous sclerosis complex (TSC) is a genetic disorder caused by mutations in the TSC1 (hamartin) or TSC2 (tuberin) tumor suppressor genes. Many children with TSC have neurological symptoms including epilepsy, mental retardation, and autism. However, the molecular mechanisms underlying these neuropathologies are unclear. Therefore, understanding the neural functions of TSC1 and TSC2 could lead to improved treatments for neurological disorders in TSC patients. Dr. Bernardo Sabatini of Harvard Medical School hypothesized that TSC1 is necessary in mature, differentiated neurons for the establishment of proper neuronal morphology and synaptic function. He believes that disruption of TSC1 results in the function of each neuron being perturbed and that, because of these defects in each brain cell, the brain as a whole does not function properly. This is in contrast to the conventional hypothesis that the presence of tumors within the brain creates a disorganized brain architecture. With funding from a Department of Defense Tuberous Sclerosis Complex Research Program Fiscal Year 2003 Idea Development Award, Dr. Sabatini and his research team examined the role of the TSC pathway in regulating the growth of post-mitotic differentiated neurons in a cell-autonomous manner. By downregulating the levels of TSC1 and TSC2 in cell culture and animal models, the research team showed that the TSC pathway regulated soma size, the density and size of dendritic spines, and the properties of excitatory synapses in hippocampal pyramidal neurons. Loss of a single copy of TSC1, as is observed in the neurons of TSC patients, was sufficient to disrupt dendritic spine structure. Interestingly, these morphological effects were independent of regulation of TSC2 by Akt, but they did require rapamycin-sensitive mTOR activity and regulation of cofilin (an actin depolymerization factor). These results indicate that the TSC pathway regulates neuronal structure and function and that cell-autonomous disruptions of synapse function contribute to neurological symptoms of TSC.
Tavazoie SF, Alvarez VA, Ridenour DA, Kwiatkowski DJ, and Sabatini BL. 2005. Nature Neuroscience 8(12):1727-1734.
Tuberous sclerosis complex is caused by inactivating mutations in TSC1 (hamartin) or TSC2 (tuberin) tumor suppressor genes. The disorder is associated with increased activity of mammalian target of rapamycin (mTOR), a central regulator of protein synthesis and cell growth that is inhibited by chronic oxygen deprivation (hypoxia). Tumor hypoxia has been associated with a negative prognosis in several types of cancers. With funding from a Department of Defense Tuberous Sclerosis Complex Research Program Fiscal Year 2003 Idea Development Award, Dr. William Kaelin of the Dana-Farber Cancer Institute has discovered that the TSC1/TSC2 protein complex regulates mTOR in response to hypoxia. Dr. Kaelin and his research team showed that an intact TSC1/TSC2 complex is required for mTOR inhibition by hypoxia. Inactivation of TSC2 conferred a proliferative advantage to cells grown under hypoxic conditions. Dr. Kaelin's group further defined the mechanisms of TSC1/TSC2-mediated mTOR inhibition under hypoxic conditions. Downregulation of mTOR by hypoxia requires expression of the hypoxia-inducible Redd1 gene but is independent of the AMP-activated protein kinase (AMPK) and Peutz-Jehgers syndrome (Lkb1) genes, which are involved in TSC1/TSC2-mediated mTOR inhibition under conditions of energy depletion. The investigators also found that TSC2 is required for Redd1 to downregulate phosphorylation of S6K, an mTOR substrate; these data indicate that Redd1 may act upstream of TSC1/TSC2 to downregulate mTOR in response to hypoxia. Improved understanding of the role of TSC2 in response to hypoxia may lead to better treatments for tumors associated with tuberous sclerosis complex.
Brugarolas J, Lei K, Hurley RL, Manning BD, Reiling JH, Hafen E, Witters LA, Ellisen LW, and Kaelin WG Jr. 2004. Regulation of mTOR function in response to hypoxia by REDD1 and the TSC1/TSC2 tumor suppressor complex. Genes and Development 18(23):2893-2904
Brugarolas J. and Kaelin WG Jr. 2004. Dysregulation of HIF and VEGF is a unifying feature of the familial hamartoma syndromes. Cancer Cell 6(1):7-10.
Brugarolas JB, Vazquez F, Reddy A, Sellers WR, Kaelin WG Jr. 2003. TSC2 regulates VEGF through mTOR-dependent and -independent pathways. Cancer Cell 4(2):147-158.
Tuberous sclerosis is an inherited disease caused by mutations in the TSC1 or TSC2 genes. Approximately 80% of people with tuberous sclerosis suffer from epileptic seizures, which are often resistant to anticonvulsive therapy. Other manifestations of the disorder, including kidney disease and tumors on vital organs, also can be severe, yet there are no treatments specific to tuberous sclerosis. A complete understanding of the functions of the TSC1 protein, hamartin, and the TSC2 protein, tuberin, is required for the development of targeted therapeutics. Dr. Elizabeth Henske, a recipient of a Department of Defense Tuberous Sclerosis Complex Research Program fiscal year 2002 Idea Development Award, is investigating hamartin and tuberin function in the yeast Schizosaccharomyces pombe. Yeast model systems provide powerful and convenient means to examine important cellular and molecular processes, as many pathways are similar in yeast and mammals and experiments can be performed more rapidly than in mice or other mammalian systems. Dr. Henske determined that the loss of either TSC1 or TSC2 in yeast resulted in decreased uptake of arginine, an amino acid used for the synthesis of proteins, polyamines (molecules required for cell growth), and nitric oxide (a signaling molecule involved in many cellular functions). Expression of putative amino acid and polyamine transporters also was lower in mutant yeast than in normal control yeast. Moreover, deficiency in either TSC1 or TSC2 caused decreases in intracellular levels of glutamate and other amino acids. This phenotype was rescued by Pas1, a G1 cyclin, suggesting a link between nutrient availability and cell cycle progression. These results indicate that hamartin and tuberin play critical roles in amino acid sensing, uptake, and metabolism and that tuberous sclerosis symptoms may be linked to defects in those key cellular functions. Importantly, diminished glutamate uptake from the synapses in the brains of TSC1-deficient mice is believed to contribute to seizure development, suggesting that the yeast model may provide a novel system for the study of tuberous sclerosis-related epilepsy and for preclinical screening of new therapeutics that may ameliorate seizures in individuals with the disease.
van Slegtenhorst M, Carr E, Stoyanova R, Kruger WD,and Henske EP. 2004. Tsc1+ and tsc2+ regulate arginine uptake and metabolism in Schizosaccharomyces pombe. Journal of Biological Chemistry 279:12706-12713.
van Slegtenhorst M, Mustafa A, and Henske EP. 2005. Pas1, a G1 cyclin, regulates amino acid uptake and rescues a delay in G1 arrest in Tsc1 and Tsc2 mutants in Schizosaccharomyces pombe. Human Molecular Genetics 14 (19): 2851-2858.
Epilepsy is one of the most devastating complications of tuberous sclerosis complex (TSC). Approximately 80% of children who have TSC develop epileptic seizures, which are often severe and refractory to available treatments. Seizures and other manifestations of TSC have been attributed to malformed areas in the brain, known as cortical tubers, that are believed to arise during embryonic development. Dr. David Gutmann and colleagues of the Washington University School of Medicine have determined new mechanisms by which TSC gene defects in the brain result in seizures. Understanding such cellular and molecular mechanisms of seizures is necessary for developing new tailored therapies.
With funding from a Department of Defense Fiscal Year 2002 Tuberous Sclerosis Complex Research Program Idea Development Award, Dr. Gutmann and colleagues used mouse models in which the Tsc1 gene was inactivated in the astrocyte class of neuroglial cells to study TSC-related epilepsy. These mice exhibit enhanced astrocyte proliferation, abnormal neuronal organization, and seizures. The investigators found abnormal expression of neuroglial differentiation markers in Tsc1-deficient astrocytes. Similar gene expression patterns were observed in cortical tubers and subependymal giant cell astrocytoma tumors from TSC patients, suggesting that both types of lesions arise from similar neuroglial progenitor cells and that Tsc1 gene inactivation in humans leads to aberrant progenitor cell differentiation. Tsc1 loss in mouse astrocytes also was associated with increased activity of the Rheb/mammalian target of rapamycin (mTOR)/p70S6 kinase (S6K) pathway, supporting recent evidence that the TSC1/TSC2 protein complex may regulate Rheb and S6K. In addition, they found that an important astrocyte adhesion molecule, adhesion molecule in glia (AMOG), also regulates the mTOR signaling pathway, but independent of TSC.
Gutmann and colleagues examined the molecular mechanisms that might underlie increased neuronal excitability and lead to seizures. They showed that Tsc1-deficient astrocytes had decreased expression of two weak inward rectifier potassium currents and exhibited diminished potassium current in functional assays. These astrocyte potassium current defects were not reversed by rapamycin, an inhibitor of mTOR. Dr. Gutmann and his team believe that the observed abnormalities in astrocyte potassium uptake could potentially lead to excessive synaptic stimulation in neurons, resulting in hyperexcitability and seizures.
Dr. Gutmann's research team has (1) discovered several genetic and cellular abnormalities that result from astrocyte-specific inactivation of Tsc1, (2) demonstrated that there is a role for astrocyte potassium homeostasis in influencing seizures in mouse models of TSC, and (3) developed a novel concept that the astrocyte may be centrally involved in the pathogenesis of neurological complications of TSC, including epilepsy. Based on these findings, innovative therapies for epilepsy could potentially target astrocytes.
Ess KC, Uhlmann EJ, Li W, Li H, DeClue JE, Crino PB, and Gutmann DH. 2004. Expression profiling in tuberous sclerosis complex (TSC) knockout mouse astrocytes to characterize human TSC brain pathology. Glia 46:28-40.
Uhlmann EJ, Li W, Scheidenhelm D, Gau CL, Tamanoi F, and Gutmann DH. 2004. Loss of tuberous sclerosis complex 1 (Tsc1) expression results in increased Rheb/S6K pathway signaling important for astrocyte cell size regulation. Glia 47:180-188.
Ess KC, Kamp KA, Tu BP, and Gutmann DH. 2005. Developmental origin of subependymal giant cell astrocytoma in tuberous sclerosis complex. Neurology 64:1446-1449.
Scheidenheim DK, Cresswell J, Haipek CA, Fleming TP, Mercer RW, and Gutmann DH. 2005. Akt-dependent cell size regulation by the adhesion molecule on glia (AMOG) occurs independently of phosphotidylinositol 3-kinase and Rheb signaling. Molecular and Cellular Biology 25:3151-3162.
Jansen LA, Uhlmann EJ, Crino PB, Gutmann DH, and Wong M. 2005. Epileptogenesis and reduced inward rectifier potassium current in tuberous sclerosis complex-1 deficient astrocytes. Epilepsia (in press).