- 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
Regulation of Neuronal Differentiation in TSC
Posted December 3, 2009
Helen McNeill, Ph.D., Mount Sinai Hospital, Samuel Lunenfeld Research InstituteChildren 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.
Publication:
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.
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Protein Synthesis-Dependent Synaptic Changes in Tuberous Sclerosis Complex
Posted July 27, 2009
Akira Yoshii, M.D., Ph.D., Massachusetts Institute of Technology, Cambridge, MassachusettsTuberous 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.
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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, MarylandPatients 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.
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TSC1 and TSC2 Variants Identified in Patients with Tuberous Sclerosis Complex
Posted June 22, 2009
Mark Nellist, Ph.D., Erasmus MC-Daniel den Hoed Cancer Center Rotterdam, The NetherlandsTuberous 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.
Publications:
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.
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The Rags Necessary to Mediate Amino Acid Signaling to mTORC1
Posted June 9, 2009
David Sabatini, M.D., Ph.D., Whitehead Institute for Biomedical Research, Cambridge, MassachusettsTuberous 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
Publications:
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.
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