1. TSC1/2 Complex and Tuberous Sclerosis
Devin Gibbs

2. Tuberous Sclerosis Causes Hamartomas
Tuberous Sclerosis is a relatively rare disease affecting 1:6000 people. Patients generally have a normal life expectancy excluding complications.
The hallmark of the disease is the formation of benign tumors known as hamartomas. These can manifest in many different tissues throughout the body. The skin, brain, kidney, heart, and lungs are all common locations of hamartomas.
Though these tumors are not malignant, they can cause other problems due to their size and location. Renal and brain hamartomas pose the greatest risk. For instance, in the brain the tumors can block the flow of cerebral spinal fluid leading to hydrocephalus. Additional neurological symptoms are often involved as a result of cerebral hamartomas including epilepsy, cognitive impairment, and autism. Renal hamartomas can pose a problem because of the risk of bleeding due to malformed blood vessels.
TS symptoms can vary widely in severity. For instance, some cases are very mild and asymptomatic. The prognosis really depends on the particular set of symptoms the patient has and their severity.

3. Mutations in TSC1/2 Cause Tuberous Sclerosis
So what is causing tuberous sclerosis?
Genetic screening points to mutations in one of two proteins: TSC1 and TSC2.
TSC follows Knudson’s two hit hypothesis. Formation of hamartomas requires the loss of both functional alleles.
Lets take a look at the structure of TSC1 and 2…
TSC2 stands for tuberous sclerosis complex 2 and encodes a protein known as tuberin. TSC1 encodes a protein known as hamartin. Tuberin and hamartin bind to each other and act as a complex. TSC1 serves to stabilize TSC2 and prevent it from ubiquitylation.
Looking at the structure of TSC1 and TSC2, both genes have interaction sites with many different proteins suggesting diverse roles in the cell. Many of the proteins the complex interacts with are involved in cell proliferation and survival such as CDK1 and ERK. Additionally, you’ll notice that TSC2 has a GAP domain. Thus, it must function to turn off a GTPase signaling molecule.

4. TSC1/2 Mediates Cell Growth
How do these various domains function in the protein?
One role of TSC2 is to regulate cell growth by indirectly inhibiting a protein called mTOR. The GAP domain of TSC2 functions to link the tuberin/hamartin complex with mTOR. GAP inhibits the GTPase Rheb (a Ras family protein) which when bound to GTP activates mTOR.
mTOR is a serine-threonine kinase that promotes cell growth and indirectly cell proliferation by enhancing ribosomal biosynthesis and protein translation.

5. TSC1/2 Mediates Cell Proliferation
TSC can also directly effect cell proliferation through its interaction with the b-catenin destruction complex. The complex can associate with GSK3 to help degrade b-catenin acting in a similar way to APC. When TSC1/2 is lost, b-catenin is upregulated.

6. TSC1/2 Integrates Growth and Energy Signals
This is what the tuberous sclerosis complex affects downstream, but what signals does it respond to?
You can see that the TSC1/2 complex is inhibited by mitogenic signals like ERK and cell cycle progression signals like CDK1. Conversely, the complex is stimulated by proteins that lead to differentiation like GSK3B or nutrient deprivation sensors like AMPK.
From these diverse signals, it looks like TSC1/2 integrates signals about energy status and mitogenic signals to decide whether growth and proliferation is the appropriate action to take.
Additionally, TSC1/2 has a role in preventing apoptosis due to energy-deprivation or unfolded protein response.
Other experiments have demonstrated increased rates of apoptosis when TSC2 is lost due to deregulation of the apoptotic machinery.
TSC2’s role in apoptosis may explain the benign nature of the tumors seen in tuberous sclerosis. Loss of TSC2 causes proliferation, but this proliferation is balanced somewhat by apoptosis.

7. A Diverse Array of Mutations can Cause Tuberous Sclerosis
for image
Going back to tuberous sclerosis, we said that TSC is caused by loss of function mutations in the tumor suppressors TSC1 and TSC2.
TSC1 mainly exhibits truncating mutations which is consistent with its role as an adaptor protein. In TSC2, missense mutations are clustered in the GAP domain
To date, over 1000 allelic variants in the TSC1 and TSC2 genes have been found. There is little genotype to phenotype correlation so it is difficult to know what disease progression will look like just by knowing the particular mutation.
So we know the effect of these mutation at the organismal level (TSC), but what do they do at the cellular and molecular levels?

8. TSC1/2 Knockout Results in Cell Growth and Neuronal Disorganization
Knocking out both alleles of either TSC1 or TSC2 is embryonic lethal, so scientists use conditional knockouts in mice to model TS. In the top panels, deletion of either TSC1 or TSC2 resulted in enlarged cells in the brain and greater disorganization as a result. These findings were amplified in the TSC2 knockout which is correlated with TSC2 causing more severe neurological symptoms.
In Panel B, brain weight is measured in the knockouts as compared to WT. TSC1 and TSC2 exhibited larger brain weights.
Additional experiments not shown here also showed an increase in cell number.
These results suggest that TSC1/2 serves as a tumor suppressor, since its loss results in growth and proliferation.

9. TSC1/TSC2 Knockout Increases mTOR Activation
Now we know that knocking out TSC1/2 results in increased cell growth. How does the complex accomplish this at the molecular level?
Here we are looking at a western blot of proteins in the brain.
When TSC1/2 are knocked out there are higher levels of p-s6 so that means mtor is more active.
s6 is phosphorylated by mTOR. The presence of p-S6 signifies mTOR activity.
In both this data set and the one I just showed you, mutated TSC2 produces the more severe phenotype. This is likely a result of greater mTOR activation when TSC2 function is lost. Looking back at the structure of the genes, TSC2 contains the GAP domain. Perhaps TSC2 has some minimal GAP activity even without TSC1.
Given this mTOR activation a possible therapy is to inhibit mTOR.

10. mTOR Inhibitors Are Promising Therapies
Rapamycin
A natural mTOR inhibitor called Rapamycin was already in existence, so researchers have been exploring rapamycin as a therapy for TSC. The exact mechanism is unknown but Rapamycin first binds an endogenous inhibitor of mTOR called FKBP. This complex then binds a domain of mTOR that causes allosteric inhibition of kinase activity in mTOR.
In mice, rapamycin and other mTOR inhibitors have been shown to prevent seizures and cognitive defects in young animals and also reverse these symptoms in older animals.
Also causes tumor regression though not permanently.
Rapamycin may be good therapy, but not actual cure. Limitations is that it needs to be given prior to onset of neurological symptoms to have best effect. Later treatment seems to require chronic therapy or tumors come back. Thus, it appears like the damage done by TSC is currently more preventable than it is curable. Additionally, mTOR is not the only target of TSC so mTOR inhibitors cannot be the complete answer.
FKBP

11. Summary
The TSC1/2 complex acts as a tumor suppressor to monitor growth and energy signals and inhibit cell cycle progression through inhibiting mTOR and β-catenin.
Loss of function mutations in TSC1 and/or TSC2 lead to constitutive activation of mTOR and upregulation of β-catenin.
mTOR inhibitors show promise in treating TSC.

12. References
Ballou, L., & Lin, R. (2008). Rapamycin and mTOR kinase inhibitors. J Chem Biol., 1(1-4),
Crino, P. B., Nathanson, K. L., & Henske, E. P. (2006). The tuberous sclerosis complex. N Engl J Med, 355(13), doi: /NEJMra055323
Jones, A. C., Shyamsundar, M. M., Thomas, M. W., Maynard, J., Idziaszczyk, S., Tomkins, S., Cheadle, J. P. (1999). Comprehensive mutation analysis of TSC1 and TSC2-and phenotypic correlations in 150 families with tuberous sclerosis. American Journal of Human Genetics, 64(5), doi: /302381
Mak, B. C., Kenerson, H. L., Aicher, L. D., Barnes, E. A., & Yeung, R. S. (2005). Aberrant β-catenin signaling in tuberous sclerosis. The American Journal of Pathology, 167(1), doi: /S (10)
März, A. M., Fabian, A., Kozany, C., Bracher, A., & Hausch, F. (2013). Large FK506-binding proteins shape the pharmacology of rapamycin. Molecular and Cellular Biology, 33(7), doi: /MCB
Tomasoni, R., & Mondino, A. (2011). The tuberous sclerosis complex: Balancing proliferation and survival. Biochemical Society Transactions, 39(2), doi: /BST ; /BST
Yao, J., Phan, A., Jehl, V., Shah, G., & Meric-Bernstam, F. (2013). Everolimus in advanced pancreatic neuroendocrine tumors: The clinical experience.  . Cancer Res., 73(5),
Zeng, L., Rensing, N. R., Zhang, B., Gutmann, D. H., Gambello, M. J., & Wong, M. (2011). Tsc2 gene inactivation causes a more severe epilepsy phenotype than Tsc1 inactivation in a mouse model of tuberous sclerosis complex. Human Molecular Genetics, 20(3), doi: /hmg/ddq491