**6. Molecular pathogenesis of TSC**

Up to 85% of TSC cases are due to mutations in either *TSC1* or *TSC2* genes which lead to a truncated protein with a loss of function mechanism. Investigation of somatic mutations in a variety of TSC hamartomas supports classification of the *TSC1* and *TSC2* as tumor suppressor genes (Cheadle et al., 2000). Mutations in *TSC1* and *TSC2* affect neuronal proliferation, differentiation, and migration (Crino et al., 1999). The identification of the *TSC1* and *TSC2* genes and their encoded proteins, hamartin and tuberin respectively, has aided in understanding the molecular pathogenesis of TSC where hamartomatous formation is the outcome.

Both hamartin and tuberin are widely expressed in normal tissues including brain, liver, and kidney. Hamartin is highly expressed in G0-arrested cells and throughout the ongoing cell cycle (Crino, 2004). Alterations in tuberin expression have been reported in patients with TSC. Immunoreactivity of tuberin is reduced in the brain with TSC. Loss of hamartin and tuberin formation due to *TSC1* and *TSC2* mutations can enhance proliferation of neural and astrocytic precursor cells and increased in cell size characteristic of dysplastic neurons and giant cells. When either of the *TSC1* or *TSC2* genes is inactivated, G1 is shortened and tissues become hypertropic (Potter et al., 2001). Over-expression of either hamartin or tuberin can lengthen G1 and inhibits cell proliferation (Tapon et al., 2001).

Many studies indicated that hamartin and tuberin, encoded by *TSC1* and *TSC2* genes respectively, function as a complex. The complex has a stable interaction with stoichiometry of 1:1. The tight binding interaction between the two proteins formed a tumour suppressor heterodimer (Kwiatkowaki, 2008). Hamartin and tuberin have been found to physically associate with one another in vivo. Disruption in either one of the two genes may result in a truncated protein with the loss in controlling the cell growth and proliferation. The 130 kDa hamartin contains a putative transmembrane domain. Hamartin's membrane bound protein

Perhaps because of more chances of the variations to occur along considerable length of intronic portions within *TSC2*, less pathogenicity variations can be conclusively determined. The tables showed that of 1690 *TSC1* and *TSC2* variations reported, only 27.7% (468) were found in *TSC1*. There are some explanations for the fact that up to date more *TSC2* 

a. According to Knudson's hypothesis, loss of heterozygosity (LOH) of a tumor suppressor gene is necessary for tumor progression. Recent investigations of somatic mutations in a variety of TSC hamartomas support classification of the TSC genes as tumor suppressor genes. Loss of Heterozygosity (LOH) in *TSC1* hamartomas are rare compared to that in *TSC2* hamartomas (Cheadle et al., 2000). This may reflect the low

b. Several large studies reported that *TSC1* mutations are presented with less severe phenotype than *TSC2* mutations. In 1991, Osborne and colleagues outlined that although TSC incidence is reported as 1 in approximately 6000, the true incidence of TSC is not known because of a number of undiagnosed cases consisting mostly of mildly affected or asymptomatic individuals. It is probable that this portion of patients harbored *TSC1*

mutations, accounting for the small number of *TSC1* mutations found until today. c. *TSC2* coding region is about 1.5 times longer than *TSC1* and has approximately twice the number of splice sites, affording a proportionally increased opportunity for all

Up to 85% of TSC cases are due to mutations in either *TSC1* or *TSC2* genes which lead to a truncated protein with a loss of function mechanism. Investigation of somatic mutations in a variety of TSC hamartomas supports classification of the *TSC1* and *TSC2* as tumor suppressor genes (Cheadle et al., 2000). Mutations in *TSC1* and *TSC2* affect neuronal proliferation, differentiation, and migration (Crino et al., 1999). The identification of the *TSC1* and *TSC2* genes and their encoded proteins, hamartin and tuberin respectively, has aided in understanding the molecular pathogenesis of TSC where hamartomatous formation

Both hamartin and tuberin are widely expressed in normal tissues including brain, liver, and kidney. Hamartin is highly expressed in G0-arrested cells and throughout the ongoing cell cycle (Crino, 2004). Alterations in tuberin expression have been reported in patients with TSC. Immunoreactivity of tuberin is reduced in the brain with TSC. Loss of hamartin and tuberin formation due to *TSC1* and *TSC2* mutations can enhance proliferation of neural and astrocytic precursor cells and increased in cell size characteristic of dysplastic neurons and giant cells. When either of the *TSC1* or *TSC2* genes is inactivated, G1 is shortened and tissues become hypertropic (Potter et al., 2001). Over-expression of either hamartin or

Many studies indicated that hamartin and tuberin, encoded by *TSC1* and *TSC2* genes respectively, function as a complex. The complex has a stable interaction with stoichiometry of 1:1. The tight binding interaction between the two proteins formed a tumour suppressor heterodimer (Kwiatkowaki, 2008). Hamartin and tuberin have been found to physically associate with one another in vivo. Disruption in either one of the two genes may result in a truncated protein with the loss in controlling the cell growth and proliferation. The 130 kDa hamartin contains a putative transmembrane domain. Hamartin's membrane bound protein

tuberin can lengthen G1 and inhibits cell proliferation (Tapon et al., 2001).

variations were found compared to *TSC1*:

frequency of *TSC1* diseases.

manner of small mutations.

is the outcome.

**6. Molecular pathogenesis of TSC** 

and two coiled-coil domains are necessary for its association with tuberin (van Slegtenhorst et al., 1997 and van Slegtenhorst et al., 1998).

Although *TSC1*- or *TSC2*-specific functions are possible, it seems that the predominant biochemical activity of these proteins is exerted by an equimolar complex, which regulates the state of GTP-loading of the rheb GTPase, and thereby regulates mTOR activation in the cell. As most hamartomas in TSC develop through a two-hit inactivation mechanisms (Knudson's hypothesis for tumor suppressor genes, including *TSC1* and *TSC2*), it appears likely that somatic mutations in *TSC1* are less common than those in *TSC2*, just as the rate of germline mutation in *TSC1* is much lower than that in *TSC2*. Thus, fewer and/or less severe clinical manifestations would be seen in *TSC1* patients.

LOH is very common within TSC hamartomas, except for cardiac or brain. In both organs, study says that wildtype hamartin and tuberin are present. LOH is an event by which within the affected cells, the genomic DNA loss its heterozygosity, becoming homozygous for the mutation. In other cells (unaffected cells) the genomic DNA shows heterozygous for the mutation. To analyze the occurrence of LOH it is necessary to perform mutation analysis on the genomic DNA extracted from the affected cells as well as from the unaffected cells.

After the discovery of *TSC1* and *TSC2* and their encoded proteins, several downstream protein cascades that might be affected by the pathogenesis of the disease, such as the pathway of mTOR (mammalian target of rapamycin), were identified.

Fig. 1. Hamartin and tuberin as tumor suppressor gene.

Tuberous Sclerosis Complex 21

interaction with ERM or actin binding proteins. It was also shown to activate the GTPase Rho and regulates focal adhesion and stress fiber formation. Hamartin activates the GTPase Rho via the overlapping region of Rhos's amino acid and hamartin's tuberin-interaction domain (Lamb et al., 2000). The dysregulation of signalling by the Rho family of GTPase is said to have a critical role in cancer cell migration, invasion, and metastasis (Clark et al.,

It has also been recently shown that the functional complex interact with G2/M cyclindependant kinase 1 and its regulatory cyclins. Thus, mutation in either both genes may alter the kinetics of cell divisions (Catania et al., 2001). The functional heteromeric complex of hamartin and tuberin also plays important role in modulating the pathways of insulin receptor-or insulin-like growth factor-mediated signalling. The pathway functions downstream of the cell signalling molecule Akt, also play roles in regulating cell growth and

At present, the management of TSC is symptomatic. Some of TSC manifestations have been subjected to drug therapies but they are still in the developmental stage (Yates et al., 2006). Table 5 summarized several drugs under investigation for their efficacy towards Tuberous

The discovery of mTOR (mammalian target of rapamycin) pathway upregulation in tuberous-sclerosis-associated tumours presents new possibilities for treatment strategies. A TSC mouse treated with rapamycin, also known as sirolimus, was found to have its learning

Sirolimus is a macrolide antibiotic that acts as an mTOR kinase inhibitor. It is isolated from *Streptomyces hygroscopicus*. Sirolimus and its analogs have been shown to make the dysregulated mTOR pathway return to normal in cells that lack *TSC1* or *TSC2*. Several results from in-vitro or in-vivo animal studies suggest that sirolimus or its analogues might be effective in the treatment of various manifestations of tuberous sclerosis such as skin lesions (Rauktys et al., 2008), lymphangioleiomyomatosis (Goncharova et al., 2006 and Bissler et al., 2008), renal angiomyolipomas (Lee et al., 2006; Herry et al., 2007 and Wienecke et al., 2006), renal-cell carcinoma (Robb et al., 2007), subependymal giant-cell astrocytomas

However, angiomyolipomas increased in volume after the therapy was discontinued, and some patients taking sirolimus experienced serious adverse events (Bissler et al., 2008; Herry

Recently, other classes of drugs have also been found to be possible therapeutic options for TSC. Interferon gamma and interferon alpha interact with mTOR, leading to deactivation of the translational repressor 4E-BP1, which could be beneficial for the treatment of tuberous sclerosis (Kaur et al., 2007). Other classes of drugs ranging from those which can alter amino acids metabolism, inhibit VEGF signalling and inhibit microtubules were also studied. Presence or absence of amino acids is an important regulator of mTOR pathway

For example, L-asparaginase, a hydrolase enzyme and one of the most important agents used in multidrug chemotherapy for the treatment of cancer. It is mainly used to treat human leukemic cells in acute lymphoblastic leukemic. L-asparaginase has been found to

(Franz et al., 2006) or even polycystic kidney disease (Weimbs et al., 2006).

2000; Evers et al., 2000; Royal et al., 2000 and Schmitz et al., 2000).

**7. Studies on therapeutics options for TSC** 

and memory deficits improved (Ehninger et al., 2008).

et al., 2007 and Wienecke et al., 2006).

signalling (Avruch et al., 2006).

potentially cell size.

Sclerosis Complex.

Figure 1 illustrated the roles of hamartin and tuberin in cell metabolism describing that disruption in either of both genes may result in loss of the control of cell growth and proliferation. Hamartin-tuberin complex inhibits the mTOR which is a key regulator in the signalling pathway of cell proliferation and organ size (Kwiatkowski, 2008). It has been reported that the complex regulates mTOR via hydrolysis of Rheb-GTP into its inactive GDP bound state, Rheb-GDP (Rosner et al., 2004 and Tee et al., 2003).

Tuberin and hamartin form an intracellular complex which activates GTPase, reducing stimulation of mTOR. mTOR detects signals of nutrient availability, hypoxia, or growth factor stimulation, and is part of many cell processes, such as cell-cycle progression, transcription and translation control, and nutrient uptake. It phosphorylates, among other proteins, S6K1 and eukaryotic translation initiation factor 4E-BP1. S6K1 is a kinase that activates ribosomal subunit protein S6, leading to ribosome recruitment and protein translation. 4E-BP1 inhibits activity of eukaryotic translation initiation factor 4E (eIF4E) and, when phosphorylated by mTOR, releases eIF4E from its control.

The complex inhibits mTOR by acting as a GAP toward Rheb, which promotes hydrolysis of Rheb-GTP, converting it to an inactive GDP bound state. Without its active GTP bound state, Rheb cannot stimulate mTOR-mediated signalling to downstream components S6K1 and 4E-BP1. The mechanism is reversed with the presence of amino acids which activates Rheb-GEF. RhebGEF converts Rheb-GDP to its active Rheb-GTP and promotes mTOR signalling. Akt inactivates TSC tumor suppressor complex by phosphorylation of *TSC2* (Tee et al., 2003).

Common *TSC2* mutations result in the loss of the GAP domain of tuberin through Cterminal truncations, whereas some point mutations are clustered within the GAP domain. It is also reported that, an intact GAP domain of tuberin is crucial for association with hamartin in the formation of tuberin-hamartin heterodimers. The heterodimers will inhibit Rheb-induced mTOR signalling and can also function as a GAP toward Rheb. Higher proportion of the active GTP bound form of Rheb can likely be found within TSC patients. It is the result of non-functional tuberin-hamartin heterodimers where the genes failed to encodes for a functional protein (van Slegtenhorst et al., 1998).

The 200kDa (1806 amino acids) tuberin is homologous to the GTPase activating proteins (GAP) rap1GAP and mSpa1 where it contains relatively hydrophobic N-terminal domain and conserved 163 amino acids region close to the C-terminus. Rap1GAP is the member of Ras-related protein and functions in regulation of DNA synthesis and cell-cycle transition. The GAP activity of functional tuberin can regulate the effects of Rap1 on G to S phase transition during cell division. Thus, it implies that *TSC2* mutations may result in constitutive activation of Rap1 (Wienecke et al., 1996 and Wienecke et al., 1997).

Tuberin also has been demonstrated to interact with rab5. Rab5 is a cytosolic protein, is an effector for the endosomal small GTPase and therefore involved in endocytic fusion events (Stenmark et all., 1995). Consistently with the finding, tuberin has also been shown to act as a GTPase activating protein for rab5 and reduce the fluid-phase endocytosis (Xiao et al., 1997).

As for 130 kDa (1164 amino acids) hamartin, it has hydrophilic protein with no significant homology to tuberin or other known vertebrate protein. Van Slegtenhorst and colleagues have investigated the association between endogenous hamartin and tuberin and they found out that both proteins play a closely related role (van Slegtenhorst et al., 1998). The methods used in the study suggest that inactivation of hamartin and tuberin may prevent the formation of a functional protein complex.

Hamartin was recently identified as an interactor with the cytoskeletal proteins, ERM family (Lamb et al., 2000). The function loss can alternatively compromise neural migration via

Figure 1 illustrated the roles of hamartin and tuberin in cell metabolism describing that disruption in either of both genes may result in loss of the control of cell growth and proliferation. Hamartin-tuberin complex inhibits the mTOR which is a key regulator in the signalling pathway of cell proliferation and organ size (Kwiatkowski, 2008). It has been reported that the complex regulates mTOR via hydrolysis of Rheb-GTP into its inactive GDP

Tuberin and hamartin form an intracellular complex which activates GTPase, reducing stimulation of mTOR. mTOR detects signals of nutrient availability, hypoxia, or growth factor stimulation, and is part of many cell processes, such as cell-cycle progression, transcription and translation control, and nutrient uptake. It phosphorylates, among other proteins, S6K1 and eukaryotic translation initiation factor 4E-BP1. S6K1 is a kinase that activates ribosomal subunit protein S6, leading to ribosome recruitment and protein translation. 4E-BP1 inhibits activity of eukaryotic translation initiation factor 4E (eIF4E) and,

The complex inhibits mTOR by acting as a GAP toward Rheb, which promotes hydrolysis of Rheb-GTP, converting it to an inactive GDP bound state. Without its active GTP bound state, Rheb cannot stimulate mTOR-mediated signalling to downstream components S6K1 and 4E-BP1. The mechanism is reversed with the presence of amino acids which activates Rheb-GEF. RhebGEF converts Rheb-GDP to its active Rheb-GTP and promotes mTOR signalling. Akt inactivates TSC tumor suppressor complex by phosphorylation of *TSC2* (Tee et al., 2003). Common *TSC2* mutations result in the loss of the GAP domain of tuberin through Cterminal truncations, whereas some point mutations are clustered within the GAP domain. It is also reported that, an intact GAP domain of tuberin is crucial for association with hamartin in the formation of tuberin-hamartin heterodimers. The heterodimers will inhibit Rheb-induced mTOR signalling and can also function as a GAP toward Rheb. Higher proportion of the active GTP bound form of Rheb can likely be found within TSC patients. It is the result of non-functional tuberin-hamartin heterodimers where the genes failed to

The 200kDa (1806 amino acids) tuberin is homologous to the GTPase activating proteins (GAP) rap1GAP and mSpa1 where it contains relatively hydrophobic N-terminal domain and conserved 163 amino acids region close to the C-terminus. Rap1GAP is the member of Ras-related protein and functions in regulation of DNA synthesis and cell-cycle transition. The GAP activity of functional tuberin can regulate the effects of Rap1 on G to S phase transition during cell division. Thus, it implies that *TSC2* mutations may result in

Tuberin also has been demonstrated to interact with rab5. Rab5 is a cytosolic protein, is an effector for the endosomal small GTPase and therefore involved in endocytic fusion events (Stenmark et all., 1995). Consistently with the finding, tuberin has also been shown to act as a GTPase activating protein for rab5 and reduce the fluid-phase endocytosis (Xiao et al., 1997). As for 130 kDa (1164 amino acids) hamartin, it has hydrophilic protein with no significant homology to tuberin or other known vertebrate protein. Van Slegtenhorst and colleagues have investigated the association between endogenous hamartin and tuberin and they found out that both proteins play a closely related role (van Slegtenhorst et al., 1998). The methods used in the study suggest that inactivation of hamartin and tuberin may prevent

Hamartin was recently identified as an interactor with the cytoskeletal proteins, ERM family (Lamb et al., 2000). The function loss can alternatively compromise neural migration via

constitutive activation of Rap1 (Wienecke et al., 1996 and Wienecke et al., 1997).

bound state, Rheb-GDP (Rosner et al., 2004 and Tee et al., 2003).

when phosphorylated by mTOR, releases eIF4E from its control.

encodes for a functional protein (van Slegtenhorst et al., 1998).

the formation of a functional protein complex.

interaction with ERM or actin binding proteins. It was also shown to activate the GTPase Rho and regulates focal adhesion and stress fiber formation. Hamartin activates the GTPase Rho via the overlapping region of Rhos's amino acid and hamartin's tuberin-interaction domain (Lamb et al., 2000). The dysregulation of signalling by the Rho family of GTPase is said to have a critical role in cancer cell migration, invasion, and metastasis (Clark et al., 2000; Evers et al., 2000; Royal et al., 2000 and Schmitz et al., 2000).

It has also been recently shown that the functional complex interact with G2/M cyclindependant kinase 1 and its regulatory cyclins. Thus, mutation in either both genes may alter the kinetics of cell divisions (Catania et al., 2001). The functional heteromeric complex of hamartin and tuberin also plays important role in modulating the pathways of insulin receptor-or insulin-like growth factor-mediated signalling. The pathway functions downstream of the cell signalling molecule Akt, also play roles in regulating cell growth and potentially cell size.
