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Folia Neuropathologica
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vol. 46

Expression of tuberin and hamartin in tuberous sclerosis complex-associated and sporadic cortical dysplasia of Taylor\'s balloon cell type

Wiesława Grajkowska
Katarzyna Kotulska
Ewa Matyja
Magdalena Larysz-Brysz
Marek Mandera
Marcin Roszkowski
Dorota Domańska-Pakieła
Joanna Lewin-Kowalik
Sergiusz Jóźwiak

Folia Neuropathol 2008; 46 (1): 43-48
Online publish date: 2008/03/21
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Malformations due to abnormal cortical development are now recognized as one of the major aetiologies causing intractable epilepsy in children [7]. A recent classification divides neuronal migration disorders into mild cortical dysplasias (MCDs) and focal cortical dysplasias (FCDs), with respect to cytoarchitectural criteria [26]. Cortical dysplasias with isolated architectural abnormalities such as dyslamination are classified as FCD type I. FCD type II is restricted to specimens with architectural abnormalities and prominent dysmorphic cellular components. FCD type IIB, so-called Taylor's type, is characterized by presence of balloon cells in the deep cortex and white matter [5,26]. Balloon cells are increased in size and have an eosinophilic cytoplasm and an eccentric, pleomorphic nucleus. The pathogenesis of balloon cells is not clear. It was shown that balloon cells demonstrate a mixture of immunohistochemical markers of both neuronal and glial origin [8,14,32].
Balloon cells are also a hallmark of tuberous sclerosis complex (TSC) brain pathology, namely cortical tubers and subependymal giant cell astrocytomas (SEGAs). TSC is an autosomal dominant genetic disorder seen in about 1 : 6000 live births, that is characterized by formation of tumour-like growths (hamartomas) in the brain, skin, kidneys, heart and other organs [4,23]. TSC occurs due to germline or mosaic mutation in either of two genes: TSC1 or TSC2 [6]. The protein product of TSC1 gene, hamartin, consists of 1164 amino acids with a calculated mass of 130 kDa [19]. TSC2 gene encodes tuberin, a protein of 1784 amino acids and 198 kDa [13]. Both proteins are widely expressed in human tissues, including brain, and they appear to co-localize in most cells [10,11,27,28]. The mechanisms through which TSC1 and TSC2 control cellular growth and proliferation have been partially elucidated, with light being shed into the dynamic interaction between hamartin and tuberin, forming a tumour suppressor heterodimer. This complex inhibits the mammalian target of rapamycin (mTOR). mTOR is a key regulator in the signalling pathway of cell proliferation and organ size, phosphorylating two downstream target proteins, S6K1 and 4E-BP1 (eukaryotic translation initiation factor 4E binding protein 1). S6K1 is a kinase that activates ribosomal subunit protein S6, leading to ribosome recruitment and protein translation. It was recently demonstrated that Rheb (Ras homologue enriched in brain) is the direct target of TSC1/TSC2 complex, leading to the inactivation of mTOR.
Rheb belongs to the family of G signal transducing proteins. The TSC1/TSC2 heterodimer switches Rheb from an active to an inactive state leading to cell growth arrest [4,15].
Elimination of tuberin or hamartin from cells leads to activation of S6K1 and 4E-BP1 kinases, and
a resulting cell growth advantage. In most TSC-related hamartomas both tuberin and hamartin expression is reduced or, more often, absent [4,16,17,20-22].
Cortical tubers in TSC share virtually all neuropathological features of classical Taylor-type cortical dysplasia type IIB [24,26]. However, the molecular mechanisms underlying FCD type IIB are not clear. It is also not known whether FCD type IIB found in TSC patients differs from sporadic cases. Our preliminary report suggested that balloon cells in TSC and FCD type IIB may share the same origin [14]. We postulated that FCD type IIB may in fact present the focal form of TSC. To elucidate further this issue, we studied tuberin and hamartin expression in
TSC-related as well as sporadic FCD type IIB.

Material and Methods

The study was approved by the local Ethics Committee, The Children's Memorial Health Institute, Warsaw. The 6 cases included in this study were from the Department of Pathology, The Children's Memorial Health Institute, Warsaw. Patients underwent resection of FCD for medically intractable epilepsy. In each case, the histological diagnosis of Taylor's balloon cell type FCD was confirmed by standard methods. Three patients met the clinical criteria for TSC. In two of them, genetic examination was performed and TSC2 mutation was found in both cases. In 3 other patients, TSC was not confirmed on the basis of negative skin examination and kidney, liver and heart USG as well as the neuroimaging study revealing FCD as the sole pathology. All samples were fixed overnight in 4% formalin and routinely processed into paraffin.
Two paraffin embedded blocks of cases with FCD type I and 3 samples of normal brain tissue, gained from the same Department of Pathology, were used as a reference group.
Sections of 10 µm were made by means of Reichert Jung Microtome. The sections were mounted on slides covered with NovoBond (Novocastra, England), air dried for 24 hours and deparaffinized through xylene and graded alcohols to PBS (phosphate-buffered saline; SIGMA). To enable antibody binding, the high-temperature antigen unmasking method using citrate buffer was applied. The unspecific binding sites were blocked in 10% BSA (bovine serum albumin) and 10% goat serum (both SIGMA). The sections were then treated simultaneously with anti-tuberin (Novocastra; 1:25 dilution) and anti-hamartin (Santa Cruz, USA;
1:50 dilution) primary antibodies. We used anti-tuberin mouse monoclonal antibody recognizing the 240 amino acid N-terminal region of human tuberin and anti-hamartin rabbit polyclonal antibody directed against the 300 amino acid N-terminal region of human hamartin. The specimens were kept in a humid chamber, 37°C, for 1 hour. After triple rinsing in PBS, the mixture of secondary antibodies was applied. These antibodies were goat anti-mouse IgG conjugated with AlexaFluor 488 and goat anti-rabbit IgG conjugated with AlexaFluor 568 (both Molecular Probes, USA). Staining was performed in a moist chamber, room temperature, for 2 hours. Then, after triple rinsing in PBS, the slides were covered with VectaShield (Vector, USA) and coverslipped. The specimens were examined under an Olympus Fluoview confocal microscope (Olympus, Japan). Merged pictures of both excitation wavelengths were made and analyzed.


Histopathologically, the surgical specimens with FCD type I exhibited abnormal microcolumnar arrangement of cortical neurons and presence of ectopic neurons in white matter (Fig. 1). The cases diagnosed as FCD type IIB showed advanced disorganization of laminar architecture accompanied by numerous dysplastic neurons and typical balloon cells (Fig. 2). The balloon cells displayed enlarged, round cell bodies with glassy, eosinophilic cytoplasm and eccentrically located nucleus (Fig. 3). Multinucleated cells were seen and sometimes exhibited intranuclear pseudoinclusions.
In all control normal brains, as well as in FCD type I samples, strong tuberin and hamartin expression was documented in the majority of neuronal cells and other cellular elements. Both these proteins were often co-localized. Tuberin and hamartin immunoreactive cells were found in the cortex and in white matter (Fig. 4).
In FCD type IIB specimens from patients with TSC, there was no expression of either tuberin or hamartin. Neither balloon cells nor other cellular elements of the specimen showed immunoreactivity for tuberin and hamartin (Fig. 5). In sporadic FCD type IIB cases, a few tuberin and hamartin positive cells were detected only at the white-grey matter junction and in deeper parts of the white matter, whereas cortical balloon cells showed distinct loss of both tuberin and hamartin (Fig. 6).
Interestingly, the dysplastic cortex in non-TSC patients was at times bordered by normally appearing brain tissue that was immunopositive for both tuberin and hamartin. However, in TSC patients, normal-appearing brain tissue surrounding the dysplastic region of the cortex was negative for hamartin and tuberin.


In the present study we used confocal microscopy to detect the expression and co-expression of
both TSC-related proteins, hamartin and tuberin, in TSC-related and sporadic focal cortical dysplasia type IIB. We found strong tuberin and hamartin expression in normal brain tissue and in FCD type I. Tuberin and hamartin positive cells were seen both in the cortex and in the white matter. In FCD type IIB, both sporadic and TSC-related, tuberin and hamartin expression was markedly reduced or even absent. Cortical balloon cells were negative for tuberin and hamartin in all FCD type IIB samples.
Tuberin and hamartin are widely expressed in most human tissues and they usually share their localization; however, some differences have been noticed [11,13,29]. Tuberin demonstrates a diffuse cytoplasmic expression pattern, while hamartin distribution is more punctate. In the brain, hamartin is predominantly found along neuronal and astrocytic processes, whereas in the perinuclear region of Purkinje cells tuberin is distributed solely [11]. Tuberin and hamartin are tumour suppressor proteins which have been found mutated in TSC. However, investigations on both TSC-related proteins in
TSC-related hamartomas are limited in the available literature. Moreover, while immunohistochemical studies in TSC-related extra-neuronal tumours showed loss of both TSC1 and TSC2 products expression, the data obtained from brain lesions were conflicting. Henske et al. [12] found loss of tuberin in a few analyzed SEGAs. In SEGAs and cortical tubers, Mizuguchi [20,21] found loss of both proteins expression using Western blot and, surprisingly, reduced but present labelling in the same tumour, when using immunohistochemistry. Some authors found loss of tuberin immunostaining in spindle and epithelioid but not giant cells in SEGA [1,21,28].
Our previous study revealed loss of both tuberin
and hamartin expression in ten SEGAs samples. Interestingly, loss of both tuberin and hamartin expression did not depend on TSC1 or TSC2 mutation [16].
In cortical dysplasia, the data concerning tuberin and hamartin are scant and even more confusing. Becker et al. studied the alterations in TSC1 and TSC2 genes in a cohort of patients with focal cortical dysplasia [3]. By means of microdissection and laser-assisted isolation they obtained DNA from balloon cells and dysplastic neurons. Sequence alterations resulting in amino acid exchange in the TSC1 gene were increased in FCD samples when compared to normal brain tissue. Moreover, they found loss of heterozygosity at the TSC1 gene locus in nearly half of FCD samples. Ljungberg et al. [18] reported similar activation of the mTOR pathway in cytomegalic neurons in cortical dysplasia and TSC lesions. These findings further support our previous work [14] describing the molecular similarities between balloon cells in TSC and cortical dysplasia. On the other hand, Baybis et al. [2] found reduced tuberin mRNA levels in balloon cells obtained from TSC cortical tubers but not in giant cells obtained from cortical dysplasia. They used cDNA array analysis to study mRNA from single microdissected cells obtained from TSC cortical tubers and cortical dysplasia. Several differences in gene expression were observed between tubers and dysplasia samples; thus the authors suggested that these lesions differ in pathogenesis.
The occurrence of hamartomas in tuberous sclerosis is commonly ascribed for the loss of heterozygosity (LOH) phenomenon. In contrast to heart, kidney and lung tumours, brain lesions in TSC rarely present LOH for TSC1 or TSC [4,15]. Nevertheless, tuberin and hamartin are usually lost or markedly reduced in these lesions. Moreover, regardless of TSC1 or TSC2 mutation found in a particular patient, loss
of both tuberin and hamartin proteins was reported in SEGAs [16]. In the present study, we also found loss
of both proteins in TSC-related and sporadic FCD
type IIB. Loss of both proteins seems to be a unique feature of TSC lesions as in some sporadic astrocytomas and gangliogliomas, tuberin was reduced, but no disturbances in hamartin expression were found [27,31]. These findings suggest separable roles of tuberin and hamartin in sporadic tumour development.
In conclusion, our results confirmed the suggestion that FCD type IIB with balloon cells and TSC brain lesions with classical Taylor-type cortical dysplasia type IIB might represent the same entity or that FCD with balloon cells might simply be
a solitary form of TSC [30]. This similar pattern of TSC1 and TSC2 products expression indicates that TSC brain lesions and FCD type IIB may share common pathogenesis. The problem whether FCD type IIB is truly a focal form of tuberous sclerosis requires further studies using more samples and genetic analyses. Recently, some animal as well as clinical trials of rapamycin in brain lesions associated with tuberous sclerosis complex have been reported [9]. The use of this immunosuppressant acting by inhibition of mTOR seems to be promising in TSC. If FCD type IIB is derived by the same pathomechanism, such treatment might also be reasonable in cortical dysplasia associated with drug-resistant epilepsy.


The work was supported by Polish Ministry of Science grant No. PB0916/P01/2006/31 and The Children's Memorial Health Institute grant No. 112/06.


1. Arai Y, Ackerley C, Becker L. Loss of the TSC2 product tuberin
in subependymal giant-cell tumors. Acta Neuropathol 1999;
98: 233-239.
2. Baybis M, Yu J, Lee A, Golden JA, Weiner H, McKhann G 2nd, Aronica E, Crino PB. mTOR cascade activation distinguishes tubers from focal cortical dysplasia. Ann Neurol 2004; 56: 478-487.
3. Becker AJ, Urbach H, Scheffler B, Baden T, Normann S, Lahl R, Pannek HW, Tuxhorn I, Elger CE, Schramm J, Wiestler OD, Blumcke I. Focal cortical dysplasia of Taylor's balloon cell type: mutational analysis of the TSC1 gene indicates a pathogenic relationship to tuberous sclerosis. Ann Neurol 2002; 52: 29-37.
4. Chan JA, Zhang H, Roberts PS, Jozwiak S, Wieslawa G, Lewin-
-Kowalik J, Kotulska K, Kwiatkowski DJ. Pathogenesis of Tuberous Sclerosis Subependymal Giant Cell Astrocytomas: biallelic inactivation of TSC1 or TSC2 leads to mTOR activation. J Neuropathol Exp Neurol 2004; 63: 1236-1242.
5. Colombo N, Tassi L, Galli C, Citterio A, Lo Russo G, Scialfa G, Spreafico R. Focal cortical dysplasias: MR imaging, histopathologic, and clinical correlations in surgically treated patients with epilepsy. AJNR Am J Neuroradiol 2003; 24: 724-733.
6. Dabora SL, Jozwiak S, Franz DN, Roberts PS, Nieto A, Chung J, Choy YS, Reeve MP, Thiele E, Egelhoff JC, Kasprzyk-Obara J, Domanska-Pakiela D, Kwiatkowski DJ. Mutational analysis in
a cohort of 224 tuberous sclerosis patients indicates increased severity of TSC2, compared with TSC1, disease in multiple organs. Am J Hum Genet 2001; 68: 64-80.
7. Emich-Widera E, Larysz D, Kluczewska E, Larysz P, Adamek D, Mandera M, Marszał E. Malformations of cortical development in children: clinical manifestation, neuroimaging, and neuropathology in selected cases. Folia Neuropathol 2006; 44: 307-313.
8. Englund C, Folkerth RD, Born D, Lacy JM, Hevner RF. Aberrant neuronal-glial differentiation in Taylor-type focal cortical dysplasia (type IIA/B). Acta Neuropathol 2005; 109: 519-533.
9. Franz DN, Leonard J, Tudor C, Chuck G, Care M, Sethuraman G, Dinopoulos A, Thomas G, Crone KR. Rapamycin causes regression of astrocytomas in tuberous sclerosis complex. Ann Neurol 2006; 59: 490-498.
10. Geist RT, Reddy AJ, Zhang J, Gutmann DH. Expression of the tuberous sclerosis 2 gene product, tuberin, in adult and developing nervous system tissues. Neurobiol Dis 1996; 3: 111-120.
11. Gutmann DH, Zhang Y, Hasbani MJ, Goldberg MP, Plank TL, Petri Henske E. Expression of the tuberous sclerosis complex gene products, hamartin and tuberin, in central nervous system tissues. Acta Neuropathol 2000; 99: 223-230.
12. Henske EP, Wessner LL, Golden J, Scheithauer BW, Vortmeyer AO, Zhuang Z, Klein-Szanto AJ, Kwiatkowski DJ, Yeung RS. Loss of tuberin in both subependymal giant cell astrocytomas and angiomyolipomas supports a two-hit model for the pathogenesis of tuberous sclerosis tumors. Am J Pathol 1997; 151: 1639-1647.
13. Johnson MW, Kerfoot C, Bushnell T, Li M, Vinters HV. Hamartin and tuberin expression in human tissues. Mod Pathol 2001;
14: 202-210.
14. Jozwiak J, Kotulska K, Jozwiak S. Similarity of balloon cells in focal cortical dysplasia to giant cells in tuberous sclerosis. Epilepsia 2006; 47: 805.
15. Jóźwiak J, Grajkowska W, Kotulska K, Jóźwiak S, Zalewski W, Zajaczkowska A, Roszkowski M, Słupianek A, Włodarski P. Brain Tumor Formation in Tuberous Sclerosis Depends on Erk Activation. Neuromol Med 2007 (epub ahead of print).
16. Jóźwiak S, Kwiatkowski D, Kotulska K, Larysz-Brysz M, Lewin-Kowalik J, Grajkowska W, Roszkowski M. Tuberin and hamartin expression is reduced in the majority of subependymal giant cell astrocytomas in tuberous sclerosis complex consistent with
a two-hit model of pathogenesis. J Child Neurol 2004; 19: 102-106.
17. Kerfoot C, Wienecke R, Menchine M, Emelin J, Maize JC Jr, Welsh CT, Norman MG, DeClue JE, Vinters HV. Localization of tuberous sclerosis 2 mRNA and its protein product tuberin in normal human brain and in cerebral lesions of patients with tuberous sclerosis. Brain Pathol 1996; 6: 367-375.
18. Ljungberg MC, Bhattacharjee MB, Lu Y, Armstrong DL, Yoshor D, Swann JW, Sheldon M, D'Arcangelo G. Activation of mammalian target of rapamycin in cytomegalic neurons of human cortical dysplasia. Ann Neurol 2006; 60: 420-429.
19. Miloloza A, Rosner M, Nellist M, Halley D, Bernaschek G, Hengstschläger M. The TSC1 gene product, hamartin, negatively regulates cell proliferation. Hum Mol Genet 2000; 9: 1721-1727.
20. Mizuguchi M, Ikeda K, Takashima S. Simultaneous loss of hamartin and tuberin from the cerebrum, kidney and heart with tuberous sclerosis. Acta Neuropathol 2000; 99: 503-510.
21. Mizuguchi M, Kato M, Yamanouchi H, Ikeda K, Takashima S. Loss of tuberin from cerebral tissues with tuberous sclerosis and astrocytoma. Ann Neurol 1996; 40: 941-944.
22. Mizuguchi M, Kato M, Yamanouchi H, Ikeda M, Takashima S. Tuberin immunohistochemistry in brain, kidneys and heart
with or without tuberous sclerosis. Acta Neuropathol (Berl) 1997; 94: 525-531.
23. Mizuguchi M, Takashima S. Neuropathology of tuberous sclerosis. Brain Dev 2001; 23: 508-515.
24. Montenegro MA. Focal cortical dysplasia. Arch Neurol 2003;
60: 634-636.
25. Murthy V, Stemmer-Rachamimov AO, Haddad LA, Roy JE, Cutone AN, Beauchamp RL, Smith N, Louis DN, Ramesh V. Developmental expression of the tuberous sclerosis proteins tuberin and hamartin. Acta Neuropathol 2001; 101: 202-210.
26. Palmini A, Najm I, Avanzini G, Babb T, Guerrini R, Foldvary-
-Schaefer N, Jackson G, Lüders HO, Prayson R, Spreafico R, Vinters HV. Terminology and classification of the cortical dysplasias. Neurology 2004; 62: S2-S8.
27. Platten M, Meyer-Puttlitz B, Blümcke I, Waha A, Wolf HK, Nöthen MM, Louis DN, Sampson JR, von Deimling A. A novel splice site associated polymorphism in the tuberous sclerosis 2 (TSC2) gene may predispose to the development of sporadic gangliogliomas. J Neuropathol Exp Neurol 1997; 56: 806-810.
28. Vinters HV, Kerfoot C, Catania M, Emelin JK, Roper SN, DeClue JE. Tuberous sclerosis-related gene expression in normal and dysplastic brain. Epilepsy Res 1998; 32: 12-23.
29. Wienecke R, Guha A, Maize JC Jr, Heideman RL, DeClue JE, Gutmann DH. Reduced TSC2 RNA and protein in sporadic astrocytomas and ependymomas. Ann Neurol 1997; 42: 230-235.
30. Yagishita A, Arai N. Cortical tubers without other stigmata of tuberous sclerosis: imaging and pathological findings. Neuroradiology 1999; 41: 428-432.
31. Yamamoto Y, Jones KA, Mak BC, Muehlenbachs A, Yeung RS. Multicompartmental distribution of tuberous sclerosis gene products, hamartin and tuberin. Arch Biochem Biophys 2002; 404: 210-217.
32. Yamanouchi H. Activated remodeling and N-methyl-D-aspartate (NMDA) receptors in cortical dysplasia. J Child Neurol 2005; 20: 303-307.
Copyright: © 2008 Mossakowski Medical Research Centre Polish Academy of Sciences and the Polish Association of Neuropathologists. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0) License (http://creativecommons.org/licenses/by-nc-sa/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material, provided the original work is properly cited and states its license.

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