Authors: van Scheppingen J., Broekaart D.W.M., Scholl T., Zuidberg M.R.J., Anink J.J., Spliet W.G., van Rijen P.C., Czech T., Hainfellner J.A., Feucht M., Mühlebner A., van Vliet E.A., Aronica E.

J Neuroinflammation. 2016 Aug 26;13(1):202. doi: 10.1186/s12974-016-0662-z.


Background: The proteasome is a multisubunit enzyme complex involved in protein degradation, which is essential for many cellular processes. During inflammation, the constitutive subunits are replaced by their inducible counterparts, resulting in the formation of the immunoproteasome. 
Methods: We investigated the expression pattern of consitutive (B1, B5) and immunoproteasome (B1i, B5i) subunits using immunohistochemistry in malformations of cortical development (MCD; focal cortical dysplasia (FCD) IIa and b, cortical tubers from patients with tuberous sclerosis complex (TSC) and mild MCD (mMCD)). Glial cells in culture were used to elucidate the mechanisms regulating immunoproteasome subunit expression. 
Results: Increased expression was observed in both FCDII and TSC; B1, B1i, B5 and B5i were detected (within cytosol and nucleus) in dysmorphic neurons, balloon/giant cells, and reactive astrocytes. Glial and neuronal nuclear expression positively correlated with seizure frequency. Positive correlation was also observed between the glial expression of constitutive and immunoproteasome subunits and IL-1B. Accordingly, the proteasome subunit expression was modulated by IL-1B in human astrocyrtes in vitro. Expression of both constitutive and immunoproteasome subunits in FCD II-derived astroglial cultures was negatively regulated by treatment with the immunomodulatory drug rapamycin (inhibitor of the mammalian target of rapamycin (mTOR) pathway, which is activated in both TSC and FCD II).
Conclusions: These observations support the dysregulation of the proteasome system in both FCD and TSC and provide new insights on the mechanism of regulation the (immuno)proteasome in astrocytes and the molecular links between inflammation, mTOR activation and epilepsy. 


Authors: Mühlebner A., van Scheppingen J., Hulshof H.M., Scholl T., Iyer A.M., Aninck J.J., van den Ouweland A.M.W., Nellist M.D., Jansen F.E., Spliet W.G.M., Krsek P., Benova B., Zamecnik J., Crino P.B., Prayer D., Czech T., Wöhrer A., Rahimi J., Höftberger R., Hainfeller J.A., Feucht M., Aronica E.

PLOS One. 2016 Jun 13;11(6):e0157396. doi: 10.1371/journal.pone.0157396. eCollection 2016.


Tuberous Sclerosis Complex (TSC) is a genetic hamartoma syndrome frequently associated with severe intractable epilepsy. In some TSC patients epilepsy surgery is a promising treatment option provided that the epileptogenic zone can be precisely delineated. TSC brain lesions (cortical tubers) contain dysmorphic neurons, brightly eosinophilic giant cells and white matter alterations in various proportions. However, a histological classification system has not been established for tubers. Therefore, the aim of this study was to define distinct histological patterns within tubers based on semi-automated histological quantification and to find clinically significant correlations. In total, we studied 28 cortical tubers and seven samples of perituberal cortex from 28 TSC patients who had undergone epilepsy surgery. We assessed mammalian target of rapamycin complex 1 (mTORC1) activation, the number of giant cells, dysmorphic neurons, neurons, and oligodendrocytes, and calcification, gliosis, angiogenesis, inflammation and myelin content. Three distinct histological profiles emerged based on the proportion of calcifications, dysmorphic neurons and giant cells designated types A, B, and C. In the latter two types we were able to subsequently associate them with specific features on presurgical MRI. Therefore, these histopathological patterns provide consistent criteria for improved definition of the clinico-pathological features of cortical tubers identified by MRI and provide a basis for further exploration of the functional and molecular features of cortical tubers in TSC.


Authors: Fuso A., Iyer A.M., van Scheppingen J., Maccarrone M., Scholl T., Hainfeller J.A., Feucht M., Jansen F.E., Spliet W.G., Krsek P., Zamecnik J., Mühlebner A., Aronica E.

J Mol Neurosci. 2016 Aug;59(4): 464-70. doi: 10.1007/s12031-016-0750-7. Epub 2016 Apr 28.


In tuberous sclerosis complex (TSC), overexpression of numerous genes associated with inflammation has been observed. Among different proinflammatory cytokines, interleukin-1B (IL-1B) has been shown to be significantly involved in epileptogenesis and maintenance of seizures. Recent evidence indicates that IL-1B gene expression can be regulated by DNA methylation of its promoter. In the present study, we hypothesized that hypomethylation in the promoter region of the IL-1B gene may underlie its overexpression observed in TSC brain tissue. Bisulfite sequencing was used to study the methylation status of the promoter region of the IL-1B gene in TSC and control samples. IL-1B is overexpressed in tubers, and gene expression is correlated with promoter hypomethylation at CpG and non-CpG sites. Our results provide the first evidence of epigenetic modulation of the IL-1B signaling in TSC. Thus, strategies that target epigenetic alterations could offer new therapeutic avenues to control the persistent activation of interleukin-1B-mediated inflammatory signaling in TSC brain.

Authors: Blazejczyk M., Macias M., Korostynski M., Firkowska M., Piechota M., Skalecka A., Tempes A., Koscielny A., Urbanska M., Przewlocki R., Jaworski J.

Mol Neurobiol 2016 March 19; doi: 10.1007/s12035-016-9821-6


Epileptogenesis is a process triggered by initial environmental or genetic factors that result in epilepsy and may continue during disease progression. Important parts of this process include changes in transcriptome and the pathological rewiring of neuronal circuits that involves changes in neuronal morphology. Mammalian/mechanistic target of rapamycin (mTOR) is upregulated by proconvulsive drugs, e.g., kainic acid, and is needed for progression of epileptogenesis, but molecular aspects of its contribution are not fully understood. Since mTOR can modulate transcription, we tested if rapamycin, an mTOR complex 1 inhibitor, affects kainic acid-evoked transcriptome changes. Using microarray technology, we showed that rapamycin inhibits the kainic acid-induced expression of multiple functionally heterogeneous genes. We further focused on engulfment and cell motility 1 (Elmo1), which is a modulator of actin dynamics and therefore could contribute to pathological rewiring of neuronal circuits during epileptogenesis. We showed that prolonged overexpression of Elmo1 in cultured hippocampal neurons increased axonal growth, decreased dendritic spine density, and affected their shape. In conclusion, data presented herein show that increased mTORC1 activity in response to kainic acid has no global effect on gene expression. Instead, our findings suggest that mTORC1 inhibition may affect development of epilepsy, by modulating expression of specific subset of genes, including Elmo1, and point to a potential role for Elmo1 in morphological changes that accompany epileptogenesis.


authors: Domanska-Pakiela D., Kaczorowska M., Jurkiewicz E., Kotulska K., Dunin-Wasowicz D., Jozwiak S.

Eur J Paediatr Neurol. 2014 Jul;18(4):458-68. doi: 10.1016/j.ejpn.2013.12.006.


Tuberous sclerosis complex (TSC) is a multisystem, autosomal dominant disorder characterized by multiple hamartomas development. Epilepsy is the most common symptom appearing in 80-90% of the patients mainly in the first year of life. A prompt and early seizure control is crucial and can prevent development of an epileptic encephalopathy and secondary mental retardation. Therefore the very early identification of seizures seems to be of a great importance. We present the cases of 5patients diagnosed with TSC prenatally or perinatally and regularly monitored (at 4-6 weeks intervals) with EEG before theepilepsyonset. The patientsage at baseline varied from 9 days to 9 weeks. In all of the patients epileptiform discharges preceded the epilepsyonset. The time interval between abnormality detection on EEG and the epilepsyonset varied from 1 to 8 days. The patient's age at the epilepsyonset ranged from the 17th day to the 5th month of life. In one patient the EEG was abnormal from the beginning and in this patient the epileptic seizures started from the neonatal period. In the rest of thepatients (4/5) the EEG remained normal throughout the first months of life. In all of the children epilepsy started with focal motor seizures. Our study is the first prospective one showing the results of the EEG monitoring in TSC patients and the natural evolution of the EEG patterns in patients with the seizures types other than infantile spasms.