Authors: Scheldeman C., Mills J.D., Siekierska A., Serra I., Copmans D., Iyer A.M., et al. 

Neurobiol Dis, 2017


Tuberous sclerosis complex (TSC) is a rare, genetic disease caused by loss-of-function mutations in either TSC1 or TSC2. Patients with TSC are neurologically characterized by the presence of abnormal brain structure, intractable epilepsy and TSC-associated neuropsychiatric disorders. Given the lack of effective long-term treatments for TSC, there is a need to gain greater insight into TSC-related pathophysiology and to identify and develop new treatments. In this work we show that homozygous tsc2-/- mutant zebrafish larvae, but nog tsc2+/- and WT larvae display enlarged brains, reduced locomotor behavior and epileptiform discharges at 7 dpf. In addition, we pharmacologically validated the TSC model by demonstrating the dramatic rescue effect of pericardially injected rapamycin, a well-known mTOR inhibitor, on selected behavioral read-outs and at the molecular level. By means of transcriptome profiling we also acquired more insight into the neuropathology of TSC, and as a result were able to highight possible new treatment targets. The gene expression profiles of WT and  tsc2+/- larvae revealed 117 differentially expressed genes (DEGs), while between WT and  tsc2-/- larvae and  tsc2+/- and  tsc2-/- larvae there were 1414 and 1079 DEGs, respectively. Pathway enrichment analysis from the WT and  tsc2-/- DEGs, identified 14 enriched pathways from the up-regulated genes and 6 enriched pathways from the down-regulated genes. Moreover, genes related to inflammation and immune response were up-regulated in the heads of  tsc2-/- larvae, in line with the findings in human brain tissue where inflammatory and immune responses appear to be major hallmarks of TSC. Taken together, our phenotypic, transcriptomic and pharmacological analysis identified the  tsc2-/- zebrafish as a preclinical model that mirrors well aspects of the human condition and delineated relevant TSC-related biological pathways. The model may be of value for future TSC-related drug discovery and development programs. 

Authors: Moavero R., Carai A., Mastronuzzi A., Marciano S., Graziola F., Vigevano F., et al. 

Pediatric Neurology, 2017;68:59-63


Background: Subependymal giant cell astrocytomas (SEGAs) are low-grade tumors affecting up to 20% of patients with tuberous sclerosis complex (TSC). Early neurosurgical resection has been the only standard treatment until few years ago when a better understanding of the molecular pathogenesis of TSC led to the use of mammalian target of rapamycin (mTOR) inhibitors. Surgical resection of SEGAs is still considered as the first line treatment in individuals with symptomatic hydrocephalus and intratumoral hemorrhage. We describe four patients with symptomatic or asymptomatic hydrocephalus who were successfully treated with the mTOR inhibitor everolimus. 

Methods: We collected the clinical data of four consecutive patients presenting with symptomatic or asymptomatic hydrocephalus due to a growth of subependymal giant cell astrocytomas and who could not undergo surgery for different reasons. 

Results: All patients experienced a clinically significant response to everolimus and an early shrinkage of the SEGA with improvement in ventricular dilatation. Everolimus was well tolerated by all individuals. 

Conclusions: Our clinical series demonstrate a possible expanding indication for mTOR inhibition in TSC, which can be considered in patients with asymptomatic hydrocephalus or even when the symptoms already appeared. It offers a significant therapeutic alternative to individuals that once would have undergone immediate surgery. Everolimus might also allow postponement of a neurosurgical resection, making it elective with an overall lower risk. 

Authors: Mills J.D., Iyer A.M., van Scheppingen J., Bongaarts A., Anink J.J., Janssen B. et al. 

Scientific reports, 2017;7(1):8089


Tuberous Sclerosis Complex (TSC) is a rare genetic disorder that results from a mutation in the TSC1 or TSC2 genes leading to constitutive activation of the mechanistic target of rapamycin complex 1 (mTORC1). TSC is associated with autism, intellectual disability and severe epilepsy. Cortical tubers are believed to represent the neuropathological substrates of these disabling manifestations in TSC. In the presented study we used high-throughput RNA sequencing in combination with systems-based computational approaches to investigate the complexity of the TSC molecular network. Overall we detected 438 differentially expressed genes and 991 differentially expressed small non-coding RNAs in cortical tubers compared to autopsy control brain tissue. We observed increased expression of genes associated with inflammatory, innate and adaptive immune responses. In contrast, we observed a down-regulation of genes associated with neurogenesis and glutamate receptor signaling. MicroRNAs represented the largest class of over-expressed small non-coding RNA species in tubers. In particular, our analysis revealed that the miR-34 family (including miR-34a, miR-34b and miR-34c) was significantly over-expressed. Functional studies demonstrated the ability of miR-34b to modulate neurite outgrowth in mouse primary hippocampal neuronal cultures. This study provied new insights into the TSC transcriptomic network along with the identification of potential new treatment targets. 


Authors: Broekaart D.W.M., van Scheppingen J., Geijtenbeek K.W., Zuidberg M.R.J., Anink J.J., Baayen J.C., et al. 

Epilepsia, 2017;58(8):1462-72. 


Objective: Inhibition of the mammalian target of rapamycin (mTOR) pathway reduces epileptogenesis in various epilepsy models, possibly by inhibition of inflammatory processes, which may include the proteasome system. To study the role of mTOR inhibition in the regulation of the proteasome system, we investigated (immuno) proteasome expression during epileptogenesis, as well as the effects of the mTOR inhibitor rapamycin. 

Methods: The expression of constitutive (β1, β5) and immunoproteasome (β1i, β5i) subunits was investigated during epileptogenesis using immunohistochemistry in the electrical post-status epilepticus (SE) rat model for temporal lobe epilepsy (TLE). The effect of rapamycin was studied on (immuno)proteasome subunit expression in post-SE rats that were treated for 6 weeks. (Immuno)proteasome expression was validated in the brain tissue of patients who had SE or drug-resistant TLE and the effect of rapamycin was studied in primary human astrocyte cultures. 

Results: In post-SE rats, increased (immuno)proteasome expression was detected throughout epileptogenesis in neurons and astrocytes within the hippocampus and piriform cortex and was most evident in rats that developed a progressive form of epilepsy. Rapamycin-treated post-SE rats had reduced (immuno)proteasome protein expression and a lower number of spontaneous seizures compared to vehicle-treated rats. (Immuno)proteasome expression was also increased in neurons and astrocytes within the human hippocampus after SE and in patients with drug-resistant TLE. In vitro studies using cultured human astrocytes showed that interleukin (IL)-1β-induced (immuno)proteasome gene expression could be attenuated by rapamycin. 

Significance: Because dysregulation of the (immuno)proteasome system is observed before the occurence of spontaneous seizures in rats, is associated with progression of epilepsy and can be modulated via the mTOR pathway, it may represent an interesting novel target for drug treatment in epilepsy. 

Authors: Aronica E., Bauer S., Bozzi Y., Caleo M., Dingledine R., Gorter J.A., et al. 

Epilepsia, 2017;58 Suppl 3:27-38


A large body of evidence that has accumulated over the past decade strongly supports the role of inflammation in the pathophysiology of human epilepsy. Specific inflammatory molecules and pathways have been identified that influence various pathologic outcomes in different experimental models of epilepsy. New antiseizure therapies may be derived from these novel potential targets. An essential and crucial question is whether targeting these molecules and pathways may result in anti-ictogenesis, antiepileptogenesis, and/or disease-modification effects. Therefore, preclinical testing in models mimicking relevant aspects of epileptogenesis is needed to guide integrated experimental and clinical trial designs. We discuss the most recent preclinical proof-of-concept studies validating a number of therapeutic approaches against inflammatory mechanisms in animal models that could represent novel avenues for drug development in epilepsy. Finally, we suggest future directions to accelerate preclinical to clinical translation of these recent discoveries.