Abstracts

Rationale: Tuberous sclerosis complex (TSC) is a multisystemic disease associated with seizures and inflammation in the brain. We have shown that epileptogenic tubers have increased tryptophan uptake and evidence that this uptake is related to increased tryptophan metabolism by the kynurenine pathway (KP). Products of the KP include the neurotoxic metabolite quinolinic acid, and the end product of the KP is nicotinamide adenine dinucleotide (NAD), a substrate for many cell reactions including poly(ADP-ribosyl)ation. Recently, epileptogenic tubers were found to have increased expression of a protein associated with drug resistance called major vault protein (MVP), which constitutes the majority of the ribonucleoprotein vault particle. The vault particle also contains a poly(ADP-ribose) polymerase termed vPARP. Here we investigate whether there is association between MVP and the KP in epileptogenic tubers. We localized the presence of MVP and the rate-limiting enzyme of the KP, indoleamine 2,3-dioxygenase (IDO), in epileptogenic tubers. Then we studied whether there is an association between the expression of MVP and IDO and the downstream KP enzyme 3-hydroxyanthranilate-3,4-dioxygenase (HAO), which leads to the production of the pro-convulsant metabolite quinolinic acid. Methods: Immunocytochemistry for IDO and MVP (N=8) and western blots for IDO (isoforms 1 and 2), HAO, and MVP (N=25) were performed on surgical specimens from patients with TSC who underwent epilepsy surgery for whom epileptogenic tuber and relatively normal tissue areas were available. Actin was used as endogenous control. Results: IDO expression was present in dysplastic cells and neuropil (Figure 1A and 1B), while some cells did not stain (Figure 1C). The perivascular tissue also showed positive immunoreaction (Figure 1D). MVP demonstrated similar pattern, staining dysplastic cells and neuropil (Figure 1E and 1F). Similarly to IDO, some dysplastic cells were not stained (Figure 1G). The vascular bed was also positive (Figure 1H). Epileptogenic tubers had higher expression of MVP, IDO2 and HAO compared to control areas (all p<0.01), but not IDO1 (p=0.25). Expression of MVP correlated with HAO in the epileptogenic tissue (r =0.7, p<0.001) and in the control area (r=0.8, p<0.001) (Figure 2A and 2B). A trend was observed between expression of MVP and IDO1 in the epileptogenic tissue (r=0.4, p=0.086), while expression of MVP in the control tissues correlated with IDO1 (r=0.44, p=0.028). Conclusions: Coordinate expression of KP enzymes and MVP may be due to common regulatory mechanisms such as IFN-γ/Jak-STAT signaling related to inflammation in TSC. Alternatively, increased demand for NAD due to vPARP activity of the vault particle may drive KP activity including the formation of quinolinic acid. Further study of the interplay between MVP and the KP may help better understand how lesions in TSC become epileptogenic and drug resistant, providing further targets for pharmacological treatment.