Abstract by Christina Kjær

Background: One of the most frequent brain circuit disorders, mesial temporal lobe epilepsy (mTLE), causes patient disability and, despite intensive research, ⁓ 30% of the patients do not achieve sustained seizure freedom with current antiseizure medications. Thus, there is a need for identification of new drug targets in mTLE.

Objective: To identify differentially expressed genes associated with drug-resistant mTLE and to identify and validate putative novel lead drug targets among them. Secondly, to investigate the influence of preanalytical factors on messenger ribonucleic acid (mRNA) yield and integrity.

Method: Differentially expressed gene identification was done by 1) Transcriptome analysis of preexisting mTLE microarray datasets and a novel mTLE ribonucleic acid sequencing (RNA-Seq) dataset, 2) Bioinformatics-driven lead drug target identification, and 3) Follow up lead target validation attempts. This was done on mRNA and protein isolated from fresh hippocampal and temporal lobe neocortical tissues using quantitative polymerase chain reaction (qPCR), protein immunoblots, and immunohistochemical analysis, respectively. Preanalytical factors affecting mRNA yield and integrity were examined for the following conditions: Time-before-collection, tissue specimen size, tissue collection method, and RNA isolation method.

Results: In this thesis, 3,040 significant mTLE-associated, differentially expressed genes were identified. Comparing this dataset with the 5,523 differentially expressed genes identified in a preexisting mTLE microarray dataset, a 93.9% agreement on direction of regulation was identified – corresponding to a robust list of 1,069 differentially expressed genes. Five possible novel mTLE lead drug targets were identified using an unbiased bioinformatics strategy followed by a range of structured bioinformatics selection criteria. The lead drug target validation attempt found that CACNB3 was significantly regulated in mTLE at mRNA and protein level. Also, brain-derived mRNA integrity was largely unaffected by preanalytical factors.

Conclusion: The results provide new insights into gene expression associated with drugresistant mTLE by identification of 3,040 mTLE-regulated genes. Notably, CACNB3 – the β3 subunit of voltage gated Ca2+channels – may be a possible novel drug target in mTLE.