Authors :
Presenting Author: Chaitali Ghosh, PhD, FAES – Cleveland Clinic Lerner Research Institute
Rosemary Westcott, BS – Biomedical Engineering – Cleveland Clinic Lerner Research Institute, Cleveland, Ohio, USA; David Skvasik, BS – Biomedical Engineering – Cleveland Clinic Lerner Research Institute, Cleveland, Ohio, USA; Ishant Khurana, PhD – Epigenetics in Human Health and Disease Program – Baker Heart and Diabetes Institute, Melbourne, Australia; Assam El-Osta, PhD – Epigenetics in Human Health and Disease Program – Baker Heart and Diabetes Institute, Melbourne, Australia; Imad Najm, MD – Charles Shor Epilepsy Center – Neurological Institute, Cleveland Clinic, Cleveland, Ohio, USA
Rationale:
Focal cortical dysplasia (FCD) is a major cause of refractory epilepsy and associated with compromised neurovascular and metabolic function. The current study examines the regulation of glucose-transporter1 (GLUT1) and brain hypometabolism in FCD patient subtypes that could lead to early diagnosis and intervention.
Methods:
Studied surgically resected human brain tissues were characterized (n=55) into the following FCD subtypes: FCD2A, FCD2B, FCD3A, FCD3B, and non-lesional. Using methyl-CpG capture associated with massive parallel sequencing, DNA methylation analysis was performed on a subset of samples (n=14, matched brain and blood samples) to determine target genes linked to GLUT1 and glucose metabolism. We evaluated the brain glucose-lactate levels by biochemical assay, and GLUT1, vascular endothelial growth factor (VEGFα), lactate transporter 2 (MCT2), mammalian target of rapamycin (mTOR) by western blot analysis across FCD subtypes, which were later correlated to age and gender of these individuals (n=55). The GLUT1 levels were also assessed in dysplastic and non-dysplastic brain tissues of same subjects by immunohistochemistry and isolated epileptic brain endothelial cells (EPI-ECs) by western blot. As “proof-of concept” validation, the effect of a DNA methylation inhibitor, 5-Aza-2′-deoxycytidine (5Aza, 10µM) was tested on glucose uptake of [3H]-2-deoxyglucose and GLUT1, VEGFα levels in EPI-ECs and mTOR signaling in human embryonic kidney cells (HEK), post-5Aza treatment for 24h.
Results:
We observe hypermethylation of GLUT1 and other key genes (BDNF and mTOR) responsible for glucose metabolism that distinguishes FCD and non-FCD in both the brain and blood samples. Suppressed GLUT1 and low glucose-lactate ratios correspond to elevated VEGFα in FCD2A and FCD2B brain tissues independent of age and gender. Dysplastic regions with cortical disorganization show GLUT1 suppression compared to non-dysplastic brain tissues and endothelial cells from the same subject. Increased mTOR signaling (mTOR, p-mTOR and p-70S6K) is predominant pathway in dysplastic brains (FCD vs. non-FCD). The DNA methylation inhibitor did not influence cytotoxicity, increased GLUT1 levels (*p< 0.05) and decreased VEGFα (*p< 0.05) in EPI-ECs. In addition, 5Aza restored glucose uptake function (*p< 0.05) under low glucose-high lactate condition when compared to untreated cells, suggesting DNA methylation influences GLUT1 and cellular response to altered glucose uptake in EPI-EC. Similarly, in HEK cells stimulated by low glucose-high lactate state, inhibition of DNA methylation with 5Aza reduced mTOR and MCT2 levels when compared to 5Aza-untreated cells.