Our multidimensional research integrating epigenetic modulators and multidrug resistance factors with the analysis of DRE patients and BBB model systems provides insight into the possible molecular mechanisms involved in DRE.
As chromosomal translocation partners, TET family members, including TET1 and TET2, were initially found in leukaemia and proven to be key regulators of DNA demethylation owing to their dioxygenase activity. Recently, numerous studies have indicated the significance of TET2 proteins and 5-hmC in epigenetic regulation in neurodegenerative conditions such as Parkinson's disease (PD) and Alzheimer's disease (AD)[35–37]. Similar to previous approaches searching for methylated markers, such as DNMT, Reelin and BDNF, we first observed the expression and location of TET1 and TET2 in the HS and non-HS temporal neocortex in DRE, which resulted in the significant discovery that TET2 expression is extensively induced in focal lesions of DRE. 5-hmC is the most stable and abundant product of TET enzymatic activity, which can be well recognized by the fact that the active TET methylation machinery correlates with chromatin accessibility. Our result of 5-hmC in the DRE neocortex was in line with de Nijs, L. et al.’s finding and did not show specific changes in DRE patients compared with controls. We think there may be other demethylation pathways that offset the corresponding effects or cannot be evaluated merely by the 5-hmC level. For example, a recent study indicated that the fC/caC pathway promotes rapid DNA demethylation at reprogramming loci, even though 5-hmC is maintained at a steady-state abundance.
In addition, TET2’s role in the innate immune response allows it to function in a large number of pathophysiological processes associated with inflammatory diseases, which establish a “bridge” connecting it to cerebrovascular inflammation. However, whether it exerts protective or deteriorative effects in various diseases appears entirely distinct, e.g., decreased TET2 expression may exacerbate vasculitis and adverse vascular remodelling of pulmonary arterial hypertension (PAH), while elevated TET2 expression causes neuronal damage and loss in PD. Advances in our understanding of the mechanisms that govern neuroinflammation in epilepsy, particularly in the BBB, also raised some considerations concerning its importance in the clinical management of seizures. Neuroinflammation can provoke BBB dysfunction and P-gp induction in seizure models, which focus on COX-2[45, 46] and IL-1β signals in vascular endothelial cells. Further BBB disruption might have functional effects on the therapeutic effects of antiepileptic drugs (AEDs), thereby causing DRE. This is why we shifted our attention to the transporter thesis. Consequently, to investigate the potential association between TET2 and P-gp, we tentatively examined the expression status in histologic samples and isolated cerebral vessels of the DRE temporal lobe. Both TET2 and P-gp had higher expression than the control. In a BBB-simulated model constructed with hCMEC/D3 cells, TET2 depletion led to a reduction in the transcription, protein expression, and efflux function of P-gp, which suggested that TET2 has a positive regulatory impact on P-gp in the brain endothelium.
TET2-mediated 5mC oxidation can occur in a locus-specific manner. Additionally, methylation of ABCB1 can be induced by individualized variance, drugs induction, and disease progression, thereby changing the expression of ABCB1. However, our results indicated that the catalytic function of TET2 cannot contrapose the specific methylation region of ABCB1. Naturally, it does not mean that TET2 cannot catalyse demethylation in this process. It may also function by affecting the methylation of mRNA or other cis-acting elements like enhancer.
Our research also has limitations related to sample size and the failure to explore specific mechanisms, such as inflammatory pathways, in the seizure model. Interestingly, N-methyl D-aspartate (NMDA) antagonists and COX-2 inhibitors, such as celecoxib, have been shown to prevent the seizure-induced increase in P-gp functionality, thereby reversing AED resistance in rats. The rapidly increase of glutamate stimulants in seizure in vitro and in vivo models and overactivation of glutamate receptors were considered to be an important trigger for the increase of P-gp in brain capillary endothelial cells. Thus, the chronic epilepsy model exposed to glutamate or NMDA seem to be a reasonable option to investigate the latent inflammatory framework of TET2 and its relationship with pharmacoresistance and BBB dysfunction [52–54].
In summary, our results support TET2 as a possible epigenetic marker in DRE. In addition, the manipulation of P-gp expression and functionality by TET2 in a BBB model underlies its involvement in the progression of pharmacoresistant epilepsy. The next steps for research in future studies could focus on identifying inflammatory mechanisms in combination with epigenetic targeting of TET2 in epileptogenesis and structural alterations of BBB.