Reduced endogenous KYNA synthesis was due to altered levels of KAT II and PLP
From the above experiments, it was evident that endogenous KYNA synthesis was reduced and QUIN synthesis was elevated in the hippocampal samples obtained from patients with MTLE-HS which could be attributed to the dysfunctional machinery responsible for synthesis of those. KYNA is synthesized from KYN in presence of enzyme KAT II. To address whether the altered expression of this enzyme was correlated with the reduced synthesis of its product, we investigated its mRNA and protein expression by quantitative reverse transcriptase polymerase chain reaction (qPCR) and western blotting respectively. We found no alteration of mRNA expression in the hippocampal samples (Figure 6a). However, protein expression was significantly downregulated in the hippocampal samples (Figure 6b). However, the magnitude of this reduction was not proportional to that of reduction in concentration of KYNA. Given the fact that function of KAT II depends on the co-factor PLP, we estimated its concentration by HPLC and found that the concentration of PLP was significantly reduced in the hippocampal samples (Figure 6g). Then we investigated mRNA and protein expression of the enzyme PNPO which synthesizes PLP from pyridoxine. We found that both mRNA (Figure 6h) as well as protein expression of this enzyme (Figure 6i) were significantly downregulated in the hippocampal samples.
We investigated the expression profile of IDO, the rate-limiting enzyme for TRP to KYN conversion. Both mRNA (Figure 6c) as well as protein level expression (Figure 6d) were significantly upregulated in the hippocampal samples. We focused on the rate-limiting enzyme for QUIN synthesis which is KMO because as per our results, concentration of QUIN was higher in the hippocampal samples. Both mRNA (Figure 6e) as well as protein expression (Figure 6f) were not significantly altered in the hippocampal samples.