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.