Reduction in endogenous KYNA synthesis contributes to the hyperexcitability in MTLE-HS
Astrocyte-neuron interaction play a crucial role in the regulation of glutamatergic activity. One of the regulation mechanisms is the conversion of KYN to KYNA in astrocytes which inhibits glutamate receptor-mediated synaptic transmission on to neurons (Moroni et al., 2012). Pyridoxine and PNPO has long history with epilepsy treatment (Watanabe, 1995; Ito et al., 2000; Kuo and Wang, 2002; Jaeger et al., 2016). We observed that the mRNA and protein expression of the PNPO enzyme were significantly downregulated in MTLE-HS patients leading to reduced PLP synthesis (Figure 7g, h, i). Taken together, reduction in both KAT II expression as well as its co-factor PLP might be affectingde novo synthesis of KYNA from KYN. Our observation that tissue slices incubated with saturating concentration of KYN (200µM) showed negligible change in the frequency and amplitude of spontaneous EPSCs in the hippocampal samples of patients with MTLE-HS compared to that in non-seizure control samples(Figure 4e) can be attributed to both presynaptic as well as postsynaptic glutamatergic activity. KYNA inhibits action potential dependent spontaneous glutamatergic activities (Banerjee et al., 2012) and reduction of endogenous KYNA synthesis enhances hippocampal extracellular glutamate release as evidenced in animal models (Pocivavsek et al., 2011; Potter et al., 2010). Further experiments involving isolation of miniature EPSCs in the presence of KYNA may further help us understand the regulation of glutamatergic activity by quantal currents in the hippocampal samples obtained from patients with MTLE-HS. We suspect that the amount of KYNA synthesizedde novo in the hippocampal samples was less than IC50 value of KYNA for α7 nAChR (1-8 µM) (Hilmas et al., 2001) and glycine site of NMDA receptor (10-30µM) (Parsons et al., 1997). Consequently, presynaptic α7 nAChR dependent glutamate release was not regulated and action potential dependent presynaptic inputs were enhanced, leading to increase in the frequency of sEPSCs (Banerjee et al., 2020). The postsynaptic NMDA receptors were upregulated and functionally hyperactive in MTLE-HS hippocampus (Banerjee et al., 2017). As, postsynaptic glycine sites of NMDA receptors were also not competitively inhibited by KYNA, the amplitude of spontaneous EPSCs was also increased. Future studies involving incubating the brain slices with either the KAT II or PLP or PLP synthesizing enzyme PNPO will be beneficial to prove the central hypothesis more accurately by assessing the extent of recovery of KYNA synthesis in MTLE-HS.
We could not see significant alterations in GABAergic activities under resting state. This suggests that, under resting conditions, hyperexcitatory activities were primarily mediated by glutamate receptors in the hippocampal samples. Moreover, these MTLE-HS patients were on AEDs (Supplementary table 1), most of which are GABAA receptor modulators, may have compensated for the altered GABAergic activity in the hippocampal samples.