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(Abst. 1.087), 2019

Dysregulation of Glutamate-Glutamine Cycle Transporters in Human Drug-Resistant Epilepsy
Authors: Scott Massa, University of Pennsylvania; Kimberly Sansalone, University of Pennsylvania; Sarah Gourmaud, University of Pennsylvania; Timothy Lucas, Hospital of the Univ. of Pennsylvania; Frances E. Jensen, University of Pennsylvania; Kathryn A. Davis, Hospital of the Univ. of Pennsylvania; Delia M. Talos, University of Pennsylvania
Content: Rationale: Temporal lobe epilepsy (TLE) is one of the most common localization-related epilepsies and is highly associated with resistance to medications. Dysregulated levels of the excitatory neurotransmitter glutamate (GLU) and its precursor, glutamine (GLN), have been implicated in both human and experimental TLE. Extracellular and intracellular GLU and GLN concentrations are controlled by inter-conversion enzymes and transporters. Prior studies in human chronic epilepsy have consistently found decreased expression levels of glutamine synthetase (GS), the astrocytic enzyme responsible for the conversion of GLU into GLN, along with increased expression of the neuronal phosphate-activated glutaminase (PAG), the GLN to GLU converting enzyme. On the contrary, there has been greater variability in expression level results from analyses of the glial GLU transporters, the excitatory amino acid transporter (EAAT) subtypes 1 and 2, while the GLN transporter levels have yet to be studied in human TLE. The sodium-coupled neutral amino acid transporter (SNAT) subtypes 3 and 5 are responsible for GLN efflux from astrocytes, while the SNAT1 and 2 transporters mediate the uptake of GLN into neurons. Investigation of these transporters can provide key insights into the dysregulation of glutamatergic neurotransmission in TLE. Methods: Hippocampal and temporal lobe cortical tissues were obtained via surgical resection from drug-resistant TLE patients (n=15, mean age = 26.3 years) at the Hospital of the University of Pennsylvania, the Children's Hospital of Philadelphia and the Children’s Hospital Boston. Postmortem samples from age and region-matched control subjects (n=9, mean age = 19.9 years) were obtained from the University of Maryland Brain and Tissue Bank, a brain and tissue repository of the NIH NeuroBioBank. This investigation was approved by the local Institutional Review Board. Expression levels of EAAT1, EAAT2, SNAT1 and SNAT5 were quantified through Western blotting and compared between groups. Statistical significance (p < 0.05) was assessed by either Student’s t-tests or Mann-Whitney U tests. Results: In TLE hippocampus, there was a significant upregulation of EAAT1 (1036 ± 486% of control, p < 0.05), EAAT2 (159 ± 15% of control, p < 0.01) and SNAT1 (203 ± 18% of control, p < 0.001), with no significant change in SNAT5 (202 ± 68% of control, p > 0.05) expression. In TLE cortex, there was a significant upregulation of EAAT1 (800 ± 196% of control, p < 0.01) and SNAT5 (218 ± 47% of control, p < 0.05) but no significant change in EAAT2 (163 ± 22% of control, p > 0.05) and SNAT1 (112 ± 8% of control, p > 0.05) expression. Conclusions: The coordinated upregulation of GLU and GLN transporters in human TLE hippocampus and temporal lobe cortex reported here is consistent with enhanced GLU availability for GLN synthesis in astrocytes (EAAT1 and EAAT2 increase), which is correspondingly associated with increased GLN availability for enhanced GLU generation in neurons (SNAT1 and SNAT5 elevation). These results suggest a potential novel mechanism leading to enhanced glutamatergic neurotransmission in TLE and identify the GLN transporters SNAT1 and SNAT5 as novel targets for anti-seizure therapies in this widely drug-resistant form of epilepsy. Funding: This work was supported by grants from the National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS): R01NS101156 (DMT), K23NS092973 (KAD), R21NS105437 (FEJ) and R01NS080565 (FEJ).