Abstract
This thesis describes the research that was undertaken to find peripheral markers for epilepsy and ALS. Changes in the glutamatergic system and excitotoxicity are suggested to play a role in the pathogenesis of epilepsy and amyotrophic lateral sclerosis (ALS) (chapter 1) and therefore research was focused on proteins important in
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maintaining low extracellular glutamate concentrations: glutamate transporters (EAATs) and the enzyme glutamine synthetase (GS).
Human blood platelets take-up glutamate in a Na+-dependent manner, suggesting the involvement of EAATs (chapter 2). EAAT2 protein was found in human blood platelets, but uptake in resting platelets can be inhibited only by high concentrations of DHK. EAAT2 is likely to contribute to glutamate uptake in resting human platelets, but possibly other transporters are involved as well. Thrombin activation of blood platelets, involving α-granule secretion, increased glutamate uptake 9-fold. Uptake was DHK sensitive and kinetic studies showed an increase in active transporters. EAAT2 protein was present on α-granules. Thus, the recruitment of EAAT2 from á-granules is most likely the cause of the increase in glutamate uptake.
EAAT2 and excitotoxicity have been implicated in the pathogenesis of ALS. Platelets of ALS patients had a normal basal and thrombin-stimulated glutamate uptake and a normal expression of EAAT2 (chapter 3). However, the amount of GS protein in ALS patients' platelets was increased. Thus, GS expression in platelets may provide a blood marker for ALS and for the effectiveness of therapy.
As platelets are cell fragments and have no active transcription and translation, we examined the expression of glutamatergic transcripts in leukocytes (chapter 4). Microarray and PCR analysis showed that leukocytes express a number of genes implicated in glutamatergic signaling, including EAAT1, 2 and 3 and GS. Also GS protein and enzyme activity was demonstrated. GS mRNA was approximately 50% decreased in newly diagnosed epileptic children and after 1.5 years and appeared not to be affected by AED treatment. Thus, leukocyte GS expression seems to be an early marker of epilepsy.
To study the possible correlation between blood and brain parameters in TLE, we studied EAAT expression in the hippocampus of animals with experimentally induced TLE, using the juvenile pilocarpine model (chapter 5). Increases in glutamate transporter GLAST and GLT1 protein were found in the hilus (distinctly stained cells) and the stratum lacunosum-moleculare, in animals 2 weeks after the inductions of SE, but not at later time-points. GS protein expression was only examined 2 weeks after SE and was not different from controls. Vimentin staining, a marker for reactive gliosis, was increased in the same regions, suggesting that the changes in GLAST and GLT1 are related to gliosis (chapter 5). These changes might reflect temporary changes in glutamatergic transmission and might be part of the mechanism leading to epilepsy. Using the same rats, leukocyte mRNA expression was quantified also (chapter 6). EAAT1 and EAAT3 transcripts were present in rat leukocytes but were expressed at a level too low for qPCR analysis. GS transcripts were abundantly present in rat leukocytes. QPCR showed no difference in animals after SE. Thus, GS seems not to be a peripheral marker of the epileptogenesis in animals of the juvenile pilocarpine model.
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