Ph.D Thesis

Ph.D StudentShleper Maria
SubjectRole of D-serine Release in Neuronal Cell Death Mediated
by NMDA Receptor Activation
DepartmentDepartment of Medicine
Supervisor PROF. Herman Wolosker
Full Thesis textFull thesis text - English Version


High levels of D-serine were discovered in the mammalian brain, where it plays a role as an endogenous ligand at the "glycine site" of N-methyl-D-aspartate (NMDA) receptor. However, D-serine action has not been previously compared with that of endogenous glycine, and the relative importance of the two coagonists remains unclear. We now investigated the efficiencies of the two coagonists in mediating NMDA receptor neurotoxicity in organotypic hippocampal slices. Removal of endogenous D-serine from slices was achieved by pretreating the tissue with recombinant D-serine deaminase enzyme. This enzyme is several orders of magnitude more efficient than previous methods to remove D-serine. We report that complete removal of D-serine virtually abolished NMDA-elicited neurotoxicity but did not protect against kainate. Although levels of glycine were 10-fold higher than D-serine, endogenous glycine was ineffective in mediating NMDA receptor neurotoxicity. The effect of endogenous glycine could be observed only after simultaneous removal of endogenous D-serine and blockage of the glycine transporter GlyT1. Our data indicate that D-serine is the dominant coagonist for NMDA receptor-elicited neurotoxicity, mediating all cell death elicited by NMDA in organotypic slices.

Mechanisms that regulate D-serine concentration in a synapse are not known. We also investigated both in vitro and in vivo the D-serine release pathways in neurons and astrocytes. We developed new technique, utilizing glutaminase enzyme, which allows the detection of endogenous D-serine release from cell cultures. Using this technique we demonstrate robust release of endogenous neuronal D-serine mediated by a-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), confirming previous study employing D-[3H]serine. We also found that astrocytes release D-serine under AMPA stimulation, but not under veratridine, ouabain and KCl stimulation. This difference in astrocyte and neuronal response allowed us to utilize veratridine as tool to monitor neuronal D-serine release in vivo by brain microdyalisis.  We demonstrate that neuronal release of D-serine upon depolarization of neurons may play a role in vivo and that benzamil can be used as a tool to block D-serine release. Release of endogenous D-serine was mostly through a cytosolic route, since it was insensitive to bafilomycin A1, which is a potent inhibitor of the vesicular neurotransmitter release. Additionally, we now report a new pathway for D-serine release via volume-regulated anion channels (VRAC), which can be activated either by AMPA or hypotonicity. We demonstrate that astrocytes, but not neurons release D-serine under hypotonic conditions and that this release is inhibited by specific VRAC inhibitors.