Ph.D Thesis

Ph.D StudentKartvelishvily Elena
SubjectIdentification of a Neuronal Pool of D-Serine and its
Role in NMDA Receptor Regulation
DepartmentDepartment of Medicine
Supervisor PROF. Herman Wolosker
Full Thesis textFull thesis text - English Version



D-Serine is a coagonist of N-methyl-D-aspartate (NMDA) receptors that occurs at high levels in the brain. Biosynthesis of D-Serine is carried out by the serine racemase enzyme that converts L- into D-serine. D-serine has been demonstrated to occur in glial cells, which led to the proposal that astrocytes are the only source of D-serine. We now report significant amounts of serine racemase and D-serine in primary neuronal cultures and neurons in vivo. Several neuronal culture types express serine racemase in which D-serine synthesis is comparable to that of glial cultures. Immunohistochemical staining of brain sections with new antibodies revealed the presence of serine racemase and D-serine in neurons. Cortical neurons expressing serine racemase also expressed NMDA receptor subunit 2a in situ.  Neuron-derived D-serine contributes for NMDA receptor activation in cortical neuronal cultures. Degradation of endogenous D-serine by addition of the recombinant enzyme D-serine deaminase diminished NMDA-elicited excitotoxicity.  Release of neuronal D-serine was mediated by ionotropic glutamate receptor agonists, such as NMDA, a-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and kainate. L-homocysteic amino acid, an endogenous glutamate receptor agonist also mediates robust D-serine release. Removal of either external Ca2+ or Na+ blocked D-serine release. Veratridine, which opens sodium channels, also induced robust release of D-serine from neurons. Release of D-serine was mostly through a cytosolic route, since it was insensitive to bafilomycin A1 and tetanus toxin, which are potent inhibitors of the vesicular neurotransmitter release. D-Serine was also not transported into purified synaptic vesicles under conditions that are optimal for the uptake of known transmitters. We found that AMPA-induced D-serine release is blocked by benzamil. Benzamil blocks the entry of Na+ through Na+/Ca2+-exchanger,  as well as other targets, such as Na+/H+-exchanger. We suggest that inhibition of Na+ influx by benzamil drops basal intracellular Na+ concentration, which is necessary for D-serine release. Our results suggest that neurons are a major source of D-serine. Glutamate-induced neuronal D-serine release provides a novel mechanism for activating NMDA receptors by an autocrine or paracrine way.