Serine racemase: activation by glutamate neurotransmission via glutamate receptor interacting protein and mediation of neuronal migration

Abstract

Serine racemase (SR), localized to astrocytic glia that ensheathe synapses, converts L-serine to D-serine, an endogenous ligand of the NMDA receptor. We report the activation of SR by glutamate neurotransmission involving alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors via glutamate receptor interacting protein (GRIP) and the physiologic regulation of cerebellar granule cell migration by SR. GRIP physiologically binds SR, augmenting SR activity and D-serine release. GRIP infection of neonatal mouse cerebellum in vivo enhances granule cell migration. Selective degradation of D-serine by D-amino acid oxidase and pharmacologic inhibition of SR impede migration, whereas D-serine activates the process. Thus, in neuronal migration, glutamate stimulates Bergmann glia to form and release D-serine, which, together with glutamate, activates NMDA receptors on granule neurons, chemokinetically enhancing migration.

Fig. 1.

GRIP binds SR in yeast two-hybrid analysis and by immunoprecipitation. (A) Of one million clones screened from rat hippocampus and cortex cDNA library, two clones were both His- and β-gal-positive. The two positive clones correspond to GRIP PDZ domains 4, 5, and 6. (B) Yeast two-hybrid analysis establishes selective binding of GRIP's PDZ domain 6 to SR. (C and D) Yeast two-hybrid analysis was used to determine which region of SR interacts with GRIP. The last amino acid of SR, Val-339, was mutated to glycine, which abolishes the interaction between SR and GRIP. (E) SR(WT) interacts with GRIP in HEK-293 cells, but SR(V339G) does not bind to GRIP. GRIP was coimmunoprecipitated from mouse brain with SR antibody. (F) Colocalization of GRIP and GFAP, a glial marker, in mouse brain. (G) Colocalization of GRIP, GluR2/3, and SR with GFAP. Primary mixed glial cultures were double-labeled by using anti-GRIP, anti-GluR2/3, anti-SR, and anti-GFAP. GRIP, SR, GluR2/3, and GRIP expression in primary glial culture is also shown by Western blot analysis.

Fig. 2.

SR interaction with GRIP increases d-serine synthesis and release. (A) Incubation of SR(WT)-transfected cell lysate with l-serine markedly augments d-serine synthesis, whereas incubation with cell lysate transfected with SR(V339G), which does not bind to GRIP, provides 65% less d-serine formation compared with SR(WT)-transfected cell lysate; *, P < 0.005. (Inset) C6 glioma cells express GRIP. (B) AMPA stimulation elicits 2.5 times more d-serine release than control; *, P < 0.005. d-serine release in control samples reflects endogenous AMPA receptor activation, as it is markedly reduced by the AMPA receptor antagonist NBQX; **, P < 0.001. (C) All GRIP virus-infected cultures display two to three times more d-serine release in the media than empty virus-infected cultures; **, P < 0.001. AMPA stimulates and NBQX inhibits d-serine release in GRIP and empty virus-infected cultures; *, P < 0.005 and ***, P < 0.0005. (D) With empty adenovirus, AMPA treatment increases cellular d-serine >10-fold, an effect blocked by NBQX. GRIP infection increases d-serine levels to the same values in all conditions. d-serine levels for control, NBQX-, and AMPA/NBQX-treated cell lysates were undetectable.

Fig. 3.

GRIP augments d-serine cerebellar concentrations in intact mice and accelerates cerebellar granule cell migration. (A) GRIP adenoviral infection in P11 mice increases cerebellar d-serine levels. GRIP PDZ-6 adenovirus-infected cerebellum displays a 2-fold increase in d-serine levels compared with the empty adenoviral-infected cerebellum; *, P < 0.001. (B) BrdUrd staining of P11 cerebellum infected with WT and GRIP adenovirus. BrdUrd-labeled granule cells are stained and can be observed in the EGL, ML, and IGL. (C) There are two times more granule cells in the ML and 30% more granule cells in the IGL for GRIP-infected cerebellum compared with WT virus-infected cerebellum; *, P < 0.001. Granule cell density in the EGL is correspondingly reduced; *, P < 0.001.

Fig. 4.

d-serine increases neuronal migration.. (A) DAO and SR inhibitors decrease granule cell migration. Cerebellar slices were pretreated with DAO. When DAO is removed during the migration period, d-serine increases the granule cell migration distance by 2.5 times that of untreated slices; *, P < 0.005. Similar results were obtained for continuous treatment with DAO (**, P < 0.005) compared with DAO alone. Thus, d-serine or sodium benzoate (NaBENZ) rescue the granule cell migration from DAO's inhibitory effect. (B) Met-Phen and Et-Phen, inhibitors of SR block granule cell migration are shown; **, P < 0.005. Phen inhibits activity of SR only very weakly and exerts modest inhibitory effects on granule cell migration (*, P < 0.05) compared with control. Et-Phen's effects on granule cell migration are reversed by adding d-serine. (C) Inhibiting SR blocks granule cell calcium transients. Representative traces of spontaneous calcium transients in granule cells (3 days in vitro) that are inhibited by Et-Phen are shown. (D) AMPA receptor blockade by NBQX prevents granule cell migration. When NBQX was removed, granule cell migration could be rescued by adding AMPA or d-serine to the media (*, P < 0.005) compared with NBQX alone. Similar results were obtained with continuous treatment with NBQX during the migration period (**, P < 0.005) compared with NBQX alone. (E) d-serine increases granule cell migration in cerebellar explant cultures. Granule cells on HEK-293 cells transfected with empty plasmid and SR(K56G), which does not bind to pyridoxal 5′phosphate, do not migrate far from the cerebellar explant, whose border is shown by a dashed line. In SR(WT)-transfected condition, granule cells migrate further than control or SR(K56G) cells. Granule cells were stained with NeuN, a neuron specific marker. (F) Granule cells (n = 30) with SR(WT) transfection migrate more than twice the distance of control and SR(K56G) mutant preparations (*, P < 0.005).

Fig. 5.

Model for d-serine and glutamate regulation of cerebellar granule cell migration. Glutamate activates AMPA receptors (AMPA-R) on Bergmann glia, leading to receptor phosphorylation and dissociation of receptor-bound GRIP, which then binds to SR. GRIP activates SR with release of newly formed d-serine to join glutamate in stimulating NMDA receptor (NMDA-R) on granule cells, releasing intracellular calcium and facilitating migration from the EGL to the IGL.