Abstract
D-Serine is a physiological co-agonist that activates N-methyl D-aspartate receptors (NMDARs) and is essential for neurotransmission, synaptic plasticity, and behavior. D-Serine may also trigger NMDAR-mediated neurotoxicity, and its dysregulation may play a role in neurodegeneration. D-Serine is synthesized by the enzyme serine racemase (SR), which directly converts L-serine to D-serine. However, many aspects concerning the regulation of D-serine production under physiological and pathological conditions remain to be elucidated. Here, we investigate possible mechanisms regulating the synthesis of D-serine by SR in paradigms relevant to neurotoxicity. We report that SR undergoes nucleocytoplasmic shuttling and that this process is dysregulated by several insults leading to neuronal death, typically by apoptotic stimuli. Cell death induction promotes nuclear accumulation of SR, in parallel with the nuclear translocation of GAPDH and Siah proteins at an early stage of the cell death process. Mutations in putative SR nuclear export signals (NESs) elicit SR nuclear accumulation and its depletion from the cytosol. Following apoptotic insult, SR associates with nuclear GAPDH along with other nuclear components, and this is accompanied by complete inactivation of the enzyme. As a result, extracellular D-serine concentration is reduced, even though extracellular glutamate concentration increases severalfold. Our observations imply that nuclear translocation of SR provides a fail-safe mechanism to prevent or limit secondary NMDAR-mediated toxicity in nearby synapses.
Highlights
D-Serine is a physiological co-agonist that activates N-methyl D-aspartate receptors (NMDARs) and is essential for neurotransmission, synaptic plasticity, and behavior
serine racemase (SR) Targeting Is Altered by Pathological Stimuli—We evaluated the effects of cell death-inducing agents that include endoplasmic reticulum (ER) stressors and apoptotic drugs on SR localization and D-serine synthesis
We described a novel mechanism of SR regulation in neurons, linking a variety of insults leading to neuronal death with D-serine production
Summary
Materials—L- and D-serine were purchased from Bachem. L-[3H]Serine and D-[3H]serine were purchased from American Radiolabeled Chemicals. Co-immunoprecipitation Experiments—A crude nuclear fraction of neuronal culture cells was obtained, and nuclei were disrupted by sonication in 10 mM Tris-HCl (pH 7.4), 10 mM KCl, 1.5 mM MgCl2, 2 mM DTT, and a Mini-complete protease inhibitor mixture. For GAPDH immunoprecipitation, the nuclear fraction was processed as above, except that 10 g of purified anti-GAPDH (Sigma) or a rabbit IgG control was covalently coupled to protein A magnetic beads, as described previously [46]. To determine the effect of soluble nucleoplasmic proteins on SR activity, HEK293 cells were transfected with mouse SR in pRK5-KS plasmid, and after 48 h, the cells were lysed by three cycles of freezing and thawing in lysis buffer containing 10 mM Tris-HCl (pH 7.4), 10 mM KCl, 1.5 mM MgCl2, 2 mM DTT, and a protease inhibitor mixture (mini complete EDTA-free, Roche Applied Science). Statistical Analysis—Statistics were performed by means of repeated-measures analysis of variance and the Tukey multiple comparison test or by the two-tailed, paired Student’s t test using GraphPad Prism software version 6.03 (GraphPad Inc.)
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