Abstract
D-serine is an endogenous coagonist at the glycine site of synaptic NMDA receptors (NMDARs), synthesized by serine racemase (SR) through conversion of L-serine. It is crucial for synaptic plasticity and is implicated in schizophrenia. Our previous studies demonstrated specific loss of SR, D-serine-responsive synaptic NMDARs, and glutamatergic synapses in cortical neurons lacking α7 nicotinic acetylcholine receptors, which promotes glutamatergic synapse formation and maturation during development. We thus hypothesize that D-serine and SR (D-serine/SR) are associated with glutamatergic synaptic development. Using morphological and molecular studies in cortical neuronal cultures, we demonstrate that D-serine/SR are associated with PSD-95 and NMDARs in postsynaptic neurons and with glutamatergic synapse stability during synaptic development. Endogenous D-serine and SR colocalize with PSD-95, but not presynaptic vesicular glutamate transporter 1 (VGLUT1), in glutamatergic synapses of cultured cortical neurons. Low-density astrocytes in cortical neuronal cultures lack SR expression but contain enriched D-serine in large vesicle-like structures, suggesting possible synthesis of D-serine in postsynaptic neurons and storage in astrocytes. More interestingly, endogenous D-serine and SR colocalize with PSD-95 in the postsynaptic terminals of glutamatergic synapses during early and late synaptic development, implicating involvement of D-serine/SR in glutamatergic synaptic development. Exogenous application of D-serine enhances the interactions of SR with PSD-95 and NR1, and increases the number of VGLUT1- and PSD-95-positive glutamatergic synapses, suggesting that exogenous D-serine enhances postsynaptic SR/PSD-95 signaling and stabilizes glutamatergic synapses during cortical synaptic development. This is blocked by NMDAR antagonist 2-amino-5-phosphonopentanoic acid (AP5) and 7-chlorokynurenic acid (7-CK), a specific antagonist at the glycine site of NMDARs, demonstrating that D-serine effects are mediated through postsynaptic NMDARs. Conversely, exogenous application of glycine has no such effects, suggesting D-serine, rather than glycine, modulates postsynaptic events. Taken together, our findings demonstrate that D-serine/SR are associated with PSD-95 and NMDARs in postsynaptic neurons and with glutamatergic synapse stability during synaptic development, implicating D-serine/SR as regulators of cortical synaptic and circuit development.
Highlights
NMDA receptors (NMDARs) are glutamate-gated ionotropic channels that are crucial for many physiological processes including neurotransmission, synaptic plasticity, and learning and memory (Waxman and Lynch, 2005; Hardingham and Bading, 2010; Vyklicky et al, 2014)
Triple immunostaining with α-serine racemase (SR) or α-D-amino acid oxidase (DAAO) and α-MAP2 and α-GLT1 show that SR is abundantly distributed in the soma, nucleus and dendrites of MAP2-positive cortical neurons (N) (Figure 1A), but absent in the soma and astrocytic terminals of GLT1positive astrocytes (AS) (Figure 1B) in cortical neuronal cultures
The present study uses in vitro cultured cortical neurons to show that D-serine and SR are associated with PSD-95 and NMDARs in postsynaptic neurons and with glutamatergic synapse stability
Summary
NMDARs are glutamate-gated ionotropic channels that are crucial for many physiological processes including neurotransmission, synaptic plasticity, and learning and memory (Waxman and Lynch, 2005; Hardingham and Bading, 2010; Vyklicky et al, 2014). D-serine is recognized as an important physiological modulator in many NMDAR-dependent processes and functions, including brain development, synaptic transmission and plasticity, learning and memory, and social interactions (Mothet et al, 2000, 2015; Yang et al, 2003; Kim et al, 2005a; Labrie et al, 2008; DeVito et al, 2011; Papouin et al, 2012; Li and Wang, 2013; Rosenberg et al, 2013). Glutamatergic synapses and D-serine/SR are decreased in the forebrain of α7 nicotinic acetylcholine receptor knockout mice (Lin et al, 2014b) These changes resemble the major neurochemical deficits in schizophrenia (Lin et al, 2014a,b). We hypothesize that D-serine/SR may be involved in glutamatergic synaptic development, and that D-serine/SR deficiency may thereby disrupt cortical synaptic and circuit development, contributing to permanent deficits in schizophrenia
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