Ursula Staubli

D-Serine Is a Substrate for Neutral Amino Acid Transporters ASCT1/SLC1A4 and ASCT2/SLC1A5, and Is Transported by Both Subtypes in Rat Hippocampal Astrocyte Cultures.

N-methyl-D-aspartate (NMDA) receptors play critical roles in synaptic transmission and plasticity. Activation of NMDA receptors by synaptically released L-glutamate also requires occupancy of co-agonist binding sites in the tetrameric receptor by either glycine or D-serine. Although D-serine appears to be the predominant co-agonist at synaptic NMDA receptors, the transport mechanisms involved in D-serine homeostasis in brain are poorly understood. In this work we show that the SLC1 amino acid transporter family members SLC1A4 (ASCT1) and SLC1A5 (ASCT2) mediate homo- and hetero-exchange of D-serine with physiologically relevant kinetic parameters. In addition, the selectivity profile of D-serine uptake in cultured rat hippocampal astrocytes is consistent with uptake mediated by both ASCT1 and ASCT2. Together these data suggest that SLC1A4 (ASCT1) may represent an important route of Na-dependent D-serine flux in the brain that has the ability to regulate extracellular D-serine and thereby NMDA receptor activity.

Activity of the enantiomers of erythro-3-hydroxyaspartate at glutamate transporters and NMDA receptors.

The enantiomers of erythro‐3‐hydroxyaspartate were tested for activity at glutamate transporters and NMDA receptors. Both enantiomers inhibited glutamate transporters in rat hippocampal crude synaptosomes and elicited substrate‐like activity at excitatory amino acid transporter 1, 2, and 3 as measured by voltage clamp in the Xenopus oocyte expression system. The enantiomers had similar affinities, but the D‐enantiomer showed a lower maximal effect at excitatory amino acid transporter 1, 2, and 3 than the L‐enantiomer. Surprisingly, D‐erythro‐3‐hydroxyaspartate was a potent NMDA receptor agonist with an EC50 value in rat hippocampal neurons of 320 nM, whereas the L‐enantiomer was 100‐fold less potent. L‐erythro‐3‐hydroxyaspartate showed activity at both glutamate transporters and NMDA receptors at concentrations that are reported to inhibit serine racemase, indicating a lack of selectivity. This enantiomeric pair may assist in shedding further light on the structural requirements for substrate activity at glutamate transporters and for agonist activity at NMDA receptors.

Phenylglycine analogs are inhibitors of the neutral amino acid transporters ASCT1 and ASCT2 and enhance NMDA receptor-mediated LTP in rat visual cortex slices

&NA; The N‐methyl‐D‐aspartate receptor (NMDA) co‐agonist D‐serine is a substrate for the neutral amino acid transporters ASCT1 (SLC1A4) and ASCT2 (SLC1A5). We identified L‐phenylglycine (PG) and its analogs as inhibitors of ASCT1 and ASCT2. PG analogs were shown to be non‐substrate inhibitors of ASCT1 and ASCT2 with a range of activities relative to other amino acid transport systems, including sodium‐dependent glutamate transporters, the sodium‐independent D‐serine transporter asc‐1 and system L. L‐4‐chloroPG was the most potent and selective ASCT1/2 inhibitor identified. The PG analogs facilitated theta‐burst induced long‐term potentiation in rat visual cortex slices in a manner that was dependent on extracellular D‐serine. For structurally‐related PG analogs, there was an excellent correlation between ASCT1/2 transport inhibition and enhancement of LTP which was not the case for inhibition of asc‐1 or system L. The ability of PG analogs to enhance LTP is likely due to inhibition of D‐serine transport by ASCT1/2, leading to elevated extracellular levels of D‐serine and increased NMDA receptor activity. These results suggest that ASCT1/2 may play an important role in regulating extracellular D‐serine and NMDA receptor‐mediated physiological effects and that ASCT1/2 inhibitors have the potential for therapeutic benefit. HighlightsD‐serine is a substrate for the amino acid transporters ASCT1 and ASCT2.L‐phenylglycine (PG) analogs inhibited ASCT1 and ASCT2 and related transporters.PG analogs enhanced theta‐burst induced LTP in a D‐serine‐dependent manner.LTP enhancement by PG analogs correlated strongly with ASCT1/2 inhibition.ASCT1 and ASCT2 may regulate extracellular D‐serine and NMDA receptor activity.

Restoration of visual performance by d-serine in models of inner and outer retinal dysfunction assessed using sweep VEP measurements in the conscious rat and rabbit

The NMDA subtype of glutamate receptor and its co-agonist d-serine play a key role in synaptic function in the central nervous system (CNS), including visual cortex and retina. In retinal diseases such as glaucoma and macular degeneration, a loss of vision arises from malfunction of retinal cells, resulting in a glutamate hypofunctional state along the visual pathway in the affected parts of the visual field. An effective strategy to remedy this loss of function might be to increase extracellular levels of d-serine and thereby boost synaptic NMDA receptor-mediated visual transmission and/or plasticity to compensate for the impairment. We tested this idea in brain slices of visual cortex exhibiting long-term potentiation, and in rodent models of visual dysfunction caused by retinal insults at a time when the injury had stabilized to look for neuroenhancement effects. An essential aspect of the in vivo studies involved adapting sweep VEP technology to conscious rats and rabbits and combining it with intracortical recording while the animals were actively attending to visual information. Using this technology allowed us to establish complete contrast sensitivity function curves. We found that systemic d-serine dose-dependently rescued the contrast sensitivity impairment in rats with blue light-induced visual dysfunction. In rabbits with inner retinal dysfunction, both systemic and intravitreal routes of d-serine provided a rescue of visual function. In sum, we show that co-agonist stimulation of the NMDA receptor via administration of exogenous d-serine might be an effective therapeutic strategy to enhance visual performance and compensate for the loss of vision resulting from retinal disease.

Inhibitors of the Neutral Amino Acid Transporters ASCT1 and ASCT2 Are Effective in In Vivo Models of Schizophrenia and Visual Dysfunction

The N-methyl-d-aspartate receptor coagonist d-serine is a substrate for the neutral amino acid transporters ASCT1 and ASCT2, which may regulate its extracellular levels in the central nervous system (CNS). We tested inhibitors of ASCT1 and ASCT2 for their effects in rodent models of schizophrenia and visual dysfunction, which had previously been shown to be responsive to d-serine. L-4-fluorophenylglycine (L-4FPG), L-4-hydroxyPG (L-4OHPG), and L-4-chloroPG (L-4ClPG) all showed high plasma bioavailability when administered systemically to rats and mice. L-4FPG showed good brain penetration with brain/plasma ratios of 0.7–1.4; however, values for L-4OHPG and L-4ClPG were lower. Systemically administered L-4FPG potently reduced amphetamine-induced hyperlocomotion in mice, whereas L-4OHPG was 100-fold less effective and L-4ClPG inactive at the doses tested. L-4FPG and L-4OHPG did not impair visual acuity in naive rats, and acute systemic administration of L-4FPG significantly improved the deficit in contrast sensitivity in blue light–treated rats caused by retinal degeneration. The ability of L-4FPG to penetrate the brain makes this compound a useful tool to further evaluate the function of ASCT1 and ASCT2 transporters in the CNS.