Vesicular uptake and exocytosis of L‐aspartate is independent of sialin

Abstract

The mechanism of release and the role of l-aspartate as a central neurotransmitter are controversial. A vesicular release mechanism for l-aspartate has been difficult to prove, as no vesicular l-aspartate transporter was identified until it was found that sialin could transport l-aspartate and l-glutamate when reconstituted into liposomes. We sought to clarify the release mechanism of l-aspartate and the role of sialin in this process by combining l-aspartate uptake studies in isolated synaptic vesicles with immunocyotchemical investigations of hippocampal slices. We found that radiolabeled l-aspartate was taken up into synaptic vesicles. The vesicular l-aspartate uptake, relative to the l-glutamate uptake, was twice as high in the hippocampus as in the whole brain, the striatum, and the entorhinal and frontal cortices and was not inhibited by l-glutamate. We further show that sialin is not essential for exocytosis of l-aspartate, as there was no difference in ATP-dependent l-aspartate uptake in synaptic vesicles from sialin-knockout and wild-type mice. In addition, expression of sialin in PC12 cells did not result in significant vesicle uptake of l-aspartate, and depolarization-induced depletion of l-aspartate from hippocampal nerve terminals was similar in hippocampal slices from sialin-knockout and wild-type mice. Further, there was no evidence for nonvesicular release of l-aspartate via volume-regulated anion channels or plasma membrane excitatory amino acid transporters. This suggests that l-aspartate is exocytotically released from nerve terminals after vesicular accumulation by a transporter other than sialin.

Motor Axon Synapses on Renshaw Cells Contain Higher Levels of Aspartate than Glutamate

“…It is possible that all three antibodies are capable of immunodetecting SLC17A5, but each displays either different cross-reactivities with other proteins in tissue, or might detect SLC17A5 in different compartments depending on differential exposure or masking of their different epitopes. Similar specificity concerns with these antibodies were recently raised in another study [33]. Therefore, these antibodies rendered “dirty” immunoreactions in the spinal cord and require further investigation before the exact nature of SLC17A5 immunoreactivity can be reported with confidence.…”

Are vesicular neurotransmitter transporters potential treatment targets for temporal lobe epilepsy?

“…Moreover, a proteomic study (Takamori et al, 2006) did not detect sialin among the other synaptic vesicle proteins, purified from rat brain. On the other hand, no evidence was found of non-vesicular release of L-aspartate, which confirms the exocytotic release in hippocampus, though, after vesicular accumulation by another transporter than sialin (Morland et al, 2013). …”

“…Only recently, Morland and coworkers observed ATP-dependent vesicular uptake of L-aspartate in the brain. Moreover, vesicular L-aspartate uptake, relative to the L-glutamate uptake, was twice as high in the hippocampus as in the whole brain, indicating a crucial role of aspartate signaling in this brain region (Morland et al, 2013). In vitro data suggest that NMDA receptor-mediated excitotoxicity is likely caused by extrasynaptic NR1-NR2B NMDA receptor activation by aspartate, which implicates an important role of aspartate signaling in pathologies marked by excitotoxicity.…”

“…Moreover, the involvement of sialin in the vesicular loading of aspartate has recently been questioned, based on in vitro data from sialin (−/−) mice (Morland et al, 2013). Of course, it cannot be excluded that these transgenic mice develop compensatory mechanisms to counteract the loss of sialin.…”

3-aminoglutarate is a “silent” false transmitter for glutamate neurons

“…In our own experiments, Daspartate was unable to reduce potassium-evoked, calciumdependent L-glutamate release from synaptosomes indicating that it does not behave as a false transmitter in this system. However, a vesicular transporter for aspartate, sialin, has been described (Miyaji et al, 2008, but also see Morland et al, 2013) and so it is possible that these agents are false transmitters for a system separate from the classical L-glutamate neurotransmitter system. Land D-aspartate and 3-hydroxyaspartate all have significant affinity as agonists for NMDA receptors (Grimwood et al, 1991), a property that would complicate their use as "silent" false transmitters in physiological studies.…”