Down-regulation of glutamate release from hippocampal neurons by sialidase.

Sialidase, which removes sialic acid residues in sialylglycoconjugates, is essential for hippocampal memory and synaptic plasticity. Enzyme activity of sialidase is rapidly increased in response to neural excitation. Because sialic acid bound to gangliosides such as the tetra-sialoganglioside GQ1b is crucial for calcium signalling and neurotransmitter release, neural activity-dependent removal of sialic acid may affect hippocampal neurotransmission. In the present study, we found that 2-deoxy-2, 3-didehydro-D-N-acetylneuraminic acid (DANA), a sialidase inhibitor, increased expression of ganglioside GQ1b/GT1a in hippocampal acute slices. Extracellular glutamate level in the rat hippocampus measured by using in vivo microdialysis was increased by the sialidase inhibitor 2, 3-dehydro-2-deoxy-N-glycolylneuraminic acid as well as DANA. Synaptic vesicle exocytosis and intracellular Ca2+ increase evoked by high-K+ were also enhanced by DANA in primary cultured hippocampal neurons. Expression of GQ1b/GT1a was rapidly decreased by depolarization with high-K+, suggesting that the increase in sialidase activity by neural excitation is sufficient for cleavage of sialic acid. Our findings indicate that sialidase down-regulates glutamate release from hippocampal neurons via Ca2+ signalling modulation. Neural activity-dependent desialylation by sialidase may be a negative-feedback factor against presynaptic activity.

Rapid regulation of sialidase activity in response to neural activity and sialic acid removal during memory processing in rat hippocampus.

Sialidase cleaves sialic acids on the extracellular cell surface as well as inside the cell and is necessary for normal long-term potentiation (LTP) at mossy fiber-CA3 pyramidal cell synapses and for hippocampus-dependent spatial memory. Here, we investigated in detail the role of sialidase in memory processing. Sialidase activity measured with 4-methylumbelliferyl-α-d-N-acetylneuraminic acid (4MU-Neu5Ac) or 5-bromo-4-chloroindol-3-yl-α-d-N-acetylneuraminic acid (X-Neu5Ac) and Fast Red Violet LB was increased by high-K+-induced membrane depolarization. Sialidase activity was also increased by chemical LTP induction with forskolin and activation of BDNF signaling, non-NMDA receptors, or NMDA receptors. The increase in sialidase activity with neural excitation appears to be caused not by secreted sialidase or by an increase in sialidase expression but by a change in the subcellular localization of sialidase. Astrocytes as well as neurons are also involved in the neural activity-dependent increase in sialidase activity. Sialidase activity visualized with a benzothiazolylphenol-based sialic acid derivative (BTP3-Neu5Ac), a highly sensitive histochemical imaging probe for sialidase activity, at the CA3 stratum lucidum of rat acute hippocampal slices was immediately increased in response to LTP-inducible high-frequency stimulation on a time scale of seconds. To obtain direct evidence for sialic acid removal on the extracellular cell surface during neural excitation, the extracellular free sialic acid level in the hippocampus was monitored using in vivo microdialysis. The free sialic acid level was increased by high-K+-induced membrane depolarization. Desialylation also occurred during hippocampus-dependent memory formation in a contextual fear-conditioning paradigm. Our results show that neural activity-dependent desialylation by sialidase may be involved in hippocampal memory processing.

Catalytic preference of Salmonella typhimurium LT2 sialidase for N-acetylneuraminic acid residues over N-glycolylneuraminic acid residues.

In a comparison of sialidase activities toward N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), we found that Salmonella typhimurium LT2 sialidase (STSA) hardly cleaved 4-methylumbelliferyl Neu5Gc (4MU-Neu5Gc). The k cat/K m value of STSA for 4MU-Neu5Gc was found to be 110 times lower than that for 4-methylumbelliferyl Neu5Ac (4MU-Neu5Ac). Additionally, STSA had remarkably weak ability to cleave α2-3-linked-Neu5Gc contained in gangliosides and equine erythrocytes. In silico analysis based on first-principle calculations with transition-state analogues suggested that the binding affinity of Neu5Gc2en is 14.3 kcal/mol more unstable than that of Neu5Ac2en. The results indicated that STSA preferentially cleaves Neu5Ac residues rather than Neu5Gc residues, which is important for anyone using this enzyme to cleave α2-3-linked sialic acids.