Precise and tight regulation of the number of synapses is a crucial factor for healthy nervous system development. Atypical abundance or disposal of synapses in the brain may lead to neurodevelopmental disorders, such as autism, schizophrenia, or epilepsy [1]. Microglia, the main phagocytes of CNS, regulate synapse pruning to selectively eliminate or strengthen the subset of synapses by "eat me" – "spear me" distinction determined by specific neuronal markers. Sialic acid – a monosaccharide, which typically terminates the structures of glycoconjugates that compose neuronal glycocalyx, has a particular importance in neuron-microglia interaction. In the presence of neuronal sialic acid microglial phagocytic activity is actively inhibited, while desialylation of neurons promotes their phagocytosis in vitro [2]. However, the potential role of sialic acid in synapse pruning as a local specific marker on synapses has not been determined yet.
We used mouse organotypic hippocampal slice cultures as a platform to define how sialylation provides a selective mechanism for the maintenance and strengthening of necessary synapses. We applied bioorthogonal CLICK chemistry to metabolically label de novo synthesised sialic acid on the surface of excitatory neurons [3]. We used chemical inhibitors (1S,2R)-1-((3S,4R,5R,6S)-3-acetamido-4,6-diacetoxy-5-fluoro-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)propane-1,2,3-triyl triacetate (3FAX) and 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA) targeting either sialic acid synthesis or desialylation, respectively, to alter neuronal sialylation.
Mouse organotypic hippocampal slice cultures were established and used as an ex vivo system allowing to study metabolic processes of neuronal sialylation. The visualization and quantitative analysis of newly synthesized sialic acids on dendrites of excitatory neurons showed specificity of metabolic sialic acid labelling. The inhibition of sialyltransferases by 3FAX significantly reduced neuronal sialylation, while sialidase inhibition by DANA led to increased sialylation levels. Our results indicated that neuronal sialylation can be chemically modulated ex vivo allowing the precise assessment of sialic acid abundance on the neuronal structures, such as dendrites and individual synapses.
In conclusion, we demonstrated the applicability of mouse organotypic hippocampal slice cultures as a robust model to investigate de novo synthesis and the turnover of sialic acid in the mouse developing brain ex vivo. Moreover, our system enables to target and characterise the exact role of the enzymes involved in neuronal sialylation and desialylation in synaptic pruning.