Figure 2. Cultured CNS neurons from w¹¹¹⁸ and 4-4, but not those from 2d/2d, incorporate sialic acids.

(A) Molecular structure of Ac₄ManNAc (NAC) and Ac₄ManNAz (NAZ); and a schematic representation of the Click-iT reaction between the azide-modified sialoglycoconjugates and alkyne group on the fluorescent agent. (B) General pathway for sialic acid incorporation into glycoconjugates in Dm. Once in the cytoplasm non-specific esterases remove the acetyl groups in NAC and NAZ converting them into ManNAc and ManNAz. ManNAc (or ManNAz) is processed to become CMP-Sia (cytidine monophosphate-sialic acid) (or CMP-Sia5NAz) which is the sialic acid donor molecule. DmSAS: Dm sialic acid synthetase; DmCSAS: Dm CMP sialic acid synthetase; DmSialT: Dm sialyltransferase; PEP: phosphoenolpyruvate; Sia-9-P: sialic acid 9-phosphate; Pi: phosphate; CTP: cytidine triphosphate; PPi: pyrophosphate. (C-G) Dissociated CNS cultures (1-3CNS/coverslip) were cultured in either serum containing medium (C-F) or in SFM (G) for one to three days in the presence of NAZ or NAC (30-50μM). (C-E) Show pictures of neurons that were immunostained with anti-HRP and the nuclei stained with Hoechst (middle panels). Staining with the Alexa-Fluor-488-Alkyne (left panels). The red dotted lines outline the soma (light pictures, right panels). Incorporation of Sia5NAz (fluorescent intensity labeling) was measured in individual neuronal somas. (63x objective, bar=10μm). (F) The mean percentage of neurons that incorporated Sia5NAz when cultured in serum containing medium; n=3-5 separate experiments. One-way analysis of variance (P<0.001); post test Bonferroni, #P<0.05 between the 2d/2d group and the w¹¹¹⁸ group. No difference was found between the 4-4 group and the w¹¹¹⁸ group. Sia5NAz incorporation was measured in 25-100 neurons per condition in one experiment; and in 102-150 neurons per condition in four experiments. (G) The mean percentage of neurons that incorporated Sia5NAz when cultured in SFM; n=3-4 separate experiments. No significant difference was found between any of the groups (One-way analysis of variance). Sia5NAz incorporation was measured in 103-146 neurons per condition in the four experiments.