Fig. 6: GlcNAc supplementation rescues N-glycosylation and downstream pathogenic phenotypes in PD iPSn. | Nature Communications

Fig. 6: GlcNAc supplementation rescues N-glycosylation and downstream pathogenic phenotypes in PD iPSn.

From: The hexosamine biosynthetic pathway rescues lysosomal dysfunction in Parkinson’s disease patient iPSC derived midbrain neurons

Fig. 6

A Western blot of N-glycosylated proteins using biotinylated Con-A, in iPSn (Corr and A53T iPSn at day 122) treated with 10 mM GlcNAc for 7 days. (−) indicates Vehicle control, which is PBS. GAPDH is a loading control. Quantification is shown on the right (n = 3 for A53T Vehicle control and n = 4 for all other groups). B Western blot analysis of GCase from Triton X-100 soluble (T-sol) and insoluble (T-insol) fractions in Corr and A53T iPSn in the presence or absence of GlcNAc, using coomassie brilliant blue (CBB) as a loading control. Quantification is shown on the right (n = 3). C GCase maturation was measured by western blot using endoglycosidase H (endo H) digestion of lysates from Veh and GlcNAc supplemented A53T iPSn. GAPDH is a loading control. Quantification is shown on the right (n = 3). D Analysis of lysosomal GCase activity in living A53T iPSn after supplementation with GlcNAc. Fluorescent substrate degradation was evaluated in a microplate reader for 3 h. Activity in acidic compartments was determined by measuring the response to a lysosomal inhibitor bafilomycin A1 n = 4. E, F Western blot analysis of Hex B in Corr and A53T iPSn at day 122 in the presence or absence of GlcNAc, using coomassie brilliant blue (CBB) as loading control. Quantifications of mature and immature Hex B are shown in (F) (n = 3). Note that the Immature Hex B bands are shown at a higher intensity compared to the mature Hex B bands, to avoid saturation of the mature forms. G, H Western blot analysis of α-syn in Corr and A53T iPSn from Triton X-100 soluble (T-sol) and insoluble (T-insol) fractions using C20 or syn303 antibodies, in the presence or absence of GlcNAc. Coomassie brilliant blue (CBB) was used as loading control. α-Syn knock-out (SNCA KO) iPSn was used as a negative control. * indicates non-α-syn bands. Quantification is shown in (H) (n = 3). I Neuron viability was assessed in iPSn A53T at day 122 supplemented with GlcNAc, by quantification of neurofilament (NFM) content and normalized to cell volume using cell-tag. Quantification is shown below (n = 10). Scatter plots represent individual culture well replicates. For all quantifications, values are the mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001. Student’s two-sided t-test was used for (A, B, C, D, E, F, I) and ANOVA with Tukey’s post hoc test was used for panel H. Source data are provided as a Source Data file.

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