Journal: The Journal of Neuroscience
Article Title: Loss of Christianson Syndrome Na + /H + Exchanger 6 (NHE6) Causes Abnormal Endosome Maturation and Trafficking Underlying Lysosome Dysfunction in Neurons
doi: 10.1523/JNEUROSCI.1244-20.2021
Figure Lengend Snippet: Loss of NHE6 increases MVB fusion with the PM and exosome secretion. A , Representative TIRF image depicting MVB-PM fusion and exosome release as developed from . Widefield image of neuron cotransfected with mCherry and CD63-pHluorin expression constructs. White inset, Location of MVB-PM fusion and the zoomed in panels on the right. Each panel represents the progression of a CD63-pHluorin fusion event with the PM with the number of seconds indicated below the panel. Scale bars: large, 10 µm; small, 1 µm. B , Quantification of full MVB-PM fusion/exosome release events per cell over 5 min in WT and Nhe6 -/Y male littermate mouse primary hippocampal neurons at 14 DIV (WT n = 28 cells from 7 mice, Nhe6 -/Y n = 18 cells from 7 mice, 5 litters, p = 0.009, Glass's Δ = 2.27). C , Quantification of full MVB-PM fusion/exosome release events per cell over 5 min in WT and Nhe6 -/Y male mouse primary hippocampal neurons at 14 DIV under the following conditions: untreated (same as in B ), U18666A (positive control) (WT n = 14 cells from 5 mice, Nhe6 -/Y n = 14 cells from 5 mice, 3 litters), bafilomycin A1 (positive control) (WT n = 14 cells from 7 mice, Nhe6 -/Y n = 16 cells from 6 mice, 4 litters, Kruskal–Wallis test with Dunn's test: WT untreated compared with WT bafilomycin A1 p = 0.002, Glass's Δ = 3.04). D , E , CD63 western blot ( D ) and quantification ( E ) in WT and Nhe6 -/Y male littermate mouse primary hippocampal neurons at 14 DIV (WT n = 5 cultures, Nhe6 -/Y n = 5 cultures, 5 litters, p = 0.02, Glass's Δ = 1.68). F , Released β-Hex enzyme activity following short-term incubation in Tyrode's solution followed by treatment with either ionomycin or DMSO (WT n = 9, Nhe6 -/Y n = 9, 8 litters). G , Released CatD enzyme activity following short-term incubation in Tyrode's solution followed by treatment with either ionomycin or DMSO (WT n = 6, Nhe6 -/Y n = 6, 5 litters). H , Released LDH activity across all β-Hex (WT n = 5, Nhe6 -/Y n = 5, 5 litters) and CatD (WT n = 4, Nhe6 -/Y n = 4, 3 litters) experiments. Data are mean ± SEM. Unpaired two-tailed Student's t test ( C , WT- Nhe6 -/Y : bafilomycin A1) with Welch's correction ( E ), Mann–Whitney test ( B , C , WT- Nhe6 -/Y : U18666A), Kruskal–Wallis test with Dunn's test ( C , differences between treatments by genotype), two-way ANOVA with Tukey's multiple comparisons test ( F , G , H ).
Article Snippet: The following antibodies were used for western blot: actin (Sigma, A3853, Ms, 1:1000), CatD (R&D Systems, AF1029, Gt, 1:1000), CD63 (Abcam, EPR21151-ab217345, Rb, 1:1000), ci-mannose 6-phosphate receptor (M6PR) (Cell Signaling Technology, 14364S, Rb, 1:500), GAPDH (Sigma, G8795, Ms, 1:40 000), LAMP1 (DSHB, 1D4B, Rt, 1:1000), RAB5 (Cell Signaling Technology, 3547, Rb, 1:1000), and RAB7 (Sigma, R8779, Ms, 1:1000).
Techniques: Expressing, Construct, Positive Control, Western Blot, Activity Assay, Incubation, Two Tailed Test, MANN-WHITNEY
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