Fig. 10: Incorporation of extracellular Hexβ into neuronal lysosomes.

a Schematic depicting the clathrin-mediated endocytosis (CME) pathway and mechanism of action of inhibitors, Cytochalasin D (CytD), Chlorpromazine (CPZ), Dynasore and IGFII, a competitive allosteric inhibitor of the cation-independent mannose-6-phosphate receptor (CI-MPR). b Bar graph of Hexβ activity assay measured by absorbance value in media only and media containing his-tagged recombinant Hexβ protein, demonstrating that the his-tagged Hexβ protein is enzymatically active. Groups compared using a two-tailed unpaired Student’s T test, p < 0.0001. c Confocal images of mouse hippocampal neurons treated with media +/− his-tagged Hexβ protein immunolabeled for neurons (NeuN, magenta), lysosomal-associated membrane protein 1 (LAMP1, cyan), his-tagged Hexβ protein (HIS-TAG, yellow), and merged image showing orthogonal x/z and z/y projections at top and right of image showing colocalization of LAMP1⁺ and HIS-TAG⁺ staining within NeuN⁺ neurons after treatment with Hexβ (white). d Bar graph representing the percentage of neurons with intracellular incorporation of his-tagged Hexβ protein as identified by orthogonal imaging of HIS-TAG staining within NeuN⁺ neurons showing percentage of imaged neurons without intracellular his-tagged Hexβ staining and neurons with intracellular his-tagged Hexβ staining +/− endocytosis inhibitors/CI-MPR inhibitor. p < 0.0001 for all significant comparisons. Schematic created in BioRender. Butler, C. (2025) https://BioRender.com/nuiylri. Source data are provided as a Source Data file. Data are represented as mean ± SEM (n = 4 biological replicates for neuronal cultures/experiment with at least n = 10 neurons imaged per condition/ per experiment); groups compared by one-way ANOVA with Tukey’s post-hoc test to examine biologically relevant interactions unless otherwise noted; statistics derived from statistical significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.