Fig. 2: OPC contact facilitates neuronal lysosome release at the contact site.

A–C Immunostaining and 3D-reconstruction of neurons, OPCs and lysosomes in NG2^EYFP mouse cortex using NeuN, GFP and Lamp1 antibodies. B OPC processes contacting neuronal somata were indicated in yellow. C Neuronal lysosomes shown in colors based on their volume. D Display of lysosome volume (indicated by circle size) and their proximity to OPC and neuronal surfaces (5925 lysosomes from 17 cells/3 mice). The color indicates the frequency of lysosomal appearance at corresponding location. E Correlation of lysosome volume and proximity to OPC surface (2077 lysosomes from 17 cells/3 mice). F Live-imaging of a lysosome (dotted circle) trafficking in neuron-OPC co-culture and analysis of maximum fluorescence intensity (MAX FI) of lysosomes over the period. Dotted line indicates neuronal membrane. G Live-imaging and quantification of lysosome exocytosis in neurons from pure neuronal culture and neurons with (neuron^OPC-contact) or without OPC contact (neurons^{w/o-OPC-contact}) from co-culture. In neurons^OPC-contact, the Alexa647⁺ puncta were classified into proximal (<1 µm, magenta) and distal (>1 µm, green) based on their distance to OPC contact site (Pure neuron=9 cells/4 independent experiments; neurons^OPC-contact = 9 cells/4 independent experiments, neurons^{w/o-OPC-contact} = 3 cells/3 independent experiments; 4 pups/experiment, two-way ANOVA, multiple comparisons). H Lysosome exocytosis in acute brain slices was imaged and the number of Alexa647⁺ puncta in each neuron was quantified (ctl = 10 cells/3 mice; dKO=6 cells/3 mice). I Immunostaining and 3D-reconstruction of OPCs (PDGFRα⁺), neurons (NeuN⁺) and lysosomes (Cathepsin D, CTSD⁺). I₁ Quantification of relative volume of OPC processes contacting neuronal somata (ctl = 190 cells/5 mice, dKO = 116 cells/5 mice). I₂, I₃ Analysis of the relative number and volume of neuronal lysosomes. (ctl = 112 cells/7 mice, dKO = 74 cells/4 mice). J Immunolabeling and 3D reconstruction of neurons, OPCs and lysosomes with NeuN, PDGFRα and Lamp1 antibodies. The relative number and volume of neuronal lysosomes were quantified. (ctl = 80 cells/5 mice, DTA = 52 cells/4 mice). In (H–J), small dots represent cells while big dots represent mice, two-sided unpaired t-tests were used. R.U. = relative units, normalized to the controls. Data are shown as mean ± SEM in (G–J). Source data are provided as a Source Data file. Created in BioRender. Fang, L. (2025) https://BioRender.com/a00p319 and https://BioRender.com/v85j019.