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. 2022 Sep 23;13(1):5583.
doi: 10.1038/s41467-022-33200-4.

CNS myelination requires VAMP2/3-mediated membrane expansion in oligodendrocytes

Mable Lam #  1 , Koji Takeo #  1   2 , Rafael G Almeida  3 , Madeline H Cooper  1 , Kathryn Wu  1 , Manasi Iyer  1 , Husniye Kantarci  1 , J Bradley Zuchero  4
Affiliations

CNS myelination requires VAMP2/3-mediated membrane expansion in oligodendrocytes

Mable Lam et al. Nat Commun. .

Abstract

Myelin is required for rapid nerve signaling and is emerging as a key driver of CNS plasticity and disease. How myelin is built and remodeled remains a fundamental question of neurobiology. Central to myelination is the ability of oligodendrocytes to add vast amounts of new cell membrane, expanding their surface areas by many thousand-fold. However, how oligodendrocytes add new membrane to build or remodel myelin is not fully understood. Here, we show that CNS myelin membrane addition requires exocytosis mediated by the vesicular SNARE proteins VAMP2/3. Genetic inactivation of VAMP2/3 in myelinating oligodendrocytes caused severe hypomyelination and premature death without overt loss of oligodendrocytes. Through live imaging, we discovered that VAMP2/3-mediated exocytosis drives membrane expansion within myelin sheaths to initiate wrapping and power sheath elongation. In conjunction with membrane expansion, mass spectrometry of oligodendrocyte surface proteins revealed that VAMP2/3 incorporates axon-myelin adhesion proteins that are collectively required to form nodes of Ranvier. Together, our results demonstrate that VAMP2/3-mediated membrane expansion in oligodendrocytes is indispensable for myelin formation, uncovering a cellular pathway that could sculpt myelination patterns in response to activity-dependent signals or be therapeutically targeted to promote regeneration in disease.

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Conflict of interest statement

K.T. is an employee of Toray Industries, Inc. The remaining authors declare no competing interests.

Figures

Fig. 1
Fig. 1. VAMP2/3-mediated exocytosis is required for CNS myelination.
a Genetic cross for Cre-induced botulinum toxin light chain B (BoNT/B; or “iBot”) under the constitutive CAG promoter (CAGpr) in pre-myelinating oligodendrocytes. Created with BioRender.com. b iBot inactivates v-SNAREs VAMP1 (not depicted), VAMP2, and VAMP3, blocking vesicular fusion to the plasma membrane (exocytosis). c Immunolabeling of P12 spinal cord cross sections from control (iBot/+, left) and iBot;Cnp-Cre (right) mice for MBP (magenta), neurofilament heavy chain 200 (NF200, white), and GFP to mark iBot expression (green). Scale bar, 500 μm. Bottom: insets of dorsal column containing parallel tracts of axons. Scale bar, 100 μm. See Supplementary Fig. 2h for MBP staining of n = 5 biologically independent replicates. d Quantification of white matter area defined as the percent of P12 spinal cord cross section area immunolabeled by the myelin marker MBP. Squares and circles denote males and females, respectively. Average ± SEM for n = 5; control: 28.1 ± 2.97%; iBot;Cnp-Cre: 11.9 ± 1.63%, statistical measurement (p-value) determined by an unpaired, two-tailed t-test. Source data are provided in the Source Data file. e Quantification of the percent of P12 spinal cord cross section area immunolabeled by the axon marker NF200. Squares and circles denote males and females, respectively. Average ± SEM for n = 5; control: 14.6 ± 2.41%; iBot;Cnp-Cre: 9.76 ± 1.70%, p-value determined by an unpaired, two-tailed t-test. f Transmission electron microscopy images from P12 mouse spinal cord dorsal column cross sections (top: control (iBot/+); bottom: iBot;Cnp-Cre), with similar results for n = 5 biologically independent replicate quantified in h. Scale bar, 1 μm. Note the dramatic hypomyelination in iBot;Cnp-Cre mice. g Top: categories of myelination stages observed from cross sections of myelinated axons, with the axon in gray and myelin in magenta. Bottom: electron microscopy images of each myelination stage representative of n = 5 biologically independent replicates quantified in h. Scale bar 1 μm. h Quantification of the percent of axons in each myelination stage from electron microscopy in f, showing reduced wrapping and increased unmyelinated axons in iBot;Cnp-Cre mice. and circles denote males and females, respectively. Average ± SEM for n = 5; unmyelinated: control (61.0 ± 3.17%), iBot;Cnp-Cre (90.2 ± 2.76%); partially/fully ensheathed: control (10.3 ± 2.06%), iBot;Cnp-Cre (6.38 ± 1.68%); wrapped/wrapping: control (28.6 ± 1.96%), iBot;Cnp-Cre (3.28 ± 1.23%), p-value determined by an unpaired, two-tailed t-test. Source data are provided in the Source Data file.
Fig. 2
Fig. 2. VAMP2/3-mediated exocytosis occurs preferentially in myelin sheaths.
a Top: diagram depicting oligodendrocyte differentiation in culture; bottom: transfection of oligodendrocyte precursors with pHluorin-tagged VAMP2 or VAMP3 to visualize exocytosis. Created with BioRender.com. b Representative image of a pre-myelinating oligodendrocyte expressing VAMP2-pHluorin. Montage shows an exocytotic event over time, and the corresponding plot of intensity vs. time shows the characteristic fluorescent increase and decay (fitted by the green dotted line). c Frequency of VAMP2- or VAMP3-pHluorin events localized to the soma or to the processes/sheets in cultured rat oligodendrocyte precursors (gray), pre-myelinating (yellow), or mature oligodendrocytes (magenta). Each pair of points connected by a line shows events for one cell. See Supplementary Table 1 for average event frequencies ± SEM. Statistical significance was determined using the mean of each biologically independent replicate (n = 3) by an ordinary one-way ANOVA with Tukey correction for multiple comparisons. Source data are provided in the Source Data file. d Top: representative images of oligodendrocyte-lineage cells expressing VAMP2-pHluorin in the larval zebrafish spinal cord. Bottom: examples of VAMP2 exocytotic events within oligodendrocyte processes and sheaths. e Frequency of VAMP2- or VAMP3-pHluorin events in oligodendrocytes of the zebrafish spinal cord localized to the soma or to the processes/sheaths in precursors/pre-myelinating (yellow), early myelinating (pale pink), or mature oligodendrocytes (magenta). Each pair of points connected by a line shows events for one cell (typically from one fish). See Supplementary Table 2 for average event frequencies ± SEM. Statistical significance was determined by an ordinary one-way ANOVA with Tukey correction for multiple comparisons. f Spatial frequency of VAMP2 and VAMP3 exocytotic events within myelin sheaths, where the paranode is defined as 3 μm from the sheath edge. The measured frequencies at paranodes (mean ± SEM) were (50 ± 22.4)% for VAMP2 and (24.9 ± 12.7)% for VAMP3, each with n = 25 sheaths from five experiments. See Supplementary Table 3.
Fig. 3
Fig. 3. VAMP2/3-mediated exocytosis is required for oligodendrocyte membrane expansion.
a Oligodendrocyte precursors purified from transgenic mouse brains (control vs. iBot;Cnp-Cre) and differentiated in culture to investigate cell-intrinsic effects of VAMP2/3 inactivation. Created with BioRender.com. b Thresholded masks of primary oligodendrocytes stained for GalCer lipid during a differentiation time course of 3, 5, and 7 days from control (top) and iBot;Cnp-Cre (bottom) mice to show cell morphologies. Scale bar, 50 μm. See Supplementary Fig. 6e for examples of GalCer staining. Source data are provided in the Source Data file. c Quantification of membrane surface area marked by GalCer lipid, where each light-shaded point corresponds to a single cell, each dark-shaded point represents the mean area of cells from one biologically independent replicate, and the line represents the mean area from n = 5 biologically independent replicates for each condition. See Supplementary Table 4 for descriptive statistics. Statistical measurements (p-value) were determined by an unpaired, two-tailed t-test. Source data are provided in the Source Data file.
Fig. 4
Fig. 4. VAMP2/3-mediated membrane expansion is required for myelin sheath elongation.
a Schematic of co-cultures between oligodendrocytes (magenta) and CNS-derived axons from retinal ganglion cell aggregates with radially protruding axons (gray). Created with BioRender.com. b Primary oligodendrocytes purified from control (top) or iBot;Cnp-Cre mouse brains (bottom) cultured on CNS-derived axons (retinal ganglion cell re-aggregates) for 7 days and stained for MBP (magenta) and NF200 (white), with GFP to mark iBot expression (green). All scale bars, 200 μm. See Supplementary Fig. 8 for examples of sheath quantification. Source data are provided in the Source Data file. Quantification of the number of sheaths per oligodendrocyte (c) and the length of sheaths (d) from co-cultures, where each light-shaded point represents a single sheath, each dark-shaded point represents the mean length of all sheaths from one biologically independent replicate, and the line represents the mean sheath length from n = 4 biologically independent replicates with 33–63 cells each. Mean number of sheaths per cell body ± SEM: control 9.8 ± 1.8, iBot 6.9 ± 1.2. Mean sheath length ± SEM: control 90.9 ± 5.2 μm, iBot 60.5 ± 7.4 μm. Statistical measurements (p-value) were determined by an unpaired, two-tailed t-test. Source data are provided in the Source Data file. e Schematic of a brain region containing sparsely myelinated fibers at P12 examined in f–h. Created with BioRender.com. f Immunolabeling of P12 mouse sagittal brain slices from control (top) and iBot;Cnp-Cre (bottom) littermates for MBP (magenta) and GFP (green). Control samples do not show GFP signal, since iBot is not expressed. Images are representative of n = 5 control and n = 4 iBot;Cnp-Cre mice quantified in g, h. Scale bar, 20 μm. Source data are provided in the Source Data file. Quantification of sheath length from P12 brain slices depicting scatter plots (g) and histogram distribution (h) between control (gray) and iBot;Cnp-Cre (green). Squares and circles denote males and females, respectively. Mean sheath length ± SEM: control 54.2 ± 3.81 μm, iBot 39.6 ± 2.38 μm from n = 5 (control) or 4 (iBot;Cnp-Cre) biologically independent replicates with 441–472 total sheaths quantified per condition. Statistical measurement (p-value) was determined by an unpaired, two-tailed t-test. Source data are provided in the Source Data file.
Fig. 5
Fig. 5. Vesicles accumulate at the inner tongue of myelin sheaths following inactivation of VAMP2/3.
a Super-resolution (Airyscan confocal) images of P12 mouse cingulate cortex from control (top) and iBot;Cnp-Cre (bottom) littermates immunostained for MBP. Scale bar, 20 μm (inset, 2 μm). b 3D reconstructed Airyscan confocal z-stack of a myelinated axon cross section from a control (top) and iBot;Cnp-Cre (bottom) mouse spinal cord immunostained for MBP. See also Supplementary Videos 3 and 4. c Quantification of sheath diameter on thresholded regions of high MBP intensity from a. Squares and circles denote males and females, respectively. Mean sheath diameter ± SEM: control 0.89 ± 0.13 μm from 161 regions quantified within n = 3 biologically independent replicates; iBot 1.46 ± 0.08 μm from 165 regions quantified within n = 5 biologically independent replicates. See Supplementary Fig. 11 for quantification methodology. Statistical measurement (p-value) was determined by an unpaired, two-tailed t-test. Source data are provided in the Source Data file. d Top: electron microscopy of myelinated axons from control and iBot;Cnp-Cre spinal cords, where the axon is colored blue and myelin vesicles are colored in yellow/orange (scale bar 1 μm). Bottom: insets for myelin vesicles from top row (scale bar 0.5 μm). Images are representative of myelinated axons observed from n = 5 biologically independent replicate pairs. e Quantification of electron microscopy data from mouse spinal cord cross sections for the presence of vesicles within myelinated axons. Control vs. iBot;Cnp-Cre mean ± SEM, respectively from n = 5 biologically independent replicates: at the inner tongue 6.87 ± 1.01% vs. 26.5 ± 5.19%; within non-compacted wraps 1.12 ± 0.34% vs. 5.24 ± 1.80%, p-values determined by an unpaired, two-tailed t-test. Created with BioRender.com. Source data are provided in the Source Data file.
Fig. 6
Fig. 6. Oligodendrocyte VAMP2/3 is required for delivery of myelin adhesion proteins and node of Ranvier formation.
a Biotinylation of surface proteins on mature oligodendrocytes differentiated in culture followed by immunoprecipitation (IP) with streptavidin beads for mass spectrometry. Created with BioRender.com. b Proteomic analysis of immunoprecipitated surface proteins and interactors from control vs. iBot;Cnp-Cre oligodendrocytes. Data points exclude non-specific interactors of the streptavidin IP identified in the non-biotinylated samples. Statistical measurements were determined from two-sample T-test with a Benjamini-Hochberg false discovery rate adjustment (padj). The negative log10 padj of each specific interactor was plotted against its average log2 fold change between iBot;Cnp-Cre and control samples. The dotted curve corresponds to the threshold cutoff for a 2-fold change between samples and a permutation-based false discovery rate correction for multiple comparisons with p < 0.05. Magenta points highlight notable myelin proteins that are significantly dependent on VAMP2/3 for surface delivery. c Selected gene ontology (GO) annotations using gProfiler for VAMP2/3-dependent hits from the surface proteomic analysis (b) with a dotted line marking padj = 0.05. Adjusted p-values were determined by the gProfiler website, which uses the cumulative hypergeometric test with the g:SCS correction method. d Annotated localization of top VAMP2/3-dependent proteins in oligodendrocytes. The area of each circle scales linearly to the log2 fold depletion in iBot;Cnp-Cre oligodendrocytes. Lines connecting proteins denote reported and predicted protein interactions from the STRING database. Created with BioRender.com. e Immunohistochemistry of axonal node components Caspr (cyan) and AnkG (red) for longitudinal sections of the spinal cord harvested from control (top) and iBot;Cnp-Cre (bottom) littermates at P12. Images are representative of n = 4 biologically independent replicates quantified in g. Scale bar, 20 μm. f Representative images of node classifications. Scale bar, 2 μm. g Quantification of the number of nodes in each category over 48,000–72,000 μm2 of spinal cord longitudinal section for each of n = 4 biologically independent replicates. Square and circle symbols denote males and females, respectively. Mean number of nodes per 1000 μm2 ± SEM for control vs. iBot;Cnp-Cre, respectively: mature nodes 2.85 ± 0.52 vs. 0.28 ± 0.09; heminodes 2.03 ± 0.76 vs. 0.70 ± 0.19; clusters 3.93 ± 0.66 vs. 1.48 ± 0.50. Statistical significance was determined by one-way ANOVA with multiple comparisons correction. Source data are provided in the Source Data file.
Fig. 7
Fig. 7. Model figure of how VAMP2/3-mediated exocytosis drives CNS myelination.
a In nascent myelin sheaths, VAMP2/3-mediated exocytosis occurs at the inner tongue to drive wrapping and at sheath edges to drive elongation. VAMP2/3-mediated exocytosis results in full-vesicle fusion that incorporates myelin membrane material and axon-myelin adhesion proteins. Created with BioRender.com. b VAMP2/3-mediated exocytosis coordinates sheath elongation with surface delivery of axon-myelin adhesion proteins to form nodes of Ranvier in the CNS. Created with BioRender.com.
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