Oligodendroglial macroautophagy is essential for myelin sheath turnover to prevent neurodegeneration and death

Abstract

Although macroautophagy deficits are implicated across adult-onset neurodegenerative diseases, we understand little about how the discrete, highly evolved cell types of the central nervous system use macroautophagy to maintain homeostasis. One such cell type is the oligodendrocyte, whose myelin sheaths are central for the reliable conduction of action potentials. Using an integrated approach of mouse genetics, live cell imaging, electron microscopy, and biochemistry, we show that mature oligodendrocytes require macroautophagy to degrade cell autonomously their myelin by consolidating cytosolic and transmembrane myelin proteins into an amphisome intermediate prior to degradation. We find that disruption of autophagic myelin turnover leads to changes in myelin sheath structure, ultimately impairing neural function and culminating in an adult-onset progressive motor decline, neurodegeneration, and death. Our model indicates that the continuous and cell-autonomous maintenance of the myelin sheath through macroautophagy is essential, shedding insight into how macroautophagy dysregulation might contribute to neurodegenerative disease pathophysiology.

Keywords: Alfy/Wdfy3; CP: Neuroscience; amphisome; autophagosome; myelin; neurodegeneration; oligodendrocyte; selective autophagy.

Figure 1.. The inactivation of autophagy in OLGs results in an adult-onset accumulation of myelin, increased myelin sheath thickness, and abnormal myelin structures

(A–H) Ultrastructural images of myelin and g-ratio calculation from optic nerve at (A) 2, (C) 9, and (E) 16 months old and from (G) corpus callosum (CC) at 16 months old. n = 3 brains/genotype. (B, D, F, H) Mean g-ratios are plotted per quartile of axon diameter (μm). Bars represent average values across the 3 brains. The number of axons per quartile also shown. ANOVA reveals that in the optic nerve, there is no difference in mean g-ratios across quartiles at 2 months old (p = 0.5746; p = 0.9952; p = 0.3783; 0.5196). In contrast, g-ratios are significantly lower across the first 3 and 2 quartiles for 9 (p = 0.0473; p = 0.0179; p = 0.0256; p = 0.1951) and 16 months old (p = 0.0174; p = 0.0467; p = 0.072; p = 0.4738), respectively. CC also shows a similar difference (p = 0.0139; p = 0.0455; p = 0.0304; p = 0.2661). The axon diameters represented per quartile are as follows (upper value indicated): 2 (Q1 = 0.43, Q2 = 0.55, Q3 = 0.69, Q4 = 1.8), 9 (Q1 = 0.46, Q2 = 0.59, Q3 = 0.78, Q4 = 2.08), and 16 months old (Q1 = 0.50; Q2 = 0.63, Q3 = 0.80, Q4 = 1.77). (I) Redundant myelin and myelin whorls in 16-month-old cKO CC (top) and optic nerve (bottom). (J) Probability values of finding myelin g-ratio less than 0.6 of 225 axons examined at 2, 9, and 16 months old.

Figure 2.. Basal autophagy in OLGs in vivo and in culture

(A and B) GFP-LC3⁺ autophagosomes in OLGs in the adult brain under basal conditions. CC of GFP-LC3 transgenic mice was examined at 2, 9, and 12 months old. Acute treatment with chloroquine prevented autophagosome maturation. (A) Representative image from 9-month-old CC. (B) Quantification of raw integrated density value, sum of pixel intensity data (NIH ImageJ), of GFP-LC3 corrected for measured area (perinuclear of Olig2⁺ cells) reveals that basal autophagy is active in OLGs across time, with the highest activity at 2 months old. n = 3 brains/age. (C–H) Immunofluorescence in different stages of OLG maturation. Staining against phalloidin (white) and (C and D) Atg16 (yellow), (E and F) p62 (cyan), and (G and H) LC3 (green) reveal a notable presence of all of these proteins throughout the cells during different stages of maturation. Mature OLGs with the boxed areas enlarged and shown at right highlight the presence of these proteins in the cytoplasmic channels of the myelin sheath. Representative images from n = 100 cells/3 litters.

Figure 3.. Autophagosome formation and maturation occurs throughout the mature OLs as revealed by live-cell imaging to capture selectively MBP

(A–C) GFP-LC3 in green, LysoTracker red in magenta, and overlay in white. Arrows indicate particle of interest. Time indicated in minutes. (A) Regions of the mature OLG, taken from an individual cell. The cell is visually divided into discrete regions as indicated: cell body, artery, branch, artery/branch (A/B) intersection, bulb, and edge. Image taken from Video S2. (B and C) Selected frames from Video S3. Additional particles are found in Video S3 and summarized in Figure S3. (D–F) Summary of all movies analyzed. (D) Each individual punctum analyzed across time. Green indicates the appearance of GFP-LC3 puncta, and magenta indicates positive signal for LysoTracker red. Co-localization represented in yellow. The formation of 185 GFP-LC3+ puncta analyzed across 6 movies monitored throughout the cell, then analyzed for time through maturation. Structures that were only Lysotracker+ were not analyzed. (E) Quantification of the frequency of autophagic vacuole (AV) appearance corrected for area. (F) Time to acidify versus cell region. A/B Int., Artery-Branch intersection. Data from 143 cells/2 cultures.

Figure 4.. The oligodendrocyte protein MBP is degraded by selective autophagy

(A) Co-localization of MBP in autophagosomes. MBP (magenta) and endogenous LC3 (green) staining revealed upon lysosome inhibition. OLGs are treated with Leupeptin overnight to mildly impede lysosome function and promote autophagosome accumulation. Representative images from n = 90 cells/5 cultures. (B–D) Increased presence of MBP in the detergent-insoluble fraction of Atg5cKO OLGs. (B) Schematic representation. (C and D) Western blot analysis of 1% Tx-100 (C) -soluble and (D) -insoluble Ctrl and cKO cell lysates. Probing the detergent insoluble pellet (resolubilized in 8 M urea) indicates greater accumulation of MBP in the absence of Atg5. Representative blots from n = 3 cultures/genotype. (E and F) MBP (magenta) and NeuN (blue) in adult cortex from Atg5Ctrl and cKO mice. (G and H) p62 and MBP in Atg5cKO OLGs. Accumulation of p62 (cyan) can be detected throughout the cKO cells. Co-staining for MBP (magenta) reveals that regions of p62 accumulation also have high MBP (shown in inset). Representative images from n = 60 cells/3 cultures/genotype. (I) Co-immunoprecipitation (coIP) of endogenous Alfy, p62, and LC3 with MBP from wild-type brain tissue. (J) Transmission electron microscopy analyses of mature OLGs in culture reveals the presence of large double-membrane structures filled with electron-dense material, but devoid of bulk cytosol, suggestive of aggregated cargoes captured via selective autophagy. Representative images from 20 cells/4 cultures.

Figure 5.. Integral membrane myelin proteins require macroautophagy for their degradation

(A and B) Endogenous (A) PLP and (B) MOG in Atg5Ctrl and cKO OLGs. Both proteins accumulate more frequently in Atg5cKO OLGs, as indicated by the number of punctate structures quantified per cell (PLP: Ctrl = 10.85 ± 6.15 versus cKO = 22.17 ± 8.42; two-tailed, Student’s t test: p = 0.017; MOG: Ctrl = 9.11 ± 8.70 versus cKO = 46.43 ± 23.5; two-tailed, Student’s t test: p < 0.001). Higher magnification shows areas of interest. n = 60 cells/3 cultures/genotype. (C) MOG co-localizes with LC3⁺ structures in wild-type (WT) OLGs. (D–G) Fractionation of the adult brain reveals the presence of myelin associated proteins in AVs. (D) AV isolation. Fractionation of the post nuclear supernatant (PNS, S1) across a step gradient of nycodenz (nyc) segregates S1 into 3 fractions (F1, F2, F3) and a pellet. F1 represents cytosol (Cyto), and F2 represents the light membrane (LM) fraction. F3 is further fractionation across a Percoll gradient into F4 and F5. The densest fraction, F5, enriches for AVs. (E) Immunoblotting reveals the presence of myelin proteins in the AV fractions. (F) Immuno-isolation (I-I) of LC3⁺ AVs leads to a further enrichment of myelin proteins. (G) PK protection reveals that the myelin proteins are within AVs. 30 μg AVs are exposed to the noted amount of PK and incubated for the times shown. In the presence of detergent, and thereby upon membrane disruption, the proteins are susceptible to degradation by PK. Fractions from n = 5 brains/fractionation. Data representing n = 3 fractionations from 3- to 6-month-old mice.

Figure 6.. Myelin-associated integral membrane proteins such as MOG are internalized by endocytosis and then form an amphisome to be degraded

(A) Endocytosis of MOG as shown by overnight exposure to the surface membrane dye, mCLING. Atg5 Ctrl and cKO cultures reveal similar co-localization of MOG (magenta) and mCLING (cyan). n = 60 cells/3 cultures/genotype. Chi-square test reveals no significant difference between cKO and Ctrl cultures in the likelihood that MOG co-localizes to mCLING (chi-square statistic = 0.60594, p = 0.607). (B) Co-staining of mCLING, MOG, and MBP. Triple staining shows that while MOG (magenta) and MBP (blue) can simultaneously co-localize with mCLING (cyan), MOG-mCLING co-localization can also occur without MBP. Dashed-line boxes highlight MOG-mCLING localization events, whereas solid line boxes indicate sites of triple co-localization. n = 40 cells/2 cultures. (C and D) EM analyses of mature OLGs in culture treated with Leupeptin overnight. Structures reminiscent of (C) amphisomes, double-membrane structures that appear fused with single-membrane vesicles filled with myelin-like membranes, are found throughout the cell, as well as (D) unilamellar autolysosomes. 20 cells/4 cultures. (E–G) Live-cell imaging of GFP-LC3⁺ cells (green) treated with mCLING (magenta). (E and F) Selected frames from Video S5 of individual puncta. Arrows indicate particle of interest. Time in minutes. A complete cell can be viewed in Video S4, and additional particles in Video S5, and summarized in Figure S6. (G) Each individual puncta analyzed across time. Green indicates the appearance of GFP-LC3 puncta, and magenta indicates positive signal for mCLING. Co-localization represented in yellow. The formation of 187 GFP-LC3⁺ puncta analyzed across 6 movies monitored throughout the cell, then analyzed for mCLING fusion. Structures that were only mCLING⁺ were not analyzed. (H) Schematic model of the cell-autonomous turnover of myelin by macroautophagy. Aggregated MBP is taken up in a p62- and Alfy-dependent manner into an autophagosome via selective autophagy. These structures meet with endocytosed integral membrane myelin proteins to form an amphisome. The amphisome then fuses with a lysosome (yellow) to permit content degradation (dark green).

Figure 7.. The inactivation of oligodendroglial autophagy results in behavioral deficits, neurodegeneration, and death

(A) Survival plot of Atg7cHet and cKO mice. n = 30 littermate pairs. (B) Atg7cKO mice demonstrate locomotor deficits starting at 5 months old. The same cohort of mice (n = 15/genotype) are monitored for 5 min across time. Repeated measures ANOVA (RM-ANOVA) reveals a significant difference between genotype (F_(1,26) = 5.212, p = 0.0308), with a significant decline starting at 5 months old in Atg7cKO (p = 0.0215) but not Ctrl (p = 0.5809). No difference was observed in rearing behavior at young ages (not shown). (C) Rearing behavior monitored for 1 h at 12 or 16 months old. RM-ANOVA revealed a difference between genotype (F_{(1, 29)} = 1.420; p = 0.0002), and Fisher’s posthoc Least Significant Difference (LSD) reveals that it is different at 12 (p = 0.0041) and 16 months old (p = 0.0134). (D–H) Loss of oligodendroglial macroautophagy leads to glial degeneration. (D) Z projections of CNPase staining of the same volume of tissue from region matched areas of Atg7cHet and cKO mice at 16 months old (n = 3/genotype). No differences were observed at 9 or 12 months old (not shown, n = 3/genotype/age). The CNPase signal in cKO is noticeably decreased and patchier than in cHet. (E and F) Higher magnification views of cKO tissue. CNPase (magenta) is co-stained with neurofilament heavy chain (NFH; cyan) and Dapi (blue). Ragged myelin sheaths (white arrow), accumulations within OL cell bodies (yellow arrow), and myelin blebs (green arrow) are observed in the cKO but not in the Ctrl (Figure S7A). (G and H) Staining for activated caspase-3 (yellow) in the CC and cortex of 12-month-old Atg7Ctrl, 4-month-old Atg7cKO, and 12-month-old Atg7cKO mice. Staining reveals caspase-3⁺ signal in 12-month-old Atg7cKO mice within the (G) MBP⁺ (magenta) CC and (H) NeuN⁺ cortical neurons, with an occasional signal in the 4-month-old Atg7cKO and no signal in control brains. n = 3/genotype/age. (I) EM micrographs of CC from 16-month-old Atg7cKO mice. Axonal spheroids (yellow arrows), aberrant OL structures (cyan arrows), and myelin debris (green v-arrowhead).