Internalization of exogenous myelin by oligodendroglia promotes lineage progression

Oligodendrocytes, traditionally recognized for their role in central nervous system myelination, have emerged during the last decades as key participants maintaining brain homoeostasis in response to metabolic demands and stress. In addition, injury to myelin prompts a regenerative response that leads to the format ion of new myelin sheaths. However, the signals regulating effective remyelination by oligodendrocytes are still not completely understood. Here, we report that oligodendrocytes can internalize exogenous myelin both in vitro and in vivo, which leads to an increase in their proliferation and differentiation when their functions are not compromised. RNA sequencing reveals that myelin debris alters oligodendrocyte transcriptional profile, suppressing immune -related pathways and de novo cholesterol and fatty acid biosynthesis, while inducing lipid droplet formation to store and process internalized myelin particles. As a result, progression of the oligodendroglial lineage is enhanced in primary cell cultures, as shown by increased viabil ity, proliferation and differentiation . Oligodendrocytes also acquire a more differentiated phenotype, with larger cell areas, a more complex morphology and myelination of synthetic nanofibers. Stereotaxic injection of fluorescent myelin into mouse cortex shows internalization by microglia and, to a lesser extent, by oligodendroglia. Notably, in the zebrafish model, ventricular injections of myelin also increase the number of ventral oligodendrocytes in the spinal cord, further supporting that myelin can promote lineage progression. These findings challenge the classical view that myelin debris intrinsically inhibits oligodendrocyte proliferation , suggesting instead that oligodendrocytes can use myelin to support self-renewal and maturation, acting as a trop hic factor in the absence of pathological cues. .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted July 23, 2025. ; https://doi.org/10.1101/2025.07.18.665370doi: bioRxiv preprint 3

Oligodendrocytes (OLs) are a highly specialised class of glial cells of central nervous system (CNS), best known for their role in producing myelin sheaths following their differentiation from oligodendrocyte progenitor cells (OPCs) during early development (Bradl and Lassmann 2010; Emery 2010; Nave and Werner 2014). Myelin is a multilamellar, lipid-rich structure composed of approximately 70–80% lipids, 2 0% proteins and a small fraction of mRNA (Simons and Nave 2016; Stadelmann et al. 2019) . Traditionally, OLs were considered primarily responsible for axonal myelination, a process that enables saltatory conduction, in which electrical impulses jump between nodes of Ranvier along the axon, allowing rapid propagation of action potentials (Bradl and Lassmann 2010; Nave and Werner 2014; Simons and Nave 2016). However, research over the last decades has expanded this view, revealing additional mechanisms through which OLs contribute to the maintenance of neuronal function. Beyond their role in myelination, OLs also play a key role in the metabolic support of axons (Nave, Asadollahi, and Sasmita 2023; Philips and Rothstein 2017) . Neurons have high energy demands due to constant ATP -dependant activity of ion pumps, synaptic vesicle recycling and generation of membrane potentials (Faria-Pereira and Morais 2022). Paradoxically, their lack of intrinsic energy reserves makes them reliant on surrounding glial cells for metabolic support. In addition to astrocytes (Tekkök et al. 2005), under homeostatic conditions OLs contribute to this energy supply primarily by transferring lactate to axons through the monoc arboxylate transporter 1 (MCT1) (Lee et al. 2012; R inholm et al. 2011) . Furthermore, it has recently been suggested that in response to stress, OLs can modulate their own metabolic machinery to obtain energy from myelin-derived fatty acids. Genetically starved mice, lacking GLUT1 expression in OLs, exhib it a reduction in the thickness of their myelin sheaths (Asadollahi et al. 2024) , a phenomenon that correlates with the loss of myelin in specific brain areas in humans following the extreme physical stress of running a marathon (Ramos-Cabrer et al. 2025). Likewise, the use of myelin lipids as an energy source has been proposed in the context of aging as a compensatory mechanism for age-related metabolic decline (Klosinski et al. 2015). In experimental au toimmune encephalomyelitis , a mouse model of multiple sclerosis (MS) , single-cell RNA analysis demonstrates that OLs exhibit a form of non -professional phagocytic behaviour, upregulating interferon response genes and expressing major histocompa tibility complex type 1 and 2 (Falcão et al. 2018). Conventionally, clearance of myelin debris has been attributed to microglia and infiltrating macrophages, which actively engulf and degrade lipid - rich remnants during injury or disease (Franklin and Ffrench -Constant 2017; Kent and Miron .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted July 23, 2025. ; https://doi.org/10.1101/2025.07.18.665370doi: bioRxiv preprint 4 2024). It has been p roposed that OL -lineage cells can internalize extracellular myelin debris , activating memory and effector CD4 + cells (Falcão et al. 2018) . Therefore, there is emerging evidence that OLs may have alternative roles in pathological conditions. Further research is needed to delineate the molecular mechanisms involved in this phagocytic response and the extent to which these non -canonical properties of OLs occur in physiologi cal and pathological conditions. In this study, we demonstrate that oligodendroglia can internalize extracellular myelin debris both in vitro and in vivo and that this process promotes their proliferation and differentiation. By demonstrating the capacity of OLs to engage in debris clearance and that this process is associated w ith increased OL proliferation, we highlight the importance of a previously underappreciated aspect of their biology. This insight underscores the evolving view of OLs not just as passive support cells, but as dynamic regulators of CNS integrity and repair. .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted July 23, 2025. ; https://doi.org/10.1101/2025.07.18.665370doi: bioRxiv preprint 5

1. Animals 1.1. Rodents Primary cell culture experiments were conducted using Sprague Dawley rats, sourced from the Animal Facility of the University of the Basque Country (UPV/EHU) (Leioa, Spain). C57BL/6J mice obtained from Jackson Laboratories (Bar Harbor, Maine, USA) were used to perform stereotaxic injections. Animals were housed under standard conditions, maintaining a 12 h light/dark cycle and ad libitum access to food and water. Experiments were reviewed and approved by the internal Animal Ethics Committee of the UPV/EHU, according to the guidelines set by the European Communities Council Directive 2010/63/EU. 1.2. Zebrafish Zebrafish (Danio rerio) were maintained under standard laboratory conditions at the University of Edinburgh, in compliance with UK Home Office regulations (PP0103366) and institutional ethical guidelines. Adult fish were housed on a 14-hour light/10-hour dark cycle. Embryos were raised at 28.5 °C in 90 mm Petri dish with 10 mM HEPES -buffered E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl₂, 0.33 mM MgSO₄), staged by days post-fertilization (dpf) according to Kimmel et al. (1995), and analyzed up to 4 dpf, prior to the onset of sexual differentiation. The following transgenic lines were used in this study: Tg(mbp:EGFP-CAAX) (Almeida et al. 2011), Tg(mbp:EGFP) (Almeida et al. 2011) , Tg(mpeg1:EGFP) (Ellett et al. 2011) and Tg(olig1:nls- mApple) (Marisca et al. 2020). 2. Oligodendrocyte cell culture Primary oligodendrocyte cultures were obtained as previously described (Barres et al. 1992) , with modifications (Sánchez-Gómez et al. 2018). Briefly, optic nerves were extracted from P11– 12 rats. After enzymatic and mechanical digestion, cells were filtered and seeded on 1 μg/ml poly-D-lysine–coated (PDL) coatings (Sigma-Aldrich). Otherwise stated, OLs were maintained in differentiation media, described in (Sánchez-Gómez et al. 2018). Oligodendrocytes were seeded at 10,000 cells/well for immunocytochemical (ICC), cell viability experiments and on Ibidi μ-Dishes for time -lapse imaging. For RNAseq analysis, 300,000 cells/well were seeded. For myelination assays, cells were seeded on coverslips with polycaprolactone nanofibers (Sigma-Aldrich) at a 20,000 cells/well density and CNTF and NT -3 were omitted from the medium. .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted July 23, 2025. ; https://doi.org/10.1101/2025.07.18.665370doi: bioRxiv preprint 6 3. Cortical glial cell cultures Primary mixed glial cultures were obtained from the cortical lobes of P0 -2 rats, according to previously described by Mcarthy & De Vellis (1980) with modifications (Sánchez-Gómez et al. 2018). Briefly, forebrains were dissected to isolate the cortical lobes and after enzymatic and mechanical digestions, cell suspension was seeded in 75 cm 2 flasks coated with PDL (Sigma - Aldrich). The resulting glial culture contained OPCs, microglia and astrocytes. After 14 days, the different cell types were isolated and seeded using different mechanical shakings, based on the different adhesion properties as described in Domercq et al. (2007) for microglial ce lls and in Sánchez-Gómez et al. (2018) for OPCs. After detachment and isolation of microglia and OP Cs, the astrocyte monolayer was trypsinized with Trypsin-EDTA (0.5 g/l porcine trypsin and 0.2 g/l EDTA, Sigma-Aldrich) during 7 minutes and the cell suspension was seeded. All cell types were seeded on PDL coatings and cell density was adjusted to experimental requirements. For ICC and cell viabi lity assays, 10,000 OLs or microglia and 250,000 astrocytes were seeded per well and in Ibidi μ-Dishes for time-lapse imaging. Myelin was added at 1 DIV. 4. Hippocampal neuronal cultures Hippocampal neurons were prepared from embryonic day 18 rat embryos. Hip pocampi were dissected from embryonic brains and dissociated in TrypLE Express (Thermo -Fisher) for 10 min at 37°C. Cells were resuspended and homogenized in Neurobasal (Gibco) with 10% FBS Hyclone, 2 mM L -glutamine (both from Sigma -Aldrich) and 50 U/ml pen icillin-streptomycin (Gibco). Hippocampal neurons were cultured on PDL-coated coverslips at 20.000 cells/well. The medium was supplemented with B27 (Gibco) and 20 μM 5 -fluorodeoxyuridine and uridine (Sigma Aldrich) and changed every 3 days. Myelin was added at 7 DIV. 5. Myelin extraction and labelling Myelin was isolated from adult rat or mouse brains following the protocol established by Norton & Poduslo (1973) . Briefly, brain tissue was mechanically homogenized in 0.32 M sucrose and subjected to rounds of ultracentrifugation using 0.85 M sucrose gradients. Concentration of isolated myelin was determined using Bradford protein assay (ThermoFisher) and labelled with Alexa488-NHS or Alexa594-NHS dye (Invitrogen) for 1 hour at room temperature in PBS (pH=8). Excess dye was removed by 24-hour dialysis, and labeled myelin was resuspended in sterile PBS (pH=7.4) and frozen at -80oC. For in vitro assays, myelin was thawed and vortexed for 3 0 seconds to obtain uniformly sized aggregates and added to culture medium at 5 µg/ml for immunofluorescence and cell viability .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted July 23, 2025. ; https://doi.org/10.1101/2025.07.18.665370doi: bioRxiv preprint 7 experiments and 1 µg/ml for time -lapse recordings. For in vivo experiments, myelin was sonicated during 30 minutes before use and injected at 200 µg/ml. 6. Cell viability assays Cell viability was assessed using the Calcein-AM dye (Invitrogen). C ells were incubated with 1 μM of dye for 30 minutes and fluorescence was measured with a Synergy HT fluorimeter reader (Bio-Tek Instruments). Results are expressed as the relative percentage of cellular viability with respect to control conditions. 7. Time-lapse imaging Alexa-488 labelled myelin was added to the culture media 15 minutes prior to the beginning of the recording to let myelin debris to settle and avoid out -of-focus images. Bright -field and fluorescence images (488nm laser) were acquired every 10 minutes in 8-10 localized points using a BioStation IM -Q microscope (Nikon). Cells were maintained at 37ºC and 5% CO 2 during the recording. 8. Immunochemical analysis For ICC analysis, primary cultures were fixed in 4 % paraformaldehyde (PFA) diluted in 0.1 M phosphate buffer (PB) for 20 min. IHC was performed in free -floating sections obtained as described in section 12.2. A standard immunofluorescenc e protocol was used consisting of a 1 hour incubation at room tempera ture (RT) in blocking solution containing 4 % normal goat serum (Vector Labs) and 0.1 % triton X -100 in PBS, except for PDGFR -α antibody for which normal donkey serum was used. Cells were incubated overnight at 4 ºC with primary antibodies, followed by an hour incubation at RT with AlexaFluor-conjugated secondary antibodies and DAPI (4 μg/ml; Sigma-Aldrich) for nuclei staining. After washings, coverslips were mounted on glass slides using Dako Glycergel Mounting Medium (Agilent). Primary antibodies used for immunohistochemistry were as follows: mouse anti‐MBP (1:500; Biolegend), goat anti -PDGFR-α (1:250, R&D Systems), rabbit anti -NG2 (1:500; Abcam), mouse anti-Olig2 (1:500; Milipore), rabbit anti-Ki67 SP6 (1:250, Abcam) and guinea pig anti-Iba1 (1:300; Synaptic Systems). The corresponding Alexa Fluor secondary antibodies (ThermoFisher) were used accordingly to the host species of primary antibodies at a 1:500 dilution. 9. Lipid droplet staining and imaging Fixed cells were stained with Oil Red O (BioVision), fol lowing manufacturer’s instructions: cells were incubated in 60% isopropanol for 5 minutes and then stained with Oil Red O (1.8 mg/ml in dH2O) for 20 minutes. After consecutive washings in dH 2O, nuclei were stained with DAPI (4 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted July 23, 2025. ; https://doi.org/10.1101/2025.07.18.665370doi: bioRxiv preprint 8 μg/ml; Sigma-Aldrich) and coverslips were mounted. Images were acquired in a Zeiss LSM880 Airyscan microscope using a 40x objective. 10. Confocal imaging and analysis Immunofluorescence imaging was conducted on a Zeiss LSM880 Airyscan or Leica TCS SP8. Imaging parameters were kept constan t within each experimental set. Image processing and quantification were carried out using FIJI (ImageJ) software. To assess oligodendrocyte morphology, we performed Sholl analysis. Individual cells were segmented as regions of interest and skeletonized. Morphological complexity was measured as the number of intersections between cell processes and concentric circle templates centered on the soma. 11. Bulk RNA sequencing Total RNA from OLs cultures was isolated using NZY Total RNA Isolation kit (NZYTech), according to the manufacturer’s instructions. The quantity and quality of the RNAs were evaluated using Qubit RNA HS Assay Kit (Thermo Fisher) and Agilent RNA 6000 Nano Chips (Agilent Technologies), respectively. Sequencing libraries were prepared followin g “TruSeq Stranded mRNA Sample Preparation Guide (Part # 15031058 Rev. E)” using the “TruSeq® Stranded mRNA Library Prep” kit and TruSeq RNA CD Index Plate (Illumina). From 400ng of total RNA, mRNA was purified, fragmented and primed for cDNA synthesis using Illumina and ThermoFisher reagents. Then, A -tailing and adaptor ligation were performed. Finally, enrichment of libraries was achieved by PCR and quantified using Qubit dsDNA HS DNA Kit (Thermo Fisher Scientific) and visualized on an Agilent 2100 Bioana lyzer using Agilent High Sensitivity DNA kit (Agilent Technologies). Using STAR software version 2.7.10b (Dobin et al. 2012), FASTQ files were aligned to Rattus norvegicus genome data base “rn6” and reads for analysed features were assigned and counted from the processed BAM files using SubRead's FeatureCounts version v2.0.3 (Liao, Smyth, and Shi 2014). Differential expression analysis was performed with the R library DESeq2 version 1.44.0 (Love, Huber, and Anders 2014). GSEA were performed with described gene sets using gene set permutations (n = 1000) for the assessment of significance and signal -to-noise metric for ranking genes from the Molecular Signature Database ( https://www.gsea- msigdb.org/gsea/msigdb/index.jsp) (Liberzon et al. 2015). 12. Exogenous myelin internalization in mice 12.1. Stereotaxic injections Brain stereotaxic injections were performed in 8 -10 weeks old C57BL/6J mic e. Two 0.5 μl injections of mouse-derived Alexa488-labelled myelin diluted in saline solution (0.9% NaCl) were .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted July 23, 2025. ; https://doi.org/10.1101/2025.07.18.665370doi: bioRxiv preprint 9 performed at the following coordinates: Bregma 0.5 mm AP, 1.0 mm LM, 1.3 mm VD and Bregma 2.5 mm AP, 0.8 mm LM, 1.0 mm VD. The content was injected at a constant rate of 0.1 μl/min using a Hamilton syringe, leaving the needle in place for an additional 3 minutes. Both injections were made in the same hemisphere. In sham -operated control mice, saline was injected using the equivalent procedures. Animals were perfused 24 or 48 hours after the injection. 12.2. Tissue processing for immunohistochemistry Mice were anesthetized and perfused with 4 % PFA in 0.1 M PB. Dissected brains were post - fixed overnight at 4 °C in the same solution. Then, coronal 40 µm -thick sections were obtained using a Microm HM 650V Microtome (ThermoFisher). Sections were selected for further staining based on the presence of fluorescent signal from myelin and/or the tissue scar from the surgery in the case of sham animals. 12.3. Transmission electron microscopy Mice were anesthetized and perfused with 4 % paraformaldehyde and 0.1% glutaraldehyde in 0.1 M PB. Coronal 50 μm-thick brain sections were obtained using a Microm HM650V microtome (Thermo Fisher). To stain Alexa -488 labelled myelin using immunogold, slices were incubated with blocking solution (10% BSA, 0.1% sodium azide, 0.05% triton X-100 in TBS) for 60 min at RT. Subsequently, sections were incubated with rabbit IgG anti -Alexa 488 antibody (1:100, ThermoFisher) in blocking solution for 4 days at 4°C. Following several washes, sections were incubated for 2 h at RT with 1.4 nm gold -labeled goat anti -rabbit IgG (1:200; Nanoprobes Inc.) in blocking buffer. Sections were postfixed in 1% glutaraldehyde for 10 min at RT and washed in ddH20. Gold particles were silver -intensified with a HQ Silver kit (Nanoprobes) in the dark for 12 min and tissue was washed with ddH20 followed by 0.1 M PB. The day after, sections were osmicated (1% OsO4 in 0.1 M PB) for 30 min and dehydrated in graded ethanol concentrations (50 –100%) to propylene oxide and embedde d in epoxy resin (Sigma-Aldrich) by immersion in decreasing concentration of propylene oxide. Tissue was then embedded in fresh resin overnight and allowed to polymerize at 60°C for 2 days. Semithin sections (500 nm thick) were cut using a PowerTome ultramicrotome (RMC Boeckeler) and stained with 1% toluidine blue for sample orientation. Ultrathin sections (50 –60 nm thick) were cut with a diamond knife (Diatome), collected on nickel mesh grids, and stained with 4% uranyl acetate for 30 min followed by 2.5% lead citrate for 10 min. .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted July 23, 2025. ; https://doi.org/10.1101/2025.07.18.665370doi: bioRxiv preprint 10 TEM images were obtained using the JEOL JEM 1400 Plus electron microscope (SGiker, UPV/EHU) at magnifications ranging from 1000x to 8000x. 13. Myelin internalization in zebrafish 13.1. Intracerebroventricular injections At 3 dpf, larvae wer e anesthetized using 600 μM Tricaine (Sigma -Aldrich) and embedded in a 2.5% agarose drop. Larvae were oriented ventrally, with the yolk sac oriented to the bottom of the drop, allowing access to the dorsal area. The needle was positioned through the thin r oof plate of the hindbrain without damaging the brain tissue beneath. Each larva received two sequential 0.5 nl microinjections of Alexa -594 or -488 labelled myelin in nuclease -free water, which was also injected as a control. Following the injection, hear tbeat and circulation were checked and larvae were gently released from the agarose. Injected fish were transferred to 12- well plates with E3, where they were maintained until imaging. Fish were monitored until full recovery from anaesthesia, and only thos e exhibiting normal swimming behaviour were included in subsequent analyses. 13.2. Live imaging and quantification of zebrafish larvae For live imaging, 3 –4 dpf zebrafish larvae were anesthetized with 600 μM Tricaine (Sigma - Aldrich) and mounted laterally in 2.5% agarose as described by Vagionitis & Czopka (2018) . Imaging was performed using a Zeiss LSM880 confocal microscope equipped with Airyscan detection and a Zeiss W Plan -Apochromat 20×/1.0 NA water -dipping objective or Zeiss Axio Imager Z1 equipped with an Apotome.2 unit and using a 10× objective. Larvae injected with labelled myelin were visually scored for distribution of fluorescence over the spinal cord area as high (continuous fluorescence >10 clusters), low (sparse <5 clusters), or medium with intermediate amounts of fluorescence. Animals lacking detectable fluorescence were excluded from experimental groups. For internalization experiments, fluorescence was assessed throughout the spinal cord and brain, with specific regions of interest sampled for an alysis. For figure preparation, maximum - intensity projections of z-stacks were generated and representative regions were cropped. For cell counting, the anal pore was used as a reference point to image a consistent anterior region covering the full depth o f the spinal cord. Individual oligodendrocytes were manually counted in each z -stack and classified as ventral or dorsal based on their contact with the Mauthner axon and normalized to the imaged length. A minimum of two independent injection .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted July 23, 2025. ; https://doi.org/10.1101/2025.07.18.665370doi: bioRxiv preprint 11 rounds were analyzed. All zebrafish images are shown as lateral views of the spinal cord, with anterior to the left and dorsal at the top. 13.3. Statistical analysis Data are presented as mean ± standard error of mean (SEM) and n represents the number of animals, cultures or cells analyzed, as specified in figure legends. Statistical analyses were performed using GraphPad Prism 8 (GraphPad Software Inc). Comparisons between two groups were analysed using paired Student’s two-tailed t-test in in vitro experiments and unpaired in in vivo experiments. p values < 0.05 were considered statistically significant. .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted July 23, 2025. ; https://doi.org/10.1101/2025.07.18.665370doi: bioRxiv preprint 12

1. Oligodendroglia internalize exogenous myelin debris in vitro In order to address the internalization capacity of oligodendroglial cells along the lineage , we first exposed different p rimary rat cell cultures to Alexa -labelled myelin debris for 48 hours (Figure 1B) . Myelin was isolated from rats (Figure 1A) to prevent cross -species reactivity. Notably, both OLs and OPCs were found to internalize exogenous myelin (Figure 1C). Confocal imaging of OLs labelled with the membrane-linked Calcein-AM dye confirmed the internalization of myelin debris by oligodendroglia (Suppl. Figure 1). Interestingly, our results suggest a maturation -dependent enhancement of phagocytic or endocytic activity within the oligodendroglial lineage (Figure 1D). Specifically, OLs were more efficient at engulfing extracellular myelin, with more than 90% of cells containing internalized myelin particles. In contrast, OPCs presented significantly lower levels of internalization, with only about 20% of the cells showing uptake, potentially indicating a more limited role in debris clearance at this developmental stage. In order to further explore the temporal dynamics of myelin internalization, we performed time lapse imaging of cultured OLs over 48 hours (Figure 1E). Interestingly, the processes of OLs were the first cellular region contacting the myelin debris and some particles were observed migrating along the processes toward the soma (Figure 1E, bottom section) . The relative size of myelin debris compared to the thin OL processes strongly suggests that complete internalization occurs upon reaching the soma. After 48 hours, myelin accumulated predominantly in clusters within the cytoplasm and only small particles remained in the processes. In phagocytic cell types, such as microglia and macrophages, internalized material typically accumulates in the soma, where it is processed and degraded by lysosomal enzymes (Trivedi et al., 2020; Yu et al., 2022). Thus, the clustering of myelin within this region in OLs further supports the idea that internalization is completed upon arrival of myelin debris to the cell body. During the 48 -hour time -lapse imaging period, OLs remained static while their processes extended radially. Notably, OL processes did not exhibit directed growth toward the nearby myelin particles, suggesting that they do not actively pursue it. Instead, they appear to maintain their intrinsic growth patterns and may initiate internalization upon passive contact . Importantly, compared t o controls, OLs exposed to myelin appeared more ramified and exhibited an increased cell size. Morphological sholl analysis confirmed that myelin-exposed OLs were larger and exhibited higher process complexity, as determined by increased branching. Moreover, exposure to myelin enhanced cell survival, as indicated by a reduction in cell death .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted July 23, 2025. ; https://doi.org/10.1101/2025.07.18.665370doi: bioRxiv preprint 13 compared to control conditions during the time -lapse imaging period (Figure 1F , green arrowheads). Building on these observations, we next investigate d the effects of myelin exposure on microglia, astrocytes and neurons in vitro. As expected, microglial cells (Suppl. Figure 2A) actively phagocytosed myelin particles and over 1 hour most of microglia cell bodies visible by phase contrast progressively became Alexa -Fluor+. The microglial re sponse was characterized by a higher and more rapid uptake of myelin when compared to OLs , supporting the ir established role in the clearance of myelin debris as professional phagocytic cells (Franklin and Ffrench - Constant 2017; Kotter et al. 2006) . In contrast, astrocytes (Suppl. Figure 2B) exhibited minimal uptake and some myelin clusters were observed around the cells. Moreover, exposure to myelin did not appear to disrupt the astrocytic monolayer . Similarly, neurons showed no significant internalization of myelin (Suppl. Figure 2C) . In both cases, exposure to myelin did not affect cellular survival (Suppl. Figure 2D). These findings highlight the distinct contributions of different cell types in the internalization and clearance of myelin debris . Moreover, myelin appears to signal back specifically to OLs, promoting lineage progression and survival. These results lead us to hypothesize that myelin debris may alter metabolic and signalling pathways, thereby promoting OL differentiation and/or myelination. Our results demonstrate that OLs, in addition to microglia, are also capable of internalizing myelin, particularly as they mature. Based on these findings, we directed subsequent experiments towards myelinating OLs to further elucidate pathways triggered by myelin internalization. 2. Exposure to myelin alters the metabolic transcriptional profile of oligodendrocytes We performed RNA sequencing of OLs cultured in the presence of myelin for 48 hours , which depicted 63 differentially expressed genes (Figure 2B). Gene Set Enrichment Analysis (GSEA) (Figure 2C) revealed that myelin exposure triggered a downregulation of immune -related signaling, including tumor necrosis factor alpha (TNF α), interleukin signaling, interferon response and inflammatory re sponse and complement pathways (Figure 2D) . This transcriptomic profile suggests a distinct cellular phenotype, diverging from the disease-specific state activated in OLs under experimental MS conditions (Falcão et al. 2018). In addition, several metabol ic pathways were significantly altered. Notably, genes involved in cholesterol homeostasis were downregulated (Figure 2E). This included reduced expression of key enzymes critical to the mevalonate pathway for cholesterol biosynthesis , such as mevalonate diphosphate decarboxylase ( Mvd), phosphomevalonate kinase ( Pmvk) and .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted July 23, 2025. ; https://doi.org/10.1101/2025.07.18.665370doi: bioRxiv preprint 14 emopamil binding protein ( Ebp), along with acyl-CoA synthetase short-chain family member 2 (Acss2), which contributes to precursor synthesis . These findings suggest that internalized myelin may serve as an exogenous source of cholesterol, thereby reducing the need for de novo synthesis and potentially facilitating lipid recycling for new myelin production . Indeed, Oil Red O staining revealed that internalized myelin induced an increase in lipid droplets (LDs) formation, which colocalize with myelin, suggesting that internalized myelin is stored within lipid droplets (Figure 3). LDs are key organelles that maintain lipid homeostasis, prevent cholesterol overload (Nugent et al. 2020; Ralhan et al. 2021) and regulate lipid intermediates necessary for myelin synthesis (S. A. Berghoff et al. 2021; Hayashi and Su 2004) . Therefore, the presence of LDs in our model further supports the notion that internalized myelin is actively being processed and recycled. Within the “Cholesterol Homeostasis” gene signature, we also found a downregulation of genes involved in fatty acid metabolism. This included fatty acid synthase ( Fasn), a key enzyme involved in de novo fatty acid synthesis, along with stearoyl-CoA desaturase (Scd) and fatty acid desaturase 2 ( Fads2), which mediate fatty acid desaturation and elongation, respectively. Additionally, genes linked to lipid anabolism within the “Lipid Metabolic Process” signature, such as fatty acyl -CoA redu ctase 2 ( Far2) and fatty acid transporter 6 ( Slc27a6), were also downregulated (Figure 2F). These finding s suggest that myelin -derived metabolites may influence the lipid pool in OLs, thereby downregulating endogenous lipid biosynthesis pathways. In contr ast, pathways associated with lipid catabolism showed a tendency towards an enrichment, including fatty acid metabolism and oxidative phosphorylation (Figure Suppl. 3A- B). This result supports the hypothesis that OLs may be activating the molecular machine ry required to process the lipid components present in internalized myelin. Among the top 15 upregulated genes within the “Fatty Acid Metabolism” signature, we identified key lipid transporters and activators, such as carnitine palmitoyltransferase 1A (Cpt1a) and 2 (Cpt2) and acyl-CoA synthetase medium -chain family member 3 ( Acsm3). Notably, critical enzymes involved in the β-oxidation pathway, including 3-ketoacyl-CoA thiolase (Acaa2), 2,4-dienoyl-CoA reductase 1 ( Decr1) and acyl -CoA thioesterase 2 ( Acot2) and 8 ( Acot8) were also found to be upregulated. Finally, p athways associated with cell proliferation and differentiation, such as transforming growth factor β ( Tgfβ) signaling, showed a non-significant enriched expression, as well as, the “Oligodendrocyte differentiation ” signature (Figure Suppl. 3C-D). Altogether, transcriptomic .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted July 23, 2025. ; https://doi.org/10.1101/2025.07.18.665370doi: bioRxiv preprint 15 analysis revealed that myelin exposure upregulates metabolic and signaling pathways, which could promote OL differentiation and myelination. 3. Myelin internalization triggers oligodendrocyte proliferation in vitro We next assessed the effects of myelin internalization on oligodendroglial lineage progression. After exposing primary OL cultures to myelin, we found that myelin debris increased cell viability by ~80%, as assessed by Calcein -AM, which could be attributed either to a greater number of viable cells and/or to an increase in cell size (Figure 4B). To better understand the underlying cause, we conducted additional analyses using immunocytoc hemistry. Consistent with cell viability assays, i mmunostaining of the oligodendroglial lineage marker Olig2 confirmed an increase in the total number of cells (Figure 4C). Interestingly, proliferation marker Ki67 was enhanced in cultures exposed to myelin (Figure 4C), suggesting a boost in proliferation. This observation likely reflects an expansion of the OPC pool (Dimou and Götz 2014; Young et al. 2013), which was further confirmed by the increase in NG2+ cells (Figure 4D). In parallel, we detected higher numbers of MBP+ myelinating oligodendrocytes, while the ratio of NG2 + to MBP+ cells remained constant (Figure 4 D). Thus, myelin internalization accelerates lineage progression maintaining the dynamics between precursor and mature stages. These changes prompted us to investigate whether internalized myelin boosted myelination capacity of mature OLs, typically associated to a more differentiated state (Bradl and Lassmann 2010). To this end, we seeded mature OLs onto coverslips containing synthetic nanofibers. We found that exposure to myelin significantly enhanced nanofiber myelination , as shown by an increase in OL area (Figure 4E). Taken together, our results suggest that myelin internalization triggers OL proliferation, as well as morphological complexity and differentiation. Thus, myelin debris may serve as a positive regulator that activates the intrinsic program of OL development and myelination, promoting both expansion and progression of the oligodendroglial lineage. 4. Internalization of exogenously injected myelin in the mouse brain In vitro experiments were conducted in the absence of other cell types and their associated signalling interactions present in vivo. Therefore, it was important to investiga te whether the internalization of myelin by OLs and the accompanying increase in proliferation was also observed in a more complex cellular environment. We aimed to determine first, whether the capacity of OLs to internalize myel in is sufficiently robust to be observed in the presence of active microglia, and second, whether microglial uptake of myelin debris trigger s signalling cascades that modulate OL behaviour. To this end, we performed stereotaxic injections of .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted July 23, 2025. ; https://doi.org/10.1101/2025.07.18.665370doi: bioRxiv preprint 16 fluorescently labelled myelin into the cerebral cortex of adult mice. Although myelin debris is typically associated with demyelinating pathologies or injury, we sought to isolate the internalization process from confounding factors, such as OL dysfunction. While the stereotaxic procedure inherently induces a local inflammatory response , this approach enabled us to evaluate OL internalization capacity under non -demyelinating conditions. Moreover, myelin turnover may also involve clearance mechanisms beyond microglial phagocytosis, potentially implicating OLs themselves. As expected, microglial cells rapidly migrated towards the injection site. Within the first 24 hours post-injection, most microglial cells accumulated in the injection core (Figure 5A), temporarily depleting microglia from the immediate surroundings and forming a circular gap with a radius of 150–300 µm, which was subsequently repopulated over the following 24 hours (Figure 5B). Immunohistochemistry confirmed that microglia were the predominant cell type within the injection site and internalized debris was clearly localized within phagocytic pouches (Figure 5C). Microglia adopted a rounded, amoeboid morphology typical of activated, phagocytic cells. These findings were corroborated by electron microscopy (EM), which visualized Alexa-labelled myelin debris within intracellular compartments in microglial cytoplasm (Figure 5 D). This expected behaviour validated the approach and served as a positive control that the surgery procedure did not impair the capacity of phagocytic cells to internalize exogenous myelin. Interestingly, 48 hours post-injection we also identified a subset of PDGRα+ OPCs (Figure 5E) and APC+ OLs (Figure 5G) containing myelin particles, as confirmed by TEM analysis (Figure 5F and 5H). This was observed in a more limited and structurally distinct manner compared to microglia. Unlike the well -formed phagocytic vesicles seen in microglia, internalized myelin within OLs appeared in smaller, s cattered inclusions without the formation of large degradation compartments. This could also reflect the lower myelin load within OLs, which may allow them to degrade and process debris without activating the same specialized degradation pathways observed in professional phagocytes. We next sought to determine OL proliferation. The stereotaxic procedure itself triggers a local proliferative response in the cortical area surrounding the injection , masking any additional effect that myelin may exert. As a result, the proliferation marker Ki67 increased compared to the contralateral hemisphere, but no significant difference was detected between myelin- and sham-injected animals (Figure 5I). Therefore, in order to address whether myelin internalization influences OLs proliferation in vivo without injury -related confounds, we complemented our analysis using the zebrafish model. .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted July 23, 2025. ; https://doi.org/10.1101/2025.07.18.665370doi: bioRxiv preprint 17 5. Exogenously injected myelin increases oligodendrocyte number in the zebrafish Zebrafish larvae offer a transparent and genetically tractable system allowing for high spatial- temporal resolution for live imaging (Chia et al. 2022; Doszyn, Dulski, and Zmorzynska 2024) . This model enables for in vivo visualization of myelin internalization dynamics, while preserving the complexity of a living organism. We performed brain ventricle injections in transgenic zebrafish larvae expressing membrane - targeted EGFP under the myelin basic protein promoter ( Tg(mbp:EGFP-CAAX)), which allows selective labelling of OLs (Almeida et al. 2011) . Membrane localization of EGFP facilitates the assessment of potential internalization of myelin, as engulfed debris can be clearly distinguished within the boundaries of myelinating oligodendrocytes, including fine processes and soma. Labelled myelin was injected into the brain ventricle at 3 days post-fertilization (dpf) and to avoid a potential confounding localized response to the injection, the spinal cord was imaged, rather than the injection site at different time points (Figure 6A). At this developmental s tage, brain ventricles are already connected to the spinal canal, allowing the movement of injected material directly into the spinal cord. Control animals were injected with vehicle solution. To determine the distribution and diffusion of Alexa-labelled myelin following injection, we first imaged 2 hours post-injection. The spinal cord was divided into anterior, central and posterior regions for analysis. The anterior region (Figure 6B), located closer to the brain ventricle, showed clear presence of labelled myelin debris dispersed along and outside the central canal, reaching both the ventral and dorsal myelinated tracts of the spinal cord. T he central and p osterior regions (Figure Suppl 4A), situated more caudally along the tail, showed little to no detectable myelin at this time point . This confirmed that the injected material diffused through the cerebrospinal fluid and reached the anterior spinal cord, allowing for potential interaction with resident oligodendrocytes. At this early time point, however, we did not observe internalization of myelin by oligodendrocytes . Importantly, zebrafish larvae did not exhibit any visible behavioural abnormalities or morphological defects following injection and the introduced myelin was well tolerated, indicating that it was not toxic at the administered concentration. By 24 hours post -injection, myelin fragments had formed more discrete clusters, typically 7 to 10 per fish, distributed along the spinal cord. To determine the identity of the cells containing these clusters, we repeated the injections in Tg(mpeg1:EGFP) zebrafish (Ellett et al. 2011) , labelling microglia and macrophages. Colocalization analysis revealed that large myelin clusters were predominantly found within microglial cells, confirming their ac tive role in mye lin phagocytosis (Figure Suppl 4B). In addition to microglial uptake, we also detected smaller myelin .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted July 23, 2025. ; https://doi.org/10.1101/2025.07.18.665370doi: bioRxiv preprint 18 particles contained within the soma of OLs (Figure 6C) and associated with their myelin tracts (Figure 6D). These findings demonstrate tha t, consistent with our in vitro data and murine model, OLs in zebrafish are capable of internalizing exogenous myelin. We next evaluated the impact of myelin internalization on the oligodendroglial lineage in the zebrafish spinal cord. Remarkably, 24 hours after the injection of myelin, we observed a modest yet significant increase in the number of ventral OLs (Figure 6E), which are located closer to the central canal where the injected myelin accumulates. In contrast, dorsal OLs, potentially less exposed to myelin, did not show a significant change in cell number. This spatially restricted response suggests a direct, contact-dependent effect, potentially driven by direct internalization of myelin debris. Moreover, OPCs, imaged in Tg(olig1:nlsmApple) zebrafish (Marisca et al. 2020), showed a non-significant increase following injection of myelin (Figure 6F). Taken together, and considering that zebrafish development is characterized by low levels of oligodendroglial cell death (Almeida and Lyons 2016) , these findings suggest that myelin uptake could locally be enhancing proliferation, rather than promoting oligodendroglial survival. .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted July 23, 2025. ; https://doi.org/10.1101/2025.07.18.665370doi: bioRxiv preprint 19

In this study, we found that OLs have the capaci ty to internalize myelin debris, leading to transcriptional changes and promoting cellular proliferation and differentiation. Traditionally, the role of myelin debris in regulating oligodendroglial dynamics has been investigated in the context of demyelination, where it is known to inhibit OPC proli feration and hinder their recruitment to lesion site s (Kotter et al. 2006; Plemel et al. 2013) . However, the molecular mechanisms underlying this inhibitory effect remain incompletely understood (Baer et al. 2009; Ji et al. 2024; Molina -Gonzalez et al. 2 023). Moreover, demyelination involves massive tissue damage, microglia activation and immune infiltration. Thus, signaling from damaged tissue and immune cells could potentially affect the OL population. Our findings support the idea that the deleterious impact of myelin debris is highly context -dependent, shaped by the health of OLs and signals from surrounding cellular environment. In contrast , in the absence of overt pathology, myelin may function as a trophic factor that enhances OL lineage progression. Despite some insights, the extent to which OLs can internalize myelin and the underlying mechanisms re main insufficiently understood. Evidence suggest s that OPCs may internalize myelin particles via phagocytosis (Falcão et al. 2018). Similarly, Schwann cells, the myelinating cells of the peripheral nervous system, have been shown to internalize myelin following nerve injury through specific autophagy pathways (Gomez-Sanchez et al., 2015) . In the case of myelinating OLs, several studies have demonstrated that, under pathological and physiological conditions, they express several molecular components known to be involved in endocytic and phagocytic pathways. These include receptors such as low-density lipoprotein receptor-related protein 1 (LRP1), implicated in myelin uptake (Fernández-Castañeda et al. 2020; Gaultier et al. 2009; Lin et al. 2017) ; antigen presentation molecules (Falcão et al. 2018; Zeis, Enz, and Schaeren-Wiemers 2015); and members of the tetraspanin family (Terada et al. 2002) . In this study, we demonstrated that OLs not only have the capacity to internalize myelin debris but also to transport it retrogradely from their processes to the soma, where internalization is completed and degradation begins. This observation suggest the presence of a surface-based trafficking mechanism along their processes. Consistent with this, microvesicles interacting with microglia are known to exhibit directional motion along the external plasma membrane toward the soma, a process dependent on phosphatidylserine-mediated recognition (Prada et al. 2016). Experimental models of demyelination often involve direct targeting and damage to OLs (Blakemore and Franklin 2008; Lassmann and Bradl 2017; Torre-Fuentes et al. 2020; Zirngibl et al. 2021), which inherently disrupts their function and may obscure broader roles . In response .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted July 23, 2025. ; https://doi.org/10.1101/2025.07.18.665370doi: bioRxiv preprint 20 to injury, OPCs become activated, undergoing transcriptomic reprogramming to an immature state that pro motes proliferation and migration by upregulation of immune -related genes (Moyon et al. 2015). OLs also upregulate inflammatory mediators, stress response genes, anti- oxidant enzymes, cholesterol metabolism and growth factors (Hou et al. 2023) . This signature largely overlaps with the disease -associated OLs recently reported in different neurodegenerative models (Pandey et al. 2022) . Importantly, TREM2 deficiency in microglia impaired transcriptomic changes in disease associated OLs, suggesting that OL reprogramming is driven by microglial activation rather than by exposure to myelin debris (Hou et al. 2023). Our data shows that exposure to myelin debris, in the absence of an inflammatory insult, induces i) an opposite transcriptional signature in OLs characterized by a reduced expression of immune- related pathways and cholesterol biosynthesis and ii) an increase in OLs proliferation and differentiation. It is therefore possible that during demyelination pa thological cues directly or indirectly influence OL behavior. During CNS development, reduced expression of the cholesterol transporters Abca1 and Abcg1, transporters of cholesterol efflux to the extracellular space, is associated with the onset of active myelination (Nelissen et al. 2011) . Transcriptomic analysis also suggested predominant upregulation of the cholesterol-synthesis pathway in OLs during remyelination (Voskuhl, R.R et al., 2019; Berghoff, S.A et al., 2022). This downregulation facilitates cholesterol retention within the cell, essential for myelin synthesis. A similar regulatory pattern emerges under demyelinating conditions, wherein neurons upregulate cholesterol synthesis, while OLs downregulate Abca1 and Abcg1 expression to minimize chol esterol efflux and prioritize lipid availability for remyelination (S. Berghoff et al. 2021; Meffre et al. 2015). Consistent with these observations, our findings demonstrate that when OLs are exposed to myelin debris, they adopt a lipid -conserving pheno type that promotes cholesterol retention and supports myelin synthesis. Similarly, lipid droplets, that regulate lipid intermediates necessary for myelin synthesis during development (Hayashi and Su 2007), are increased after myelin exposure. This adaptive response mirrors the molecular mechanisms observed during developmental myelination, suggesting a conserved strategy for optimizing lipid utilization. While this study did not aim to dissect the molecular mechanisms through which myelin promotes OL proliferation and differentiation, this remains a key area for future research. Based on its lipid-rich composition and previous findings (Asadollahi et al. 2024; Ramos -Cabrer et al. 2025), OLs could be using myelin to buffer energetic stress. Although β-oxidation capacity of OLs is considered limited (Fünfschilling et al. 2012; Rao et al. 2017; Tepavčevic 2021) , there is evidence that suggests it is still functionally active (Hofmann et al. 2017). Importantly, in contrast .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted July 23, 2025. ; https://doi.org/10.1101/2025.07.18.665370doi: bioRxiv preprint 21 to previous studies, our experimental paradigm did not restrict glucose availability . Therefore, the observed effects are unlikely to be driven by a metabolic shift away from their preferentially glycolytic activity, but rath er as a response to excess lipid availability. Additionally, both lipids and proteins in myelin are known to exert signaling functions. Lipid receptors such as CPT1A (Tang et al. 2022) , sphingosine-1-phosphate receptor ( S1PR) (Jaillard et al. 2005) or Lysophosphatidic acid receptor 1 (LPAR1) (Nogaroli et al. 2009; Yung et al. 2015) play essential roles in regulating lipid homeostasis and myelination. These pathways may be activated upon myelin internalization and could contribute to the observed proliferative and morphological responses in OLs. In conclusion , our findings identify that OLs can internalize and respond to myelin de bris, suggesting that myelin debris itself is not intrinsically hindering the progression of the oligodendroglial lineage. Rather, detrimental effects associated with myelin debris may stem from concomitant OL damage. These results underscore the critical role of OL integrity during pathology. .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted July 23, 2025. ; https://doi.org/10.1101/2025.07.18.665370doi: bioRxiv preprint 22

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It is The copyright holder for this preprintthis version posted July 23, 2025. ; https://doi.org/10.1101/2025.07.18.665370doi: bioRxiv preprint 31 Figures and legends Control Myelin 0 2,000 4,000 6,000 8,000 10,000 Area MBP+ (m2/cell) ✱✱✱✱ OPCs OLs 0 20 40 60 80 100 Cell internalization of exogenous myelin (%) Cells with internalized myelin Cells without internalized myelin ✱✱✱✱ 0 20 40 60 80 100 0 20 40 60 Radial distance to center (m) Number of intersections Control Myelin YZ XZ OligodendrocytesOPCs YZ A B C D Alexa-labellingAdult rat brain Isolation of myelin 0h 12h 24h Myelin 6h ControlMyelin 30h 35h 40h 45h XZ Control Myelin 48hE F OligodendrocytesOPCs My PDGFRα Olig2 DAPI Myelin MBP DAPI Myelin PDGFRα DAPI My MBP Olig2 DAPI .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted July 23, 2025. ; https://doi.org/10.1101/2025.07.18.665370doi: bioRxiv preprint 32 Figure 1. Internalization of exogenous myelin by oligodendroglial cells . (A) Schematic illustrating the isolation and fluorescent labelling of myelin debris. ( B) Experimental design for evaluating myelin uptake. ( C) Orthogonal confocal views showing internalized exogenous myelin within oligodendrocytes (OLs) and oligodendrocyte precursor cells (OPCs). Scale bar: 10 µm. (D) Comparative analysis of myelin internalization between OPC and OL cultures. Scale bar: 20 µm. (E) Time-lapse imaging of OLs reveals increased morphological complexity and cell abundance in response to myelin exposure (top), and transport of internalized myelin along OL processes (bottom). Scale bar: 50 µm; zoomed -in panel: 20 µm. (F) Sholl analysis of OLs after 48 hours of myelin exposure. Scale bar: 20 µm. Area under the curve: Control, 1052 ± 78.7; Myelin, 2303 ± 139; ****p < 0.0001. Peak number of intersections: Control, 26.2; Myelin, 49.9. Each dot represents a single cell (n = 15). .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted July 23, 2025. ; https://doi.org/10.1101/2025.07.18.665370doi: bioRxiv preprint 33 Figure 2. Transcriptional profile of oligodendrocytes exposed to exogenous myelin debris. OLs were treated with Alexa -labelled myelin for 48 hours, from DIV 1 to 3, followed by transcriptomic analysis (n = 4 cultures). ( A) Principal component analysis (PCA) showing clear separation between treated and untreated samples. ( B) Volcano plot displaying significantly upregulated genes (red) and downregulated genes (blue) in OLs exposed to myelin compared to control conditions. Differentially expressed genes (DEGs) were defined by │fold change│ > 1.5 and adjusted p-value < 0.05. Representative DEGs are labelled. (C) Gene Set Enrichment Analysis (GSEA) of Hallmark pathways, showing the top 15 upregulated (red) and downregulated (blue) biological processes ranked by normalized enrichment score (NES). Significantly enriched pathways (FDR q-value < 0.25) are highlighted. Metabolic, differentiation-related and immune- related pathways are indicated in green, red, and blue, respectively. (D-E) Enrichment plots (left) for selected Hallmark gene sets with corresponding NES, FDR q -values, and Core Enrichment Genes (CEGs), defined as the percentage o f significantly enriched genes in the set. Heatmaps (right) show the top 15 most enriched genes for each pathway. (F) Enrichment plot for the “Lipid metabolic process” gene signature. -2 0 2 Normalized Enrichment Score DNA repair Adipogenesis Oxidative phosphorilation Xenobiotic metabolism Hypoxia UV response Fatty acid metabolism Apical junction Myogenesis Wnt β-catening signaling Bile acid metabolism TGFβ signaling Apical surface Hedgehog signaling Notch signaling Angiogenesis PI3K AKT MTOR signaling MTORC1 signaling Cholesterol homeostasis Coagulation Complement KRAS signaling IL6 JAK STAT3 signaling Allograft rejection IL2 STAT5 signaling Pancreas β cells Interferon α response Interferon γ response Inflammatory response TNFα signaling via NFKβ D A PCA Dim 1 (30.6%) 0 50-50 0 50 -50 Dim 2 (27.4%) B C GSEA - Hallmarks -1 0 1 0 2 4 Log2FoldChange -Log10padj Abca1 Abcg1 IL6 Slc43a3 Cldn10 St8sia3 Control Myelin INFLAMMATORY RESPONSE Trib3 Plaur Atf5 Fads2 Mvd Acss2 Fabp5 Fasn Scd Acat2 Ebp Pcyt2 Cxcl16 Pmvk Sema3 NES = -1.2941 FDR q-value = 0.2337 LIPID METABOLIC PROCESSCHOLESTEROL HOMEOSTASIS MyelinControl FE Clec5a Ccl22 Edn1 Tnfrsf9 Il18rap Ccrl2 Csf3 Il6 Inhba Il10 Scarf1 Ptger2 Lta Lif Ccl5 MyelinControl NES = -1.0832 FDR q-value = 0.1754 NES = -1.8736 FDR q-value = 0.0005 Aoah Dgkb Etfbkmt Far2 Ptgr1 Gal3st4 Rbp3 Aldh3a1 St8sia3 Acsm3 Dpep1 Slc27a6 Adh1c Neu4 Fmo5 MyelinControl CEG = 17.97% CEG = 46.75% CEG = 50.75% .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted July 23, 2025. ; https://doi.org/10.1101/2025.07.18.665370doi: bioRxiv preprint 34 Figure 3. Lipid transport and storage following myelin internalization in oligodendrocytes. OLs were cultured with Alexa Fluor 488 -labeled myelin for 48 hours. Myelin internalization led to increased formation of lipid droplets, visualized by Oil Red O staining, which labels neutral lipids. Scale bar: 10 µm. *p < 0.05. Control Myelin 0 200 400 600 800 1,000 1,200 Oil Red+ area (m2/cell) ✱ DAPIMerge Oil Red Myelin ControlMyelin .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted July 23, 2025. ; https://doi.org/10.1101/2025.07.18.665370doi: bioRxiv preprint 35 Figure 4. Exogenous myelin promotes oligodendroglial lineage progression . (A) Schematic of the experimental design. (B) Calcein-AM viability assay showing fluorometric values normalized to control mean. ( C) Immunocytochemical (ICC) analysis of OL cultures indicates that myelin exposure increases the number of oligodendrocytes (Olig2⁺), associated with enhanced proliferation (Ki67⁺). (D) This increase results in higher numbers of myelinating oligodendrocytes (MBP⁺) and oligodendrocyte precursor cells (NG2⁺), without affecting the proportion of progenitors to mature OLs. ( E) Enhanced myelination of nanofibers by myelin -exposed OLs, reflected by an increased cellular area. Scale bars: A–C 50 µm; D-E 25 µm. *p < 0.05, **p < 0.01, ***p < 0.001 Control Myelin 0 10 20 30 40 NG2+ cells/mm2 ✱ Control Myelin 0 100 200 300 400 500 MBP+ cells/mm2 ✱✱ Control Myelin 0 200 400 600 800 1,000 1,200 Olig2+ cells/mm2 0.0802 ControlMyelin C A Control MyelinB D MBP NG2 Myelin DAPI MergeMyelin Olig2 Calcein-AM ControlMyelin Ki67 Control Myelin 0 10 20 30 40 Ki67+ Olig2+/ Olig2+ (%) ✱ Control Myelin 0 100 200 300 Cell viability (%) ✱✱✱ MBP Myelin Olig2 ControlMyelin Control Myelin 0 20 40 60 80 100 MBP or NG2 / MBP + NG2 (% of cells) OLs OPCs 0.9858 Control Myelin 0 20 40 60 80 100 MBP or NG2 / MBP + NG2 (% of cells) OLs OPCs 0.9858 E Control Myelin 0 1,000 2,000 3,000 MBP+ area (µm2/cell) ✱ .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted July 23, 2025. ; https://doi.org/10.1101/2025.07.18.665370doi: bioRxiv preprint 36 Figure 5. Internalization of Alexa Fluor 488 -labeled myelin debris in adult mice following stereotaxic injection. (A) Cortical section showing microglial migration to the injection site 24 hours post-surgery, resulting in a cell -free gap around the injecti on core indicated by a dotted line. Scale bars: 300 µm (main), 100 µm (zoom). ( B) Repopulation of surrounding areas by microglia 48 hours after injection. Scale bar: 50 µm. (C) Formation of phagocytic pouches within microglia actively internalizing myelin debris. Scale bar: 10 µm. (D,F,H) Immunogold labelling of Alexa Fluor 488 -labeled myelin (white arrows) confirms internalization by microglia ( D), OPCs (F), and oligodendrocytes (H), as observed by transmission electron microscopy (TEM). Microglia exhibit large vesicles (white), indicative of professional phagocytosis, whereas oligodendroglia show more diffuse internalized structures. Scale bar: 1 µm. ( E,G) Internalization of exogenous myelin by OPCs (E) and OLs (G) in vivo. Scale bars: 20 µm (main), 10 µm (zoom). (I) Quantification of proliferating cells at the injection site 48 hours after injection in both sham - and myelin- injected animals in a radius of 300 µm around the lesion site . A dashed line indicates basal proliferation levels in a corresponding a rea of the contralateral hemisphere. No significant differences in basal proliferation were detected. Each dot represents an individual injection. Sham Myelin 0 100 200 300 400 500 Ki67+ cells / mm2 0.1999 A MBP Iba-1 Myelin B C D I Iba-1 Myelin DAPI Ki67 Control Myelin Injected hemisphere Contralateral hemisphere OPCs OLs Microglia E E F G H PDGFRα Myelin DAPI Injected hemisphere Contralateral hemisphere APC Myelin DAPI Ki67 Myelin .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted July 23, 2025. ; https://doi.org/10.1101/2025.07.18.665370doi: bioRxiv preprint 37 Figure 6. Oligodendrocytes internalize exogenous myelin in zebrafish. (A) Schematic representation of cereb roventricular injections of Alexa Fluor -labelled myelin performed at 3 days post-fertilization (dpf) in Tg(mbp:EGFP-CAAX) zebrafish, which express membrane-bound GFP in oligodendrocytes. (B) Confocal imaging of the spinal cord 2 hours post -injection shows the diffusion of fluorescent myelin into the central canal in the anterior region. (C–D) 24 hours post-injection, myelin signal is largely cleared from the canal, but a small fraction of exogenous myelin remains visible within the somata ( C) and processes (D) of oligodendrocytes. (E) Representative spinal cord images from Tg(mbp:EGFP) (somatic fluorescence in OLs) and Tg(Olig1:nls-mApple) (nuclear marker for OPCs) zebrafish at 24 h post -injection, with quantification of myelin-positive cells. Scale bar = 10 µm. ControlMyelin Myelin MBP A B C XZMyelin MBP E XZ YZ YZ Myelin MBP Myelin MBP ControlMyelin Myelin Olig1 Dorsal Ventral F D ControlMyelin Control Myelin 0 5 10 15 Dorsal OLs OLs / 100m Control Myelin 0 5 10 15 20 Ventral OLs OLs / 100m ✱ Control Myelin 0 10 20 30 40 Total OLs OLs / 100m Control Myelin 0 10 20 30 40 Total OPCs OPCs / 100m 0.0607 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted July 23, 2025. ; https://doi.org/10.1101/2025.07.18.665370doi: bioRxiv preprint