Article Figures & Data
Figures
- Figure 1.
LPC 18:1 protects against hyperdeimination of MBP in vitro. A, UK Biobank International Classification of Diseases PheWeb Analysis points toward genes associated with ocular manifestations of MS (UK BioBank: Multiple Sclerosis, accession number, 335; Optic Neuritis, accession number, 377.3). Gene expression analysis during different myelin conditions points toward elevated levels of LPGAT1 and LIPC in demyelinating conditions [GEO (Gene Expression Omnibus) accession number: GSE19403]. *Higher levels of these enzymes would metabolize LPC 18:1, resulting in lower levels of LPC 18:1. B, Genes reported to have been associated in MS and NMO using the DisGeNET database [Multiple Sclerosis, concept unique identifier (CUI), C0026769; and Neuromyelits Optica, CUI C0027873]. Genes were curated for genes involved in phospholipid metabolism and subsequently compared for genes shared between MS and NMO. Shared genes were associated with plasma membrane synthesis. *Phospholipid profile comparison was taken from our previous study (Valdivia et al., 2020). ON, Optic nerve. Based on our previous study LPC 18:1 is the only lipid to protect against aberrant hyperdeimination. C, Structural difference between LPC 18:0 and LPC 18:1. Arrow indicates the position of unsaturation in the acyl chain of LPC 18:1. D, Dot blot analysis of levels of deimination in EAE brain and spinal cord (S.C.). Levels of deimination increase as the EAE clinical score increases (brain score 0, one-way ANOVA n.s.; brain score 1, one-way ANOVA n.s.; brain score 2, one-way ANOVA n.s.; brain score 3, one-way ANOVA, **p ≤ 0.01; S.C. score 0, one-way ANOVA n.s.; S.C. score 1, one-way ANOVA, **p ≤ 0.01; S.C. score 2, one-way ANOVA, **p ≤ 0.01; S.C. score 3, one-way ANOVA, **p ≤ 0.01). Citrulline, Millipore-MABS54887. n = 3. E, Mass spectrometry analysis of mouse and human MBP isoform 5 deimination reaction in the presence of LPC 18:0 and LPC 18:1. Deiminated cites, Bold-red arginine (R). F, Mass spectrometry analysis of mouse MBP isoform 5 deimination reaction in the presence of LPC 18:0 and LPC 18:1 and lipid rafts isolated from C57BL/6J WT or EAE mouse brain. Deiminated cites, Bold-red arginine (R). See Extended Data Figure 1-1.
- Figure 2.
LPC 18:1 optic nerve injections improve visual function in EAE mice. A, Surgical procedure for ON injections. Image created using BioRender. B, Dose–response curve analysis for determining LPC 18:1 dose: 150 μm was the inflection point at which the control lipid LPC 18:0 decreased visual function while LPC 18:1 retained normal visual function. C, Visual function analysis using PERG for optic nerve injections determined that 150 μm LPC 18:1 does not affect visual function (****p ≤ 0.0001; one-way ANOVA, ****p ≤ 0.0001). D, PERG waves demonstrate that 150 μm optic nerve injection of LPC 18:1 does not affect visual function 4 days-post-injection (DPI). E, Immunohistochemistry of C57BL/6J WT animals injected with LPC 18:0 and LPC 18:1 at 150 μm. MBP, Abcam, ab7349. Blue, DAPI; green, MBP. F, Immunohistochemistry of C57BL/6J WT animals injected with 150 μm Cy5-LPC 18:1 1 h postinjection (HPI) and 4 DPI. MBP: catalog #ab7349 Abcam. Blue, DAPI; green, MBP; pink, Cy5-LPC 18:1. G, Dot blot analysis of levels of deimination in the C57BL/6J-MOG35-55 EAE optic nerves injected with either LPC 18:0 or LPC 18:1. Levels of deimination are attenuated when injected with LPC 18:1 (***p ≤ 0.001; one-way ANOVA, ***p ≤ 0.001). Citrulline: MABS54887, Millipore. H, PERG analysis of LPC 18:1 optic nerve injections in the C57BL/6J-MOG35-55 EAE animals at a clinical score of 2. The duration of the PERG measurements stopped 12 d post-LPC 18:1 optic nerve injection (DPI) when tissue was harvested. PERG analysis at 12 DPI demonstrates a statistical significance among all of the groups (***p ≤ 0.001; ****p ≤ 0.0001; one-way ANOVA, ****p ≤ 0.0001). I, Composition of animals in each of the groups. J, Flash ERG analysis demonstrates that the loss in visual function is not because of changes in inner retinal activity (photoreceptors) n.s., No-significance. K, Immunohistochemistry of EAE animals injected with LPC 18:1 at 150 μm demonstrates increased myelination in the treated groups. MBP, catalog #ab7349, Abcam. Blue, DAPI; green, MBP. L, PERG analysis of LPC 18:0 optic nerve injections in the C57BL/6J-MOG35-55 EAE animals at a clinical score of 2. Duration of the PERG measurements stopped 12 d post-LPC 18:1 optic nerve injection (DPI) when tissue was harvested. M, PERG analysis of LPC 18:1 optic nerve injections in the SJL/J- PLP139-151 EAE animals at a clinical score of 2. The duration of the measurements stopped 12 DPI when tissue was harvested. PERG analysis at the 12 DPI demonstrates a statistical significance between the two groups (****p ≤ 0.0001; one-way ANOVA, ****p ≤ 0.0001). See Extended Data Figures 2-1, 2-2, and 2-3.
- Figure 3.
LPC 18:1 optic nerve injections promote remyelination in EAE mice. A, Multivariable dimension reduction analysis (principal component analysis, left panel) and bilinear factor model analysis (partial least-squares discriminant analysis, right panel) demonstrating the separation of samples into four distinct groups. NI, No-injection control; LR, low-recovery group; HR, high-recovery group; SN, stay-normal group. B, Quantitative iTRAQ mass spectrometry analysis of EAE optic nerves injected with LPC 18:1 (no-injection control, low-recovery, high-recovery, and stay-normal groups). One-way ANOVA demonstrating 88 significant proteins. Adjusted p-value cutoff = 0.001; post hoc analysis with Fisher’s LSD. C, Correlation pattern analysis demonstrating the top 10 proteins that either increase (red) or decrease (blue) starting with the no-injection control group and progressively moving toward the SN group. Distance measure: Pearson R. D, Reactome pathway analysis of proteins that are shared between the ANOVA and correlation pattern analysis (50 proteins for positive correlation and 38 proteins for negative correlation). Reactome pathways interactors were excluded, pathway p-value ≤ 0.05. E, Western blot analysis demonstrating a negative correlation between oligodendrocyte progenitor cell markers and mature oligodendrocyte markers (PDGFR-α: one-way ANOVA, ***p ≤ 0.001; MBP: one-way ANOVA, ***p ≤ 0.001; MOG: one-way ANOVA, ***p ≤ 0.001). PDGFR-α, catalog #ab134123, Abcam; MBP: catalog #ab7349 Abcam; MOG, catalog #ab28766, Abcam. n = 3. NI, No-injection control group; LR, low-recovery group; HR, high-recovery group; SN, stay-normal group. F, Immunohistochemistry of EAE animals injected with LPC 18:1 at 150 μm demonstrating mature oligodendrocyte localization (yellow arrowhead). MBP: catalog #ab7349, Abcam; OSP: catalog #ab7474, Abcam. Blue, DAPI; green, MBP; red, OSP. G, Dose curve analysis for determining histidine dose: 10 μm was the concentration used for C13-histidine + LPC 18:1 EAE optic nerve injections. H, Mass spectrometry C13-histidine + LPC 18:1 EAE optic nerve injections demonstrating the incorporation of isobaric C13-histidine in myelin basic protein. See Extended Data Figures 3-1, 3-2, 3-3, 3-4, and 3-5.
- Figure 4.
Oligodendrocyte maturation is mediated by LPC 18:1. A, Rat oligodendrocyte progenitor cells culture depicts with typical OPC morphology (bright field). Arrows point toward OPCs, inlet panel represents the area in dotted square at a higher magnification. B, Immunocytochemistry of rat OPCs treated with LPC 18:1 or LPC 18:0 at 10 μm, and C13-Histidine at 10 μm demonstrating LPC 18:1-mediated oligodendrocyte maturation. MBP: catalog #ab7349, Abcam; OSP: catalog #ab7474, Abcam. Blue, DAPI; green, MBP; red, OSP. Scale bar, 25 μm. C, Reactome pathway analysis of proteins labeled with C13-histidine in the LPC 18:1 and LPC 18:0 OPC-treated groups. Reactome pathway interactors were excluded, pathway p-value ≤ 0.05. D, OPC groups incubated with Cy5-LPC 18:1 at different time points followed by UV cross-linking. PhastGel identification of proteins crossed linked with Cy5-LPC 18:1 (pink band). E, Relative abundance of LPC 18:1 in rat OPC, rat oligodendrocyte and mouse retinal ganglion cell cultures transfected with siRNAs. LPCAT1: catalog #s102346, Ambion; PLA2G4C: catalog #s107315, Ambion; LIPC: catalog #s67780, Ambion. ±SEM; *p ≤ 0.05, **p ≤ 0.01. F, Relative abundance of LPC 18:1 rat OPC, rat oligodendrocyte, and mouse retinal ganglion cell cultures transfected with overexpression constructs. LPGAT1: catalog #Ex-mm15104-M61, Genecopoeia; LIPC: catalog #Ex-mm03119-M61, Genecopoeia. ±SEM; *p ≤ 0.05, **p ≤ 0.01. G, OPC cell cultures treated with exogenous LPC 18:1 or LPC 18:0 followed by cellular fractionation. SNX4: catalog #ab198504, Abcam; PPAR-δ: catalog #ab23673, Abcam; CHD7: catalog #6505S, Cell Signaling Technology; PCLO: catalog #ab20664, Abcam. EF, Extracellular fraction; CF, cytosolic fraction; NF, nuclear fraction. H, Nuclear fraction electrophoretic mobility shift assay (EMSA) of OPC cell cultures treated with exogenous LPC 18:1 or LPC 18:0. I, protein–lipid overlay assay demonstrating the binding of selected proteins with LPC 18:1 and LPC 18:0. SNX4: catalog #ab198504, Abcam; PPAR-δ: catalog #ab23673, Abcam; CHD7: catalog #6505S, Cell Signaling Technology; PCLO: catalog #ab20664, Abcam. J, Proposed model for LPC 18:1-mediated remyelination through oligodendrocyte maturation. See Extended Data Figures 4-1, 4-2, 4-3, 4-4, 4-5, and 4-6.
Extended Data
Figure 1-1
Lipid raft isolation from mouse spinal cord and brain tissue. A, EAE clinical score graph demonstrating the progression of clinical symptoms. LPC 18:1 injections were delivered to EAE animals when they reached a score of 2. B, Immunoprecipitation of MBP from BL21 cells. MBP: catalog #ab7349, Abcam. MBP isoform 5 from both mouse and human were isolated. P, Pellet; W, wash discard; E, eluent. C, Gradient setup for isolation of lipid rafts using sucrose density centrifugation. Sucrose fractions were collected in 18 fractions, but only 1–15 were used as fractions 16–18 are not typically considered raft fractions. D, Western blot analysis of sucrose fractions 1–15 demonstrates that only fractions 1 and 2 of brain tissue are (+) for Flot1 and (–) for TfR. *This criterion is typical for lipid raft identification. E, Total protein quantification and total cholesterol quantification for lipid raft fraction. Lipid rafts typically have low protein content and high cholesterol content, supporting the presence of lipid rafts in sucrose fractions 1 and 2. Download Figure 1-1, TIF file.
Figure 2-1
EAE induction and optic nerve injection timeline. Animals were injected intraperitoneally with pertussis toxin (catalog #181, Biological Laboratories) a day before and a day after immunization with MOG35-55 (catalog #12668-05, BioSynthesis) emulsified in complete Freund’s adjuvant. Visual function was assessed using PERG. Animals were monitored for the development of EAE clinical scores until they reached an EAE score of 2, followed by optic nerve injections of LPC 18:1 or LPC 18:0 or PBS. Animal visual function was monitored 4, 6, 8, 10, and 12 d post-optic nerve injection (DPI). Optic nerves were harvested the day after and processed for further analysis. Download Figure 2-1, TIF file.
Figure 2-2
Immunohistochemistry (IHC) of optic nerves in WT and EAE mice. A, Optic nerve of C57BL/6J WT mice injected with either LPC 18:0 or LPC 18:1 at 150 μm. MBP: catalog #ab7349, Abcam. Blue, DAPI; green, MBP. B, Optic nerve of C57BL/6J WT mice injected with LPC 18:1-Cy5 at 150 μm. Tissue was harvested 1 h post injection (HPI) and 4 DPI. MBP: catalog #ab7349, Abcam. Blue, DAPI; green, MBP; pink, Cy5-LPC 18:1. C, Optic nerve of EAE mice injected with LPC 18:1 at 150 μm. Tissue was harvested 12 d post-LPC 18:1 injection (DPI). MBP, catalog #ab7349 Abcam. Blue, DAPI; green, MBP. Download Figure 2-2, TIF file.
Figure 2-3
Electron microscopy of EAE optic nerves injected with LPC 18:1. Left, Representative coronal cross section for WT optic nerves (noninjected). Middle, Representative optic nerve for EAE mice in the high-recovery group (injected with LPC 18:1). Right, Representative optic nerve for EAE mice in the no-injection group. Yellow arrow points towards myelin sheath (dark rings). Myelin is observed to be compacted in the left and middle panels, whereas its less compact in the right panel. Download Figure 2-3, TIF file.
Figure 3-1
Isolation of myelin proteins from EAE optic nerves injected with isobaric C13-histidine. A, Schematic experimental setup for isobaric optic nerve injections. Only newly synthesized protein should incorporate isobaric C13-histidine in its sequence. B, Sucrose gradient ultracentrifugation of optic nerves (one optic nerve per tube). Myelin fraction is localized to the top of the gradient. C, Validation of the presence of myelin proteins in myelin fraction of the sucrose gradient ultracentrifugation. MBP: catalog #ab7349, Abcam; MOG: catalog #ab28766, Abcam. D, MBP enrichment for mass spectrometry analysis. MBP: catalog #ab7349, Abcam. E, Mass spectrometry spectra for demonstrating incorporation of C13-histidine into MBP peptide sequence. Download Figure 3-1, TIF file.
Figure 3-2
LPC 18:1 optic nerve injections promote myelination and neuroprotection. Proteins identified using the liposome flotation assay complex directly with micelles made from LPC 18:1. Proteins labeled during the isobaric C13-histidine experiment are proteins that are newly synthesized as a consequence of LPC 18:1 signaling. iTRAQ analysis identifies proteins that have a gradual increase in abundance in the following pattern: No-Injection → Low-Recovery → High-Recovery → Stay-Normal. Cell-specific protein expression was referenced using the EMBL-EBI Expression Atlas database. Reactome pathway analysis demonstrated protective and beneficial pathways being activated. Download Figure 3-2, TIF file.
Figure 3-3
iTRAQ NI-LR-HR-SN. Download Figure 3-3, DOC file.
Figure 3-4
Isobaric C13-histidine. Download Figure 3-4, DOC file.
Figure 3-5
Proteins complexing with LPC 18:1 and LPC 18:0 micelles. Download Figure 3-5, DOC file.
Figure 4-1
LPC 18:1 oligodendrocyte progenitor cell differentiation experimental setup. Experimental setup for rat OPC cultures. OPC were plated at 3 × 104 cells per well/chamber (10 μl for 96-well plate and 30 μl for slide chamber). OPCs were incubated at 37°C for 24 h (incubated >24 h if cells were not fully attached). Three different experimental setups included the following: (1) plating and exposing OPCs to LPC 18:1-Cy5 (10 μm) followed by UV cross-linking, cell lysis, and PhastGel separation; (2) plating and exposing OPCs to differentiation supplement as a positive control, LPC 18:0 (10 μm), LPC 18:1 (10 μm), and PAD followed by cell lysis and mass spectrometry analysis. Only the treated groups included isobaric C13-histidine; and (3) plating and exposing OPCs to LPC 18:0 (10 μm), LPC 18:1 (10 μm), and PAD followed by fixation using 4% paraformaldehyde (PFA) and imagining. Download Figure 4-1, TIF file.
Figure 4-2
Rat OPCs treated with LPC 18:1. A, Immunohistochemistry of rat OPCs treated with LPC 18:1. MBP: catalog #ab7349, Abcam; OSP: catalog #ab7474, Abcam. Blue, DAPI; green, MBP; red, OSP. Scale bar, 25 μm. B, Rat OPC cell culture treated with LPC 18:1 (bright field). Download Figure 4-2, TIF file.
Figure 4-3
Lyso-lipid metabolic enzymes mediate levels of LPC 18:1 experimental setup. A, Experimental setup for silencing of LPC metabolic enzymes via siRNA. Rat OPCs and mouse RGCs were plated at 3 × 104 cells/well/chamber (10 μl for 96-well plate and 30 μl for slide chamber). OPCs and RGCs were incubated at 37°C for 24 h (incubated >24 h if cells were not fully attached). OPCs were either differentiated into oligodendrocytes through the addition of differentiation supplement. OPCs, oligodendrocytes and RGCs were transfected with siRNAs for LPCAT1, PLA2G4C, and LIPC followed by cell lysis and mass spectrometry lipidomic analysis for LPC 18:1 levels. B, The same experimental setup as described above was implemented with the exception of cell transfection. Cells were transfected with an overexpression construct for LPGAT1 and LIPC. Cells were lysed and analyzed by mass spectrometry for levels of LPC 18:1. Download Figure 4-3, TIF file.
Figure 4-4
Cellular fractionation and identification of proteins of interest experimental setup. Experimental setup for rat OPC cultures. OPCs were plated at 3 × 104 cells/well (10 μl for 96-well plate). OPCs were incubated at 37°C for 24 h (incubated >24 h if cells were not fully attached) followed by treatment with differential supplement control, LPC 18:0 (10 μm) or LPC 18:1 (10 μm). Cells were incubated for 24 h followed by media collection (extracellular fraction) and NE-PER cellular fractionation. Cellular fractions were analyzed using Western blot for the presence of proteins of interest (SNX4, PPAR-δ, CHD7, and PCLO). The nuclear fraction was further analyzed via electrophoretic mobility shift assay. Download Figure 4-4, TIF file.
Figure 4-5
OPC cell culture with siRNA and overexpression construct. A, Representative cell culture morphology for rat OPCs treated with siRNA, rat oligodendrocytes (OL) treated with siRNA and mouse RGCs treated with siRNA. B, Representative cell culture morphology for rat OPCs treated with overexpression construct, rat oligodendrocytes (OLs) treated with overexpression construct and mouse RGCs treated with overexpression. OPC and OL cultures were validated using mRNA and protein analyses following published articles (Jäkel et al., 2019). Download Figure 4-5, TIF file.
Figure 4-6
Proposed model for LPC 18:1-mediated immune response. Top left model depicts the levels of protein (P) and deimination (D) during healthy conditions. Both levels are equal. Bottom left model depicts the levels of P and D during multiple sclerosis. Protein levels are stable; however, the levels of deimination increase. A, Increase in deimination and the presence of LPC 18:1 permit the development of oligodendrocyte progenitor cells into mature oligodendrocytes. This process is permissible for myelination. B, During myelination, MBP maintains baseline levels of deimination and a close conformation. This close conformation allows for the formation of a stable compact myelin sheath. C, In the absence of LPC 18:1, oligodendrocyte progenitor cells cannot fully differentiate, which leads to an accumulation of aberrant hyperdeimination. Increased hyperdeimination on MBP is associated with priming of the immune system. D, Aberrant hyperdeimination of MBP is also associated with an open conformation, which makes MBP more susceptible to proteolysis. This increase in proteolysis depletes MBP from the myelin sheath, making it unstable and susceptible to demyelination. All of these events contribute to the autoimmune response observed in multiple sclerosis. Download Figure 4-6, TIF file.
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