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. 2014 Sep 25;9(9):e108646.
doi: 10.1371/journal.pone.0108646. eCollection 2014.

Myelin basic protein induces neuron-specific toxicity by directly damaging the neuronal plasma membrane

Jie Zhang  1 , Xin Sun  2 , Sixin Zheng  1 , Xiao Liu  1 , Jinghua Jin  1 , Yi Ren  3 , Jianhong Luo  1
Affiliations

Myelin basic protein induces neuron-specific toxicity by directly damaging the neuronal plasma membrane

Jie Zhang et al. PLoS One. .

Abstract

The central nervous system (CNS) insults may cause massive demyelination and lead to the release of myelin-associated proteins including its major component myelin basic protein (MBP). MBP is reported to induce glial activation but its effect on neurons is still little known. Here we found that MBP specifically bound to the extracellular surface of the neuronal plasma membrane and induced neurotoxicity in vitro. This effect of MBP on neurons was basicity-dependent because the binding was blocked by acidic lipids and competed by other basic proteins. Further studies revealed that MBP induced damage to neuronal membrane integrity and function by depolarizing the resting membrane potential, increasing the permeability to cations and other molecules, and decreasing the membrane fluidity. At last, artificial liposome vesicle assay showed that MBP directly disturbed acidic lipid bilayer and resulted in increased membrane permeability. These results revealed that MBP induces neurotoxicity through its direct interaction with acidic components on the extracellular surface of neuronal membrane, which may suggest a possible contribution of MBP to the pathogenesis in the CNS disorders with myelin damage.

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Conflict of interest statement

Competing Interests: The authors have declared no competing interests exist.

Figures

Figure 1
Figure 1. MBP is toxic to primary hippocampal neurons in vitro.
(A) Morphology of MBP-treated neurons. Primary cultured neurons with or without eGFP overexpression (green) were incubated with 50 µg/mL MBP for 24 h. HBSS was used as control. Degeneration of soma and neurites is indicated with arrows. Scale bar, 50 µm. (B) Neuronal degeneration induced by 24-h MBP incubation at the indicated concentrations. Details are shown in the enlarged images. Scale bar, 100 µm. (C) Time-lapse imaging of neurons incubated with HBSS or 50 µg/mL MBP. Normal morphology is indicated by arrows, and degeneration by arrowheads. Scale bar, 100 µm. (D) Neuronal death induced by 24-h MBP incubation at the indicated concentrations, assessed by DAPI/PI double-staining. Dead cells are PI-positive. Scale bar, 100 µm. (E) Statistical analysis of (D). Incubation with 30 and 50 µg/mL MBP induced cell death (***P<0.001; ns, not significant; one-way ANOVA with Dunnett's post-test; n = 5). (F) Statistical analysis of PI-positive neurons after HBSS or 50 µg/mL MBP incubation for different times (**P<0.01, ***P<0.001; two-way ANOVA with Bonferroni's post-test; 6 h, n = 6; 12 h, n = 10; 24 h, n = 6). (G) Statistical analysis of PI-positive neurons after 24-h incubation with HBSS, mouse IgG (mIgG), BSA or MBP (50 µg/mL each). In contrast to MBP, no cell death was induced by mIgG or BSA (***P<0.001; ns, not significant; one-way ANOVA with Dunnett's post-test; n = 5). Data are mean ± SEM.
Figure 2
Figure 2. Toxic effect of MBP is neuron-specific.
Primary astrocytes, microglia, oligodendrocytes and the endothelial cell line bEND.3 were incubated with HBSS or 50 µg/mL MBP for 24 h. No cell death was indicated by DAPI/PI double-staining. Scale bar, 100 µm.
Figure 3
Figure 3. MBP binds to the extracellular surface of neuronal plasma membrane.
(A) Neurons were incubated with HBSS, 10 µg/mL or 50 µg/mL MBP for 5 min, and washed for three times with HBSS, 5 min each time. The cells were fixed in 4% paraformaldehyde without permeabilization and stained for surface-bound MBP on plasma membrane (green). Nuclei were indicated by DAPI (blue). General morphology of neurons was observed by differential interference contrast (DIC) microscopy. MBP was shown to bind to the extracellular surface on both soma (arrowheads) and neurites (arrows). Scale bar, 50 µm. (B) Primary astrocytes, microglia, oligodendrocytes and the endothelial cell line bEND.3 were incubated with HBSS or 50 µg/mL MBP for 30 min, washed with HBSS for three times, 5 min each time, and then stained for MBP surface binding (without permeabilization). No surface-bound MBP was detected (green). GFAP and CD11b were used as markers of astrocytes and microglia, respectively (red). Scale bar, 50 µm. (C) Primary microglia were incubated with 50 µg/mL MBP for the indicated times and the intracellular distribution of MBP was shown by staining with permeabilization (green). Iba1 (red) was used as a microglial marker. Scale bar, 50 µm. Similar results were obtained in three independent experiments.
Figure 4
Figure 4. Surface binding and toxicity of MBP rely on polypeptide basicity.
(A) MBP surface binding on neurons after 30-min pre-incubation in ECS at pH 7.4 or pH 10.8 (equal to the isoelectric point of MBP) and subsequent treatment with 10 µg/mL MBP for 5 min. MBP surface binding was abolished in ECS at pH 10.8. Scale bar, 50 µm. (B) MBP surface binding on neurons after 30-min pre-incubation with 5 µg/mL PRM or 50 µg/mL BSA and subsequent 5-min treatment with 10 µg/mL MBP. Surface binding of MBP was blocked by pre-incubation of basic protein PRM. Scale bar, 50 µm. (C) MBP surface binding on neurons after 5-min incubation with 10 µg/mL MBP alone, or MBP-lipid mixtures. To obtain the MBP-lipid mixtures, 5 µM phosphatidylinositol (PtdIns, an acidic lipid) or 1000 µM phosphatidylethanolamine (PE, a neutral lipid) were pre-mixed with 10 µg/mL MBP for 30 min. Surface binding of MBP was blocked by the PtdIns pre-mixing. Scale bar, 50 µm. (D) Neurons were incubated with 50 µg/mL MBP alone or a MBP-PtdIns mixture (5 µM PtdIns with 50 µg/mL MBP) for 24 h. No neuronal degeneration was found by morphology with MBP-PtdIns treatment. Scale bar, 50 µm. (E) Statistical analysis of neuronal death by DAPI/PI double-staining after treatment in (D) (***P<0.001; one-way ANOVA with Dunnett's post-test; n = 5). (F) Binding of MBP with the neuronal P2 fraction. The P2 fraction, with or without trypsin pre-treatment (0.25%, 37°C for 1 h), was incubated with 10 µg of MBP for 30 min, washed and then assessed for MBP association by western blot. MBP was shown to bind with the P2 fraction independent of trypsinization (ns, not significant; unpaired, two-tailed t-test; n = 3). Data are mean ± SEM.
Figure 5
Figure 5. MBP depolarizes the resting membrane potential.
(A) Representative neuronal resting membrane potentials (RMPs) recorded before and after HBSS or 50 µg/mL MBP treatment in current-clamp mode. (B) Statistical analysis of RMP changes. Compared to those in HBSS treatment (ns, not significant; paired, two-tailed t-test; n = 10), neuronal RMPs were depolarized after MBP treatment (***P<0.001; paired, two-tailed t-test; n = 11).
Figure 6
Figure 6. MBP induces cation influx.
(A) Ca2+ influx in neurons measured by Fluo-4 fluorescence. Fluo-4-loaded neurons were treated as indicated and monitored for 90 sec after 30 sec of baseline recording. The representative images are shown before (27 sec) and after (96 sec) drug administration. Scale bar, 25 µm. Intracellular Ca2+ was increased by 600 µM glutamate (n = 20) or 50 µg/mL MBP (n = 21). “Glut+Inhi” (n = 18) and “MBP+Inhi” (n = 21) represent the presence of APV (100 µM), CNQX (20 µM) and nimodipine (10 µM) during treatment. “MBP+0 Ca” (n = 17) represents neurons incubated with MBP in a Ca2+-free solution. (B) Time course of the Ca2+ increase (Fluo-4 F/F0) in different conditions. Note that a plateau was reached shortly after treatment. (C) Time course of Zn2+ influx (FluoZin F/F0) in neurons treated with 50 µg/mL BSA (n = 38), 50 µg/mL MBP (n = 40) or 600 µM glutamate (n = 26) in the presence of 10 µM external Zn2+. (D) Statistical analysis of Ca2+ increase after treatment with HBSS (n = 17), 50 µg/mL MBP (n = 24), PRM (n = 23) or BSA (n = 21) (***P<0.001; ns, not significant; one-way ANOVA with Dunnett's post-test). (E) Statistical analysis of neuronal death by DAPI/PI double-staining after 24-h incubation with 50 µg/mL MBP in the presence of APV (100 µM), MK801 (40 µM), CNQX (20 µM), nimodipine (10 µM) or EGTA (1 mM) (***P<0.001; unpaired, two-tailed t-test; n = 4). Data are mean ± SEM.
Figure 7
Figure 7. MBP interferes with the fluidity of the neuronal plasma membrane.
(A) Hippocampal neurons expressing pDisplay-GFP plasmid were treated with HBSS (n = 31) or 50 µg/mL MBP (n = 41) and then subject to FRAP assays. Representative images showing membrane-bound GFP were acquired before bleaching (pre-bleach) and 0.4, 4, 8, 12 and 16 sec after bleaching (post-bleach), with arrows indicating the photobleached areas. Scale bar, 5 µm. (B) Recovery curves measured by GFP intensity in (A). (C) Diffusion constant of the FRAP curve (***P<0.001; unpaired, two-tailed t-test). (D) Fluorescence recovery time t1/2 of the FRAP curve (***P<0.001; unpaired, two-tailed t-test). Data are mean ± SEM.
Figure 8
Figure 8. MBP damages neuronal membrane integrity.
(A) Damage to the neuronal plasma membrane was assessed by the intracellular presence of living cell-impermeable calcein. Neurons were pre-treated with HBSS, 50 µg/mL BSA, PRM or MBP for 30 min and then incubated with calcein for additional 10 min. Details are shown in the timeline. Scale bar, 100 µm. (B) Plasma membrane damage after pre-treatment with 50 or 100 µg/mL MBP for 0, 10, 30 and 60 min. Scale bar, 100 µm. Note that neurons were still treated with MBP for 10 min in the calcein step of the “0 minute” group. Similar results were obtained in three independent experiments.
Figure 9
Figure 9. MBP at a high concentration induces the loss of cellular contents.
(A) and (B) Representative images of hippocampal neurons pre-loaded with calcein-AM (cell-permeable calcein), before and after 2000 µg/mL BSA, 2000 µg/mL MBP or 1000 µg/mL PRM incubation. MBP and PRM incubation induced significant loss of intracellularly loaded calcein. Scale bar, 100 µm.
Figure 10
Figure 10. MBP directly disrupts the integrity of the lipid bilayer in vitro.
(A) and (B) Release of calcein from calcein-loaded liposomes after 50 µg/mL BSA or MBP treatment. Liposomes were prepared from the indicated components. 10% Triton X-100 (from c to d) was used to induce 100% calcein release. (C) Effect of different concentrations of MBP on calcein release from calcein-loaded liposomes containing a 1∶1 mass ratio of PC∶PS. (D) Effect of MBP treatment (50 µg/mL) on calcein release from liposomes containing different mass ratios (7∶3 or 9∶1) of PC∶PS. Data are from three independent experiments and indicated as mean ± SEM.
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