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. 2023 Apr;18(4):832-839.
doi: 10.4103/1673-5374.353493.

Poly-L-ornithine blocks the inhibitory effects of fibronectin on oligodendrocyte differentiation and promotes myelin repair

Ya-Jie Xiong  1 , Shahid Hussain Soomro  1 , Zhong-Hai Huang  2 , Pan-Pan Yu  2 , Jie Ping  3 , Hui Fu  1
Affiliations

Poly-L-ornithine blocks the inhibitory effects of fibronectin on oligodendrocyte differentiation and promotes myelin repair

Ya-Jie Xiong et al. Neural Regen Res. 2023 Apr.

Abstract

The extracellular matrix surrounding oligodendrocytes plays an important role during myelination and remyelination in the brain. In many cases, the microenvironment surrounding demyelination lesions contains inhibitory molecules, which lead to repair failure. Accordingly, blocking the activity of these inhibitory factors in the extracellular matrix should lead to more successful remyelination. In the central nervous system, oligodendrocytes form the myelin sheath. We performed primary cell culture and found that a natural increase in fibronectin promoted the proliferation of oligodendrocyte progenitors during the initial stage of remyelination while inhibiting oligodendrocyte differentiation. Poly-L-ornithine blocked these inhibitory effects without compromising fibronectin's pro-proliferation function. Experiments showed that poly-L-ornithine activated the Erk1/2 signaling pathway that is necessary in the early stages of differentiation, as well as PI3K signaling pathways that are needed in the mid-late stages. When poly-L-ornithine was tested in a lysolecithin-induced animal model of focal demyelination, it enhanced myelin regeneration and promoted motor function recovery. These findings suggest that poly-L-ornithine has the potential to be a treatment option for clinical myelin sheath injury.

Keywords: Erk1/2; PI3K; differentiation; extracellular matrix; fibronectin; lysolecithin-induced demyelination; oligodendrocyte; poly-L-ornithine; proliferation; remyelination.

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Conflict of interest statement

None

Figures

Figure 1
Figure 1
The flow chart. LPC: Lysolecithin; MCM: mixed cell-medium; MOGM: modified oligodendrocyte precursor cell growth-medium; ODM: oligodendrocyte precursor cell differentiation-medium; OPC: oligodendrocyte precursor cells; PDL: poly-D-lysine; PLO: poly-L-ornithine.
Figure 2
Figure 2
FN promotes the proliferation of NG2+ cells. (A) Representative images of OPCs plated on glass coverslips coated with FN or PDL and cultured for 2 days in proliferation medium. The number of cells was significantly higher in the FN group than in the PDL group. Scale bar: 100 µm. (B) Quantitative analysis of A. Data were from three independent experiments. (C) Representative immunofluorescence images of OPCs stained for NG2 (an OPC marker, red, stained with Alexa Fluor555), Ki67 (a proliferating-cell marker, green, stained with Alexa Fluor488), DAPI (blue, nucleus staining), cultured for 2 days in proliferation medium. Compared with the PDL group, the number of NG2+ cells and the proportion of Ki67+ cells in NG2+ cells were significantly higher in the FN group. Scale bar: 50 µm. (D) Quantitative analysis of NG2+ cells in C. (E) Quantitative analysis of proliferating OPCs (Ki67+ NG2+ cells/NG+ cells) in the different treatment groups. Data are expressed as mean ± SD from at least three independent experiments. **P < 0.01, ***P < 0.001 (Student’s t-test). DAPI: 4′,6-Diamidino-2-phenylindole; FN: fibronectin; NG2: nerve-glia antigen 2; OPC: oligodendrocyte precursor cell; PDL: poly-D-lysine.
Figure 3
Figure 3
FN suppresses the differentiation of oligodendrocytes. (A) The diagram illustrates changes in cell morphology revealed by typical markers during oligodendrocyte differentiation in vitro. OPCs cultured for 2 days in proliferation medium (2 d PM) were stained with NG2 antibody (red, stained with Alexa Fluor555). CNP antibody was used after 1 and 3 days in the differentiation medium (1/3 d DM). MBP antibody was used to mark OLs cultured for 5 days in the differentiation medium (5 d DM). (B) Representative immunofluorescence images of OLs stained for Olig2 (green, stained with Alexa Fluor488) and CNP (red, stained with Alexa Fluor555), cultured for 3 days in differentiation medium. Arrows point to cells with ring-like structure. The proportion of OLs with ring-like structure was significantly lower in the FN group than in the PDL group. (C) Quantification of the percentage of cells in B having ring-like structure or non-ring-like structure. (D) Quantification of mRNA expression levels (normalized by the PDL group) of MBP and CNP from cells cultured for 3 days in differentiation medium. (E) Representative western blots of MBP and CNP proteins from OPCs cultured for 3 days in differentiation medium. (F) Quantification of the protein expression levels (normalized by the PDL group) of MBP and CNP in E. (G) Representative immunofluorescence images of OLs stained for Olig2 (green, stained with Alexa Fluor488) and MBP (red, stained with Alexa Fluor555) after 5 days in differentiation medium. The OLs in the FN group exhibited less membranous and myelin-sheet structures than did those in the PDL group. Scale bars: 50 µm (A), 100 µm (B, G). (H) Quantification of G, showing the percentages of cells bearing non-membranous, membranous, or myelin sheet structures. Data are expressed as mean ± SD from three independent experiments. **P < 0.01, ***P < 0.001 (Student’s t-test). CNP: 2′,3′-Cyclic nucleotide 3′-phosphodiesterase; DM: differentiation medium; FN: fibronectin; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; MBP: myelin basic protein; NG2: nerve-glia antigen 2; Olig2: oligodendrocyte transcription factor 2; OPC: oligodendrocyte precursor cell; PDL: poly-D-lysine; PM: proliferation medium.
Figure 4
Figure 4
PLO blocks the negative effects of FN on oligodendrocyte differentiation and promotes oligodendrocyte maturation. (A) Representative immunofluorescence images of OLs (stained with Alexa Fluor555) cultured for 1 day in differentiation medium (1 d DM) under different coating conditions. After treatment with the PLO and FN combination, OLs showed more complex morphology than those in the PDL or FN groups. (B) Quantitative analysis of D values on cells in A. (C) Representative immunofluorescence images of OLs stained for Olig2 (green, stained with Alexa Fluor488), and CNP (red, stained with Alexa Fluor555) after being cultured for 3 days in differentiation medium (3 d DM) under different coating conditions. Compared with the PDL group, OLs in the FN group exhibited less ring-like structure, while those in the PLO + FN group exhibited more ring-like structure. (D) Quantification of the ratios of cells in A with/without ring-like structure. Data were collected from three independent experiments. (E) Representative western blots of MBP and CNP proteins from oligodendrocytes cultured for 3 days in differentiation medium under different coating conditions. (F) Quantification of C (normalized by the PDL group), with data from at least three independent experiments. (G) Images of OLs stained for Olig2 (green, stained with Alexa Fluor488) and MBP (red, stained with Alexa Fluor555) after being cultured for 5 days in differentiation medium (5 d DM) under different coating conditions. Arrow 1: cells with non-membranous structure; arrow 2: cells with membranous structure; arrow 3: cells with myelin sheets. Compared with the PDL group, OLs in the FN group exhibited less membranous structure and no myelin sheets, while those in the PLO + FN group exhibited more membranous structure and myelin sheets. Scale bars: 50 µm in A, 100 µm in C and G. (H) Quantification of the percentages of cells having non-membranous/membranous structure or myelin sheets, as revealed by MBP immunostaining in E. Data are expressed as mean ± SD of at least 10 cells from each of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way analysis of variance followed by Tukey’s multiple comparison test). CNP: 2′,3′-Cyclic nucleotide 3′-phosphodiesterase; DAPI: 4′,6-diamidino-2-phenylindole; DM: differentiation medium; FN: fibronectin; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; MBP: myelin basic protein; NG2: nerve-glia antigen 2; OL: oligodendrocyte; Olig2: oligodendrocyte transcription factor 2; PDL: poly-D-lysine; PLO: poly-L-ornithine.
Figure 5
Figure 5
PLO has no significant effects on OPC proliferation and does not interfere with the proliferative effect of FN. (A) Representative immunofluorescence images of OPCs stained for NG2 (red, stained with Alexa Fluor555, an OPC marker), Ki67 (green, stained with Alexa Fluor488, a proliferation marker), and DAPI (blue, staining for nuclei), cultured for 2 days in proliferation medium (2 d PM) under different coating conditions. The proliferation rate of OPCs in the PLO + FN group was similar to that in the FN group, which was higher than that in the PDL or PLO groups. Scale bar: 100 µm. (B) Quantitative analysis of A, showing the proportion of Ki67 and NG2 co-labeled cells to NG2 cells. (C) Representative western blots of CyclinD1 protein from oligodendrocytes cultured for 2 days in proliferation medium (2 d PM). (D) Quantification of the CyclinD1 protein expression level (normalized by the PDL group) in C. Data were collected from at least three independent western blots. Data are expressed as mean ± SD from three independent experiments. *P < 0.05, **P < 0.01 (one-way analysis of variance followed by Tukey’s multiple comparison test). DAPI: 4′,6-Diamidino-2-phenylindole; FN: fibronectin; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; MBP: myelin basic protein; NG2: nerve-glia antigen 2; Olig2: oligodendrocyte transcription factor 2; OPC: oligodendrocyte precursor cell; PDL: poly-D-lysine; PLO: poly-L-ornithine; PM: proliferation medium.
Figure 6
Figure 6
PLO and FN function through ERK and PI3K/Akt signaling pathways. (A) The diagram shows the integrins (αV, β1, β3, β5) expressed in oligodendrocytes that specifically bind to FN. Data are collected from published literature. (B) Quantitative analysis of the expression levels (normalized by the PDL group) of integrins (αV, β1, β3, β5) during the proliferation and differentiation stages of oligodendrocytes by real-time quantitative polymerase chain reaction. (C) Representative western blots of Akt, P-Akt (Thr308), S6K, P-S6K, Erk1/2, and P-Erk1/2 proteins from OPCs cultured with different coating substances for 2 days in proliferation medium (2 d PM). (D) Quantification of the western blot results (normalized by the PDL group) showed in C. (E) Representative western blots of GSK3β, P-GSK3β, CREB, P-CREB, Akt, P-Akt, Erk, and P-Erk proteins from cells cultured for 1 day in differentiation medium (1d DM). (F) Quantification (normalized by the PDL group) of the results in E. (G) Representative western blots of Akt, P-Akt, Erk, P-Erk, GSK3β, P-GSK3β, CREB, and P-CREB proteins from cells cultured for 3 days in differentiation medium (3d DM). (H) Quantification (normalized by the PDL group) of the results in G. All data are expressed as mean ± SD from at least three independent experiments. *P < 0.05, **P < 0.01 (one-way analysis of variance followed by Tukey’s multiple comparison test). Akt: Protein kinase B; CREB: cyclic adenosine monophosphate response element binding protein; DM: differentiation medium; Erk1/2: extracellular signal-regulated kinases 1 and 2; FN: fibronectin; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GSK3β: glycogen synthase kinase 3 beta; P-Akt; phospho-protein kinase B; P-CREB: phospho-cyclic adenosine monophosphate response element binding protein; PDL: poly-D-lysine; P-Erk1/2: phospho-extracellular signal-regulated kinases 1 and 2; P-GSK3β: phospho-glycogen synthase kinase 3 beta; PI3K: phosphoinositide 3-kinase; PLO: poly-L-ornithine; PM: proliferation medium; P-S6k: phospho-ribosomal S6 kinase; S6k: ribosomal S6 kinase.
Figure 7
Figure 7
PLO promotes functional myelin repair after 14 days of lysolecithin-induced demyelination injury. (A) Illustration of the mouse model of LPC-induced demyelination. Demyelination was restricted to the ventrolateral white matter, approximately 0.6 mm lateral to the midline. Black arrowheads on the steric spinal cord model indicate the injection sites, blue dots on the transection plane indicate the lesion sites, and the white dashed lines mark off the lesion area. (B) RNA in situ hybridization for MBP and PLP1 on spinal cords with different treatments. The white dashed lines mark off the lesion area. PLP1+/MBP+ OLs were significantly greater in the PLO-treated group. Scale bar: 50 µm. (C) Quantification of PLP1+ OLs in the lesion area with different treatments as shown in B (n = 3). (D) Representative MBP (red, stained with Alexa Fluor555) and CC1 (red, stained with Alexa Fluor555) immunofluorescence staining. Compared with control groups, MBP-stained areas and the number of CC1 positive cells were significantly greater in the PLO-treated group. The white dashed lines mark off areas of increased cell density (as shown by DAPI staining) representing the location of LPC demyelination. Scale bar: 50 µm. (E) Quantification of CC1+ OLs in C (n = 3). (F) EC staining showing myelin in the spinal cord after different treatments. The area of demyelination was significantly lower in the PLO-treated group than in the control groups. The white dashed lines mark off the demyelinated area. Scale bar: 100 µm. (G) Quantification of the size of the demyelinated region in F. Three animals were used for each group (n = 3). (H) Representative immunostaining for astrocytes (GFAP+ cell, red, stained with Cy3) and microglia (Iba1+ cell, green, stained with Alexa Fluor488) in lesion areas 14 days after injury. The number of astrocytes or microglia in the PLO-treated group was similar to that in the control groups. The white dashed lines mark off the lesion area. Scale bar: 50 µm. (I) Quantification of GFAP+ cells and Iba1+ cells in H. Three animals were used for each group (n = 3). (J) Scores for “base of support” were compared among different groups one day before surgery and 14 days after surgery (n = 5). (K) Regularity index values for the different groups (n = 4). All data are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t-test was used to compare two groups, and one-way analysis of variance followed by Tukey’s multiple comparison test was performed for comparisons of three or more groups). CC1: Adenomatous polyposis coli; DAPI: 4′,6-diamidino-2-phenylindole; dpl: days post-lesion; EC: eriochrome cyanine; GFAP: glial fibrillary acidic protein; Iba-1: ionized calcium binding adaptor molecule 1; LPC: lysolecithin; MBP: myelin basic protein; PDL: poly-D-lysine; PLO: poly-L-ornithine; PLP: proteolipid protein.
Figure 8
Figure 8
PLO exhibits dose-dependent effects in the animal model of demyelination. (A) Eriochrome cyanine staining 14 days after the different treatments. The area of demyelination in the ventrolateral funiculus is indicated by the dashed outline. The area of demyelination in the LPC + 100 µg/mL group was significantly smaller than that in the LPC, LPC + 20 µg/mL, or LPC + 500 µg/mL groups. (B) Quantification of the demyelinated area in A. (C) Representative CC1 (red, stained with Alexa Fluor555) immunofluorescence staining 14 days after injury. The number of CC1+ OLs was greater in the LPC + 100 µg/mL group than in the LPC, LPC + 20 µg/mL, or LPC + 500 µg/mL groups. Scale bars: 100 µm in A, 50 µm in C. (D) Quantification of CC1+ cells in C. All data are expressed as mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t-test). CC1: Adenomatous polyposis coli; DAPI: 4′,6-diamidino-2-phenylindole; LPC: lysolecithin.
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