Identifying Lanthionine Ketimine Derivatives for Maturation and Proliferative Effects in Oligodendrocyte Progenitor Cells

Abstract

Previous studies have shown that lanthionine ketimine ethyl ester (LKE) reduces clinical scores in the experimental autoimmune encephalomyelitis (EAE) mouse model of Multiple Sclerosis, induces differentiation of oligodendrocyte progenitor cells (OPCs) in vitro, and accelerates remyelination following cuprizone induced demyelination. In a search for derivatives with greater efficacy to induce OPC maturation or proliferation, we screened a panel of 2-alkyl and 3-phosphonate substituted LK derivatives. Incubation of Oli-neu oligodendrocyte cells with 2-n-butyl- or 2-n-hexyl-LKE-phosphonate reduced spontaneous cell death, increased proliferation, and increased maturation. These were associated with changes in corresponding mRNA levels of Olig2, PLP, and O4. These derivatives also reduced cell death and increased proliferation and maturation in primary mouse OPCs. The increased hydrophobicity of these derivatives suggests these will be better candidates for testing effects in animal models of Multiple Sclerosis and other demyelinating diseases.

Keywords: Oli-neu cells; differentiation; myelin; oligodendrocyte; proliferation.

Figure 1.

Chemical Structures of Lanthionine Ketimine analogues. (A) Structures of LK, LKE, and phosphonate derivatives with possible 2-substitutents. (B) LK analogues used in this study with calculated LogP values

Figure 2.

Relative toxicity of LK derivatives in Oli-neu cells. Oli-neu cells were incubated with 10 µM of (A) LKE and LKE-P derivatives and (B) LK-P derivatives, then cell death measured after indicated days by LDH release assay. For each day data is presented as the % cell death compared to untreated (CTL) cells, which was 16%, 18%, 14% on days 1, 2, and 3 respectively. Data is mean ± se of n = 5 per group. On day 2 cell death was reduced by 2-n-butyl LKE-P (BUT); and on day 3 by LKE, BUT, and 2-n-hexyl LKE-P (HEX). *, p < 0.05 versus CTL, 2-way repeated measures ANOVA with Dunnett’s multiple comparison test.

Figure 3.

Incubation with LKE or HEX increases Oli-neu cell proliferation. (A) Representative images of Oli-neu cells after incubation with 10 µM LKE, HEX, BUT, or nothing (CTL) for 72 hours, then stained for Ki67 (red) or PDGFRα (green), and counterstained with DAPI (blue). Incubation with LKE or HEX increased both (B) Ki67 and (C) PDGRFα staining versus CTL, whereas only LKE (D) increased the % of PDGFRα+:Ki67+ double labeled cells. Data is mean ± se percent staining derived from 10 fields of view from each of 8 wells (CTL, LKE) or 4 wells (HEX, BUT), and normalized to staining in CTL cells in which of the total (DAPI stained) cells 52% were Ki67+, 43% were PDGFRα+, and 50% were doubled-labeled cells for Ki67 and PDGFRα. ****, p < 0.0001 versus CTL, 1-way ANOVA with Dunnett’s multiple comparison test.

Figure 4.

Incubation with LKE or HEX increases total oligodendrocyte numbers. (A) Representative images of Oli-neu cells following incubation with 10 µM LKE, HEX, BUT, or nothing (CTL) for 72 hours, then stained for Olig2 (red) or O4 (green), and counterstained with DAPI (blue). (B) Incubation with LKE or HEX increased Olig2 staining. (C) Incubation with BUT decreased O4 staining. Data is mean ± se percent staining derived from 10 fields of view from each of 8 wells (CTL, LKE) or 4 wells (HEX, BUT), and normalized to staining in the CTL cells in which of the total (DAPI stained) cells 33% were O4+ and 30% were Olig2+. ***, p < 0.0005; ****, p < 0.0001 versus CTL, 1-way ANOVA with Dunnett’s multiple comparison test.

Figure 5.

Incubation with LKE or HEX increases Oli-neu cell maturation. (A) Representative images of Oli-neu cells following incubation with 10 µM LKE, HEX, BUT, or nothing (CTL) for 72 hours, then stained for PLP (red) and counterstained with DAPI (blue). (B) Incubation with LKE or HEX significantly PLP staining. Data is mean ± se percent staining derived from each of 30 fields of view from 4 wells and normalized to staining in the CTL cells in which only 4.0% of the cells stained for PLP. ****, p < 0.0001 versus CTL, 1-way ANOVA with Dunnett’s multiple comparison test. (C) Representative images from a second study showing the presence of processes in LKE, HEX, and BUT cells. The brightness was increased on these to allow visualization of processes, but results in higher background staining.

Figure 6.

Effects of LKE, HEX, and BUT on Oli-neu relative mRNA expression. Oli-neu cells were incubated with 10 µM LKE, HEX, BUT, or nothing (CTL) for 24 hours, then mRNA isolated, converted to cDNA, and used to quantify mRNA levels of (A) Olig2, (B) PDGFRα, (C) O4, and (D) PLP relative to β-actin mRNA levels measured in the same sample. Data is mean ± se of n = 4 per group, normalized to values measured in CTL cells. Both Olig2 and PLP relative mRNA levels were increased by HEX and BUT; while O4 mRNA was increased by HEX. *, p < 0.05; **, p < 0.005 versus CTL, 1-way ANOVA with Dunnett’s multiple comparison test.

Figure 7.

Relative toxicity of LK derivatives in primary OPCs. Primary mouse OPCs cells were incubated with 10 µM of (A) LKE and LKE-P derivatives and (B) LK-P derivatives, then cell death measured after indicated days by LDH release assay. For each day the data is presented as % cell death compared to untreated (CTL) cells, which was 13%, 16%, and 16% on days 1, 2, and 3 respectively. Data is mean ± se of n = 5 per group. On days 1 cell death was reduced by LKE, all 3 LKE-P and all 3 LK-P analogs. Cell death was reduced by LKE and the 3 LKE-P analogs on day 2, but only by LKE on day 3, and only by 2-n-butyl-LK-P on day 2. *, p < 0.05 versus CTL, 2-way repeated measures ANOVA with Dunnett’s multiple comparison test.

Figure 8.

Effects of LKE, HEX, and BUT on OPC proliferation and total cell numbers. (A) Representative images of primary mouse OPCs after incubation with 25 µM LKE, HEX, BUT, or nothing (CTL) for 48 hours, then stained for Ki67 (green) or Olig2 (red), and counterstained with DAPI (blue). (B) Incubation with all analogs increased) Ki67 staining. (C) Only LKE significantly increased Olig2 staining. Data is mean ± se of the number of positively stained cells from 10 fields of view from 4 wells, normalized to number of cells in CTL (61.5 ± 4.9 per mm² for Ki67; 296 ± 8.3 per mm² for Olig2). *, p < 0.05; ****, p < 0.0001 versus CTL, 1-way ANOVA with Dunnett’s multiple comparison test.

Figure 9.

Effects of LKE, HEX, and BUT on OPC maturation. (A) Representative images of primary mouse OPCs after incubation with 25 µM LKE, HEX, BUT, or nothing (CTL) for 48 hours, then stained for PLP (red), and counterstained with DAPI (blue). (B) Incubation with LKE or HEX significantly increased the number of PLP stained cells. Data is mean ± se the number of positively stained cells derived from 10 fields of view from 4 wells, normalized to the number cells of PLP stained cells in CTL (9.5 ± 0.9 per mm²). ***, p < 0.001; ****, p < 0.0001 versus CTL, 1-way ANOVA with Dunnett’s multiple comparison test. (C) Co-staining of CTL and LKE-treated OPCs does not reveal co-express of PDGFRα (green) and PLP (red).