The Effects of NLY01, a Novel Glucagon-Like Peptide-1 Receptor Agonist, on Cuprizone-Induced Demyelination and Remyelination: Challenges and Future Perspectives

Abstract

Recent evidence suggests that the glucagon-like peptide-1 receptor (GLP-1R) agonists have neuroprotective activities in the CNS in animal models of Parkinson's disease, Alzheimer's disease, and multiple sclerosis (MS). This study aimed to investigate whether a novel long-acting GLP-1R agonist, NLY01, could limit demyelination or improve remyelination as occurs in MS using the cuprizone (CPZ) mouse model. Herein, we assessed the expression of GLP-1R on oligodendrocytes in vitro and found that mature oligodendrocytes (Olig2⁺PDGFRa^-) express GLP-1R. We further confirmed this observation in the brain by immunohistochemistry and found that Olig2⁺CC1⁺ cells express GLP-1R. We next administered NLY01 twice per week to C57B6 mice while on CPZ chow diet and found that NLY01 significantly reduced demyelination with greater weight loss than vehicle-treated controls. Because GLP-1R agonists are known to have anorexigenic effect, we then administered CPZ by oral gavage and treated the mice with NLY01 or vehicle to ensure the dose consistency of CPZ ingestion among mice. Using this modified approach, NLY01 was no longer effective in reducing demyelination of the corpus callosum (CC). We next sought to examine the effects of NLY01 treatment on remyelination after CPZ intoxication and during the recovery period using an adoptive transfer-CPZ (AT-CPZ) model. We found no significant differences between the NLY01 and vehicle groups in the amount of myelin or the number of mature oligodendrocytes in the CC. In summary, despite the promising anti-inflammatory and neuroprotective effects of GLP-1R agonists that have been previously described, our experiments provided no evidence to support a beneficial effect of NLY01 on limiting demyelination or enhancing remyelination. This information may be useful in selecting proper outcome measures in clinical trials of this promising class of drugs in MS.

Keywords: Cuprizone; GLP-1R; MS; Myelin; NLY01; Oligodendrocytes.

Fig. 1

Experimental design to test NLY01 effect on CPZ-induced de- and remyelination. a CPZ only-demyelination model;Mice were fed by CPZ for 4 weeks; CPZ-remyelination model: mice were fed by CPZ and then put on the regular diet (normal chow) for 2 weeks; Adoptive transfer-CPZ (AT-CPZ) model: After CPZ excision and starting regular diet (normal chow), myelin-specific effector T cells (Th17) were injected intraperitoneally to the mice. NLY01 or vehicle was injected to the mice subcutaneously in different models as described in the text and shown in the following figures (2-6). b Three brain sections per mouse (anterior, middle, and posterior regions identified relative to Bregma) were stained and examined using Black-Gold staining and immunohistochemistry. The box shows the central region of corpus callosum that was quantified in stained sections

Fig. 2

Mature oligodendrocytes, but not OPCs, express GLP-1R. a Representative immunofluorescent images of mouse OPCs in vitro stained with Olig2, GLP-1R, and PDGFRα. b White arrows show Olig2⁺PDGFRα⁻ cells that are GLP-1R positive in the box area, scale 20 µm. c Representative in vitro images of mature oligodendrocytes stained with Olig2, GLP-1R, and CC1. d White arrows show Olig2⁺CC1⁺ cells that are GLP-1R positive in the enlarged figure of the box area, scale = 20 µm. e A representative region of the corpus callosum showing mature oligodendrocytes express GLP-1R, scale = 50 µm. f White arrows show Olig2⁺CC1⁺GLP-1R⁺ cells in enlarged figure for the box area

Fig. 3

NLY01 effect on the demyelination induced by Cuprizone chow. a Schematic of the NLY01 treatment plan at the early stage of CPZ-induced demyelination. Mice were fed CPZ and treated with NLY01 or vehicle. The brains were collected after 4 weeks of CPZ feeding. Black-Gold staining was quantified in the representative images of CC shown in panel a, scale 200 µm. Number of mice per treatment group including both females and males, vehicle, n = 7; NLY01, n = 7. Each dot represents one mouse. b Schematic of the NLY01 treatment plan at the middle stage of CPZ-induced demyelination. Quantification of percent area positive for Black-Gold in each respective region of the CC shown in panel b, scale 200 µm. Number of mice per treatment group (all females), vehicle, n = 5; NLY01, n = 5. Each dot represents one mouse. c The daily weigh records and comparison of weight changes before and after treatment in NLY01 or vehicle group. All the data are presented as mean ± SEM and analyzed by Student’s t-test (panels a and b) or paired t-test (panel c). p value ≤ 0.05 was considered significant

Fig. 4

NLY01 effect on the demyelination induced by Cuprizone gavage. a Schematic of the NLY01 treatment in the cohort of mice receiving CPZ gavage. Mice were fed CPZ and treated with NLY01 or vehicle on day 0 of CPZ initiation. The brains were collected after 4 weeks of CPZ. b The daily record of body weight of mice treated with NLY01 or vehicle. c The body weight of the mice before and after treatment (14 and 28 days) with NLY01 or vehicle analyzed by paired t-test. Number of mice per treatment group including both males and females, vehicle, n = 8; NLY01, n = 6. Each dot represents one mouse. d Representative images of Black-Gold staining and the quantification of the percentage area positive for myelin in each respective region of the CC, scale 200 µm. Number of mice per treatment group including both males and females, vehicle, n = 8; NLY01, n = 6. Each dot represents one mouse. Data were analyzed by Student’s t-test. All the data are presented as mean ± SEM. p value ≤ 0.05 was considered significant

Fig. 5

NLY01 does not affect the trafficking of effector T cells to the brain or onset of EAE following adoptive transfer in the AT-CPZ model. a Representative flow cytometric plots of CD4⁺Vβ11⁺ T cells polarized to effector T cells and produced inflammatory cytokines in vitro. b The percentage of CD4⁺Vβ11⁺ T cells that produce IL-17 and/or IFN in vitro. c The number of T cells trafficking into the CC in the AT-CPZ model. d IHC image of the CC stained with MBP and CD3 + T cells after adoptive transfer compared to no adoptive transfer condition (scale = 100 µm). The boxed regions are shown as enlarged split color images of the CC (Scale = 20 µm). e Schematic of the NLY01 treatment. Mice were treated with NLY01 or vehicle at remyelination phase 4 days after adoptive transfer. The brains were collected 2 weeks after adoptive transfer. f The behavioral plot of the mice in AT-CPZ cohort that received effector T cells and treated with NLY01 or vehicle. The behavior scores are presented as mean ± SEM from one representative experiment. The experiment was repeated 3 times. Number of mice per treatment group (all females), vehicle, n = 6; NLY01, n = 7. Each dot represents one mouse. g Quantification of the CD3⁺ T cells in the anterior, middle, and posterior regions of the CC. All the data are presented as mean ± SEM and analyzed by Student’s t-test. p value ≤ 0.05 was considered significant

Fig. 6

NLY01 has no effect on the remyelination of the corpus callosum. a Schematic of the remyelination plan and NLY01 treatment in AT-CPZ model. Mice were fed CPZ and treated with NLY01 or vehicle 4 days after CPZ withdrawal and adoptive transfer. Number of mice per treatment group (all females), vehicle, n = 8; NLY01, n = 7. Quantification of percent area positive for Black-Gold in each respective region of the CC. Representative images of Black-Gold staining in the anterior, middle, and posterior regions of the CC from the cohort shown in panel a, scale 200 µm. b Schematic of the remyelination plan and NLY01 treatment in the CPZ-remyelination model. Mice were fed CPZ and treated with NLY01 or vehicle 4 days after CPZ withdrawal. Number of mice per treatment group (all females), vehicle, n = 7; NLY01, n = 7. Quantification of percent area positive for Black-Gold in each respective region of the CC. Representative images of Black-Gold staining in the anterior, middle, and posterior regions of the CC from the cohort are shown in panel, scale 200 µm. c Quantification of oligo lineage cells in the boxed regions of the CC from the AT-CPZ cohort shown in panel a. The plot shows the counts of total oligo lineage cells (Olig2⁺), mature oligodendrocytes (Olig2⁺CC1⁺), or OPCs (Olig2⁺PDGFRα⁺) between NLY01- or vehicle-treated mice. Each dot represents an average of counts in 5 boxed regions of the CC as shown in the large image. All the data are presented as mean ± SEM and analyzed by Student’s t-test (panels a and b) or one-way ANOVA (panel c). p value ≤ 0.05 was considered significant