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. 2023 Aug 17:17:1201317.
doi: 10.3389/fncel.2023.1201317. eCollection 2023.

Nicotinamide enhances myelin production after demyelination through reduction of astrogliosis and microgliosis

Stefanos Ioannis Kaplanis  1   2 , Despoina Kaffe  3 , Niki Ktena  1   2 , Andriani Lygeraki  3 , Ourania Kolliniati  2   4   5 , Maria Savvaki  1   2 , Domna Karagogeos  1   2
Affiliations

Nicotinamide enhances myelin production after demyelination through reduction of astrogliosis and microgliosis

Stefanos Ioannis Kaplanis et al. Front Cell Neurosci. .

Abstract

Caloric restriction is the chronic reduction of total caloric intake without malnutrition and has attracted a lot of attention as, among multiple other effects, it attenuates demyelination and stimulates remyelination. In this study we have evaluated the effect of nicotinamide (NAM), a well-known caloric restriction mimetic, on myelin production upon demyelinating conditions. NAM is the derivative of nicotinic acid (vitamin B3) and a precursor of nicotinamide adenine dinucleotide (NAD+), a ubiquitous metabolic cofactor. Here, we use cortical slices ex vivo subjected to demyelination or cultured upon normal conditions, a lysolecithin (LPC)-induced focal demyelination mouse model as well as primary glial cultures. Our data show that NAM enhances both myelination and remyelination ex vivo, while it also induces myelin production after LPC-induced focal demyelination ex vivo and in vivo. The increased myelin production is accompanied by reduction in both astrogliosis and microgliosis in vivo. There is no direct effect of NAM on the oligodendrocyte lineage, as no differences are observed in oligodendrocyte precursor cell proliferation or differentiation or in the number of mature oligodendrocytes. On the other hand, NAM affects both microglia and astrocytes as it decreases the population of M1-activated microglia, while reducing the pro-inflammatory phenotype of astrocytes as assayed by the reduction of TNF-α. Overall, we show that the increased myelin production that follows NAM treatment in vivo is accompanied by a decrease in both astrocyte and microglia accumulation at the lesion site. Our data indicate that NAM influences astrocytes and microglia directly, in favor of the remyelination process by promoting a less inflammatory environment.

Keywords: astrocytes; caloric restriction; microglia; myelin; nicotinamide (NAM); remyelination.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Nicotinamide (NAM) increases myelination of axons in ex vivo organotypic brain slices. (A) Representative confocal images of the cortical area of organotypic brain slices from postnatal day 4 mice, immunolabeled with an antibody against NF200 (red) to mark axons and an antibody against MBP (green) to mark myelin. DAPI was used for nuclear staining (blue). White arrows point at the myelinated axons (yellow). (B) Quantification of the percentage of NF200-positive area that co-localized with MBP, indicates the percentage of myelinated axons. Two groups were used: vehicle-treated (EtOH) for the control and NAM-treated (0.1 mM) for 6 days. For both groups n = 4. Data are shown as mean ± SEM. Student’s t-test was used to determine statistical significance. *p < 0.05. Scale bar: 50 μm.
FIGURE 2
FIGURE 2
Nicotinamide (NAM) induces remyelination of axons in ex vivo organotypic brain slices after LPC demyelination protocol. (A) Representative confocal images of the cortical area of organotypic brain slices from postnatal day 4 mice, immunolabeled with an antibody against NF200 (red) to mark axons and an antibody against MBP (green) to mark myelin. DAPI was used for nuclear staining (blue). EtOH was used as control and compared with NAM treatment at 0.1 mM for 6 days. In both groups LPC demyelination protocol was applied prior to EtOH or NAM treatment. White arrows point at the myelinated axons (yellow). (B) Quantification of the percentage of NF200-positive area that co-localized with MBP, indicates the percentage of myelinated axons. Two groups were used: LPC and EtOH treatment for the control and LPC and NAM treatment at 0.1 mM for 6 days. For both groups n = 4. Data are shown as mean ± SEM. Student’s t-test was used to determine statistical significance. ***p ≤ 0.001. Scale bar: 50 μm.
FIGURE 3
FIGURE 3
Nicotinamide (NAM) increases myelin production in the lesion site of the LPC-induced demyelination mouse model at 14 dpi. (A) Representative immunohistochemical confocal images of the corpus callosum labeled for MBP (green) and DAPI (blue). The area surrounded by dotted line denotes the lesion area. (B) Quantification of MBP density in control mice (LPC), in mice which received the lower dose of NAM after LPC (40 mg/kg/day) and in mice which received the higher dose of NAM after LPC (400 mg/kg/day) for 14 days. For all 3 groups n = 5. Data are shown as mean ± SEM. Student’s t-test was used to determine statistical significance. *p < 0.05. Scale bar: 200 μm.
FIGURE 4
FIGURE 4
Nicotinamide (NAM) reduces both microgliosis and astrogliosis in the lesion area of LPC-induced demyelination at 14 dpi. (A) Representative immunohistochemical images of the corpus callosum labeled for MBP (green), IBA1 (red) and DAPI (blue). (B) Quantification of densitometric analysis for IBA1 in control mice (LPC), in mice which received the lower dose of NAM after LPC (40 mg/kg/day) and in mice which received the higher dose of NAM after LPC (400 mg/kg/day) for 14 days. (C) Representative confocal images of the corpus callosum labeled for MBP (green), GFAP (red) and DAPI (blue). (D) Quantification of densitometric analysis for GFAP in control mice (LPC), in mice which received the lower dose of NAM after LPC (40 mg/kg/day) and in mice which received the higher dose of NAM after LPC (400 mg/kg/day) for 14 days. The area surrounded by dotted lines denotes the lesion area. A total of 14 days of treatment after LPC stereotactic injection. Three groups of mice (vehicle with only LPC injection, NAM 40 mg/kg/day and NAM 400 mg/kg/day with LPC) were tested. For all groups n = 5. Data are shown as mean ± SEM. Student’s t-test was used to determine statistical significance. ***p ≤ 0.001. Scale bar: 200 μm.
FIGURE 5
FIGURE 5
Nicotinamide (NAM) suppresses microglial activation in vitro. (A) Representative immunocytochemical confocal images of primary microglia cultures. Microglia labeled with IBA1 (green) marker and nuclear DAPI (blue). Four groups were used: vehicle, NAM 0.1 mM, NAM 0.2 mM, and NAM 0.4 mM. (B) Quantification of percentage of ramified microglia in the four different conditions. (C) Western blot analysis for iNOS in microglial lysates after activation with LPS. Four groups were used: vehicle, NAM 0.1 mM, NAM 0.2 mM, and NAM 0.4 mM. (D) Quantification of Western blot analysis for the four different conditions. (E) ELISA analysis for the secretion of anti-inflammatory cytokine IL-10. Two groups were used: vehicle and 0.4 mM NAM. For immunocytochemical experiments, n = 4. For biochemical experiments, n = 3. Data are shown as mean ± SEM. Student’s t-test was used to determine statistical significance. **p ≤ 0.01, ****p ≤ 0.0001. Scale bar: 150 μm.
FIGURE 6
FIGURE 6
NAM induces both autophagy and the anti-inflammatory phenotype of astrocytes in vitro. (A) Quantification of IL-10 secretion from astrocytes after NAM treatment at concentrations of 0.1, 0.2, and 0.4 mM compared with the vehicle. (B) ELISA analysis for the secretion of the pro-inflammatory factor TNF-α in activated with LPS, primary astrocyte cultures. Two groups were used: vehicle and 0.4 mM NAM treated. (C) Western blot analysis for LC3 in lysates from primary cultures of astrocytes under four different conditions (vehicle, NAM 0.1 mM, NAM 0.2 mM, and NAM 0.4 mM). All groups were also treated for 4 h with 10 nM Bafilomycin A1 (BafA1) before cells were collected. (D) Quantification of LC3II/LC3I protein levels. For all procedures, in each group n = 3. Data are shown as mean ± SEM. Student’s t-test was used to determine statistical significance. *p < 0.05, **p ≤ 0.01, ***p ≤ 0.001.
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Grants and funding

This project received funding from the Hellenic Foundation for Research and Innovation (HFRI), under grant agreement No. 1676, from the National Multiple Sclerosis Society (NMSS, pilot Research Grant), the Hellenic Academy of Neuroimmunology (HELANI) and the Fondation Sante (The Sidney Altman Scholarship Program). DeK was supported by an Onassis Foundation Scholarship (G ZS 004-1/2022-2023).

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