PDK4 Inhibition Ameliorates Melatonin Therapy by Modulating Cerebral Metabolism and Remyelination in an EAE Demyelinating Mouse Model of Multiple Sclerosis

Abstract

We recently showed that melatonin ameliorates the severity of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. However, efficiency of melatonin therapy was associated with side effects, manifested by slowing down of remyelination, through increasing the inhibitory effects of brain pyruvate dehydrogenase kinase-4 (PDK-4) on pyruvate dehydrogenase complex (PDC), a key enzyme in fatty acid (FA) synthesis during remyelination. In this study, we investigated the metabolic profile of FA synthesis using combination therapy of melatonin and diisopropylamine dichloroacetate (DADA), a PDK4 inhibitor, in EAE mice. Disease progression was monitored by recording the disability scores. Immunological, oligodendrogenesis and metabolic factors were also evaluated. Results showed that combination therapy of melatonin and DADA significantly reduced EAE disability scores, compared to melatonin, whereas DADA alone did not have any effect. In addition, co-therapy inhibited pro-inflammatory while increasing anti-inflammatory cytokines, significantly better than melatonin alone. Moreover, administration of combination drugs recovered the declined expression of oligodendrocytic markers in EAE, more potently than melatonin. Furthermore, co-therapy affected cerebral energy metabolism by significantly reducing lactate levels while increasing N-acetylaspartate (NAA) and 3-hydroxy-3-methyl-glutaryl-coenzyme-A reductase (HMGCR) levels. Finally, while melatonin increased lactate and PDK4 expression levels and greatly reduced PDC activity, co-therapy significantly restored PDC function while reducing the lactate levels. In summary, administration of melatonin with DADA increased the efficiency of melatonin treatment by eliminating the inhibitory effects of PDK4 on PDC's function, a critical step for proper FA synthesis during remyelination.

Trial registration: ClinicalTrials.gov NCT03498131.

Keywords: PDC; PDK4; diisopropylamine dichloroacetate; experimental autoimmune encephalomyelitis (EAE); fatty acids; melatonin; multiple scleorsis (MS); neuroinflammation.

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.

Copyright © 2022 Ghareghani, Farhadi, Rivest and Zibara.

Figures

Figure 1

(A) Schematic representation of experiment procedures and (B–G) daily assessment of EAE severity. Neurological disability analysis displayed an amelioration in EAE disease severity in melatonin (EAE+MEL) and combined therapy of melatonin and DADA (EAE-MEL+DADA), but not DADA alone. Treatment was initiated from day 13 post immunization for 10 consecutive days. (n=8 mice/group).

Figure 2

The effects of melatonin, DADA, and combination therapy on cytokine levels in the brain. Brain homogenates were used to quantify the levels of pro-inflammatory cytokines (A) IL-1β and (B) TNF and anti-inflammatory cytokines (C) IL-4 and (D) IL-10. Significance is indicated by *p < 0.05; **p < 0.01; ***p < 0.001 and ****p < 0.0001.

Figure 3

Oligodendrocyte’s expression levels. (A) Western blot analysis of myelin basic protein (MBP) and myelin-associated oligodendrocytic basic protein (MOBP) in the brain. The two markers (MBP and MOBP) were run alongside the same β-actin as housekeeping control. (B, C) Quantitative analysis for MBP and MOBP proteins, respectively. Values are expressed as the Mean ± SEM. Each group included 8 mice (n = 8). Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Tukey’s test. Significance is indicated by **p < 0.01; and ****p < 0.0001.

Figure 4

Brain levels of (A) lactate and (B) N-acetylaspartate (NAA), using HPLC. Values are expressed as the Mean ± SEM. Each group included 8 mice (n = 8). Statistical analysis was performed by two-way analysis of variance (ANOVA) followed by Tukey’s test. Significance is indicated by *p < 0.05; **p < 0.01; and ****p < 0.0001.

Figure 5

mRNA expression levels of 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMGCR) in brain homogenates. β-actin was used as an internal control. Quantification of HMGCR expression levels was normalized to controls. Values are expressed as the Mean ± SEM. Each group included 8 mice (n = 8). Statistical analysis was performed by two-way analysis of variance (ANOVA) followed by Tukey’s test. Significance is indicated by **p

Figure 6

Expression of PDK4. PDK4 levels after administration of melatonin with DADA in brain homogenates (A) mRNA expression levels of PDK4 assessed by real time-PCR. (B, C) Protein expression levels of PDK4 assessed by western blot. β-actin was used as an internal control for normalization for both real time-PCR and western blot. PDK4 expression levels was normalized to controls. Significance is indicated by **p < 0.01 and ***p < 0.001.

Figure 7

Activity of PDC. The change in the activity of PDC enzyme was measured either at the (A) “active” or (B) “total” forms of PDC. Values are expressed as the Mean ± SEM. Each group included 8 mice (n = 8). Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Tukey’s test. Significance is indicated by **p < 0.01; ***p < 0.001 and ****p < 0.0001.

Figure 8

The models of Melatonin and DADA effects on FA synthesis during remyelination. (A) In physiological conditions, citrate is produced from imported glucose or pyruvate and from imported NAA into the oligodendrocyte to be utilized as a substrate for FA synthesis required from myelination. Proper function of PDC warranty the synthesis of this citrate. (B) During EAE, PDC function remains intact, which is involved in pyruvate conversion into citrate. The other precursor of citrate, NAA, shows significant reduction, which is suggested to reduce the citrate level, which might slow down FA synthesis and subsequently the remyelination in EAE mice. (C) Melatonin therapy recovers the reduced level of NAA in EAE mice in order to increase the citrate levels required for FA synthesis. On the other hand, melatonin increases PDK4 expression which acts as an inhibitor of PDC, leading to suppression of citrate synthesis. However, the oligodendrocyte still imports the NAA from axons or any other unknown sources to have enough citrate for FA synthesis. (D) Since PDC inhibition by melatonin could be considered as a side effect of melatonin therapy, DADA administered in combination with melatonin would suppress the function of PDK4 and rescue the PDC from inhibiting PDK4 function. This leads to the recovery of citrate synthesis by the PDC-dependent pathway, while NAA, another source of citrate, is already high due to melatonin, resulting together into the fast formation of sufficient sources for FA synthesis in remyelination. Yellow arrow shows reduction, green arrow shows increase, red arrow shows conversions; blue rectangle shows the enzymes, Pyr, pyruvate; FA, Fatty acid; PDK-4, pyruvate dehydrogenase kinase-4; PDC, pyruvate dehydrogenase complex;, Ac-CoA, acetyl-CoA; HMGCR, 3-hydroxy3-methylglutaryl-coenzyme-A reductase; TCA, tricarboxylic acid cycle; NAA, N-acetylaspartate.