Increased evidence suggests that dysregulation of cholesterol metabolism may be a key event contributing to progression of multiple sclerosis (MS). Using an experimental autoimmune encephalomyelitis (EAE) model of MS we revealed specific changes in the mRNA and protein expression of key molecules involved in the maintaining of cholesterol homeostasis in the rat spinal cord: 3-hydroxy-3-methylglutaryl-coenzyme-A reductase (HMGCR), apolipoprotein E (ApoE) and cholesterol 24-hydroxylase (CYP46A1) during the course of disease. The presence of myelin lipid debris was seen only at the peak of EAE in demyelination loci being efficiently removed during the recovery period. Since CYP46A1 is responsible for removal of cholesterol excess, we performed a detailed profiling of CYP46A1 expression and revealed regional and temporal specificities in its distribution. Double immunofluorescence staining demonstrated CYP46A1 localization with neurons, infiltrated macrophages, microglia and astrocytes in the areas of demyelination, suggesting that these cells play a role in cholesterol turnover in EAE. We propose that alterations in the regulation of cholesterol metabolism at the onset and peak of EAE may add to the progression of disease, while during the recovery period may have beneficial effects contributing to the regeneration of myelin sheath and restoration of neuronal function.

Neuroinflammation and microglial activation, common components of most neurodegenerative diseases, can be imitated in vitro by challenging microglia cells with Lps. We here aimed to evaluate the effects of agmatine pretreatment on Lps-induced oxidative stress in a mouse microglial BV-2 cell line. Our findings show that agmatine suppresses nitrosative and oxidative burst in Lps-stimulated microglia by reducing iNOS and XO activity and decreasing O₂⁻ levels, arresting lipid peroxidation, increasing total glutathione content, and preserving GR and CAT activity. In accordance with these results, agmatine suppresses inflammatory NF-kB, and stimulates antioxidant Nrf2 pathway, resulting in decreased TNF, IL-1 beta, and IL-6 release, and reduced iNOS and COX-2 levels. Together with increased ARG1, CD206 and HO-1 levels, our results imply that, in inflammatory conditions, agmatine pushes microglia towards an anti-inflammatory phenotype. Interestingly, we also discovered that agmatine alone increases lipid peroxidation end product levels, induces Nrf2 activation, increases total glutathione content, and GPx activity. Thus, we hypothesize that some of the effects of agmatine, observed in activated microglia, may be mediated by induced oxidative stress and adaptive response, prior to Lps stimulation.

Modulating metabolic pathways in activated microglia can alter their phenotype, which is relevant in uncontrolled neuroinflammation as a component of various neurodegenerative diseases. Here, we investigated how pretreatment with agmatine, an endogenous polyamine, affects metabolic changes in an in vitro model of neuroinflammation, a murine microglial BV‐2 cell line exposed to lipopolysaccharide (LPS). Hence, we analyzed gene expression using qPCR and protein levels using Western blot and ELISA. Microglial metabolic status was assessed by measuring lactate release and cellular ATP by enzymatic and luminescence spectrophotometry. Mitochondrial functionality was analyzed by fluorescent probes detecting mitochondrial membrane potential (mtMP) and superoxide production. Our findings suggest that kinase pathways associated with hypoxia‐inducible factor‐1α (HIF‐1α) regulate energy metabolism in pro‐inflammatory activated microglia. We have shown that LPS induces HIF‐1α and genes for glucose transporter and glycolytic rate, increases lactate production and causes mitochondrial dysfunction, suggesting a metabolic shift towards glycolysis. Agmatine inhibits the PI3K/Akt pathway and negatively regulates mammalian target of rapamycin (mTOR) phosphorylation and HIF‐1α levels, reducing lactate and tumor necrosis factor (TNF) production, which is supported by pharmacological blockade of PI3K. Pretreatment with agmatine also rescues mitochondrial function by counteracting the LPS‐induced decline in mtMP and increase in mitochondrial superoxide, resulting in an anti‐apoptotic effect. Agmatine alone increases intracellular ATP levels and maintains this effect even under pro‐inflammatory conditions. Our study emphasizes the ability of agmatine to engage in metabolic reprogramming of pro‐inflammatory microglia through increased ATP production and modulation of signaling pathway involved in promoting glycolysis and cytokine release.

Inflammation, demyelination, and axonal damage to the central nervous system (CNS) are the hallmarks of multiple sclerosis (MS) and its representative animal model, experimental autoimmune encephalomyelitis (EAE). There is scientific evidence for the involvement of growth hormone (GH) in autoimmune regulation. Previous data on the relationship between the GH/insulin like growth factor-1 (IGF-1) axis and MS/EAE are inconclusive; therefore, the aim of our study was to investigate the changes in the GH axis during acute monophasic EAE. The results show that the gene expression of Ghrh and Sst in the hypothalamus does not change, except for Npy and Agrp, while at the pituitary level the Gh, Ghrhr and Ghr genes are upregulated. Interestingly, the cell volume of somatotropic cells in the pituitary gland remains unchanged at the peak of the disease. We found elevated serum GH levels in association with low IGF-1 concentration and downregulated Ghr and Igf1r expression in the liver, indicating a condition resembling GH resistance. This is likely due to inadequate nutrient intake at the peak of the disease when inflammation in the CNS is greatest. Considering that GH secretion is finely regulated by numerous central and peripheral signals, the involvement of the GH/IGF-1 axis in MS/EAE should be thoroughly investigated for possible future therapeutic strategies, especially with a view to improving EAE disease.