RAM-589.555 favors neuroprotective and anti-inflammatory profile of CNS-resident glial cells in acute relapse EAE affected mice.

BACKGROUND: Targeting RNA polymerase-1 (POL1) machinery is a new strategy for suppression of multiple sclerosis (MS) relapse activity. Oral administration of POL1 inhibitor RAM-589.555, which is characterized by high permeability and bioavailability in naïve mice, ameliorates proteolipid protein (PLP)-induced experimental autoimmune encephalomyelitis (EAE) by suppressing activated autoreactive lymphocytes. We assessed the accessibility of RAM-589.555 to the central nervous system (CNS) of EAE-mice and further investigated its immunomodulatory effects on CNS-resident astro- and micro-glial cells in-vitro and in-vivo. METHODS: Effects of RAM-589.555 on activated microglia and astrocyte viability, proliferation, and secretion of neurotrophic factors were assessed in-vitro. The pharmacokinetic of RAM-589.555 was evaluated in the blood and central nervous system (CNS) of EAE-affected mice. High-dimensional single-cell mass cytometry was applied to characterize the effect of RAM-589.555 on EAE-affected mice's CNS-resident micro- and astroglial cells and CNS-infiltrating immune cells, which were obtained seven days after RAM-589.555 administration at EAE onset. Simultaneously, the expression level of pre-rRNA, the POL1 end product, was assessed in blood cells, microglia, and astrocytes to monitor RAM-589.555 effects. RESULTS: RAM-589.555 demonstrated blood and CNS permeability in EAE mice. In-vitro, incubation with 400 nM of RAM-589.555 significantly reduced viability and proliferation of lipopolysaccharide (LPS)-activated microglia by 70% and 45% (p < 0.05), respectively, while tumor necrosis factor α (TNFα)-activated astrocytes were not affected. The secretion of neurotrophic factors was preserved. Furthermore, 7 days after administration of RAM-589.555 at EAE onset, the level of pre-rRNA transcript in peripheral blood mononuclear cells (PBMC) was decreased by 38.6% (p = 0.02), while levels of pre-rRNA transcript in microglia and astrocytes remained unchanged. The high-dimensional single-cell mass cytometry analysis showed decreased percentages of CNS-resident microglia and astrocytes, diminished pro-inflammatory cytokines (IL-1ß, IL-6, IL-12, IL-17, TNFα, and IFNγ), and an increase of their anti-inflammatory cytokines (IL-4, IL-10, and TGFß) in RAM-589.555-treated compared to vehicle-treated mice (p < 0.05). CONCLUSIONS: These data correlate RAM-589.555-induced clinical amelioration and its CNS-permeability to decreased CNS-inflammation, and decreased micro- and astrogliosis, while restoring micro- and astroglial anti-inflammatory and neuroprotective capacity.

Kinetic anomalies in the interactions of Nile red with microalgae.

Nile red (NR) is a popular fluorescent indicator to visualize lipid bodies in intact cells and has been extensively utilized to monitor triglyceride accumulation in microalgae. Typically, addition of NR to algae results in a rapid fluorescence enhancement followed by fluorescence quenching. NR fluorescence rise can be resolved into two kinetic phases: a fast phase (P1, sec), monitored at 525 nm/630 nm, followed by a slower phase (P2, min), monitored at 488 nm/575 nm. Studies with isolated plasma membrane (PM) and lipid globule (LG) preparations, suggest that P1 and P2 represent entry to the PM and transfer to LG, respectively. High NaCl slows down the interactions of NR with algae and with lipid globules. The onset of NR fluorescence quenching varies in different algae species between 5 min to 1h, and is observed in intact cells and in isolated LG. NR fluorescence quenching depends on NR concentration and is almost eliminated at low NR/cell ratios, indicating that it results from self-interactions of LG-associated dye. Glycerol has a dual effect on NR fluorescence: it eliminates kinetic anomalies resulting from limited solubility and self-interactions, but it also quenches NR fluorescence. NR fluorescence quenching by glycerol, as well as NR fluorescence enhancement by iodide anions, was observed only at high NR/LG ratios. These findings suggest that lipid-associated NR is more exposed to hydrophilic quenchers at high than at low NR concentrations. The results emphasize the importance of defining the optimal time window and NR concentrations for monitoring lipid accumulation in microalgae by NR fluorescence and clarify the origin of spectral anomalies resulting from self-interactions of dye molecules.

Regulation of the GPR40 locus: towards a molecular understanding.

GPR40 {FFAR1 [non-esterified ('free') fatty acid receptor 1]} is a G-protein-coupled receptor expressed preferentially in pancreatic beta-cells. GPR40 functions as a receptor for medium and long-chain fatty acids, and has been implicated in mediating both physiological and pathological effects of fatty acids on beta-cells. The GPR40 gene is encoded at an interesting chromosomal locus that contains several genes: at the 5'-end of the locus, located approximately 4 kb upstream of GPR40, is CD22, a gene encoding a receptor expressed selectively in lymphocytes and involved in B-lymphocyte maturation and function. At the 3'-end of the locus are the GPR41 (FFAR3) and GPR43 (FFAR2) genes encoding receptors activated by short-chain fatty acids. The intergenic region between CD22 and GPR40 contains several evolutionarily conserved sequence blocks, among them HR2 and HR3. beta-Cell-specific expression of GPR40 is controlled at the transcriptional level through HR2, a potent beta-cell-specific enhancer. The mechanisms controlling cell-specific expression of the remaining genes in the cluster are unknown. Given the divergent modes of expression of the genes within the locus and their demonstrated physiological significance, it is important to analyse further the locus with a view to fully understanding the basis for transcriptional regulation of the encoded genes.