Analiza działania modulatorów receptorów dla sfingozyno-1-fosforanu w eksperymentalnych modelach choroby Alzheimera

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Title: Analiza działania modulatorów receptorów dla sfingozyno-1-fosforanu w eksperymentalnych modelach choroby Alzheimera

Level of degree:

Degree discipline :

medical sciences

Degree grantor:

Mossakowski Medical Research Institute Polish Academy of Sciences

Abstract:

The dissertation aimed to investigate the effect of sphingosine-1-phosphate receptor (S1PR) modulators on changes at the transcriptional and translational levels in selected proteins involved in: synaptic transmission, apoptosis, inflammation and AKT kinase-dependent prosurvival pathway in experimental models of Alzheimer's disease. In vivo studies were conducted on 3- and 12-month-old (3M and 12M) FVB-Tg mice with the London mutation in the APP gene (APP+) and mice without the transgene (APP-), which were administered i.p. FTY720 (1 mg/kg b.w.) or 0.9% NaCl for 14 days. The brain cortex and hippocampus were used for analysis. A reduction in Vamp1 expression was demonstrated in the brain cortex in both age groups and in the hippocampus of 3M APP+ mice. Decreased expression of Stxa1, Snap25, Syt1, Cplx1 and Nrxn1 was observed in the hippocampus of 12M APP+ mice. Reduced Nrxn1 mRNA level was also found in the cortex of 12M APP+ mice, and lower Syt1 expression in the hippocampus of 3M APP+ mice. An increase in the of SYP and SYT immunoreactivity was observed in the brain cortex of 3M APP+ mice. Administration of FTY720 to 12M APP+ animals increased the reduced expression of Stx1a, Snap25, Cplx1 and Nrxn1 in the hippocampus and Vamp1 in the cerebral cortex. Changes in the ratio pAKT/AKT were demonstrated in the hippocampus – a decrease in 3M APP+ mice and an increase in 12M APP+ mice. In the hippocampus of 12M APP+ animals, an increase in the phosphorylation of GSK-3β kinase and Tau protein on serine 416 was observed. The administration of FTY720 had no significant effect on the observed changes. In vitro studies were performed on mouse hippocampal neurons (HT22) and microglia (BV2) treated for 24 h with 1 μM Aβ1-42 oligomers (Aβo) and S1PR modulators: 0.1 μM ponesimod, 1 μM CYM5541, 0.1 μM CYM50308, 0.1 μM A971432, 0.1 μM siponimod and 1 nM pFTY720. Aβo significantly reduced S1pr1 mRNA level in BV2 cells, which was counteracted by A971432. A decrease in S1pr4 expression was found in HT22 cells treated with Aβo + CYM5541, and an increase – in BV2 cells treated with Aβo + pFTY720 and siponimod. Aβo significantly reduced the mitochondrial membrane potential and cell viability, and the tested modulators at the concentrations used had no effect on the observed changes. In the BV2 line, Aβo induced a reduction in Bad expression, which was not counteracted by the tested modulators. Administration of Aβo + ponesimod, CYM5541, and CYM50308 increased Bax expression in BV2 cells. In both cell lines, after administration of Aβo, decreased Bcl2 expression was found, which in HT22 cells was restored to control values after treatment with CYM50308 and A971432. In the BV2 line treated with Aβo, an increase in Il1b expression, enhanced by most of the modulators tested, and an increase in Tnf expression were observed. In HT22 cells, an increase in the Il1b mRNA level was induced by the administration of Aβo + siponimod, and an increase in Tnf expression was induced by the administration of Aβo + ponesimod. In both cell lines, an Aβo-induced increase in the mRNA level of Il6 was found, which was reduced in HT22 cells after pFTY720 administration, as well as a decrease in Il18 expression, which was not influenced by the S1PR modulators. The results of in vivo studies indicate that the effect of the London mutation in the APP gene depends on the brain structure and age of the animals and may be visible at the transcriptional level or at the level of posttranslational modifications. Age-dependent effect of fingolimod in reducing negative changes in the expression of genes encoding presynaptic proteins was also found. The results of in vitro studies indicate a negative effect of Aβo on the expression of genes for apoptotic proteins, proinflammatory cytokines and mitochondrial function, and that the effect of S1PR modulation in Aβo toxicity may vary depending on the cell line.