Fingolimod Affects Transcription of Genes Encoding Enzymes of Ceramide Metabolism in Animal Model of Alzheimer's Disease.

The imbalance in sphingolipid signaling may be critically linked to the upstream events in the neurodegenerative cascade of Alzheimer's disease (AD). We analyzed the influence of mutant (V717I) amyloid ß precursor protein (AßPP) transgene on sphingolipid metabolism enzymes in mouse hippocampus. At 3 months of age AßPP/Aß presence upregulated enzymes of ceramide turnover on the salvage pathway: ceramide synthases (CERS2, CERS4, CERS6) and also ceramidase ACER3. At 6 months, only CERS6 was elevated, and no ceramide synthase was increased at 12 months. However, sphingomyelin synthases, which utilize ceramide on the sphingomyelinase pathway, were reduced (SGMS1 at 12 and SGMS2 at 6 months). mRNAs for sphingomyelin synthases SGMS1 and SGMS2 were also significantly downregulated in human AD hippocampus and neocortex when compared with age-matched controls. Our findings suggest early-phase deregulation of sphingolipid homeostasis in favor of ceramide signaling. Fingolimod (FTY720), a modulator of sphingosine-1-phosphate receptors countered the AßPP-dependent upregulation of hippocampal ceramide synthase CERS2 at 3 months. Moreover, at 12 months, FTY720 increased enzymes of ceramide-sphingosine turnover: CERS4, ASAH1, and ACER3. We also observed influence of fingolimod on the expression of the sphingomyelinase pathway enzymes. FTY720 counteracted the AßPP-linked reduction of sphingomyelin synthases SGMS1/2 (at 12 and 6 months, respectively) and led to elevation of sphingomyelinase SMPD2 (at 6 and 12 months). Therefore, our results demonstrate potentially beneficial, age-specific effects of fingolimod on transcription of sphingolipid metabolism enzymes in an animal model of AD.

Alterations of Transcription of Genes Coding Anti-oxidative and Mitochondria-Related Proteins in Amyloid ß Toxicity: Relevance to Alzheimer's Disease.

A growing body of evidence indicates that pathological forms of amyloid beta (Aß) peptide contribute to neuronal degeneration and synaptic loss in Alzheimer's disease (AD). In this study, we investigated the impact of exogenous Aß1-42 oligomers (AßO) and endogenously liberated Aß peptides on transcription of genes for anti-oxidative and mitochondria-related proteins in cell lines (neuronal SH-SY5Y and microglial BV2) and in brain cortex of transgenic AD (Tg-AD) mice, respectively. Our results demonstrated significant AßO-evoked changes in transcription of genes in SH-SY5Y cells, where AßO enhanced expression of Sod1, Cat, mt-Nd1, Bcl2, and attenuated Sirt5, Sod2 and Sdha. In BV2 line, AßO increased the level of mRNA for Sod2, Dnm1l, Bcl2, and decreased for Gpx4, Sirt1, Sirt3, mt-Nd1, Sdha and Mfn2. Then, AßO enhanced free radicals level and impaired mitochondrial membrane potential only in SH-SY5Y cells, but reduced viability of both cell types. Inhibitor of poly(ADP-ribose)polymerase-1 and activator of sirtuin-1 more efficiently enhanced viability of SH-SY5Y than BV2 affected by AßO. Analysis of brain cortex of Tg-AD mice confirmed significant downregulation of Sirt1, Mfn1 and mt-Nd1 and upregulation of Dnm1l. In human AD brain, changes of microRNA pattern (miRNA-9, miRNA-34a, miRNA-146a and miRNA-155) seem to be responsible for decrease in Sirt1 expression. Overall, our results demonstrated a diverse response of neuronal and microglial cells to AßO toxicity. Alterations of genes encoding Sirt1, Mfn1 and Drp1 in an experimental model of AD suggest that modulation of mitochondria dynamics and Sirt1, including miRNA strategy, may be crucial for improvement of AD therapy.

Modulatory Effects of Fingolimod (FTY720) on the Expression of Sphingolipid Metabolism-Related Genes in an Animal Model of Alzheimer's Disease.

Sphingolipid signaling disturbances correlate with Alzheimer's disease (AD) progression. We examined the influence of FTY720/fingolimod, a sphingosine analog and sphingosine-1-phosphate (S1P) receptor modulator, on the expression of sphingolipid metabolism and signaling genes in a mouse transgenic AD model. Our results demonstrated that AßPP (V717I) transgene led with age to reduced mRNA expression of S1P receptors (S1PRs), sphingosine kinase SPHK2, ceramide kinase CERK, and the anti-apoptotic Bcl2 in the cerebral cortex and hippocampus, suggesting a pro-apoptotic shift in 12-month old mice. These changes largely emulated alterations we observed in the human sporadic AD hippocampus: reduced SPHK1, SPHK2, CERK, S1PR1, and BCL2. We observed that the responses to FTY720 treatment were modified by age and notably differed between control (APP-) and AD transgenic (APP+) animals. AßPP (V717I)-expressing 12-month-old animals reacted to fingolimod with wide changes in the gene expression program in cortex and hippocampus, including increased pro-survival SPHKs and CERK. Moreover, BCL2 was elevated by FTY720 in the cortex at all ages (3, 6, 12 months) while in hippocampus this increase was observed at 12 months only. In APP- mice, fingolimod did not induce any significant mRNA changes at 12 months. Our results indicate significant effect of FTY720 on the age-dependent transcription of genes involved in sphingolipid metabolism and pro-survival signaling, suggesting its neuroprotective role in AD animal model.

Inhibition of poly(ADP-ribose) polymerase-1 alters expression of mitochondria-related genes in PC12 cells: relevance to mitochondrial homeostasis in neurodegenerative disorders.

Alzheimer's disease (AD) is characterized by the release of amyloid beta peptides (Aß) in the form of monomers/oligomers which may lead to oxidative stress, mitochondria dysfunction, synaptic loss, neuroinflammation and, in consequence, to overactivation of poly(ADP-ribose) polymerase-1 (PARP-1). However, Aß peptides are also released in the brain ischemia, traumatic injury and in inflammatory response. PARP-1 is suggested to be a promising target in therapy of neurodegenerative disorders. We investigated the impact of PARP-1 inhibition on transcription of mitochondria-related genes in PC12 cells. Moreover, the effect of PARP-1 inhibitor (PJ34) on cells subjected to Aß oligomers (AßO) - evoked stress was analyzed. Our data demonstrated that inhibition of PARP-1 in PC12 cells enhanced the transcription of genes for antioxidative enzymes (Sod1, Gpx1, Gpx4), activated genes regulating mitochondrial fission/fusion (Mfn1, Mfn2, Dnm1l, Opa1, Fis1), subunits of ETC complexes (mt-Nd1, Sdha, mt-Cytb) and modulated expression of several TFs, enhanced Foxo1 and decreased Nrf1, Stat6, Nfkb1. AßO elevated free radicals concentration, decreased mitochondria membrane potential (MMP) and cell viability after 24h. Gene transcription was not affected by AßO after 24h, but was significantly downregulated after 96h. In AßO stress, PJ34 exerted stimulatory effect on expression of several genes (Gpx1, Gpx4, Opa1, Mfn2, Fis1 and Sdha), decreased transcription of numerous TFs (Nrf1, Tfam, Stat3, Stat6, Trp53, Nfkb1) and prevented oxidative stress. Our results indicated that PARP-1 inhibition significantly enhanced transcription of genes involved in antioxidative defense and in regulation of mitochondria function, but was not able to ameliorate cells viability affected by Aß.

Inhibition of Poly(ADP-ribose) Polymerase-1 Enhances Gene Expression of Selected Sirtuins and APP Cleaving Enzymes in Amyloid Beta Cytotoxicity.

Poly(ADP-ribose) polymerases (PARPs) and sirtuins (SIRTs) are involved in the regulation of cell metabolism, transcription, and DNA repair. Alterations of these enzymes may play a crucial role in Alzheimer's disease (AD). Our previous results indicated that amyloid beta (Aß) peptides and inflammation led to activation of PARP1 and cell death. This study focused on a role of PARP1 in the regulation of gene expression for SIRTs and beta-amyloid precursor protein (ßAPP) cleaving enzymes under Aß42 oligomers (AßO) toxicity in pheochromocytoma cells (PC12) in culture. Moreover, the effect of endogenously liberated Aß peptides in PC12 cells stably transfected with human gene for APP wild-type (APPwt) was analyzed. Our results demonstrated that AßO enhanced transcription of presenilins (Psen1 and Psen2), the crucial subunits of γ-secretase. Aß peptides in APPwt cells activated expression of ß-secretase (Bace1), Psen1, Psen2, and Parp1. The inhibitor of PARP1, PJ-34 in the presence of AßO upregulated transcription of α-secretase (Adam10), Psen1, and Psen2, but also Bace1. Concomitantly, PJ-34 enhanced mRNA level of nuclear Sirt1, Sirt6, mitochondrial Sirt4, and Parp3 in PC12 cells subjected to AßOs toxicity. Our data indicated that Aß peptides through modulation of APP secretases may lead to a vicious metabolic circle, which could be responsible for maintaining Aß at high level. PARP1 inhibition, besides activation of nuclear SIRTs and mitochondrial Sirt4 expression, enhanced transcription of enzyme(s) involved in ßAPP metabolism, and this effect should be considered in its application against Aß peptide toxicity.