Alterations in the transcriptional profile of genes related to glutamatergic signalling in animal models of Alzheimer's disease. The effect of fingolimod.

Alzheimer's disease (AD) is a multi-factorial illness that leads to progressive cognitive impairment. A glutamatergic system dysfunction has been reported to be implicated in the pathomechanism of AD. Therefore, in the current study we characterized the transcriptional profile of glutamate-related genes in transgenic AbPP V717I (TgAD) and sporadic (SAD, streptozotocin-induced) models of AD. Genes encoding glutamate membrane-bound (GLAST, GLT1, EAAC1) and vesicular (VGLUT1-3) transporters as well as ionotropic (AMPA, NMDA) and metabotropic (mGluR3, mGluR5) receptors were analysed. Based on qPCR analysis, we observed a discrepancy between TgAD and SAD mice in the profile of targeted genes. We noticed age-dependent upregulation of genes encoding VGLUT1, NMDAR1 and mGluR3 in 12-month-old TgAD mice. In the SAD model upregulation of genes encoding AMPAR1 and NMDAR1 as well as downregulation of GLAST, VGLUT3 and mGluR5 were found. Next, the effect of fingolimod (FTY720) was indicated. In the TgAD model, the drug reversed altered transcription of the mGluR3 glutamate receptor to the control level, whereas in the SAD model it downregulated the genes encoding VGLUT1, AMPAR2 and mGluR3. Interestingly, FTY720 influenced mGluR3 mRNA in both examined models. Observed alterations of gene transcription and the effects of FTY720 may potentially constitute an interesting target for further pharmacological studies.

Age-Related Transcriptional Deregulation of Genes Coding Synaptic Proteins in Alzheimer's Disease Murine Model: Potential Neuroprotective Effect of Fingolimod.

Alzheimer's disease (AD) induces time-dependent changes in sphingolipid metabolism, which may affect transcription regulation and neuronal phenotype. We, therefore, analyzed the influence of age, amyloid ß precursor protein (AßPP), and the clinically approved, bioavailable sphingosine-1-phosphate receptor modulator fingolimod (FTY720) on the expression of synaptic proteins. RNA was isolated, reverse-transcribed, and subjected to real-time PCR. Expression of mutant (V717I) AßPP led to few changes at 3 months of age but reduced multiple mRNA coding for synaptic proteins in a 12-month-old mouse brain. Complexin 1 (Cplx1), SNAP25 (Snap25), syntaxin 1A (Stx1a), neurexin 1 (Nrxn1), neurofilament light (Nefl), and synaptotagmin 1 (Syt1) in the hippocampus, and VAMP1 (Vamp1) and neurexin 1 (Nrxn1) in the cortex were all significantly reduced in 12-month-old mice. Post mortem AD samples from the human hippocampus and cortex displayed lower expression of VAMP, synapsin, neurofilament light (NF-L) and synaptophysin. The potentially neuroprotective FTY720 reversed most AßPP-induced changes in gene expression (Cplx1, Stx1a, Snap25, and Nrxn1) in the 12-month-old hippocampus, which is thought to be most sensitive to early neurotoxic insults, but it only restored Vamp1 in the cortex and had no influence in 3-month-old brains. Further study may reveal the potential usefulness of FTY720 in the modulation of deregulated neuronal phenotype in AD brains.

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.

The Role of Ceramide and Sphingosine-1-Phosphate in Alzheimer's Disease and Other Neurodegenerative Disorders.

Bioactive sphingolipids-ceramide, sphingosine, and their respective 1-phosphates (C1P and S1P)-are signaling molecules serving as intracellular second messengers. Moreover, S1P acts through G protein-coupled receptors in the plasma membrane. Accumulating evidence points to sphingolipids' engagement in brain aging and in neurodegenerative disorders such as Alzheimer's, Parkinson's, and Huntington's diseases and amyotrophic lateral sclerosis. Metabolic alterations observed in the course of neurodegeneration favor ceramide-dependent pro-apoptotic signaling, while the levels of the neuroprotective S1P are reduced. These trends are observed early in the diseases' development, suggesting causal relationship. Mechanistic evidence has shown links between altered ceramide/S1P rheostat and the production, secretion, and aggregation of amyloid ß/α-synuclein as well as signaling pathways of critical importance for the pathomechanism of protein conformation diseases. Sphingolipids influence multiple aspects of Akt/protein kinase B signaling, a pathway that regulates metabolism, stress response, and Bcl-2 family proteins. The cross-talk between sphingolipids and transcription factors including NF-κB, FOXOs, and AP-1 may be also important for immune regulation and cell survival/death. Sphingolipids regulate exosomes and other secretion mechanisms that can contribute to either the spread of neurotoxic proteins between brain cells, or their clearance. Recent discoveries also suggest the importance of intracellular and exosomal pools of small regulatory RNAs in the creation of disturbed signaling environment in the diseased brain. The identified interactions of bioactive sphingolipids urge for their evaluation as potential therapeutic targets. Moreover, the early disturbances in sphingolipid metabolism may deliver easily accessible biomarkers of neurodegenerative disorders.

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.

The role of ceramide and SEW 2871 in the transcription of enzymes involved in amyloid b precursor protein metabolism in an experimental model of Alzheimer's disease.

Alzheimer's disease (AD) is characterized by alterations of amyloid precursor protein (APP) metabolism, accumulation of amyloid  peptides (A), hyperphosphorylation of Tau proteins and also by sphingolipids disturbances. These changes lead to oxidative stress, mitochondria dysfunction, synaptic loss and neuro-inflammation. It is known that A may promote ceramides formation and reversely, ceramides could stimulate A peptides release. However, the effect of ceramide and sphingosine-1-phosphate (S1P) on APP metabolism has not been fully elucidated. In this study we investigated the role of ceramide and S1P on APP metabolism. Moreover, the effect of ceramide and SEW 2871 (agonist for S1P receptor-1) on Sirt1 (NAD+-dependent nuclear enzyme responsible for stress response) gene expression under A toxicity was analyzed. Experiments were carried out using pheochromocytoma cells (PC-12) transfected with: an empty vector (used as a control), human wild-type APP gene (APPwt) and Swedish mutated (K670M/N671L) APP gene (APPsw). Our results indicated that C2-ceramide significantly decreased the viability of the APPwt, APPsw as well as empty vector-transfected PC12 cells. It was observed that C2-ceramide had no significant effect on the mRNA level of - and -secretase in APPwt and APPsw cells. However, it significantly decreased transcription of -secretase in control cells. Results also showed a significant increase in Psen1 (crucial subunit of -secretase) gene expression in APPsw cells after incubation with C2-ceramide. We observed that SEW 2871 significantly upregulated the mRNA level of -secretase in control-empty vector-transfected cells subjected to C2-ceramide toxicity. The same tendency, though insignificant, was observed in APPwt and APPsw cells. Moreover, SEW 2871 enhanced the mRNA level of -secretase and Psen1 in APPsw cells after C2-ceramide treatment. Additionally, SEW 2871 significantly upregulated a gene expression of Sirt1 in APPwt and also APPsw cells subjected to C2-ceramide toxicity. Furthermore, it was observed that SEW 2871 significantly enhanced the viability of all investigated cells' lines probably through its positive influence on Sirt1.

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.

Sirtuins and Their Roles in Brain Aging and Neurodegenerative Disorders.

Sirtuins (SIRT1-SIRT7) are unique histone deacetylases (HDACs) whose activity depends on NAD+ levels and thus on the cellular metabolic status. SIRTs regulate energy metabolism and mitochondrial function. They orchestrate the stress response and damage repair. Through these functions sirtuins modulate the course of aging and affect neurodegenerative diseases. SIRTSs interact with multiple signaling proteins, transcription factors (TFs) and poly(ADP-ribose) polymerases (PARPs) another class of NAD+-dependent post-translational protein modifiers. The cross-talk between SIRTs TFs and PARPs is a highly promising research target in a number of brain pathologies. This review describes updated results on sirtuins in brain aging/neurodegeneration. It focuses on SIRT1 but also on the roles of mitochondrial SIRTs (SIRT3, 4, 5) and on SIRT6 and SIRT2 localized in the nucleus and in cytosol, respectively. The involvement of SIRTs in regulation of insulin-like growth factor signaling in the brain during aging and in Alzheimer's disease was also focused. Moreover, we analyze the mechanism(s) and potential significance of interactions between SIRTs and several TFs in the regulation of cell survival and death. A critical view is given on the application of SIRT activators/modulators in therapy of neurodegenerative diseases.

The Synthesis of 1,3,5-triazine Derivatives and JNJ7777120 Analogues with Histamine H4 Receptor Affinity and Their Interaction with PTEN Promoter.

The involvement of histamine and H4 receptor (H4 R) in cancer has been investigated recently using the H4 R agonists and antagonists. The scope of the research project was synthesis and exploration of the consequences of a group of compounds with histamine H4 receptor (H4 R) affinity on the promoter of PTEN gene encoding the antitumor PTEN protein. The series of novel compounds based either on H4 R antagonists JNJ7777120 structure or 1,3,5-triazine scaffold were synthesized, evaluated for histamine H4 R affinity and used in this study. Compounds 5 and 7 belonging to the group of JNJ7777120 analogues showed the highest interaction with the promoter of PTEN gene and weak affinity against H4 R with Ki value >100 µm. These compounds showed no significant effect on neuroblastoma IMR-32 cells viability indicating no correlation between PTEN gene promoter affinity and antitumor activity. Compound 6, another JNJ7777120 analogue, showed the highest effect on IMR-32 viability with calculated IC50 = 23.27 µm. The 1,3,5-triazine derivatives exhibited generally low or medium interaction with PTEN gene promoter. However, the 1,3,5-triazine derivative 11 with the para-bromo substituent showed the highest affinity against H4 R with Ki value of 520 nm and may be considered as a new lead structure.