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.

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.

Levels of selected pro- and anti-inflammatory cytokines in cerebrospinal fluid in patients with hydrocephalus.

Cytokines are widely known mediators of inflammation accompanying many neurodegenerative disorders including normal pressure hydrocephalus (NPH). NPH is caused by impaired cerebrospinal fluid (CSF) absorption and treated by surgical shunt insertion. The early diagnosis of NPH is difficult because of various manifestations of the disease. One of the most promising research directions is biochemical CSF analysis. The aim of this study was to determine the CSF levels of cytokines. The levels of various cytokines (IL-6, IL-8, IL-12, IL-10 and TNF-α) were measured in patients with idiopathic active normal pressure hydrocephalus, arrested hydrocephalus and hydrocephalus with brain atrophy compared to controls. Our study showed that the concentrations of IL-6 and IL-8 were significantly elevated in the group with idiopathic active hydrocephalus compared to control patients. Moreover, we observed that the levels of IL-6 and IL-8 in the group with idiopathic active hydrocephalus were significantly higher compared to patients with arrested hydrocephalus and hydrocephalus with brain atrophy..

The Lipoxygenases: Their Regulation and Implication in Alzheimer's Disease.

Inflammatory processes and alterations of lipid metabolism play a crucial role in Alzheimer's disease (AD) and other neurodegenerative disorders. Polyunsaturated fatty acids (PUFA) metabolism impaired by cyclooxygenases (COX-1, COX-2), which are responsible for formation of several eicosanoids, and by lipoxygenases (LOXs) that catalyze the addition of oxygen to linolenic, arachidonic (AA), and docosahexaenoic acids (DHA) and other PUFA leading to formation of bioactive lipids, significantly affects the course of neurodegenerative diseases. Among several isoforms, 5-LOX and 12/15-LOX are especially important in neuroinflammation/neurodegeneration. These two LOXs are regulated by substrate concentration and availability, and by phosphorylation/dephosphorylation through protein kinases PKA, PKC and MAP-kinases, including ERK1/ERK2 and p38. The protein/protein interaction also is involved in the mechanism of 5-LOX regulation through FLAP protein and coactosin-like protein. Moreover, non-heme iron and calcium ions are potent regulators of LOXs. The enzyme activity significantly depends on the cell redox state and is differently regulated by various signaling pathways. 5-LOX and 12/15-LOX convert linolenic acid, AA, and DHA into several bioactive compounds e.g. hydroperoxyeicosatetraenoic acids (5-HPETE, 12S-HPETE, 15S-HPETE), which are reduced to corresponding HETE compounds. These enzymes synthesize several bioactive lipids, e.g. leucotrienes, lipoxins, hepoxilins and docosahexaenoids. 15-LOX is responsible for DHA metabolism into neuroprotectin D1 (NPD1) with significant antiapoptotic properties which is down-regulated in AD. In this review, the regulation and impact of 5-LOX and 12/15-LOX in the pathomechanism of AD is discussed. Moreover, we describe the role of several products of LOXs, which may have significant pro- or anti-inflammatory activity in AD, and the cytoprotective effects of LOX inhibitors.

Sphingosine-1-phosphate and its effect on glucose deprivation/glucose reload stress: from gene expression to neuronal survival.

Sphingosine kinase-1 (Sphk1-1, EC 2.7.1.91) is a regulator of pro-survival signalling, and its alterations have been observed in Alzheimer's disease, brain ischemia and other neurological disorders. In this study we addressed the question whether Sphk1 and its product, sphingosine-1-phosphate (S1P), play a significant role in glucose deprivation (GD)/glucose reload (GR) stress in hippocampal neuronal cells (HT22). It was found that GD (6 h) followed by 24 h of GR evoked enhancement of the free radical level and neuronal HT22 cell death. Moreover, the significantly stronger gene expression for the pro-apoptotic Bax protein and down-regulation of the anti-apoptotic Bcl-2 and Bcl-XL proteins were observed. Concomitantly, this stress up-regulated: gene expression, protein level and activity of Sphk1. Exogenous S1P at 1 µM concentration and the other agonists of the S1P1 receptor (SEW 2871 and P-FTY720) enhanced HT22 cell viability affected by GD/GR stress. This mechanism is mediated by S1P receptor(s) signalling and by the activation of gene expression for Bcl-2 and Bcl-XL. Summarising, our data suggest that sphingolipid metabolism may play an important role in the early events that take place in neuronal cell survival/death under GD/GR stress. Our data demonstrate that exogenous S1P, through the activation of specific receptors S1P1 and S1P3 signalling pathways, regulates the gene expression for anti-apoptotic proteins and enhances neuronal cell survival affected by GD/GR stress.

Ceramide in the molecular mechanisms of neuronal cell death. The role of sphingosine-1-phosphate.

Ceramide and sphingosine-1-phosphate (S1P), two important bioactive sphingolipids, have been suggested as being key players in the pathology of Alzheimer's disease in inflammation and cancer. However, their role in the molecular mechanisms of neuronal death has not been fully elucidated. Our study indicated that ceramide significantly enhanced the level of free radicals and decreased the viability of the human neuroblastoma cell line (SH-SY5Y) through inhibition of the prosurvival PI3-K/Akt pathway. Ceramide also decreased anti-apoptotic (Bcl-2) and increased pro-apoptotic (Bax, Hrk) mRNA/protein levels. Concomitantly, our study indicated that ceramide induced poly(ADP-ribose) polymerase-1 (PARP-1) activation and accumulation of poly(ADP-ribose) PAR, a signalling molecule involved in mitochondria-nucleus cross-talk and mitochondria integrity. Ceramide treatment significantly decreased the level of apoptosis-inducing factor (AIF) in the mitochondria. The PARP-1 inhibitor (PJ-34) prevented AIF release from the mitochondria. In addition, our data showed that exogenously added S1P increased the viability of SH-SY5Y through the S1P (1,3) receptor-dependent mechanism. It was also revealed that the S1P and PARP-1 inhibitor (PJ-34) decreased oxidative stress, gene expression of the pro-apoptotic Hrk protein and up-regulated the anti-apoptotic Bcl-2 protein. Our data demonstrate that neuronal cell death evoked by ceramide is regulated by PARP/PAR/AIF and by S1P receptor signalling. In summary, our results suggest that PARP-1 inhibitor(s) and modulators of sphingosine-1-phosphate receptor(s) should be considered in potential therapeutic strategies directed at neurodegenerative diseases.

Evaluation of the antioxidative properties of lipoxygenase inhibitors.

BACKGROUND: Oxidative stress is a component of many pathological conditions including neurodegenerative diseases and inflammation. An important source of reactive oxygen species (ROS) are lipoxygenases (LOX) - enzymes responsible for the metabolism of arachidonic acid and other polyunsaturated fatty acids. LOX inhibitors have a protective effect in inflammatory diseases and in neurodegenerative disorders because of their anti-inflammatory activity. However, the molecular mechanism of the protective action of LOX inhibitors has not yet been fully elucidated. METHODS: The aim of this study was to compare the antioxidative potential of widely used LOX inhibitors: BWB70C, AA-861, zileuton, baicalein and NDGA. The antioxidative properties were evaluated in cell-free systems. We measured the effect of the tested compounds on iron/ascorbate-induced lipid peroxidation and on carbonyl group formation in the rat brain homogenate. Direct free radical scavenging was analyzed by using DPPH assay. RESULTS: Our data showed that the inhibitor of all LOXs, i.e., NDGA, 5-LOX inhibitor BWB70C and the inhibitor of 12/15-LOX, baicalein, significantly decreased the level of lipid and protein oxidation. The free radical scavenging activity of these inhibitors was comparable to known ROS scavengers, i.e., resveratrol and trolox. Zileuton (the inhibitor of 5-LOX) slightly prevented lipid and protein oxidation, it also scavenged the DPPH radical. AA-861 (the inhibitor of 5 and 12/15-LOX) slightly protected lipids against Fe/asc-evoked lipid peroxidation at high concentrations, but had no effect on carbonyl group formation and DPPH scavenging. CONCLUSIONS: Our results indicate that some LOX inhibitors demonstrate potent anti-oxidative, free radical scavenging properties. AA-861, whose antioxidative potential is very weak, may be a specific tool to be used in experimental and perhaps even clinical applications.

Poly(ADP-ribose) metabolism in brain and its role in ischemia pathology.

The biological roles of poly(ADP-ribose) polymers (PAR) and poly(ADP-ribosyl)ation of proteins in the central nervous system are diverse. The homeostasis of PAR orchestrated by poly(ADP-ribose) polymerase-1 (PARP-1) and poly(ADP-ribose) glycohydrolase (PARG) is crucial for cell physiology and pathology. Both enzymes are ubiquitously distributed in neurons and glia; however, they are segregated at the subcellular level. PARP-1 serves as a "nick sensor" for single- or double-stranded breaks in DNA and is involved in long and short patch base-excision repair, while PARG breaks down PAR. The stimulation of PARP-1 and PAR formation can activate proinflammatory transcription factors, including nuclear factor kappa B. However, hyperactivation of PARP-1 can result in depletion of NAD/ATP, and in PAR-dependent mitochondrial pore formation leading to release of apoptosis inducing factor and cell death. The role of PAR as a death signaling molecule in brain ischemia-reperfusion and inflammation as well as the effect of gender and aging is presented in this review. Modulating the PAR level through pharmacological or genetic intervention on PARP-1/PARG activity and gene expression should be a valuable way for neuroprotective strategy.

Lipoxygenase inhibitors protect brain cortex macromolecules against oxidation evoked by nitrosative stress.

Lipoxygenases (LOX) are a family of enzymes that are responsible for the metabolism of arachidonic and docosahexaenoic acid and for the formation of several eicosanoids and docosanoids, including leukotrienes, lipoxins and neuroprotectins. Depending on cells' redox state and other milieu conditions, these enzymes are engaged in oxidative stress and cell death mechanisms or in cell protection. In this study the antioxidative properties of several inhibitors of LOX isoforms were evaluated. We investigated the effect of a non-selective inhibitor of all LOXs and selective inhibitors of 5-LOX and 12-LOX on lipid and protein oxidation in brain cortex subjected to nitrosative stress, on dityrosine formation and on survival of an immortalized clonal mouse hippocampal cell line (HT22). The nitrosative stress was induced by nitric oxide (NO) donor, 0.5 mM sodium nitroprusside (SNP) and peroxynitrite (0.03 mM). Our data showed that nitrosative stress led to significant enhancement of lipid peroxidation and carbonyl group formation in brain cortex homogenate compared to control. Inhibitor of all LOXs nordihydroguaiaretic acid, 5-LOX inhibitors (zileuton, BWB70C), and inhibitors of 12/15-LOX baicalein and AA-861 (also an inhibitor of 5-LOX) significantly reduced, in a concentration dependent manner (1-10 µM), the level of lipid and protein oxidation. However, AA-861 and zileuton had no effect on carbonyl group formation. Moreover, we observed that LOX inhibitors protected a significant pool of HT22 cells against death evoked by 0.5 mM SNP. In summary, our results indicate that all LOX inhibitors in concentrations above 1-2.5 M demonstrated antioxidative properties. These results should be taken into consideration during evaluation of experimental and clinical effects of LOX inhibitors.