Recent Insights into the Interplay of Alpha-Synuclein and Sphingolipid Signaling in Parkinson's Disease.

Molecular studies have provided increasing evidence that Parkinson's disease (PD) is a protein conformational disease, where the spread of alpha-synuclein (ASN) pathology along the neuraxis correlates with clinical disease outcome. Pathogenic forms of ASN evoke oxidative stress (OS), neuroinflammation, and protein alterations in neighboring cells, thereby intensifying ASN toxicity, neurodegeneration, and neuronal death. A number of evidence suggest that homeostasis between bioactive sphingolipids with opposing function-e.g., sphingosine-1-phosphate (S1P) and ceramide-is essential in pro-survival signaling and cell defense against OS. In contrast, imbalance of the "sphingolipid biostat" favoring pro-oxidative/pro-apoptotic ceramide-mediated changes have been indicated in PD and other neurodegenerative disorders. Therefore, we focused on the role of sphingolipid alterations in ASN burden, as well as in a vast range of its neurotoxic effects. Sphingolipid homeostasis is principally directed by sphingosine kinases (SphKs), which synthesize S1P-a potent lipid mediator regulating cell fate and inflammatory response-making SphK/S1P signaling an essential pharmacological target. A growing number of studies have shown that S1P receptor modulators, and agonists are promising protectants in several neurological diseases. This review demonstrates the relationship between ASN toxicity and alteration of SphK-dependent S1P signaling in OS, neuroinflammation, and neuronal death. Moreover, we discuss the S1P receptor-mediated pathways as a novel promising therapeutic approach in PD.

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.

The Cross-Talk Between Sphingolipids and Insulin-Like Growth Factor Signaling: Significance for Aging and Neurodegeneration.

Bioactive sphingolipids: sphingosine, sphingosine-1-phosphate (S1P), ceramide, and ceramide-1-phosphate (C1P) are increasingly implicated in cell survival, proliferation, differentiation, and in multiple aspects of stress response in the nervous system. The opposite roles of closely related sphingolipid species in cell survival/death signaling is reflected in the concept of tightly controlled sphingolipid rheostat. Aging has a complex influence on sphingolipid metabolism, disturbing signaling pathways and the properties of lipid membranes. A metabolic signature of stress resistance-associated sphingolipids correlates with longevity in humans. Moreover, accumulating evidence suggests extensive links between sphingolipid signaling and the insulin-like growth factor I (IGF-I)-Akt-mTOR pathway (IIS), which is involved in the modulation of aging process and longevity. IIS integrates a wide array of metabolic signals, cross-talks with p53, nuclear factor κB (NF-κB), or reactive oxygen species (ROS) and influences gene expression to shape the cellular metabolic profile and stress resistance. The multiple connections between sphingolipids and IIS signaling suggest possible engagement of these compounds in the aging process itself, which creates a vulnerable background for the majority 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.

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.

Opportunities for the repurposing of PARP inhibitors for the therapy of non-oncological diseases.

The recent clinical availability of the PARP inhibitor olaparib (Lynparza) opens the door for potential therapeutic repurposing for non-oncological indications. Considering (a) the preclinical efficacy data with PARP inhibitors in non-oncological diseases and (b) the risk-benefit ratio of treating patients with a compound that inhibits an enzyme that has physiological roles in the regulation of DNA repair, we have selected indications, where (a) the severity of the disease is high, (b) the available therapeutic options are limited, and (c) the duration of PARP inhibitor administration could be short, to provide first-line options for therapeutic repurposing. These indications are as follows: acute ischaemic stroke; traumatic brain injury; septic shock; acute pancreatitis; and severe asthma and severe acute lung injury. In addition, chronic, devastating diseases, where alternative therapeutic options cannot halt disease development (e.g. Parkinson's disease, progressive multiple sclerosis or severe fibrotic diseases), should also be considered. We present a preclinical and clinical action plan for the repurposing of PARP inhibitors. LINKED ARTICLES: This article is part of a themed section on Inventing New Therapies Without Reinventing the Wheel: The Power of Drug Repurposing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.2/issuetoc.

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..

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 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.

Sphingosine kinases modulate the secretion of amyloid ß precursor protein from SH-SY5Y neuroblastoma cells: the role of α-synuclein.

Sphingosine kinases (SphK 1&2) are involved in the regulation of cell survival, differentiation and neurotransmitter secretion. Current data suggest potential links between sphingolipid signalling, α-synuclein (ASN) and Alzheimer's disease (AD). Our aim was to investigate the possible role of SphKs and ASN in the regulation of the production and secretion of the amyloid ß precursor protein (APP). We have previously shown that ASN intensified the secretion and toxicity of amyloid ß (Aß) to the point where it caused cell death. Our current results show that APP, the precursor protein for Aß, is also influenced by ASN. The stable overexpression of wtASN in SH-SY5Y cells caused a three-fold, significant increase of the cellular APP level. This suggests that the influence of ASN on Aß metabolism may actually occur at the level of APP protein rather than only through the changes of its cleavage into Aß. To elucidate the mechanisms of APP modulation the cells were exposed to S1P and an SphK inhibitor (SKI). 72 h S1P treatment at 5 µM caused a nearly 50% reduction of the cellular APP signal. S1P also caused a tendency towards higher APP secretion, though the results were insignificant. The inhibition of SphKs decreased medium APP levels in a dose-dependent manner, reaching significance at 5 µM SKI with a correspondingly elevated intracellular level. Thus, it is reasonable to expect that in fact the influence of SphK activity on APP might be pro-secretory. This would also be in agreement with numerous articles on SphK-dependent secretion in the literature. The chronic nature of AD further suggests that subtle alterations in APP metabolism could have the potential to drive important changes in brain condition.

Expression and activity of PARP family members in the hippocampus during systemic inflammation: their role in the regulation of prooxidative genes.

Poly(ADP-ribose)polymerase-1 (PARP-1) plays an important role in DNA repair processes during oxidative/genotoxic stress, in regulation of transcription factors and in cell death mechanisms. However, little is known about the physiopathological role of other PARP family members. In this study we analyzed, for the first time, expression of PARP family genes in the hippocampus of mice subjected to lipopolysaccharide (LPS)-evoked systemic inflammatory response (SIR). Moreover, the effect of SIR on PARP activity and on its role in gene expression was evaluated. Our data indicated that SIR enhances PARP-1, -3, -9, -12 and -14 gene expressions in mouse hippocampus. PARP activity in the hippocampus was also considerably elevated during SIR with a maximum value at 12h after LPS administration, indicating significant formation and accumulation of poly(ADP-ribose) (PAR) - this reaction's product. Concomitantly, higher expression of genes for inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), TNF-α and NADPH oxidase subunits occurred in the hippocampus during the first hours of SIR. The inhibitor of PARP, i.e. 3-aminobenzamide (3-AB; 30mg/kg b.w.), protected the hippocampal cells against PAR formation and expression of the gene for iNOS. However, PARP activity had no effect on the mRNA level of COX-2, TNF-α and NADPH oxidase in the hippocampus. Bioinformatic analysis predicted that interaction between PARP/PAR and 41 transcription factors may be important for regulation of expression of 139 genes from the group of 262 that changed expression in the hippocampus during SIR. Summarizing, enhancement of gene expression and activity of PARP family members by SIR could lead, through modification of the protein poly(ADP-ribosylation) process, to changes in the proteins' activity. A higher level of PAR and PARP/PAR protein interaction may affect the functioning of several transcription factors. PARP activity plays a role in regulation of expression of iNOS, the main enzyme responsible for oxidative/nitrosative stress in SIR.

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.

Natural inhibitors of poly(ADP-ribose) polymerase-1.

Poly(ADP-ribose) polymerases (PARPs) are enzymes that catalyze the transfer of ADP-ribose units from ß-nicotinamide adenine dinucleotide (NAD(+)) to acceptor proteins. PARP-1 is responsible for more than 90 % of protein poly-ADP-ribosylation in the brain and may play a role as a molecular switch for cell survival and death. The functional roles of PARP-1 are largely mediated by its activation after binding to damaged DNA. Upon binding, PARP-1 activity increases rapidly and cleaves NAD(+) into ADP-ribose and nicotinamide. Increased activity of PARP-1 can promote DNA repair and its interaction with several transcription factors, whereas hyperactivation of PARP-1 can result in poly(ADP-ribose) accumulation and depletion of NAD(+) and ATP which may lead to caspase independent apoptotic or necrotic cell death, respectively. Excessive PARP-1 activity has been implicated in the pathogenesis of numerous clinical conditions such as stroke, myocardial infarction, inflammation, diabetes, and neurodegenerative disorders. Therefore, it is not surprising that the search for PARP-1 inhibitors with specific therapeutic uses (e.g., brain ischemia, cancer) has been an active area of research. Beyond medicinal uses, naturally occurring PARP-1 inhibitors may also offer a unique preventative means at attenuating chronic inflammatory diseases through dietary supplementation. This possibility has prompted research for specific, naturally occurring inhibitors of PARP-1. Though fewer investigations focus on identifying endogenous inhibitors/modulators of PARP-1 activity in vivo, these activities are very important for better understanding the complex regulation of this enzyme and the potential long-term benefits of supplementation with PARP-1 inhibitors. With this in mind, the focus of this article will be on providing a state-of-the-science review on endogenous and naturally occurring compounds that inhibit PARP-1.

Poly(ADP-ribose) polymerase-1 in amyloid beta toxicity and Alzheimer's disease.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a key enzyme responsible for the maintenance of genome stability, transcriptional regulation, and long-term potentiation in neurons. However, the excessive activation of PARP-1 under pathological conditions may lead to an accumulation of poly(ADP-ribose) (PAR), a novel signaling molecule that induces programmed cell death, or to NAD depletion that induces energy crisis and necrotic cell death. PARP-1 is thought to be primarily a nuclear enzyme, but some data indicate that it can also be localized to the mitochondria where it is responsible for posttranslational modification of electron transport chain complexes and alteration of mitochondria function. The enhancement of PARP-1 activity and the accumulation of PAR were demonstrated in the brain of patients with Alzheimer's disease (AD), particularly in neurons of the frontal and temporal lobes and in skin fibroblasts and lymphoblasts. Moreover, it has been reported that PARP-1 gene polymorphisms are highly associated with the development of AD. The activation of PARP-1 by oxidative stress seems to be an early and important event in the pathogenesis of AD. It is now widely accepted that the overproduction and oligomerization of amyloid ß (Aß) are responsible for the activation of a free radical cascade and oxidative stress in AD. Interestingly, the activity of PARP-1 is enhanced in AD and is also increased by Aß peptides. The activation of PARP-1 by Aß can lead to the PAR-mediated release of apoptosis-inducing factor from the mitochondria and its translocation to the nucleus, which leads to death of some populations of cells. A role of PARP-1 in the regulation of Aß precursor protein metabolism processing and Aß liberation has not been described previously. The study presented in this review indicated the relationship between PARP-1 activation, alteration of mitochondria function, and Aß toxicity. The presented data should stimulate further studies on the role of PARP-1 in AD pathogenesis and thereby engage a new perspective regarding AD therapy.

Inhibition of poly(ADP-ribose) polymerase-1 attenuates the toxicity of carbon tetrachloride.

Carbon tetrachloride (CCl(4)) is routinely used as a model compound for eliciting centrilobular hepatotoxicity. It can be bioactivated to the trichloromethyl radical, which causes extensive lipid peroxidation and ultimately cell death by necrosis. Overactivation of poly(ADP-ribose) polymerase-1 (PARP-1) can rapidly reduce the levels of ß-nicotinamide adenine dinucleotide and adenosine triphosphate and ultimately promote necrosis. The aim of this study was to determine whether inhibition of PARP-1 could decrease CCl(4)-induced hepatotoxicity, as measured by degree of poly(ADP-ribosyl)ation, serum levels of lactate dehydrogenase (LDH), lipid peroxidation, and oxidative DNA damage. For this purpose, male ICR mice were administered intraperitoneally a hepatotoxic dose of CCl(4) with or without 6(5H)-phenanthridinone, a potent inhibitor of PARP-1. Animals treated with CCl(4) exhibited extensive poly(ADP-ribosyl)ation in centrilobular hepatocytes, elevated serum levels of LDH, and increased lipid peroxidation. In contrast, animals treated concomitantly with CCl(4) and 6(5H)-phenanthridinone showed significantly lower levels of poly(ADP-ribosyl)ation, serum LDH, and lipid peroxidation. No changes were observed in the levels of oxidative DNA damage regardless of treatment. These results demonstrated that the hepatotoxicity of CCl(4) is dependent on the overactivation of PARP-1 and that inhibition of this enzyme attenuates the hepatotoxicity of CCl(4).

Lipoxygenases and poly(ADP-ribose) polymerase in amyloid beta cytotoxicity.

The 12/15-lipoxygenase(s) (LOX), poly(ADP-ribose) polymerase (PARP-1) activity and mitochondrial apoptosis inducing factor (AIF) protein in the amyloid ß (Aß) toxicity were investigated in PC12 cells that express either wild-type (APPwt) or double Swedish mutation (APPsw) forms of human Aß precursor protein. Different levels of Aß secretion and free radicals formation characterize these cells. The results demonstrated a relationship between the Aß levels and LOX protein expression and activity. High Aß concentration in APPsw cells correlated with a significant increase in free radicals and LOX activation, which leads to translocation of p65/NF-κB into the nucleus. An increase in AIF expression in mitochondria was observed concurrently with inhibition of PARP-1 activity in the nuclear fraction of APPsw cells. We suggested that AIF accumulation in mitochondria may be involved in adaptive/protective processes. However, inhibition of PARP-1 may be responsible for the disturbances in transcription and DNA repair as well as the degeneration of APP cells. Under conditions of increased nitrosative stress, evoked by the nitric oxide donor, sodium nitroprusside (SNP, 0.5 mM), 70-80% of all cells types died after 24 h, significantly more in APPsw cells. There was no further significant change in mitochondrial AIF level and PARP-1 activity compared to corresponding non-treated cells. Only one exception was observed in PC12 control, where SNP significantly inhibits PARP-1 activity. Moreover, SNP significantly activated gene expression for 12/15-LOX in all types of investigated cells. Inhibitors of all LOX isoforms and specific inhibitor of 12-LOX enhanced the survival of cells that were subjected to SNP. We conclude that the LOX pathways may play a role in Aß toxicity and in nitrosative-stress-induced cell death and that inhibition of these pathways offers novel protective strategies.

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.