Robert P. Strosznajder

Poly(ADP-ribose) polymerase: the nuclear target in signal transduction and its role in brain ischemia-reperfusion injury.

Poly(ADP-ribose) polymerase (PARP)-1 is a DNA nick sensor that transforms ADP-ribose from βNAD+ in the form of polymer to over 40 nuclear proteins, particularly to histones, several transcription factors, and PARP itself, modulating their activities and functions. PARP-1 activated by DNA breaks facilitates transcription, replication, and DNA base excision repair. The last studies indicate that PARP-1 is the new nuclear target for fast signals evoked in cell membranes by depolarization and cholinergic and glutaminergic receptors stimulation. Excessive activation of PARP-1 by peroxynitrate-evoked DNA damage during oxidative stress can cause cell death by NAD+/ATP depletion after ischemia-reperfusion injury, inflammation, and diabetes mellitus. The PARP-1 through interaction with nuclear factor-κB, p53, and other transcription factors might significantly modulate cell survival and death and a type of death pathway. The pharmacological modulation of PARP-1 might offer a new effective approach for neuroprotection.

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 Alzheimers 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 β (Aβ) 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 Aβ peptides. The activation of PARP-1 by Aβ 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 Aβ precursor protein metabolism processing and Aβ liberation has not been described previously. The study presented in this review indicated the relationship between PARP-1 activation, alteration of mitochondria function, and Aβ 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.

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.

Poly(ADP-ribose) polymerase during reperfusion after transient forebrain ischemia: its role in brain edema and cell death.

The activation of poly(ADP-ribose) polymerase (PARP) in the reperfused brain after ischemia has been assumed but never has been directly presented. Our studies indicate a different dynamic of PARP activity alteration in hippocampus during reperfusion after 3 and 10 min of transient forebrain ischemia in gerbils. The phasic stimulation of PARP activity was observed during reperfusion 15 min, 120 min, and 4 d after 3 min of ischemia with subsequent lowering of its activity close to control value on the seventh day of reperfusion. After 10 min of ischemic insult, PARP activity significantly increased from the third to the seventh day of reperfusion. The protein level of PARP was not significantly changed during reperfusion after 3 and 10 min of ischemia, with one exception: On the third day after 10 min of ischemia, PARP protein level was 28% lower compared to control; however, no enhancement of 85-kDa protein immunoreactivity was observed. These data indicate the lack of PARP cleavage in hippocampus of gerbils subjected to ischemia-reperfusion injury. The inhibitor of PARP, 3-aminobenzamide (3-AB) in a dose of 30 mg/kg b.w. (body weight) injected intravenously directly after 3 min of ischemia protects >60% of neuronal cells against death in the CA1 layer of hippocampus but has no effect after 10 min of ischemic episode. 3-AB decreased forebrain edema significantly after 3 and 10 min of ischemia. Our data indicate that PARP inhibitor(s) might offer a potent therapeutic strategy for short global ischemia. The combination of PARP inhibitor with potent antioxidant might enhance its ameliorating effect.

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.

t-Butyl hydroperoxide and oxidized low density lipoprotein enhance phospholipid hydrolysis in lipopolysaccharide-stimulated retinal pericytes

Free radicals induced by organic peroxides or oxidized low density lipoprotein (oxLDL) play a critical role in the development of atherosclerosis. In investigating this process, and the concomitant inflammatory response, the role of pericytes, cells supporting the endothelial ones in blood vessels, has received little attention. In this study we tested the hypothesis that tert-butyl hydroperoxide (t-BuOOH) and oxLDL, administered in sublethal doses to the culture medium of retinal pericytes, function as prooxidant signals to increase the stimulation of the peroxidation process induced by lipopolysaccharide (LPS). Confluent cell monolayers were exposed to t-BuOOH (25-400 microM), native LDL or oxLDL (3.4-340 nmol hydroperoxides/mg protein, 1-100 micro). LPS (1 microg/ml), t-BuOOH (200 microM), and oxLDL (100 microM), but not native LDL, incubated for 24 h with cells, markedly increased lipid peroxidation, cytosolic phospholipase A2 (cPLA2) activity and arachidonic acid (AA) release in a time- and dose-dependent manner. AACOCF(3), a potent cPLA2 inhibitor, and the antioxidant alpha-tocopherol strongly inhibited the prooxidant-stimulated AA release. Long-term exposure to maximal concentrations of t-BuOOH (400 microM) or oxLDL (100 microM) had a sharp cytotoxic effect on the cells, described by morphological and biochemical indices. The presence of t-BuOOH or oxLDL at the same time, synergistically increased phospholipid hydrolysis induced by LPS alone. 400 microM t-BuOOH or 100 microM oxLDL had no significant effect on the stimulation of an apoptosis process estimated by DNA laddering and light and electron microscopy. The results indicate that (i) pericytes may be the target of extensive oxidative damage; (ii) activation of cPLA2 mediates AA liberation; (iii) as long-term regulatory signals, organic peroxide and specific constituents of oxLDL increase the pericyte ability to degrade membrane phospholipids mediated by LPS which was used, in the present study, to simulate in vitro an inflammatory burst in the retinal capillaries.

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.

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.

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.

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. Parkinsons 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.