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A growing body of evidence indicates that pathological forms of amyloid beta (Aβ) peptide contribute to neuronal degeneration and synaptic loss in Alzheimer’s disease (AD). In this study, we investigated the impact of exogenous Aβ1-42 oligomers (AβO) and endogenously liberated Aβ 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 AβO-evoked changes in transcription of genes in SH-SY5Y cells, where AβO enhanced expression of Sod1, Cat, mt-Nd1, Bcl2, and attenuated Sirt5, Sod2 and Sdha. In BV2 line, AβO increased the level of mRNA for Sod2, Dnm1l, Bcl2, and decreased for Gpx4, Sirt1, Sirt3, mt-Nd1, Sdha and Mfn2. Then, AβO 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 AβO. 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 AβO 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.

In the current study, we analyzed the effects of the systemic inflammatory response (SIR) and amyloid β (Aβ) peptide on the expression of genes encoding cyclins and cyclin-dependent kinase (Cdk) in: (i) PC12 cells overexpressing human beta amyloid precursor protein (βAPP), wild-type (APPwt-PC12), or carrying the Swedish mutantion (APPsw-PC12); (ii) the murine hippocampus during SIR; and (iii) Alzheimer’s disease (AD) brain. In APPwt-PC12 expression of cyclin D2 (cD2) was exclusively reduced, and in APPsw-PC12 cyclins cD2 and also cA1 were down-regulated, but cA2, cB1, cB2, and cE1 were up-regulated. In the SIR cD2, cB2, cE1 were found to be significantly down-regulated and cD3, Cdk5, and Cdk7 were significantly up-regulated. Cyclin cD2 was also found to be down-regulated in AD neocortex and hippocampus. Our novel data indicate that Aβ peptide and inflammation both significantly decreased the expression of cD2, suggesting that Aβ peptides may also contribute to downregulation of cD2 in AD brain.

Maternal immune activation (MIA) is a risk factor for neurodevelopmental disorders in offspring, but the pathomechanism is largely unknown. The aim of our study was to analyse the molecular mechanisms contributing to synaptic alterations in hippocampi of adolescent rats exposed prenatally to MIA. MIA was evoked in pregnant female rats by i.p. administration of lipopolysaccharide at gestation day 9.5. Hippocampi of offspring (52–53-days-old rats) were analysed using transmission electron microscopy (TEM), qPCR and Western blotting. Moreover, mitochondrial membrane potential, activity of respiratory complexes, and changes in glutathione system were measured. It was found that MIA induced changes in hippocampi morphology, especially in the ultrastructure of synapses, including synaptic mitochondria, which were accompanied by impairment of mitochondrial electron transport chain and decreased mitochondrial membrane potential. These phenomena were in agreement with increased generation of reactive oxygen species, which was evidenced by a decreased reduced/oxidised glutathione ratio and an increased level of dichlorofluorescein (DCF) oxidation. Activation of cyclin-dependent kinase 5, and phosphorylation of glycogen synthase kinase 3β on Ser9 occurred, leading to its inhibition and, accordingly, to hypophosphorylation of microtubule associated protein tau (MAPT). Abnormal phosphorylation and dysfunction of MAPT, the manager of the neuronal cytoskeleton, harmonised with changes in synaptic proteins. In conclusion, this is the first study demonstrating widespread synaptic changes in hippocampi of adolescent offspring prenatally exposed to MIA.

Maternal immune activation (MIA), induced by infection during pregnancy, is an important risk factor for neuro-developmental disorders, such as autism. Abnormal maternal cytokine signaling may affect fetal brain development and contribute to neurobiological and behavioral changes in the offspring. Here, we examined the effect of lipopolysaccharide-induced MIA on neuro-inflammatory changes, as well as synaptic morphology and key synaptic protein level in cerebral cortex of adolescent male rat offspring. Adolescent MIA offspring showed elevated blood cytokine levels, microglial activation, increased pro-inflammatory cytokines expression and increased oxidative stress in the cerebral cortex. Moreover, pathological changes in synaptic ultrastructure of MIA offspring was detected, along with presynaptic protein deficits and down-regulation of postsynaptic scaffolding proteins. Consequently, ability to unveil MIA-induced long-term alterations in synapses structure and protein level may have consequences on postnatal behavioral changes, associated with, and predisposed to, the development of neuropsychiatric disorders.

Autism spectrum disorders (ASD) are a heterogeneous group of neurodevelopmental conditions categorized as synaptopathies. Environmental risk factors contribute to ASD aetiology. In particular, prenatal exposure to the anti-epileptic drug valproic acid (VPA) may increase the risk of autism. In the present study, we investigated the effect of prenatal exposure to VPA on the synaptic morphology and expression of key synaptic proteins in the hippocampus and cerebral cortex of young-adult male offspring. To characterize the VPA-induced autism model, behavioural outcomes, microglia-related neuroinflammation, and oxidative stress were analysed. Our data showed that prenatal exposure to VPA impaired communication in neonatal rats, reduced their exploratory activity, and led to anxiety-like and repetitive behaviours in the young-adult animals. VPA-induced pathological alterations in the ultrastructures of synapses accompanied by deregulation of key pre- and postsynaptic structural and functional proteins. Moreover, VPA exposure altered the redox status and expression of proinflammatory genes in a brain region-specific manner. The disruption of synaptic structure and plasticity may be the primary insult responsible for autism-related behaviour in the offspring. The vulnerability of specific synaptic proteins to the epigenetic effects of VPA may highlight the potential mechanisms by which prenatal VPA exposure generates behavioural changes.

α-Synuclein (ASN) and parkin, a multifunctional E3 ubiquitin ligase, are two proteins that are associated with the pathophysiology of Parkinson’s disease (PD). Excessive release of ASN, its oligomerization, aggregation, and deposition in the cytoplasm contribute to neuronal injury and cell death through oxidative-nitrosative stress induction, mitochondrial impairment, and synaptic dysfunction. In contrast, overexpression of parkin provides protection against cellular stresses and prevents dopaminergic neural cell loss in several animal models of PD. However, the influence of ASN on the function of parkin is largely unknown. Therefore, the aim of this study was to investigate the effect of extracellular ASN oligomers on parkin expression, S-nitrosylation, as well as its activity. For these investigations, we used rat pheochromocytoma (PC12) cell line treated with exogenous oligomeric ASN as well as PC12 cells with parkin overexpression and parkin knock-down. The experiments were performed using spectrophotometric, spectrofluorometric, and immunochemical methods. We found that exogenous ASN oligomers induce oxidative/nitrosative stress leading to parkin S-nitrosylation. Moreover, this posttranslational modification induced the elevation of parkin autoubiquitination and degradation of the protein. The decreased parkin levels resulted in significant cell death, whereas parkin overexpression protected against toxicity induced by extracellular ASN oligomers. We conclude that lowering parkin levels by extracellular ASN may significantly contribute to the propagation of neurodegeneration in PD pathology through accumulation of defective proteins as a consequence of parkin degradation. Electronic supplementary material The online version of this article (10.1007/s12035-018-1082-0) contains supplementary material, which is available to authorized users.

Alzheimer's disease (AD) is characterized by the release of amyloid beta peptides (Aβ) 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, Aβ 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 Aβ oligomers (AβO) - 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. AβO elevated free radicals concentration, decreased mitochondria membrane potential (MMP) and cell viability after 24h. Gene transcription was not affected by AβO after 24h, but was significantly downregulated after 96h. In AβO 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 Aβ.

Abnormalities of alpha-synuclein (ASN), the main component of protein deposits (Lewy bodies), were observed in Parkinson’s disease (PD), dementia with Lewy bodies, Alzheimer’s disease, and other neurodegenerative disorders. These alterations include increase in the levels of soluble ASN oligomers in the extracellular space. Numerous works have identified several mechanisms of their toxicity, including stimulation of the microglial P2X7 receptor leading to oxidative stress. While the significant role of purinergic signaling—particularly, P2 family receptors—in neurodegenerative disorders is well known, the interaction of extracellular soluble ASN with neuronal purinergic receptors is yet to be studied. Therefore, in this study, we have investigated the effect of ASN on P2 purinergic receptors and ATP-dependent signaling. We used neuroblastoma SH-SY5Y cell line and rat synaptoneurosomes treated with exogenous soluble ASN. The experiments were performed using spectrofluorometric, radiochemical, and immunochemical methods. We found the following: (i) ASN-induced intracellular free calcium mobilization in neuronal cells and nerve endings depends on the activation of purinergic P2X7 receptors; (ii) activation of P2X7 receptors leads to pannexin 1 recruitment to form an active complex responsible for ATP release; and (iii) ASN greatly decreases the activity of extracellular ecto-ATPase responsible for ATP degradation. Thus, it is concluded that purinergic receptors might be putative pharmacological targets in the molecular mechanism of extracellular ASN toxicity. Interference with P2X7 signaling seems to be a promising strategy for the prevention or therapy of PD and other neurodegenerative disorders.

Cyclin-dependent kinase 5 (Cdk5) is critical for nervous system's development and function, and its aberrant activation contributes to pathomechanism of Alzheimer's disease and other neurodegenerative disorders. It was recently suggested that Cdk5 may participate in regulation of inflammatory signalling. The aim of this study was to analyse the mechanisms involved in regulating Cdk5 activity in the brain during systemic inflammatory response (SIR) as well as the involvement of Cdk5 in controlling the expression of inflammatory genes. Genetic and biochemical alterations in hippocampus were analysed 3 and 12 h after intraperitoneal injection of lipopolysaccharide. We observed an increase in both Cdk5 gene expression and protein level. Moreover, phosphorylation of Cdk5 on Ser159 was significantly enhanced. Also transcription of Cdk5-regulatory protein (p35/Cdk5r1) was augmented, and the level of p25, calpain-dependent cleavage product of p35, was increased. All these results demonstrated rapid activation of Cdk5 in the brain during SIR. Hyperactivity of Cdk5 contributed to enhanced phosphorylation of tau and glycogen synthase kinase 3β. Inhibition of Cdk5 with Roscovitine reduced activation of NF-κB and expression of inflammation-related genes, demonstrating the critical role of Cdk5 in regulation of gene transcription during SIR.

Free radical-induced oxidative damage is implicated in the pathogenesis of neurodegenerative disorders, and antioxidants are presumably of therapeutic value in such diseases. Our previous data indicated that free radicals are strongly associated with brain aging and also play an important role in cytotoxicity of amyloidogenic proteins including α-synuclein and amyloid β, which accumulate in brains during Parkinson’s and Alzheimer’s diseases. Disruption of the equilibrium of pro-oxidants and antioxidants results in oxidative stress that leads to the modification of DNA, proteins, carbohydrates, and lipids. It is widely accepted that antioxidants acting as radical scavengers protect the brain against oxidative damage in neurodegenerative diseases. Plant products are rich sources of phytochemicals and have been found to possess a variety of biological activities, including antioxidative potential. The aim of this study was to analyse the antioxidative potential of alkaloid fractions from Huperzia selago and Diphasiastrum complanatum to protect macromolecules against oxidative damage. Thin layer chromatography (TLC) and high-performance liquid chromatography with diode array (HPLC-DAD) and electrospray ionisation mass spectrometric detection (ESI-MS/MS) were used to carry out a comprehensive characterization of alkaloids isolated from the plant material. The effect of the tested compounds on iron/ascorbate-induced lipid peroxidation and car bonyl group formation was analysed in the rat brain homogenate. Direct free radical scavenging (DPPH assay) and the effect on dityrosine formation were measured in cell-free systems. Our results indicated that a number of alkaloid extracts at concentration of 25 µg/ml exhibited antioxidant activity as indicated by DPPH radical scavenging potential (up to 59% inhibition) and inhibition of dityrosine formation. Selected alkaloid fractions provided significant protection against lipid peroxidation and protein oxidation in rat brain tissue homogenate, reducing iron/ascorbate-induced damage by about 20% and 76%, respectively. Overall, the results indicated that selected alkaloids isolated from Huperzia selago effectively protect macromolecules from oxidative stress injury, which will give us an insight into the potential of alkaloids in terms of opening up a new therapeutic approach for oxidative stress-dependent disorders.

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 12 h 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; 30 mg/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.

Neuroinflammation and oxidative stress are key intertwined pathological factors in many neurological, particularly neurodegenerative diseases, such as Alzheimer's and Parkinson's disorders as well as autism. The present study was conducted to evaluate the protective effects of Selol, an organic selenium donor, against lipopolysaccharide (LPS)-mediated inflammation in rat brain. The results demonstrated that the peripheral administration of LPS in a dose of 100 μg/kg b.w. evoked typical pathological reaction known as systemic inflammatory response. Moreover, we observed elevated blood levels of thiobarbituric acid-reactive substances (TBARS), a marker of oxidative stress, as well as increased concentration of tumor necrosis factor-α (TNF-α) in LPS-treated animals. Selol significantly prevented these LPS-evoked changes. Subsequently, Selol protected against LPS-induced up-regulation of proinflammatory cytokines (Tnfa, Ifng, Il6) in rat brain cortex. The molecular mechanisms through which Selol prevented the neuroinflammation were associated with the inhibition of oxidized glutathione (GSSG) accumulation and with an increase of glutathione-associated enzymes: glutathione peroxidase (Se-GPx), glutathione reductase (GR) as well as thioredoxin reductase (TrxR) activity and expression. Finally, we observed that Selol administration effectively protected against LPS-induced changes in the expression of brain-derived neurotrophic factor (Bdnf). In conclusion, our studies indicated that Selol effectively protects against LPS-induced neuroinflammation by inhibiting pro-inflammatory cytokine release, by boosting antioxidant systems, and by augmenting BDNF level. Therefore, Selol could be a multi-potent and effective drug useful in the treatment and prevention of brain disorders associated with neuroinflammation.

The aim of the present study was to analyse the alterations of Cyclin dependent kinase 5 (Cdk5) expression and phosphorylation in PC12 cells overexpressing Amyloid precursor protein (APP). Our results demonstrated enhanced cell death and increased levels of mRNA for the Cdk5 gene in APP-transfected cells. Significantly decreased phosphorylation of Cdk5 at Tyr15 was observed in APPsw cells, which is responsible for a reduction in Cdk5 activity. Cdk5-dependent phosphorylation of Glycogen synthase kinase-3β (Gsk-3β) at Ser9 was also decreased, which can lead to the increase of Gsk-3β activity and hyperphosphorylation of MAP tau. Our results demonstrate for the first time, a deregulation of Cdk5 phosphorylation in APP-transfected cells.

Although the inducible isoform of nitric oxide synthase (iNOS) is a well-established source of nitric oxide (NO*) during inflammation of the central nervous system (CNS), little is known about the involvement of constitutive isoforms of NOS (cNOS) in the inflammatory process. The aim of this study was to compare the responses of the expression and activity of iNOS and the two cNOS isoforms, neuronal and endothelial (nNOS and eNOS, respectively), in the brain to systemic inflammation and their roles in the cascade of events leading to degeneration and apoptosis. A systemic inflammatory response in C57BL/6 mice was induced by intraperitoneal injection of lipopolysaccharide [LPS; 1 mg/kg body weight (b.w.)]. The relative roles of the NOS isoforms were evaluated after injection of NG-nitro-L-arginine (NNLA; 30 mg/kg b.w.), which preferentially inhibits cNOS, or 1400W (5 mg/kg b.w.), an inhibitor of iNOS. Biochemical and morphological alterations were analyzed up to 48 hr after administration of LPS. Systemic LPS administration evoked significant ultrastructural alterations in brain capillary vessels, neuropils, and intracellular organelles of neurons, astrocytes, and microglia. Apoptotic/autophagic processes occurred in many neurons of the substantia nigra (SN), which coincided with exclusive enhancement of iNOS expression and activity in this brain region. Moreover, inhibitors of both iNOS and cNOS prevented LPS-evoked release of apoptosis-inducing factor (AIF) from SN mitochondria. Collectively, the results indicate that synthesis of NO* by both the inducible and constitutive NOS isoforms contribute to the activation of apoptotic pathways in the brain during systemic inflammation.

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.

Cyclin-dependent kinase 5 (Cdk5) is involved in proper neurodevelopment and brain function and serves as a switch between neuronal survival and death. Overactivation of Cdk5 is associated with many neurodegenerative disorders such as Alzheimer's or Parkinson's diseases. It is believed that in those diseases Cdk5 may be an important link between disease-initiating factors and cell death effectors. A common hallmark of neurodegenerative disorders is incorrect folding of specific proteins, thus leading to their intra- and extracellular accumulation in the nervous system. Abnormal Cdk5 signaling contributes to dysfunction of individual proteins and has a substantial role in either direct or indirect interactions of proteins common to, and critical in, different neurodegenerative diseases. While the roles of Cdk5 in α-synuclein (ASN) - tau or β-amyloid peptide (Aβ) - tau interactions are well documented, its contribution to many other pertinent interactions, such as that of ASN with Aβ, or interactions of the Aβ - ASN - tau triad with prion proteins, did not get beyond plausible hypotheses and remains to be proven. Understanding of the exact position of Cdk5 in the deleterious feed-forward loop critical for development and progression of neurodegenerative diseases may help designing successful therapeutic strategies of several fatal neurodegenerative diseases. Cyclin-dependent kinase 5 (Cdk5) is associated with many neurodegenerative disorders such as Alzheimer's or Parkinson's diseases. It is believed that in those diseases Cdk5 may be an important factor involved in protein misfolding, toxicity and interaction. We suggest that Cdk5 may contribute to the vicious circle of neurotoxic events involved in the pathogenesis of different neurodegenerative diseases.

Free radical-induced oxidative damage is implicated in the pathogenesis of neurodegenerative disorders, and antioxidants are presumably of therapeutic value in such diseases. Our previous data indicated that free radicals are strongly associated with brain aging and also play an important role in cytotoxicity of amyloidogenic proteins including -synuclein and amyloid , which accumulate in brains during Parkinson's and Alzheimer's diseases. Disruption of the equilibrium of pro-oxidants and antioxidants results in oxidative stress that leads to the modification of DNA, proteins, carbohydrates, and lipids. It is widely accepted that antioxidants acting as radical scavengers protect the brain against oxidative damage in neurodegenerative diseases. Plant products are rich sources of phytochemicals and have been found to possess a variety of biological activities, including antioxidative potential. The aim of this study was to analyse the antioxidative potential of alkaloid fractions from Huperzia selago and Diphasiastrum complanatum to protect macromolecules against oxidative damage. Thin layer chromatography (TLC) and high-performance liquid chromatography with diode array (HPLC-DAD) and electrospray ionisation mass spectrometric detection (ESI-MS/MS) were used to carry out a comprehensive characterization of alkaloids isolated from the plant material. The effect of the tested compounds on iron/ascorbate-induced lipid peroxidation and carbonyl group formation was analysed in the rat brain homogenate. Direct free radical scavenging (DPPH assay) and the effect on dityrosine formation were measured in cell-free systems. Our results indicated that a number of alkaloid extracts at concentration of 25 µg/ml exhibited antioxidant activity as indicated by DPPH radical scavenging potential (up to 59% inhibition) and inhibition of dityrosine formation. Selected alkaloid fractions provided significant protection against lipid peroxidation and protein oxidation in rat brain tissue homogenate, reducing iron/ascorbate-induced damage by about 20% and 76%, respectively. Overall, the results indicated that selected alkaloids isolated from Huperzia selago effectively protect macromolecules from oxidative stress injury, which will give us an insight into the potential of alkaloids in terms of opening up a new therapeutic approach for oxidative stress-dependent disorders.

Poly(ADP-ribose) polymerase-1 (PARP-1) is involved in DNA repair, but its overactivation can induce cell death. Our aim was to investigate the role of PARP-1 in activation of programmed cell death processes in the brain during systemic inflammation. Our data indicated that lipopolysaccharide (1mg/kgb.w., i.p.)-evoked systemic inflammation enhanced PARP-1 activity in the mouse brain, leading to the lowering of beta-NAD(+) concentration, to translocation of apoptosis inducing factor from mitochondria to the nucleus, and to enhanced lipid peroxidation. Inhibitor of PARP-1, 3-aminobenzamide (30 mg/kgb.w., i.p.), protected the brain against prooxidative and cell death processes, suggesting involvement of PARP-1 in systemic inflammation-related processes in the brain.

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.

Protein oxidation of human umbilical-vein endothelial cells (HUVEC) in culture was examined under various conditions of oxidative stress. Extracellular protein (ECP) oxidation was assessed by determining dityrosine bond formation, which is indicated by the covalent coupling of the membrane-impermeable tyramine-fluorescein conjugate (TyrFluo) to HUVEC proteins. The acetylated membrane-permeable form of TyrFluo (acetylTyrFluo) was used for the determination of intracellular protein (ICP) oxidation. Oxidative stress was induced by exposing the HUVEC to PMA-activated human neutrophils, to a horseradish peroxidase/hydrogen peroxide (HRP/H(2)O(2)) system or to H(2)O(2) alone. Coupling of the probes was determined by confocal laser scanning microscopy and by Western blotting using anti-fluorescein antibody. Diethylamine nitric oxide (DEANO) was used to determine the effect of NO on the tyrosyl radical formation in proteins. The oxidative burst generated by activated neutrophils for 15 min, resulted in inducing dityrosine formation in ECP of HUVEC. Similar results were obtained with HRP/H(2)O(2), but H(2)O(2) alone did not have any effect on ECP. In the presence of DEANO (0.1 mM or higher), ECP oxidation was almost completely inhibited. This indicates that NO may protect endothelial cells against protein oxidation by activated neutrophils under pro-inflammatory conditions. Activated neutrophils did not oxidize ICP of HUVEC, which strongly suggests that the effect of the oxidative burst was restricted to the proteins exposed to the medium.

α-Synuclein (ASN) plays an important role in pathogenesis of Parkinson's disease (PD) and other neurodegenerative disorders. Novel and most interesting data showed elevated tauopathy in PD and suggested relationship between ASN and Tau protein. However, the mechanism of ASN-evoked Tau protein modification is not fully elucidated. In this study we investigated the role of extracellular ASN in Tau hyperphosphorylation in rat pheochromocytoma (PC12) cells and the involvement of glycogen synthase kinase-3β (GSK-3β) and cyclin-dependent kinase 5 (CDK5) in ASN-dependent Tau modification. Our results indicated that exogenously added ASN increases Tau phosphorylation at Ser396. Accordingly, the GSK-3β inhibitor (SB-216763) prevented ASN-evoked Tau hyperphosphorylation, but the CDK5 inhibitor had no effect. Moreover, western blot analysis showed that ASN affected GSK-3β via increasing of protein level and activation of this enzyme. GSK-3β activity evaluated by its phosphorylation status assay showed that ASN significantly increased the phosphorylation of this enzyme at Tyr216 with parallel decrease in phosphorylation at Ser9, indicative of stimulation of GSK-3β activity. Moreover, the effect of ASN on microtubule (MT) destabilization and cell death with simultaneous the involvement of GSK-3β in these processes were analyzed. ASN treatment increased the amount of free tubulin and concomitantly reduced the amount of polymerized tubulin and SB-216763 suppressed these ASN-induced changes in tubulin, indicating that GSK-3β is involved in ASN-evoked MT destabilization. ASN-induced apoptotic processes lead to decrease in PC12 cells viability and SB-216763 protected those cells against ASN-evoked cytotoxicity. Concluding, extracellular ASN is involved in GSK-3β-dependent Tau hyperphosphorylation, which leads to microtubule destabilization. GSK-3β inhibition may be an effective strategy for protecting against ASN-induced cytotoxicity.

The determination of free radicals and their reaction products in biological systems is discussed, with an emphasis on the use of liquid chromatographic methods for the analysis. Free radicals formed from oxygen and its reduction products, nitric oxide, and lipid radicals are considered. Whilst reversed-phase high-performance liquid chromatography has found wide application for these analyses, ion-exclusion chromatography also has significant potential and some developments in this area are described.

In this study we evaluated biochemical blood serum parameters and the association of Cyclin-dependent kinase 5 (CDK5) gene polymorphisms with the risk of Alzheimer's disease (AD) in the Polish population. We observed an elevated total cholesterol, low-density lipoproteins (LDL) and homocysteine levels and lower concentrations of high-density lipoproteins (HDL) and vitamin B12 in AD patients. However, the analyzed CDK5 polymorphisms were not associated with the biochemical parameters. Moreover, we found no association between the studied polymorphisms and the risk of AD in the Polish population. The meta-analysis of previously published and current study was performed. In conclusion, our study demonstrated that alteration of cholesterol, LDL, HDL, homocysteine and B12 concentration may be an important factor in pathogenesis of AD.

Mitochondria are intracellular organelles that play a crucial role in energy metabolism. Most cell energy is obtained through mitochondrial metabolic pathways, especially the Krebs cycle and electron transport chain which is the main site for production of reactive oxygen species such as superoxide, hydrogen peroxide, and hydroxyl radicals. Brain tissue is highly sensitive to oxidative stress due to its high oxygen consumption, iron and lipid contents, and low activity of antioxidant defenses. Thus, energy metabolism impairment and oxidative stress are important events that have been related to the pathogenesis of diseases affecting the central nervous system. In the present issue, the pathogenesis of common Alzheimer's (AD) and Parkinson's (PD) diseases is addressed in five papers. Mondragon-Rodriguez et al. proposed that phosphorylated tau protein could play the role of potential connector and that a combined therapy involving antioxidants and check points for synaptic plasticity during early stages of the disease could become a viable therapeutic option for AD treatment. This paper is accompanied by a study by T. Rohn that explores the potential role that the triggering receptor expressed on myeloid cells 2 (TREM2) normally plays and how loss of function may contribute to AD pathogenesis by enhancing oxidative stress and inflammation within the central nervous system. Additionally, O. Myhre et al. review the possible impact of environmental exposures in metal dyshomeostasis and inflammation in AD and PD. Furthermore, T. Omura et al. explore recent studies on the mechanism of endoplasmic reticulum stress-induced neuronal death related to PD, focusing on the involvement of human ubiquitin ligase HRD1 in the prevention of neuronal death as well as a potential therapeutic approach for PD based on the upregulation of HRD1. Lastly, S. Matsuda et al. showed a concise overview on the cellular functions of the mitochondrial kinase PINK1 and the relationship between Parkinsonism and mitochondrial dynamics, with particular emphasis on a mitochondrial damage response pathway and mitochondrial quality control. Three of the papers deal with aspects of oxidative stress implicated in the pathogenesis of neurodegenerative diseases. W. Liu et al. reviewed the current literature on the effects of oxidative stress due to exhaustive training on uncoupling protein 2 (UCP2) and Bcl-2/Bax in rat skeletal muscles. A.-M. Enciu et al. explore the possibility of oxidative-induced molecular mechanisms of blood-brain barrier disruption and tight junction protein expression alteration, in relation to aging and neurodegeneration. Moreover, M. Tajes et al. suggest that peroxynitrite induces cell death and is a very harmful agent in brain ischemia. A. Hosseini and M. Abdollahi review the pathogenesis of diabetic neuropathy with a focus on oxidative stress and introduced therapies dependent or independent of oxidative stress. A. Sekigawa et al. review the currently available evidence that neither mitochondria nor leucine-rich repeat kinase 2 (LRRK2) was present in the swellings of mice expressing P123H β-synuclein, suggesting that α- and β-synucleins might play differential roles in the mitochondrial pathology of α-synucleinopathies. The paper by P. F. Schuck et al. showed that trans-glutaconic acid is toxic to brain cells in vitro, by causing alterations in cell ion balance and probably neurotransmission, as well as oxidative stress in rat cerebral cortex. To complete the issue, M. J. Rodriguez et al. discuss the mitochondrial KATP channel as a new target to control microglia activity, avoid its toxic phenotype, and facilitate a positive disease outcome. Fortes et al. showed that 5TIO1 can protect the brain against neuronal damage regularly observed during neuropathologies. These papers are accompanied by a review by Z. Yu et al. on how the neuroglobin's neuroprotection is related to mitochondria function and regulation. By compiling these papers, we hope to enrich our readers and researchers with respect to mitochondrial dysfunction, energy metabolism impairment, and oxidative stress in the pathophysiology of neurodegenerative diseases. Emilio L. Streck Grzegorz A. Czapski Cleide Goncalves da Silva

The aim of this paper is to examine if pre- and neonatal exposure to lead (Pb) may intensify or inhibit apoptosis or necroptosis in the developing rat brain. Pregnant experimental females received 0.1% lead acetate (PbAc) in drinking water from the first day of gestation until weaning of the offspring; the control group received distilled water. During the feeding of pups, mothers from the experimental group were still receiving PbAc. Pups were weaned at postnatal day 21 and the young rats of both groups then received only distilled water until postnatal day 28. This treatment protocol resulted in a concentration of Pb in rat offspring whole blood (Pb-B) below the threshold of 10 μg/dL, considered safe for humans.We studied Casp-3 activity and expression, AIF nuclear translocation, DNA fragmentation, as well as Bax, Bcl-2 mRNA and protein expression as well as BDNF concentration in selected structures of the rat brain: forebrain cortex (FC), cerebellum (C) and hippocampus (H). The microscopic examinations showed alterations in hippocampal neurons.Our data shows that pre- and neonatal exposure of rats to Pb, leading to Pb-B below 10 μg/dL, can decrease the number of hippocampus neurons, occurring concomitantly with ultrastructural alterations in this region. We observed no morphological or molecular features of severe apoptosis or necrosis (no active Casp-3 and AIF translocation to nucleus) in young brains, despite the reduced levels of BDNF. The potential protective factor against apoptosis was probably the decreased Bax/Bcl-2 ratio, which requires further investigation. Our findings contribute to further understanding of the mechanisms underlying Pb neurotoxicity and cognition impairment in a Pb-exposed developing brain.

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 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.

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.

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.

Our previous studies indicated that exogenous alpha-synuclein (ASN) activates neuronal nitric oxide (NO) synthase (nNOS) in rat brain slices. The present study, carried out on immortalized hippocampal neuronal cells (HT22), was designed to extend the previous results by showing the molecular pathway of NO-mediated cell death induced by exogenous ASN. Extracellular ASN (10 microM) was found to stimulate nitric oxide synthase (NOS) and increase caspase-3 activity in HT22 cells, leading to poly(ADP-ribose) polymerase (PARP-1) cleavage. The inhibitor of Ca2+-dependent NOS (N-nitro-L-arginine, 100 microM) prevented ASN-evoked caspase-3 activation and PARP-1 degradation. ASN exposure resulted in apoptotic death of HT22 cells and this effect was reversed by inhibition of NO synthesis and caspase-3 activity. Our results demonstrated that extracellular ASN induces neuronal cell death by NO-mediated caspase-3 activation.

A systemic inflammatory reaction may have detrimental effects on the organism, including the central nervous system. Previous studies have indicated that lipopolysaccharide (LPS)-evoked systemic inflammation induces pathological alterations in the mouse midbrain, especially in the substantia nigra. The aim of the present study was to investigate whether the hippocampus is also affected after an intraperitoneal (i.p.) injection of LPS. We focussed on the dynamics of proinflammatory gene expression and the processes leading to neuronal cell death. A systemic inflammatory response in C57BL/6 mice was induced by an i.p. injection of LPS (1mg/kg b.w.). The genetic, biochemical and morphological alterations were analysed up to 96h after LPS administration using quantitative PCR, immunochemical, immunocytochemical and electron microscopic methods. Real-time PCR analysis indicated an altered expression of several genes, mainly responsible for arachidonic acid release and metabolism, in the hippocampus 96h after the systemic administration of LPS. Three hours after LPS treatment, the level of mRNA for iNOS, COX-2 and TNFα was increased; then, after 6-24h, it rose for TLR4 and cPLA2. The expression of 5-LOX and 12-LOX was increased at 12-24 and 24-48h after LPS injection, respectively. Our data demonstrate for the first time the sequential activation of the expression of several pro-inflammatory genes responsible for the maintenance of the inflammatory response. Moreover, the electron microscopy studies presented the stimulation of apoptosis-inducing factor (AIF)-mediated death signalling and cathepsin B-related autophagy or necrosis. These biochemical and morphological alterations in the hippocampus, which were induced by systemic inflammation, may be responsible for the impairment of cognition function observed previously.

Our previous data demonstrated that systemic inflammation evoked by intraperitoneal injection of lipopolysaccharide (LPS; 1 mg/kg b.w.) induces morphological and biochemical changes in the brain, including alterations of poly(ADP-ribose) polymerase-1 (PARP-1) activity and expression of several genes. In this study, the effect of systemic inflammatory response (SIR) on glutathione redox state and on cognition, spatial memory and locomotor activity was evaluated by using spectrophotometric method, object recognition test, Morris water-maze and open-field tests, respectively. The effect of PARP-1 inhibitor was included in this study. Our data indicated that SIR significantly decreases reduced glutathione (GSH) level, enhances its disulfide form (GSSG) and decreases glutathione reductase activity. Moreover, SIR affects the object recognition and locomotor activity but has negligible effect on spatial memory. PARP-1 inhibitor protects against LPS-evoked recognition impairment and significantly improves spatial memory in LPS-treated mice. The effect of PARP-1 inhibitor could be in part connected with lowering of PARP-1 involvement in regulation of transcription of several pro-inflammatory genes. Moreover, PARP-1 inhibitors may modulate glutamatergic receptor signaling that plays an important role in learning and memory.

The only well confirmed genetic risk factor for sporadic Alzheimer's disease (AD) is the possession of apolipoprotein E (APOE) epsilon4 allele. As it contributes to 40-70% of AD cases, a large proportion of genetic variance may be determined by additional loci. Our aim was to estimate how reported genetic factors (APOE, NOS3, MTHFR) interact to increase the risk for AD and combine them with environmental factors (homocysteine, vitamin B12, cholesterol). Genotyping was performed in 154 AD patients and 176 healthy controls. Levels of homocysteine, vitamin B12 and cholesterol were assessed in subgroups of 100 AD patients and 100 controls. We found a difference in APOE epsilon4 and NOS3 G/G distribution between groups (p0.005). Plasma total homocysteine was increased and vitamin B12 decreased in AD patients (p0.001). The influence of APOE epsilon4 and NOS3 G alleles on the risk of AD was independent of homocysteine, vitamin B12 levels and MTHFR status.

Our study focused on the relationship between amyloid β 1-42 (Aβ), sphingosine kinases (SphKs) and mitochondrial sirtuins in regulating cell fate. SphK1 is a key enzyme involved in maintaining sphingolipid rheostat in the brain. Deregulation of the sphingolipid metabolism may play a crucial role in the pathogenesis of Alzheimer's disease (AD). Mitochondrial function and mitochondrial deacetylases, i.e. sirtuins (Sirt3,-4,-5), are also important for cell viability. In this study, we evaluated the interaction between Aβ1-42, SphKs and Sirts in cell survival/death, and we examined several compounds to indicate possible target(s) for a strategy protecting against cytotoxicity of Aβ1-42. PC12 cells were subjected to Aβ1-42 oligomers and SphK inhibitor SKI II for 24-96 h. Our data indicated that Aβ1-42 enhanced SphK1 expression and activity after 24 h, but down-regulated them after 96 h and had no effect on Sphk2. Aβ1-42 and SKI II induced free radical formation, disturbed the balance between pro- and anti-apoptotic proteins and evoked cell death. Simultaneously, up-regulation of anti-oxidative enzymes catalase and superoxide dismutase 2 was observed. Moreover, the total protein level of glycogen synthase kinase-3β was decreased. Aβ1-42 significantly increased the level of mitochondrial proteins: apoptosis-inducing factor AIF and Sirt3, -4, -5. By using several pharmacologically active compounds we showed that p53 protein plays a significant role at very early stages of Aβ1-42 toxicity. However, during prolonged exposure to Aβ1-42, the activation of caspases, MEK/ERK, and alterations in mitochondrial permeability transition pores were additional factors leading to cell death. Moreover, SphK product, sphingosine-1-phosphate (S1P), and Sirt activators and antioxidants, resveratrol and quercetin, significantly enhanced viability of cells subjected to Aβ1-42. Our data indicated that p53 protein and inhibition of SphKs may be early key events responsible for cell death evoked by Aβ1-42. We suggest that activation of S1P-dependent signalling and Sirts may offer a promising cytoprotective strategy.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme involved in DNA repair, replication and cell cycle. However, its overactivation leads to nicotinamide adenine dinucleotide and ATP depletion and cell death. The inhibitors of PARP-1 were successfully used in the basic studies and in animal models of different diseases. For this reason, it is important to discriminate between specific and non-specific antioxidant properties of PARP-1 inhibitors. The aim of this study was to investigate the effect of PARP-1 inhibitors on the free radical level and oxidation of macromolecules and to compare their properties with the efficacy of antioxidants. Oxidative stress was induced in the brain cortex homogenate by FeCl(2) or CuSO(4) at 25 microM during 15 min incubation at 37 degrees C. PARP-1 inhibitors 3-aminobenzamide (3-AB), 1,5-dihydroxyisoquinoline (DHIQ) and 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (DPQ), and the antioxidants alpha-tocopherol, resveratrol and Tempol were used at 0-5 mM. Free radical contents were estimated by spin-trapping using HPLC. Lipid and protein oxidation were determined by measuring thiobarbituric acid reactive substances and carbonyl groups or using fluorescent probe TyrFluo, respectively. Our data indicate that 3-AB and DHIQ are potent hydroxyl radical scavengers and inhibitors of protein oxidation. DHIQ additionally decreases lipid peroxidation. DPQ has no antioxidant properties and seems to be a specific PARP-1 inhibitor, however, it is a water insoluble compound. Among the investigated antioxidants, the most potent was resveratrol and then alpha-tocopherol and Tempol. These results indicate that 3-A beta, benzamide and DHIQ are potent hydroxyl radical scavengers and antioxidants. These data ought to be taken into consideration when properties of these compounds as PARP inhibitors are evaluated.

The ionotropic glutamatergic receptor system, especially the subtype mediated by N-methyl-D-aspartic acid (NMDA), is known to exhibit special sensitivity to the effect of ethanol. This is due partly to the ability of ethanol to modulate the production of nitric oxide through the NMDA-nitric oxide synthase (NOS) pathway. In this study, we examined the effects of ethanol on basal and NMDA-stimulated NOS activity in rat hippocampal slices by measuring the conversion of [(14)C]-arginine into [(14)C]-citrulline in an incubation system containing the necessary cofactors. Stimulation of hippocampal slices with NMDA (100 microM) enhanced NOS activity by 43% (n = 12). Although ethanol did not alter NOS activity when added to the incubation system during NMDA stimulation, it dose-dependently inhibited NMDA-NOS activity when added to the slices during the 90-min preincubation period. Further assay of NOS activity with brain cytosolic fraction indicated an inhibitory effect of ethanol (200 mM) when the assay was carried out in the absence of exogenous tetrahydrobiopterin (BH4), a redox-active cofactor for NOS. Incubation of brain homogenates resulted in a time-dependent increase in the levels of lipid peroxidation products, but ethanol did not further enhance these products. Taken together, these results provide evidence for the role of BH4 but not oxidative stress in the inhibitory effect of ethanol on NMDA-NOS activity in rat hippocampal slices.