Biocompatibility and proteomic profiling of DMSA-coated iron nanocubes in a human glioblastoma cell line.

Background: Superparamagnetic iron core iron oxide shell nanocubes have previously shown superior performance in magnetic resonance imaging T2 contrast enhancement compared with spherical nanoparticles. Methods: Iron core iron oxide shell nanocubes were synthesized, stabilized with dimercaptosuccinic acid (DMSA-NC) and physicochemically characterized. MRI contrast enhancement and biocompatibility were assessed in vitro. Results: DMSA-NC showed a transverse relaxivity of 122.59 mM-1·s-1 Fe. Treatment with DMSA-NC did not induce cytotoxicity or oxidative stress in U-251 cells, and electron microscopy demonstrated DMSA-NC localization within endosomes and lysosomes in cells following internalization. Global proteomics revealed dysregulation of iron storage, transport, transcription and mRNA processing proteins. Conclusion: DMSA-NC is a promising T2 MRI contrast agent which, in this preliminary investigation, demonstrates favorable biocompatibility with an astrocyte cell model.

MRI is a powerful tool used in the diagnosis of cancer, strokes and other injuries. An MRI scan can be improved with the use of iron oxide nanoparticles, which enhance the contrast of the image. In this study we have developed cube-shaped iron nanoparticles (nanocubes), which have been previously shown to be more effective at inducing contrast. We demonstrated that iron-based nanocubes do not damage or induce stress in cells and work effectively as an MRI contrast agent. We further analyzed how the nanocubes may affect cell functioning by investigating changes to protein levels in the cells. The results of this study are promising steps towards using iron-based nanocubes as a tool to improve the clarity of MRI scans for medical imaging and diagnosis. Future work must determine whether these nanocubes work effectively and safely in an animal model, which is a critical step in progressing to their use in clinical settings.

Evaluation of Dimercaptosuccinic Acid-Coated Iron Nanoparticles Immunotargeted to Amyloid Beta as MRI Contrast Agents for the Diagnosis of Alzheimer's Disease.

Nanoparticle-based magnetic contrast agents have opened the potential for magnetic resonance imaging (MRI) to be used for early non-invasive diagnosis of Alzheimer's disease (AD). Accumulation of amyloid pathology in the brain has shown association with cognitive decline and tauopathy; hence, it is an effective biomarker for the early detection of AD. The aim of this study was to develop a biocompatible magnetic nanoparticle targeted to amyloid beta (Aß) plaques to increase the sensitivity of T2-weighted MRI for imaging of amyloid pathology in AD. We presented novel iron core-iron oxide nanoparticles stabilized with a dimercaptosuccinic acid coating and functionalized with an anti-Aß antibody. Nanoparticle biocompatibility and cellular internalization were evaluated in vitro in U-251 glioblastoma cells using cellular assays, proteomics, and transmission electron microscopy. Iron nanoparticles demonstrated no significant in vitro cytotoxicity, and electron microscopy results showed their movement through the endocytic cycle within the cell over a 24 h period. In addition, immunostaining and bio-layer interferometry confirmed the targeted nanoparticle's binding affinity to amyloid species. The iron nanoparticles demonstrated favourable MRI contrast enhancement; however, the addition of the antibody resulted in a reduction in the relaxivity of the particles. The present work shows promising preliminary results in the development of a targeted non-invasive method of early AD diagnosis using contrast-enhanced MRI.

Macrophage- and Microglia-Related Chemokines Are Associated with Small Vessel (White Matter) Vascular Dementia: A Case-Control Study.

INTRODUCTION: Little is known about the role of inflammation in the process of small vessel vascular dementia (VaD). Recently, the notion that small vessel VaD is caused solely by vascular pathology has been challenged by new evidence of concomitant breakdown of the blood-brain barrier and dysregulation of neuroinflammation in the white matter. METHODS: We examined selected inflammatory cytokines and chemokines in the plasma from patients with small vessel VaD (n = 41) and from age-matched controls (n = 131) using multiplex bead-based assays. Participants were recruited from a memory disorder clinic and from a hospital or community. RESULTS: When compared to controls, patients with small vessel VaD had a highly significant increase in the plasma interferon-γ-inducible protein 10 (IP-10) level (p < 0.0001) and a highly significant decrease in plasma macrophage inflammatory protein 1-beta (MIP-1ß) level (p < 0.0001). We also observed a significant increase in patients' levels of interleukin-10 (IL-10) (p = 0.022) as well as decreases in interleukin-8 (IL-8) (p = 0.004) and interleukin-7 (IL-7) (p = 0.011) when compared to age-matched controls. CONCLUSION: Both IP-10 and MIP-1ß are macrophage-related chemokines. The significant differences between cases and controls suggest a potential role for macrophages in small vessel VaD neuroinflammation. Although it remains unclear whether there is a causal effect of their alteration for small vessel VaD, a better understanding of these molecules in the pathogenesis of small vessel VaD may lead to improved diagnosis and future treatment outcomes against this disease.

Mechanisms of impaired mitochondrial homeostasis and NAD+ metabolism in a model of mitochondrial heart disease exhibiting redox active iron accumulation.

Due to the high redox activity of the mitochondrion, this organelle can suffer oxidative stress. To manage energy demands while minimizing redox stress, mitochondrial homeostasis is maintained by the dynamic processes of mitochondrial biogenesis, mitochondrial network dynamics (fusion/fission), and mitochondrial clearance by mitophagy. Friedreich's ataxia (FA) is a mitochondrial disease resulting in a fatal hypertrophic cardiomyopathy due to the deficiency of the mitochondrial protein, frataxin. Our previous studies identified defective mitochondrial iron metabolism and oxidative stress potentiating cardiac pathology in FA. However, how these factors alter mitochondrial homeostasis remains uncharacterized in FA cardiomyopathy. This investigation examined the muscle creatine kinase conditional frataxin knockout mouse, which closely mimics FA cardiomyopathy, to dissect the mechanisms of dysfunctional mitochondrial homeostasis. Dysfunction of key mitochondrial homeostatic mechanisms were elucidated in the knockout hearts relative to wild-type littermates, namely: (1) mitochondrial proliferation with condensed cristae; (2) impaired NAD+ metabolism due to perturbations in Sirt1 activity and NAD+ salvage; (3) increased mitochondrial biogenesis, fusion and fission; and (4) mitochondrial accumulation of Pink1/Parkin with increased autophagic/mitophagic flux. Immunohistochemistry of FA patients' heart confirmed significantly enhanced expression of markers of mitochondrial biogenesis, fusion/fission and autophagy. These novel findings demonstrate cardiac frataxin-deficiency results in significant changes to metabolic mechanisms critical for mitochondrial homeostasis. This mechanistic dissection provides critical insight, offering the potential for maintaining mitochondrial homeostasis in FA and potentially other cardio-degenerative diseases by implementing innovative treatments targeting mitochondrial homeostasis and NAD+ metabolism.

The parasite-derived peptide FhHDM-1 activates the PI3K/Akt pathway to prevent cytokine-induced apoptosis of ß-cells.

Type 1 diabetes (T1D) is an autoimmune disease characterised by the destruction of the insulin-producing beta (ß)-cells within the pancreatic islets. We have previously identified a novel parasite-derived molecule, termed Fasciola hepatica helminth defence molecule 1 (FhHDM-1), that prevents T1D development in non-obese diabetic (NOD) mice. In this study, proteomic analyses of pancreas tissue from NOD mice suggested that FhHDM-1 activated the PI3K/Akt signalling pathway, which is associated with ß-cell metabolism, survival and proliferation. Consistent with this finding, FhHDM-1 preserved ß-cell mass in NOD mice. Examination of the biodistribution of FhHDM-1 after intraperitoneal administration in NOD mice revealed that the parasite peptide localised to the pancreas, suggesting that it exerted a direct effect on the survival/function of ß-cells. This was confirmed in vitro, as the interaction of FhHDM-1 with the NOD-derived ß-cell line, NIT-1, resulted in increased levels of phosphorylated Akt, increased NADH and NADPH and reduced activity of the NAD-dependent DNA nick sensor, poly(ADP-ribose) polymerase (PARP-1). As a consequence, ß-cell survival was enhanced and apoptosis was prevented in the presence of the pro-inflammatory cytokines that destroy ß-cells during T1D pathogenesis. Similarly, FhHDM-1 protected primary human islets from cytokine-induced apoptosis. Importantly, while FhHDM-1 promoted ß-cell survival, it did not induce proliferation. Collectively, these data indicate that FhHDM-1 has significant therapeutic applications to promote ß-cell survival, which is required for T1D and T2D prevention and islet transplantation. KEY MESSAGES: FhHDM-1 preserves ß-cell mass in NOD mice and prevents the development of T1D. FhHDM-1 enhances phosphorylation of Akt in mouse ß-cell lines. FhHDM-1 increases levels of NADH/NADPH in mouse ß-cell lines in vitro. FhHDM-1 prevents cytokine-induced cell death of mouse ß-cell lines and primary human ß-cells in vitro via activation of the PI3K/Akt pathway.

The kynurenine pathway in chronic diseases: a compensatory mechanism or a driving force?

The kynurenine (KYN) pathway (KP) of tryptophan (TRP) metabolism is dysregulated in inflammation-driven pathologies including oncological and brain diseases [e.g., multiple sclerosis (MS), depression] and thus is a promising therapeutic target. Both pathological and compensatory mechanisms underlie disease-associated KP activation. There is growing evidence for bioenergetic roles of certain KP metabolites such as kynurenic acid (KA), or quinolinic acid (QA) as an NAD+ precursor, which may explain its frequently observed 'pathological' overactivation. Disease- and tissue-specific aspects, negative feedback on inflammatory signals, and the balance of downstream metabolites are likely to be decisive factors in the interpretation of an imbalanced KP. Therapeutic strategies should consider the compensatory actions and bioenergetic roles of KP metabolites to successfully design future theragnostic approaches aimed at attenuating disease progression.

Plasma lipidome is dysregulated in Alzheimer's disease and is associated with disease risk genes.

Lipidomics research could provide insights of pathobiological mechanisms in Alzheimer's disease. This study explores a battery of plasma lipids that can differentiate Alzheimer's disease (AD) patients from healthy controls and determines whether lipid profiles correlate with genetic risk for AD. AD plasma samples were collected from the Sydney Memory and Ageing Study (MAS) Sydney, Australia (aged range 75-97 years; 51.2% male). Untargeted lipidomics analysis was performed by liquid chromatography coupled-mass spectrometry (LC-MS/MS). We found that several lipid species from nine lipid classes, particularly sphingomyelins (SMs), cholesterol esters (ChEs), phosphatidylcholines (PCs), phosphatidylethanolamines (PIs), phosphatidylinositols (PIs), and triglycerides (TGs) are dysregulated in AD patients and may help discriminate them from healthy controls. However, when the lipid species were grouped together into lipid subgroups, only the DG group was significantly higher in AD. ChEs, SMs, and TGs resulted in good classification accuracy using the Glmnet algorithm (elastic net penalization for the generalized linear model [glm]) with more than 80% AUC. In general, group lipids and the lipid subclasses LPC and PE had less classification accuracy compared to the other subclasses. We also found significant increases in SMs, PIs, and the LPE/PE ratio in human U251 astroglioma cell lines exposed to pathophysiological concentrations of oligomeric Aß42. This suggests that oligomeric Aß42 plays a contributory, if not causal role, in mediating changes in lipid profiles in AD that can be detected in the periphery. In addition, we evaluated the association of plasma lipid profiles with AD-related single nucleotide polymorphisms (SNPs) and polygenic risk scores (PRS) of AD. We found that FERMT2 and MS4A6A showed a significantly differential association with lipids in all lipid classes across disease and control groups. ABCA7 had a differential association with more than half of the DG lipids (52.63%) and PI lipids (57.14%), respectively. Additionally, 43.4% of lipids in the SM class were differentially associated with CLU. More than 30% of lipids in ChE, PE, and TG classes had differential associations with separate genes (ChE-PICALM, SLC24A4, and SORL1; PE-CLU and CR1; TG-BINI) between AD and control group. These data may provide renewed insights into the pathobiology of AD and the feasibility of identifying individuals with greater AD risk.

Role of Nitric Oxide in Neurodegeneration: Function, Regulation, and Inhibition.

Reactive nitrogen species (RNS) and reactive oxygen species (ROS), collectively known as reactive oxygen and nitrogen species (RONS), are the products of normal cellular metabolism and interact with several vital biomolecules including nucleic acid, proteins, and membrane lipids and alter their function in an irreversible manner which can lead to cell death. There is an imperative role for oxidative stress in the pathogenesis of cognitive impairments and the development and progression of neural injury. Elevated production of higher amounts of nitric oxide (NO) takes place in numerous pathological conditions, such as neurodegenerative diseases, inflammation, and ischemia, which occur concurrently with elevated nitrosative/oxidative stress. The enzyme nitric oxide synthase (NOS) is responsible for the generation of NO in different cells by conversion of Larginine (Arg) to L-citrulline. Therefore, the NO signaling pathway represents a viable therapeutic target. Naturally occurring polyphenols targeting the NO signaling pathway can be of major importance in the field of neurodegeneration and related complications. Here, we comprehensively review the importance of NO and its production in the human body and afterwards highlight the importance of various natural products along with their mechanisms against various neurodegenerative diseases involving their effect on NO production.

Alteration in Gene Pair Correlations in Tryptophan Metabolism as a Hallmark in Cancer Diagnosis.

Tryptophan metabolism plays essential roles in both immunomodulation and cancer development. Indoleamine 2,3-dioxygenase, a rate-limiting enzyme in the metabolic pathway, is overexpressed in different types of cancer. To get a better understanding of the involvement of tryptophan metabolism in cancer development, we evaluated the expression and pairwise correlation of 62 genes in the metabolic pathway across 12 types of cancer. Only gene AOX1, encoding aldehyde oxidase 1, was ubiquitously downregulated, Furthermore, we observed that the 62 genes were widely and strongly correlated in normal controls, however, the gene pair correlations were significantly lost in tumor patients for all 12 types of cancer. This implicated that gene pair correlation coefficients of the tryptophan metabolic pathway could be applied as a prognostic and/or diagnostic biomarker for cancer.

Clinical Assessment of the NADome as Biomarkers for Healthy Aging.

Nicotinamide adenine dinucleotide (NAD+) and its related metabolites (NADome) are important endogenous analytes that are thought to play important roles in cellular metabolism, inflammation, oxidative stress, cancer, neurodegeneration, and aging in mammals. However, these analytes are unstable during the collection of biological fluids, which is a major limiting factor for their quantitation. Herein, we describe a highly robust and quantitative method using liquid chromatography coupled to tandem mass spectrometry to quantify the NADome in whole blood, plasma, mononuclear cells, platelets, cerebrospinal fluid (CSF), and urine. This methodology represents a "gold standard" of measure for understanding biological pathways and developing targeted pharmacological interventions to modulate NAD+ biosynthesis and NAD-dependent mediators in health and disease.

The potential of the novel NAD+ supplementing agent, SNH6, as a therapeutic strategy for the treatment of Friedreich's ataxia.

Friedreich's ataxia (FA) is due to deficiency of the mitochondrial protein, frataxin, which results in multiple pathologies including a deadly, hypertrophic cardiomyopathy. Frataxin loss leads to deleterious accumulations of redox-active, mitochondrial iron, and suppressed mitochondrial bioenergetics. Hence, there is an urgent need to develop innovative pharmaceuticals. Herein, the activity of the novel compound, 6-methoxy-2-salicylaldehyde nicotinoyl hydrazone (SNH6), was assessed in vivo using the well-characterized muscle creatine kinase (MCK) conditional frataxin knockout (KO) mouse model of FA. The design of SNH6 incorporated a dual-mechanism mediating: (1) NAD+-supplementation to restore cardiac bioenergetics; and (2) iron chelation to remove toxic mitochondrial iron. In these studies, MCK wild-type (WT) and KO mice were treated for 4-weeks from the asymptomatic age of 4.5-weeks to 8.5-weeks of age, where the mouse displays an overt cardiomyopathy. SNH6-treatment significantly elevated NAD+ and markedly increased NAD+ consumption in WT and KO hearts. In SNH6-treated KO mice, nuclear Sirt1 activity was also significantly increased together with the NAD+-metabolic product, nicotinamide (NAM). Therefore, NAD+-supplementation by SNH6 aided mitochondrial function and cardiac bioenergetics. SNH6 also chelated iron in cultured cardiac cells and also removed iron-loading in vivo from the MCK KO heart. Despite its dual beneficial properties of supplementing NAD+ and chelating iron, SNH6 did not mitigate cardiomyopathy development in the MCK KO mouse. Collectively, SNH6 is an innovative therapeutic with marked pharmacological efficacy, which successfully enhanced cardiac NAD+ and nuclear Sirt1 activity and reduced cardiac iron-loading in MCK KO mice. No other pharmaceutical yet designed exhibits both these effective pharmacological properties.

Zero valent iron core-iron oxide shell nanoparticles as small magnetic particle imaging tracers.

Nanoparticle tracers with small sizes and large magnetization are critical for biomedical imaging and especially for magnetic particle imaging (MPI). Small size is important for accessing future intracellular and neurological in vivo applications Here, we show <15 nm nanoparticles made of zero valent iron cores, iron oxide shells and coated with a strongly binding brush co-polymer are effective MPI tracers. The small nanoparticle cores create a hydrodynamic diameter that is half of the state-of-the-art iron oxide tracers while the strongly magnetic zero valent iron maintains similar MPI signal magnitude and resolution.

Novel multifunctional iron chelators of the aroyl nicotinoyl hydrazone class that markedly enhance cellular NAD+ /NADH ratios.

BACKGROUND AND PURPOSE: Alzheimer's disease (AD) is a multifactorial condition leading to cognitive decline and represents a major global health challenge in ageing populations. The lack of effective AD therapeutics led us to develop multifunctional nicotinoyl hydrazones to target several pathological characteristics of AD. EXPERIMENTAL APPROACH: We synthesised 20 novel multifunctional agents based on the nicotinoyl hydrazone scaffold, which acts as a metal chelator and a lipophilic delivery vehicle, donating a NAD+ precursor to cells, to target metal dyshomeostasis, oxidative stress, ß-amyloid (Aß) aggregation, and a decrease in the NAD+ /NADH ratio. KEY RESULTS: The most promising compound, 6-methoxysalicylaldehyde nicotinoyl hydrazone (SNH6), demonstrated low cytotoxicity, potent iron (Fe)-chelation efficacy, significant inhibition of copper-mediated Aß aggregation, oxidative stress alleviation, effective donation of NAD+ to NAD-dependent metabolic processes (PARP and sirtuin activity) and enhanced cellular NAD+ /NADH ratios, as well as significantly increased median Caenorhabditis elegans lifespan (to 1.46-fold of the control); partly decreased BACE1 expression, resulting in significantly lower soluble amyloid precursor protein-ß (sAPPß) and Aß1-40 levels; and favourable blood-brain barrier-permeation properties. Structure-activity relationships demonstrated that the ability of these nicotinoyl hydrazones to increase NAD+ was dependent on the electron-withdrawing or electron-donating substituents on the aldehyde- or ketone-derived moiety. Aldehyde-derived hydrazones containing the ONO donor set and electron-donating groups were required for NAD+ donation and low cytotoxicity. CONCLUSIONS AND IMPLICATIONS: The nicotinoyl hydrazones, particularly SNH6, have the potential to act as multifunctional therapeutic agents and delivery vehicles for NAD+ precursors for AD treatment.

NAD+ therapy in age-related degenerative disorders: A benefit/risk analysis.

Nicotinamide adenine dinucleotide (NAD+) is an essential pyridine nucleotide that is present in all living cells. NAD+ acts as an important cofactor and substrate for a multitude of biological processes including energy production, DNA repair, gene expression, calcium-dependent secondary messenger signalling and immunoregulatory roles. The de novo synthesis of NAD+ is primarily dependent on the kynurenine pathway (KP), although NAD+ can also be recycled from nicotinic acid (NA), nicotinamide (NAM) and nicotinamide riboside (NR). NAD+ levels have been reported to decline during ageing and age-related diseases. Recent studies have shown that raising intracellular NAD+ levels represents a promising therapeutic strategy for age-associated degenerative diseases in general and to extend lifespan in small animal models. A systematic review of the literature available on Medline, Embase and Pubmed was undertaken to evaluate the potential health and/or longevity benefits due to increasing NAD+ levels. A total of 1545 articles were identified and 147 articles (113 preclinical and 34 clinical) met criteria for inclusion. Most studies indicated that the NAD+ precursors NAM, NR, nicotinamide mononucleotide (NMN), and to a lesser extent NAD+ and NADH had a favourable outcome on several age-related disorders associated with the accumulation of chronic oxidative stress, inflammation and impaired mitochondrial function. While these compounds presented with a limited acute toxicity profile, evidence is still quite limited and long-term human clinical trials are still nascent in the current literature. Potential risks in raising NAD+ levels in various clinical disorders using NAD+ precursors include the accumulation of putative toxic metabolites, tumorigenesis and promotion of cellular senescence. Therefore, NAD+ metabolism represents a promising target and further studies are needed to recapitulate the preclinical benefits in human clinical trials.

Antioxidant, antimicrobial and neuroprotective effects of Octaviania asterosperma in vitro.

Octaviania asterosperma (hypogeous Basidiomycota) We investigated the phenolic composition, and antioxidant, antimicrobial and antigenotoxic effects of methanol extracts of fruiting bodies from Octaviania asterosperma. The total phenolic content (ppm) of O. asterosperma was found to be catechin (54.73 ± 4.68), epicatechin (123.90 ± 8.52), caffeic acid (4.23 ± 0.97), p-hydroxybenzoic acid (37.72 ± 3.84), cinnamic acid (58.07 ± 5.40), gallic acid (56.64 ± 6.39), clorogenic acid (80.76 ± 4.92) and coumaric acid (2.45 ± 0.15). The total antioxidant status (TAS), total oxidant status (TOS) and oxidative stress index (OSI) were 3.410 ± 0.099 mmol/L, 7.548 ± 0.147 µmol/L and 0.221 ± 0.005 respectively. O. asterosperma showed some promising antimicrobial activity. The extract showed no genotoxic potential and attenuated hydrogen peroxide (H2O2)-induced oxidative DNA damage in neurons. Pre-treatment with O. asterosperma maintained mitochondrial function, reduced expression levels of cleaved-caspase-3 and apoptosis-inducing factor (AIF) when HT22 cells were exposed to pathophysiological concentrations of GLU (25 mM) and modulated protein kinase B (Akt), the mammalian target of rapamycin (mTOR), and the phosphotase and tensin homolog on chromosome ten (PTEN). O. asterosperma is an important food for the treatment or management of neurodegenerative disorders due to its phenolic content and potent antioxidant and anti-excitotoxic effects.

A Pilot Study Investigating Changes in the Human Plasma and Urine NAD+ Metabolome During a 6 Hour Intravenous Infusion of NAD.

Accumulating evidence suggests that active maintenance of optimal levels of the essential pyridine nucleotide, nicotinamide adenine dinucleotide (NAD+) is beneficial in conditions of either increased NAD+ turnover or inadequate synthesis, including Alzheimer's disease and other neurodegenerative disorders and the aging process. While studies have documented the efficacy of some NAD+ precursors such as nicotinamide riboside (NR) in raising plasma NAD+, no data are currently available on the fate of directly infused NAD+ in a human cohort. This study, therefore, documented changes in plasma and urine levels of NAD+ and its metabolites during and after a 6 h 3 µmol/min NAD+ intravenous (IV) infusion. Surprisingly, no change in plasma (NAD+) or metabolites [nicotinamide, methylnicotinamide, adenosine phosphoribose ribose (ADPR) and nicotinamide mononucleotide (NMN)] were observed until after 2 h. Increased urinary excretion of methylnicotinamide and NAD+ were detected at 6 h, however, no significant rise in urinary nicotinamide was observed. This study revealed for the first time that: (i) at an infusion rate of 3 µmol/min NAD+ is rapidly and completely removed from the plasma for at least the first 2 h; (ii) the profile of metabolites is consistent with NAD+ glycohydrolase and NAD+ pyrophosphatase activity; and (iii) urinary excretion products arising from an NAD+ infusion include NAD+ itself and methyl nicotinamide (meNAM) but not NAM.

The Precursor to Glutathione (GSH), γ-Glutamylcysteine (GGC), Can Ameliorate Oxidative Damage and Neuroinflammation Induced by Aß40 Oligomers in Human Astrocytes.

Glutathione (GSH) is one of the most abundant thiol antioxidants in cells. Many chronic and age-related diseases are associated with a decline in cellular GSH levels or impairment in the catalytic activity of the GSH biosynthetic enzyme glutamate cysteine ligase (GCL). γ-glutamylcysteine (GGC), a precursor to glutathione (GSH), can replenish depleted GSH levels under oxidative stress conditions, by circumventing the regulation of GSH biosynthesis and providing the limiting substrate. Soluble amyloid-ß (Aß) oligomers have been shown to induce oxidative stress, synaptic dysfunction and memory deficits which have been reported in Alzheimer's disease (AD). Calcium ions, which are increased with age and in AD, have been previously reported to enhance the formation of Aß40 oligomers, which have been casually associated with the pathogenesis of the underlying neurodegenerative condition. In this study, we examined the potential beneficial effects of GGC against exogenous Aß40 oligomers on biomarkers of apoptosis and cell death, oxidative stress, and neuroinflammation, in human astrocytes. Treatment with Aß40 oligomers significantly reduced the cell viability and apoptosis of astrocyte brain cultures and increased oxidative modifications of DNA, lipids, and protein, enhanced pro-inflammatory cytokine release and increased the activity of the proteolytic matrix metalloproteinase enzyme, matric metalloproteinase (MMP)-2 and reduced the activity of MMP-9 after 24 h. Co-treatment of Aß40 oligomers with GGC at 200 µM increased the activity of the antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx) and led to significant increases in the levels of the total antioxidant capacity (TAC) and GSH and reduced the GSSG/GSH ratio. GGC also upregulated the level of the anti-inflammatory cytokine IL-10 and reduced the levels of the pro-inflammatory cytokines (TNF-α, IL-6, and IL-1ß) and attenuated the changes in metalloproteinase activity in oligomeric Aß40-treated astrocytes. Our data provides renewed insight on the beneficial effects of increased GSH levels by GGC in human astrocytes, and identifies yet another potential therapeutic strategy to attenuate the cytotoxic effects of Aß oligomers in AD.

Multi-copper ferroxidase deficiency leads to iron accumulation and oxidative damage in astrocytes and oligodendrocytes.

Accumulation of iron has been associated with the pathobiology of various disorders of the central nervous system. Our previous work has shown that hephaestin (Heph) and ceruloplasmin (Cp) double knockout (KO) mice induced iron accumulation in multiple brain regions and that this was paralleled by increased oxidative damage and deficits in cognition and memory. In this study, we enriched astrocytes and oligodendrocytes from the cerebral cortex of neonatal wild-type (WT), Heph KO and Cp KO mice. We demonstrated that Heph is highly expressed in oligodendrocytes, while Cp is mainly expressed in astrocytes. Iron efflux was impaired in Cp KO astrocytes and Heph KO oligodendrocytes and was associated with increased oxidative stress. The expression of Heph, Cp, and other iron-related genes was examined in astrocytes and oligodendrocytes both with and without iron treatment. Interestingly, we found that the expression of the mRNA encoding ferroportin 1, a transmembrane protein that cooperates with CP and HEPH to export iron from cells, was positively correlated with Cp expression in astrocytes, and with Heph expression in oligodendrocytes. Our findings collectively demonstrate that HEPH and CP are important for the prevention of glial iron accumulation and thus may be protective against oxidative damage.

Resveratrol Enhances Apoptotic and Oxidant Effects of Paclitaxel through TRPM2 Channel Activation in DBTRG Glioblastoma Cells.

Numerous studies have reported a strong association between increased production of reactive oxygen species (ROS) and the pathobiology of several diseases, and cancer in particular. Therefore, manipulation of cellular oxidative stress levels represents an important therapeutic target. Recently, resveratrol (RESV), a naturally occurring phytochemical, has been shown to sensitize several cell lines to the anticancer effects of other chemotherapeutic agents, including paclitaxel (PAX). However, the molecular mechanisms of action of RESV through oxidative sensitive TRPM2 channel activation remain unclear. The aim of this study was to evaluate the effect of combination therapy of RESV and PAX on activation of TRPM2 in DBTRG glioblastoma cells. DBTRG cells were divided into four treatment groups: control, RESV (50 µM), PAX (50 µM), and PAX + RESV for 24 hours. Our data shows that markers for apoptosis, mitochondrial membrane depolarization and mitochondrial function, intracellular steady-state ROS levels, caspase 3 activity, TRPM2 current density, and Ca2+ florescence intensity were significantly increased in DBTRG cells following treatment with PAX and RESV, respectively, although cell viability was also decreased by these treatments. These biochemical markers were further increased to favor the anticancer effects of PAX in DBTRG cells in combination with RESV. The PAX and RESV-mediated increase in current density and Ca2+ florescence intensity was decreased with a TRPM2 blocker. This suggests that for this combination therapy to have a substantial effect on apoptosis and cell viability, the TRPM2 channel must be stimulated.

Novel therapeutic strategies for stroke: The role of autophagy.

Autophagy is an important biological mechanism involved in the regulation of numerous fundamental cellular processes that are mainly associated with cellular growth and differentiation. Autophagic pathways are vital for maintaining cellular homeostasis by enhancing the turnover of nonfunctional proteins and organelles. Neuronal cells, like other eukaryotic cells, are dependent on autophagy for neuroprotection in response to stress, but can also induce cell death in cerebral ischemia. Recent studies have demonstrated that autophagy may induce neuroprotection following acute brain injury, including ischemic stroke. However in some special circumstances, activation of autophagy can induce cell death, playing a deleterious role in the etiology and progression of ischemic stroke. Currently, there are no therapeutic options against stroke that demonstrate efficient neuroprotective abilities. In the present work, we will review the significance of autophagy in the context of ischemic stroke by first outlining its role in ischemic neuronal death. We will also highlight the potential therapeutic applications of pharmacological modulators of autophagy, including some naturally occurring polyphenolic compounds that can target this catabolic process. Our findings provide renewed insight on the mechanism of action of autophagy in stroke together with potential neuroprotective compounds, which may partially exert their function through enhancing mitochondrial function and attenuating damaging autophagic processes.