Browsing by Author "Guillemin, Gilles J."
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Item Central Kynurenine Pathway Shift with age in Women(2016-03-01) Grant, Ross; Guillemin, Gilles J.; Guest, Jade; de Bie, JosienAge is considered a dominant risk factor in the development of most neurodegenerative disorders. The kynurenine pathway, a major metabolic pathway of tryptophan is altered in the majority of neurodegenerative disorders. In this study, we have analysed CSF samples from 49 healthy women across a wide age range (0–90) for kynurenine pathway metabolites and the inflammatory marker neopterin. Our results show central tryptophan metabolism is increased with age in women, with an apparent shift towards the neurotoxin quinolinic acid. We also observed an increase in central levels of the inflammatory marker neopterin with age and a positive correlation between neopterin and kynurenine pathway activation. We conclude that, the changes that occur in the kynurenine pathway as a result of normal ageing are mechanistically linked to increased inflammatory signalling and have some explanatory potential with regard to age‐associated degenerative diseases in the CNS. Management of health in ageing and (preventative) treatment would do well to look to the kynurenine pathway for potentially novel solutions.
Item Characterisation of the Kynurenine Pathway in Skin‐Derived Fibroblasts and Keratinocytes(2015-06-01) Braidy, Nady; Guillemin, Gilles J.; Lovejoy, David; Bustamente, Sonia; Grant, Ross; Sheipouri, DibaAcute UVB exposure triggers inflammation leading to the induction of indoleamine 2,3 dioxygenase (IDO1), one of the first enzymes in the kynurenine pathway (KP) for tryptophan degradation. However, limited studies have been undertaken to determine the catabolism of tryptophan within the skin. The aim of this study was two fold: (1) to establish if the administration of the proinflammatory cytokine interferon‐gamma (IFN‐γ) and/or UVB radiation elicits differential KP expression patterns in human fibroblast and keratinocytes; and (2) to evaluate the effect of KP metabolites on intracellular nicotinamide adenine dinucleotide (NAD+) levels, and cell viability. Primary cultures of human fibroblasts and keratinocytes were used to examine expression of the KP at the mRNA level using qPCR, and at the protein level using immunocytochemistry. Cellular responses to KP metabolites were assessed by examining extracellular lactate dehydrogenase (LDH) activity and intracellular NAD+ levels. Major downstream KP metabolites were analyzed using GC/MS and HPLC. Our data shows that the KP is fully expressed both in human fibroblasts and keratinocytes. Exposure to UVB radiation and/or IFN‐γ causes significant changes in the expression pattern of downstream KP metabolites and enzymes. Exposure to various concentrations of KP metabolites showed marked differences in cell viability and intracellular NAD+ production, providing support for involvement of the KP in the de novo synthesis of NAD+ in the skin. This new information will have a significant impact on our understanding of the pathogenesis of UV related skin damage and the diagnosis of KP related disease states.
Item Differential Expression of Sirtuins in the Aging rat Brain(2015-05-08) Guillemin, Gilles J.; Sachdev, Perminder; Smythe, George; Chan-Ling, Tailoi; Mansour, Hussein; Jayasena, Tharusha; Grant, Ross; Poljak, Anne; Braidy, NadyAlthough there are seven mammalian sirtuins (SIRT1-7), little is known about their expression in the aging brain. To characterize the change(s) in mRNA and protein expression of SIRT1-7 and their associated proteins in the brain of “physiologically” aged Wistar rats. We tested mRNA and protein expression levels of rat SIRT1-7, and the levels of associated proteins in the brain using RT-PCR and western blotting. Our data shows that SIRT1 expression increases with age, concurrently with increased acetylated p53 levels in all brain regions investigated. SIRT2 and FOXO3a protein levels increased only in the occipital lobe. SIRT3-5 expression declined significantly in the hippocampus and frontal lobe, associated with increases in superoxide and fatty acid oxidation levels, and acetylated CPS-1 protein expression, and a reduction in MnSOD level. While SIRT6 expression declines significantly with age acetylated H3K9 protein expression is increased throughout the brain. SIRT7 and Pol I protein expression increased in the frontal lobe. This study identifies previously unknown roles for sirtuins in regulating cellular homeostasis and healthy aging.
Item Lycopene Pretreatment Ameliorates Acute Ethanol Induced NAD+ Depletion in Human Astroglial Cells(2015-05-14) Grant, Ross; Heng, Benjamin; Guillemin, Gilles J.; Guest, JadeExcessive alcohol consumption is associated with reduced brain volume and cognition. While the mechanisms by which ethanol induces these deleterious effects in vivo are varied most are associated with increased inflammatory and oxidative processes. In order to further characterise the effect of acute ethanol exposure on oxidative damage and NAD+ levels in the brain, human U251 astroglioma cells were exposed to physiologically relevant doses of ethanol (11 mM, 22 mM, 65 mM, and 100 mM) for≤30 minutes. Ethanol exposure resulted in a dose dependent increase in both ROS and poly(ADP-ribose) polymer production. Significant decreases in total NAD(H) and sirtuin 1 activity were also observed at concentrations≥22 mM. Similar to U251 cells, exposure to ethanol (≥22 mM) decreased levels of NAD(H) in primary human astrocytes. NAD(H) depletion in primary astrocytes was prevented by pretreatment with 1𝜇M of lycopene for 3.5 hours. Unexpectedly, in U251 cells lycopene treatment at concentrations≥5𝜇M resulted in significant reductions in [NAD(H)]. This study suggests that exposure of the brain to alcohol at commonly observed blood concentrations may cause transitory oxidative damage which may be at least partly ameliorated by lycopene.
Item Mapping NAD+ Metabolism in the Brain of Ageing Wistar Rats: Potential Targets for Influencing Brain Senescence(2014-04-01) Sachdev, Perminder; Smythe, George; Guillemin, Gilles J.; Chan-Ling, Tailoi; Mansour, Hussein; Jayasena, Tharusha; Grant, Ross; Poljak, Anne; Braidy, NadyOver the last decade, the importance of NAD+ has expanded beyond its role as an essential cofactor for energy metabolism. NAD+ has emerged as a major signalling molecule that serves as the sole substrate for several enzymatic reactions including the DNA repair enzyme, poly(ADP-ribose) polymerase (PARP), NAD-dependent protein deacetylases or CD38, and transcriptional factors by a new class of histone deacetylases known as sirtuins. NAD+ Levels are regulated by the metabolic status and cellular stress caused by oxidative stress and DNA damage. Since a detailed study of NAD+ metabolism in the healthy ageing mammalian brain is nascent, we examined the effect of ageing on intracellular NAD+ metabolism in different brain regions in female Wistar rats in young (3 months), middle aged (12 months) and older adults (24 months). Our results are the first to show a significant decline in intracellular NAD+ levels and NAD:NADH ratio with ageing in the CNS, occurring in parallel to an increase in lipid peroxidation and protein oxidation (o- and m-tyrosine) and a decline in total antioxidant capacity. Hyperphosphorylation of H2AX levels was also observed together with increased PARP-1 and PARP-2 expression, and CD38 activity, concomitantly with reduced NAD+ and ATP levels and SIRT1 function in the cortex, brainstem, hippocampus and cerebellum. Reduced activity of mitochondrial complex I–IV and impaired maximum mitochondrial respiration rate were also observed in the ageing rat brain. Among the multiple physiological pathways associated with NAD+ catabolism, our discovery of CD38 as the major regulator of cellular NAD+ levels in rat neurons indicates that CD38 is a promising therapeutic target for the treatment of age-related neurodegenerative diseases.
Item Mechanism for Quinolinic Acid Cytotoxicity in Human Astrocytes(2009-01-01) Guillemin, Gilles J.; Brew, Bruce J.; Adams, Seray; Grant, Ross; Braidy, NadyThere is growing evidence implicating the kynurenine pathway (KP) and particularly one of its metabolites, quinolinic acid (QUIN), as important contributors to neuroinflammation in several brain diseases. While QUIN has been shown to induce neuronal and astrocytic apoptosis, the exact mechanisms leading to cell death remain unclear. To determine the mechanism of QUIN-mediated excitotoxicity in human brain cells, we measured intracellular levels of nicotinamide adenine dinucleotide (NAD+) and poly(ADP-ribose) polymerase (PARP) and extracellular lactate dehydrogenase (LDH) activities in primary cultures of human neurons and astrocytes treated with QUIN. We found that QUIN acts as a substrate for NAD+ synthesis at very low concentrations (nM) in both neurons and astrocytes, but is cytotoxic at sub-physiological concentrations (>150 nM) in both the cell types. We have shown that the NMDA ion channel blockers, MK801 and memantine, and the nitric oxide synthase (NOS) inhibitor, L-NAME, significantly attenuate QUIN-mediated PARP activation, NAD+ depletion, and LDH release in both neurons and astrocytes. An increased mRNA and protein expression of the inducible (iNOS) and neuronal (nNOS) forms of nitric oxide synthase was also observed following exposure of both cell types to QUIN. Taken together these results suggests that QUIN-induced cytotoxic effects on neurons and astrocytes are likely to be mediated by an over activation of an NMDA-like receptor with subsequent induction of NOS and excessive nitric oxide (NO•)-mediated free radical damage. These results contribute significantly to our understanding of the pathophysiological mechanisms involved in QUIN neuro- and gliotoxicity and are relevant for the development of therapies for neuroinflammatory diseases.