Nursing & Health
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Item Application of Targeted Mass Spectrometry for the Quantification of Sirtuins in the Central Nervous System(2016-10-20) Sachdev, Perminder; Raftery, M; Teo, C; Smythe, George; Grant, Ross; Muenchhoff, J; Rowlands, B; Zhong, L; Braidy, Nady; Poljak, Anne; Jayasena, TharushaSirtuin proteins have a variety of intracellular targets, thereby regulating multiple biological pathways including neurodegeneration. However, relatively little is currently known about the role or expression of the 7 mammalian sirtuins in the central nervous system. Western blotting, PCR and ELISA are the main techniques currently used to measure sirtuin levels. To achieve sufficient sensitivity and selectivity in a multiplex-format, a targeted mass spectrometric assay was developed and validated for the quantification of all seven mammalian sirtuins (SIRT1-7). Quantification of all peptides was by multiple reaction monitoring (MRM) using three mass transitions per protein-specific peptide, two specific peptides for each sirtuin and a stable isotope labelled internal standard. The assay was applied to a variety of samples including cultured brain cells, mammalian brain tissue, CSF and plasma. All sirtuin peptides were detected in the human brain, with SIRT2 being the most abundant. Sirtuins were also detected in human CSF and plasma, and guinea pig and mouse tissues. In conclusion, we have successfully applied MRM mass spectrometry for the detection and quantification of sirtuin proteins in the central nervous system, paving the way for more quantitative and functional studies.
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 Resveratrol as a Potential Therapeutic Candidate for the Treatment and Management of Alzheimer's Disease(2016-04-28) Grant, Ross; Sachdev, Perminder; Mohammad Nabavi, Seyed; Mansour, Hussein; Jayasena, Tharusha; Poljak, Anne; Jugder, Bat-Erdene; Braidy, NadyResveratrol (3,4',5-trihydroxystilbene) is a naturally occurring phytochemical present in red wine, grapes, berries, chocolate and peanuts. Clinically, resveratrol has exhibited significant antioxidant, anti-inflammatory, anti-viral, and anti-cancer properties. Although resveratrol was first isolated in 1940, it was not until the last decade that it was recognised for its potential therapeutic role in reducing the risk of neurodegeneration, and Alzheimer's disease (AD) in particular. AD is the primary cause of progressive dementia. Resveratrol has demonstrated neuroprotective effects in several in vitro and in vivo models of AD. Apart from its potent antioxidant and anti-inflammatory roles, evidence suggests that resveratrol also facilitates non-amyloidogenic breakdown of the amyloid precursor protein (APP), and promotes removal of neurotoxic amyloid beta (Aβ) peptides, a critical step in preventing and slowing down AD pathology. Resveratrol also reduces damage to neuronal cells via a variety of additional mechanisms, most notably is the activation of NAD+-dependent histone deacetylases enzymes, termed sirtuins. However in spite of the considerable advances in clarifying the mechanism of action of resveratrol, it is unlikely to be effective as monotherapy in AD due to its poor bioavailability, biotransformation, and requisite synergism with other dietary factors. This review summarizes the relevance of resveratrol in the pathophysiology of AD. It also highlights why resveratrol alone may not be an effective single therapy, and how resveratrol coupled to other compounds might yet prove an effective therapy with multiple targets.
Item Nicotinamide Adenine Dinucleotide and its Related Precursors for the Treatment of Alzheimer's Disease(2018-03-01) Sachdev, Perminder; Grant, Ross; Braidy, NadyPurpose of review The current review discusses the biology and metabolism of the essential pyridine nucleotide nicotinamide adenine dinucleotide (NAD+) in the central nervous system. We also review recent work suggesting important neuroprotective effects that may be associated with the promotion of NAD+ levels through NAD+ precursors against Alzheimer's disease.
Recent findings Perturbations in the physiological homoeostatic state of the brain during the ageing process can lead to impaired cellular function, and ultimately leads to loss of brain integrity and accelerates cognitive and memory decline. Increased oxidative stress has been shown to impair normal cellular bioenergetics and enhance the depletion of the essential nucleotides NAD+ and ATP. NAD+ and its precursors have been shown to improve cellular homoeostasis based on association with dietary requirements, and treatment and management of several inflammatory and metabolic diseases in vivo. Cellular NAD+ pools have been shown to be reduced in the ageing brain, and treatment with NAD+ precursors has been hypothesized to restore these levels and attenuate disruption in cellular bioenergetics.
Summary NAD+ and its precursors may represent an important therapeutic strategy to maintain optimal cellular homoeostatic functions in the brain. NAD+ precursors are available in a variety of foods and may be translated to the clinic in the form of supplements.
Item Role of Nicotinamide Adenine Dinucleotide and Related Precursors as Therapeutic Targets for Age-Related Degenerative Diseases: Rationale, Biochemistry, Pharmacokinetics, and Outcomes(2019-01-10) Sachdev, Perminder; Grant, Ross; Jayasena, Tharusha; Poljak, Anne; Khorshidi, Fatemeh; Clement, James; Berg, Jade; Braidy, NadySignificance: Nicotinamide adenine dinucleotide (NAD+) is an essential pyridine nucleotide that serves as an essential cofactor and substrate for a number of critical cellular processes involved in oxidative phosphorylation and ATP production, DNA repair, epigenetically modulated gene expression, intracellular calcium signaling, and immunological functions. NAD+ depletion may occur in response to either excessive DNA damage due to free radical or ultraviolet attack, resulting in significant poly(ADP-ribose) polymerase (PARP) activation and a high turnover and subsequent depletion of NAD+, and/or chronic immune activation and inflammatory cytokine production resulting in accelerated CD38 activity and decline in NAD+ levels. Recent studies have shown that enhancing NAD+ levels can profoundly reduce oxidative cell damage in catabolic tissue, including the brain. Therefore, promotion of intracellular NAD+ anabolism represents a promising therapeutic strategy for age-associated degenerative diseases in general, and is essential to the effective realization of multiple benefits of healthy sirtuin activity. The kynurenine pathway represents the de novo NAD+ synthesis pathway in mammalian cells. NAD+ can also be produced by the NAD+ salvage pathway.
Recent Advances: In this review, we describe and discuss recent insights regarding the efficacy and benefits of the NAD+ precursors, nicotinamide (NAM), nicotinic acid (NA), nicotinamide riboside (NR), and nicotinamide mononucleotide (NMN), in attenuating NAD+ decline in degenerative disease states and physiological aging.
Critical Issues: Results obtained in recent years have shown that NAD+ precursors can play important protective roles in several diseases. However, in some cases, these precursors may vary in their ability to enhance NAD+ synthesis via their location in the NAD+ anabolic pathway. Increased synthesis of NAD+ promotes protective cell responses, further demonstrating that NAD+ is a regulatory molecule associated with several biochemical pathways.
Future Directions: In the next few years, the refinement of personalized therapy for the use of NAD+ precursors and improved detection methodologies allowing the administration of specific NAD+ precursors in the context of patients' NAD+ levels will lead to a better understanding of the therapeutic role of NAD+ precursors in human diseases.
Item Quantitation of NAD+: Why Do We Need to Measure it?(2018-12-01) Braidy, Nady; Sachdev, Perminder; Grant, Ross; Clement, James; Liu, YueBackground:
Nicotinamide adenine dinucleotide (NAD+) is an essential pyridine nucleotide that is currently investigated as an important target to extend lifespan and health span. Age-related NAD+ depletion due to the accumulation of oxidative stress is associated with reduced energy production, impaired DNA repair and genomic instability.
Scope of review: NAD+ levels can be elevated therapeutically using NAD+ precursors or through lifestyle modifications including exercise and caloric restriction. However, high amounts of NAD+ may be detrimental in cancer progression and may have deleterious immunogenic roles.
Major conclusions: Standardized quantitation of NAD+ and related metabolites may therefore represent an important component of NAD+ therapy.
General significance: Quantitation of NAD+ may serve dual roles not only as an ageing biomarker, but also as a diagnostic tool for the prevention of malignant disorders.
Item Inhibition of Indoleamine 2,3 Dioxygenase Activity by H2O2(2006-06-01) Smythe, George A.; Sachdev, Perminder; Walker, Mark J.; Truscott, Roger J.; Littlejohn, Tamantha K.; Takikawa, Osamu; Willows, Robert D.; Jamie, Joanne F.; Austin, Chris J.; Grant, Ross; Poljak, AnneIndoleamine 2,3-dioxygenase is the first and rate limiting enzyme of the kynurenine pathway of tryptophan metabolism, has potent effects on cell proliferation and mediates antimicrobial, antitumorogenic, and immunosuppressive effects. As a potent cytotoxic effector, the mechanisms of indoleamine 2,3-dioxygenase inhibition deserve greater attention. The work presented here represents the first systematic study exploring the mechanisms by which low levels of hydrogen peroxide (10–100 μM) inhibit indoleamine 2,3-dioxygenase in vitro. Following brief peroxide exposure both enzyme inhibition and structural changes were observed. Loss of catalysis was accompanied by oxidation of several cysteine residues to sulfinic and sulfonic acids, observed by electrospray and MALDI mass spectrometry. Enzyme activity could in part be preserved in the presence of sulfhydryl containing compounds, particularly DTT and methionine. However, these structural alterations did not prevent substrate (l-tryptophan) binding. Some enzyme activity could be recovered in the presence of thioredoxin, indicating that the inhibitory effect of H2O2 is at least partially reversible in vitro. We present evidence that cysteine oxidation represents one mechanism of indoleamine 2,3-dioxygenase inhibition.
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.