Hussein Mansour

Age Related Changes in NAD+ Metabolism Oxidative Stress and Sirt1 Activity in Wistar Rats

The cofactor nicotinamide adenine dinucleotide (NAD+) has emerged as a key regulator of metabolism, stress resistance and longevity. Apart from its role as an important redox carrier, NAD+ also serves as the sole substrate for NAD-dependent enzymes, including poly(ADP-ribose) polymerase (PARP), an important DNA nick sensor, and NAD-dependent histone deacetylases, Sirtuins which play an important role in a wide variety of processes, including senescence, apoptosis, differentiation, and aging. We examined the effect of aging on intracellular NAD+ metabolism in the whole heart, lung, liver and kidney of female wistar rats. Our results are the first to show a significant decline in intracellular NAD+ levels and NAD:NADH ratio in all organs by middle age (i.e.12 months) compared to young (i.e. 3 month old) rats. These changes in [NAD(H)] occurred in parallel with an increase in lipid peroxidation and protein carbonyls (o- and m- tyrosine) formation and decline in total antioxidant capacity in these organs. An age dependent increase in DNA damage (phosphorylated H2AX) was also observed in these same organs. Decreased Sirt1 activity and increased acetylated p53 were observed in organ tissues in parallel with the drop in NAD+ and moderate over-expression of Sirt1 protein. Reduced mitochondrial activity of complex I-IV was also observed in aging animals, impacting both redox status and ATP production. The strong positive correlation observed between DNA damage associated NAD+ depletion and Sirt1 activity suggests that adequate NAD+ concentrations may be an important longevity assurance factor.

Changes in kynurenine pathway metabolism in the brain, liver and kidney of aged female Wistar rats.

The kynurenine pathway of tryptophan catabolism plays an important role in several biological systems affected by aging. We quantified tryptophan and its metabolites kynurenine (KYN), kynurenine acid (KYNA), picolinic acid (PIC) and quinolinic acid (QUIN), and activity of the kynurenine pathway enzymes indoleamine 2,3‐dioxygenase (IDO), tryptophan 2,3‐dioxygenase (TDO) and quinolinic acid phosphoribosyltransferase (QPRTase), in the brain, liver and kidney of young, middle‐aged and old female Wistar rats. Tryptophan levels and TDO activity decreased in all tissues with age. In contrast, brain IDO activity increased with age, while liver and kidney IDO activity decreased with age. The levels of KYN, KYNA, QUIN and PIC in brain all increased with age, while the levels of KYN in the liver and kidney showed a tendency to decrease. The levels of KYNA in the liver did not change, but the levels of KYNA in the kidney increased. The levels of PIC and QUIN increased significantly in the liver but showed a tendency to decrease in the kidney. QPRTase activity in both brain and liver decreased with age but was elevated in the kidney in middle‐aged (12‐month‐old) rats. These age‐associated changes in tryptophan metabolism have the potential to impact upon major biological processes, including lymphocyte function, pyridine (NAD(P)(H)) synthesis and N‐methyl‐d‐aspartate (NMDA)‐mediated synaptic transmission, and may therefore contribute to several degenerative changes of the elderly.

Aging‐related changes in astrocytes in the rat retina: imbalance between cell proliferation and cell death reduces astrocyte availability

The aim of this study was to investigate changes in astrocyte density, morphology, proliferation and apoptosis occurring in the central nervous system during physiological aging. Astrocytes in retinal whole‐mount preparations from Wistar rats aged 3 (young adult) to 25 months (aged) were investigated qualitatively and quantitatively following immunofluorohistochemistry. Glial fibrillary acidic protein (GFAP), S100 and Pax2 were used to identify astrocytes, and blood vessels were localized using Griffonia simplicifolia isolectin B4. Cell proliferation was assessed by bromodeoxyuridine incorporation and cell death by TUNEL‐labelling and immunolocalization of the apoptosis markers active caspase 3 and endonuclease G. The density and total number of parenchymal astrocytes in the retina increased between 3 and 9 months of age but decreased markedly between 9 and 12 months. Proliferation of astrocytes was detected at 3 months but virtually ceased beyond that age, whereas the proportion of astrocytes that were TUNEL positive and relative expression of active caspase 3 and endonuclease G increased progressively with aging. In addition, in aged retinas astrocytes exhibited gliosis‐like morphology and loss of Pax2 reactivity. A small population of Pax2+/GFAP− cells was detected in both young adult and aged retinas. The reduction in the availability of astrocytes in aged retinas and other aging‐related changes reported here may have a significant impact on the ability of astrocytes to maintain homeostasis and support neuronal function in old age.

Differential expression of sirtuins in the aging rat brain

Although 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.

Accelerating Alzheimer's research through 'natural' animal models.

Purpose of review Alzheimers disease is a complex multifactorial age-related neurodegenerative disorder. Current transgenic animal models do not fully recapitulate human Alzheimers disease at the molecular, cellular and behavioural levels. This review aims to address the clinical relevance of using ‘physiologically’ aged rats, dogs and Octodon degus, as more representative ‘natural’ ecologically valid models to elucidate mechanistic aspects of Alzheimers disease, and for the development of therapeutic agents to attenuate age-related cognitive decline. Recent findings Aged rats, dogs and O. degus decline cognitively and ultimately develop Alzheimers disease-like symptoms in response to the natural ageing process. Aged rats provide a tractable and popular model to examine the neurobiological basis underlying cognitive decline with age, but they do not develop Alzheimers disease pathology. Progressive accumulation of abnormal amyloid-beta in extracellular plaques and surrounding cerebral vasculature is a common feature in human Alzheimers disease, aged canine model and most nonhuman primates. Interestingly, the O. degus develops amyloid-beta deposits, neurofibrillary tangles containing hyperphosphorylated tau protein, altered cholinergic transmission and cognitive deficits analogous to those observed in Alzheimers disease. Natural animal models better represent the full pathophysiology of Alzheimers disease and are not only a viable alternative to transgenic models, but also are arguably the preferable model. Summary ‘Natural’ models are useful to elucidate the neurobiological basis of Alzheimers disease and develop effective therapeutic strategies that can be translated into human clinical trials.

Connexin 30 Expression and Frequency of Connexin Heterogeneity in Astrocyte Gap Junction Plaques Increase with Age in the Rat Retina

We investigated age-associated changes in retinal astrocyte connexins (Cx) by assaying Cx numbers, plaque sizes, protein expression levels and heterogeneity of gap junctions utilizing six-marker immunohistochemistry (IHC). We compared Wistar rat retinal wholemounts in animals aged 3 (young adult), 9 (middle-aged) and 22 months (aged). We determined that retinal astrocytes have gap junctions composed of Cx26, -30, -43 and -45. Cx30 was consistently elevated at 22 months compared to younger ages both when associated with parenchymal astrocytes and vascular-associated astrocytes. Not only was the absolute number of Cx30 plaques significantly higher (P<0.05) but the size of the plaques was significantly larger at 22 months compared to younger ages (p<0.05). With age, Cx26 increased significantly initially, but returned to basal levels; whereas Cx43 expression remained low and stable with age. Evidence that astrocytes alter connexin compositions of gap junctions was demonstrated by the significant increase in the number of Cx26/Cx45 gap junctions with age. We also found gap junctions comprised of 1, 2, 3 or 4 Cx proteins suggesting that retinal astrocytes use various connexin protein combinations in their gap junctions during development and aging. These data provides new insight into the dynamic and extensive Cx network utilized by retinal astrocytes for communication within both the parenchyma and vasculature for the maintenance of normal retinal physiology with age. This characterisation of the changes in astrocytic gap junctional communication with age in the CNS is crucial to the understanding of physiological aging and age-related neurodegenerative diseases.

Diagnostic and Prognostic Potential of Retinal Biomarkers in Early On-Set Alzheimer’s Disease

OBJECTIVE Accumulating evidence suggests that the eye can be used in the assessment of early on-set Alzheimers disease (AD). The eye offers a natural window to the brain through the retina. The retina and brain share common developmental origins and patho-physiological origins and mechanisms, having been sequestered from it during early development, but retaining its connections with the brain via the optic nerve. Therefore, it is well understood that neurological abnormalities have a direct profound impact on the retina. Recent studies suggest an array of physiological and pathological changes in the retina in dementia and specifically in AD. There are also reports on imaging the two hallmark proteins of the disease, extracellular amyloid beta peptides and intracellular hyper phosphorylated tau protein, as a proxy to neuroimaging. RESULTS In this review, we summarise retinal structural, functional and vascular changes reported to be associated with AD. We also review techniques employed to image these two major hall mark proteins of AD and their relevance for early detection of AD.

Age-related changes in NAD+ metabolism in the brain of aged Wister rats: Relevance for Alzheimer's disease

Background: Aging is characterised by elevated levels of oxidative stress. DNA damage and reduced energy production are hallmarks of this degenerative process. Nicotinamide adenine dinucleotide (NAD+) provides a link between energy production, resistance to stress and longevity. NAD+ serves as a redox carrier, and as a substrate for the NAD-dependent DNA nick sensor poly(ADP-ribose) polymerase (PARP), and the Sirtuins, a family of transcription regulators that play crucial roles in cellular resistance to stress and increased lifespan. Methods: We examined age associated effects on intracellular NAD+ metabolism in selected brain regions in female wistar rats. Formation of protein carbonyls were detected using oand m-tyrosine assay using GC/MS. Lipid peroxidation was observed using western blotting for the detection of 4-hydroxynonenal protein expression. DNA damage and SIRT1 activity were measured using well established flourometric assays. Intracellular NAD+, NADH levels, and PARP activities were measured spectrophotometrically using well established assays. Results: Our results are the first to show a significant decline in intracellular NAD+ levels and NAD:NADH ratio after 12 and 24 months of age compared to young 3 month old rats, in parallel with an increase in lipid peroxidation, and increased formation of protein carbonyls and decline in total antioxidant capacity in the cortex, brainstem, hippocampus and cerebellum. We also found elevated levels of phosphorylated H2AX levels, a measure of DNA damage, in these same brain regions. Reduced mitochondrial complex I activity was also observed in aged rats, suggesting an accumulation of NADH and enhanced potential for production of free radicals by Fenton chemistry. A strong positive correlation was found between oxidative stress, PARP activity, ADP-ribose polymers and NAD+ depletion suggesting a role for NAD+ as a longevity assurance factor. While decreased Sirt1 activity was observed in response to NAD+ depletion, our observed moderate over-expression of Sirt1 may be a response to retard aging and confer resistance to oxidative stress in the brain. Conclusions: Chronic oxidative stress may lead to significant depletion of cellular NAD+ levels. Therefore, therapeutic targets aimed at promoting cellular NAD+ anabolism may prove efficacious in the protection of age-dependent cellular damage, in general, and in neurodegenerative diseases such as Alzheimer’s disease.