The Effect of Stress Induced Premature Senescence on the Expression of Heterogeneous RibonucleoIeoprotein

The role of heterogeneous nuclear ribonucleoproteins (hnRNP) in cellular senescence is yet to be defined. Cellular senescence is a terminal growth arrest in somatic cells. It is thought to be the consequence of telomeric shortening that acts as a DNA damage signal. Conversely, cells induced into premature senescence (SIPS) by oxidative stress, is independent of telomere attrition. Premature senescence has been proposed to be physiologically relevant as it can be induced by treatment with chemotherapeutic agents. In particular, we are studying the roles of hnRNP A1 and A2 in the maintenance of the senescence phenotype. hnRNPs are a family of RNA binding proteins that play key roles in various metabolic functions of the RNA such as splicing. hnRNP A1 in addition to its biochemical functions in RNA metabolism can bind to mRNAs for transport through the nuclear envelope. Our studies have shown that the protein level of hnRNP A1 is lower in senescent cells than in young cells. Thus, we have hypothesized that the altered expression of hnRNP A1 potentially modulates gene expression profiles and may maintain the senescent phenotype. We have observed that varying the levels of hnRNP A1 alters the expression levels of p21, p16 and ARF, all cell cycle regulators. To determine if this is the case during stress induced premature senescence (SIPS), IMR-90 fibroblast diploid normal lung cells were treated with a sub-lethal concentration of hydrogen peroxide that has been shown to induce premature senescence. To determine if hnRNPs may play a role in vivo, we measured their expression in rat hippocampal lysates isolated from rats treated with a combination of the chemotherapeutic drugs, doxorubicin and cyclophosphamide. We have shown here that stress induced premature senescence is not modulated in the same hnRNP dependent fashion as replicative senescence and possibly independent of p38MAPK

Testosterone Deficiency Accelerates Neuronal and Vascular Aging of SAMP8 Mice: Protective Role of eNOS and SIRT1

Oxidative stress and atherosclerosis-related vascular disorders are risk factors for cognitive decline with aging. In a small clinical study in men, testosterone improved cognitive function; however, it is unknown how testosterone ameliorates the pathogenesis of cognitive decline with aging. Here, we investigated whether the cognitive decline in senescence-accelerated mouse prone 8 (SAMP8), which exhibits cognitive impairment and hypogonadism, could be reversed by testosterone, and the mechanism by which testosterone inhibits cognitive decline. We found that treatment with testosterone ameliorated cognitive function and inhibited senescence of hippocampal vascular endothelial cells of SAMP8. Notably, SAMP8 showed enhancement of oxidative stress in the hippocampus. We observed that an NAD+-dependent deacetylase, SIRT1, played an important role in the protective effect of testosterone against oxidative stress-induced endothelial senescence. Testosterone increased eNOS activity and subsequently induced SIRT1 expression. SIRT1 inhibited endothelial senescence via up-regulation of eNOS. Finally, we showed, using co-culture system, that senescent endothelial cells promoted neuronal senescence through humoral factors. Our results suggest a critical role of testosterone and SIRT1 in the prevention of vascular and neuronal aging

The interaction between the circadian clock, temperature entrainment and the insoluble protein content

The elderly and patients suffering from age-associated neurodegenerative diseases (ND) often present with disrupted circadian timing systems such as alterations in their wake-sleep cycle. As aging and ND are associated with an increase of protein aggregates in the brain tissue, a correlation between protein aggregation and a disrupted circadian timing system seems likely. This study aims to investigate the effect of the circadian clock on protein aggregation in aging neuronal cells. We established an aging neuronal cell model by inducing senescence with administration of a histone deacetylase inhibitor (LBH589) in mouse neuroblastoma cells (N2A cells). One hallmark of senescent cells in an increase of protein aggregates. Therefore, senescent neurons represent a low complexity in vitro model for aging cells in which to test discrete hypotheses. In order to investigate the effect of circadian entrainment on protein aggregation in senescent cells, we kept senescent N2A cells in 24 temperature cycles with 34 °C (12 hours) and 37 °C (12 hours). A comparison with control cells kept in 24h temperature cycles or constant temperature showed effects of a 24h zeitgeber cycle on aggregation. Analyses of the protein content in N2A cells kept in temperature cycles revealed a decrease in insoluble protein content compared to cells kept in constant temperature condition. However, we observed no differences in the insoluble protein content of cells kept in 24h versus non-24h temperature cycles. Therefore, we suggest a direct effect of temperature on the insoluble protein content in N2A cells, without a significant influence of circadian clock oscillation. These findings support the hypothesis that the master pacemaker of the circadian timing system, the suprachiasmatic nucleus (SCN), influences protein aggregation through regulating temperature amplitudes and not as initially expected through circadiantemperature amplitudes. An impaired SCN and temperature alterations in elderly and ND patients, suggested by several studies, may influence the proteostasis network adverse. Therefore, a better rhythmicity (like activity at day time, sleep during night) in elderly could lead to a better entrainment of the SCN and higher temperature amplitudes, which would protect the cell from protein aggregates and thus could be a therapy option for ND in future. Furthermore, the effect of protein aggregates on cell survival could be a focus of future projects. If decreasing the insoluble protein content of a cell is beneficial, this could represent a target for new therapeutic approaches to treat or even prevent neurodegenerative diseases

Reactive and Senescent Astroglial Phenotypes as Hallmarks of Brain Pathologies

Astrocytes, as the most abundant glial cells in the central nervous system, are tightly integrated into neural networks and participate in numerous aspects of brain physiology and pathology. They are the main homeostatic cells in the central nervous system, and the loss of astrocyte physiological functions and/or gain of pro-inflammatory functions, due to their reactivation or cellular senescence, can have profound impacts on the surrounding microenvironment with pathological outcomes. Although the importance of astrocytes is generally recognized, and both senescence and reactive astrogliosis have been extensively reviewed independently, there are only a few comparative overviews of these complex processes. In this review, we summarize the latest data regarding astrocyte reactivation and senescence, and outline similarities and differences between these phenotypes from morphological, functional, and molecular points of view. A special focus has been given to neurodegenerative diseases, where these phenotypic alternations of astrocytes are significantly implicated. We also summarize current perspectives regarding new advances in model systems based on astrocytes as well as data pointing to these glial cells as potential therapeutic targets

The role of antioxidants in the interplay between oxidative stress and senescence

Cellular senescence is an irreversible state of cell cycle arrest occurring in response to stressful stimuli, such as telomere attrition, DNA damage, reactive oxygen species, and oncogenic proteins. Although beneficial and protective in several physiological processes, an excessive senescent cell burden has been involved in various pathological conditions including aging, tissue dysfunction and chronic diseases. Oxidative stress (OS) can drive senescence due to a loss of balance between pro-oxidant stimuli and antioxidant defences. Therefore, the identification and characterization of antioxidant compounds capable of preventing or counteracting the senescent phenotype is of major interest. However, despite the considerable number of studies, a comprehensive overview of the main antioxidant molecules capable of counteracting OS-induced senescence is still lacking. Here, besides a brief description of the molecular mechanisms implicated in OS-mediated aging, we review and discuss the role of enzymes, mitochondria-targeting compounds, vitamins, carotenoids, organosulfur compounds, nitrogen non-protein molecules, minerals, flavonoids, and non-flavonoids as antioxidant compounds with an anti-aging potential, therefore offering insights into innovative lifespan-extending approaches

Cholestasis-Induced Cognitive Decline: Neurological Mechanisms and Therapeutic Implications

Ph. D. ThesisCognitive dysfunction occurs during the cholestatic liver disease Primary biliary Cholangitis (PBC). Patients experience with short-term memory and concentration deficit, termed ‘brain fog’. This can be debilitating and severely affect quality of life, with no beneficial treatments (Newton, Hollingsworth et al. 2008) In this thesis, using the murine Bile Duct Ligation (BDL) model and in vitro modelling with human neuronal cells, I investigated the effects of cholestasis on the blood-brain barrier (BBB) and resultant changes within the brain. This study focussed on the hippocampus due to its crucial role in learning and memory. Changes to BBB could be observed from day 6, including astrocyte detachment, and permeability using MRI. By day 10 mice had visual spatial memory deficits, neuroinflammation was seen within the hippocampus, and changes were observed in gamma frequency oscillations. Interestingly, neurons in the hippocampus possessed features of senescence in cholestatic mice (telomere associated DNA damage, and P21+ RNA), highlighting parallels in pathology between the liver (where senescence is known to occur during cholestasis) and the brain. This effect was mirrored during in vitro studies, where neurons treated with PBC patient serum (to simulate pathological exposure to bile acids) also displayed increased sen-B-gal, a feature of senescence. In another strand, I examined the ability of both approved (ursodeoxycholic acid, Obeticholic Acid) and experimental (Bezafibrate) therapies to modify cognitive processes. Only Obeticholic Acid provides a potential therapeutic due to its in vivo and in vitro effects. Strikingly, OCA improve cognition and reduces neuronal senescence in BDL mice and in vitro when neurons are pre-treated before serum treatments. Therefore, the data presented in this thesis implicates senescence as a key pathological feature of cholestatic disease not just in the liver but also in brain where it has been previously linked to poor cognition (Baker, Wijshake et al. 2011, Fielder, Tweedy et al. 2020). Further investigation of early intervention with OCA may prove beneficial to patients experiencing cognitive deficit

Investigating the expression of Topoisomerase II Beta in aged neurons: development of a Murine Cell Line and Drosophila model

The enzyme Topoisomerase II Beta (Top2B) has previously been shown to be a crucial component of neuronal differentiation and development in mammals. It is also expressed in adult neuronal tissue where it plays important roles in facilitating transcription of long genes and early response genes and may also be involved in DNA repair. To date, studies investigating age-related changes in Top2B expression in neuronal tissue are limited and the importance of Top2B in the maintenance of neuronal function and integrity during ageing has not yet been fully elucidated, thus this study aimed to further investigate Top2B during the ageing process. The development of an in vitro murine model of neuronal ageing was successfully achieved using the Cath.a-differentiated (CAD) cell line. A neuronal-like phenotype in CAD cells was achieved through serum starvation and cells were then chronologically aged. Levels of Top2B mRNA and protein were seen to decline significantly during ageing of the cells in RT-qPCR and western blotting experiments, respectively. Concomitant increases in protein levels of the tumour suppressor gene p21 were also observed as well as a significant accumulation of double strand breaks as shown by γH2AX assays. In addition, preliminary in vivo experiments also revealed age-related declines of Top2B in mouse hippocampus. The development of an equivalent human in vitro model using the human neuroblastoma cell line SH-SY5Y was unsuccessful. Further in vivo experiments using Drosophila brain tissue also revealed significant age-related declines in Topoisomerase II (Top2) protein levels with ageing in both males and females, which was accompanied by a decline in locomotor function and increases in advanced glycation end-products (AGEs) in females. Interestingly, in Drosophila this was not accompanied by a reduction in Top2 mRNA levels. Reduction in the levels of mouse Top2B and Drosophila Top2 with age may have profound effects on transcription and the ability of cells to repair DNA damage and may result in increased vulnerability to oxidative stress, ultimately having detrimental effects on longevity and normal ageing. Thus, these models offer an opportunity to further elucidate the functional effect of this loss, its causes and potential pharmaceutical interventions to reverse these effects. Importantly, they also illustrate the need for such research to be carried out in human neuronal cells and brain tissues

A study of the polycomb group complexes in the maintenance of heterochromatic genome stability and Alzheimer's disease

La démence d'Alzheimer est une maladie neurodégénérative caractérisée par une perte progressive et irreversible des fonctions cognitives et des compétences intellectuelles. La maladie d’Alzheimer se présente sous deux formes: la forme familiale ou précoce (EOAD) qui représente 5% des cas et elle est liée à des mutations génétiques affectant le métabolisme des peptides amyloïde; et la forme tardive ou sporadique (LOAD) qui représente 95% des cas mais son étiologie est encore mal définie. Cependant, le vieillissement reste le principal facteur de risque pour développer LOAD. Les changements épigénétiques impliquant des modifications des histones jouent un rôle crucial dans les maladies neurodégénératives et le vieillissement lié à l'âge. Des données récentes ont décrit LOAD comme un désordre de l'épigénome et ont associé ce trouble à l'instabilité génomique. Les protéines Polycomb sont des modificateurs épigénétiques qui induisent le remodelage de la chromatine et la répression des gènes à l'hétérochromatine facultative. Nous rapportons que les souris hétérozygotes pour une protéine Polycomb développent avec l'âge un trouble neurologique ressemblant à LOAD caractérisé par l’altération des fonctions cognitives, la phosphorylation de la protéine tau, l'accumulation des peptides amyloïde, et le dysfonctionnement synaptique. Ce phénotype pathologique est précédé par la décondensation de l’hétérochromatine neuronale et l'activation de la réponse aux dommages à l'ADN. Parallèlement, une réduction d’expression de polycomb, malformations de l'hétérochromatine neuronale, et l'accumulation de dommages à l'ADN étaient également présents dans les cerveaux de patients LOAD. Remarquablement, les dommages de l'ADN ne sont pas distribués de façon aléatoire sur le génome mais sont enrichis au niveau des séquences répétitives. Les conclusions présentées dans cette thèse ont identifié des modifications épigénétiques spécifiques qui conduisent à une instabilité génomique aberrante menant à la formation de LOAD. Ces résultats vont aider au développement de nouveaux traitements qui peuvent potentiellement ralentir la neurodégénérescence.Alzheimer’s disease (AD) is the most common neurodegenerative disorder characterized by progressive and irreversible decline in cognitive functions and thinking skills. There are two types of AD: early-onset or familial AD (EOAD) that accounts for 5% of cases and is linked to mutations affecting the amyloid metabolism and late-onset or sporadic AD (LOAD), which accounts for 95% of cases, however the etiology of this type remains poorly delineated with ageing presenting the main risk factor. Epigenetic changes involving histone modifications play a critical role in ageing and age- related neurodegenerative diseases. Recent evidence describing LOAD as an epigenetic disorder has accrued, associating this disorder to global genomic instability. Polycomb group proteins are epigenetic modifiers initiating chromatin remodeling and gene repression at facultative heterochromatin. We report that mice heterozygous for a polycomb protein develop with advancing age a neurological disorder resembling LOAD characterized by impaired memory behaviour, tau phosphorylation, amyloid accumulation, and synaptic dysfunction. Interestingly, this phenotype was preceded by neuronal heterochromatin decondensation and activation of DNA damage response. Concomitantly, polycomb deficiency, de-compaction of neuronal heterochromatin, and accumulation of DNA damage machinery were also characteristic of LOAD brains. Remarkably, DNA damage was not randomly distributed on the genome but enriched at heterochromatin. The findings presented in this thesis identified specific epigenetic modifications that lead to aberrant genomic instability in LOAD and will aid in the development of novel therapeutics, which may potentially slow neurodegeneration

Blocking the Notch Pathway with Gamma-Secretase Inhibitors Enhances Temozolomide Treatment of Gliomas through Therapy-Induced Senescence: A Dissertation

Glioma therapy relies on induction of cytotoxicity; however, the current combination of surgery, irradiation (IR) and temozolomide (TMZ) treatment does not result in a long-term cure. Our lab previously demonstrated that a small population of glioma cells enters a transient cell cycle arrest in response to chemotherapy. Treatment with TMZ significantly decreases initial neurosphere formation; however, after a short recovery period, a small number of cells resume neurosphere formation and repopulate the culture. This recovery of neurosphere growth recapitulates the inevitable glioma recurrence in the clinic. The focus of our laboratory is to study direct-target therapies that can be combined with TMZ to inhibit neurosphere recovery. The Notch pathway is a promising target because it is involved in cell growth and survival. Here, we demonstrate that blocking the Notch pathway using gamma-secretase inhibitors (GSIs) enhances TMZ treatment. The combination of TMZ and GSI treatments targets the cells capable of recovery. TMZ + GSI treated cells do not recover and are no longer capable of self-renewal. Interestingly, recovery is inhibited when the GSI is administered 24 hrs after TMZ treatment, demonstrating a sequence-dependent mechanism. TMZ + GSI treatment also decreases tumorigenicity. When glioma cell lines were treated in vitro and implanted in NU/NU nude mice, TMZ + GSI treatment extended latency and greatly increased survival. In addition, in vivo TMZ + GSI treatment completely blocked tumor progression and resulted in the loss of a palpable tumor in 50% of mice, while none of the TMZ-only treated mice survived. TMZ + GSI treated cultures and xenografts display a senescent phenotype. Cultures treated with TMZ + GSI have decreased proliferation, but no increase in cell death. We observed an increase in the number of cells expressing senescence-associated β-galactosidase in vitro and in vivo. This demonstrates that inhibition of the Notch pathway shifts TMZ-treated cells from a transient cell cycle arrest into a permanent senescent state. Senescent cells can stimulate the innate immune system. Here we demonstrate that TMZ + GSI treatment increases phagocytosis in vitro. New therapy combinations, such as TMZ + GSI, are arising in the field of therapy-induced senescence (TIS). Overall, this data demonstrates the importance of the Notch pathway in chemoprotection and maintenance of TMZ-treated gliomas. The addition of GSIs to current treatments is a promising target-directed therapy to decrease the rate of brain tumor recurrence by inducing senescence and tumor clearance