Human Neural Stem Cell Aging Is Counteracted by Î±

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Human neural stem cell ageing is counteracted by #-glyceryl-phosphoryl-ethanolamine Simona Daniele, Eleonora Da Pozzo, Caterina Iofrida, and Claudia Martini ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00078 • Publication Date (Web): 11 May 2016 Downloaded from http://pubs.acs.org on May 13, 2016

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ACS Chemical Neuroscience

Human neural stem cell ageing is counteracted by α-glyceryl-phosphorylethanolamine

Simona Danielea,b, Eleonora Da Pozzoa, Caterina Iofridaa, Claudia Martinia*. a

Dept. of Pharmacy, University of Pisa, Italy

b

Dept. of Pharmacological and Biomolecular Sciences, University of Milan, Italy.

*Corresponding Author: Department of Pharmacy, University of Pisa, Via Bonanno, 6, Pisa 56126, Italy. Tel: +390502219509; Fax: +39050 2210680; [email protected]

1

ACS Paragon Plus Environment


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ABSTRACT Neural stem cells (NSCs) represent a subpopulation of cells, located in specific regions of the adult mammalian brain, with the ability of self-renewing and generating neurons and glia. In aged NSCs, modifications in the amount and composition of membrane proteins/lipids, which lead to a reduction in membrane fluidity and cholinergic activities, have been reported. In this respect, molecules that are effective at normalising the membrane composition and cholinergic signalling could counteract stem cell ageing. Alpha-glycerylphosphorylethanolamine (GPE), a nootropic drug, plays a role in phospholipid biosynthesis and acetylcholine release. Herein, GPE was assayed on human NSC cultures and on hydroxyurea-aged

cells.

Using

cell

counting,

colorimetric

and

fluorimetric

analyses,

immunoenzymatic assays and real time PCR experiments, NSC culture proliferation, senescence, reactive oxygen species and ADP/ATP levels were assessed. Aged NSCs exhibited cellular senescence, decreased proliferation, and an impairment in mitochondrial metabolism. These changes included a substantial induction in the nuclear factor NFκB, a key inflammatory mediator. GPE cell treatment significantly protected the redox state and functional integrity of mitochondria, and counteracted senescence and NF-κB activation. In conclusion, our data show the beneficial properties of GPE in this model of stem cell ageing. Key words: α-Glycerylphosphorylethanolamine; neural stem cell; aging; oxidative stress; phospholipid precursor; inflammation; cellular membrane.

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Introduction Ageing is characterised by a gradual physical and functional decline of cells; common features of senescence are telomere attrition, epigenetic modifications, reduction in protein synthesis, mitochondrial dysfunction, and deregulation of intercellular communication.1,2 In particular, the brain ageing process is characterised by significant structural and functional changes,3-5 which include degeneration of neurons3 and astrocytes,4 alterations of the plasma membrane composition,5 and neuroinflammation.6 The beneficial effects of phospholipid (PL) have been widely described in brain ageing. For example, phosphatidylcholine (PC) administration has been shown to increase explicit memory, through the improvement of choline,7 which plays a key role in brain development serving as an acetylcholine (ACh) precursor and as a constituent of the cellular membranes.7 Moreover, phosphatidylethanolamine (PE) and its precursor ethanolamine have been shown to regulate positively autophagy and to extend the lifespan of yeast and human cultures.8 Alpha-glycerylphosphorylethanolamine (GPE) is a precursor of the main constituents of the cellular membrane, PE and PC.9,10 Moreover, GPE acts through the PC pathway to improve ACh synthesis. Cholinergic neuronal circuits are critically compromised in ageing and neuronal diseases. Thus, GPE acts as a nootropic compound11 by improving the neuronal structures related to memorisation processes; in addition, GPE has been demonstrated to protect astrocytes from amyloid-induced reactive gliosis.9 However, the effects of GPE on stem cell niches remain unexplored. Neural Stem Cells (NSCs) represent a subpopulation of cells located in dentate gyrus of the hippocampus and in the subventricular zone of the lateral ventricles, which is capable of selfrenewal and differentiation (adult neurogenesis). Although neurogenesis proceeds throughout life, its rate decreases with increasing age due to an intrinsic decline in NSC responsiveness.12-15 Moreover, these aged-dependent modifications of NSC proliferation16,17 and differentiation18 cause the loss of cellular repair and regeneration capacity, thus contributing to brain disorders.19,20 In particular, specific alterations in the neurogenic niches correlate with early symptoms of neurodegenerative diseases (NDs), including Parkinson's and Huntington's.20,21 The decreased hippocampal neurogenesis has been related to memory retention in a mouse model of synucleinopathies, thus establishing a direct link between NSC differentiation and Parkinson's disease.22 An altered adult neurogenesis has been reported in transgenic models of Alzheimer's disease (AD),20,23-25 correlating NSC impairment with the presence of amyloid plaques.26 In AD patients, the majority of the studies has described a decreased neurogenesis,4,20,27 nevertheless an increased NSC differentiation has been also reported.28 3



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Impaired NSC self-renewal and neurogenesis have been also associated to an impairment of fatty acid and lipid pathways.29,30 For example, a decline in memory and learning abilities of elder brain has been related to a reduced amount of PC and/or polyunsaturated fatty acids (PUFAs) in neurogenic areas.29,31 Moreover, restoring the NSC membrane composition by PC supplementation has been shown to improve hippocampal neurogenesis and to decrease soluble tumour necrosis factor-alpha

(TNF-α)

levels,32

counteracting

systemic

inflammation.

Furthermore,

the

glycerophospholipid phosphatidylserine has been shown to improve NSC functionality and cholinergic transmission, leading to the final improvement of memory and learning.33,34 In this line, molecules that are effective in restoring PL content and cholinergic activities could constitute therapeutic tools to counteract NSC ageing. In the current study, a hydroxyurea (HU) treatment protocol2 was used to effectively induce human NSC senescence. The human cellular model was optimized and carefully characterized, and the putative protective and reparative effects of GPE were investigated. The present data demonstrated that GPE is efficacious in recovering NSCs from the cellular oxidative stress and defective mitochondrial metabolism that occur during the HU-induced ageing. This study further supports current investigations of new molecules candidate in counteracting the molecular features of the stem cell senescence.35 Further data are needed to translate these in vitro protective effects into in vivo models of NSC senescence and global brain ageing.

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Results and Discussion

Nevertheless NSC pivotal role in brain “well-being” has been extensively demonstrated, NSC ageing studies are obstructed by their in vivo limited availability, and by the lack of exhaustive models to examine ageing-related molecular mechanisms.2,36 In this paper, the first aim was to establish a human cellular model with a broad utility for the molecular investigation of NSC ageing, and then to dissect the effects of the PL analogue, GPE, on the most common parameters impaired in cellular ageing. Establishing the NSC ageing model An HU treatment protocol, previously reported in rat cells,2 was used in order to set up the cellular model. In order to determine the best dose of HU to induce cellular senescence, NSCs were challenged with different HU concentrations (0.5, 8 and 20 mM) for 1 and 16 h. The cell incubation with 20 mM HU caused cell death, as demonstrated by apoptotic staining (Figure 1A, B). Moreover, this HU concentration induced a significant reduction in the number of living cells and an increase of dead NSCs (Figure 1C). These data confirmed that the highest HU concentration induced NSC apoptosis/death, consistent to previously reported data on rat NSCs.2 In contrast, 0.5 and 8 mM HU did not affect cellular viability (Figure 1A and B). Then, a parameter of cellular ageing, the Double-strand break (DSB) extent in DNA, was examined by quantifying the phosphorylated histone γH2AX.2 Challenging cells with HU for 16 h induced a concentration-dependent increase in γH2AX phosphorylation (Figure 1D). However, DSBs induced by 0.5 mM HU returned to the starting point following 36 h cell wash out (data not shown). In contrast, 8 mM HU triggered a long-lasting γH2AX activation (Figure 1D and E). Similar to previous studies in rat cells,2 our data confirmed that a low dose of HU caused a transitory DNA damage, whereas 20 mM HU induced NSC cell death. Thus, the HU mild dose (8 mM) treatment for 16 h was chosen for the experimental model of NSC ageing. Globally, our results in human NSCs are consistent with those reported by Xu and co-workers on rat cells,2 and suggest that our experimental system can represent an useful human NSC model to investigate the effects of putative therapeutic anti-ageing compounds.

FIGURE 1 NEAR HERE 5



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GPE reverses DNA damage and restores NSC proliferative activity To investigate GPE-mediated effects on the NSC ageing model, two GPE treatments were designed (Figure 2A): one consisting in a 36 h pre-challenge with GPE before HU treatment (aimed at preventing HU-induced ageing), and the other consisting in a 36 h GPE challenging after HU exposure (aimed at activating reparative processes). Using these treatment protocols, the common parameters associated to cellular ageing (i.e., DNA damage, cellular senescence, mitochondrial impairment) were examined. The GPE pre-treatment significantly reduced γH2AX phosphorylation in aged cells. Similar results were obtained by treating NSCs with GPE following HU removal (Figure 2B), demonstrating that the drug can both prevent and reverse DNA damage. At the two tested concentrations (10 µg/ml or 100 µg/ml), GPE did not affect the basal level of DSBs (Figure 2B). Consistent with these data, a semisynthetic PC derivative, alpha-glycerylphosphorylethanocholine, has been shown to reduce DNA fragmentation in rat astroglial cultures,37 and an endogenous PL metabolite has inhibited ultraviolet light-induced DNA damage in human keratinocytes.38 Our results suggest that the previously demonstrated beneficial effects of PL-related molecules in preserving DNA integrity in differentiated cells could concern the stem cell pool too. Next, the effects of GPE on aged NSC self-renewal were investigated. The marked reduction in living cells induced by HU (Figure 2C) was counteracted by a pre-challenge with GPE (Figure 2C). Comparable results were gained treating the cells with GPE after HU exposure (Figure 2C). These data are concordant with those obtained by Bisaglia et al., showing that GPE prevented amyloid-induced impairment of astrocyte proliferation,9 thus confirming that the molecule under studies can rescue the proliferative arrest of both differentiated glial cells and NSCs.

FIGURE 2 NEAR HERE

GPE blocks NSC senescence To determine if the mild HU-mediated DSBs can trigger cell senescence, the established senescence marker β-galactosidase (SA-β-gal) was used. The HU exposure induced a significant β-galactosidase staining (Figure 3A) that is consistent with previously reports on HU-treated rat NSCs,2 and with data on mammalian NSCs following ionizing radiation-induced DNA damage,39 further supporting the validity of our cellular model to investigate the beneficial effects of new molecules. 6


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The percentage of SA-β-gal-positive cells were 5.66 ± 0.54 % in the control group and 29.45 ± 2.54 % in the HU-treated group. A significantly reduced β-galactosidase expression was shown in NSCs pre-treated with 10 or 100 µg/ml GPE for 36 h before the induction of cellular ageing (6.75 ± 1.19 % and 5.60 ± 0.45 %, respectively, Figure 3B). Similar effects were obtained in post-treatment samples. Higher protective effects were obtained in pre-treatment protocol with respect to the posttreatment, suggesting a preventive protective effect of GPE. The obtained data on NSC senescence are in agreement with those reported for the Ginsenoside Rg1 in a rat model of D-galactose-induced ageing.40

FIGURE 3 NEAR HERE

GPE restores mitochondrial energy metabolism Mitochondria show a pivotal role in ageing-related signalling pathways; specifically, the quiescent state of stem cells is characterised by a low cell metabolism.41 ATP is produced by the mitochondrial electron transport chain as an important source of cellular energy. A fall in ATP production, caused by electron transport chain defects, can trigger cellular senescence.42 In this respect, a decline in ATP content can also rise the ratio AMP/ATP (or ADP/ATP), thus generating a cellular bioenergetic imbalance.42-44 Herein, challenging NSCs with 8 mM HU significantly decreased ATP levels (Figure 4A) and slightly increased ADP content (Figure 4B), which resulted in an increase in the ADP/ATP ratio (Figure 4C). These data confirm that cellular ageing impairs energy metabolism:45 indeed, different animal or cellular models of ageing have shown an increase of ADP or (AMP) over ATP.41,44,45 GPE did not significantly affect ATP or ADP content (Figure 4A, B and C). In contrast, pre- or post-treatment with GPE (particularly at the highest concentration) significantly blocked HUmediated effects (i.e., ADP increase and ATP decrease) and restored the physiological ADP/ATP ratio (Figure 4A, B and C). Globally, GPE effects on HU-induced alteration of ADP/ATP content suggest that the compound can exert protective actions on NSCs; accordingly to this hypothesis, human neuronal mitochondrial uncoupling protein-5 has been demonstrated to protect neuronal cells

by

preserving

mitochondrial

ATP

levels

and

membrane

potential.46

GPE blocks the production of reactive oxygen species (ROS)

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Multiple ageing-related impairments ultimately converge to negatively impact cellular respiration via ROS overproduction. According to the “free radical theory of ageing”, oxygen-derived free radicals are responsible for the cellular age-associated impairment.12 As shown in Figure 4D, challenging NSCs with HU enhanced cellular ageing by increasing ROS production. The drug alone did not significantly affect ROS content (Figure 4D). Pre-treatment with GPE counteracted HU-mediated effects in a concentration-dependent manner (Figure 4D). Similar results were obtained for NSCs post-treated with GPE (Figure 4D). Consistent with our findings, challenging erythrocytes or rat brain with PC markedly prevented oxidative stress,47 thus confirming that PL precursors can block age-related accumulation of free radicals.

FIGURE 4 NEAR HERE

The effects of GPE on ageing-related genes Ageing is associated with alterations in the p53/p21 signalling, a pivotal response pathway to DNA damage that controls cell cycle arrest and senescence.48 Thus, the effects of GPE on the expression of ageing-related genes were investigated. Challenging NSCs with GPE alone (10 µg/ml or 100 µg/ml) did not significantly affect p53 transcription after 36 h of incubation (Figure 5A). Notably, p21 mRNA was slightly increased at both doses of GPE; however, this increase reached significance only at the highest GPE concentration. Consistent with previous studies in HU-treated rat NSCs,2 a mild exposure to HU for 16 h increased the mRNA levels of p53 (Figure 5A) and p21 (Figure 5B). A pre- or post-treatment with GPE did not attenuate the HU- mediated increase in p53 transcription (Figure 5A). The p53 transcriptional network is related to several cellular transduction pathways, including positive and negative feedback mechanisms.49 Thus, one hypothesis to explain our findings are the feedback mechanisms that occurred during the 36 h GPE treatment. In contrast to the lack of effects on p53 mRNA levels, GPE pre-treatment completely counteracted the HU-mediated increase in p21 mRNA (Figure 5B). A significant decrease in p21 mRNA was observed at the highest GPE concentration in cells treated with GPE after HU exposure (Figure 5B). Globally, these data suggest that GPE-mediated action on p21 may contribute to its anti-ageing effects; consistent with this hypothesis, the mRNA expression of cellular senescence associated genes p53, p21 and p19 in the hippocampus of aged rats.40 8


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FIGURE 5 NEAR HERE

GPE counteracts ageing-mediated inflammation Brain inflammation and apoptosis are under the transcriptional control of several factors, including NF-κB.50 In particular, NF-κB has been emerging as hub of ageing inflammatory network, to the point that inductors of this signalling pathway have been proposed as potential ageing biomarkers.51 HU strongly enhanced NF-kB mRNA levels (Figure 6A), thus confirming the involvement of this signalling pathway in cellular ageing. Indeed, chronic NF-kB activation has been shown to determine an impairment of NSC proliferation and differentiation, and energy balance, finally contributing to neurodegenerative mechanisms.52 Challenging NSCs with GPE alone (10 µg/ml or 100 µg/ml) did not significantly affect NF-kB mRNA expression after 36 h of incubation (Figure 6A). Both pre- and the post-treatment with GPE almost completely reversed the increase in NF-κB transcription (Figure 6A). The attenuation of the NF-κB signalling pathway was confirmed at the protein level using western blotting (Figure 6B and C). Globally, these data suggest that the anti-ageing effects of GPE involve a block of the NF-kB pathway, thus suggesting an anti-inflammatory action. Consistent with our data, PLs blocked NF-κB pathway and amyloid or tau toxicity in neuronal-like cells.53 Moreover, NT-020, a proprietary blend of polyphenols, has been shown to increase NSC proliferation and improve cognitive function in aged rats by attenuating NF-κB signalling,54 thus confirming that blocking the inflammatory pathway restores neurogenesis. Future studies will investigate the intracellular pathway related to the marked anti-inflammatory effects of GPE on aged NSCs. Taken together, these results show that the HU-treatment protocol efficaciously induced the common features of NSC ageing, including cellular senescence, decreased proliferation, ROS accumulation, an increased ATP/ADP ratio, and an activation of ageing-related genes/proteins, including the key inflammatory mediator NF-κB. GPE pre- and post-treatment protocols were shown to significantly prevent and counteract the aforementioned parameters of cellular ageing, thus suggesting this molecule as a putative tool to counteract NSC ageing.

FIGURE 6 NEAR HERE

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GPE effectively produces phospholipids and acetylcholine in NSCs To shed light on the putative mechanisms involved in GPE-mediated effects, PL and ACh levels were measured. GPE has been tested in rat astrocytes9 and rat septal nucleus;10 however, we are the first to report on the effects of GPE on PL content in human NSCs. GPE produced a significant increase in PC content after 36 h of treatment (Figure 7A). Moreover, challenging NSCs with GPE (10 µg/ml or 100 µg/ml) effectively enhanced ACh content (Figure 7B), thus confirming GPE as a PC precursor and an ACh donor. These results confirm that the GPE nootropic action can be related to both PC production and ACh release. Indeed, in addition to the described beneficial effects of PL precursors, literature reports that the direct production of ACh and the restoration of cholinergic neuronal integrity in human NSCs improve aged-mice cognitive function or enhance NSC proliferation.55-57 Finally, to investigate the putative effects of GPE alone on NSC proliferation/viability, cells were incubated with the two GPE concentrations for 36 h. The results (Figure 7C) showed that GPE did not significant effect NSC proliferation. Consistent with these results, GPE did not alter the viability of astrocytes.7 These data suggest that the antiageing effects of GPE do not involve an enhancement on NSC proliferation.

FIGURE 7 NEAR HERE

Conclusions To our knowledge, this is the first study to establish and characterise an ageing model of human neural stem cells. In these aged cells, GPE was demonstrated to recover the cell's proliferative potential and mitochondrial metabolism, decrease ROS production, and block the inflammatory pathway. The multiple signalling pathways affected by GPE treatment, suggest its beneficial actions on agedmediated alterations of the stem cell subpopulation. The data suggest that the aforementioned protective properties may be associated, at least in part, with GPE ability to enhance PC and ACh production in NSCs. The results are consistent with the valuable roles of PL or ACh precursors in preserving membrane fluidity and functionality, as well as other “well-being” pathways of cells. These beneficial properties suggest that these molecules play a pivotal role in reverting the stem cell niche modifications that contribute to age-related neurodegenerative disorders.19,20 In fact, ageing is a 10


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major risk factor for such diseases:58 as a consequence, NDs may impinge on agents like GPE able to preserve the NSC pool, whose integrity is essential in preserving brain homeostasis. These results represent an interesting starting point to translate these protective effects, shown on the cellular model, into in vivo models of NSC ageing and global brain ageing.

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Methods

Cell culture and pharmacological treatments H9-derived human NSCs were purchased from GIBCO (Life Technologies, Milan, Italy). ELISA kits for cytokine determinations were from Thermo Fisher Scientific (Rodano, Milan, Italy). GPE was from Angelini Acraf S.p.A. Stock solutions of varying GPE concentrations were created by dilution with phosphate-buffered saline (PBS 1X). All other reagents were of the highest commercially available grade and obtained from standard commercial sources. H9-derived NSCs were cultured as previously described.59 Experiments were performed with cultured cells from passages 1 to 4. To establish the ageing model, NSCs were treated for 1 or 16 h with HU at a concentration of 0.5, 8 or 20 mM. To verify the protective effects of GPE, NSCs were pre-treated with GPE (10 µg/ml or 100 µg/ml) for 36 h. Next, NSCs were washed and incubated with 8 mM HU for 16 h. To study post treatment effects, NSCs were treated with HU for 16 h, washed and then treated with GPE (10 µg/ml or 100 µg/ml) for 36 h (Figure 2A).

PC, ACh and choline release NSCs were incubated (10 µg/ml or 100 µg/ml) for 36 h. Then, NSCs were collected and lysed. PC, ACh or choline content was determined by colourimetric (Cayman Chemical, USA) or fluorimetric (Abnova, Taiwan) assays.

Apoptosis assessment NSCs were seeded in 24-multiwell plates and then treated for 1 or 16 h with HU at concentrations of 0.5, 8 or 20 mM. Following treatment, early and late apoptotic NSCs were estimated by Muse Apo Assays® (Merck-Millipore) as reported before.60-62 Apoptotic and dead cells were distinguished by using the annexin V conjugated with fluorescein isothiocyanate (FITC) and amino-actinomycin D (7-AAD).60-62

Quantification of DNA damage NSCs were seeded in 6-multiwell plates (1 x 105 cells/well) and grown until they reached semiconfluence. NSCs were treated for 1 or 16 h with HU at a concentration of 0.5, 8 or 20 mM. After HU removal, NSCs were cultured in growth medium for an additional 36 h. When indicated, NSCs were pre-treated with GPE (10 µg/ml or 100 µg/ml) for 36 h, washed and then incubated with 8 12


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mM of HU for 16 h. All other NSCs were challenged with HU for 16 h, washed, and then treated with GPE (10 µg/ml or 100 µg/ml) for 36 h. Following treatments, cells were lysed. Equal amounts of protein were incubated in pre-coated wells.

DSBs were evaluated by quantifying phosphorylated γH2AX (gamma

H2AX

Pharmacodynamic Assay, Trevigen Inc., Gaithersburg, MD, USA). The chemioluminescence data were reported as the fold change from control cells.

Cell proliferation/viability assays NSCs were seeded in 24-multiwell plates, pre-treated with GPE (10 µg/ml or 100 µg/ml) for 36 h, washed and then incubated with 8 mM of HU for 16 h. Alternatively, NSCs were challenged with HU for 16 h, washed, and then treated with GPE (10 µg/ml or 100 µg/ml) for 36 h. When indicated, NSCs were treated with 20 mM HU for 16 h. After incubation, the number of living and dead cells was determined by a Muse™ Cell Analyser (MUSE Cell Count and Viability Assay, MerckMillipore). The data are reported as living and dead cells in each well.

ROS production ROS activity within the cells was determined using the fluorogenic dye 2',7'-dichlorofluorescin diacetate (H2DCFDA, Molecular Probes, Invitrogen).63 NSCs were seeded in black 96-multiwell plates (5 x 103 cells/well). NSCs were treated with GPE in DMEM without phenol red in the presence or absence of HU. One hour prior to treatment completion, 50 µM H2DCFDA was added to the same media in the dark at 37°C.63 As a positive control, H2O2 was added at a concentration of 100 µM. The fluorescence intensity (excitation 485 nm and emission 520 nm) was normalised based on the number of cells stained with crystal violet. 64

ATP and ADP content NSCs were seeded in 6-multiwell plates (1 x 105 cells/well) and grown until they reached semiconfluence. The cells were then incubated with GPE and/or HU. After treatment, the amount of ATP and ADP were estimated using a luminescence assay kit (ApoSENSOR ADP/ATP Ratio Assay Kit, Enzo Life Sciences, Vinci-Biochem, Florence, Italy).64

Senescence analysis

13



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NSCs were seeded in 24-multiwell plates (2.5 x 104 cells/well), grown until semi-confluence, and treated or pre-treated with GPE and/or HU. SA-β-Gal expression was evaluated as previously described.60

RNA extraction and real time PCR analysis NSCs were treated as previously described. After treatment, NSCs were collected, and the mRNA levels of p53, p21 and NF-kB were evaluated by quantitative real time PCR using Fluocycle® II SYBR® (Euroclone, Milan, Italy).59 β-actin mRNA levels were used to normalise mRNA levels of each sample.60 Gel electrophoresis and the analysis of melting curves were used to ensure the specificity of PCR products.60 The nucleotide sequences, annealing temperature and product size of the primers have been previously reported.59,65,66

NF-κB activation NSCs were treated as described above. Then, NSCs were lysed in the "subcellular fractionation buffer" as previously reported.66 Next, the nuclear levels of NF-kB p65 protein were estimated using western blot analysis as described.66 ImageJ program was used to quantify immunoreactive bands.66

Statistical analysis Data analysis was performed using the t-test and one-way analysis of variance (ANOVA) with Bonferroni's corrected t-tests for post-hoc pair-wise comparisons. P

Human Neural Stem Cell Aging Is Counteracted by Î±

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