Neuroprotection by Cocktails of Dietary Antioxidants under Conditions of Nerve Growth Factor Deprivation.

Dietary antioxidants may be useful in counteracting the chronic inflammatory status in neurodegenerative diseases by reducing oxidative stress due to accumulation of reactive oxygen species (ROS). In this study, we newly described the efficacy of a number of dietary antioxidants (polyphenols, carotenoids, thiolic compounds, and oligoelements) on viability of neuronal PC12 cells following Nerve Growth Factor (NGF) deprivation, a model of age-related decrease of neurotrophic support that triggers neuronal loss. Neuroprotection by antioxidants during NGF deprivation for 24 h was largely dependent on their concentrations: all dietary antioxidants were able to efficiently support cell viability by reducing ROS levels and restoring mitochondrial function, while preserving the neuronal morphology. Moreover, ROS reduction and neuroprotection during NGF withdrawal were also achieved with defined cocktails of 3-6 different antioxidants at concentrations 5-60 times lower than those used in single treatments, suggesting that their antioxidant activity was preserved also at very low concentrations. Overall, these data indicate the beneficial effects of antioxidants against oxidative stress induced by decreased NGF availability and suggest that defined cocktails of dietary factors at low concentrations might be a suitable strategy to reduce oxidative damage in neurodegenerative diseases, while limiting possible side effects.

In vivo and in silico analysis of PCNA ubiquitylation in the activation of the Post Replication Repair pathway in S. cerevisiae.

BACKGROUND: The genome of living organisms is constantly exposed to several damaging agents that induce different types of DNA lesions, leading to cellular malfunctioning and onset of many diseases. To maintain genome stability, cells developed various repair and tolerance systems to counteract the effects of DNA damage. Here we focus on Post Replication Repair (PRR), the pathway involved in the bypass of DNA lesions induced by sunlight exposure and UV radiation. PRR acts through two different mechanisms, activated by mono- and poly-ubiquitylation of the DNA sliding clamp, called Proliferating Cell Nuclear Antigen (PCNA). RESULTS: We developed a novel protocol to measure the time-course ratios between mono-, di- and tri-ubiquitylated PCNA isoforms on a single western blot, which were used as the wet readout for PRR events in wild type and mutant S. cerevisiae cells exposed to acute UV radiation doses. Stochastic simulations of PCNA ubiquitylation dynamics, performed by exploiting a novel mechanistic model of PRR, well fitted the experimental data at low UV doses, but evidenced divergent behaviors at high UV doses, thus driving the design of further experiments to verify new hypothesis on the functioning of PRR. The model predicted the existence of a UV dose threshold for the proper functioning of the PRR model, and highlighted an overlapping effect of Nucleotide Excision Repair (the pathway effectively responsible to clean the genome from UV lesions) on the dynamics of PCNA ubiquitylation in different phases of the cell cycle. In addition, we showed that ubiquitin concentration can affect the rate of PCNA ubiquitylation in PRR, offering a possible explanation to the DNA damage sensitivity of yeast strains lacking deubiquitylating enzymes. CONCLUSIONS: We exploited an in vivo and in silico combinational approach to analyze for the first time in a Systems Biology context the events of PCNA ubiquitylation occurring in PRR in budding yeast cells. Our findings highlighted an intricate functional crosstalk between PRR and other events controlling genome stability, and evidenced that PRR is more complicated and still far less characterized than previously thought.

RNase H and postreplication repair protect cells from ribonucleotides incorporated in DNA.

The chemical identity and integrity of the genome is challenged by the incorporation of ribonucleoside triphosphates (rNTPs) in place of deoxyribonucleoside triphosphates (dNTPs) during replication. Misincorporation is limited by the selectivity of DNA replicases. We show that accumulation of ribonucleoside monophosphates (rNMPs) in the genome causes replication stress and has toxic consequences, particularly in the absence of RNase H1 and RNase H2, which remove rNMPs. We demonstrate that postreplication repair (PRR) pathways-MMS2-dependent template switch and Pol ζ-dependent bypass-are crucial for tolerating the presence of rNMPs in the chromosomes; indeed, we show that Pol ζ efficiently replicates over 1-4 rNMPs. Moreover, cells lacking RNase H accumulate mono- and polyubiquitylated PCNA and have a constitutively activated PRR. Our findings describe a crucial function for RNase H1, RNase H2, template switch, and translesion DNA synthesis in overcoming rNTPs misincorporated during DNA replication, and may be relevant for the pathogenesis of Aicardi-Goutières syndrome.

Mind the gap: keeping UV lesions in check.

Cells respond to genotoxic insults by triggering a DNA damage checkpoint surveillance mechanism and by activating repair pathways. Recent findings indicate that the two processes are more related than originally thought. Here we discuss the mechanisms involved in responding to UV-induced lesions in different phases of the cell cycle and summarize the most recent data in a model where Nucleotide Excision Repair (NER) and exonucleolytic activities act in sequence leading to checkpoint activation in non replicating cells. The critical trigger is likely represented by problematic intermediates that cannot be completely or efficiently repaired by NER. In S phase cells, on the other hand, the replicative polymerases, blocked by bulky UV lesions, re-initiate DNA synthesis downstream of the lesions, leaving behind a ssDNA tract. If these gaps are not rapidly refilled, checkpoint kinases will be activated.

Cross-talk between cell cycle induction and mitochondrial dysfunction during oxidative stress and nerve growth factor withdrawal in differentiated PC12 cells.

Neuronal death has been reported to involve mitochondrial dysfunction and cell cycle reentry. In this report, we used Nerve Growth Factor (NGF)-differentiated PC12 cells to investigate mechanisms linking mitochondrial dysfunction and cell cycle activation during neuronal death induced by NGF withdrawal and/or oxidative stress. We found that loss of survival following H(2) O(2) -induced oxidative stress or NGF deprivation was preceded by a decrease in mitochondrial membrane potential (ΔΨm), increase in reactive oxygen species (ROS), and up-regulation of cyclin D1 and phosphorylation (Ser-780) of protein retinoblastoma (P-pRb), without an increase of proliferation rates. Treatment with H(2) O(2) , but not NGF deprivation, also induced the phosporylation (Ser-10) of p27(kip1) and the appearance of a cleaved P-p27(kip1) fragment of about 15 kDa. The extent of cell cycle activation appeared to be inversely correlated to the duration of toxic stimuli (pulse/continuous). H(2) O(2) -induced mitogenic responses appeared to be mediated by induction of P-MAPK and P-Akt and were blocked by p38MAPK and JNK inhibitors as well as by the CDK inhibitor flavopiridol (Flav) and by sodium selenite (Sel), a component of selenoproteins, including glutathione peroxidases. Inhibition of p38MAPK and JNK, instead, did not affect cyclin D1 changes following NGF deprivation. Finally, both Flav hydrochloride and Sel partially prevented mitochondrial dysfunction and cell death following NGF withdrawal or H(2) O(2) toxicity, but not during oxidative stress in the absence of NGF. Taken together, these data suggest that H(2) O(2) -induced oxidative stress can determine distinct patterns of mitogenic responses as a function of mitochondrial dysfunction depending on 1) intensity/duration of stress stimuli and/or 2) presence of NGF.