Modulation of DNA base excision repair during neuronal differentiation.

Neurons are terminally differentiated cells with a high rate of metabolism and multiple biological properties distinct from their undifferentiated precursors. Previous studies showed that nucleotide excision DNA repair is downregulated in postmitotic muscle cells and neurons. Here, we characterize DNA damage susceptibility and base excision DNA repair (BER) capacity in undifferentiated and differentiated human neural cells. The results show that undifferentiated human SH-SY5Y neuroblastoma cells are less sensitive to oxidative damage than their differentiated counterparts, in part because they have robust BER capacity, which is heavily attenuated in postmitotic neurons. The reduction in BER activity in differentiated cells correlates with diminished protein levels of key long patch BER components, flap endonuclease-1, proliferating cell nuclear antigen, and ligase I. Thus, because of their higher BER capacity, proliferative neural progenitor cells are more efficient at repairing DNA damage compared with their neuronally differentiated progeny.

Mitochondrial base excision repair in mouse synaptosomes during normal aging and in a model of Alzheimer's disease.

Brain aging is associated with synaptic decline and synaptic function is highly dependent on mitochondria. Increased levels of oxidative DNA base damage and accumulation of mitochondrial DNA (mtDNA) mutations or deletions lead to mitochondrial dysfunction, playing an important role in the aging process and the pathogenesis of several neurodegenerative diseases. Here we have investigated the repair of oxidative base damage, in synaptosomes of mouse brain during normal aging and in an AD model. During normal aging, a reduction in the base excision repair (BER) capacity was observed in the synaptosomal fraction, which was associated with a decrease in the level of BER proteins. However, we did not observe changes between the synaptosomal BER activities of presymptomatic and symptomatic AD mice harboring mutated amyolid precursor protein (APP), Tau, and presinilin-1 (PS1) (3xTgAD). Our findings suggest that the age-related reduction in BER capacity in the synaptosomal fraction might contribute to mitochondrial and synaptic dysfunction during aging. The development of AD-like pathology in the 3xTgAD mouse model was, however, not associated with deficiencies of the BER mechanisms in the synaptosomal fraction when the whole brain was analyzed.

Novel DNA mismatch-repair activity involving YB-1 in human mitochondria.

Maintenance of the mitochondrial genome (mtDNA) is essential for proper cellular function. The accumulation of damage and mutations in the mtDNA leads to diseases, cancer, and aging. Mammalian mitochondria have proficient base excision repair, but the existence of other DNA repair pathways is still unclear. Deficiencies in DNA mismatch repair (MMR), which corrects base mismatches and small loops, are associated with DNA microsatellite instability, accumulation of mutations, and cancer. MMR proteins have been identified in yeast and coral mitochondria; however, MMR proteins and function have not yet been detected in human mitochondria. Here we show that human mitochondria have a robust mismatch-repair activity, which is distinct from nuclear MMR. Key nuclear MMR factors were not detected in mitochondria, and similar mismatch-binding activity was observed in mitochondrial extracts from cells lacking MSH2, suggesting distinctive pathways for nuclear and mitochondrial MMR. We identified the repair factor YB-1 as a key candidate for a mitochondrial mismatch-binding protein. This protein localizes to mitochondria in human cells, and contributes significantly to the mismatch-binding and mismatch-repair activity detected in HeLa mitochondrial extracts, which are significantly decreased when the intracellular levels of YB-1 are diminished. Moreover, YB-1 depletion in cells increases mitochondrial DNA mutagenesis. Our results show that human mitochondria contain a functional MMR repair pathway in which YB-1 participates, likely in the mismatch-binding and recognition steps.

DNA base excision repair activities in mouse models of Alzheimer's disease.

Alzheimer's disease (AD) has been correlated with elevated levels of oxidative DNA damage. Base excision repair (BER) is the main repair pathway for the removal of oxidative DNA base modifications. We have recently found significant functional deficiencies in BER in brains of sporadic AD and amnestic mild cognitive impairment patients. In this study we tested whether altered BER activities are associated with appearance of symptoms in different brain regions of pre-symptomatic and symptomatic mice harboring mutant APP alone or in combination with Tau and PS1. Our results suggest that unlike in humans, the development of AD-like pathology in the studied mouse models is not associated with deficiencies in BER.

Mitochondrial DNA damage and repair in neurodegenerative disorders.

By producing ATP and regulating intracellular calcium levels, mitochondria are vital for the function and survival of neurons. Oxidative stress and damage to mitochondrial DNA during the aging process can impair mitochondrial energy metabolism and ion homeostasis in neurons, thereby rendering them vulnerable to degeneration. Mitochondrial abnormalities have been documented in all of the major neurodegenerative disorders-Alzheimer's, Parkinson's and Huntington's diseases, and amyotrophic lateral sclerosis. Mitochondrial DNA damage and dysfunction may be downstream of primary disease processes such as accumulation of pathogenic proteins. However, recent experimental evidence demonstrates that mitochondrial DNA damage responses play important roles in aging and in the pathogenesis of neurodegenerative diseases. Therapeutic interventions that target mitochondrial regulatory systems have been shown effective in cell culture and animal models, but their efficacy in humans remains to be established.

Defective DNA base excision repair in brain from individuals with Alzheimer's disease and amnestic mild cognitive impairment.

Oxidative stress is thought to play a role in the pathogenesis of Alzheimer's disease (AD) and increased oxidative DNA damage has been observed in brain tissue from AD patients. Base excision repair (BER) is the primary DNA repair pathway for small base modifications such as alkylation, deamination and oxidation. In this study, we have investigated alterations in the BER capacity in brains of AD patients. We employed a set of functional assays to measure BER activities in brain tissue from short post-mortem interval autopsies of 10 sporadic AD patients and 10 age-matched controls. BER activities were also measured in brain samples from 9 amnestic mild cognitive impairment (MCI) subjects. We found significant BER deficiencies in brains of AD patients due to limited DNA base damage processing by DNA glycosylases and reduced DNA synthesis capacity by DNA polymerase beta. The BER impairment was not restricted to damaged brain regions and was also detected in the brains of amnestic MCI patients, where it correlated with the abundance of neurofibrillary tangles. These findings suggest that BER dysfunction is a general feature of AD brains which could occur at the earliest stages of the disease. The results support the hypothesis that defective BER may play an important role in the progression of AD.

Responses of antioxidants in the lichen Ramalina lacera may serve as an early-warning bioindicator system for the detection of air pollution stress.

The aim of this study was to identify, in the lichen Ramalina lacera, antioxidants that could provide indications of air pollution stress, and respond earlier than traditionally used structural/physiological parameters. The pollution-sensitive lichen R. lacera was transplanted from its relatively unpolluted natural habitat to two air-polluted sites for a period of up to 6 months. The superoxide dismutase and catalase activities, total water- and lipid-soluble low-molecular-weight antioxidant capacities and chlorophyll b/chlorophyll a ratios were assessed every 6 weeks. The earliest signs of oxidative stress were detected in the activities of fungal copper/zinc-superoxide dismutase, algal iron-superoxide dismutase and water-soluble low-molecular-weight antioxidants, which increased significantly as early as 42 days after exposure to pollution. Catalase activity increased in lichens transplanted to the polluted sites after 90 days. All activities decreased towards the end of the experiment. The impact of air pollution on R. lacera, using the traditionally employed parameter of chlorophyll b/chlorophyll a ratio, was only detected after 6 months of exposure to air pollution. Our results indicate that antioxidant parameters may serve as improved early-warning indicators of air pollution stress in lichens.

Characterization of enzymatic antioxidants in the lichen Ramalina lacera and their response to rehydration.

Lichens are slow-growing associations of fungi and green algae or cyanobacteria. This symbiotic association forms a common thallus that does not possess roots or a waxy cuticle and depends mainly on atmospheric input of mineral nutrients. The lifestyle of most lichens is composed of alternating periods of desiccation with low metabolic activity and hydration that induces increase in their metabolism. We have previously shown that rehydration of the naturally desiccated lichen Ramalina lacera resulted in a rapid increase in photosynthesis and was accompanied by a burst of intracellular production of reactive oxygen species and nitric oxide, as well as a transient decrease in water-soluble antioxidant capacity. We report here on enzymatic antioxidants of R. lacera and their response to rehydration. Native gel electrophoresis of crude extracts of R. lacera stained for superoxide dismutase (SOD) activity revealed four Fe-SOD and four Mn-SOD electromorphs that are synthesized by the alga, a Cu/Zn-SOD and a Mn-SOD that are the product of the fungus, and two catalases synthesized one by the fungus and the other by the algae. In addition, we detected glutathione reductase and glucose-6-phosphate dehydrogenase activities in crude extracts of R. lacera. Rehydration of the thalli resulted in a decrease in SOD activity of all forms, and a transient decrease in total catalase activity, as well as a decrease in the antioxidant auxiliary enzymes glutathione reductase and glucose-6-phosphate dehydrogenase.

Rehydration of the lichen Ramalina lacera results in production of reactive oxygen species and nitric oxide and a decrease in antioxidants.

Lichens are slow-growing associations of fungi and unicellular green algae or cyanobacteria. They are poikilohydric organisms whose lifestyle in many cases consists of alternating periods of desiccation, with low metabolic activity, and hydration, which induces increase in their metabolism. Lichens have apparently adapted to such extreme transitions between desiccation and rehydration, but the mechanisms that govern these adaptations are still poorly understood. In this study, the effect of rehydration on the production of reactive oxygen species and nitric oxide as well as low-molecular-weight antioxidants was investigated with the lichen Ramalina lacera. Rehydration of R. lacera resulted in the initiation of and a rapid increase in photosynthetic activity. Recovery of photosynthesis was accompanied by bursts of intracellular production of reactive oxygen species and nitric oxide. Laser-scanning confocal microscopy using dichlorofluorescein fluorescence revealed that formation of reactive oxygen species following rehydration was associated with both symbiotic partners of the lichen. The rate and extent of reactive oxygen species production were similar in the light and in the dark, suggesting a minor contribution of photosynthesis. Diaminofluorescein fluorescence, indicating nitric oxide formation, was detected only in fungal hyphae. Activities associated with rehydration did not have a deleterious effect on membrane integrity as assessed by measurement of electrolyte leakage, but water-soluble low-molecular-weight antioxidants decreased significantly.