Lewy bodies (LBs), which are the hallmark pathologic features of Parkinson's disease and of dementia with LBs, have several morphologic and molecular similarities to aggresomes. Whether such cytoplasmic inclusions contribute to neuronal death or protect cells from the toxic effects of misfolded proteins remains controversial. In this report, the role of aggresomes in cell viability was addressed in the context of over-expressing ␣-synuclein and its interacting partner synphilin-1 using engineered 293T cells. Inhibition of proteasome activity elicited the formation of juxtanuclear aggregates with characteristics of aggresomes including immunoreactivity for vimentin, ␥-tubulin, ubiquitin, proteasome subunit, and hsp70. As expected from the properties of aggresomes, the microtubule disrupting agents, vinblastin and nocodazole, markedly prevented the formation of these inclusions. Similar to LBs, the phosphorylated form of ␣-synuclein co-localized in these synphilin-1-containing aggresomes. Although the caspase inhibitor z-VAD-fmk significantly reduced the number of apoptotic cells, it had no impact on the percentage of aggresome-positive cells. Finally, quantitative analysis revealed aggresomes in 60% of nonapoptotic cells but only in 10% of apoptotic cells. Additionally, ␣-synuclein-induced apoptosis was not coupled with increased prevalence of aggresomebearing cells. Taken together, these observations indicate a disconnection between aggresome formation and apoptosis, and support a protective role for these inclusions from the toxicity associated with the combined over-expression of ␣-synuclein and synphilin-1.

To investigate the effect of RNA oxidation on normal cellular functions, we studied the translation of nonoxidized and oxidized luciferase mRNA in both rabbit reticulocyte lysate and human HEK293 cells. When HEK293 cells transfected with nonoxidized mRNA encoding the firefly luciferase protein were cultured in the presence of paraquat, there was a paraquat concentration-dependent increase in the formation of luciferase short polypeptides (SPs) concomitant with an increase in 8-oxoguanosine. Short polypeptides were also formed when the mRNA was oxidized in vitro by the Fe-ascorbate-H2O2 metal-catalyzed oxidation system before its transfection into cells. Translation of the in vitro oxidized mRNA in rabbit reticulocyte lysate also led to formation of SPs. The SPs formed by either procedure contained the N-terminal and the C-terminal portions of the tagged luciferase. In addition, the oxidized mRNA was able to associate with ribosomes to form polysomes similar to those formed with nonoxidized mRNA preparations, indicating that the oxidized mRNAs are mostly intact; however, their translation fidelity was significantly reduced. Nevertheless, our results indicate that the SPs were derived from both premature termination of the translation process of the oxidized mRNA and the proteolytic degradation of the modified full-length luciferase resulting from translation errors induced by oxidized mRNA. In light of these findings, the physiological consequences of mRNA oxidation are discussed.8-oxoguanosine ͉ firefly luciferase ͉ premature termination ͉ RNA oxidation ͉ reactive oxygen species T he generation of reactive oxygen species (ROS) is an unavoidable cellular event in normal respiring cells. ROS are known to play an important role in normal physiological functions and they can also oxidize cellular macromolecules, such as nucleic acids, proteins, and lipids, and lead to the loss of their physiological functions (1, 2). Growing evidence indicates that increased oxidative damage appears to be associated with a wide range of age-related neurodegenerative conditions. Although oxidation of DNA may ultimately have deleterious consequences on cellular homeostasis, multiple DNA repair systems are available to minimize such damage (3). Because of the fact that RNAs are less protected against ROS under oxidative stress conditions, intracellular RNA was found to be more susceptible to oxidation than DNA (4, 5). Similarly, irradiation of human skin fibroblasts with 765-kJ/m 2 UV A induced more extensive RNA oxidation than DNA oxidation (6). The oxidation of rRNA, tRNA, and mRNA may have serious effects on cellular homeostasis, because oxidation of these RNAs could impair the overall integrity of translational processes. Moreover, it was recently shown that oxidation of RNA is implicated in several neurodegenerative diseases (7-9). Nunomura et al. (8) reported that cytoplasmic RNA oxidation occurs to a greater extent in neurons that are implicated in the development of Alzheimer's disease (AD). Shan et al. (10) found that signif...

Mammalian apurinic/apyrimidinic endonuclease 1 is a DNA repair enzyme involved in genome stability and expression of genes involved in oxidative stress responses, tumor progression and chemoresistance. However, the molecular mechanisms underlying the role of apurinic/apyrimidinic endonuclease 1 in these processes are still unclear. Recent findings point to a novel role of apurinic/apyrimidinic endonuclease 1 in RNA metabolism. Through the characterization of the interactomes of apurinic/apyrimidinic endonuclease 1 with RNA and other proteins, we demonstrate here a role for apurinic/apyrimidinic endonuclease 1 in pri-miRNA processing and stability via association with the DROSHA-processing complex during genotoxic stress. We also show that endonuclease activity of apurinic/apyrimidinic endonuclease 1 is required for the processing of miR-221/222 in regulating expression of the tumor suppressor PTEN. Analysis of a cohort of different cancers supports the relevance of our findings for tumor biology. We also show that apurinic/apyrimidinic endonuclease 1 participates in RNA-interactomes and protein-interactomes involved in cancer development, thus indicating an unsuspected post-transcriptional effect on cancer genes.