Aracely Garcia-Garcia

DNA damage and autophagy.

Both exogenous and endogenous agents are a threat to DNA integrity. Exogenous environmental agents such as ultraviolet (UV) and ionizing radiation, genotoxic chemicals and endogenous byproducts of metabolism including reactive oxygen species can cause alterations in DNA structure (DNA damage). Unrepaired DNA damage has been linked to a variety of human disorders including cancer and neurodegenerative disease. Thus, efficient mechanisms to detect DNA lesions, signal their presence and promote their repair have been evolved in cells. If DNA is effectively repaired, DNA damage response is inactivated and normal cell functioning resumes. In contrast, when DNA lesions cannot be removed, chronic DNA damage triggers specific cell responses such as cell death and senescence. Recently, DNA damage has been shown to induce autophagy, a cellular catabolic process that maintains a balance between synthesis, degradation, and recycling of cellular components. But the exact mechanisms by which DNA damage triggers autophagy are unclear. More importantly, the role of autophagy in the DNA damage response and cellular fate is unknown. In this review we analyze evidence that supports a role for autophagy as an integral part of the DNA damage response.The induction of inherited DNA sequence mutations arising in the germline (i.e., sperm or egg) of mice exposed in utero to diesel exhaust particles (DEPs) via maternal inhalation compared to unexposed controls was investigated in this study. Previous work has shown that particulate air pollutants (PAPs) from industrial environments cause DNA damage and mutations in the sperm of adult male mice. Effects on the female and male germline during critical stages of development (in utero) are unknown. In mice, previous studies have shown that expanded simple tandem repeat (ESTR) loci exhibit high rates of spontaneous mutation, making this endpoint a valuable tool for studying inherited mutation and genomic instability. In the present study, pregnant C57Bl/6 mice were exposed to 19mg/m(3) DEP from gestational day 7 through 19, alongside air exposed controls. Male and female F1 offspring were raised to maturity and mated with control CBA mice. The F2 descendents were collected and ESTR germline mutation rates were derived from full pedigrees (mother, father, offspring) of F1 male and female mice. We found no evidence for increased ESTR mutation rates in females exposed in utero to DEP relative to control females. In contrast, a statistically significant increase in the mutation frequency of male mice exposed in utero to DEP was observed (2-fold; Fishers exact p<0.05). Thus, maternal exposure to DEP results in increased mutation in sperm during development.

Adenoviruses induce autophagy to promote virus replication and oncolysis.

Adenoviruses with deletion of E1b have been used in clinical trials to treat cancers that are resistant to conventional therapies. The efficacy of viral replication within cancer cells determines the results of oncolytic therapy, which remains poorly understood and requires further improvement. In this report, we show that adenoviruses induce autophagy by increasing the conversion of LC3-I to LC3-II and the formation of the Atg12-Atg5 complex. Inhibition of autophagy with 3-methyladenine (3MA) resulted in a decreased synthesis of adenovirus structural proteins, and thereby a poor viral replication; promotion of autophagy with rapamycin increased adenovirus yield. This study indicates that adenovirus-induced autophagy correlates positively with virus replication and oncolytic cell death, and that autophagy may generate nutrients that can be used for building viral progeny particles. These results further suggest that chemotherapeutic agents that increase cancer cell autophagy may improve the efficacy of oncolytic virotherapy.

Thiol-redox signaling, dopaminergic cell death, and Parkinson's disease.

SIGNIFICANCE Parkinsons disease (PD) is characterized by the selective loss of dopaminergic neurons of the substantia nigra pars compacta, which has been widely associated with oxidative stress. However, the mechanisms by which redox signaling regulates cell death progression remain elusive. RECENT ADVANCES Early studies demonstrated that depletion of glutathione (GSH), the most abundant low-molecular-weight thiol and major antioxidant defense in cells, is one of the earliest biochemical events associated with PD, prompting researchers to determine the role of oxidative stress in dopaminergic cell death. Since then, the concept of oxidative stress has evolved into redox signaling, and its complexity is highlighted by the discovery of a variety of thiol-based redox-dependent processes regulating not only oxidative damage, but also the activation of a myriad of signaling/enzymatic mechanisms. CRITICAL ISSUES GSH and GSH-based antioxidant systems are important regulators of neurodegeneration associated with PD. In addition, thiol-based redox systems, such as peroxiredoxins, thioredoxins, metallothioneins, methionine sulfoxide reductases, transcription factors, as well as oxidative modifications in protein thiols (cysteines), including cysteine hydroxylation, glutathionylation, and nitrosylation, have been demonstrated to regulate dopaminergic cell loss. FUTURE DIRECTIONS In this review, we summarize major advances in the understanding of the role of thiol-redox signaling in dopaminergic cell death in experimental PD. Future research is still required to clearly understand how integrated thiol-redox signaling regulates the activation of the cell death machinery, and the knowledge generated should open new avenues for the design of novel therapeutic approaches against PD.

Impairment of Atg5-dependent autophagic flux promotes paraquat- and MPP+-induced apoptosis but not rotenone or 6-hydroxydopamine toxicity.

Controversial reports on the role of autophagy as a survival or cell death mechanism in dopaminergic cell death induced by parkinsonian toxins exist. We investigated the alterations in autophagic flux and the role of autophagy protein 5 (Atg5)-dependent autophagy in dopaminergic cell death induced by parkinsonian toxins. Dopaminergic cell death induced by the mitochondrial complex I inhibitors 1-methyl-4-phenylpyridinium (MPP⁺) and rotenone, the pesticide paraquat, and the dopamine analog 6-hydroxydopamine (6-OHDA) was paralleled by increased autophagosome accumulation. However, when compared with basal autophagy levels using chloroquine, autophagosome accumulation was a result of impaired autophagic flux. Only 6-OHDA induced an increase in autophagosome formation. Overexpression of a dominant negative form of Atg5 increased paraquat- and MPP⁺-induced cell death. Stimulation of mammalian target of rapamycin (mTOR)-dependent signaling protected against cell death induced by paraquat, whereas MPP⁺-induced toxicity was enhanced by wortmannin, a phosphoinositide 3-kinase class III inhibitor, rapamycin, and trehalose, an mTOR-independent autophagy activator. Modulation of autophagy by either pharmacological or genetic approaches had no effect on rotenone or 6-OHDA toxicity. Cell death induced by parkinsonian neurotoxins was inhibited by the pan caspase inhibitor (Z-VAD), but only caspase-3 inhibition was able to decrease MPP⁺-induced cell death. Finally, inhibition of the lysosomal hydrolases, cathepsins, increased the toxicity by paraquat and MPP⁺, supporting a protective role of Atg5-dependent autophagy and lysosomes degradation pathways on dopaminegic cell death. These results demonstrate that in dopaminergic cells, Atg5-dependent autophagy acts as a protective mechanism during apoptotic cell death induced by paraquat and MPP⁺ but not during rotenone or 6-OHDA toxicity.

Compartmentalized oxidative stress in dopaminergic cell death induced by pesticides and complex I inhibitors: Distinct roles of superoxide anion and superoxide dismutases

The loss of dopaminergic neurons induced by the parkinsonian toxins paraquat, rotenone, and 1-methyl-4-phenylpyridinium (MPP(+)) is associated with oxidative stress. However, controversial reports exist regarding the source/compartmentalization of reactive oxygen species (ROS) generation and its exact role in cell death. We aimed to determine in detail the role of superoxide anion (O2(•-)), oxidative stress, and their subcellular compartmentalization in dopaminergic cell death induced by parkinsonian toxins. Oxidative stress and ROS formation were determined in the cytosol, intermembrane (IMS), and mitochondrial matrix compartments, using dihydroethidine derivatives and the redox sensor roGFP, as well as electron paramagnetic resonance spectroscopy. Paraquat induced an increase in ROS and oxidative stress in both the cytosol and the mitochondrial matrix prior to cell death. MPP(+) and rotenone primarily induced an increase in ROS and oxidative stress in the mitochondrial matrix. No oxidative stress was detected at the level of the IMS. In contrast to previous studies, overexpression of manganese superoxide dismutase (MnSOD) or copper/zinc SOD (CuZnSOD) had no effect on alterations in ROS steady-state levels, lipid peroxidation, loss of mitochondrial membrane potential (ΔΨm), and dopaminergic cell death induced by MPP(+) or rotenone. In contrast, paraquat-induced oxidative stress and cell death were selectively reduced by MnSOD overexpression, but not by CuZnSOD or manganese-porphyrins. However, MnSOD also failed to prevent ΔΨm loss. Finally, paraquat, but not MPP(+) or rotenone, induced the transcriptional activation of the redox-sensitive antioxidant response elements (ARE) and nuclear factor kappa-B (NF-κB). These results demonstrate a selective role of mitochondrial O2(•-) in dopaminergic cell death induced by paraquat, and show that toxicity induced by the complex I inhibitors rotenone and MPP(+) does not depend directly on mitochondrial O2(•-) formation.

Alterations in energy/redox metabolism induced by mitochondrial and environmental toxins: a specific role for glucose-6-phosphate-dehydrogenase and the pentose phosphate pathway in paraquat toxicity.

Parkinson’s disease (PD) is a multifactorial disorder with a complex etiology including genetic risk factors, environmental exposures, and aging. While energy failure and oxidative stress have largely been associated with the loss of dopaminergic cells in PD and the toxicity induced by mitochondrial/environmental toxins, very little is known regarding the alterations in energy metabolism associated with mitochondrial dysfunction and their causative role in cell death progression. In this study, we investigated the alterations in the energy/redox-metabolome in dopaminergic cells exposed to environmental/mitochondrial toxins (paraquat, rotenone, 1-methyl-4-phenylpyridinium [MPP+], and 6-hydroxydopamine [6-OHDA]) in order to identify common and/or different mechanisms of toxicity. A combined metabolomics approach using nuclear magnetic resonance (NMR) and direct-infusion electrospray ionization mass spectrometry (DI-ESI-MS) was used to identify unique metabolic profile changes in response to these neurotoxins. Paraquat exposure induced the most profound alterations in the pentose phosphate pathway (PPP) metabolome. 13C-glucose flux analysis corroborated that PPP metabolites such as glucose-6-phosphate, fructose-6-phosphate, glucono-1,5-lactone, and erythrose-4-phosphate were increased by paraquat treatment, which was paralleled by inhibition of glycolysis and the TCA cycle. Proteomic analysis also found an increase in the expression of glucose-6-phosphate dehydrogenase (G6PD), which supplies reducing equivalents by regenerating nicotinamide adenine dinucleotide phosphate (NADPH) levels. Overexpression of G6PD selectively increased paraquat toxicity, while its inhibition with 6-aminonicotinamide inhibited paraquat-induced oxidative stress and cell death. These results suggest that paraquat “hijacks” the PPP to increase NADPH reducing equivalents and stimulate paraquat redox cycling, oxidative stress, and cell death. Our study clearly demonstrates that alterations in energy metabolism, which are specific for distinct mitochondiral/environmental toxins, are not bystanders to energy failure but also contribute significant to cell death progression.

Combining DI-ESI–MS and NMR datasets for metabolic profiling

Metabolomics datasets are commonly acquired by either mass spectrometry (MS) or nuclear magnetic resonance spectroscopy (NMR), despite their fundamental complementarity. In fact, combining MS and NMR datasets greatly improves the coverage of the metabolome and enhances the accuracy of metabolite identification, providing a detailed and high-throughput analysis of metabolic changes due to disease, drug treatment, or a variety of other environmental stimuli. Ideally, a single metabolomics sample would be simultaneously used for both MS and NMR analyses, minimizing the potential for variability between the two datasets. This necessitates the optimization of sample preparation, data collection and data handling protocols to effectively integrate direct-infusion MS data with one-dimensional (1D) 1H NMR spectra. To achieve this goal, we report for the first time the optimization of (i) metabolomics sample preparation for dual analysis by NMR and MS, (ii) high throughput, positive-ion direct infusion electrospray ionization mass spectrometry (DI-ESI–MS) for the analysis of complex metabolite mixtures, and (iii) data handling protocols to simultaneously analyze DI-ESI–MS and 1D 1H NMR spectral data using multiblock bilinear factorizations, namely multiblock principal component analysis (MB-PCA) and multiblock partial least squares (MB-PLS). Finally, we demonstrate the combined use of backscaled loadings, accurate mass measurements and tandem MS experiments to identify metabolites significantly contributing to class separation in MB-PLS-DA scores. We show that integration of NMR and DI-ESI–MS datasets yields a substantial improvement in the analysis of metabolome alterations induced by neurotoxin treatment.Graphical abstract

Biomarkers of protein oxidation in human disease

Oxidative stress is caused by an imbalance between the production of reactive species of oxygen and nitrogen (RS) and the ability to either detoxify the reactive intermediates produced or repair the resulting damage. Ultimately, oxidative stress conveys the alteration in cellular function caused by the reaction of RS with cellular constituents. Oxidative stress has been extensively reported to participate in the progression of a variety of human diseases including cancer, neurodegenerative disorders and diabetes. Oxidation of proteins is thought to be one of the major mechanisms by which oxidative stress is integrated into cellular signal transduction pathways. Thus, recent research efforts have been aimed to identify the role of specific oxidative protein modifications in the signal transduction events mediating the etiology of human diseases progression. The identification of these oxidative modifications has also raised the possibility of using this knowledge to develop new methods to diagnose diseases before they are clinically evident. In this work, we summarize the mechanisms by which RS generate distinct oxidative modifications. Furthermore, we also review the potential of these oxidative modifications to be used as early biomarkers of human disease.

E2F-1 lacking the transcriptional activity domain induces autophagy.

The transcription factor E2F-1 plays a crucial role in the control of cell proliferation. E2F-1 has tumor suppressive properties by inducing apoptosis and autophagy. In this study, E2F-1 and its truncated form (E2Ftr), lacking the transactivation domain (TAD), were compared for their ability to induce autophagy. In Gaussia luciferase-based assays, both E2F-1 and E2Ftr induced the proteolytic cleavage of the autophagic marker LC3. In addition, LC3 and autophagy protein 5 (Atg5) were upregulated by E2F-1 and E2Ftr. Likewise, both E2F proteins induced a punctate pattern of GFP-tagged LC3, indicating autophagosome formation. The presence of double-membrane autophagic vesicles induced by E2F-1 and E2Ftr was confirmed by transmission electron microscopy (TEM). The application of z-VAD-fmk, a caspase inhibitor, partially blocked both E2F-1 and E2Ftr-mediated cytotoxicity. Moreover, Atg5−/− cells were more resistant to the E2F-1 or E2Ftr-induced cell killing effect than Atg5 wt cells. The TAD of E2F-1 is not essential for induction of autophagy; apoptosis and autophagy cooperate for an efficient cancer cell killing effect induced by E2F-1 or E2Ftr. E2Ftr-induced autophagy is a promising approach to destroy tumors that are resistant to conventional treatments.

Adenovirus-Mediated Expression of Truncated E2F-1 Suppresses Tumor Growth In Vitro and In Vivo

Adenovirus (Ad)‐mediated E2F‐1 gene transfer induces apoptosis in cancer cells in vitro and in vivo, but clinical application of E2F‐1 in cancer gene therapy remains controversial because of the oncogenic potential of E2F‐1. This barrier can be circumvented by using the truncated form of the E2F‐1 gene (E2Ftr) (amino acids 1 through 375), which lacks the E2F‐1 transactivation domain and cell cycle‐promoting effects.