Transcriptomics analysis of human iPSC-derived dopaminergic neurons reveals a novel model for sporadic Parkinson's disease.

Parkinson's disease (PD) is a progressive, neurodegenerative disease affecting over 1% of the population beyond 65 years of age. Although some PD cases are inheritable, the majority of PD cases occur in a sporadic manner. Risk factors comprise next to heredity, age, and gender also exposure to neurotoxins from for instance pesticides and herbicides. As PD is characterized by a loss of dopaminergic neurons in the substantia nigra, it is nearly impossible to access and extract these cells from patients for investigating disease mechanisms. The emergence of induced pluripotent stem (iPSC) technology allows differentiating and growing human dopaminergic neurons, which can be used for in vitro disease modeling. Here, we differentiated human iPSCs into dopaminergic neurons, and subsequently exposed the cells to increasing concentrations of the neurotoxin MPP+. Temporal transcriptomics analysis revealed a strong time- and dose-dependent response with genes over-represented across pathways involved in PD etiology such as "Parkinson's Disease", "Dopaminergic signaling" and "calcium signaling". Moreover, we validated this disease model by showing robust overlap with a meta-analysis of transcriptomics data from substantia nigra from post-mortem PD patients. The overlap included genes linked to e.g. mitochondrial dysfunction, neuron differentiation, apoptosis and inflammation. Our data shows, that MPP+-induced, human iPSC-derived dopaminergic neurons present molecular perturbations as observed in the etiology of PD. Therefore we propose iPSC-derived dopaminergic neurons as a foundation for a novel sporadic PD model to study the pathomolecular mechanisms of PD, but also to screen for novel anti-PD drugs and to develop and test new treatment strategies.

Acetaminophen Overdose as a Potential Risk Factor for Parkinson's Disease.

Four complementary approaches were used to investigate acetaminophen overdose as a risk factor for Parkinson's disease (PD). Circulating microRNAs (miRNAs) serum profiles from acetaminophen-overdosed patients were compared with patients with terminal PD, revealing four shared miRNAs. Similarities were found among molecular structures of dopamine (DA), acetaminophen, and two known PD inducers indicating affinity for dopaminergic transport. Potential interactions between acetaminophen and the human DA transporter were confirmed by molecular docking modeling and binding free energy calculations. Thus, it is plausible that acetaminophen is taken up by the dopaminergic transport system into the substantia nigra (SN). A ChEMBL query identified proteins that are similarly targeted by DA and acetaminophen. Here, we highlight CYP3A4, present in the SN, a predominant metabolizer of acetaminophen into its toxic metabolite N-acetyl-p-benzoquinone imine and shown to be regulated in PD. Overall, based on our results, we hypothesize that overdosing of acetaminophen is a potential risk factor for parkinsonism.

Genes associated with Parkinson's disease respond to increasing polychlorinated biphenyl levels in the blood of healthy females.

Polychlorinated biphenyls (PCBs) are a class of widespread environmental pollutants, commonly found in human blood, that have been suggested to be linked to the occurrence of sporadic Parkinson's disease (PD). It has been reported that some non-coplanar PCBs accumulate in the brains of female PD patients. To improve our understanding of the association between PCB exposure and PD risk we have applied whole transcriptome gene expression analysis in blood cells from 594 PCB-exposed subjects (369 female, 225 male). Interestingly, we observe that in females, blood levels of non-coplanar PCBs appear to be associated with expression levels of PD-specific genes. However, no such association was detected in males. Among the 131 PD-specific genes affected, 39 have been shown to display similar changes in expression levels in the substantia nigra of deceased PD patients. Especially among the down-regulated genes, transcripts of genes involved in neurotransmitter vesicle-related functions were predominant.

Both the concentration and redox state of glutathione and ascorbate influence the sensitivity of arabidopsis to cadmium.

BACKGROUND AND AIMS: Cadmium (Cd) is a non-essential trace element that elicits oxidative stress. Plants respond to Cd toxicity via increasing their Cd-chelating and antioxidative capacities. They predominantly chelate Cd via glutathione (GSH) and phytochelatins (PCs), while antioxidative defence is mainly based on the use and recycling of both GSH and ascorbate (AsA), complemented by superoxide dismutase (SOD) and catalase (CAT). In addition, both metabolites act as a substrate for the regeneration of other essential antioxidants, which neutralize and regulate reactive oxygen species (ROS). Together, these functions influence the concentration and cellular redox state of GSH and AsA. In this study, these two parameters were examined in plants of Arabidopsis thaliana exposed to sub-lethal Cd concentrations. METHODS: Wild-type plants and mutant arabidopsis plants containing 30-45 % of wild-type levels of GSH (cad2-1) or 40-50 % of AsA (vtc1-1), together with the double-mutant (cad2-1 vtc1-1) were cultivated in a hydroponic system and exposed to sub-lethal Cd concentrations. Cadmium detoxification was investigated at different levels including gene expression and metabolite concentrations. KEY RESULTS: In comparison with wild-type plants, elevated basal thiol levels and enhanced PC synthesis upon exposure to Cd efficiently compensated AsA deficiency in vtc1-1 plants and contributed to decreased sensitivity towards Cd. Glutathione-deficient (cad2-1 and cad2-1 vtc1-1) mutants, however, showed a more oxidized GSH redox state, resulting in initial oxidative stress and a higher sensitivity to Cd. In order to cope with the Cd stress to which they were exposed, GSH-deficient mutants activated multiple alternative pathways. CONCLUSIONS: Our observations indicate that GSH and AsA deficiency differentially alter plant GSH homeostasis, resulting in opposite Cd sensitivities relative to wild-type plants. Upon Cd exposure, GSH-deficient mutants were hampered in chelation. They experienced phenotypic disturbances and even more oxidative stress, and therefore activated multiple alternative pathways such as SOD, CAT and ascorbate peroxidase, indicating a higher Cd sensitivity. Ascorbate deficiency, however, was associated with enhanced PC synthesis in comparison with wild-type plants after Cd exposure, which contributed to decreased sensitivity towards Cd.

Differential response of Arabidopsis leaves and roots to cadmium: glutathione-related chelating capacity vs antioxidant capacity.

This study aims to uncover the spatiotemporal involvement of glutathione (GSH) in two major mechanisms of cadmium (Cd)-induced detoxification (i.e. chelation and antioxidative defence). A kinetic study was conducted on hydroponically grown Arabidopsis thaliana (L. Heyhn) to gain insight into the early events after exposure to Cd. Cadmium detoxification was investigated at different levels, including gene transcripts, enzyme activities and metabolite content. Data indicate a time-dependent response both within roots and between plant organs. Early on in roots, GSH was preferentially allocated to phytochelatin (PC) synthesis destined for Cd chelation. This led to decreased GSH levels, without alternative pathways activated to complement GSH's antioxidative functions. After one day however, multiple antioxidative pathways increased including superoxide dismutase (SOD), ascorbate (AsA) and catalase (CAT) to ensure efficient neutralization of Cd-induced reactive oxygen species (ROS). As a consequence of Cd retention and detoxification in roots, a delayed response occurred in leaves. Together with high leaf thiol contents and possibly signalling responses from the roots, the leaves were protected, allowing them sufficient time to activate their defence mechanisms.

Problems inherent to a meta-analysis of proteomics data: a case study on the plants' response to Cd in different cultivation conditions.

This meta-analysis focuses on plant-proteome responses to cadmium (Cd) stress. Initially, some general topics related to a proteomics meta-analysis are discussed: (1) obstacles encountered during data analysis, (2) a consensus in proteomic research, (3) validation and good reporting practices for protein identification and (4) guidelines for statistical analysis of differentially abundant proteins. In a second part, the Cd responses in leaves and roots obtained from a proteomics meta-analysis are discussed in (1) a time comparison (short versus long term exposure), and (2) a culture comparison (hydroponics versus soil cultivation). Data of the meta-analysis confirmed the existence of an initial alarm phase upon Cd exposure. Whereas no metabolic equilibrium is established in hydroponically exposed plants, an equilibrium seems to be manifested in roots of plants grown in Cd-contaminated soil after long term exposure. In leaves, the carbohydrate metabolism is primarily affected independent of the exposure time and the cultivation method. In addition, a metabolic shift from CO2-fixation towards respiration is manifested, independent of the cultivation system. Finally, some ideas for the improvement of proteomics setups and for comparisons between studies are discussed. BIOLOGICAL SIGNIFICANCE: This meta-analysis focuses on the plant responses to Cd stress in leaves and roots at the proteome level. This meta-analysis points out the encountered obstacles when performing a proteomics meta-analysis related to inherent technologies, but also related to experimental setups. Furthermore, the question is addressed whether an extrapolation of results obtained in hydroponic cultivation towards soil-grown plants is possible.

A physiological and proteomic study of poplar leaves during ozone exposure combined with mild drought.

The occurrence of high-ozone concentrations during drought episodes is common considering that they are partially caused by the same meteorological phenomena. It was suggested that mild drought could protect plants against ozone-induced damage by causing the closure of stomata and preventing the entry of ozone into the leaves. The present experiment attempts to create an overview of the changes in cellular processes in response to ozone, mild drought and a combined treatment based on the use of 2D-DiGE to compare the involved proteins, and a number of supporting analyses. Morphological symptoms were worst in the combined treatment, indicating a severe stress, but fewer proteins were differentially abundant in the combined treatment than for ozone alone. Stomatal conductance was slightly lowered in the combined treatment. Shifts in carbon metabolism indicated that the metabolism changed to accommodate for protective measures and changes in the abundance of proteins involved in redox protection indicated the presence of an oxidative stress. This study allowed identifying a set of proteins that changed similarly during ozone and drought stress, indicative of crosstalk in the molecular response of plants exposed to these stresses. The abundance of other key proteins changed only when the plants are exposed to specific conditions. Together this indicates the coexistence of generalized and specialized responses to different conditions.

Metal-induced oxidative stress and plant mitochondria.

A general status of oxidative stress in plants caused by exposure to elevated metal concentrations in the environment coincides with a constraint on mitochondrial electron transport, which enhances ROS accumulation at the mitochondrial level. As mitochondria are suggested to be involved in redox signaling under environmental stress conditions, mitochondrial ROS can initiate a signaling cascade mediating the overall stress response, i.e., damage versus adaptation. This review highlights our current understanding of metal-induced responses in plants, with focus on the production and detoxification of mitochondrial ROS. In addition, the potential involvement of retrograde signaling in these processes will be discussed.

A difference gel electrophoresis study on thylakoids isolated from poplar leaves reveals a negative impact of ozone exposure on membrane proteins.

Populus tremula L. x P. alba L. (Populus x canescens (Aiton) Smith), clone INRA 717-1-B4, saplings were subjected to 120 ppb ozone exposure for 28 days. Chloroplasts were isolated, and the membrane proteins, solubilized using the detergent 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC), were analyzed in a difference gel electrophoresis (DiGE) experiment comparing control versus ozone-exposed plants. Extrinsic photosystem (PS) proteins and adenosine triphosphatase (ATPase) subunits were detected to vary in abundance. The general trend was a decrease in abundance, except for ferredoxin-NADP(+) oxidoreductase (FNR), which increased after the first 7 days of exposure. The up-regulation of FNR would increase NAPDH production for reducing power and detoxification inside and outside of the chloroplast. Later on, FNR and a number of PS and ATPase subunits decrease in abundance. This could be the result of oxidative processes on chloroplast proteins but could also be a way to down-regulate photochemical reactions in response to an inhibition in Calvin cycle activity.

Differential impact of chronic ozone exposure on expanding and fully expanded poplar leaves.

Populus tremula L. × Populus alba L. (Populus ×c anescens (Aiton) Smith) - clone INRA 717-1-B4 saplings (50 cm apex to base and carrying 19 leaves on average) - were followed for 28 days. Half of the trees were grown in charcoal-filtered air while the other half were exposed to 120 ppb ozone for 11 h a day during the light period. The expanding leaf number 4 was tagged at the beginning of the experiment and finished expansion between 7 and 14 days. These leaves were harvested weekly for biochemical and proteome analyses using quantitative bidimensional electrophoresis (DiGE). Independent of the ozone treatment, all the analyses allowed a distinction between expanding and adult leaves. The results indicate that during the expansion phase (Days 0-7) the enzymatic machinery of the leaves is set up, and remains dynamically stable in the adult leaves (Days 14-28). Although ozone had no apparent effect on expanding leaves, the metabolic stability in fully expanded leaves observed in ozone-free plants was disturbed after 2 weeks of exposure and a stress-induced response became apparent.

The impact of atmospheric composition on plants: a case study of ozone and poplar.

Tropospheric ozone is the main atmospheric pollutant that causes damages to trees. The estimation of the threshold for ozone risk assessment depends on the evaluation of the means that this pollutant impacts the plant and, especially, the foliar organs. The available results show that, before any visible symptom appears, carbon assimilation and the underlying metabolic processes are decreased under chronic ozone exposure. By contrast, the catabolic pathways are enhanced, and contribute to the supply of sufficient reducing power necessary to feed the detoxification processes. Reactive oxygen species delivered during ozone exposure serve as toxic compounds and messengers for the signaling system. In this review, we show that the contribution of genomic tools (transcriptomics, proteomics, and metabolomics) for a better understanding of the mechanistic cellular responses to ozone largely relies on spectrometric measurements.

A DIGE analysis of developing poplar leaves subjected to ozone reveals major changes in carbon metabolism.

Tropospheric ozone pollution is described as having major negative effects on plants, compromising plant survival. Carbon metabolism is especially affected. In the present work, the effects of chronic ozone exposure were evaluated at the proteomic level in developing leaves of young poplar plants exposed to 120 ppb of ozone for 35 days. Soluble proteins (excluding intrinsic membrane proteins) were extracted from leaves after 3, 14 and 35 days of ozone exposure, as well as 10 days after a recovery period. Proteins (pI 4 to 7) were analyzed by 2-D DIGE experiments, followed by MALDI-TOF-TOF identification. Additional observations were obtained on growth, lesion formation, and leaf pigments analysis. Although treated plants showed large necrotic spots and chlorosis in mature leaves, growth decreased only slightly and plant height was not affected. The number of abscised leaves was higher in treated plants, but new leaf formation was not affected. A decrease in chlorophylls and lutein contents was recorded. A large number of proteins involved in carbon metabolism were identified. In particular, proteins associated with the Calvin cycle and electron transport in the chloroplast were down-regulated. In contrast, proteins associated with glucose catabolism increased in response to ozone exposure. Other identified enzymes are associated with protein folding, nitrogen metabolism and oxidoreductase activity.