Transcriptomics uncovers substantial variability associated with alterations in manufacturing processes of macrophage cell therapy products.

Gene expression plasticity is central for macrophages' timely responses to cues from the microenvironment permitting phenotypic adaptation from pro-inflammatory (M1) to wound healing and tissue-regenerative (M2, with several subclasses). Regulatory macrophages are a distinct macrophage type, possessing immunoregulatory, anti-inflammatory, and angiogenic properties. Due to these features, regulatory macrophages are considered as a potential cell therapy product to treat clinical conditions, e.g., non-healing diabetic foot ulcers. In this study we characterized two differently manufactured clinically relevant regulatory macrophages, programmable cells of monocytic origin and comparator macrophages (M1, M2a and M0) using flow-cytometry, RT-qPCR, phagocytosis and secretome measurements, and RNA-Seq. We demonstrate that conventional phenotyping had a limited potential to discriminate different types of macrophages which was ameliorated when global transcriptome characterization by RNA-Seq was employed. Using this approach we confirmed that macrophage manufacturing processes can result in a highly reproducible cell phenotype. At the same time, minor changes introduced in manufacturing resulted in phenotypically and functionally distinct regulatory macrophage types. Additionally, we have identified a novel constellation of process specific biomarkers, which will support further clinical product development.

Correction: Neurobiological roots of psychopathy.

A correction to this paper has been published and can be accessed via a link at the top of the paper.

Neurobiological roots of psychopathy.

Psychopathy is an extreme form of antisocial behavior, with about 1% prevalence in the general population, and 10-30% among incarcerated criminal offenders. Although the heritability of severe antisocial behavior is up to 50%, the genetic background is unclear. The underlying molecular mechanisms have remained unknown but several previous studies suggest that abnormal glucose metabolism and opioidergic neurotransmission contribute to violent offending and psychopathy. Here we show using iPSC-derived cortical neurons and astrocytes from six incarcerated extremely antisocial and violent offenders, three nonpsychopathic individuals with substance abuse, and six healthy controls that there are robust alterations in the expression of several genes and immune response-related molecular pathways which were specific for psychopathy. In neurons, psychopathy was associated with marked upregulation of RPL10P9 and ZNF132, and downregulation of CDH5 and OPRD1. In astrocytes, RPL10P9 and MT-RNR2 were upregulated. Expression of aforementioned genes explained 30-92% of the variance of psychopathic symptoms. The gene expression findings were confirmed with qPCR. These genes may be relevant to the lack of empathy and emotional callousness seen in psychopathy, since several studies have linked these genes to autism and social interaction.

Sex-specific transcriptional and proteomic signatures in schizophrenia.

It has remained unclear why schizophrenia typically manifests after adolescence and which neurobiological mechanisms are underlying the cascade leading to the actual onset of the illness. Here we show that the use of induced pluripotent stem cell-derived neurons of monozygotic twins from pairs discordant for schizophrenia enhances disease-specific signal by minimizing genetic heterogeneity. In proteomic and pathway analyses, clinical illness is associated especially with altered glycosaminoglycan, GABAergic synapse, sialylation, and purine metabolism pathways. Although only 12% of all 19,462 genes are expressed differentially between healthy males and females, up to 61% of the illness-related genes are sex specific. These results on sex-specific genes are replicated in another dataset. This implies that the pathophysiology differs between males and females, and may explain why symptoms appear after adolescence when the expression of many sex-specific genes change, and suggests the need for sex-specific treatments.

Generation of a human induced pluripotent stem cell line (LL008 1.4) from a familial Alzheimer's disease patient carrying a double KM670/671NL (Swedish) mutation in APP gene.

A double mutation (KM670/671NL) in amyloid precursor protein gene (APP) is causative for familial Alzheimer's disease and has been shown to increase the total Aß burden. Here we report the generation and characterization of an iPSC line from a fAD patient carrying APP KM670/671NL. The generated iPSCs retained the mutation, expressed pluripotency markers, showed a normal karyotype and differentiated into all three germ layers. This iPSC line can be used, for example, in disease modeling and mechanistic studies. Resource table.

Temporal Dynamics of Gene Expression During Endothelial Cell Differentiation From Human iPS Cells: A Comparison Study of Signalling Factors and Small Molecules.

Endothelial cell (EC) therapy may promote vascular growth or reendothelization in a variety of disease conditions. However, the production of a cell therapy preparation containing differentiated, dividing cells presenting typical EC phenotype, functional properties and chemokine profile is challenging. We focused on comparative analysis of seven small molecule-mediated differentiation protocols of ECs from human induced pluripotent stem cells. Differentiated cells showed a typical surface antigen pattern of ECs as characterized with flow cytometry analysis, functional properties, such as tube formation and ability to uptake acetylated LDL. Gene expression analysis by RNA sequencing revealed an efficient silencing of pluripotency genes and upregulation of genes related to cellular adhesion during differentiation. In addition, distinct patterns of transcription factor expression were identified during cellular reprogramming providing targets for more effective differentiation protocols in the future. Altogether, our results suggest that the most optimal EC differentiation protocol includes early inhibition of Rho-associated coiled-coil kinase and activation of cyclic AMP signaling, and inhibition of transforming growth factor beta signaling after mesodermal stage. These findings provide the first systematic characterization of the most potent signalling factors and small molecules used to generate ECs from human induced pluripotent stem cells and, consequently, this work improves the existing EC differentiation protocols and opens up new avenues for controlling cell fate for regenerative EC therapy.

Structural Immaturity of Human iPSC-Derived Cardiomyocytes: In Silico Investigation of Effects on Function and Disease Modeling.

Background: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as a promising experimental tool for translational heart research and drug development. However, their usability as a human adult cardiomyocyte model is limited by their functional immaturity. Our aim is to analyse quantitatively those characteristics and how they differ from adult CMs. Methods and Results: We have developed a novel in silico model with all essential functional electrophysiology and calcium handling features of hiPSC-CMs. Importantly, the virtual cell recapitulates the immature intracellular ion dynamics that are characteristic for hiPSC-CMs, as quantified based our in vitro imaging data. The strong "calcium clock" is a source for a dual function of excitation-contraction coupling in hiPSC-CMs: action potential and calcium transient morphology vary substantially depending on the activation sequence of underlying ionic currents and fluxes that is altered in spontaneous vs. paced mode. Furthermore, parallel simulations with hiPSC-CM and adult cardiomyocyte models demonstrate the central differences. Results indicate that hiPSC-CMs translate poorly the disease specific phenotypes of Brugada syndrome, long QT Syndrome and catecholaminergic polymorphic ventricular tachycardia, showing less robustness and greater tendency for arrhythmic events than adult CMs. Based on a comparative sensitivity analysis, hiPSC-CMs share some features with adult CMs, but are still functionally closer to prenatal CMs than adult CMs. A database analysis of 3000 hiPSC-CM model variants suggests that hiPSC-CMs recapitulate poorly fundamental physiological properties of adult CMs. Single modifications do not appear to solve this problem, which is mostly contributed by the immaturity of intracellular calcium handling. Conclusion: Our data indicates that translation of findings from hiPSC-CMs to human disease should be made with great caution. Furthermore, we established a mathematical platform that can be used to improve the translation from hiPSC-CMs to human, and to quantitatively evaluate hiPSC-CMs development toward more general and valuable model for human cardiac diseases.

Creation of a library of induced pluripotent stem cells from Parkinsonian patients.

Induced pluripotent stem cells (iPSCs) are becoming an important source of pre-clinical models for research focusing on neurodegeneration. They offer the possibility for better understanding of common and divergent pathogenic mechanisms of brain diseases. Moreover, iPSCs provide a unique opportunity to develop personalized therapeutic strategies, as well as explore early pathogenic mechanisms, since they rely on the use of patients' own cells that are otherwise accessible only post-mortem, when neuronal death-related cellular pathways and processes are advanced and adaptive. Neurodegenerative diseases are in majority of unknown cause, but mutations in specific genes can lead to familial forms of these diseases. For example, mutations in the superoxide dismutase 1 gene lead to the motor neuron disease amyotrophic lateral sclerosis (ALS), while mutations in the SNCA gene encoding for alpha-synuclein protein lead to familial Parkinson's disease (PD). The generations of libraries of familial human ALS iPSC lines have been described, and the iPSCs rapidly became useful models for studying cell autonomous and non-cell autonomous mechanisms of the disease. Here we report the generation of a comprehensive library of iPSC lines of familial PD and an associated synucleinopathy, multiple system atrophy (MSA). In addition, we provide examples of relevant neural cell types these iPSC can be differentiated into, and which could be used to further explore early disease mechanisms. These human cellular models will be a valuable resource for identifying common and divergent mechanisms leading to neurodegeneration in PD and MSA.

Generation of Functional Neuromuscular Junctions from Human Pluripotent Stem Cell Lines.

Several neuromuscular diseases involve dysfunction of neuromuscular junctions (NMJs), yet there are no patient-specific human models for electrophysiological characterization of NMJ. We seeded cells of neurally-induced embryoid body-like spheres derived from induced pluripotent stem cell (iPSC) or embryonic stem cell (ESC) lines as monolayers without basic fibroblast factor (bFGF) and observed differentiation of neuronal as well as spontaneously contracting, multinucleated skeletal myotubes. The myotubes showed striation, immunoreactivity for myosin heavy chain, actin bundles typical for myo-oriented cells, and generated spontaneous and evoked action potentials (APs). The myogenic differentiation was associated with expression of MyoD1, myogenin and type I ryanodine receptor. Neurons formed end plate like structures with strong binding of α-bungarotoxin, a marker of nicotinic acetylcholine receptors highly expressed in the postsynaptic membrane of NMJs, and expressed SMI-32, a motoneuron marker, as well as SV2, a marker for synapses. Pharmacological stimulation of cholinergic receptors resulted in strong depolarization of myotube membrane and raised Ca(2+) concentration in sarcoplasm, while electrical stimulation evoked Ca(2+) transients in myotubes. Stimulation of motoneurons with N-Methyl-D-aspartate resulted in reproducible APs in myotubes and end plates displayed typical mEPPs and tonic activity depolarizing myotubes of about 10 mV. We conclude that simultaneous differentiation of neurons and myotubes from patient-specific iPSCs or ESCs results also in the development of functional NMJs. Our human model of NMJ may serve as an important tool to investigate normal development, mechanisms of diseases and novel drug targets involving NMJ dysfunction and degeneration.

Immunomodulation by interleukin-33 is protective in stroke through modulation of inflammation.

Cerebral stroke induces massive Th1-shifted inflammation both in the brain and the periphery, contributing to the outcome of stroke. A Th1-type response is neurotoxic whereas a Th2-type response is accompanied by secretion of anti-inflammatory cytokines, such as interleukin-4 (IL-4). Interleukin-33 (IL-33) is a cytokine known to induce a shift towards the Th2-type immune response, polarize macrophages/microglia towards the M2-type, and induce production of anti-inflammatory cytokines. We found that the plasma levels of the inhibitory IL-33 receptor, sST2, are increased in human stroke and correlate with a worsened stroke outcome, suggesting an insufficient IL-33-driven Th2-type response. In mouse, peripheral administration of IL-33 reduced stroke-induced cell death and improved the sensitivity of the contralateral front paw at 5days post injury. The IL-33-treated mice had increased levels of IL-4 in the spleen and in the peri-ischemic area of the cortex. Neutralization of IL-4 by administration of an IL-4 antibody partially prevented the IL-33-mediated protection. IL-33 treatment also reduced astrocytic activation in the peri-ischemic area and increased the number of Arginase-1 immunopositive microglia/macrophages at the lesion site. In human T-cells, IL-33 treatment induced IL-4 secretion, and the conditioned media from IL-33-exposed T-cells reduced astrocytic activation. This study demonstrates that IL-33 is protective against ischemic insult by induction of IL-4 secretion and may represent a novel therapeutic approach for the treatment of stroke.

Transplanted Human Induced Pluripotent Stem Cell-Derived Neural Progenitor Cells Do Not Promote Functional Recovery of Pharmacologically Immunosuppressed Mice With Contusion Spinal Cord Injury.

Improved functional recovery after spinal cord injury by transplantation of induced pluripotent stem cell-derived neural stem/progenitor cells (iPSC-NPCs) has been reported. However, beneficial effects of iPSC-based therapy have so far been produced mostly using genetically immunodeficient rodents. Because of the long time required for generation and characterization of iPSCs, the use of autologous iPSCs for treating patients with acute spinal cord injury (SCI) is not feasible. Therefore, it is of utmost importance to investigate the effect of iPSC-based therapy on functional recovery after SCI using pharmacologically immunosuppressed, immunocompetent animal models. Here we studied the functional outcome following subacute transplantation of human iPSC-derived NPCs into contused mouse spinal cord when tacrolimus was used as an immunosuppressive agent. We show that human iPSC-derived NPCs transplanted into pharmacologically immunosuppressed C57BL/6J mice exhibited poor long-term survival and failed to improve functional recovery after SCI as measured by Basso Mouse Scale (BMS) for locomotion and CatWalk gait analysis when compared to vehicle-treated animals. The scarce effect of iPSC-based therapy observed in the current study may be attributable to insufficient immunosuppressive effect, provided by monotherapy with tacrolimus in combination with immunogenicity of transplanted cells and complex microenvironment of the injured spinal cord. Our results highlight the importance of extensive preclinical studies of transplanted cells before the clinical application of iPSC-based cell therapy is achieved.

Chondrogenic differentiation of human pluripotent stem cells in chondrocyte co-culture.

Chondrogenic differentiation of human embryonic (hESCs) or induced pluripotent stem cells (hiPSCs) has been achieved in embryoid bodies (EBs) by adding selected growth factors to the medium. Also chondrocyte-secreted factors have been considered to promote the chondrogenic differentiation. Hence, we studied whether co-culture with primary chondrocytes can induce hESCs or hiPSCs to differentiate into chondrocyte lineage. Co-culture of hESCs or hiPSCs was established in a transwell insert system in feeder-free culture conditions, while hESCs or hiPSCs grown alone in the wells were used as controls. After 3-week co-culture with weekly replenished chondrocytes, the chondrogenically committed cells (hCCCs) were evaluated by morphology, immunocytochemistry, quantitative real-time RT-PCR, and analysis of chondrogenic, osteogenic and adipogenic differentiation markers. The expressions of chondrocyte- and pluripotency-associated genes were frequently measured during the monolayer expansion of hCCCs from passage 1 to 10. Human CCCs displayed morphology similar to chondrocytes, and expressed chondrocyte-associated genes, which were declined following passaging, similarly to passaged chondrocytes. They also formed a chondrogenic cell pellet, and differentiated into chondrocytic cells, which secreted abundant extracellular matrix. Human CCCs also proliferated rapidly. However, they did not show osteogenic or adipogenic differentiation capacity. Our results show that co-culture of hESCs or hiPSCs with primary chondrocytes could induce specific chondrogenic differentiation.

Improved method of producing human neural progenitor cells of high purity and in large quantities from pluripotent stem cells for transplantation studies.

Transplantation of human neural progenitor cells (hNPCs) is a promising therapeutic approach for various diseases of the central nervous system (CNS). Reliable testing of hNPC transplantation in animal models of neurological diseases requires that these cells can be produced in sufficient amounts, show consistent homogeneity as a neural cell population, and be reliably labeled for in vivo tracking. In addition, the cells should be characterized as being at the optimal state of differentiation favoring successful engraftment. Here, we show that high numbers of purified hNPCs can be produced from human embryonic stem cells (hESCs) by manually selecting specifically sized and shaped spheres followed by fluorescence-activated cell sorting based on the relative cell size. In addition, we report that labeling of hNPCs with ultra-small superparamagnetic iron oxide (USPIO) particles does not affect the cellular morphology or growth. More importantly, we show that the transduction with lentiviral vector encoding green fluorescent protein (GFP) decreases the neurality of the cell population. We conclude that our cost-effective protocol of generating hNPCs is widely applicable for preclinical studies on CNS disorders. This improved method of producing large quantities of high-purity hNPCs maybe useful also when generating hNPCs from human induced pluripotent stem (hiPS) cell lines. However, caution should be used when lenti-GFP transduction is applied for hNPC labeling.

Minocycline protects SH-SY5Y cells from 6-hydroxydopamine by inhibiting both caspase-dependent and -independent programmed cell death.

Minocycline, a tetracyclic antibiotic, exerts both antiinflammation by acting on microglia and a direct protection on neurons by inhibiting the apoptotic machinery at various levels. However, we are not aware of any study investigating the effects of minocycline on caspase-independent programmed cell death (PCD) pathways. This study investigated these alternative pathways in SH-SY5Y cells, a human dopaminergic cell line, challenged with 6-hydroxydopamine (6-OHDA). Minocycline exhibited neuroprotection and inhibition of the toxin-induced caspase-3-like activity, DNA fragmentation, and chromatin condensation, hallmarks of apoptosis. Moreover, we revealed that 6-OHDA also activated caspase-independent PCDs (such as paraptosis), which required de novo protein synthesis. Additionally, by separately monitoring caspase-dependent and caspase-independent pathways, we showed that inhibition of apoptosis only partially explained the protective effect of minocycline. Moreover, we observed that minocycline reduced the protein content of cells but, unexpectedly, increased the protein synthesis. These findings suggest that minocycline may actually increase protein degradation, so it may also accelerate the clearance of aberrant proteins. In conclusion, we report for the first time evidence indicating that minocycline may inhibit PCD pathways that are additional to conventional apoptosis.

Prolyl endopeptidase is involved in cellular signalling in human neuroblastoma SH-SY5Y cells.

Prolyl endopeptidase (PREP), probably acting through the inositol cycle, has been implicated in memory and learning. However, the physiological role of PREP is unknown. It has been shown that PREP expression, regulated in cerebellar granule cells, has probably a role in cell proliferation and differentiation. Here, we report the levels and subcellular distribution of PREP in human neuroblastoma SH-SY5Y cells in proliferating conditions and under differentiation induced by retinoic acid (RA). We analysed the levels of cell signalling intermediates, growth behavior and gene expression, and differentiation morphology changes, upon PREP inhibition. After induction of differentiation, PREP activity was found decreased in the nucleus but increased to high levels in the cytoplasm, due in part to increased PREP transcription. The levels of inositol (1,4,5)-trisphosphate revealed no correlation with PREP activity, but phosphorylated extracellular signal-regulated kinases 1 and 2 were decreased by PREP inhibition during early stages of differentiation. Morphological evaluation indicated that PREP inhibition retarded the onset of differentiation. PREP activity regulated gene expression of protein synthesis machinery, intracellular transport and kinase complexes. We conclude that PREP is a regulatory target and a regulatory element in cell signalling. This is the first report of a direct influence of a cell signalling molecule, RA, on PREP expression.

Different effects of scopolamine and inhibition of prolyl oligopeptidase on mnemonic and motility functions of young and 8- to 9-month-old rats in the radial-arm maze.

Prolyl oligopeptidase (POP) has been connected to memory and mood through regulation of the brain levels of its biologically active peptide substrates and phosphatidylinositol system. This is the first study in a radial-arm maze of the effects of a single dose of a novel potent prolyl oligopeptidase inhibitor, KYP-2047 (5 mg/kg, dissolved in 5% Tween 80), on memory and learning of scopolamine-treated (0.4 mg/kg, dissolved in saline) rats. Habituated (days 1 and 2) and trained (days 3-11) young (3 months) and old (8-9 months) male Wistar rats were given (i) saline + Tween, (ii) saline + KYP-2047, (iii) scopolamine + Tween or (iv) scopolamine + KYP-2047 30 min. prior to testing their memory. Food rewards located in four randomly chosen arms of the maze. The rat had 10 min. to find and eat the rewards. Time spent in the maze, visits to each arm and number of eaten rewards were measured. Old rats made generally more errors, spent more time and visited fewer arms per minute in the maze than young rats. The memory- and function-impairing effects of scopolamine were also seen more clearly in old than young rats. KYP-2047 had no or only a marginal effect on memory of either age group, but when given without scopolamine, it slightly increased the maze motility of young rats and decreased the motility of old rats. In a separate locomotor activity test, KYP-2047 enhanced the motility of young rats supporting a suggested role of POP in motor functions.

Induction of PUMA-alpha and down-regulation of PUMA-beta expression is associated with benzo(a)pyrene-induced apoptosis in MCF-7 cells.

Benzo(a)pyrene (BP) forms benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE)-DNA adducts in human breast adenocarcinoma MCF-7 cells, leading to p53 protein induction and phosphorylation. Although BP-induced apoptosis in rodent cells is known, it is still unclear in human cells. Here we have analyzed the effects of BP on p53 related apoptotic proteins, cell cycle and cell death in MCF-7 cells. PUMA-protein (p53 up-regulated modulator of apoptosis) levels were changed after BP exposure so that PUMA-alpha protein was statistically significantly increased whereas PUMA-beta protein was statistically significantly decreased. PUMA-protein levels were also investigated in ZR-75-1 cells, where PUMA-alpha protein was statistically significantly increased. Cytochrome c, which is released from mitochondria during apoptosis to form the apoptosome, was increased in cytoplasmic fraction after BP exposure in MCF-7 cells. Increased apoptosis was also seen after 48 and 72 h BP exposure (2.5 and 5 microM). In addition, BP decreased dose dependently cell viability (2.5 and 5 microM) and increased ROS formation (1 and 10 microM). Our results suggest that PUMA-alpha protein is involved in BP-induced cell death most likely through a p53 dependent apoptotic pathway.

Different viabilities and toxicity types after 6-OHDA and Ara-C exposure evaluated by four assays in five cell lines.

Cell viability studies are useful when screening novel drugs for the diseases that are related to either increased cell death or enhanced cell survival. There are numerous assays but the results that they produce are rarely unanimous. Here we compared the performance of (1) morphological microscopic assay with double DNA staining, (2) propidium iodide-digitonin assay, (3) MTT-assay, and (4) ATP-assay in human neuroblastoma (SH-SY5Y), rat glioma (C6), rabbit smooth muscle (SMC), Chinese hamster ovary (CHO) and monkey fibroblast cells (CV1-P) exposed to cytosine arabinoside (Ara-C) and 6-hydroxydopamine (6-OHDA). We found that neuronal SH-SY5Y cells were most sensitive to both toxins and the results in all viability tests correlated well. All the other cell lines were much more resistant, particularly to Ara-C but also to 6-OHDA. Toxicity of the compounds was best revealed by MTT and ATP assays, measuring the metabolic activity of the cells, and only occasionally by morphological observations or with the propidium iodide-digitonin assay which is based on the cell membrane integrity. In this research, Ara-C induced pure apoptosis whereas the toxicity type of 6-OHDA was dose-dependent. The use of several viability tests and cell lines is recommended when studying cell death, particularly apoptosis, and performance of antiapoptotic compounds.

Beneficial effect of prolyl oligopeptidase inhibition on spatial memory in young but not in old scopolamine-treated rats.

The effects of a novel prolyl oligopeptidase (POP) inhibitor KYP-2047 on spatial memory of young (3-month-old) and old (8- to 9-month-old) scopolamine-treated rats (0.4 mg/kg intraperitoneally) was investigated in the Morris water maze. In addition, the concentrations of promnesic neuropeptide substrates of POP, substance P and neurotensin in various brain areas after acute and chronic POP inhibition were measured in young rats. In addition, inositol-1,4,5-trisphosphate (IP(3)) levels were assayed in rat cortex and hippocampus after effective 2.5-day POP inhibition. KYP-2047 (1 or 5 mg/kg 30 min. before daily testing) dose-dependently improved the escape performance (i.e. latency to find the hidden platform and swimming path length) of the young but not the old rats in the water maze. POP inhibition had no consistent effect on substance P levels in cortex, hippocampus or hypothalamus, and only a modest increase in neurotensin concentration was observed in the hypothalamus after a single dose of KYP-2047. Moreover, IP(3) concentrations remained unaffected in cortex and hippocampus after POP inhibition. In conclusion, the behavioural data support the earlier findings of the promnesic action of POP inhibitors, but the mechanism of the memory-enhancing action remains unclear.

A prolyl oligopeptidase inhibitor, Z-Pro-Prolinal, inhibits glyceraldehyde-3-phosphate dehydrogenase translocation and production of reactive oxygen species in CV1-P cells exposed to 6-hydroxydopamine.

We studied the ability of prolyl oligopeptidase (POP) inhibitors, Z-Pro-Prolinal and JTP-4819, to prevent translocation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and formation of reactive oxygen species (ROS), in 6-hydroxydopamine (6-OHDA) and cytosine arabinoside (Ara-C) treated monkey fibroblast (CV1-P) and human neuroblastoma (SH-SY5Y) cells. The cells were pretreated with POP inhibitors (30 min) before addition of toxicants. GAPDH was analyzed by Western hybridization, ROS by fluorescent 2'7'-dichlorodihydro-fluorescein diacetate, and viability by the MTT method. Both toxicants induced GAPDH translocation to the particulate fraction (mitochondria and nuclei). Z-Pro-Prolinal was able to inhibit the translocation in 6-OHDA-exposed CV1-P cells. In SH-SY5Y cells and in JTP-4819 pretreated cells, no prevention of translocation was seen. However, the intensity of GAPDH in cytosolic fraction increased. Both inhibitors blocked 6-OHDA-induced ROS-production to the control level in CV1-P but, not in SH-SY5Y cells, without affecting their viability. In conclusion, POP inhibitors are able to prevent certain cell stress related factors such as ROS production or GAPDH translocation.