Antitumor Effects of a New Retinoate of the Fungal Cytotoxin Illudin M in Brain Tumor Models.

While the fungal metabolite illudin M (1) is indiscriminately cytotoxic in cancer and non-malignant cells, its retinoate 2 showed a greater selectivity for the former, especially in a cerebral context. Illudin M killed malignant glioma cells as well as primary neurons and astrocytes at similarly low concentrations and destroyed their microtubule and glial fibrillary acidic protein (GFAP) networks. In contrast, the ester 2 was distinctly more cytotoxic in highly dedifferentiated U87 glioma cells than in neurons, which were even stimulated to enhanced growth. This was also observed in co-cultures of neurons with U87 cells where conjugate 2 eventually killed them by induction of differentiation based on the activation of nuclear receptors, which bind to retinoid-responsive elements (RARE). Hence, illudin M retinoate 2 appears to be a promising drug candidate.

Versatility of microglial bioenergetic machinery under starving conditions.

Microglia are highly dynamic cells in the brain. Their functional diversity and phenotypic versatility brought microglial energy metabolism into the focus of research. Although it is known that microenvironmental cues shape microglial phenotype, their bioenergetic response to local nutrient availability remains unclear. In the present study effects of energy substrates on the oxidative and glycolytic metabolism of primary - and BV-2 microglial cells were investigated. Cellular oxygen consumption, glycolytic activity, the levels of intracellular ATP/ADP, autophagy, mTOR phosphorylation, apoptosis and cell viability were measured in the absence of nutrients or in the presence of physiological energy substrates: glutamine, glucose, lactate, pyruvate or ketone bodies. All of the oxidative energy metabolites increased the rate of basal and maximal respiration. However, the addition of glucose decreased microglial oxidative metabolism and glycolytic activity was enhanced. Increased ATP/ADP ratio and cell viability, activation of the mTOR and reduction of autophagic activity were observed in glutamine-supplemented media. Moreover, moderate and transient oxidation of ketone bodies was highly enhanced by glutamine, suggesting that anaplerosis of the TCA-cycle could stimulate ketone body oxidation. It is concluded that microglia show high metabolic plasticity and utilize a wide range of substrates. Among them glutamine is the most efficient metabolite. To our knowledge these data provide the first account of microglial direct metabolic response to nutrients under short-term starvation and demonstrate that microglia exhibit versatile metabolic machinery. Our finding that microglia have a distinct bioenergetic profile provides a critical foundation for specifying microglial contributions to brain energy metabolism.

Enhanced detection with spectral imaging fluorescence microscopy reveals tissue- and cell-type-specific compartmentalization of surface-modified polystyrene nanoparticles.

BACKGROUND: Precisely targeted nanoparticle delivery is critically important for therapeutic applications. However, our knowledge on how the distinct physical and chemical properties of nanoparticles determine tissue penetration through physiological barriers, accumulation in specific cells and tissues, and clearance from selected organs has remained rather limited. In the recent study, spectral imaging fluorescence microscopy was exploited for precise and rapid monitoring of tissue- and cell-type-specific distribution of fluorescent polystyrene nanoparticles with chemically distinct surface compositions. METHODS: Fluorescent polystyrene nanoparticles with 50-90 nm diameter and with carboxylated- or polyethylene glycol-modified (PEGylated) surfaces were delivered into adult male and pregnant female mice with a single intravenous injection. The precise anatomical distribution of the particles was investigated by confocal microscopy after a short-term (5 min) or long-term (4 days) distribution period. In order to distinguish particle-fluorescence from tissue autofluorescence and to enhance the detection-efficiency, fluorescence spectral detection was applied during image acquisition and a post hoc full spectrum analysis was performed on the final images. RESULTS: Spectral imaging fluorescence microscopy allowed distinguishing particle-fluorescence from tissue-fluorescence in all examined organs (brain, kidney, liver, spleen and placenta) in NP-treated slice preparations. In short-time distribution following in vivo NP-administration, all organs contained carboxylated-nanoparticles, while PEGylated-nanoparticles were not detected in the brain and the placenta. Importantly, nanoparticles were not found in any embryonic tissues or in the barrier-protected brain parenchyma. Four days after the administration, particles were completely cleared from both the brain and the placenta, while PEGylated-, but not carboxylated-nanoparticles, were stuck in the kidney glomerular interstitium. In the spleen, macrophages accumulated large amount of carboxylated and PEGylated nanoparticles, with detectable redistribution from the marginal zone to the white pulp during the 4-day survival period. CONCLUSIONS: Spectral imaging fluorescence microscopy allowed detecting the tissue- and cell-type-specific accumulation and barrier-penetration of polystyrene nanoparticles with equal size but chemically distinct surfaces. The data revealed that polystyrene nanoparticles are retained by the reticuloendothelial system regardless of surface functionalization. Taken together with the increasing production and use of nanoparticles, the results highlight the necessity of long-term distribution studies to estimate the potential health-risks implanted by tissue-specific nanoparticle accumulation and clearance.

Generation of diverse neuronal subtypes in cloned populations of stem-like cells.

BACKGROUND: The central nervous tissue contains diverse subtypes of neurons with characteristic morphological and physiological features and different neurotransmitter phenotypes. The generation of neurons with defined neurotransmitter phenotypes seems to be governed by factors differently expressed along the anterior-posterior and dorsal-ventral body axes. The mechanisms of the cell-type determination, however, are poorly understood. Selected neuronal phenotypes had been generated from embryonic stem (ES) cells, but similar results were not obtained on more restricted neural stem cells, presumably due to the lack of homogeneous neural stem cell populations as a starting material. RESULTS: In the presented work, the establishment of different neurotransmitter phenotypes was investigated in the course of in vitro induced neural differentiation of a one-cell derived neuroectodermal cell line, in conjunction with the activation of various region-specific genes. For comparison, similar studies were carried out on the R1 embryonic stem (ES) and P19 multipotent embryonic carcinoma (EC) cells. In response to a short treatment with all-trans retinoic acid, all cell lines gave rise to neurons and astrocytes. Non-induced neural stem cells and self-renewing cells persisting in differentiated cultures, expressed "stemness genes" along with early embryonic anterior-dorsal positional genes, but did not express the investigated CNS region-specific genes. In differentiating stem-like cell populations, on the other hand, different region-specific genes, those expressed in non-overlapping regions along the body axes were activated. The potential for diverse regional specifications was induced in parallel with the initiation of neural tissue-type differentiation. In accordance with the wide regional specification potential, neurons with different neurotransmitter phenotypes developed. Mechanisms inherent to one-cell derived neural stem cell populations were sufficient to establish glutamatergic and GABAergic neuronal phenotypes but failed to manifest cathecolaminergic neurons. CONCLUSION: The data indicate that genes involved in positional determination are activated along with pro-neuronal genes in conditions excluding any outside influences. Interactions among progenies of one cell derived neural stem cells are sufficient for the activation of diverse region specific genes and initiate different routes of neuronal specification.

Differentiation-Dependent Motility-Responses of Developing Neural Progenitors to Optogenetic Stimulation.

During neural tissue genesis, neural stem/progenitor cells are exposed to bioelectric stimuli well before synaptogenesis and neural circuit formation. Fluctuations in the electrochemical potential in the vicinity of developing cells influence the genesis, migration and maturation of neuronal precursors. The complexity of the in vivo environment and the coexistence of various progenitor populations hinder the understanding of the significance of ionic/bioelectric stimuli in the early phases of neuronal differentiation. Using optogenetic stimulation, we investigated the in vitro motility responses of radial glia-like neural stem/progenitor populations to ionic stimuli. Radial glia-like neural stem cells were isolated from CAGloxpStoploxpChR2(H134)-eYFP transgenic mouse embryos. After transfection with Cre-recombinase, ChR2(channelrhodopsin-2)-expressing and non-expressing cells were separated by eYFP fluorescence. Expression of light-gated ion channels were checked by patch clamp and fluorescence intensity assays. Neurogenesis by ChR2-expressing and non-expressing cells was induced by withdrawal of EGF from the medium. Cells in different (stem cell, migrating progenitor and maturing precursor) stages of development were illuminated with laser light (λ = 488 nm; 1.3 mW/mm2; 300 ms) in every 5 min for 12 h. The displacement of the cells was analyzed on images taken at the end of each light pulse. Results demonstrated that the migratory activity decreased with the advancement of neuronal differentiation regardless of stimulation. Light-sensitive cells, however, responded on a differentiation-dependent way. In non-differentiated ChR2-expressing stem cell populations, the motility did not change significantly in response to light-stimulation. The displacement activity of migrating progenitors was enhanced, while the motility of differentiating neuronal precursors was markedly reduced by illumination.

Fibronectin quantification without antibodies: a bioassay for the detection of the gelatin-captured macromolecule.

We have developed a new cell-adhesion-bioassay (CAA) for the quantitative determination of fibronectin in biological fluids. The assay is based on two particular properties of fibronectin: it specifically binds to gelatin with high affinity and simultaneously it can anchor to different surface molecules of a cell. First fibronectin, derived from very different biological fluids, is purified in situ, within the wells of the microtiter plates applied for the assay, using solid surface bound gelatin. After capturing the macromolecule, it is quantified based on its cell adhesive properties. In contrast to ELISA the CAA does not require specific antibodies, and as the Jurkat cells used as indicator cells, seem to recognize fibronectin from different species equally; species specificity of the reagent plays smaller, perhaps negligible, role in the determination of the amount of the macromolecule. The CAA method may not replace fibronectin specific ELISA-s, but using its principle, improved applications, for example a capture EIA for determining fibronectin can easily be envisioned and CAA may serve as a viable alternative for EIA-s when specific antibodies are not available or when relative measurement of not only the soluble but cell surface associated fibronectin is necessary.

Differentiation-Dependent Energy Production and Metabolite Utilization: A Comparative Study on Neural Stem Cells, Neurons, and Astrocytes.

While it is evident that the metabolic machinery of stem cells should be fairly different from that of differentiated neurons, the basic energy production pathways in neural stem cells (NSCs) or in neurons are far from clear. Using the model of in vitro neuron production by NE-4C NSCs, this study focused on the metabolic changes taking place during the in vitro neuronal differentiation. O2 consumption, H(+) production, and metabolic responses to single metabolites were measured in cultures of NSCs and in their neuronal derivatives, as well as in primary neuronal and astroglial cultures. In metabolite-free solutions, NSCs consumed little O2 and displayed a higher level of mitochondrial proton leak than neurons. In stem cells, glycolysis was the main source of energy for the survival of a 2.5-h period of metabolite deprivation. In contrast, stem cell-derived or primary neurons sustained a high-level oxidative phosphorylation during metabolite deprivation, indicating the consumption of own cellular material for energy production. The stem cells increased O2 consumption and mitochondrial ATP production in response to single metabolites (with the exception of glucose), showing rapid adaptation of the metabolic machinery to the available resources. In contrast, single metabolites did not increase the O2 consumption of neurons or astrocytes. In "starving" neurons, neither lactate nor pyruvate was utilized for mitochondrial ATP production. Gene expression studies also suggested that aerobic glycolysis and rapid metabolic adaptation characterize the NE-4C NSCs, while autophagy and alternative glucose utilization play important roles in the metabolism of stem cell-derived neurons.

Studies on the use of NE-4C embryonic neuroectodermal stem cells for targeting brain tumour.

Neural stem cells were suggested to migrate to and invade intracranial gliomas. In the presented studies, interactions of NE-4C embryonic neural stem cells were investigated with C6 and Gl261, LL and U87, glioblastoma cells or with primary astrocytes. Glioma-derived humoral factors did not influence the proliferation of stem cells. NE-4C-derived humoral factors did not alter the proliferation of Gl261 and U87 cells, but increased the mitotic activity of C6 cells and that of astrocytes. In chimera-aggregates, all types of glioma cells co-aggregated with astrocytes, but most of them segregated from stem cells. Complete intercalation of stem and tumour cells was detected only in chimera-aggregates of Gl261 glioma and NE-4C cells. If mixed suspensions of NE-4C and Gl261 cells were injected into the brain, stem cells survived and grew inside the tumour mass. NE-4C stem cells, however, did not migrate towards the tumour, if implanted near to Gl261 tumours established in the adult mouse forebrain. The observations indicate that not all types of stem cells could be used for targeting all sorts of brain tumours.

Uptake and bio-reactivity of polystyrene nanoparticles is affected by surface modifications, ageing and LPS adsorption: in vitro studies on neural tissue cells.

Because of their capacity of crossing an intact blood-brain barrier and reaching the brain through an injured barrier or via the nasal epithelium, nanoparticles have been considered as vehicles to deliver drugs and as contrast materials for brain imaging. The potential neurotoxicity of nanoparticles, however, is not fully explored. Using particles with a biologically inert polystyrene core material, we investigated the role of the chemical composition of particle surfaces in the in vitro interaction with different neural cell types. PS NPs within a size-range of 45-70 nm influenced the metabolic activity of cells depending on the cell-type, but caused toxicity only at extremely high particle concentrations. Neurons did not internalize particles, while microglial cells ingested a large amount of carboxylated but almost no PEGylated NPs. PEGylation reduced the protein adsorption, toxicity and cellular uptake of NPs. After storage (shelf-life >6 months), the toxicity and cellular uptake of NPs increased. The altered biological activity of "aged" NPs was due to particle aggregation and due to the adsorption of bioactive compounds on NP surfaces. Aggregation by increasing the size and sedimentation velocity of NPs results in increased cell-targeted NP doses. The ready endotoxin adsorption which cannot be prevented by PEG coating, can render the particles toxic. The age-dependent changes in otherwise harmless NPs could be the important sources for variability in the effects of NPs, and could explain the contradictory data obtained with "identical" NPs.

Stage-dependent effects of cell-to-cell connections on in vitro induced neurogenesis.

NE-4C, a p53-deficient, immortalized neuroectodermal progenitor cell line, was used to investigate the role and importance of cellular interactions in neural commitment and differentiation. NE-4C cells give rise to neurons and astrocytes in the presence of all-trans retinoic acid, if they can establish intercellular contacts. Aggregation per se, however, was insufficient to induce large-scale neuron formation. In the absence of RA, the majority of the aggregated cells died. For neuron formation, therefore, concerted actions of RA and cellular interaction were needed. Electron microscopic and electrophysiological studies revealed that gap junctions were formed between the cells. Persistent blockage of communication via gap junctions with gap junction blockers, however, had no effects on neuron formation. If cell-to-cell connections were disrupted on the fourth day after induction, the rate of neuron production increased significantly. The contact interactions formed between already committed progenitor cells seemed to hinder the formation of novel neurons. The process resembled the phenomenon called "lateral inhibition" first observed in the course of neurogenesis in Drosophila. Our results indicate that NE-4C cells provide a useful model system to investigate the role of contact communication during some early steps of neurogenesis.

Atomic force microscopy of height fluctuations of fibroblast cells.

We investigated the nanometer scale height fluctuations of 3T3 fibroblast cells with the atomic force microscope under physiological conditions. A correlation between these fluctuations and lateral cellular motility can be observed. Fluctuations measured on leading edges appear to be predominantly related to actin polymerization-depolymerization processes. We found fast (5 Hz) pulsatory behavior with 1-2 nm amplitude on a cell with low motility showing emphasized structure of stress fibers. Myosin driven contractions of stress fibers are thought to induce this pulsation.

Regulation of mouse microglia activation and effector functions by bone marrow-derived mesenchymal stem cells.

Mesenchymal stems or stromal cells (MSCs) are rare multipotent cells with potent regenerative and immunomodulatory properties. Microglial cells (MGs) are specialized tissue macrophages of the central nervous system (CNS) that continuously survey their environment with highly motile extensions. Recently, several studies have shown that MSCs are capable of reprogramming microglia into an "M2-like" phenotype characterized by increased phagocytic activity and upregulated expression of anti-inflammatory mediators in vitro. However, the precise polarization states of microglia in the presence of MSCs under physiological or under inflammatory conditions remain largely unknown. In this study, we found that MSCs induce a mixed microglia phenotype defined as Arg1-high, CD86-high, CD206-high, IL-10-high, PGE2-high, MCP-1/CCL2-high, IL-1ß-moderate, NALP-3-low, and TNF-α-low cells. These MSC-elicited MGs have high phagocytic activity and antigen-presenting ability. Lipopolysaccharide is able to shape this microglia phenotype quantitatively, but not qualitatively in the presence of MSCs. This unique polarization state resembles a novel regulatory microglia phenotype, which might contribute to the resolution of inflammation and to tissue repair in the CNS.

Interaction of differently functionalized fluorescent silica nanoparticles with neural stem- and tissue-type cells.

Engineered amorphous silica nanoparticles (SiO2 NPs), due to simple and low cost production, are increasingly used in commercial products and produced on an industrial scale. Despite the potential benefits, there is a concern that exposure to certain types of SiO2 NPs may lead to adverse health effects. As some NPs can cross the blood--brain barrier and may, in addition, reach the central nervous system through the nasal epithelium, this study addresses the responses of different neural tissue-type cells including neural stem cells, neurons, astrocytes and microglia cells to increasing doses of 50 nm fluorescent core/shell SiO2 NPs with different [-NH2, -SH and polyvinylpyrrolidone (PVP)] surface chemistry. The SiO2 NPs are characterized using a variety of physicochemical methods. Assays of cytotoxicity and cellular metabolism indicates that SiO2 NPs cause cell death only at high particle doses, except PVP-coated SiO2 NPs which do not harm cells even at very high concentrations. All SiO2 NPs, except those coated with PVP, form large agglomerates in physiological solutions and adsorb a variety of proteins. Except PVP-NPs, all SiO2 NPs adhere strongly to cell surfaces, but internalization differs depending on neural cell type. Neural stem cells and astrocytes internalize plain SiO2, SiO2-NH2 and SiO2-SH NPs, while neurons do not take up any NPs. The data indicates that the PVP coat, by lowering the particle-biomolecular component interactions, reduces the biological effects of SiO2 NPs on the investigated neural cells.

Dynamics of re-constitution of the human nuclear proteome after cell division is regulated by NLS-adjacent phosphorylation.

Phosphorylation by the cyclin-dependent kinase 1 (Cdk1) adjacent to nuclear localization signals (NLSs) is an important mechanism of regulation of nucleocytoplasmic transport. However, no systematic survey has yet been performed in human cells to analyze this regulatory process, and the corresponding cell-cycle dynamics have not yet been investigated. Here, we focused on the human proteome and found that numerous proteins, previously not identified in this context, are associated with Cdk1-dependent phosphorylation sites adjacent to their NLSs. Interestingly, these proteins are involved in key regulatory events of DNA repair, epigenetics, or RNA editing and splicing. This finding indicates that cell-cycle dependent events of genome editing and gene expression profiling may be controlled by nucleocytoplasmic trafficking. For in-depth investigations, we selected a number of these proteins and analyzed how point mutations, expected to modify the phosphorylation ability of the NLS segments, perturb nucleocytoplasmic localization. In each case, we found that mutations mimicking hyper-phosphorylation abolish nuclear import processes. To understand the mechanism underlying these phenomena, we performed a video microscopy-based kinetic analysis to obtain information on cell-cycle dynamics on a model protein, dUTPase. We show that the NLS-adjacent phosphorylation by Cdk1 of human dUTPase, an enzyme essential for genomic integrity, results in dynamic cell cycle-dependent distribution of the protein. Non-phosphorylatable mutants have drastically altered protein re-import characteristics into the nucleus during the G1 phase. Our results suggest a dynamic Cdk1-driven mechanism of regulation of the nuclear proteome composition during the cell cycle.

Retinoid machinery in distinct neural stem cell populations with different retinoid responsiveness.

Retinoic acid (RA) is present at sites of neurogenesis in both the embryonic and adult brain. While it is widely accepted that RA signaling is involved in the regulation of neural stem cell differentiation, little is known about vitamin A utilization and biosynthesis of active retinoids in the neurogenic niches, or about the details of retinoid metabolism in neural stem cells and differentiating progenies. Here we provide data on retinoid responsiveness and RA production of distinct neural stem cell/neural progenitor populations. In addition, we demonstrate differentiation-related changes in the expression of genes encoding proteins of the retinoid machinery, including components responsible for uptake (Stra6) and storage (Lrat) of vitamin A, transport of retinoids (Rbp4, CrbpI, CrabpI-II), synthesis (Rdh10, Raldh1-4), degradation of RA (Cyp26a1-c1) and RA signaling (Rarα,ß,γ, Rxrα,ß,γ). We show that both early embryonic neuroectodermal (NE-4C) stem cells and late embryonic or adult derived radial glia like progenitors (RGl cells) are capable to produce bioactive retinoids but respond differently to retinoid signals. However, while neuronal differentiation of RGl cells can not be induced by RA, neuron formation by NE-4C cells is initiated by both RA and RA-precursors (retinol or retinyl acetate). The data indicate that endogenous RA production, at least in some neural stem cell populations, may result in autocrine regulation of neuronal differentiation.

Optical waveguide lightmode spectroscopic techniques for investigating membrane-bound ion channel activities.

Optical waveguide lightmode spectroscopic (OWLS) techniques were probed for monitoring ion permeation through channels incorporated into artificial lipid environment. A novel sensor set-up was developed by depositing liposomes or cell-derived membrane fragments onto hydrophilic polytetrafluoroethylene (PTFE) membrane. The fibrous material of PTFE membrane could entrap lipoid vesicles and the water-filled pores provided environment for the hydrophilic domains of lipid-embedded proteins. The sensor surface was kept clean from the lipid holder PTFE membrane by a water- and ion-permeable polyethylene terephthalate (PET) mesh. The sensor set-up was tested with egg yolk lecithin liposomes containing gramicidin ion channels and with cell-derived membrane fragments enriched in GABA-gated anion channels. The method allowed monitoring the move of Na(+) and organic cations through gramicidin channels and detecting the Cl(-)-channel functions of the (α5ß2γ2) GABAA receptor in the presence or absence of GABA and the competitive GABA-blocker bicuculline.

Isolation of radial glia-like neural stem cells from fetal and adult mouse forebrain via selective adhesion to a novel adhesive peptide-conjugate.

Preferential adhesion of neural stem cells to surfaces covered with a novel synthetic adhesive polypeptide (AK-cyclo[RGDfC]) provided a unique, rapid procedure for isolating radial glia-like cells from both fetal and adult rodent brain. Radial glia-like (RGl) neural stem/progenitor cells grew readily on the peptide-covered surfaces under serum-free culture conditions in the presence of EGF as the only growth factor supplement. Proliferating cells derived either from fetal (E 14.5) forebrain or from different regions of the adult brain maintained several radial glia-specific features including nestin, RC2 immunoreactivity and Pax6, Sox2, Blbp, Glast gene expression. Proliferating RGl cells were obtained also from non-neurogenic zones including the parenchyma of the adult cerebral cortex and dorsal midbrain. Continuous proliferation allowed isolating one-cell derived clones of radial glia-like cells. All clones generated neurons, astrocytes and oligodendrocytes under appropriate inducing conditions. Electrophysiological characterization indicated that passive conductance with large delayed rectifying potassium current might be a uniform feature of non-induced radial glia-like cells. Upon induction, all clones gave rise to GABAergic neurons. Significant differences were found, however, among the clones in the generation of glutamatergic and cathecolamine-synthesizing neurons and in the production of oligodendrocytes.

Survival and differentiation of neuroectodermal cells with stem cell properties at different oxygen levels.

Freeze-lesioned regions of the forebrain cortex provide adequate environment for growth of non-differentiated neural progenitors, but do not support their neuron formation. Reduced oxygen supply, among numerous factors, was suspected to impair neuronal cell fate commitment. In the present study, proliferation and differentiation of neural stem/progenitor cells were investigated at different oxygen levels both in vitro and in vivo. Low (1% atmospheric) oxygen supply did not affect the in vitro viability and proliferation of stem cells or the transcription of "stemness" genes but impaired the viability of committed neuronal progenitors and the expression of proneural and neuronal genes. Consequently, the rate of in vitro neuron formation was markedly reduced under hypoxic conditions. In vivo, neural stem/progenitor cells survived and proliferated in freeze-lesioned adult mouse forebrains, but did not develop into neurons. Hypoperfusion-caused hypoxia in lesioned cortices was partially corrected by hyperbaric oxygen treatment (HBOT). HBOT, while reduced the rate of cell proliferation at the lesion site, resulted in sporadic neuron formation from implanted neural stem cells. The data indicate that in hypoxic brain areas, neural stem cells survive and proliferate, but neural tissue-type differentiation can not proceed. Oxygenation renders the damaged brain areas more permissive for tissue-type differentiation and may help the integration of neural stem/progenitor cells.

Lack of neuroprotection in the absence of P2X7 receptors in toxin-induced animal models of Parkinson's disease.

BACKGROUND: Previous studies indicate a role of P2X7 receptors in processes that lead to neuronal death. The main objective of our study was to examine whether genetic deletion or pharmacological blockade of P2X7 receptors influenced dopaminergic cell death in various models of Parkinson's disease (PD). RESULTS: mRNA encoding P2X7 and P2X4 receptors was up-regulated after treatment of PC12 cells with 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP). P2X7 antagonists protected against MPTP and rotenone induced toxicity in the LDH assay, but failed to protect after rotenone treatment in the MTT assay in PC12 cells and in primary midbrain culture. In vivo MPTP and in vitro rotenone pretreatments increased the mRNA expression of P2X7 receptors in the striatum and substantia nigra of wild-type mice. Basal mRNA expression of P2X4 receptors was higher in P2X7 knockout mice and was further up-regulated by MPTP treatment. Genetic deletion or pharmacological inhibition of P2X7 receptors did not change survival rate or depletion of striatal endogenous dopamine (DA) content after in vivo MPTP or in vitro rotenone treatment. However, depletion of norepinephrine was significant after MPTP treatment only in P2X7 knockout mice. The basal ATP content was higher in the substantia nigra of wild-type mice, but the ADP level was lower. Rotenone treatment elicited a similar reduction in ATP content in the substantia nigra of both genotypes, whereas reduction of ATP was more pronounced after rotenone treatment in striatal slices of P2X7 deficient mice. Although the endogenous amino acid content remained unchanged, the level of the endocannabinoid, 2-AG, was elevated by rotenone in the striatum of wild-type mice, an effect that was absent in mice deficient in P2X7 receptors. CONCLUSIONS: We conclude that P2X7 receptor deficiency or inhibition does not support the survival of dopaminergic neurons in an in vivo or in vitro models of PD.

Effects of a combretastatin A4 analogous chalcone and its Pt-complex on cancer cells: A comparative study of uptake, cell cycle and damage to cellular compartments.

The combretastatin A4 analogous chalcone (2E)-3-(3-hydroxy-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one 1 and its dichloridoplatinum(II) (6-aminomethylnicotinate) complex 2 were previously found to be highly active against a variety of cancer cell lines while differing in their apoptosis induction and long-term regrowth retardation (Schobert et al. [1]). Further differences were identified now. The cellular uptake of complex 2, like that of oxaliplatin, occurred mainly via organic cation transporters (OCT-1/2; ∼32%) and copper transporter related proteins (Ctr1; ∼24%), whereas that of chalcone 1 was dependent on endocytosis (∼80%). Complex 2 was more tumour-specific than 1 concerning neural cells. This was apparent from the ratios of IC(50)(48h) values against primary astrocytes versus human glioma cells U87 (>7000 for complex 2; 55 for compound 1). In tubulin-rich neurons and 518A2 melanoma cells complex 2 disrupted microtubules and actin filaments. Cancer cells treated with 2 could repair the cytoskeletal damage but ceased to proliferate and perished. Complex 2 was particularly cytotoxic against P-gp-rich cells. It acted as a substrate for ABC-transporters of types BCRP, MRP3, and MRP1 and so was less active against the corresponding cancer cell lines. Complex 2 arrested the cell cycle of the melanoma cells in G(1) and G(2)/M phases. A fragmentation of their Golgi apparatus was observed by TEM for incubation with complex 2 but not with 1. In conclusion, unlike chalcone 1, its platinum complex 2 is highly cell line specific, is taken up via cell-controlled transporters and induces apoptosis by triggering multiple targets.