Elena E. Pohl

A feedback loop comprising lin-28 and let-7 controls pre-let-7 maturation during neural stem-cell commitment

miRNA populations, including mammalian homologues of lin-4 (mir-125) and let-7, undergo a marked transition during stem-cell differentiation. Originally identified on the basis of their mutational phenotypes in stem-cell maturation, mir-125 and let-7 are strongly induced during neural differentiation of embryonic stem (ES) cells and embryocarcinoma (EC) cells. We report that embryonic neural stem (NS) cells express let-7 and mir-125, and investigate post-transcriptional mechanisms contributing to the induction of let-7. We demonstrate that the pluripotency factor Lin-28 binds the pre-let-7 RNA and inhibits processing by the Dicer ribonuclease in ES and EC cells. In NS cells, Lin-28 is downregulated by mir-125 and let-7, allowing processing of pre-let-7 to proceed. Suppression of let-7 or mir-125 activity in NS cells led to upregulation of Lin-28 and loss of pre-let-7 processing activity, suggesting that let-7, mir-125 and lin-28 participate in an autoregulatory circuit that controls miRNA processing during NS-cell commitment.

Neuronal Damage in Autoimmune Neuroinflammation Mediated by the Death Ligand TRAIL

Here, we provide evidence for a detrimental role of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in neural death in T cell-induced experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Clinical severity and neuronal apoptosis in brainstem motor areas were substantially reduced upon brain-specific blockade of TRAIL after induction of EAE through adoptive transfer of encephalitogenic T cells. Furthermore, TRAIL-deficient myelin-specific lymphocytes showed reduced encephalitogenicity when transferred to wild-type mice. Conversely, intracerebral delivery of TRAIL to animals with EAE increased clinical deficits, while naive mice were not susceptible to TRAIL. Using organotypic slice cultures as a model for living brain tissue, we found that neurons were susceptible to TRAIL-mediated injury induced by encephalitogenic T cells. Thus, in addition to its known immunoregulatory effects, the death ligand TRAIL contributes to neural damage in the inflamed brain.

In vivo detection limits of magnetically labeled embryonic stem cells in the rat brain using high-field (17.6 T) magnetic resonance imaging.

Stem cell transplantation is a promising therapeutic approach for several neurological disorders. However, it has yet to fulfill its high expectations, partially due to the lack of a reliable noninvasive method for monitoring the biodistribution of the grafted stem cells in vivo. We have used high-resolution magnetic resonance imaging (MRI) at 17.6 T, combined with efficient magnetic labeling of the stem cells with iron oxide nanoparticles, in order to assess the in vivo detection limit in small animal models. Injection of different concentrations of magnetically labeled stem cells in gel phantoms led to significant reductions in image intensity from small cellular clusters of less than 10 cells. To determine the detection limit in vivo, various numbers of both labeled and unlabeled cells were injected stereotactically into the striatum of rats. Significant hypointense signal changes were observed for 100 labeled cells. After injection of approximately 20 labeled cells, signal reduction at the injection site was observed but could not be assigned unambiguously to the cells. Our results show that high-field MRI allows tracking of a minimal number of cells in vivo, well below the number used in previous studies, opening the possibility of gaining new insights into cell migration and differentiation.

Origin of membrane dipole potential: Contribution of the phospholipid fatty acid chains

The large intrinsic membrane dipole potential, phi(d), is important for protein insertion and functioning as well as for ion transport across natural and model membranes. However, the origin of phi(d) is controversial. From experiments carried out with lipid monolayers, a significant dependence on the fatty acid chain length is suggested, whereas in experiments with lipid bilayers, the contribution of additional -CH(2)-groups seems negligibly small compared with that of the phospholipid carbonyl groups and lipid-bound water molecules. To compare the impact of the -CH(2)-groups of dipalmitoylphosphatidylcholine (DPPC) near and far from the glycerol backbone, we have varied the structure of DPPC by incorporation of sulfur atoms in place of methylene groups in different positions of the fatty acid chain. The phi(d) of symmetric lipid bilayers containing one heteroatom was obtained from the charge relaxation of oppositely charged hydrophobic ions. We have found that the substitution for a S-atom of a -CH(2)-group decreases phi(d). The effect (deltaphi(d) = -22.6 mV) is most pronounced for S-atoms near the lipid head group while a S-atom substitution in the C(13)- or C(14)-position of the hydrocarbon chain does not effect the bilayer dipole potential. Most probably deltaphi(d) does not originate from an altered dipole potential of the acyl chain containing an heteroatom but is mediated by the disruption of chain packing, leading to a decreased density of lipid dipoles in the membrane.

Polyunsaturated fatty acids activate human uncoupling proteins 1 and 2 in planar lipid bilayers

Uncoupling proteins 1 (UCP1) and 2 (UCP2) belong to the family of mitochondrial anion transporters and share 59% sequence identity with each other. Whereas UCP1 was shown to be responsible for the rapid production of heat in brown adipose tissue, the primary function and transport properties of ubiquitously expressed UCP2 are controversially discussed. Here, for the first time, the activation pattern of the recombinant human UCP2 in comparison to the recom‐binant human UCP1 are studied using a well‐defined system of planar lipid bilayers. It is shown that despite apparently different physiological functions, hUCP2 exhibited its protonophoric function similar to hUCP1—exclusively in the presence of long‐chain fatty acids (FA). The calculated hUCP2 transport rate of 4.5 s−1 is the same order of magnitude, as shown previously for UCP1. It leads to the conclusion that the differences in the activity of both proteins in living mitochondria are based exclusively on their different expression level. Both proteins are activated much more effectively by polyunsaturated than by saturated FA. The proton and total membrane conductances increased in the range palmitic < oleic < eicosatrie‐noic < linoleic < retinoic < arachidonic acids. The higher uncoupling protein (UCP)—dependent conductance in the presence of polyunsaturated FA is explained on the basis of the FA cycling hypothesis.—Beck, V., Jabůrek, M., Demina, T., Rupprecht, A., Porter, R. K., Ježek, P., Pohl, E. E. Polyunsaturated fatty acids activate human uncoupling proteins 1 and 2 in planar lipid bilayers. FASEB J. 21, 1137–1144 (2007)

CNS-irrelevant T-cells enter the brain, cause blood–brain barrier disruption but no glial pathology

Invasion of autoreactive T‐cells and alterations of the blood–brain barrier (BBB) represent early pathological manifestations of multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). Non‐CNS‐specific T‐cells are also capable of entering the CNS. However, studies investigating the spatial pattern of BBB alterations as well as the exact localization and neuropathological consequences of transferred non‐CNS‐specific cells have been thus far lacking. Here, we used magnetic resonance imaging and multiphoton microscopy, as well as histochemical and high‐precision unbiased stereological analyses to compare T‐cell transmigration, localization, persistence, relation to BBB disruption and subsequent effects on CNS tissue in a model of T‐cell transfer of ovalbumin (OVA)‐ and proteolipid protein (PLP)‐specific T‐cells. BBB alterations were present in both EAE‐mice and mice transferred with OVA‐specific T‐cells. In the latter case, BBB alterations were less pronounced, but the pattern of initial cell migration into the CNS was similar for both PLP‐ and OVA‐specific cells [mean (SEM), 95u2003×u2003103 (7.6u2003×u2003103) and 88u2003×u2003103 (18u2003×u2003103), respectively]. Increased microglial cell density, astrogliosis and demyelination were, however, observed exclusively in the brain of EAE‐mice. While mice transferred with non‐neural‐specific cells showed similar levels of rhodamine‐dextran extravasation in susceptible brain regions, EAE‐mice presented huge BBB disruption in brainstem and moderate leakage in cerebellum. This suggests that antigen specificity and not the absolute number of infiltrating cells determine the magnitude of BBB disruption and glial pathology.

Comparative analysis of uncoupling protein 4 distribution in various tissues under physiological conditions and during development.

UCP4 is a member of the mitochondrial uncoupling protein subfamily and one of the three UCPs (UCP2, UCP4, UCP5), associated with the nervous system. Its putative functions include thermogenesis, attenuation of reactive oxidative species (ROS), regulation of mitochondrial calcium concentration and involvement in cell differentiation and apoptosis. Here we investigate UCP4s subcellular, cellular and tissue distribution, using an antibody designed specially for this study, and discuss the findings in terms of the proteins possible functions. Western blot and immunohistochemistry data confirmed that UCP4 is expressed predominantly in the central nervous system (CNS), as previously shown at mRNA level. No protein was found in heart, spleen, stomach, intestine, lung, thymus, muscles, adrenal gland, testis and liver. The reports revealing UCP4 mRNA in kidney and white adipose tissue were not confirmed at protein level. The amount of UCP4 varies in the mitochondria of different brain regions, with the highest protein content found in cortex. We show that UCP4 is present in fetal murine brain tissue as early as embryonic days 12-14 (E12-E14), which coincides with the beginning of neuronal differentiation. The UCP4 content in mitochondria decreases as the age of mice increases. UCP4 preferential expression in neurons and its developmental expression pattern under physiological conditions may indicate a specific protein function, e.g. in neuronal cell differentiation.

Permeation of phloretin across bilayer lipid membranes monitored by dipole potential and microelectrode measurements

The transmembrane diffusion of phloretin across planar bilayer lipid membranes is studied under steady-state conditions. Diffusion restrictions and adsorption related effects are measured independently. The adsorption of aligned phloretin dipoles generates a change in the intrinsic dipole potential difference between the inner and outer leaflets of the lipid bilayer. It is monitored by capacitive current measurements carried out with a direct current (dc) bias. The variation of the intramembrane electric field indicates a saturation of the binding sites at the membrane interface. In contrast, pH profile measurements undertaken in the immediate membrane vicinity show a constant membrane permeability. If phloretin binding and transmembrane diffusion are treated as two competitive events rather than subsequent steps in the transport queue the contradictory results become explainable. A mathematical model is developed where it is assumed that diffusing phloretin molecules are randomly oriented, i.e., that they do not contribute to the intrinsic membrane potential. Only the dipoles adsorbing onto the membrane are oriented. Based on these theory the membrane permeability is calculated from the capacitive current data. It is found to agree very well with the permeability deduced from the microelectrode measurements.

The neural EGF family member CALEB/NGC mediates dendritic tree and spine complexity.

The development of dendritic arborizations and spines is essential for neuronal information processing, and abnormal dendritic structures and/or alterations in spine morphology are consistent features of neurons in patients with mental retardation. We identify the neural EGF family member CALEB/NGC as a critical mediator of dendritic tree complexity and spine formation. Overexpression of CALEB/NGC enhances dendritic branching and increases the complexity of dendritic spines and filopodia. Genetic and functional inactivation of CALEB/NGC impairs dendritic arborization and spine formation. Genetic manipulations of individual neurons in an otherwise unaffected microenvironment in the intact mouse cortex by in utero electroporation confirm these results. The EGF‐like domain of CALEB/NGC drives both dendritic branching and spine morphogenesis. The phosphatidylinositide 3‐kinase (PI3K)‐Akt‐mammalian target of rapamycin (mTOR) signaling pathway and protein kinase C (PKC) are important for CALEB/NGC‐induced stimulation of dendritic branching. In contrast, CALEB/NGC‐induced spine morphogenesis is independent of PI3K but depends on PKC. Thus, our findings reveal a novel switch of specificity in signaling leading to neuronal process differentiation in consecutive developmental events.

Neural differentiation of mouse embryonic stem cells as a tool to assess developmental neurotoxicity in vitro.

Mouse embryonic stem cells (mESCs) represent an attractive cellular system for in vitro studies in developmental biology as well as toxicology because of their potential to differentiate into all fetal cell lineages. The present study aims to establish an in vitro system for developmental neurotoxicity testing employing mESCs. We developed a robust and reproducible protocol for fast and efficient differentiation of the mESC line D3 into neural cells, optimized with regard to chemical testing. Morphological examination and immunocytochemical staining confirmed the presence of different neural cell types, including neural progenitors, neurons, astrocytes, oligodendrocytes, and radial glial cells. Neurons derived from D3 cells expressed the synaptic proteins PSD95 and synaptophysin, and the neurotransmitters serotonin and γ-aminobutyric acid. Calcium ion imaging revealed the presence of functionally active glutamate and dopamine receptors. In addition, flow cytometry analysis of the neuron-specific marker protein MAP2 on day 12 after induction of differentiation demonstrated a concentration dependent effect of the neurodevelopmental toxicants methylmercury chloride, chlorpyrifos, and lead acetate on neuronal differentiation. The current study shows that D3 mESCs differentiate efficiently into neural cells involving a neurosphere-like state and that this system is suitable to detect adverse effects of neurodevelopmental toxicants. Therefore, we propose that the protocol for differentiation of mESCs into neural cells described here could constitute one component of an in vitro testing strategy for developmental neurotoxicity.