Stem cell derived neurons : physiology of early differentiation and development

The developing nervous system of vertebrates is largely inaccessible; especially early neuronal differentiation and development are difficult to study. Early events in neurogenesis can be investigated more easily by using murine embryonic stem (mES) cells. Under suitable culture conditions mES cells pass through all phases of neural differentiation through an intermediate stage of embryoid bodies. As wild type or genetically modified ES cells can be grown in unlimited quantities, their differentiation into neurons represents an attractive model for studying neurodevelopmental diseases. Functional neuronal maturation can now be followed from immature to mature neurons forming excitatory and inhibitory synaptically connected networks. In my thesis work I used such a culture system to compare the functional development of mES cell derived neurons from different genetic backgrounds. I spent a lot of time establishing experimental conditions to study these cells using electrophysiological techniques. I quantified several key parameters such as resting membrane potential, voltage-gated channel activity, appearance of action potentials, to describe the neuronal differentiation of the cells. In the first days after seeding the precursors possess an immature physiology, with high input resistances, few voltage-sensitive conductance and immature spiking patterns. During subsequent development an increasing amount of voltage gated sodium- and potassium currents appeared, leading to more and more mature spiking patterns. Mature neurons form synaptically connected networks containing excitatory and inhibitory neurons. We investigated the development of spontaneous excitatory- and inhibitory postsynaptic currents (sEPSCs and sIPSCs) in these cultures. We then built upon this knowledge of normal development to study mES cell-derived neurons defective in MeCP2 signaling. This genetic defect causes an important neurodevelopmental defect in mice and in humans. We were able to show that key features of the developmental defect – a disturbed maturation of inhibitory synapses – could be reproduced in our system. For further characterization of inhibitory interneurons we derived stem cells from a GAD67-GFP cell line under two pharmacological conditions as KCl and TTX which change the activity level up or down. This will aid to understand more about the developmental processes of neurons and will help finding functional deficits in neurodevelopmental diseases

Reduced synaptic activity in neuronal networks derived from embryonic stem cells of murine Rett syndrome model

Neurodevelopmental diseases such as the Rett syndrome have received renewed attention, since the mechanisms involved may underlie a broad range of neuropsychiatric disorders such as schizophrenia and autism. In vertebrates early stages in the functional development of neurons and neuronal networks are difficult to study. Embryonic stem cell-derived neurons provide an easily accessible tool to investigate neuronal differentiation and early network formation. We used in vitro cultures of neurons derived from murine embryonic stem cells missing the methyl-CpG-binding protein 2 (MECP2) gene (MeCP2-/y) and from wild type cells of the corresponding background. Cultures were assessed using whole-cell patch-clamp electrophysiology and immunofluorescence. We studied the functional maturation of developing neurons and the activity of the synaptic connections they formed. Neurons exhibited minor differences in the developmental patterns for their intrinsic parameters, such as resting membrane potential and excitability; with the MeCP2-/y cells showing a slightly accelerated development, with shorter action potential half-widths at early stages. There was no difference in the early phase of synapse development, but as the cultures matured, significant deficits became apparent, particularly for inhibitory synaptic activity. MeCP2-/y embryonic stem cell-derived neuronal cultures show clear developmental deficits that match phenotypes observed in slice preparations and thus provide a compelling tool to further investigate the mechanisms behind Rett syndrome pathophysiology

C3 rho-inhibitor for targeted pharmacological manipulation of osteoclast-like cells.

The C3 toxins from Clostridium botulinum (C3bot) and Clostridium limosum (C3lim) as well as C3-derived fusion proteins are selectively taken up into the cytosol of monocytes/macrophages where the C3-catalyzed ADP-ribosylation of Rho results in inhibition of Rho-signalling and characteristic morphological changes. Since the fusion toxin C2IN-C3lim was efficiently taken up into and inhibited proliferation of murine macrophage-like RAW 264.7 cells, its effects on RAW 264.7-derived osteoclasts were investigated. C2IN-C3lim was taken up into differentiated osteoclasts and decreased their resorption activity. In undifferentiated RAW 264.7 cells, C2IN-C3lim-treatment significantly decreased their differentiation into osteoclasts as determined by counting the multi-nucleated, TRAP-positive cells. This inhibitory effect was concentration- and time-dependent and most efficient when C2IN-C3lim was applied in the early stage of osteoclast-formation. A single-dose application of C2IN-C3lim at day 0 and its subsequent removal at day 1 reduced the number of osteoclasts in a comparable manner while C2IN-C3lim-application at later time points did not reduce the number of osteoclasts to a comparable degree. Control experiments with an enzymatically inactive C3 protein revealed that the ADP-ribosylation of Rho was essential for the observed effects. In conclusion, the results indicate that Rho-activity is crucial during the early phase of osteoclast-differentiation. Other bone cell types such as pre-osteoblastic cells were not affected by C2IN-C3lim. Due to their cell-type selective and specific mode of action, C3 proteins and C3-fusions might be valuable tools for targeted pharmacological manipulation of osteoclast formation and activity, which could lead to development of novel therapeutic strategies against osteoclast-associated diseases

A recombinant fusion toxin based on enzymatic inactive C3bot1 selectively targets macrophages.

BACKGROUND: The C3bot1 protein (~23 kDa) from Clostridium botulinum ADP-ribosylates and thereby inactivates Rho. C3bot1 is selectively taken up into the cytosol of monocytes/macrophages but not of other cell types such as epithelial cells or fibroblasts. Most likely, the internalization occurs by a specific endocytotic pathway via acidified endosomes. METHODOLOGY/PRINCIPAL FINDINGS: Here, we tested whether enzymatic inactive C3bot1E174Q serves as a macrophage-selective transport system for delivery of enzymatic active proteins into the cytosol of such cells. Having confirmed that C3bot1E174Q does not induce macrophage activation, we used the actin ADP-ribosylating C2I (∼50 kDa) from Clostridium botulinum as a reporter enzyme for C3bot1E174Q-mediated delivery into macrophages. The recombinant C3bot1E174Q-C2I fusion toxin was cloned and expressed as GST-protein in Escherichia coli. Purified C3bot1E174Q-C2I was recognized by antibodies against C2I and C3bot and showed C2I-specific enzyme activity in vitro. When applied to cultured cells C3bot1E174Q-C2I ADP-ribosylated actin in the cytosol of macrophages including J774A.1 and RAW264.7 cell lines as well as primary cultured human macrophages but not of epithelial cells. Together with confocal fluorescence microscopy experiments, the biochemical data indicate the selective uptake of a recombinant C3-fusion toxin into the cytosol of macrophages. CONCLUSIONS/SIGNIFICANCE: In summary, we demonstrated that C3bot1E174Q can be used as a delivery system for fast, selective and specific transport of enzymes into the cytosol of living macrophages. Therefore, C3-based fusion toxins can represent valuable molecular tools in experimental macrophage pharmacology and cell biology as well as attractive candidates to develop new therapeutic approaches against macrophage-associated diseases

Qu'a-t-on mangé dans la grotte des Jarres ? Une étude des usures et résidus alimentaires des céramiques de la fin du Néolithique

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Application of C2IN-C3lim in the early stage of differentiation inhibits osteoclast-formation.

<p>C2IN-C3lim (0.5 and 2µg/mL) was added from day 0 on (A), from day 1 on (B), from day 2 on (C) or at day 0 only with subsequent medium change on day 1 (D). The number of multi-nucleated (at least three nuclei) and TRAP-positive osteoclasts per well (96 well plate) were determined at day 5. </p

Specific and selective uptake of C2IN-C3lim into macrophage-like RAW 264.7 cells.

<p><i>A</i>. ADP-ribosylation status of Rho in RAW 264.7 cells treated with C2IN-C3lim. Cells were incubated with 0.5 or 2 µg/mL of C2IN-C3lim or left untreated for control. The cells were lysed after 6 and 24 h and equal amounts of lysate proteins incubated with fresh C3bot1 and biotin-labelled NAD⁺. The biotinylated, i.e. ADP-ribosylated Rho is shown. Equal amounts of loaded protein were confirmed by Ponceau S staining of the blotted proteins (not shown). <i>B</i>. C2I alone is not taken up into RAW 264.7 cells. Cells were incubated with C2I (2 µg/mL) alone or with C2I (0.4 µg/mL) + C2IIa (0.8 µg/mL) or left untreated for control. After 6 h cells were lysed and equal amounts of lysate proteins incubated with fresh C2I and biotin-labelled NAD⁺. The biotinylated, i.e. ADP-ribosylated actin is shown. Equal amounts of loaded protein were confirmed by Ponceau S staining of the blotted proteins (not shown). <i>C</i>. C2IN-C3lim is not taken up into pre-osteoblastic MC3T3 cells under comparable conditions. Cells were incubated with C2IN-C3lim (5 µg/mL) or with C2IN-C3lim (1 µg/mL) + C2IIa (2 µg/mL) or left untreated for control. After 6 h the cells were lysed and the ADP-ribosylation status of Rho determined as described in A. The biotinylated, i.e. ADP-ribosylated Rho is shown. Equal amounts of loaded protein were confirmed by Ponceau S staining of the blotted proteins (not shown).</p

Uptake of C2IN-C3lim into differentiating osteoclasts and morhophological changes caused by C2IN-C3lim.

<p>RAW264.7 cells which were grown in the presence of RANKL to induce differentiation to osteoclasts. Cells were left untreated for control (A) or treated with C2IN-C3lim (2 µg/mL) at day 0 and 2 (B and C). At day 5, osteoclasts were stained for actin (red), nuclei (blue) and C3 (green) and cells analyzed by phase contrast microscopy (left row) and fluorescence microscopy. </p

Effect of C2IN-C3lim on osteoclast-formation.

<p>C2IN-C3lim (0.5 and 2µg/mL) was added to RAW264.7 cells from day 0 on and cells were treated with RANKL. For control, cells were treated with RANKL in the absence of C2IN-C3lim. At day 5, osteoclasts were stained for tartrate-resistant acid phosphatase. Osteoclasts formed in the absence (A) and presence of increasing concentration of C2IN-C3lim (B: 0.5 µg/mL; C: 2 µg/mL) are shown.</p