A fully automated high-throughput workflow for 3D-based chemical screening in human midbrain organoids

Three-dimensional (3D) culture systems have fueled hopes to bring about the next generation of more physiologically relevant high-throughput screens (HTS). However, current protocols yield either complex but highly heterogeneous aggregates ('organoids') or 3D structures with less physiological relevance ('spheroids'). Here, we present a scalable, HTS-compatible workflow for the automated generation, maintenance, and optical analysis of human midbrain organoids in standard 96-well-plates. The resulting organoids possess a highly homogeneous morphology, size, global gene expression, cellular composition, and structure. They present significant features of the human midbrain and display spontaneous aggregate-wide synchronized neural activity. By automating the entire workflow from generation to analysis, we enhance the intra- and inter-batch reproducibility as demonstrated via RNA sequencing and quantitative whole mount high-content imaging. This allows assessing drug effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow

Bacteria tracking by in vivo magnetic resonance imaging

Background: Different non-invasive real-time imaging techniques have been developed over the last decades to study bacterial pathogenic mechanisms in mouse models by following infections over a time course. In vivo investigations of bacterial infections previously relied mostly on bioluminescence imaging (BLI), which is able to localize metabolically active bacteria, but provides no data on the status of the involved organs in the infected host organism. In this study we established an in vivo imaging platform by magnetic resonance imaging (MRI) for tracking bacteria in mouse models of infection to study infection biology of clinically relevant bacteria. Results: We have developed a method to label Gram-positive and Gram-negative bacteria with iron oxide nano particles and detected and pursued these with MRI. The key step for successful labeling was to manipulate the bacterial surface charge by producing electro-competent cells enabling charge interactions between the iron particles and the cell wall. Different particle sizes and coatings were tested for their ability to attach to the cell wall and possible labeling mechanisms were elaborated by comparing Gram-positive and -negative bacterial characteristics. With 5-nm citrate-coated particles an iron load of 0.015 ± 0.002 pg Fe/bacterial cell was achieved for Staphylococcus aureus. In both a subcutaneous and a systemic infection model induced by iron-labeled S. aureus bacteria, high resolution MR images allowed for bacterial tracking and provided information on the morphology of organs and the inflammatory response. Conclusion: Labeled with iron oxide particles, in vivo detection of small S. aureus colonies in infection models is feasible by MRI and provides a versatile tool to follow bacterial infections in vivo. The established cell labeling strategy can easily be transferred to other bacterial species and thus provides a conceptual advance in the field of molecular MRI.<br

AP-1/σ1B-adaptin mediates endosomal synaptic vesicle recycling, learning and memory

Synaptic vesicle recycling involves AP-2/clathrin-mediated endocytosis, but it is not known whether the endosomal pathway is also required. Mice deficient in the tissue-specific AP-1–σ1B complex have impaired synaptic vesicle recycling in hippocampal synapses. The ubiquitously expressed AP-1–σ1A complex mediates protein sorting between the trans-Golgi network and early endosomes. Vertebrates express three σ1 subunit isoforms: A, B and C. The expressions of σ1A and σ1B are highest in the brain. Synaptic vesicle reformation in cultured neurons from σ1B-deficient mice is reduced upon stimulation, and large endosomal intermediates accumulate. The σ1B-deficient mice have reduced motor coordination and severely impaired long-term spatial memory. These data reveal a molecular mechanism for a severe human X-chromosome-linked mental retardation

C3G/Rapgef1 Is Required in Multipolar Neurons for the Transition to a Bipolar Morphology during Cortical Development

<div><p>The establishment of a polarized morphology is essential for the development and function of neurons. During the development of the mammalian neocortex, neurons arise in the ventricular zone (VZ) from radial glia cells (RGCs) and leave the VZ to generate the cortical plate (CP). During their migration, newborn neurons first assume a multipolar morphology in the subventricular zone (SVZ) and lower intermediate zone (IZ). Subsequently, they undergo a multi-to-bipolar (MTB) transition to become bipolar in the upper IZ by developing a leading process and a trailing axon. The small GTPases Rap1A and Rap1B act as master regulators of neural cell polarity in the developing mouse neocortex. They are required for maintaining the polarity of RGCs and directing the MTB transition of multipolar neurons. Here we show that the Rap1 guanine nucleotide exchange factor (GEF) C3G (encoded by the <i>Rapgef1</i> gene) is a crucial regulator of the MTB transition <i>in vivo</i> by conditionally inactivating the <i>Rapgef1</i> gene in the developing mouse cortex at different time points during neuronal development. Inactivation of C3G results in defects in neuronal migration, axon formation and cortical lamination. Live cell imaging shows that C3G is required in cortical neurons for both the specification of an axon and the initiation of radial migration by forming a leading process.</p></div

Loss of active β1 integrin in the C3G^Emx1-KO cortex.

<p>(A,-C) Coronal sections from heterozygous or homozygous E15 C3G^Emx1-KO cortex were stained with an anti- integrin β1 (VLA, green) and an antibody specific for active form of β1 integrin (9EG7, green) (A). No significant differences were found in the expression of β1 integrin when intensity values were plotted for the control and the C3G^Emx1-KO. (B) The level of active β1 integrin was reduced at the pial surface in the C3G^Emx1-KO cortex (+/-: arrows, -/-: arrowheads). Intensity profiles of 9EG7 immunofluorescence signals (arbitrary units) measured from the VZ (bottom) to the pial surface (top) show a significant reduction in the intensity of 9EG7 signals at the pial surface in the C3G^Emx1-KO mutant cortex only in the MZ (in the last 40 pixel positions that include the glial endfeet) where active β1 integrins are enriched in control sections [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154174#pone.0154174.ref020" target="_blank">20</a>]. The fluorescence intensity values (arbitrary units) for staining with the VLA and 9EG7 antibodies were quantified at each pixel position along the ventricular to pial axis in a rectangular box comprising an area from the VZ to the MZ in sections from 3 different embryos per genotype. The significance of differences was calculated between means at each pixel position (means ± SEM, Student’s t-test to measure the difference in the means, *p ≤ 0.05; ns, not significant). (C) Higher magnification images of the pial surface stained with the above mentioned antibodies show defects in the 9EG7 staining in the C3G^Emx1-KO. Dorsal is to the top. Single confocal planes are shown. Scale bar is 50 μm (A, B) and 20 μm (C).</p

v-SNAREs control exocytosis of vesicles from priming to fusion

SNARE proteins (soluble NSF-attachment protein receptors) are thought to be central components of the exocytotic mechanism in neurosecretory cells, but their precise function remained unclear. Here, we show that each of the vesicle-associated SNARE proteins (v-SNARE) of a chromaffin granule, synaptobrevin II or cellubrevin, is sufficient to support Ca(2+)-dependent exocytosis and to establish a pool of primed, readily releasable vesicles. In the absence of both proteins, secretion is abolished, without affecting biogenesis or docking of granules indicating that v-SNAREs are absolutely required for granule exocytosis. We find that synaptobrevin II and cellubrevin differentially control the pool of readily releasable vesicles and show that the v-SNARE's amino terminus regulates the vesicle's primed state. We demonstrate that dynamics of fusion pore dilation are regulated by v-SNAREs, indicating their action throughout exocytosis from priming to fusion of vesicles

C3G is required for axon formation in the cortex and hippocampus.

<p>(A) Coronal sections from the caudal brain of heterozygous or homozygous C3G^Emx1-KO E17 embryos were stained with Hoechst 33342 (blue, nuclei) and an anti-NFM antibody (red) as a marker for axons. The mutant cortex shows an extensive loss of axons in the cortex (arrowheads) and hippocampus (arrows). A higher magnification is shown on the right (n ≥ 4 embryos from different litters for each genotype). Dorsal is to the top and medial to the right. (B) Neurons from the cortex or hippocampus of heterozygous or homozygous C3G^Emx1-KO embryos were stained at 3 d.i.v. with the Tau-1 (axons, red) and an anti-MAP2 (minor neurites, green) antibody. Unpolarized neurons without an axon are marked by arrowheads. (C, D) The percentage of unpolarized neurons without an axon (0, black), polarized neurons with a single axon (1, gray) and neurons with multiple axons (>1, white) is shown (n = 3 independent experiments, 100 neurons from each group, means ± s.e.m.; *** p≤0.001 compared to control determined by two-way ANOVA with Tukey’s multiple comparison test). Single confocal planes are shown. Scale bars are 100 μm (A) and 20 μm (B).</p

Selective loss of axons in the C3G^Nex-KO hippocampus.

<p>Coronal sections from heterozygous and homozygous C3G^Nex-KO E17 embryos were stained with an anti-NFM antibody (green) and Hoechst 33342 (blue). A marked loss of axons can be seen in the hippocampus (bottom, arrows) but not the cortex (top, arrowheads). A higher magnification of the hippocampus is shown in the rightmost panels. Dorsal is to the top and medial to the right. Single confocal planes are shown. Scale bars are 100 μm. Images are representative for 3 independent experiments with 3 embryos per genotype from different litters.</p

C3G^Emx1-KO shows defects in RGCs at the pial surface.

<p>(A, B) Coronal sections of C3G^Emx1-KO heterozygous or homozygous knockout embryos were stained with an anti-nestin antibody (green (A) or red (B)) and Hoechst 33342 (blue) at E17. (A) Higher magnification images from the pial surface show a continuous arrangement of glial fibers and endfeet in the heterozygous control compared to the disrupted organization of glial fibers and a rupture of basement membrane in the C3G^Emx1-KO cortex (arrowheads). Arrows indicate the disorganized glial fiber network. No defects at the VZ were seen in C3G^Emx1-KO (B). Single confocal planes are shown. (C, D) Coronal sections from the C3G^Emx1-KO cortex were analyzed by electron microscopy at E17. Images of the pial surface show a distinct BM in heterozygous embryos (yellow arrowheads) with a compact arrangement of cells below the BM (C). The BM appears to be ruptured in the C3G^Emx1-KO cortex and the cells protrude outside. The area marked by a box is shown at a higher magnification on the right depicting the BM. The red arrows (magnified panels) mark the continuous BM. The broken BM in the C3G^Emx1-KO pial surface is marked by blue arrowheads. No defects were seen at the VZ and the AJs formed normally (C). (D) The number of AJs per 100 μm of the VZ was quantified and no significant differences were found between the heterozygous and homozygous C3G^Emx1-KO embryos (n = 3 embryos per genotype, means ± s.e.m., ns, not significant, Student’s t-test). Scale bars are 100μm (A) 50μm (magnified panels in A), 10 μm (B), 500 nm (C, VZ) and 2 μm (C and magnified panels, Pia). Images are representative for 3 independent experiments with 3 embryos per genotype from different litters.</p

C3G is required in multipolar neurons for neuronal polarization.

<p>(A-B) Wild type (WT) or <i>Rapgef1</i>^flox/flox (C3G f/f) E13.5 brains were transfected by <i>ex vivo</i> electroporation with (A) <i>pEF-Cre</i>, <i>pEF-LPL-LynN-EGFP</i> and <i>pTα-LPL-H2B-RFP</i> or (B) <i>pTα-Cre</i>, <i>pTα-LPL-LynN-EGFP</i> and <i>pTα-LPL-H2B-RFP</i> to specifically inactivate the conditional alleles and label early post-mitotic neurons. Imaging was performed 30h after electroporation. Neurons from WT coronal slices first extend a long trailing process followed by a leading process. Slices from <i>Rapgef1</i>^flox/flox brains showed a significant number of neurons that remained multipolar and did not extend a trailing or a leading process after more than 20 h of imaging. (C, D) The percentage of cells that formed only a trailing axon (unipolar (only axon), red), only a leading process (unipolar (only leading process), yellow), that became bipolar (blue), or remained multipolar (green) after transfection of <i>pEF-Cre</i> (C) or <i>pTα-Cre</i> (D) at the end of the imaging period of 20 h (means ± SEM, ***p ≤ 0.001, *p ≤ 0.05 two-way ANOVA with Tukey’s multiple comparison test; number of bipolar or multipolar neurons from <i>Rapgef1</i>^flox/flox slices compared to control slices; n = 37 (wildtype; EF), n = 29 (<i>Rapgef1</i>^flox/flox; EF) and n = 51 (wildtype; Tα), n = 45 (<i>Rapgef1</i>^flox/flox; Tα) from 3 independent experiments that each included multiple slices from different animals, n indicates the total number of neurons analyzed in all experiments). The VZ is to the bottom and the pial surface to the top. Scale bars are 20 μm.</p