Phenotypic drug discovery requires billions of cells for high-throughput screening (HTS) campaigns. Because up to several million different small molecules will be tested in a single HTS campaign, even small variability within the cell populations for screening could easily invalidate an entire campaign. Neurodegenerative assays are particularly challenging because neurons are post-mitotic and cannot be expanded for implementation in HTS. Therefore, HTS for neuroprotective compounds requires a cell type that is robustly expandable and able to differentiate into all of the neuronal subtypes involved in disease pathogenesis. Here, we report the derivation and propagation using only small molecules of human neural progenitor cells (small molecule neural precursor cells; smNPCs). smNPCs are robust, exhibit immortal expansion, and do not require cumbersome manual culture and selection steps. We demonstrate that smNPCs have the potential to clonally and efficiently differentiate into neural tube lineages, including motor neurons (MNs) and midbrain dopaminergic neurons (mDANs) as well as neural crest lineages, including peripheral neurons and mesenchymal cells. These properties are so far only matched by pluripotent stem cells. Finally, to demonstrate the usefulness of smNPCs we show that mDANs differentiated from smNPCs with LRRK2 G2019S are more susceptible to apoptosis in the presence of oxidative stress compared to wild-type. Therefore, smNPCs are a powerful biological tool with properties that are optimal for large-scale disease modeling, phenotypic screening, and studies of early human development.

The LRRK2 mutation G2019S is the most common genetic cause of Parkinson's disease (PD). To better understand the link between mutant LRRK2 and PD pathology, we derived induced pluripotent stem cells from PD patients harboring LRRK2 G2019S and then specifically corrected the mutant LRRK2 allele. We demonstrate that gene correction resulted in phenotypic rescue in differentiated neurons and uncovered expression changes associated with LRRK2 G2019S. We found that LRRK2 G2019S induced dysregulation of CPNE8, MAP7, UHRF2, ANXA1, and CADPS2. Knockdown experiments demonstrated that four of these genes contribute to dopaminergic neurodegeneration. LRRK2 G2019S induced increased extracellular-signal-regulated kinase 1/2 (ERK) phosphorylation. Transcriptional dysregulation of CADPS2, CPNE8, and UHRF2 was dependent on ERK activity. We show that multiple PD-associated phenotypes were ameliorated by inhibition of ERK. Therefore, our results provide mechanistic insight into the pathogenesis induced by mutant LRRK2 and pointers for the development of potential new therapeutics.

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 þ -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.

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.

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.

BackgroundDifferent 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.ResultsWe 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.ConclusionLabeled 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.

During the development of the mammalian neocortex, the generation of neurons by neural progenitors and their migration to the final position are closely coordinated. The highly polarized radial glial cells (RGCs) serve both as progenitor cells to generate neurons and as support for the migration of these neurons. After their generation, neurons transiently assume a multipolar morphology before they polarize and begin their migration along the RGCs. Here, we show that Rap1 GTPases perform essential functions for cortical organization as master regulators of cell polarity. Conditional deletion of Rap1 GTPases leads to a complete loss of cortical lamination. In RGCs, Rap1 GTPases are required to maintain their polarized organization. In newborn neurons, the loss of Rap1 GTPases prevents the formation of axons and leading processes and thereby interferes with radial migration. Taken together, the loss of RGC and neuronal polarity results in the disruption of cortical organization.

1 In visceral smooth muscles, both M 2 and M 3 muscarinic receptor subtypes are found, and produce two major metabolic effects: adenylyl cyclase inhibition and PLCb activation. Thus, we studied their relevance for muscarinic cationic current (mI CAT ) generation, which underlies cholinergic excitation. Experiments were performed on single guinea-pig ileal cells using patch-clamp recording techniques under conditions of weakly buffered [Ca 2 þ ] i (either using 50 mM EGTA or 50-100 mM fluo-3 for confocal fluorescence imaging) or with [Ca 2 þ ] i 'clamped' at 100 nM using 10 mM BAPTA/CaCl 2 mixture. 2 Using a cAMP-elevating agent (1 mM isoproterenol) or a membrane-permeable cAMP analog (10 mM 8-Br-cAMP), we found no evidence for mI CAT modulation through a cAMP/PKA pathway. 3 With low [Ca 2 þ ] i buffering, the PLC blocker U-73122 at 2.5 mM almost abolished mI CAT , in some cases without any significant effect on [Ca 2 þ ] i . When [Ca 2 þ ] i was buffered at 100 nM, U-73122 reduced both carbachol-and GTPgS-induced mI CAT maximal conductances (IC 50 ¼ 0.5-0.6 mM) and shifted their activation curves positively. 4 U-73343, a weak PLC blocker, had no effect on GTPgS-induced mI CAT , but weakly inhibited carbachol-induced current, possibly by competitively inhibiting muscarinic receptors, since the inhibition could be prevented by increasing the carbachol concentration to 1 mM. Aristolochic acid and D-609, which inhibit PLA 2 and phosphatidylcholine-specific PLC, respectively, had no or very small effects on mI CAT , suggesting that these enzymes were not involved. 5 InsP 3 (1 mM) in the pipette or OAG (20 mM) applied externally had no effect on mI CAT or its inhibition by U-73122. Ca 2 þ store depletion (evoked by InsP 3 , or by combined cyclopiazonic acid, ryanodine and caffeine treatment) did not induce any significant current, and had no effect on mI CAT in response to carbachol when [Ca 2 þ ] i was strongly buffered to 100 nM. 6 It is concluded that phosphatidylinositol-specific PLC modulates mI CAT via Ca 2 þ release, but also does so independently of InsP 3 , DAG, Ca 2 þ store depletion or a rise of [Ca 2 þ ] i . Our present results explain the previously established 'permissive' role of the M 3 receptor subtype in mI CAT generation, and provide a new insight into the molecular mechanisms underlying the shifts of the cationic conductance activation curve.