A reference human induced pluripotent stem cell line for large-scale collaborative studies.

Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between lines makes it challenging to replicate key findings and integrate data across research groups. To address this issue, we sub-cloned candidate human iPSC lines and deeply characterized their genetic properties using whole genome sequencing, their genomic stability upon CRISPR-Cas9-based gene editing, and their phenotypic properties including differentiation to commonly used cell types. These studies identified KOLF2.

Continuing Medical Education (CME) in time of crisis: How medical societies face challenges and adapt to provide unbiased CME.

Healthcare professionals need to maintain their knowledge and skills to deliver the best possible care to patients. Medical societies play an important role as providers of continuing medical education (CME) and have actively continued this role during the COVID-19 pandemic adapting the delivery of education to virtual meetings and courses. The Biomedical Alliance in Europe CME Experts Committee conducted two surveys to collect information on the delivery of CME, generally, and during the COVID-19 pandemic from the member medical societies.

The Interaction of Munc18-1 Helix 11 and 12 with the Central Region of the VAMP2 SNARE Motif Is Essential for SNARE Templating and Synaptic Transmission.

Sec1/Munc18 proteins play a key role in initiating the assembly of N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes, the molecular fusion machinery. Employing comparative structure modeling, site specific crosslinking by single amino acid substitutions with the photoactivatable unnatural amino acid p-Benzoyl-phenylalanine (Bpa) and reconstituted vesicle docking/fusion assays, we mapped the binding interface between Munc18-1 and the neuronal v-SNARE VAMP2 with single amino acid resolution. Our results show that helices 11 and 12 of domain 3a in Munc18-1 interact with the VAMP2 SNARE motif covering the region from layers -4 to +5.

Post-tetanic potentiation lowers the energy barrier for synaptic vesicle fusion independently of Synaptotagmin-1.

Previously, we showed that modulation of the energy barrier for synaptic vesicle fusion boosts release rates supralinearly (Schotten, 2015). Here we show that mouse hippocampal synapses employ this principle to trigger Ca-dependent vesicle release and post-tetanic potentiation (PTP). We assess energy barrier changes by fitting release kinetics in response to hypertonic sucrose.

Doc2 Proteins Are Not Required for the Increased Spontaneous Release Rate in Synaptotagmin-1-Deficient Neurons.

Regulated secretion is controlled by Ca sensors with different affinities and subcellular distributions. Inactivation of (synaptotagmin-1), the main Ca sensor for synchronous neurotransmission in many neurons, enhances asynchronous and spontaneous release rates, suggesting that Syt1 inhibits other sensors with higher Ca affinities and/or lower cooperativities. Such sensors could include Doc2a and Doc2b, which have been implicated in spontaneous and asynchronous neurotransmitter release and compete with Syt1 for binding SNARE complexes.

A Munc18-1 mutant mimicking phosphorylation by Down Syndrome-related kinase Dyrk1a supports normal synaptic transmission and promotes recovery after intense activity.

Phosphorylation of Munc18-1 (Stxbp1), a presynaptic organizer of synaptic vesicle fusion, is a powerful mechanism to regulate synaptic strength. Munc18-1 is a proposed substrate for the Down Syndrome-related kinase dual-specificity tyrosine phosphorylation-regulate kinase 1a (Dyrk1a) and mutations in both genes cause intellectual disability. However, the functional consequences of Dyrk1a-dependent phosphorylation of Munc18-1 for synapse function are unknown.

A critical evaluation of thyroid hormone measurements in OECD test guideline studies: Is there any added value?

Recently several OECD test guidelines were updated to include thyroid hormone measurements for assessing endocrine disruptor potency, which led to an imperative need to align interpretation of these results by the different stakeholders. We therefore evaluated 124 repro screening studies, which showed in 38% of the studies a statistical significant finding for T4 in at least one treatment group, probably due to disturbances of normal homeostasis causing high variation. Consequently, for a thorough evaluation it is extremely important to take the historical control range into account.

A Single-Cell Model for Synaptic Transmission and Plasticity in Human iPSC-Derived Neurons.

Synaptic dysfunction is associated with many brain disorders, but robust human cell models to study synaptic transmission and plasticity are lacking. Instead, current in vitro studies on human neurons typically rely on spontaneous synaptic events as a proxy for synapse function. Here, we describe a standardized in vitro approach using human neurons cultured individually on glia microdot arrays that allow single-cell analysis of synapse formation and function.

Tyrosine phosphorylation of Munc18-1 inhibits synaptic transmission by preventing SNARE assembly.

Tyrosine kinases are important regulators of synaptic strength. Here, we describe a key component of the synaptic vesicle release machinery, Munc18-1, as a phosphorylation target for neuronal Src family kinases (SFKs). Phosphomimetic Y473D mutation of a SFK phosphorylation site previously identified by brain phospho-proteomics abolished the stimulatory effect of Munc18-1 on SNARE complex formation ("SNARE-templating") and membrane fusion Furthermore, priming but not docking of synaptic vesicles was disrupted in hippocampal -null neurons expressing Munc18-1 Synaptic transmission was temporarily restored by high-frequency stimulation, as well as by a Munc18-1 mutation that results in helix 12 extension, a critical conformational step in vesicle priming.

Multi-level characterization of balanced inhibitory-excitatory cortical neuron network derived from human pluripotent stem cells.

Generation of neuronal cultures from induced pluripotent stem cells (hiPSCs) serve the studies of human brain disorders. However we lack neuronal networks with balanced excitatory-inhibitory activities, which are suitable for single cell analysis. We generated low-density networks of hPSC-derived GABAergic and glutamatergic cortical neurons.

Phosphorylation of synaptotagmin-1 controls a post-priming step in PKC-dependent presynaptic plasticity.

Presynaptic activation of the diacylglycerol (DAG)/protein kinase C (PKC) pathway is a central event in short-term synaptic plasticity. Two substrates, Munc13-1 and Munc18-1, are essential for DAG-induced potentiation of vesicle priming, but the role of most presynaptic PKC substrates is not understood. Here, we show that a mutation in synaptotagmin-1 (Syt1(T112A)), which prevents its PKC-dependent phosphorylation, abolishes DAG-induced potentiation of synaptic transmission in hippocampal neurons.

Synaptic Effects of Munc18-1 Alternative Splicing in Excitatory Hippocampal Neurons.

The munc18-1 gene encodes two splice-variants that vary at the C-terminus of the protein and are expressed at different levels in different regions of the adult mammalian brain. Here, we investigated the expression pattern of these splice variants within the brainstem and tested whether they are functionally different. Munc18-1a is expressed in specific nuclei of the brainstem including the LRN, VII and SOC, while Munc18-1b expression is relatively low/absent in these regions.

Inhibition of Voltage-Gated Calcium Channels After Subchronic and Repeated Exposure of PC12 Cells to Different Classes of Insecticides.

We previously demonstrated that acute inhibition of voltage-gated calcium channels (VGCCs) is a common mode of action for (sub)micromolar concentrations of chemicals, including insecticides. However, because human exposure to chemicals is usually chronic and repeated, we investigated if selected insecticides from different chemical classes (organochlorines, organophosphates, pyrethroids, carbamates, and neonicotinoids) also disturb calcium homeostasis after subchronic (24 h) exposure and after a subsequent (repeated) acute exposure. Effects on calcium homeostasis were investigated with single-cell fluorescence (Fura-2) imaging of PC12 cells.

Additive effects on the energy barrier for synaptic vesicle fusion cause supralinear effects on the vesicle fusion rate.

The energy required to fuse synaptic vesicles with the plasma membrane ('activation energy') is considered a major determinant in synaptic efficacy. From reaction rate theory, we predict that a class of modulations exists, which utilize linear modulation of the energy barrier for fusion to achieve supralinear effects on the fusion rate. To test this prediction experimentally, we developed a method to assess the number of releasable vesicles, rate constants for vesicle priming, unpriming, and fusion, and the activation energy for fusion by fitting a vesicle state model to synaptic responses induced by hypertonic solutions.

Comparison of plate reader-based methods with fluorescence microscopy for measurements of intracellular calcium levels for the assessment of in vitro neurotoxicity.

The intracellular calcium concentration ([Ca(2+)]i) is an important readout for in vitro neurotoxicity since calcium is critically involved in many essential neurobiological processes, including neurotransmission, neurodegeneration and neurodevelopment. [Ca(2+)]i is often measured with considerable throughput at the level of cell populations with plate reader-based assays or with lower throughput at the level of individual cells with fluorescence microscopy. However, these methodologies yield different quantitative and qualitative results.

Inhibition of voltage-gated calcium channels as common mode of action for (mixtures of) distinct classes of insecticides.

Humans are exposed to distinct structural classes of insecticides with different neurotoxic modes of action. Because calcium homeostasis is essential for proper neuronal function and development, we investigated the effects of insecticides from different classes (pyrethroid: (α-)cypermethrin; organophosphate: chlorpyrifos; organochlorine: endosulfan; neonicotinoid: imidacloprid) and mixtures thereof on the intracellular calcium concentration ([Ca(2+)]i). Effects of acute (20 min) exposure to (mixtures of) insecticides on basal and depolarization-evoked [Ca(2+)]i were studied in vitro with Fura-2-loaded PC12 cells and high resolution single-cell fluorescence microscopy.

Acute disturbance of calcium homeostasis in PC12 cells as a novel mechanism of action for (sub)micromolar concentrations of organophosphate insecticides.

Organophosphates (OPs) and carbamates are widely used insecticides that exert their neurotoxicity via inhibition of acetylcholine esterase (AChE) and subsequent overexcitation. OPs can induce additional neurotoxic effects at concentrations below those for inhibition of AChE, indicating other mechanisms of action are also involved. Since tight regulation of the intracellular calcium concentration ([Ca(2+)]i) is essential for proper neuronal development and function, effects of one carbamate (carbaryl) and two OPs (chlorpyrifos, parathion-ethyl) as well as their -oxon metabolites on [Ca(2+)]i were investigated.

A comparison of the in vitro cyto- and neurotoxicity of brominated and halogen-free flame retardants: prioritization in search for safe(r) alternatives.

Brominated flame retardants (BFRs) are abundant persistent organic pollutants with well-studied toxicity. The toxicological and ecological concerns associated with BFRs argue for replacement by safe(r) alternatives. Though previous research identified the nervous system as a sensitive target organ for BFRs, the (neuro) toxic potential of alternative halogen-free flame retardants (HFFRs) is largely unknown.

Molecular machines in the synapse: overlapping protein sets control distinct steps in neurosecretion.

Activity regulated neurotransmission shapes the computational properties of a neuron and involves the concerted action of many proteins. Classical, intuitive working models often assign specific proteins to specific steps in such complex cellular processes, whereas modern systems theories emphasize more integrated functions of proteins. To test how often synaptic proteins participate in multiple steps in neurotransmission we present a novel probabilistic method to analyze complex functional data from genetic perturbation studies on neuronal secretion.

Differential effects of 20 non-dioxin-like PCBs on basal and depolarization-evoked intracellular calcium levels in PC12 cells.

Non-dioxin-like polychlorinated biphenyls (NDL-PCBs) are environmental pollutants that are well known for their neurotoxic effects. Numerous in vitro studies reported PCB-induced increases in the basal intracellular calcium concentration ([Ca(2+)](i)), and in vivo NDL-PCB neurotoxicity appears at least partly mediated by these disturbances. However, effects of NDL-PCBs on depolarization-evoked calcium influx are poorly investigated, and effects of several congeners, including PCB53, on calcium homeostasis are still unknown.

Methamphetamine, amphetamine, MDMA ('ecstasy'), MDA and mCPP modulate electrical and cholinergic input in PC12 cells.

Reversal of the dopamine (DA) membrane transporter is the main mechanism through which many drugs of abuse increase DA levels. However, drug-induced modulation of exocytotic DA release by electrical (depolarization) and neurochemical inputs (e.g.