v-SNARE actions during Ca²⁺-triggered exocytosis

Assembly of SNARE proteins between opposing membranes mediates fusion of synthetic lipo-somes, but it is unknown whether SNAREs act during exocytosis at the moment of Ca²⁺ increase, providing the molecular force for fusion of secretory vesicles. Here, weshowthatexecution of pre- and postfusional Steps during chromaffin granule exocytosis depends crucially on a short molecular distance between the complex-forming SNARE motif and the transmembrane anchor of the vesicular SNARE protein synaptobrevin II. Extending the juxtamembrane region of synaptobrevin by insertion of flexible "linkers" reduces priming of granules, delays initiation of exocytosis upon stepwise elevation of intracellular calcium, attenuates fluctuations of early fusion pores, and slows rapid expansion of the pore in a linker-length dependent fashion. These observations provide evidence that v-SNARE proteins drive Ca²⁺-triggered membrane fusion at millisecond time scale and support a model wherein continuous molecular pulling by SNAREs guides the vesicle throughout the consecutive stages of exocytosis

In Vitro Recapitulation of Developmental Transitions in Human Neural Stem Cells.

During nervous system development, early neuroepithelial stem (NES) cells with a highly polarized morphology and responsiveness to regionalizing morphogens give rise to radial glia (RG) cells, which generate region-specific neurons. Recently, stable neural cell populations reminiscent of NES cells have been obtained from pluripotent stem cells and the fetal human hindbrain. Here, we explore whether these cell populations, similar to their in vivo counterparts, can give rise to neural stem (NS) cells with RG-like properties and whether region-specific NS cells can be generated from NES cells with different regional identities. In vivo RG cells are thought to form from NES cells with the onset of neurogenesis. Therefore, we cultured NES cells temporarily in differentiating conditions. Upon reinitiation of growth factor treatment, cells were found to enter a developmental stage reflecting major characteristics of RG-like NS cells. These NES cell-derived NS cells exhibited a very similar morphology and marker expression as primary NS cells generated from human fetal tissue, indicating that conversion of NES cells into NS cells recapitulates the developmental progression of early NES cells into RG cells observed in vivo. Importantly, NS cells generated from NES cells with different regional identities exhibited stable region-specific transcription factor expression and generated neurons appropriate for their positional identity. Stem Cells 2019;37:1429-1440

Wearable uBrain : Fabric Based-Spiking Neural Network

On garment intelligence influenced by artificial neural networks and neuromorphic computing is emerging as a research direction in the e-textile sector. In particular, bio inspired Spiking Neural Networks mimicking the workings of the brain show promise in recent ICT research applications. Taking such technological advancements and new research directions driving forward the next generation of e-textiles and smart materials, we present a wearable micro Brain capable of event driven artificial spiking neural network computation in a fabric based environment. We demonstrate a wearable Brain SNN prototype with multi-layer computation, enabling scalability and flexibility in terms of modifications for hidden layers to be augmented to the network. The wearable micro Brain provides a low size, weight and power artificial on-garment intelligent wearable solution with embedded functionality enabling offline adaptive learning through the provision of interchangeable resistor synaptic weightings. The prototype has been evaluated for fault tolerance, where we have determine the robustness of the circuit when certain parts are damaged. Validations were also conducted for movements to determine if the circuit can still perform accurate computation

MicroRNA-335-5p suppresses voltage-gated sodium channel expression and may be a target for seizure control

There remains an urgent need for new therapies for treatment-resistant epilepsy. Sodium channel blockers are effective for seizure control in common forms of epilepsy, but loss of sodium channel function underlies some genetic forms of epilepsy. Approaches that provide bidirectional control of sodium channel expression are needed. MicroRNAs (miRNA) are small noncoding RNAs which negatively regulate gene expression. Here we show that genome-wide miRNA screening of hippocampal tissue from a rat epilepsy model, mice treated with the antiseizure medicine cannabidiol, and plasma from patients with treatment-resistant epilepsy, converge on a single target-miR-335-5p. Pathway analysis on predicted and validated miR-335-5p targets identified multiple voltage-gated sodium channels (VGSCs). Intracerebroventricular injection of antisense oligonucleotides against miR-335-5p resulted in upregulation of Scn1a, Scn2a, and Scn3a in the mouse brain and an increased action potential rising phase and greater excitability of hippocampal pyramidal neurons in brain slice recordings, consistent with VGSCs as functional targets of miR-335-5p. Blocking miR-335-5p also increased voltage-gated sodium currents and SCN1A, SCN2A, and SCN3A expression in human induced pluripotent stem cell-derived neurons. Inhibition of miR-335-5p increased susceptibility to tonic-clonic seizures in the pentylenetetrazol seizure model, whereas adeno-associated virus 9-mediated overexpression of miR-335-5p reduced seizure severity and improved survival. These studies suggest modulation of miR-335-5p may be a means to regulate VGSCs and affect neuronal excitability and seizures. Changes to miR-335-5p may reflect compensatory mechanisms to control excitability and could provide biomarker or therapeutic strategies for different types of treatment-resistant epilepsy

Capture of Neuroepithelial-Like Stem Cells from Pluripotent Stem Cells Provides a Versatile System for In Vitro Production of Human Neurons

Human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC) provide new prospects for studying human neurodevelopment and modeling neurological disease. In particular, iPSC-derived neural cells permit a direct comparison of disease-relevant molecular pathways in neurons and glia derived from patients and healthy individuals. A prerequisite for such comparative studies are robust protocols that efficiently yield standardized populations of neural cell types. Here we show that long-term self-renewing neuroepithelial-like stem cells (lt-NES cells) derived from 3 hESC and 6 iPSC lines in two independent laboratories exhibit consistent characteristics including i) continuous expandability in the presence of FGF2 and EGF; ii) stable neuronal and glial differentiation competence; iii) characteristic transcription factor profile; iv) hindbrain specification amenable to regional patterning; v) capacity to generate functionally mature human neurons. We further show that lt-NES cells are developmentally distinct from fetal tissue-derived radial glia-like stem cells. We propose that lt-NES cells provide an interesting tool for studying human neurodevelopment and may serve as a standard system to facilitate comparative analyses of hESC and hiPSC-derived neural cells from control and diseased genetic backgrounds

v-SNARE actions during Ca2+-triggered exocytosis

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APP Processing in Human Pluripotent Stem Cell-Derived Neurons Is Resistant to NSAID-Based γ-Secretase Modulation

Increasing evidence suggests that elevated Aβ42 fractions in the brain cause Alzheimer's disease (AD). Although γ-secretase modulators (GSMs), including a set of nonsteroidal anti-inflammatory drugs (NSAIDs), were found to lower Aβ42 in various model systems, NSAID-based GSMs proved to be surprisingly inefficient in human clinical trials. Reasoning that the nonhuman and nonneuronal cells typically used in pharmaceutical compound validation might not adequately reflect the drug responses of human neurons, we used human pluripotent stem cell-derived neurons from AD patients and unaffected donors to explore the efficacy of NSAID-based γ-secretase modulation. We found that pharmaceutically relevant concentrations of these GSMs that are clearly efficacious in conventional nonneuronal cell models fail to elicit any effect on Aβ42/Aß40 ratios in human neurons. Our work reveals resistance of human neurons to NSAID-based γ-secretase modulation, highlighting the need to validate compound efficacy directly in the human cell type affected by the respective disease.publisher: Elsevier articletitle: APP Processing in Human Pluripotent Stem Cell-Derived Neurons Is Resistant to NSAID-Based γ-Secretase Modulation journaltitle: Stem Cell Reports articlelink: http://dx.doi.org/10.1016/j.stemcr.2013.10.011 content_type: article copyright: Copyright © 2013 The Authors. Published by Elsevier Inc.status: publishe

Functional expression of the ATP-gated P2X7 receptor in human iPSC-derived astrocytes

Activation of the ATP-gated P2X7 receptor (P2X7R), implicated in numerous diseases of the brain, can trigger diverse responses such as the release of pro-inflammatory cytokines, modulation of neurotransmission, cell proliferation or cell death. However, despite the known species-specific differences in its pharmacological properties, to date, most functional studies on P2X7R responses have been analyzed in cells from rodents or immortalised cell lines. To assess the endogenous and functional expression of P2X7Rs in human astrocytes, we differentiated human-induced pluripotent stem cells (hiPSCs) into GFAP and S100 β-expressing astrocytes. Immunostaining revealed prominent punctate P2X7R staining. P2X7R protein expression was also confirmed by Western blot. Importantly, stimulation with the potent non-selective P2X7R agonist 2',3'-O-(benzoyl-4-benzoyl)-adenosine 5'- triphosphate (BzATP) or endogenous agonist ATP induced robust calcium rises in hiPSC-derived astrocytes which were blocked by the selective P2X7R antagonists AFC-5128 or JNJ-47965567. Our findings provide evidence for the functional expression of P2X7Rs in hiPSC-derived astrocytes and support their in vitro utility in investigating the role of the P2X7R and drug screening in disorders of the central nervous system (CNS). </p

Neuronal differentiation of lt-NES cells.

<p>(<b>A–B</b>) Four weeks after growth factor removal, lt-NES cells differentiated predominantly into neurons expressing beta III-tubulin (90%), MAP2ab and the neurotransmitter GABA, plus a minor fraction of GFAP-positive astrocytes (10%). Error bars represent STD. (<b>C</b>) Following growth factor withdrawal the clonal line PKa-2 gave rise to glia and neurons of preferentially GABAergic phenotype, comparable to the parental line PKa. (<b>D</b>) Specific neuronal subtypes expressing TH, 5HT and HB9 could be observed after 4 weeks of differentiation; O4-positive oligodendrocytes were detected after 10 weeks of differentiation. (<b>E</b>) AF22 lt-NES cells stained for Nestin and beta III-tubulin at day 0, 5, 8, 11, 18, 22 of differentiation. (<b>F</b>) Proliferating AF22 cells expressing GFP under the control of the Nestin enhancer (Nestin-GFP) show double labeling with an antibody to the Nestin protein. (<b>G</b>) Nestin-GFP expression in AF22 cells at day 0, 5, 7, 9, 11, 13, 15 of differentiation under control conditions or after exposure to DAPT (2 µM). Scale bars: 100 µm.</p