Human iPS-derived Neurons Research Articles

TrkC Overexpression Protects Sevoflurane-Induced Neurotoxicity in Human Induced Pluripotent Stem Cell-Derived Neurons

Background: Inhaled anesthetic sevoflurane (SEVO) may induce cortical neurotoxicity and memory dysfunction in both animals and humans. In this study, we investigated the toxic effects of SEVO on human induced pluripotent stem cell (iPS)-derived neurons. Methods: Human iPS-derived neurons were exposed to SEVO in vitro. SEVO-induced toxic effects were examined with the viability, live caspase 3/7, and neurite density assays, respectively. The effects of SEVO on the receptors of the tyrosine kinases TrkA, TrkB, and TrkC were assessed by qRT-PCR. TrkA, TrkB, and TrkC were ectopically overexpressed in human iPS-derived neurons. Their functional effects on SEVO-induced human iPS-derived neuron toxicity were further investigated. Results: SEVO induced dose-dependent cell death, caspase 3/7 elevation, neurite degeneration, and the downregulation of Trk receptors in human iPS-derived neurons. Adenovirus-mediated Trk receptor overexpression selectively upregulated endogenous TrkA, TrkB, or TrkC gene expressions in human iPS-derived neurons. Specifically, TrkC overexpression, but not TrkA or TrkB overexpression was found to overcome the neurotoxic effects of SEVO in human iPS-derived neurons. Conclusions: SEVO may induce neurotoxicity in human iPS-derived neurons, and its neurotoxic damage could be protected by the overexpression of TrkC.

Evaluation of Toxic Amyloid 42 Oligomers in Rat Primary Cerebral Cortex Cells and Human iPS-derived Neurons Treated with 10-Me-Aplog-1, a New PKC Activator.

Amyloid β42 (Aβ42), a causative agent of Alzheimer’s disease (AD), is derived extracellularly from Aβ precursor protein (APP) following the latter’s cleavage by β-secretase, but not α-secretase. Protein kinase Cα (PKCα) activation is known to increase α-secretase activity, thereby suppressing Aβ production. Since Aβ42 oligomer formation causes potent neurotoxicity, APP modulation by PKC ligands is a promising strategy for AD treatment. Although bryostatin-1 (bryo-1) is a leading compound for this strategy, its limited natural availability and the difficulty of its total synthesis impedes further research. To address this limitation, Irie and colleagues have developed a new PKC activator with few side effects, 10-Me-Aplog-1, (1), which decreased Aβ42 in the conditioned medium of rat primary cerebral cortex cells. These results are associated with increased α-secretase but not PKCε-dependent Aβ-degrading enzyme. The amount of neuronal embryonic lethal abnormal vision (nELAV), a known β-secretase stabilizer, was reduced by treatment with 1. Notably, 1 prevented the formation of intracellular toxic oligomers. Furthermore, 1 suppressed toxic oligomerization within human iPS-derived neurons such as bryo-1. Given that 1 was not neurotoxic toward either cell line, these findings suggest that 1 is a potential drug lead for AD therapy.

Fabrication of Multielectrode Arrays for Neurobiology Applications.

Substrate-integrated multielectrode arrays (MEAs) enable multisite, long-term, and label-free sensing and actuation of neuronal electrical signals in reduced cell culture models for network electrophysiology. Conventional, thin-film fabricated passive MEAs typically provide a few tens of electrode sites. New generations of active CMOS-based high-resolution arrays provide the capabilities of simultaneous recordings from thousands of neurons over fields of view of several square millimeters, yet allowing extracellular electrical imaging to be achieved down to the subcellular scale. In turn, such advancement in chip-based electrical readouts can significantly complement recently developed biotechnological and bimolecular techniques for neurobiology applications. Here, we describe (1) a simple method to fabricate passive MEAs and (2) protocols for preparing and growing primary rat hippocampal neuronal cultures and human iPS-derived neurons on MEAs. The aim is to provide reliable protocols for initiating the reader to this technology and for stimulating their further development and experimental use in neurobiology.

327. Genetic Editing for Huntington's Disease

Huntington's disease (HD) is a neurodegenerative disorder caused by a pathological CAG expansion at the 3’ end of the first exon of the huntingtin gene (HTT). Currently, there is no efficient treatment for HD. Editing of the mutant HTT gene with the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system represents a new and promising approach. Recognition of the HTT target sequence by a single-guide RNA sequences (sgRNA) and the Cas9 protein is inducing DNA double-strand breaks (DSB), which activate endogenous cellular repair pathways. Non-homologous end joining (NHEJ) will introduce small insertion/deletion (indel) that alter the reading frame of HTT gene while homologous directed repair (HDR) is activated in the presence of a DNA template. To validate the approach and optimize the delivery of the CRISPR system with viral vectors, we first targeted artificial sequences containing fluorescent reporter genes in HEK 293T cells. An efficient gene disruption was measured and associated with a loss of fluorescence in neurons, astrocytes, in vitro and in vivo. Furthermore, we developed multiple strategies to disrupt the mutant HTT gene. Quantification demonstrated a high rate of indels, leading to a strong reduction of HTT protein in HEK 293T cells, mouse cortical neurons and human iPS-derived neurons. Blocking HTT expression in vitro HD models is improving several physiopathological parameters. We are currently evaluating the impact of allele or non-allele specific mutant HTT editing in human neurons from HD patients. Altogether, these data demonstrate the potential of the CRISPR technology as therapeutic strategy for HD.

5-HT2 receptors mediate functional modulation of GABAa receptors and inhibitory synaptic transmissions in human iPS-derived neurons

Neural progenitors differentiated from induced pluripotent stem cells (iPS) hold potentials for treating neurological diseases. Serotonin has potent effects on neuronal functions through multiple receptors, underlying a variety of neural disorders. Glutamate and GABA receptors have been proven functional in neurons differentiated from iPS, however, little is known about 5-HT receptor-mediated modulation in such neuronal networks. In the present study, human iPS were differentiated into cells possessing featured physiological properties of cortical neurons. Whole-cell patch-clamp recording was used to examine the involvement of 5-HT2 receptors in functional modulation of GABAergic synaptic transmission. We found that serotonin and DOI (a selective agonist of 5-HT2A/C receptor) reversibly reduced GABA-activated currents, and this 5-HT2A/C receptor mediated inhibition required G protein, PLC, PKC, and Ca2+ signaling. Serotonin increased the frequency of miniature inhibitory postsynaptic currents (mIPSCs), which could be mimicked by α-methylserotonin, a 5-HT2 receptor agonist. In contrast, DOI reduced both frequency and amplitude of mIPSCs. These findings suggested that in iPS-derived human neurons serotonin postsynaptically reduced GABAa receptor function through 5-HT2A/C receptors, but presynaptically other 5-HT2 receptors counteracted the action of 5-HT2A/C receptors. Functional expression of serotonin receptors in human iPS-derived neurons provides a pre-requisite for their normal behaviors after grafting.

Multitarget strategy of traditional Chinese medicine in treatment of Alzheimer's disease

critical brain regions is considered to be a causative factor in the cognitive decline and neuronal degeneration associated with Alzheimer’s disease (AD). Thus, reducing levels of toxic A b species by inhibiting BACE may be a viable therapeutic strategy for the treatment of AD. Effectively selecting BACE inhibitors for in vivo profiling requires high confidence in the translatability of in vitro potency. Human iPS-derived neurons, which more closely represent human neurons, have been proposed to be a superior cellular model for translating in vitro potency to in vivo (and later clinical) efficacy. Methods: BACE inhibitors representing 3 chemical series were chosen for profiling in a soluble enzyme assay, an APP over-expressing human H4 neuroglioma cell line, a rodent primary neuronal culture, and in human iPS-derived neurons. Our human iPS line was derived from adult non-diseased fibroblasts that were reprogrammed into GABAergic neurons. IC50 correlations for A b lowering were built to compare potency across these assays. Additionally, wild-type 129/SVEmicewere dosed with several of these BACE inhibitors to establish the in vivo brain IC50 for A b. For all assays, in vitro -toin vivo correlations (IVIVCs) of in vitro IC50 values and in vivo IC50 values were generated.Results:We report a high correlation in BACE inhibitor potency between all the in vitro assays examined, including the iPS-derived neurons. Additionally, IVIVCs for the various in vitro assays described translate well to in vivo rodent efficacy. Conclusions: Selecting appropriate chemical matter for in vivo profiling is essential for efficient drug discovery. While the non-diseased human iPS-derived neurons that we tested are more representative of human neurons than the other assay models examined, they do not provide any distinct advantage in translating in vitro potency to in vivo efficacy and thus do not appear to represent a superior translatable cellular model for screening. It remains to be seen if iPSderived neurons from AD patients offer any advantage in screening for Alzheimer’s disease therapeutics.

Predicting in vivo BACE inhibitor efficacy: Are human iPS-derived neurons superior?

Predicting in vivo BACE inhibitor efficacy: Are human iPS-derived neurons superior?