scholarly journals ∆9-tetrahydrocannabinol negatively regulates neurite outgrowth and Akt signaling in hiPSC-derived cortical neurons

2018 ◽  
Author(s):  
Carole Shum ◽  
Lucia Dutan ◽  
Emily Annuario ◽  
Katherine Warre-Cornish ◽  
Samuel E. Taylor ◽  
...  
Keyword(s):  
Neurite Outgrowth ◽  
Cortical Neurons ◽  
Endocannabinoid System ◽  
Neuronal Morphology ◽  
Cannabinoid Type 1 Receptor ◽  
Cellular Processes ◽  
Extracellular Signal ◽  
Human Neurons ◽  
Induced Pluripotent ◽  
Arachidonoyl Glycerol

AbstractEndocannabinoids regulate different aspects of neurodevelopment. In utero exposure to the exogenous psychoactive cannabinoid Δ9-tetrahydrocannabinol (Δ9-THC), has been linked with abnormal cortical development in animal models. However, much less is known about the actions of endocannabinoids in human neurons. Here we investigated the effect of the endogenous endocannabinoid 2-arachidonoyl glycerol (2AG) and Δ9-THC on the development of neuronal morphology and activation of signaling kinases, in cortical glutamatergic neurons derived from human induced pluripotent stem cells (hiPSCs). Our data indicate that the cannabinoid type 1 receptor (CB1R), but not the cannabinoid 2 receptor (CB2R), GPR55 or TRPV1 receptors, is expressed in young, immature hiPSC-derived cortical neurons. Consistent with previous reports, 2AG and Δ9-THC negatively regulated neurite outgrowth. Interestingly, acute exposure to both 2AG and Δ9-THC inhibited phosphorylation of serine/threonine kinase extracellular signal-regulated protein kinases (ERK1/2), whereas Δ9-THC also reduced phosphorylation of Akt (aka PKB). Moreover, the CB1R inverse agonist SR 141716A attenuated the negative regulation of neurite outgrowth and ERK1/2 phosphorylation induced by 2AG and Δ9-THC. Taken together, our data suggest that hiPSC-derived cortical neurons express CB1Rs and are responsive to both endogenous and exogenous cannabinoids. Thus, hiPSC-neurons may represent a good cellular model for investigating the role of the endocannabinoid system in regulating cellular processes in human neurons.

scholarly journals Neuronal network dysfunction in a model for Kleefstra syndrome mediated by enhanced NMDAR signaling

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Monica Frega ◽  
Katrin Linda ◽  
Jason M. Keller ◽  
Güvem Gümüş-Akay ◽  
Britt Mossink ◽  
...  
Keyword(s):  
Neuronal Network ◽  
Cortical Neurons ◽  
Neurodevelopmental Disorder ◽  
Control Networks ◽  
Human Neurons ◽  
Kleefstra Syndrome ◽  
Nmdar Subunit ◽  
Induced Pluripotent ◽  
Reduced Rate ◽  
The Impact

Abstract Kleefstra syndrome (KS) is a neurodevelopmental disorder caused by mutations in the histone methyltransferase EHMT1. To study the impact of decreased EHMT1 function in human cells, we generated excitatory cortical neurons from induced pluripotent stem (iPS) cells derived from KS patients. Neuronal networks of patient-derived cells exhibit network bursting with a reduced rate, longer duration, and increased temporal irregularity compared to control networks. We show that these changes are mediated by upregulation of NMDA receptor (NMDAR) subunit 1 correlating with reduced deposition of the repressive H3K9me2 mark, the catalytic product of EHMT1, at the GRIN1 promoter. In mice EHMT1 deficiency leads to similar neuronal network impairments with increased NMDAR function. Finally, we rescue the KS patient-derived neuronal network phenotypes by pharmacological inhibition of NMDARs. Summarized, we demonstrate a direct link between EHMT1 deficiency and NMDAR hyperfunction in human neurons, providing a potential basis for more targeted therapeutic approaches for KS.

scholarly journals Two engineered AAV capsid variants for efficient transduction of human cortical neurons directly converted from iPSC

2021 ◽  
Author(s):  
Sandra Fischer ◽  
Jonas Weinmann ◽  
Frank Gillardon
Keyword(s):  
Cortical Neurons ◽  
Transduction Efficiency ◽  
Adeno Associated Virus ◽  
Viral Genomes ◽  
Glutamatergic Neurons ◽  
Human Neurons ◽  
Human Cell Cultures ◽  
Primary Astrocytes ◽  
Induced Pluripotent ◽  
Viral Titers

Recombinant adeno-associated virus (AAV) is the most widely used vector for gene therapy in clinical trials. To increase transduction efficiency and specificity, novel engineered AAV variants with modified capsid sequences are evaluated in human cell cultures and non-human primates. In the present study, we tested two novel AAV capsid variants, AAV2-NNPTPSR and AAV9-NVVRSSS, in human cortical neurons, which were directly converted from human induced pluripotent stem cells and cocultured with rat primary astrocytes. AAV2-NNPTPSR variant efficiently transduced both induced human cortical glutamatergic neurons and induced human cortical GABAergic interneurons. By contrast, AAV9-NVVRSSS variant transduced both induced human cortical neurons and cocultured rat primary astrocytes. High viral titers (1x10E5 viral genomes per cell) caused a significant decrease in viability of induced human cortical neurons. Low viral titers (1x10E4 viral genomes per cell) lead to a significant increase in the neuronal activity marker c-Fos in transduced human neurons following treatment with a potassium channel blocker, which may indicate functional alterations induced by viral transduction and/or transgene expression.

scholarly journals Deficiency in MT5-MMP Supports Branching of Human iPSCs-Derived Neurons and Reduces Expression of GLAST/S100 in iPSCs-Derived Astrocytes

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1705
Author(s):  
Nikita Arnst ◽  
Pedro Belio-Mairal ◽  
Laura García-González ◽  
Laurie Arnaud ◽  
Louise Greetham ◽  
...  
Keyword(s):  
Physiological Role ◽  
Neuronal Morphology ◽  
App Processing ◽  
Amyloidogenic Processing ◽  
Model Of Alzheimer’S Disease ◽  
Human Neurons ◽  
Human Ipscs ◽  
Induced Pluripotent ◽  
The Impact

For some time, it has been accepted that the β-site APP cleaving enzyme 1 (BACE1) and the γ-secretase are two main players in the amyloidogenic processing of the β-amyloid precursor protein (APP). Recently, the membrane-type 5 matrix metalloproteinase (MT5-MMP/MMP-24), mainly expressed in the nervous system, has been highlighted as a new key player in APP-processing, able to stimulate amyloidogenesis and also to generate a neurotoxic APP derivative. In addition, the loss of MT5-MMP has been demonstrated to abrogate pathological hallmarks in a mouse model of Alzheimer’s disease (AD), thus shedding light on MT5-MMP as an attractive new therapeutic target. However, a more comprehensive analysis of the role of MT5-MMP is necessary to evaluate how its targeting affects neurons and glia in pathological and physiological situations. In this study, leveraging on CRISPR-Cas9 genome editing strategy, we established cultures of human-induced pluripotent stem cells (hiPSC)-derived neurons and astrocytes to investigate the impact of MT5-MMP deficiency on their phenotypes. We found that MT5-MMP-deficient neurons exhibited an increased number of primary and secondary neurites, as compared to isogenic hiPSC-derived neurons. Moreover, MT5-MMP-deficient astrocytes displayed higher surface area and volume compared to control astrocytes. The MT5-MMP-deficient astrocytes also exhibited decreased GLAST and S100β expression. These findings provide novel insights into the physiological role of MT5-MMP in human neurons and astrocytes, suggesting that therapeutic strategies targeting MT5-MMP should be controlled for potential side effects on astrocytic physiology and neuronal morphology.

scholarly journals Dichotomic Hippocampal Transcriptome After Glutamatergic vs. GABAergic Deletion of the Cannabinoid CB1 Receptor

2021 ◽  
Vol 13 ◽  
Author(s):  
Diego Pascual Cuadrado ◽  
Anna Wierczeiko ◽  
Charlotte Hewel ◽  
Susanne Gerber ◽  
Beat Lutz
Keyword(s):  
Gene Networks ◽  
Dynamic Equilibrium ◽  
Endocannabinoid System ◽  
Transcript Level ◽  
Genetically Engineered ◽  
Neuronal Morphology ◽  
Cannabinoid Type 1 Receptor ◽  
As Stress ◽  
Post Transcriptional Regulation ◽  
Mouse Lines

Brain homeostasis is the dynamic equilibrium whereby physiological parameters are kept actively within a specific range. The homeostatic range is not fixed and may change throughout the individual's lifespan, or may be transiently modified in the presence of severe perturbations. The endocannabinoid system has emerged as a safeguard of homeostasis, e.g., it modulates neurotransmission and protects neurons from prolonged or excessively strong activation. We used genetically engineered mouse lines that lack the cannabinoid type-1 receptor (CB1) either in dorsal telencephalic glutamatergic or in forebrain GABAergic neurons to create new allostatic states, resulting from alterations in the excitatory/inhibitory (E/I) balance. Previous studies with these two mouse lines have shown dichotomic results in the context of behavior, neuronal morphology, and electrophysiology. Thus, we aimed at analyzing the transcriptomic profile of the hippocampal CA region from these mice in the basal condition and after a mild behavioral stimulation (open field). Our results provide insights into the gene networks that compensate chronic E/I imbalances. Among these, there are differentially expressed genes involved in neuronal and synaptic functions, synaptic plasticity, and the regulation of behavior. Interestingly, some of these genes, e.g., Rab3b, Crhbp, and Kcnn2, and related pathways showed a dichotomic expression, i.e., they are up-regulated in one mutant line and down-regulated in the other one. Subsequent interrogation on the source of the alterations at transcript level were applied using exon-intron split analysis. However, no strong directions toward transcriptional or post-transcriptional regulation comparing both mouse lines were observed. Altogether, the dichotomic gene expression observed and their involved signaling pathways are of interest because they may act as “switches” to modulate the directionality of neural homeostasis, which then is relevant for pathologies, such as stress-related disorders and epilepsy.

scholarly journals Cellular and intracellular mechanisms involved in the cognitive impairment of cannabinoids

2012 ◽  
Vol 367 (1607) ◽  
pp. 3254-3263 ◽  
Author(s):  
Emma Puighermanal ◽  
Arnau Busquets-Garcia ◽  
Rafael Maldonado ◽  
Andrés Ozaita
Keyword(s):  
Cognitive Function ◽  
Molecular Mechanisms ◽  
Cannabinoid Receptors ◽  
Critical Role ◽  
Mammalian Target Of Rapamycin ◽  
Endocannabinoid System ◽  
Protein Translation ◽  
Signalling Pathways ◽  
Cellular Processes ◽  
Extracellular Signal

Exogenous cannabinoids, such as delta9-tetrahydrocannabinol (THC), as well as the modulation of endogenous cannabinoids, affect cognitive function through the activation of cannabinoid receptors. Indeed, these compounds modulate a number of signalling pathways critically implicated in the deleterious effect of cannabinoids on learning and memory. Thus, the involvement of the mammalian target of rapamycin pathway and extracellular signal-regulated kinases, together with their consequent regulation of cellular processes such as protein translation, play a critical role in the amnesic-like effects of cannabinoids. In this study, we summarize the cellular and molecular mechanisms reported in the modulation of cognitive function by the endocannabinoid system.

scholarly journals CRISPR interference-based platform for multimodal genetic screens in human iPSC-derived neurons

2019 ◽  
Author(s):  
Ruilin Tian ◽  
Mariam A. Gachechiladze ◽  
Connor H. Ludwig ◽  
Matthew T. Laurie ◽  
Jason Y. Hong ◽  
...  
Keyword(s):  
Cell Biology ◽  
Cell Types ◽  
Neuronal Morphology ◽  
Genetic Screens ◽  
Crispr Interference ◽  
Disease States ◽  
Differentiated Cells ◽  
Human Neurons ◽  
Longitudinal Imaging ◽  
Induced Pluripotent

SUMMARYCRISPR/Cas9-based functional genomics have transformed our ability to elucidate mammalian cell biology. However, most previous CRISPR-based screens were conducted in cancer cell lines, rather than healthy, differentiated cells. Here, we describe a CRISPR interference (CRISPRi)-based platform for genetic screens in human neurons derived from induced pluripotent stem cells (iPSCs). We demonstrate robust and durable knockdown of endogenous genes in such neurons, and present results from three complementary genetic screens. First, a survival-based screen revealed neuron-specific essential genes and genes that improved neuronal survival upon knockdown. Second, a screen with a single-cell transcriptomic readout uncovered several examples of genes whose knockdown had strikingly cell-type specific consequences. Third, a longitudinal imaging screen detected distinct consequences of gene knockdown on neuronal morphology. Our results highlight the power of unbiased genetic screens in iPSC-derived differentiated cell types and provide a platform for systematic interrogation of normal and disease states of neurons.

scholarly journals Functional expression of the ATP-gated P2X7 receptor in human iPSC-derived neurons and astrocytes

2021 ◽  
Author(s):  
Jaideep Kesavan ◽  
Orla Watters ◽  
Klaus Dinkel ◽  
Michael Hamacher ◽  
Jochen H.M. Prehn ◽  
...  
Keyword(s):  
Pluripotent Stem Cells ◽  
Cortical Neurons ◽  
P2x7 Receptor ◽  
Functional Expression ◽  
Extracellular Adenosine ◽  
Functional Studies ◽  
Human Neurons ◽  
Induced Pluripotent ◽  
Human Ipsc

AbstractThe P2X7 receptor (P2X7R) is a cation membrane channel activated by extracellular adenosine 5′-triphosphate. Activation of this receptor results in numerous downstream events including the modulation of neurotransmission, release of pro-inflammatory mediators, cell proliferation or cell death. While the expression of P2X7Rs is well documented on microglia and oligodendrocytes, the presence of functional P2X7Rs on neurons and astrocytes remains debated. Furthermore, to date, functional studies on the P2X7R are mostly limited to studies in cells from rodents and immortalised cell lines expressing human P2X7Rs. To assess the functional expression of P2X7Rs in human neurons and astrocytes, we differentiated human-induced pluripotent stem cells (hiPSCs) into forebrain cortical neurons that co-express FOXG1 and βIII-tubulin as well as S100 β-expressing astrocytes. Immunostaining revealed prominent punctate P2X7R staining on the soma and processes of hiPSC-derived neurons and astrocytes. In addition, our data show that stimulation with the potent nonselective P2X7R agonist BzATP induces robust calcium rises in hiPSC-derived neurons and astrocytes, which were blocked by the selective P2X7R antagonist AFC-5128. Together, our findings provide evidence for the functional expression of P2X7Rs in hiPSC-derived forebrain cortical neurons and astrocytes demonstrating that these cells offer the potential for investigating P2X7R-mediated pathophysiology and drug screening in vitro.

scholarly journals Transcription elongation factor AFF2/FMR2 regulates expression of expanded GGGGCC repeat-containing C9ORF72 allele in ALS/FTD

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Yeliz Yuva-Aydemir ◽  
Sandra Almeida ◽  
Gopinath Krishnan ◽  
Tania F. Gendron ◽  
Fen-Biao Gao
Keyword(s):  
Cortical Neurons ◽  
Elongation Factor ◽  
Premature Death ◽  
Partial Loss ◽  
Repeat Proteins ◽  
Rna Foci ◽  
Human Neurons ◽  
Induced Pluripotent ◽  
Lateral Sclerosis ◽  
Dipeptide Repeat

AbstractExpanded GGGGCC (G4C2) repeats in C9ORF72 cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How RNAs containing expanded G4C2 repeats are transcribed in human neurons is largely unknown. Here we describe a Drosophila model in which poly(GR) expression in adult neurons causes axonal and locomotor defects and premature death without apparent TDP-43 pathology. In an unbiased genetic screen, partial loss of Lilliputian (Lilli) activity strongly suppresses poly(GR) toxicity by specifically downregulating the transcription of GC-rich sequences in Drosophila. Knockout of AFF2/FMR2 (one of four mammalian homologues of Lilli) with CRISPR-Cas9 decreases the expression of the mutant C9ORF72 allele containing expanded G4C2 repeats and the levels of repeat RNA foci and dipeptide repeat proteins in cortical neurons derived from induced pluripotent stem cells of C9ORF72 patients, resulting in rescue of axonal degeneration and TDP-43 pathology. Thus, AFF2/FMR2 regulates the transcription and toxicity of expanded G4C2 repeats in human C9ORF72-ALS/FTD neurons.

scholarly journals P.135 Autism-associated mutations in SHANK2 increase synaptic connectivity and dendrite complexity in human neurons

2018 ◽  
Vol 45 (s2) ◽  
pp. S52-S52
Author(s):  
K Zaslavsky ◽  
W Zhang ◽  
E Deneault ◽  
M Zhao ◽  
DC Rodrigues ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Autism Spectrum ◽  
Dermal Fibroblasts ◽  
Neuronal Morphology ◽  
Synaptic Connectivity ◽  
Loss Of Function ◽  
Knock Out ◽  
Electrophysiological Properties ◽  
Dendrite Length ◽  
Human Neurons

Background: Heterozygous loss-of-function mutations in the synaptic scaffolding gene SHANK2 are strongly associated with autism spectrum disorder (ASD). However, their impact on the function of human neurons is unknown. Derivation of induced pluripotent stem cells (iPSC) from affected individuals permits generation of live neurons to answer this question. Methods: We generated iPSCs by reprogramming dermal fibroblasts of neurotypic and ASD-affected donors. To isolate the effect of SHANK2, we used CRISPR/Cas9 to knock out SHANK2 in control iPSCs and correct a heterozygous nonsense mutation in ASD-affected donor iPSCs. We then derived cortical neurons from SOX1+ neural precursor cells differentiated from these iPSCs. Using a novel assay that overcomes line-to-line variability, we compared neuronal morphology, total synapse number, and electrophysiological properties between SHANK2 mutants and controls. Results: Relative to controls, SHANK2 mutant neurons have increased dendrite complexity, dendrite length, total synapse number (1.5-2-fold), and spontaneous excitatory postsynaptic current (sEPSC) frequency (3-7.6-fold). Conclusions: ASD-associated heterozygous loss-of-function mutations in SHANK2 increase synaptic connectivity among human neurons by increasing synapse number and sEPSC frequency. This is partially supported by increased dendrite length and complexity, providing evidence that SHANK2 functions as a suppressor of dendrite branching during neurodevelopment.

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