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 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 Deficiency of the Lysosomal Protein CLN5 Alters Lysosomal Function and Movement

Biomolecules ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 1412
Author(s):  
Indranil Basak ◽  
Rachel A. Hansen ◽  
Michael E. Ward ◽  
Stephanie M. Hughes
Keyword(s):  
Batten Disease ◽  
Induced Pluripotent Stem Cell ◽  
Epileptic Seizures ◽  
Neuronal Model ◽  
Cell System ◽  
Motor Deficits ◽  
Glutamatergic Neurons ◽  
Human Neurons ◽  
Induced Pluripotent ◽  
Acidic Organelles

Batten disease is a devastating, childhood, rare neurodegenerative disease characterised by the rapid deterioration of cognition and movement, leading to death within ten to thirty years of age. One of the thirteen Batten disease forms, CLN5 Batten disease, is caused by mutations in the CLN5 gene, leading to motor deficits, mental deterioration, cognitive impairment, visual impairment, and epileptic seizures in children. A characteristic pathology in CLN5 Batten disease is the defects in lysosomes, leading to neuronal dysfunction. In this study, we aimed to investigate the lysosomal changes in CLN5-deficient human neurons. We used an induced pluripotent stem cell system, which generates pure human cortical-like glutamatergic neurons. Using CRISPRi, we inhibited the expression of CLN5 in human neurons. The CLN5-deficient human neurons showed reduced acidic organelles and reduced lysosomal enzyme activity measured by microscopy and flow cytometry. Furthermore, the CLN5-deficient human neurons also showed impaired lysosomal movement—a phenotype that has never been reported in CLN5 Batten disease. Lysosomal trafficking is key to maintain local degradation of cellular wastes, especially in long neuronal projections, and our results from the human neuronal model present a key finding to understand the underlying lysosomal pathology in neurodegenerative diseases.

scholarly journals Cadherin-13 is a critical regulator of GABAergic modulation in human stem-cell-derived neuronal networks

2021 ◽  
Author(s):  
Britt Mossink ◽  
Jon-Ruben van Rhijn ◽  
Shan Wang ◽  
Katrin Linda ◽  
Maria R. Vitale ◽  
...  
Keyword(s):  
Gabaergic Neurons ◽  
Inhibitory Synapses ◽  
Disease Genes ◽  
Electrode Arrays ◽  
Glutamatergic Neurons ◽  
Human Neurons ◽  
Induced Pluripotent ◽  
Micro Electrode Arrays ◽  
Gabaergic Modulation

AbstractActivity in the healthy brain relies on a concerted interplay of excitation (E) and inhibition (I) via balanced synaptic communication between glutamatergic and GABAergic neurons. A growing number of studies imply that disruption of this E/I balance is a commonality in many brain disorders; however, obtaining mechanistic insight into these disruptions, with translational value for the patient, has typically been hampered by methodological limitations. Cadherin-13 (CDH13) has been associated with autism and attention-deficit/hyperactivity disorder. CDH13 localizes at inhibitory presynapses, specifically of parvalbumin (PV) and somatostatin (SST) expressing GABAergic neurons. However, the mechanism by which CDH13 regulates the function of inhibitory synapses in human neurons remains unknown. Starting from human-induced pluripotent stem cells, we established a robust method to generate a homogenous population of SST and MEF2C (PV-precursor marker protein) expressing GABAergic neurons (iGABA) in vitro, and co-cultured these with glutamatergic neurons at defined E/I ratios on micro-electrode arrays. We identified functional network parameters that are most reliably affected by GABAergic modulation as such, and through alterations of E/I balance by reduced expression of CDH13 in iGABAs. We found that CDH13 deficiency in iGABAs decreased E/I balance by means of increased inhibition. Moreover, CDH13 interacts with Integrin-β1 and Integrin-β3, which play opposite roles in the regulation of inhibitory synaptic strength via this interaction. Taken together, this model allows for standardized investigation of the E/I balance in a human neuronal background and can be deployed to dissect the cell-type-specific contribution of disease genes to the E/I balance.

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 Deficiency of the lysosomal protein CLN5 alters lysosomal function and movement

2021 ◽  
Author(s):  
Indranil Basak ◽  
Rachel A Hansen ◽  
Michael E Ward ◽  
Stephanie M Hughes
Keyword(s):  
Batten Disease ◽  
Induced Pluripotent Stem Cell ◽  
Epileptic Seizures ◽  
Neuronal Model ◽  
Cell System ◽  
Motor Deficits ◽  
Glutamatergic Neurons ◽  
Human Neurons ◽  
Stem Cell System ◽  
Induced Pluripotent

Batten disease is a devastating childhood rare neurodegenerative disease characterized by rapid deterioration of cognition and movement, leading to death within ten to thirty years of age. One of the thirteen Batten disease forms, CLN5 Batten disease, is caused by mutations in the CLN5 gene leading to motor deficits, mental deterioration, cognitive impairment, visual impairment, and epileptic seizures in children. A characteristic pathology in CLN5 Batten disease is the defects in lysosomes, leading to neuronal dysfunction. In this study, we aimed to investigate the lysosomal changes in CLN5-deficient human neurons. We used an induced pluripotent stem cell system, which generates pure human cortical-like glutamatergic neurons. Using CRISPRi, we inhibited the expression of CLN5 in human neurons. The CLN5-deficient human neurons showed neutralised lysosomal acidity and reduced lysosomal enzyme activity measured by microscopy and flow cytometry. Furthermore, the CLN5-deficient human neurons also showed impaired lysosomal movement, a phenotype that has never been reported in CLN5 Batten disease. Lysosomal trafficking is key to maintain local degradation of cellular wastes, especially in long neuronal projections and our results from the human neuronal model present a key finding to understand the underlying lysosomal pathology in neurodegenerative diseases

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 Multiple Alu exonization in 3’UTR of a primate specific isoform of CYP20A1 creates a potential miRNA sponge

2020 ◽  
Author(s):  
Aniket Bhattacharya ◽  
Vineet Jha ◽  
Khushboo Singhal ◽  
Mahar Fatima ◽  
Dayanidhi Singh ◽  
...  
Keyword(s):  
Heat Shock ◽  
Cortical Neurons ◽  
Regulatory Networks ◽  
Large Scale ◽  
Neuronal Development ◽  
Random Sets ◽  
Rna Seq ◽  
Orphan Gene ◽  
Mirna Sponge ◽  
Human Neurons

Abstract Alu repeats contribute to phylogenetic novelties in conserved regulatory networks in primates. Our study highlights how exonized Alus could nucleate large-scale mRNA-miRNA interactions. Using a functional genomics approach, we characterize a transcript isoform of an orphan gene, CYP20A1 (CYP20A1_Alu-LT) that has exonization of 23 Alus in its 3’UTR. CYP20A1_Alu-LT, confirmed by 3’RACE, is an outlier in length (9 kb 3’UTR) and widely expressed. Using publically available datasets, we demonstrate its expression in higher primates and presence in single nucleus RNA-seq of 15928 human cortical neurons. miRanda predicts ∼4700 miRNA recognition elements (MREs) for ∼1000 miRNAs, primarily originated within these 3’UTR-Alus. CYP20A1_Alu-LT could be a potential multi-miRNA sponge as it harbors ≥10 MREs for 140 miRNAs and has cytosolic localization. We further tested whether expression of CYP20A1_Alu-LT correlates with mRNAs harboring similar MRE targets. RNA-seq with conjoint miRNA-seq analysis was done in primary human neurons where we observed CYP20A1_Alu-LT to be downregulated during heat shock response and upregulated in HIV1-Tat treatment. 380 genes were positively correlated with its expression (significantly downregulated in heat shock and upregulated in Tat) and they harbored MREs for nine expressed miRNAs which were also enriched in CYP20A1_Alu-LT. MREs were significantly enriched in these 380 genes compared to random sets of differentially expressed genes (p = 8.134e-12). Gene ontology suggested involvement of these genes in neuronal development and hemostasis pathways thus proposing a novel component of Alu-miRNA mediated transcriptional modulation that could govern specific physiological outcomes in higher primates.

scholarly journals Cloning of an Avian Adeno-Associated Virus (AAAV) and Generation of Recombinant AAAV Particles

2003 ◽  
Vol 77 (12) ◽  
pp. 6799-6810 ◽  
Author(s):  
Ioannis Bossis ◽  
John A. Chiorini
Keyword(s):  
Nucleotide Substitution ◽  
Amino Acid Level ◽  
The Other ◽  
Transduction Efficiency ◽  
Entire Genome ◽  
Single Nucleotide ◽  
Adeno Associated Virus ◽  
Reading Frame ◽  
Palindromic Structure ◽  
The Relationship

ABSTRACT Recent studies have proposed that adeno-associated viruses (AAVs) are not evolutionarily linked to other mammalian autonomous parvoviruses but are more closely linked to the autonomous parvoviruses of birds. To better understand the relationship between primate and avian AAVs (AAAVs), we cloned and sequenced the genome of an AAAV (ATCC VR-865) and generated recombinant AAAV particles. The genome of AAAV is 4,694 nucleotides in length and has organization similar to that of other AAVs. The entire genome of AAAV displays 56 to 65% identity at the nucleotide level with the other known AAVs. The AAAV genome has inverted terminal repeats of 142 nucleotides, with the first 122 forming the characteristic T-shaped palindromic structure. The putative Rep-binding element consists of a tandem (GAGY)4 repeat, and the putative terminal resolution site (trs), CCGGT/CG, contains a single nucleotide substitution relative to the AAV2 trs. The Rep open reading frame of AAAV displays 50 to 54% identity at the amino acid level with the other AAVs, with most of the diversity clustered at the carboxyl and amino termini. Comparison of the capsid proteins of AAAV and the primate dependoviruses indicate that divergent regions are localized to surface-exposed loops. Despite these sequence differences, we were able to produce recombinant AAAV particles carrying a lacZ reporter gene by cotransfection in 293T cells and were able to examine transduction efficiency in both chicken primary cells and several cell lines. Our findings indicate that AAAV is the most divergent AAV described to date but maintains all the characteristics unique to the genera of dependovirus.

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