Complete Disruption of Autism-Susceptibility Genes by Gene Editing Predominantly Reduces Functional Connectivity of Isogenic Human Neurons

Deneault, Éric; White, Stuart C.; Rodrigues, Deivid C.; Ross, P. Joel; Faheem, Muhammad; Zaslavsky, Kirill; Wang, Zhuozhi; Alexandrova, Roumiana; Pellecchia, Giovanna; Wei, Wei; Piekna, Alina; Kaur, Gaganjot; Howe, Jennifer; Kwan, Vickie; Thiruvahindrapuram, Bhooma; Walker, Susan; Lionel, Anath C.; Pasceri, Peter; Merico, Daniele; Yuen, Ryan K. C.; Singh, Karun K.; Ellis, James; Scherer, Stephen W.

doi:10.1016/j.stemcr.2018.10.003

Cited by 113 publications

(92 citation statements)

References 53 publications

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“…One ASD-affected (black arrow) and one sex-matched unaffected (black star) members were typically selected for iPSC reprogramming. ASD-affected children are represented with a black box; note that line 1-0019-002 (19-2) in A) was used as a control and was described previously (Deneault et al, 2018). …”

Section: Resultsmentioning

confidence: 99%

“…We used the NGN2 ectopic expression approach since highly-enriched populations of glutamatergic neurons can be obtained within a week, and they exhibit robust synaptic activity when co-cultured with glial cells (Zhang et al, 2013). Importantly, we determined that this strategy offers highly uniform differentiation levels between cell lines derived from different participants (Deneault et al, 2018). This consistency was necessary to perform suitable phenotyping assays such as network electrophysiology recordings of several different lines in the same experimental batch.…”

Section: Resultsmentioning

confidence: 99%

“…Carrying the same precise repertoire of rare and common genetic variants as the donor proband, iPSC-derived neurons represent the best genetic mimic of proband neurons for functional and mechanistic studies. Induced differentiation can be achieved with high efficiency and consistency using transient ectopic expression of the transcription factor NGN2 (Ho et al, 2016; Zhang et al, 2013), and this has been shown to be useful in diverse phenotyping projects (Deneault et al, 2018; Pak et al, 2015; Yi et al, 2016). Proband-specific iPSC-derived neuronal cells indeed provide a useful model to study disease pathology, and response to drugs, but throughput (both iPSC-derived neurons and phenotyping) is low, with costs still high.…”

Section: Introductionmentioning

confidence: 99%

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CNTN5-/+or EHMT2-/+human iPSC-derived neurons from individuals with autism develop hyperactive neuronal networks

Deneault

Faheem

White

et al. 2019

eLife

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Induced pluripotent stem cell (iPSC)-derived neurons are increasingly used to model Autism Spectrum Disorder (ASD), which is clinically and genetically heterogeneous. To study the complex relationship of penetrant and weaker polygenic risk variants to ASD, ‘isogenic’ iPSC-derived neurons are critical. We developed a set of procedures to control for heterogeneity in reprogramming and differentiation, and generated 53 different iPSC-derived glutamatergic neuronal lines from 25 participants from 12 unrelated families with ASD. Heterozygous de novo and rare-inherited presumed-damaging variants were characterized in ASD risk genes/loci. Combinations of putative etiologic variants (GLI3/KIF21A or EHMT2/UBE2I) in separate families were modeled. We used a multi-electrode array, with patch-clamp recordings, to determine a reproducible synaptic phenotype in 25% of the individuals with ASD (other relevant data on the remaining lines was collected). Our most compelling new results revealed a consistent spontaneous network hyperactivity in neurons deficient for CNTN5 or EHMT2. The biobank of iPSC-derived neurons and accompanying genomic data are available to accelerate ASD research.Editorial note: This article has been through an editorial process in which authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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CNTN5-/+or EHMT2-/+human iPSC-derived neurons from individuals with autism develop hyperactive neuronal networks

Deneault

Faheem

White

et al. 2019

eLife

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“…To examine L124W protein levels in excitatory cortical neurons we used a rapid induced Neurogenin-2 (Ngn2) over-expression protocol (Deneault et al, 2018;Zhang et al, 2013) (Fig. S1E).…”

Section: Generation Of Mecp2 L124w Ipscs and Ngn2-derived Excitatory mentioning

confidence: 99%

“…Excitatory cortical neurons were generated as previously described (Deneault et al, 2018;Hildebrandt et al, 2019). Briefly, on Day 0 iPSCs were made into single-cells by accutase and plated at 3x10 5 -1x10 6 cells per well on Matrigel-coated 6-well plates with StemMACS iPS-Brew XF media supplemented with 10 μM ROCK inhibitor.…”

Section: Ngn2 Neuronal Differentiationmentioning

confidence: 99%

Wide spectrum of neuronal and network phenotypes in human stem cell-derived excitatory neurons with Rett syndrome-associatedMECP2mutations

Mok

Zhang

Sheikh

et al. 2020

Preprint

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Rett syndrome (RTT) is a severe neurodevelopmental disorder primarily caused by heterozygous loss-of-function mutations in the X-linked gene methyl-CpG-binding protein 2 (MECP2) that is a global transcriptional regulator. Mutations in the methyl-binding domain (MBD) of MECP2 disrupt its interaction with methylated DNA required for proper function in the brain. Here, we investigate the effect of a novel MECP2 L124W missense mutation in the MBD in comparison to MECP2 null mutations. L124W protein had a limited ability to disrupt heterochromatic chromocenters due to decreased binding dynamics. We isolated two pairs of isogenic WT and L124W induced pluripotent stem cell lines. L124W induced excitatory neurons expressed stable protein, exhibited only increased input resistance and impaired voltage-gated Na+ and K+ currents, and their neuronal dysmorphology was limited to reduced dendritic complexity. Three isogenic pairs of MECP2 null neurons had the expected more pronounced morphological and electrophysiological phenotypes, exhibiting decreased soma area, dendrite length, capacitance and excitatory synaptic function. We examined development and maturation of excitatory neural networks using micro-electrode arrays to detect alterations in RTT connectivity. The L124W neurons had no detectable changes in network circuitry features, in contrast to MECP2 null neurons that suffered a significant change in synchronous network burst frequency and a transient extension of network burst duration. Our results from stem cell-derived RTT excitatory neurons reveal a wide range of morphological, electrophysiological and circuitry phenotypes that reflect the severity of the MECP2 mutation.

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Rise of the Chromodomain Helicase DNA‐Binding (CHD) Chromatin Remodelling Machines

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2023

Encyclopedia of Life Sciences

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Vital processes such as transcription, repair, replication and recombination continuously shape cellular chromatin landscape. The chromodomain helicase DNA‐binding (CHD) family of enzymes regulate chromatin dynamics by utilising energy from ATP hydrolysis to alter nucleosomal position, structure and composition, thereby modulating accessibility of the underlying DNA sequence. The CHD enzymes are highly conserved in evolution, and genetic studies in fungi, plants and animals demonstrate their critical roles in regulation of cellular identity and fate and organismal development. Advances in genomic studies over the past two decades led to the identification of frequent DNA copy‐number alterations, mutations and aberrant expression of CHDs in human malignancies and various disorders, highlighting their importance as relevant biomarkers or targets for therapeutic intervention. Key Concepts CHD family of enzymes (CHD1‐9) are evolutionary conserved ATP‐dependent chromatin remodelers that mobilise nucleosomes to regulate DNA‐templated processes, such as transcription, replication and repair. They are critical for normal organismal development and are frequently mutated in human disorders and cancers. Chromodomain helicase DNA‐binding proteins (CHD1‐9) are evolutionary conserved family of ATP‐dependent chromatin remodelers that mobilise nucleosomes to regulate DNA‐templated processes such as transcription, replication and DNA repair. CHD enzymes share a common domain structure: two N‐terminal chromodomains, a central ATPase/helicase domain, and a C‐terminal DNA‐binding domain. CHD proteins play crucial roles in diverse cellular processes, including embryonic development, stem cell maintenance and differentiation, and tissue‐specific gene expression. Dysregulation of CHD proteins has been associated with various human diseases, including cancer and neurodevelopmental disorders. CHD proteins are attractive targets for the development of new therapies for diseases associated with chromatin dysfunction. Understanding the molecular mechanisms underlying the function of CHD proteins may pave the way for the development of new drugs that target these proteins and improve the treatment of a variety of human diseases.

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Complete Disruption of Autism-Susceptibility Genes by Gene Editing Predominantly Reduces Functional Connectivity of Isogenic Human Neurons

Cited by 113 publications

References 53 publications

CNTN5-/+or EHMT2-/+human iPSC-derived neurons from individuals with autism develop hyperactive neuronal networks

CNTN5-/+or EHMT2-/+human iPSC-derived neurons from individuals with autism develop hyperactive neuronal networks

Wide spectrum of neuronal and network phenotypes in human stem cell-derived excitatory neurons with Rett syndrome-associatedMECP2mutations

Rise of the Chromodomain Helicase DNA‐Binding (CHD) Chromatin Remodelling Machines

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