Publications by authors named "Kevin Eggan"

123 Publications

Screening Method for CRISPR/Cas9 Inhibition of a Human DNA Virus: Herpes Simplex Virus.

Bio Protoc 2020 Sep 5;10(17):e3748. Epub 2020 Sep 5.

Department of Microbiology, Blavatnik Institute, Harvard Medical School, Boston, USA.

The efficiency of cleavage of individual CRISPR/Cas9-sgRNAs remains difficult to predict based on the CRISPR target sequence alone. Different intracellular environments (dependent on cell type or cell cycle state for example) may affect sgRNA efficiency by altering accessibility of genomic DNA through DNA modifications such as epigenetic marks and DNA-binding proteins (, histones) as well as alteration of the chromatin state of genomic DNA within the nucleus. We recently reported a multi-step screening method for the identification of efficient sgRNAs targeting the Herpes simplex virus (HSV-1) genome and reported a differential mechanism for viral inhibition by CRISPR-Cas9 in the latent versus lytic phase. The screening platform detailed in this protocol allows step-by-step testing of the efficiency of cleavage in a cell-free system and in the context of viral target cells such as human foreskin fibroblasts followed by functional testing of the effects of CRISPR/sgRNA on viral protein expression, replication, and reactivation. This strategy could be readily applied to other target cells such as pluripotent stem cell-derived human sensory neurons or other human DNA viruses.
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http://dx.doi.org/10.21769/BioProtoc.3748DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7842287PMC
September 2020

Mind the translational gap: using iPS cell models to bridge from genetic discoveries to perturbed pathways and therapeutic targets.

Mol Autism 2021 02 8;12(1):10. Epub 2021 Feb 8.

Department of Stem Cell and Regenerative Biology, Department of Molecular and Cellular Biology, Harvard Stem Cell Institute, Cambridge, MA, 02138, USA.

Autism spectrum disorder (ASD) comprises a group of neurodevelopmental disorders characterized by impaired social interactions as well as the presentation of restrictive and repetitive behaviors. ASD is highly heritable but genetically heterogenous with both common and rare genetic variants collaborating to predispose individuals to the disorder. In this review, we synthesize recent efforts to develop human induced pluripotent stem cell (iPSC)-derived models of ASD-related phenotypes. We firstly address concerns regarding the relevance and validity of available neuronal iPSC-derived models. We then critically evaluate the robustness of various differentiation and cell culture protocols used for producing cell types of relevance to ASD. By exploring iPSC models of ASD reported thus far, we examine to what extent cellular and neuronal phenotypes with potential relevance to ASD can be linked to genetic variants found to underlie it. Lastly, we outline promising strategies by which iPSC technology can both enhance the power of genetic studies to identify ASD risk factors and nominate pathways that are disrupted across groups of ASD patients that might serve as common points for therapeutic intervention.
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http://dx.doi.org/10.1186/s13229-021-00417-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7869517PMC
February 2021

Retraction Notice to: Cancer-Related Mutations Identified in Primed and Naive Human Pluripotent Stem Cells.

Cell Stem Cell 2021 Jan 14;28(1):173. Epub 2020 Dec 14.

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http://dx.doi.org/10.1016/j.stem.2020.11.020DOI Listing
January 2021

Cancer-Related Mutations Identified in Primed Human Pluripotent Stem Cells.

Cell Stem Cell 2021 Jan 14;28(1):10-11. Epub 2020 Dec 14.

The Azrieli Center for Stem Cells and Genetic Research, Department of Genetics, Silberman Institute of Life Sciences, The Hebrew University, Jerusalem 9190401, Israel. Electronic address:

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http://dx.doi.org/10.1016/j.stem.2020.11.013DOI Listing
January 2021

Effect of Ezogabine on Cortical and Spinal Motor Neuron Excitability in Amyotrophic Lateral Sclerosis: A Randomized Clinical Trial.

JAMA Neurol 2021 Feb;78(2):186-196

The Sean M. Healey and AMG Center for ALS and the Neurological Clinical Research Institute, Department of Neurology, Massachusetts General Hospital, Boston.

Importance: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease of the motor nervous system. Clinical studies have demonstrated cortical and spinal motor neuron hyperexcitability using transcranial magnetic stimulation and threshold tracking nerve conduction studies, respectively, although metrics of excitability have not been used as pharmacodynamic biomarkers in multi-site clinical trials.

Objective: To ascertain whether ezogabine decreases cortical and spinal motor neuron excitability in ALS.

Design, Setting, And Participants: This double-blind, placebo-controlled phase 2 randomized clinical trial sought consent from eligible participants from November 3, 2015, to November 9, 2017, and was conducted at 12 US sites within the Northeast ALS Consortium. Participants were randomized in equal numbers to a higher or lower dose of ezogabine or to an identical matched placebo, and they completed in-person visits at screening, baseline, week 6, and week 8 for clinical assessment and neurophysiological measurements.

Interventions: Participants were randomized to receive 600 mg/d or 900 mg/d of ezogabine or a matched placebo for 10 weeks.

Main Outcomes And Measures: The primary outcome was change in short-interval intracortical inhibition (SICI; SICI-1 was used in analysis to reflect stronger inhibition from an increase in amplitude) from pretreatment mean at screening and baseline to the full-dose treatment mean at weeks 6 and 8. The secondary outcomes included levels of cortical motor neuron excitability (including resting motor threshold) measured by transcranial magnetic stimulation and spinal motor neuron excitability (including strength-duration time constant) measured by threshold tracking nerve conduction studies.

Results: A total of 65 participants were randomized to placebo (23), 600 mg/d of ezogabine (23), and 900 mg/d of ezogabine (19 participants); 45 were men (69.2%) and the mean (SD) age was 58.3 (8.8) years. The SICI-1 increased by 53% (mean ratio, 1.53; 95% CI, 1.12-2.09; P = .009) in the 900-mg/d ezogabine group vs placebo group. The SICI-1 did not change in the 600-mg/d ezogabine group vs placebo group (mean ratio, 1.15; 95% CI, 0.87-1.52; P = .31). The resting motor threshold increased in the 600-mg/d ezogabine group vs placebo group (mean ratio, 4.61; 95% CI, 0.21-9.01; P = .04) but not in the 900-mg/d ezogabine group vs placebo group (mean ratio, 1.95; 95% CI, -2.64 to 6.54; P = .40). Ezogabine caused a dose-dependent decrease in excitability by several other metrics, including strength-duration time constant in the 900-mg/d ezogabine group vs placebo group (mean ratio, 0.73; 95% CI, 0.60 to 0.87; P < .001).

Conclusions And Relevance: Ezogabine decreased cortical and spinal motor neuron excitability in participants with ALS, suggesting that such neurophysiological metrics may be used as pharmacodynamic biomarkers in multisite clinical trials.

Trial Registration: ClinicalTrials.gov Identifier: NCT02450552.
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http://dx.doi.org/10.1001/jamaneurol.2020.4300DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7684515PMC
February 2021

Absence of Survival and Motor Deficits in 500 Repeat C9ORF72 BAC Mice.

Neuron 2020 11 5;108(4):775-783.e4. Epub 2020 Oct 5.

Department of Neurology, The Johns Hopkins University, Baltimore, MD 21205, USA; Brain Science Institute, The Johns Hopkins University, Baltimore, MD 21205, USA. Electronic address:

A hexanucleotide repeat expansion at C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD). Initial studies of bacterial artificial chromosome (BAC) transgenic mice harboring this expansion described an absence of motor and survival phenotypes. However, a recent study by Liu and colleagues described transgenic mice harboring a large repeat expansion (C9-500) and reported decreased survival and progressive motor phenotypes. To determine the utility of the C9-500 animals for understanding degenerative mechanisms, we validated and established two independent colonies of transgene carriers. However, extended studies of these animals for up to 1 year revealed no reproducible abnormalities in survival, motor function, or neurodegeneration. Here, we propose several potential explanations for the disparate nature of our findings from those of Liu and colleagues. Resolving the discrepancies we identify will be essential to settle the translational utility of C9-500 mice. This Matters Arising paper is in response to Liu et al. (2016), published in Neuron. See also the response by Nguyen et al. (2020), published in this issue.
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http://dx.doi.org/10.1016/j.neuron.2020.08.009DOI Listing
November 2020

C9orf72 suppresses systemic and neural inflammation induced by gut bacteria.

Nature 2020 06 13;582(7810):89-94. Epub 2020 May 13.

Harvard Stem Cell Institute, Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA.

A hexanucleotide-repeat expansion in C9ORF72 is the most common genetic variant that contributes to amyotrophic lateral sclerosis and frontotemporal dementia. The C9ORF72 mutation acts through gain- and loss-of-function mechanisms to induce pathways that are implicated in neural degeneration. The expansion is transcribed into a long repetitive RNA, which negatively sequesters RNA-binding proteins before its non-canonical translation into neural-toxic dipeptide proteins. The failure of RNA polymerase to read through the mutation also reduces the abundance of the endogenous C9ORF72 gene product, which functions in endolysosomal pathways and suppresses systemic and neural inflammation. Notably, the effects of the repeat expansion act with incomplete penetrance in families with a high prevalence of amyotrophic lateral sclerosis or frontotemporal dementia, indicating that either genetic or environmental factors modify the risk of disease for each individual. Identifying disease modifiers is of considerable translational interest, as it could suggest strategies to diminish the risk of developing amyotrophic lateral sclerosis or frontotemporal dementia, or to slow progression. Here we report that an environment with reduced abundance of immune-stimulating bacteria protects C9orf72-mutant mice from premature mortality and significantly ameliorates their underlying systemic inflammation and autoimmunity. Consistent with C9orf72 functioning to prevent microbiota from inducing a pathological inflammatory response, we found that reducing the microbial burden in mutant mice with broad spectrum antibiotics-as well as transplanting gut microflora from a protective environment-attenuated inflammatory phenotypes, even after their onset. Our studies provide further evidence that the microbial composition of our gut has an important role in brain health and can interact in surprising ways with well-known genetic risk factors for disorders of the nervous system.
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http://dx.doi.org/10.1038/s41586-020-2288-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7416879PMC
June 2020

A multiplexed gRNA piggyBac transposon system facilitates efficient induction of CRISPRi and CRISPRa in human pluripotent stem cells.

Sci Rep 2020 01 20;10(1):635. Epub 2020 Jan 20.

Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, 02142, USA.

CRISPR-Cas9-mediated gene interference (CRISPRi) and activation (CRISPRa) approaches hold promise for functional gene studies and genome-wide screens in human pluripotent stem cells (hPSCs). However, in contrast to CRISPR-Cas9 nuclease approaches, the efficiency of CRISPRi/a depends on continued expression of the dead Cas9 (dCas9) effector and guide RNA (gRNA), which can vary substantially depending on transgene design and delivery. Here, we design and generate new fluorescently labeled piggyBac (PB) vectors to deliver uniform and sustained expression of multiplexed gRNAs. In addition, we generate hPSC lines harboring AAVS1-integrated, inducible and fluorescent dCas9-KRAB and dCas9-VPR transgenes to allow for accurate quantification and tracking of cells that express both the dCas9 effectors and gRNAs. We then employ these systems to target the TCF4 gene in hPSCs and assess expression levels of the dCas9 effectors, individual gRNAs and targeted gene. We also assess the performance of our PB system for single gRNA delivery, confirming its utility for library format applications. Collectively, our results provide proof-of-principle application of a stable, multiplexed PB gRNA delivery system that can be widely exploited to further enable genome engineering studies in hPSCs. Paired with diverse CRISPR tools including our dual fluorescence CRISPRi/a cell lines, this system can facilitate functional dissection of individual genes and pathways as well as larger-scale screens for studies of development and disease.
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http://dx.doi.org/10.1038/s41598-020-57500-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6971260PMC
January 2020

Herpesviral lytic gene functions render the viral genome susceptible to novel editing by CRISPR/Cas9.

Elife 2019 12 2;8. Epub 2019 Dec 2.

Department of Microbiology, Blavatnik Institute, Harvard Medical School, Boston, United States.

Herpes simplex virus (HSV) establishes lifelong latent infection and can cause serious human disease, but current antiviral therapies target lytic but not latent infection. We screened for sgRNAs that cleave HSV-1 DNA sequences efficiently in vitro and used these sgRNAs to observe the first editing of quiescent HSV-1 DNA. The sgRNAs targeted lytic replicating viral DNA genomes more efficiently than quiescent genomes, consistent with the open structure of lytic chromatin. Editing of latent genomes caused short indels while editing of replicating genomes produced indels, linear molecules, and large genomic sequence loss around the gRNA target site. The HSV ICP0 protein and viral DNA replication increased the loss of DNA sequences around the gRNA target site. We conclude that HSV, by promoting open chromatin needed for viral gene expression and by inhibiting the DNA damage response, makes the genome vulnerable to a novel form of editing by CRISPR-Cas9 during lytic replication.
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http://dx.doi.org/10.7554/eLife.51662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6917492PMC
December 2019

Exome sequencing in amyotrophic lateral sclerosis implicates a novel gene, DNAJC7, encoding a heat-shock protein.

Nat Neurosci 2019 12 25;22(12):1966-1974. Epub 2019 Nov 25.

Analytic and Translational Genetics Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA.

To discover novel genes underlying amyotrophic lateral sclerosis (ALS), we aggregated exomes from 3,864 cases and 7,839 ancestry-matched controls. We observed a significant excess of rare protein-truncating variants among ALS cases, and these variants were concentrated in constrained genes. Through gene level analyses, we replicated known ALS genes including SOD1, NEK1 and FUS. We also observed multiple distinct protein-truncating variants in a highly constrained gene, DNAJC7. The signal in DNAJC7 exceeded genome-wide significance, and immunoblotting assays showed depletion of DNAJC7 protein in fibroblasts in a patient with ALS carrying the p.Arg156Ter variant. DNAJC7 encodes a member of the heat-shock protein family, HSP40, which, along with HSP70 proteins, facilitates protein homeostasis, including folding of newly synthesized polypeptides and clearance of degraded proteins. When these processes are not regulated, misfolding and accumulation of aberrant proteins can occur and lead to protein aggregation, which is a pathological hallmark of neurodegeneration. Our results highlight DNAJC7 as a novel gene for ALS.
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http://dx.doi.org/10.1038/s41593-019-0530-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6919277PMC
December 2019

RNA-seq as a tool for evaluating human embryo competence.

Genome Res 2019 10 23;29(10):1705-1718. Epub 2019 Sep 23.

Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts 02138, USA.

The majority of embryos created through in vitro fertilization (IVF) do not implant. It seems plausible that rates of implantation would improve if we had a better understanding of molecular factors affecting embryo competence. Currently, the process of selecting an embryo for uterine transfer uses an ad hoc combination of morphological criteria, the kinetics of development, and genetic testing for aneuploidy. However, no single criterion can ensure selection of a viable embryo. In contrast, RNA-sequencing (RNA-seq) of embryos could yield high-dimensional data, which may provide additional insight and illuminate the discrepancies among current selection criteria. Recent advances enabling the production of RNA-seq libraries from single cells have facilitated the application of this technique to the study of transcriptional events in early human development. However, these studies have not assessed the quality of their constituent embryos relative to commonly used embryological criteria. Here, we perform proof-of-principle advancement to embryo selection procedures by generating RNA-seq libraries from a trophectoderm biopsy as well as the remaining whole embryo. We combine state-of-the-art embryological methods with low-input RNA-seq to develop the first transcriptome-wide approach for assessing embryo competence. Specifically, we show the capacity of RNA-seq as a promising tool in preimplantation screening by showing that biopsies of an embryo can capture valuable information available in the whole embryo from which they are derived. Furthermore, we show that this technique can be used to generate a RNA-based digital karyotype and to identify candidate competence-associated genes. Together, these data establish the foundation for a future RNA-based diagnostic in IVF.
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http://dx.doi.org/10.1101/gr.252981.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6771404PMC
October 2019

Cancer-Related Mutations Identified in Primed and Naive Human Pluripotent Stem Cells.

Cell Stem Cell 2019 Oct 19;25(4):456-461. Epub 2019 Sep 19.

The Azrieli Center for Stem Cells and Genetic Research, Department of Genetics, Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Edmond J. Safra Campus - Givat Ram, Jerusalem, Israel, 9190401. Electronic address:

Human pluripotent stem cells (hPSCs) are known to harbor chromosomal aberrations, affecting their tumorigenic potential. We established a strategy to identify cancer-related point mutations in hPSCs, detecting recurrent mutations in over 20 genes, alongside those previously detected in p53. Importantly, naive hPSCs harbor, on average, four times more mutations than their primed counterparts, which appear primarily in pathways inhibited during naive conversion. Such cancer-related mutations should be taken into consideration in future applications, especially in clinical contexts.
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http://dx.doi.org/10.1016/j.stem.2019.09.001DOI Listing
October 2019

A High-Content Screen Identifies TPP1 and Aurora B as Regulators of Axonal Mitochondrial Transport.

Cell Rep 2019 Sep;28(12):3224-3237.e5

F.M. Kirby Neurobiology Center, Boston Children's Hospital, Boston, MA 02115, USA; Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Dysregulated axonal trafficking of mitochondria is linked to neurodegenerative disorders. We report a high-content screen for small-molecule regulators of the axonal transport of mitochondria. Six compounds enhanced mitochondrial transport in the sub-micromolar range, acting via three cellular targets: F-actin, Tripeptidyl peptidase 1 (TPP1), or Aurora Kinase B (AurKB). Pharmacological inhibition or small hairpin RNA (shRNA) knockdown of each target promotes mitochondrial axonal transport in rat hippocampal neurons and induced pluripotent stem cell (iPSC)-derived human cortical neurons and enhances mitochondrial transport in iPSC-derived motor neurons from an amyotrophic lateral sclerosis (ALS) patient bearing one copy of SOD1 mutation. Our work identifies druggable regulators of axonal transport of mitochondria, provides broadly applicable methods for similar image-based screens, and suggests that restoration of proper axonal trafficking of mitochondria can be achieved in human ALS neurons.
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http://dx.doi.org/10.1016/j.celrep.2019.08.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6937139PMC
September 2019

Comparison of three congruent patient-specific cell types for the modelling of a human genetic Schwann-cell disorder.

Nat Biomed Eng 2019 07 8;3(7):571-582. Epub 2019 Apr 8.

Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

Patient-specific human-induced pluripotent stem cells (hiPSCs) hold great promise for the modelling of genetic disorders. However, these cells display wide intra- and interindividual variations in gene expression, which makes distinguishing true-positive and false-positive phenotypes challenging. Data from hiPSC phenotypes and human embryonic stem cells (hESCs) harbouring the same disease mutation are also lacking. Here, we report a comparison of the molecular, cellular and functional characteristics of three congruent patient-specific cell types-hiPSCs, hESCs and direct-lineage-converted cells-derived from currently available differentiation and direct-reprogramming technologies for use in the modelling of Charcot-Marie-Tooth 1A, a human genetic Schwann-cell disorder featuring a 1.4 Mb chromosomal duplication. We find that the chemokines C-X-C motif ligand chemokine-1 (CXCL1) and macrophage chemoattractant protein-1 (MCP1) are commonly upregulated in all three congruent models and in clinical patient samples. The development of congruent models of a single genetic disease using somatic cells from a common patient will facilitate the search for convergent phenotypes.
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http://dx.doi.org/10.1038/s41551-019-0381-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6612317PMC
July 2019

Dysregulated protocadherin-pathway activity as an intrinsic defect in induced pluripotent stem cell-derived cortical interneurons from subjects with schizophrenia.

Nat Neurosci 2019 02 21;22(2):229-242. Epub 2019 Jan 21.

Department of Psychiatry, McLean Hospital/Harvard Medical School, Belmont, MA, USA.

We generated cortical interneurons (cINs) from induced pluripotent stem cells derived from 14 healthy controls and 14 subjects with schizophrenia. Both healthy control cINs and schizophrenia cINs were authentic, fired spontaneously, received functional excitatory inputs from host neurons, and induced GABA-mediated inhibition in host neurons in vivo. However, schizophrenia cINs had dysregulated expression of protocadherin genes, which lie within documented schizophrenia loci. Mice lacking protocadherin-α showed defective arborization and synaptic density of prefrontal cortex cINs and behavioral abnormalities. Schizophrenia cINs similarly showed defects in synaptic density and arborization that were reversed by inhibitors of protein kinase C, a downstream kinase in the protocadherin pathway. These findings reveal an intrinsic abnormality in schizophrenia cINs in the absence of any circuit-driven pathology. They also demonstrate the utility of homogenous and functional populations of a relevant neuronal subtype for probing pathogenesis mechanisms during development.
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http://dx.doi.org/10.1038/s41593-018-0313-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6373728PMC
February 2019

ALS-implicated protein TDP-43 sustains levels of STMN2, a mediator of motor neuron growth and repair.

Nat Neurosci 2019 02 14;22(2):167-179. Epub 2019 Jan 14.

Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA.

The findings that amyotrophic lateral sclerosis (ALS) patients almost universally display pathological mislocalization of the RNA-binding protein TDP-43 and that mutations in its gene cause familial ALS have nominated altered RNA metabolism as a disease mechanism. However, the RNAs regulated by TDP-43 in motor neurons and their connection to neuropathy remain to be identified. Here we report transcripts whose abundances in human motor neurons are sensitive to TDP-43 depletion. Notably, expression of STMN2, which encodes a microtubule regulator, declined after TDP-43 knockdown and TDP-43 mislocalization as well as in patient-specific motor neurons and postmortem patient spinal cord. STMN2 loss upon reduced TDP-43 function was due to altered splicing, which is functionally important, as we show STMN2 is necessary for normal axonal outgrowth and regeneration. Notably, post-translational stabilization of STMN2 rescued neurite outgrowth and axon regeneration deficits induced by TDP-43 depletion. We propose that restoring STMN2 expression warrants examination as a therapeutic strategy for ALS.
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http://dx.doi.org/10.1038/s41593-018-0300-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7153761PMC
February 2019

A Stem Cell-Based Screening Platform Identifies Compounds that Desensitize Motor Neurons to Endoplasmic Reticulum Stress.

Mol Ther 2019 01 19;27(1):87-101. Epub 2018 Oct 19.

Department of Pathology and Cell Biology, Center for Motor Neuron Biology and Disease, Columbia Stem Cell Initiative, Columbia University Irving Medical Center, 630 West 168th Street, New York, NY 10032, USA; Departments of Neuroscience, Rehabilitation and Regenerative Medicine, and Neurology, Columbia University Irving Medical Center, 630 West 168th Street, New York, NY 10032, USA. Electronic address:

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease selectively targeting motor neurons in the brain and spinal cord. The reasons for differential motor neuron susceptibility remain elusive. We developed a stem cell-based motor neuron assay to study cell-autonomous mechanisms causing motor neuron degeneration, with implications for ALS. A small-molecule screen identified cyclopiazonic acid (CPA) as a stressor to which stem cell-derived motor neurons were more sensitive than interneurons. CPA induced endoplasmic reticulum stress and the unfolded protein response. Furthermore, CPA resulted in an accelerated degeneration of motor neurons expressing human superoxide dismutase 1 (hSOD1) carrying the ALS-causing G93A mutation, compared to motor neurons expressing wild-type hSOD1. A secondary screen identified compounds that alleviated CPA-mediated motor neuron degeneration: three kinase inhibitors and tauroursodeoxycholic acid (TUDCA), a bile acid derivative. The neuroprotective effects of these compounds were validated in human stem cell-derived motor neurons carrying a mutated SOD1 allele (hSOD1). Moreover, we found that the administration of TUDCA in an hSOD1 mouse model of ALS reduced muscle denervation. Jointly, these results provide insights into the mechanisms contributing to the preferential susceptibility of ALS motor neurons, and they demonstrate the utility of stem cell-derived motor neurons for the discovery of new neuroprotective compounds.
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http://dx.doi.org/10.1016/j.ymthe.2018.10.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6318783PMC
January 2019

Convergence of independent DISC1 mutations on impaired neurite growth via decreased UNC5D expression.

Transl Psychiatry 2018 11 8;8(1):245. Epub 2018 Nov 8.

Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA.

The identification of convergent phenotypes in different models of psychiatric illness highlights robust phenotypes that are more likely to be implicated in disease pathophysiology. Here, we utilize human iPSCs harboring distinct mutations in DISC1 that have been found in families with major mental illness. One mutation was engineered to mimic the consequences on DISC1 protein of a balanced translocation linked to mental illness in a Scottish pedigree; the other mutation was identified in an American pedigree with a high incidence of mental illness. Directed differentiation of these iPSCs using NGN2 expression shows rapid conversion to a homogenous population of mature excitatory neurons. Both DISC1 mutations result in reduced DISC1 protein expression, and show subtle effects on certain presynaptic proteins. In addition, RNA sequencing and qPCR showed decreased expression of UNC5D, DPP10, PCDHA6, and ZNF506 in neurons with both DISC1 mutations. Longitudinal analysis of neurite outgrowth revealed decreased neurite outgrowth in neurons with each DISC1 mutation, which was mimicked by UNC5D knockdown and rescued by transient upregulation of endogenous UNC5D. This study shows a narrow range of convergent phenotypes of two mutations found in families with major mental illness, and implicates dysregulated netrin signaling in DISC1 biology.
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http://dx.doi.org/10.1038/s41398-018-0281-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6224395PMC
November 2018

Oligodendrocyte differentiation of induced pluripotent stem cells derived from subjects with schizophrenias implicate abnormalities in development.

Transl Psychiatry 2018 10 23;8(1):230. Epub 2018 Oct 23.

Harvard Medical School, Boston, MA, USA.

Abnormalities of brain connectivity and signal transduction are consistently observed in individuals with schizophrenias (SZ). Underlying these anomalies, convergent in vivo, post mortem, and genomic evidence suggest abnormal oligodendrocyte (OL) development and function and lower myelination in SZ. Our primary hypothesis was that there would be abnormalities in the number of induced pluripotent stem (iPS) cell-derived OLs from subjects with SZ. Our secondary hypothesis was that these in vitro abnormalities would correlate with measures of white matter (WM) integrity and myelination in the same subjects in vivo, estimated from magnetic resonance imaging. Six healthy control (HC) and six SZ iPS cell lines, derived from skin fibroblasts from well-characterized subjects, were differentiated into OLs. FACS analysis of the oligodendrocyte-specific surface, glycoprotein O4, was performed at three time points of development (days 65, 75, and 85) to quantify the number of late oligodendrocyte progenitor cells (OPCs) and OLs in each line. Significantly fewer O4-positive cells developed from SZ versus HC lines (95% CI 1.0: 8.6, F = 8.06, p = 0.02). The difference was greater when corrected for age (95% CI 5.4:10.4, F = 53.6, p < 0.001). A correlation between myelin content in WM in vivo, estimated by magnetization transfer ratio (MTR) and number of O4-positive cells in vitro was also observed across all time points (F = 4.3, p = 0.07), reaching significance for mature OLs at day 85 in culture (r = 0.70, p < 0.02). Low production of OPCs may be a contributing mechanism underlying WM reduction in SZ.
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http://dx.doi.org/10.1038/s41398-018-0284-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6199264PMC
October 2018

Comparative genomic analysis of embryonic, lineage-converted and stem cell-derived motor neurons.

Development 2018 11 21;145(22). Epub 2018 Nov 21.

Harvard Stem Cell Institute, Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA

Advances in stem cell science allow the production of different cell types either through the recapitulation of developmental processes, often termed 'directed differentiation', or the forced expression of lineage-specific transcription factors. Although cells produced by both approaches are increasingly used in translational applications, their quantitative similarity to their primary counterparts remains largely unresolved. To investigate the similarity between -derived and primary cell types, we harvested and purified mouse spinal motor neurons and compared them with motor neurons produced by transcription factor-mediated lineage conversion of fibroblasts or directed differentiation of pluripotent stem cells. To enable unbiased analysis of these motor neuron types and their cells of origin, we then subjected them to whole transcriptome and DNA methylome analysis by RNA sequencing (RNA-seq) and reduced representation bisulfite sequencing (RRBS). Despite major differences in methodology, lineage conversion and directed differentiation both produce cells that closely approximate the primary motor neuron state. However, we identify differences in Fas signaling, the Hox code and synaptic gene expression between lineage-converted and directed differentiation motor neurons that affect their utility in translational studies.
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http://dx.doi.org/10.1242/dev.168617DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6262794PMC
November 2018

All-optical synaptic electrophysiology probes mechanism of ketamine-induced disinhibition.

Nat Methods 2018 10 1;15(10):823-831. Epub 2018 Oct 1.

Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA.

Optical assays of synaptic strength could facilitate studies of neuronal transmission and its dysregulation in disease. Here we introduce a genetic toolbox for all-optical interrogation of synaptic electrophysiology (synOptopatch) via mutually exclusive expression of a channelrhodopsin actuator and an archaerhodopsin-derived voltage indicator. Optically induced activity in the channelrhodopsin-expressing neurons generated excitatory and inhibitory postsynaptic potentials that we optically resolved in reporter-expressing neurons. We further developed a yellow spine-targeted Ca indicator to localize optogenetically triggered synaptic inputs. We demonstrated synOptopatch recordings in cultured rodent neurons and in acute rodent brain slice. In synOptopatch measurements of primary rodent cultures, acute ketamine administration suppressed disynaptic inhibitory feedbacks, mimicking the effect of this drug on network function in both rodents and humans. We localized this action of ketamine to excitatory synapses onto interneurons. These results establish an in vitro all-optical model of disynaptic disinhibition, a synaptic defect hypothesized in schizophrenia-associated psychosis.
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http://dx.doi.org/10.1038/s41592-018-0142-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6204345PMC
October 2018

Hippo signaling in the ovary and polycystic ovarian syndrome.

J Assist Reprod Genet 2018 Oct 17;35(10):1763-1771. Epub 2018 Aug 17.

Boston IVF, 130 Second Ave., Waltham, MA, 02451, USA.

Purpose: To provide a commentary on our understanding of the role that the Hippo signaling pathway may play in patients with polycystic ovarian syndrome (PCOS) and how this understanding may impact the diagnosis of PCOS.

Methods: We assessed publications discussing the role of the Hippo signaling pathway in the ovary. In particular, we discuss how Hippo signaling disruption after ovarian fragmentation, combined with treating ovarian fragments with phosphatase and tensin homolog (PTEN) inhibitors and phosphoinositide-3-kinase stimulators to augment AKT signaling, has been used in treatment of patients with primary ovarian insufficiency. Furthermore, we discuss our own data on variations in Hippo signaling pathway gene expression in cumulus cells isolated from women undergoing IVF with a previous diagnosis of PCOS.

Results And Conclusions: Aberrant Hippo signaling in PCOS patients is likely a contributing mechanism to the multifactorial etiology of the disease. Given the challenge of discerning the underlying etiology of oligo-ovulation in some patients, especially those with normal body mass indices, and the need for customized stimulation protocols for PCOS patients who have an increased risk of over-response and higher percentage of immature oocyte yield, it is important to identify these patients prior to treatment. Hippo gene expression fingerprints could potentially be used to more accurately define patients with PCOS. Additionally, targeting this pathway with pharmacologic agents could lead to non-surgical therapeutic options for PCOS.
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http://dx.doi.org/10.1007/s10815-018-1235-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6150890PMC
October 2018

Dipeptide repeat proteins activate a heat shock response found in C9ORF72-ALS/FTLD patients.

Acta Neuropathol Commun 2018 07 4;6(1):55. Epub 2018 Jul 4.

Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, 02138, USA.

A hexanucleotide (GGGGCC) repeat expansion in C9ORF72 is the most common genetic contributor to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Reduced expression of the C9ORF72 gene product has been proposed as a potential contributor to disease pathogenesis. Additionally, repetitive RNAs and dipeptide repeat proteins (DPRs), such as poly-GR, can be produced by this hexanucleotide expansion that disrupt a number of cellular processes, potentially contributing to neural degeneration. To better discern which of these mechanisms leads to disease-associated changes in patient brains, we analyzed gene expression data generated from the cortex and cerebellum. We found that transcripts encoding heat shock proteins (HSPs) regulated by the HSF1 transcription factor were significantly induced in C9ORF72-ALS/FTLD patients relative to both sporadic ALS/FTLD cases and controls. Treatment of human neurons with chemically synthesized DPRs was sufficient to activate a similar transcriptional response. Expression of GGGGCC repeats and also poly-GR in the brains of Drosophila lead to the upregulation of HSF1 and the same highly-conserved HSPs. Additionally, HSF1 was a modifier of poly-GR toxicity in Drosophila. Our results suggest that the expression of DPRs are associated with upregulation of HSF1 and activation of a heat shock response in C9ORF72-ALS/FTLD.
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http://dx.doi.org/10.1186/s40478-018-0555-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6031111PMC
July 2018

The C9orf72-interacting protein Smcr8 is a negative regulator of autoimmunity and lysosomal exocytosis.

Genes Dev 2018 07 27;32(13-14):929-943. Epub 2018 Jun 27.

Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts 02138, USA.

While a mutation in is the most common genetic contributor to amyotrophic lateral sclerosis (ALS), much remains to be learned concerning the function of the protein normally encoded at this locus. To elaborate further on functions for C9ORF72, we used quantitative mass spectrometry-based proteomics to identify interacting proteins in motor neurons and found that its long isoform complexes with and stabilizes SMCR8, which further enables interaction with WDR41. To study the organismal and cellular functions for this tripartite complex, we generated loss-of-function mutant mice and found that they developed phenotypes also observed in loss-of-function animals, including autoimmunity. Along with a loss of tolerance for many nervous system autoantigens, we found increased lysosomal exocytosis in mutant macrophages. In addition to elevated surface Lamp1 (lysosome-associated membrane protein 1) expression, we also observed enhanced secretion of lysosomal components-phenotypes that we subsequently observed in loss-of-function macrophages. Overall, our findings demonstrate that C9ORF72 and SMCR8 have interdependent functions in suppressing autoimmunity as well as negatively regulating lysosomal exocytosis-processes of potential importance to ALS.
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http://dx.doi.org/10.1101/gad.313932.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6075033PMC
July 2018

TDP-43 induces p53-mediated cell death of cortical progenitors and immature neurons.

Sci Rep 2018 05 25;8(1):8097. Epub 2018 May 25.

Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058, Basel, Switzerland.

TAR DNA-binding protein 43 (TDP-43) is a key player in neurodegenerative diseases including frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Accumulation of TDP-43 is associated with neuronal death in the brain. How increased and disease-causing mutant forms of TDP-43 induce cell death remains unclear. Here we addressed the role of TDP-43 during neural development and show that reduced TDP-43 causes defects in neural stem/progenitor cell proliferation but not cell death. However, overexpression of wild type and TDP-43 proteins induce p53-dependent apoptosis of neural stem/progenitors and human induced pluripotent cell (iPS)-derived immature cortical neurons. We show that TDP-43 induces expression of the proapoptotic BH3-only genes Bbc3 and Bax, and that p53 inhibition rescues TDP-43 induced cell death of embryonic mouse, and human cortical neurons, including those derived from TDP-43 ALS patient iPS cells. Hence, an increase in wild type and mutant TDP-43 induces p53-dependent cell death in neural progenitors developing neurons and this can be rescued. These findings may have important implications for accumulated or mutant TDP-43 induced neurodegenerative diseases.
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http://dx.doi.org/10.1038/s41598-018-26397-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5970242PMC
May 2018

Combining NGN2 Programming with Developmental Patterning Generates Human Excitatory Neurons with NMDAR-Mediated Synaptic Transmission.

Cell Rep 2018 05;23(8):2509-2523

Stanley Center for Psychiatric Research, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA. Electronic address:

Transcription factor programming of pluripotent stem cells (PSCs) has emerged as an approach to generate human neurons for disease modeling. However, programming schemes produce a variety of cell types, and those neurons that are made often retain an immature phenotype, which limits their utility in modeling neuronal processes, including synaptic transmission. We report that combining NGN2 programming with SMAD and WNT inhibition generates human patterned induced neurons (hpiNs). Single-cell analyses showed that hpiN cultures contained cells along a developmental continuum, ranging from poorly differentiated neuronal progenitors to well-differentiated, excitatory glutamatergic neurons. The most differentiated neurons could be identified using a CAMK2A::GFP reporter gene and exhibited greater functionality, including NMDAR-mediated synaptic transmission. We conclude that utilizing single-cell and reporter gene approaches for selecting successfully programmed cells for study will greatly enhance the utility of hpiNs and other programmed neuronal populations in the modeling of nervous system disorders.
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http://dx.doi.org/10.1016/j.celrep.2018.04.066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6003669PMC
May 2018

All-Optical Electrophysiology for High-Throughput Functional Characterization of a Human iPSC-Derived Motor Neuron Model of ALS.

Stem Cell Reports 2018 06 17;10(6):1991-2004. Epub 2018 May 17.

Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA; Department of Physics, Harvard University, Cambridge, MA 02138, USA; Howard Hughes Medical Institute, Chevy Chase, MD, USA. Electronic address:

Human induced pluripotent stem cell (iPSC)-derived neurons are an attractive substrate for modeling disease, yet the heterogeneity of these cultures presents a challenge for functional characterization by manual patch-clamp electrophysiology. Here, we describe an optimized all-optical electrophysiology, "Optopatch," pipeline for high-throughput functional characterization of human iPSC-derived neuronal cultures. We demonstrate the method in a human iPSC-derived motor neuron (iPSC-MN) model of amyotrophic lateral sclerosis (ALS). In a comparison of iPSC-MNs with an ALS-causing mutation (SOD1 A4V) with their genome-corrected controls, the mutants showed elevated spike rates under weak or no stimulus and greater likelihood of entering depolarization block under strong optogenetic stimulus. We compared these results with numerical simulations of simple conductance-based neuronal models and with literature results in this and other iPSC-based models of ALS. Our data and simulations suggest that deficits in slowly activating potassium channels may underlie the changes in electrophysiology in the SOD1 A4V mutation.
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http://dx.doi.org/10.1016/j.stemcr.2018.04.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5993648PMC
June 2018

Publisher Correction: Whole genome sequencing in psychiatric disorders: the WGSPD consortium.

Nat Neurosci 2018 Jul;21(7):1017

Center for Neurobehavioral Genetics, Semel Institute for Neuroscience and Human Behavior, University of California Los Angeles, Los Angeles, CA, USA.

In the version of this article initially published, the consortium authorship and corresponding authors were not presented correctly. In the PDF and print versions, the Whole Genome Sequencing for Psychiatric Disorders (WGSPD) consortium was missing from the author list at the beginning of the paper, where it should have appeared as the seventh author; it was present in the author list at the end of the paper, but the footnote directing readers to the Supplementary Note for a list of members was missing. In the HTML version, the consortium was listed as the last author instead of as the seventh, and the line directing readers to the Supplementary Note for a list of members appeared at the end of the paper under Author Information but not in association with the consortium name itself. Also, this line stated that both member names and affiliations could be found in the Supplementary Note; in fact, only names are given. In all versions of the paper, the corresponding author symbols were attached to A. Jeremy Willsey, Steven E. Hyman, Anjene M. Addington and Thomas Lehner; they should have been attached, respectively, to Steven E. Hyman, Anjene M. Addington, Thomas Lehner and Nelson B. Freimer. As a result of this shift, the respective contact links in the HTML version did not lead to the indicated individuals. The errors have been corrected in the HTML and PDF versions of the article.
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http://dx.doi.org/10.1038/s41593-018-0102-8DOI Listing
July 2018