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1.
bioRxiv ; 2023 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-37961635

RESUMO

As genetic studies continue to identify risk loci that are significantly associated with risk for neuropsychiatric disease, a critical unanswered question is the extent to which diverse mutations--sometimes impacting the same gene-- will require tailored therapeutic strategies. Here we consider this in the context of rare neuropsychiatric disorder-associated copy number variants (2p16.3) resulting in heterozygous deletions in NRXN1, a pre-synaptic cell adhesion protein that serves as a critical synaptic organizer in the brain. Complex patterns of NRXN1 alternative splicing are fundamental to establishing diverse neurocircuitry, vary between the cell types of the brain, and are differentially impacted by unique (non-recurrent) deletions. We contrast the cell-type-specific impact of patient-specific mutations in NRXN1 using human induced pluripotent stem cells, finding that perturbations in NRXN1 splicing result in divergent cell-type-specific synaptic outcomes. Via distinct loss-of-function (LOF) and gain-of-function (GOF) mechanisms, NRXN1+/- deletions cause decreased synaptic activity in glutamatergic neurons, yet increased synaptic activity in GABAergic neurons. Stratification of patients by LOF and GOF mechanisms will facilitate individualized restoration of NRXN1 isoform repertoires; towards this, antisense oligonucleotides knockdown mutant isoform expression and alters synaptic transcriptional signatures, while treatment with ß-estradiol rescues synaptic function in glutamatergic neurons. Given the increasing number of mutations predicted to engender both LOF and GOF mechanisms in brain disease, our findings add nuance to future considerations of precision medicine.

2.
Nucleic Acids Res ; 51(20): 11142-11161, 2023 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-37811875

RESUMO

The human brain is a complex organ comprised of distinct cell types, and the contribution of the 3D genome to lineage specific gene expression remains poorly understood. To decipher cell type specific genome architecture, and characterize fine scale changes in the chromatin interactome across neural development, we compared the 3D genome of the human fetal cortical plate to that of neurons and glia isolated from the adult prefrontal cortex. We found that neurons have weaker genome compartmentalization compared to glia, but stronger TADs, which emerge during fetal development. Furthermore, relative to glia, the neuronal genome shifts more strongly towards repressive compartments. Neurons have differential TAD boundaries that are proximal to active promoters involved in neurodevelopmental processes. CRISPRi on CNTNAP2 in hIPSC-derived neurons reveals that transcriptional inactivation correlates with loss of insulation at the differential boundary. Finally, re-wiring of chromatin loops during neural development is associated with transcriptional and functional changes. Importantly, differential loops in the fetal cortex are associated with autism GWAS loci, suggesting a neuropsychiatric disease mechanism affecting the chromatin interactome. Furthermore, neural development involves gaining enhancer-promoter loops that upregulate genes that control synaptic activity. Altogether, our study provides multi-scale insights on the 3D genome in the human brain.


Assuntos
Encéfalo , Cromatina , Neurogênese , Adulto , Humanos , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Cromatina/metabolismo , Genoma , Neurônios
3.
bioRxiv ; 2023 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-37503149

RESUMO

Here, we construct genome-scale maps for R-loops, three-stranded nucleic acid structures comprised of a DNA/RNA hybrid and a displaced single strand of DNA, in the proliferative and differentiated zones of the human prenatal brain. We show that R-loops are abundant in the progenitor-rich germinal matrix, with preferential formation at promoters slated for upregulated expression at later stages of differentiation, including numerous neurodevelopmental risk genes. RNase H1-mediated contraction of the genomic R-loop space in neural progenitors shifted differentiation toward the neuronal lineage and was associated with transcriptomic alterations and defective functional and structural neuronal connectivity in vivo and in vitro. Therefore, R-loops are important for fine-tuning differentiation-sensitive gene expression programs of neural progenitor cells.

4.
Nat Neurosci ; 25(10): 1366-1378, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36171428

RESUMO

To characterize the dysregulation of chromatin accessibility in Alzheimer's disease (AD), we generated 636 ATAC-seq libraries from neuronal and nonneuronal nuclei isolated from the superior temporal gyrus and entorhinal cortex of 153 AD cases and 56 controls. By analyzing a total of ~20 billion read pairs, we expanded the repertoire of known open chromatin regions (OCRs) in the human brain and identified cell-type-specific enhancer-promoter interactions. We show that interindividual variability in OCRs can be leveraged to identify cis-regulatory domains (CRDs) that capture the three-dimensional structure of the genome (3D genome). We identified AD-associated effects on chromatin accessibility, the 3D genome and transcription factor (TF) regulatory networks. For one of the most AD-perturbed TFs, USF2, we validated its regulatory effect on lysosomal genes. Overall, we applied a systematic approach to understanding the role of the 3D genome in AD. We provide all data as an online resource for widespread community-based analysis.


Assuntos
Doença de Alzheimer , Cromatina , Doença de Alzheimer/genética , Humanos , Regiões Promotoras Genéticas , Fatores de Transcrição/genética
5.
Nat Genet ; 54(10): 1493-1503, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36163279

RESUMO

Identification of risk variants for neuropsychiatric diseases within enhancers underscores the importance of understanding population-level variation in enhancer function in the human brain. Besides regulating tissue-specific and cell-type-specific transcription of target genes, enhancers themselves can be transcribed. By jointly analyzing large-scale cell-type-specific transcriptome and regulome data, we cataloged 30,795 neuronal and 23,265 non-neuronal candidate transcribed enhancers. Examination of the transcriptome in 1,382 brain samples identified robust expression of transcribed enhancers. We explored gene-enhancer coordination and found that enhancer-linked genes are strongly implicated in neuropsychiatric disease. We identified expression quantitative trait loci (eQTLs) for both genes and enhancers and found that enhancer eQTLs mediate a substantial fraction of neuropsychiatric trait heritability. Inclusion of enhancer eQTLs in transcriptome-wide association studies enhanced functional interpretation of disease loci. Overall, our study characterizes the gene-enhancer regulome and genetic mechanisms in the human cortex in both healthy and diseased states.


Assuntos
Estudo de Associação Genômica Ampla , Locos de Características Quantitativas , Encéfalo , Elementos Facilitadores Genéticos/genética , Humanos , Locos de Características Quantitativas/genética , Sequências Reguladoras de Ácido Nucleico , Transcriptoma/genética
6.
Cell Stem Cell ; 29(2): 181-183, 2022 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-35120616

RESUMO

Aberrant migration of GABAergic interneurons during cortical neurodevelopment is implicated in Timothy Syndrome, yet the underlying mechanisms remain elusive. In this issue of Cell Stem Cell, Birey et al. model developing brain circuitry using "assembloids" from patients, characterizing a bimodal mechanism of mechano-chemically driven interneuron migration inefficiencies.


Assuntos
Transtorno Autístico , Síndrome do QT Longo , Sindactilia , Humanos , Interneurônios/fisiologia , Sindactilia/genética
7.
Biol Psychiatry ; 90(6): 362-372, 2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34176589

RESUMO

A key challenge in psychiatry research is the development of high-fidelity model systems that can be experimentally manipulated to explore and test pathophysiological mechanisms of illness. In this respect, the emerging capacity to derive neural cells and circuits from human induced pluripotent stem cells (iPSCs) has generated significant excitement. This review aims to provide a critical appraisal of the potential for iPSCs in illuminating pathophysiological mechanisms in the context of other available technical approaches. We discuss the selection of iPSC phenotypes relevant to psychiatry, the information that researchers can draw on to help guide these decisions, and how researchers choose between the use of 2-dimensional cultures and the use of more complex 3-dimensional model systems. We discuss the strengths and limitations of current models and the challenges and opportunities that they present. Finally, we discuss the potential of iPSC-based model systems for clarifying the mechanisms underlying genetic risk for psychiatry and the steps that will be needed to ensure that robust and reliable conclusions can be drawn. We argue that while iPSC-based models are ideally placed to study fundamental processes occurring within and between neural cells, they are often less well suited for case-control studies, given issues relating to statistical power and the challenges in identifying which cellular phenotypes are meaningful at the level of the whole individual. Our aim is to highlight the importance of considering the hypotheses of a given study to guide decisions about which, if any, iPSC-based system is most appropriate to address it.


Assuntos
Células-Tronco Pluripotentes Induzidas , Psiquiatria , Humanos , Modelos Biológicos , Neurônios , Fenótipo
8.
Stem Cell Reports ; 16(3): 505-518, 2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33636110

RESUMO

The host response to SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, demonstrates significant interindividual variability. In addition to showing more disease in males, the elderly, and individuals with underlying comorbidities, SARS-CoV-2 can seemingly afflict healthy individuals with profound clinical complications. We hypothesize that, in addition to viral load and host antibody repertoire, host genetic variants influence vulnerability to infection. Here we apply human induced pluripotent stem cell (hiPSC)-based models and CRISPR engineering to explore the host genetics of SARS-CoV-2. We demonstrate that a single-nucleotide polymorphism (rs4702), common in the population and located in the 3' UTR of the protease FURIN, influences alveolar and neuron infection by SARS-CoV-2 in vitro. Thus, we provide a proof-of-principle finding that common genetic variation can have an impact on viral infection and thus contribute to clinical heterogeneity in COVID-19. Ongoing genetic studies will help to identify high-risk individuals, predict clinical complications, and facilitate the discovery of drugs.


Assuntos
COVID-19/genética , Predisposição Genética para Doença/genética , Polimorfismo de Nucleotídeo Único/genética , Regiões 3' não Traduzidas/genética , Adolescente , Adulto , Animais , COVID-19/virologia , Linhagem Celular , Chlorocebus aethiops , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Feminino , Furina/genética , Interações Hospedeiro-Patógeno/genética , Humanos , Células-Tronco Pluripotentes Induzidas/virologia , Masculino , Neurônios/virologia , Peptídeo Hidrolases/genética , SARS-CoV-2/patogenicidade , Células Vero
10.
Neuron ; 109(2): 257-272.e14, 2021 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-33238137

RESUMO

To identify the molecular mechanisms and novel therapeutic targets of late-onset Alzheimer's Disease (LOAD), we performed an integrative network analysis of multi-omics profiling of four cortical areas across 364 donors with varying cognitive and neuropathological phenotypes. Our analyses revealed thousands of molecular changes and uncovered neuronal gene subnetworks as the most dysregulated in LOAD. ATP6V1A was identified as a key regulator of a top-ranked neuronal subnetwork, and its role in disease-related processes was evaluated through CRISPR-based manipulation in human induced pluripotent stem cell-derived neurons and RNAi-based knockdown in Drosophila models. Neuronal impairment and neurodegeneration caused by ATP6V1A deficit were improved by a repositioned compound, NCH-51. This study provides not only a global landscape but also detailed signaling circuits of complex molecular interactions in key brain regions affected by LOAD, and the resulting network models will serve as a blueprint for developing next-generation therapeutic agents against LOAD.


Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/terapia , Encéfalo/fisiologia , Bases de Dados Genéticas , Redes Reguladoras de Genes/fisiologia , Transdução de Sinais/fisiologia , Doença de Alzheimer/patologia , Animais , Animais Geneticamente Modificados , Encéfalo/patologia , Bases de Dados Genéticas/tendências , Drosophila melanogaster , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Masculino , Análise de Sequência de RNA/métodos
11.
bioRxiv ; 2020 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-32995783

RESUMO

The host response to SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, demonstrates significant inter-individual variability. In addition to showing more disease in males, the elderly, and individuals with underlying comorbidities, SARS-CoV-2 can seemingly render healthy individuals with profound clinical complications. We hypothesize that, in addition to viral load and host antibody repertoire, host genetic variants also impact vulnerability to infection. Here we apply human induced pluripotent stem cell (hiPSC)-based models and CRISPR-engineering to explore the host genetics of SARS-CoV-2. We demonstrate that a single nucleotide polymorphism (rs4702), common in the population at large, and located in the 3'UTR of the protease FURIN, impacts alveolar and neuron infection by SARS-CoV-2 in vitro. Thus, we provide a proof-of-principle finding that common genetic variation can impact viral infection, and thus contribute to clinical heterogeneity in SARS-CoV-2. Ongoing genetic studies will help to better identify high-risk individuals, predict clinical complications, and facilitate the discovery of drugs that might treat disease.

12.
Nat Genet ; 52(4): 363-369, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32203467

RESUMO

The genetic architecture of each individual comprises common and rare variants that, acting alone and in combination, confer risk of disease. The cell-type-specific and/or context-dependent functional consequences of the risk variants linked to brain disease must be resolved. Coupling human induced pluripotent stem cell (hiPSC)-based technology with CRISPR-based genome engineering facilitates precise isogenic comparisons of variants across genetic backgrounds. Although functional-validation studies are typically performed on one variant in isolation and in one cell type at a time, complex genetic diseases require multiplexed gene perturbations to interrogate combinations of genes and resolve physiologically relevant disease biology. Our aim is to discuss advances at the intersection of genomics, hiPSCs and CRISPR. A better understanding of the molecular mechanisms underlying disease risk will improve genetic diagnosis, drive phenotypic drug discovery and pave the way toward precision medicine.


Assuntos
Encefalopatias/genética , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Genoma/genética , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Medicina de Precisão/métodos
13.
J Neurosci Methods ; 334: 108548, 2020 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-32065989

RESUMO

BACKGROUND: Somatic cell reprogramming is routinely used to generate donor-specific human induced pluripotent stem cells (hiPSCs) to facilitate studies of disease in a human context. The directed differentiation of hiPSCs can generate large quantities of patient-derived cells; however, such methodologies frequently yield heterogeneous populations of neurons and glia that require extended timelines to achieve electrophysiological maturity. More recently, transcription factor-based induction protocols have been show to rapidly generate defined neuronal populations from hiPSCs. NEW METHOD: In a manner similar to our previous adaption of NGN2-glutamatergic neuronal induction from hiPSC-derived neural progenitor cells (NPCs), we now adapt an established protocol of lentiviral overexpression of ASCL1 and DLX2 to hiPSC-NPCs. RESULTS: We demonstrate induction of a robust and highly pure population of functional GABAergic neurons (iGANs). Importantly, we successfully applied this technique to hiPSC-NPCs derived from ten donors across two independent laboratories, finding it to be an efficient and highly reproducible approach to generate induced GABAergic neurons. Our results show that, like hiPSC-iGANs, NPC-iGANs exhibit increased GABAergic marker expression, electrophysiological maturity, and have distinct transcriptional profiles that distinguish them from other cell-types of the brain. Nonetheless, until donor-matched hiPSCs-iGANs and NPC-iGANs are directly compared, we cannot rule out the possibility that subtle differences in patterning or maturity may exist between these populations; one should always control for cell source in all iGAN experiments. CONCLUSIONS: This methodology, relying upon an easily cultured starting population of hiPSC-NPCs, makes possible the generation of large-scale defined co-cultures of induced glutamatergic and GABAergic neurons for hiPSC-based disease models and precision drug screening.

14.
J Neurosci ; 40(6): 1176-1185, 2020 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-32024766

RESUMO

Human induced pluripotent stem cells (hiPSCs) have revolutionized research on human diseases, particularly neurodegenerative and psychiatric disorders, making it possible to study mechanisms of disease risk and initiation in otherwise inaccessible patient-specific cells. Today, the integration of CRISPR engineering approaches with hiPSC-based models permits precise isogenic comparisons of human neurons and glia. This review is intended as a guideline for neuroscientists and clinicians interested in translating their research to hiPSC-based studies. It offers state-of-the-art approaches to tackling the challenges that are unique to human in vitro disease models, particularly interdonor and intradonor variability, and limitations in neuronal maturity and circuit complexity. Finally, we provide a detailed overview of the immense possibilities the field has to offer, highlighting efficient neural differentiation and induction strategies for the major brain cell types and providing perspective into integrating CRISPR-based methods into study design. The combination of hiPSC-based disease modeling, CRISPR technology, and high-throughput approaches promises to advance our scientific knowledge and accelerate progress in drug discovery.Dual Perspectives Companion Paper: Studying Human Neurodevelopment and Diseases Using 3D Brain Organoids, by Ai Tian, Julien Muffat, and Yun Li.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes/métodos , Edição de Genes/tendências , Células-Tronco Pluripotentes Induzidas , Modelos Genéticos , Humanos
15.
Nat Genet ; 51(12): 1679-1690, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31784728

RESUMO

NRXN1 undergoes extensive alternative splicing, and non-recurrent heterozygous deletions in NRXN1 are strongly associated with neuropsychiatric disorders. We establish that human induced pluripotent stem cell (hiPSC)-derived neurons well represent the diversity of NRXN1α alternative splicing observed in the human brain, cataloguing 123 high-confidence in-frame human NRXN1α isoforms. Patient-derived NRXN1+/- hiPSC-neurons show a greater than twofold reduction in half of the wild-type NRXN1α isoforms and express dozens of novel isoforms from the mutant allele. Reduced neuronal activity in patient-derived NRXN1+/- hiPSC-neurons is ameliorated by overexpression of individual control isoforms in a genotype-dependent manner, whereas individual mutant isoforms decrease neuronal activity levels in control hiPSC-neurons. In a genotype-dependent manner, the phenotypic impact of patient-specific NRXN1+/- mutations can occur through a reduction in wild-type NRXN1α isoform levels as well as the presence of mutant NRXN1α isoforms.


Assuntos
Processamento Alternativo , Proteínas de Ligação ao Cálcio/genética , Células-Tronco Pluripotentes Induzidas/fisiologia , Moléculas de Adesão de Célula Nervosa/genética , Esquizofrenia/genética , Animais , Transtorno do Espectro Autista/genética , Transtorno Bipolar/genética , Estudos de Casos e Controles , Transtorno Depressivo Maior/genética , Feminino , Expressão Gênica , Heterozigoto , Humanos , Masculino , Camundongos , Isoformas de Proteínas/genética , Deleção de Sequência
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