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1.
Comput Struct Biotechnol J ; 23: 638-647, 2024 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-38283851

RESUMEN

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas nucleases and human induced pluripotent stem cell (iPSC) technology can reveal deep insight into the genetic and molecular bases of human biology and disease. Undesired editing outcomes, both on-target (at the edited locus) and off-target (at other genomic loci) hinder the application of CRISPR-Cas nucleases. We developed Off-flow, a Nextflow-coded bioinformatic workflow that takes a specific guide sequence and Cas protein input to call four separate off-target prediction programs (CHOPCHOP, Cas-Offinder, CRISPRitz, CRISPR-Offinder) to output a comprehensive list of predicted off-target sites. We applied it to whole genome sequencing (WGS) data to investigate the occurrence of unintended effects in human iPSCs that underwent repair or insertion of disease-related variants by homology-directed repair. Off-flow identified a 3-base-pair-substitution and a mono-allelic genomic deletion at the target loci, KCNQ2, in 2 clones. Unbiased WGS analysis further identified off-target missense variants and a mono-allelic genomic deletion at the targeted locus, GNAQ, in 10 clones. On-target substitution and deletions had escaped standard PCR and Sanger sequencing analysis, while missense variants at other genomic loci were not detected by Off-flow. We used these results to filter out iPSC clones for subsequent functional experiments. Off-flow, which we make publicly available, works for human and mouse genomes currently and can be adapted for other genomes. Off-flow and WGS analysis can improve the integrity of studies using CRISPR/Cas-edited cells and animal models.

2.
Stem Cell Reports ; 19(2): 187-195, 2024 Feb 13.
Artículo en Inglés | MEDLINE | ID: mdl-38242131

RESUMEN

Amyotrophic lateral sclerosis (ALS) is a fatal, adult-onset neurodegenerative disorder characterized by progressive muscular weakness due to the selective loss of motor neurons. Mutations in the gene Fused in Sarcoma (FUS) were identified as one cause of ALS. Here, we report that mutations in FUS lead to upregulation of synaptic proteins, increasing synaptic activity and abnormal release of vesicles at the synaptic cleft. Consequently, FUS-ALS neurons showed greater vulnerability to glutamate excitotoxicity, which raised neuronal swellings (varicose neurites) and led to neuronal death. Fragile X mental retardation protein (FMRP) is an RNA-binding protein known to regulate synaptic protein translation, and its expression is reduced in the FUS-ALS lines. Collectively, our data suggest that a reduction of FMRP levels alters the synaptic protein dynamics, leading to synaptic dysfunction and glutamate excitotoxicity. Here, we present a mechanistic hypothesis linking dysregulation of peripheral translation with synaptic vulnerability in the pathogenesis of FUS-ALS.


Asunto(s)
Esclerosis Amiotrófica Lateral , Células Madre Pluripotentes Inducidas , Adulto , Humanos , Esclerosis Amiotrófica Lateral/patología , Células Madre Pluripotentes Inducidas/metabolismo , Neuronas Motoras/metabolismo , Mutación , Glutamatos/metabolismo , Proteína FUS de Unión a ARN/genética
3.
BMC Med Genomics ; 16(1): 5, 2023 01 12.
Artículo en Inglés | MEDLINE | ID: mdl-36635662

RESUMEN

BACKGROUND: The X-linked PTCHD1 locus is strongly associated with autism spectrum disorder (ASD). Males who carry chromosome microdeletions of PTCHD1 antisense long non-coding RNA (PTCHD1-AS)/DEAD-box helicase 53 (DDX53) have ASD, or a sub-clinical form called Broader Autism Phenotype. If the deletion extends beyond PTCHD1-AS/DDX53 to the next gene, PTCHD1, which is protein-coding, the individuals typically have ASD and intellectual disability (ID). Three male siblings with a 90 kb deletion that affects only PTCHD1-AS (and not including DDX53) have ASD. We performed a functional analysis of DDX53 to examine its role in NGN2 neurons. METHODS: We used the clustered regularly interspaced short palindromic repeats (CRISPR) gene editing strategy to knock out DDX53 protein by inserting 3 termination codons (3TCs) into two different induced pluripotent stem cell (iPSC) lines. DDX53 CRISPR-edited iPSCs were differentiated into cortical excitatory neurons by Neurogenin 2 (NGN-2) directed differentiation. The functional differences of DDX53-3TC neurons compared to isogenic control neurons with molecular and electrophysiological approaches were assessed. RESULTS: Isogenic iPSC-derived control neurons exhibited low levels of DDX53 transcripts. Transcriptional analysis revealed the generation of excitatory cortical neurons and DDX53 protein was not detected in iPSC-derived control neurons by western blot. Control lines and DDX53-3TC neurons were active in the multi-electrode array, but no overt electrophysiological phenotype in either isogenic line was observed. CONCLUSION: DDX53-3TC mutation does not alter NGN2 neuronal function in these experiments, suggesting that synaptic deficits causing ASD are unlikely in this cell type.


Asunto(s)
Trastorno del Espectro Autista , ARN Helicasas DEAD-box , Células Madre Pluripotentes Inducidas , Humanos , Masculino , Trastorno del Espectro Autista/genética , ARN Helicasas DEAD-box/genética , Células Madre Pluripotentes Inducidas/metabolismo , Mutación , Neuronas/metabolismo
4.
medRxiv ; 2023 Dec 27.
Artículo en Inglés | MEDLINE | ID: mdl-38234782

RESUMEN

Autism Spectrum Disorder (ASD) exhibits an ~4:1 male-to-female sex bias and is characterized by early-onset impairment of social/communication skills, restricted interests, and stereotyped behaviors. Disruption of the Xp22.11 locus has been associated with ASD in males. This locus includes the three-exon PTCHD1 gene, an adjacent multi-isoform long noncoding RNA (lncRNA) named PTCHD1-AS (spanning ~1Mb), and a poorly characterized single-exon RNA helicase named DDX53 that is intronic to PTCHD1-AS. While the relationship between PTCHD1/PTCHD1-AS and ASD is being studied, the role of DDX53 has not been examined, in part because there is no apparent functional murine orthologue. Through clinical testing, here, we identified 6 males and 1 female with ASD from 6 unrelated families carrying rare, predicted-damaging or loss-of-function variants in DDX53. Then, we examined databases, including the Autism Speaks MSSNG and Simons Foundation Autism Research Initiative, as well as population controls. We identified 24 additional individuals with ASD harboring rare, damaging DDX53 variations, including the same variants detected in two families from the original clinical analysis. In this extended cohort of 31 participants with ASD (28 male, 3 female), we identified 25 mostly maternally-inherited variations in DDX53, including 18 missense changes, 2 truncating variants, 2 in-frame variants, 2 deletions in the 3' UTR and 1 copy number deletion. Our findings in humans support a direct link between DDX53 and ASD, which will be important in clinical genetic testing. These same autism-related findings, coupled with the observation that a functional orthologous gene is not found in mouse, may also influence the design and interpretation of murine-modelling of ASD.

5.
Cell ; 185(23): 4409-4427.e18, 2022 11 10.
Artículo en Inglés | MEDLINE | ID: mdl-36368308

RESUMEN

Fully understanding autism spectrum disorder (ASD) genetics requires whole-genome sequencing (WGS). We present the latest release of the Autism Speaks MSSNG resource, which includes WGS data from 5,100 individuals with ASD and 6,212 non-ASD parents and siblings (total n = 11,312). Examining a wide variety of genetic variants in MSSNG and the Simons Simplex Collection (SSC; n = 9,205), we identified ASD-associated rare variants in 718/5,100 individuals with ASD from MSSNG (14.1%) and 350/2,419 from SSC (14.5%). Considering genomic architecture, 52% were nuclear sequence-level variants, 46% were nuclear structural variants (including copy-number variants, inversions, large insertions, uniparental isodisomies, and tandem repeat expansions), and 2% were mitochondrial variants. Our study provides a guidebook for exploring genotype-phenotype correlations in families who carry ASD-associated rare variants and serves as an entry point to the expanded studies required to dissect the etiology in the ∼85% of the ASD population that remain idiopathic.


Asunto(s)
Trastorno del Espectro Autista , Trastorno Autístico , Humanos , Trastorno del Espectro Autista/genética , Predisposición Genética a la Enfermedad , Variaciones en el Número de Copia de ADN/genética , Genómica
6.
Brain Behav Immun ; 105: 82-97, 2022 10.
Artículo en Inglés | MEDLINE | ID: mdl-35716830

RESUMEN

Maternal immune activation (MIA) during prenatal development is an environmental risk factor for psychiatric disorders including schizophrenia (SZ). Converging lines of evidence from human and animal model studies suggest that elevated cytokine levels in the maternal and fetal compartments are an important indication of the mechanisms driving this association. However, there is variability in susceptibility to the psychiatric risk conferred by MIA, likely influenced by genetic factors. How MIA interacts with a genetic profile susceptible to SZ is challenging to test in animal models. To address this gap, we examined whether differential gene expression responses occur in forebrain-lineage neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (hiPSC) generated from three individuals with a diagnosis of schizophrenia and three healthy controls. Following acute (24 h) treatment with either interferon-gamma (IFNγ; 25 ng/µl) or interleukin (IL)-1ß (10 ng/µl), we identified, by RNA sequencing, 3380 differentially expressed genes (DEGs) in the IFNγ-treated control lines (compared to untreated controls), and 1980 DEGs in IFNγ-treated SZ lines (compared to untreated SZ lines). Out of 4137 genes that responded significantly to IFNγ across all lines, 1223 were common to both SZ and control lines. The 2914 genes that appeared to respond differentially to IFNγ treatment in SZ lines were subjected to a further test of significance (multiple testing correction applied to the interaction effect between IFNγ treatment and SZ diagnosis), yielding 359 genes that passed the significance threshold. There were no differentially expressed genes in the IL-1ß-treatment conditions after Benjamini-Hochberg correction. Gene set enrichment analysis however showed that IL-1ß impacts immune function and neuronal differentiation. Overall, our data suggest that a) SZ NPCs show an attenuated transcriptional response to IFNγ treatment compared to controls; b) Due to low IL-1ß receptor expression in NPCs, NPC cultures appear to be less responsive to IL-1ß than IFNγ; and c) the genes differentially regulated in SZ lines - in the face of a cytokine challenge - are primarily associated with mitochondrial, "loss-of-function", pre- and post-synaptic gene sets. Our findings particularly highlight the role of early synaptic development in the association between maternal immune activation and schizophrenia risk.


Asunto(s)
Células Madre Pluripotentes Inducidas , Células-Madre Neurales , Esquizofrenia , Animales , Citocinas/metabolismo , Femenino , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células-Madre Neurales/metabolismo , Embarazo , Prosencéfalo , Esquizofrenia/genética , Esquizofrenia/metabolismo
7.
Biol Psychiatry ; 89(5): 486-496, 2021 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-32826066

RESUMEN

BACKGROUND: Autism is a heterogeneous collection of disorders with a complex molecular underpinning. Evidence from postmortem brain studies have indicated that early prenatal development may be altered in autism. Induced pluripotent stem cells (iPSCs) generated from individuals with autism with macrocephaly also indicate prenatal development as a critical period for this condition. But little is known about early altered cellular events during prenatal stages in autism. METHODS: iPSCs were generated from 9 unrelated individuals with autism without macrocephaly and with heterogeneous genetic backgrounds, and 6 typically developing control individuals. iPSCs were differentiated toward either cortical or midbrain fates. Gene expression and high throughput cellular phenotyping was used to characterize iPSCs at different stages of differentiation. RESULTS: A subset of autism-iPSC cortical neurons were RNA-sequenced to reveal autism-specific signatures similar to postmortem brain studies, indicating a potential common biological mechanism. Autism-iPSCs differentiated toward a cortical fate displayed impairments in the ability to self-form into neural rosettes. In addition, autism-iPSCs demonstrated significant differences in rate of cell type assignment of cortical precursors and dorsal and ventral forebrain precursors. These cellular phenotypes occurred in the absence of alterations in cell proliferation during cortical differentiation, differing from previous studies. Acquisition of cell fate during midbrain differentiation was not different between control- and autism-iPSCs. CONCLUSIONS: Taken together, our data indicate that autism-iPSCs diverge from control-iPSCs at a cellular level during early stage of neurodevelopment. This suggests that unique developmental differences associated with autism may be established at early prenatal stages.


Asunto(s)
Trastorno Autístico , Células Madre Pluripotentes Inducidas , Diferenciación Celular , Femenino , Humanos , Neurogénesis , Neuronas , Embarazo
8.
Mol Cell Neurosci ; 103: 103463, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-31917333

RESUMEN

Endocannabinoids 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 endocannabinoid 2-arachidonoyl glycerol (2AG) and Δ9-THC on the development of neuronal morphology and activation of signaling kinases, in cortical 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 decrease in 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 exogenous cannabinoids. Thus, hiPSC-neurons may represent a good cellular model for investigating the role of the endocannabinoid system in regulating cellular processes in developing human neurons.


Asunto(s)
Células Madre Pluripotentes Inducidas/efectos de los fármacos , Proyección Neuronal/efectos de los fármacos , Neuronas/efectos de los fármacos , Rimonabant/farmacología , Agonistas de Receptores de Cannabinoides/farmacología , Cannabinoides/farmacología , Dronabinol/metabolismo , Dronabinol/farmacología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Neuronas/metabolismo , Proteínas Proto-Oncogénicas c-akt/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo
9.
Biol Psychiatry ; 82(1): 49-61, 2017 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-27837918

RESUMEN

BACKGROUND: Variation in the gene encoding zinc finger binding protein 804A (ZNF804A) is associated with schizophrenia and bipolar disorder. Evidence suggests that ZNF804A is a regulator of gene transcription and is present in nuclear and extranuclear compartments. However, a detailed examination of ZNF804A distribution and its neuronal functions has yet to be performed. METHODS: The localization of ZNF804A protein was examined in neurons derived from human neural progenitor cells, human induced pluripotent stem cells, or in primary rat cortical neurons. In addition, small interfering RNA-mediated knockdown of ZNF804A was conducted to determine its role in neurite formation, maintenance of dendritic spine morphology, and responses to activity-dependent stimulations. RESULTS: Endogenous ZNF804A protein localized to somatodendritic compartments and colocalized with the putative synaptic markers in young neurons derived from human neural progenitor cells and human induced pluripotent stem cells. In mature rat neurons, Zfp804A, the homolog of ZNF804A, was present in a subset of dendritic spines and colocalized with synaptic proteins in specific nanodomains, as determined by super-resolution microscopy. Interestingly, knockdown of ZNF804A attenuated neurite outgrowth in young neurons, an effect potentially mediated by reduced neuroligin-4 expression. Furthermore, knockdown of ZNF804A in mature neurons resulted in the loss of dendritic spine density and impaired responses to activity-dependent stimulation. CONCLUSIONS: These data reveal a novel subcellular distribution for ZNF804A within somatodendritic compartments and a nanoscopic organization at excitatory synapses. Moreover, our results suggest that ZNF804A plays an active role in neurite formation, maintenance of dendritic spines, and activity-dependent structural plasticity.


Asunto(s)
Espinas Dendríticas/fisiología , Factores de Transcripción de Tipo Kruppel/metabolismo , Factores de Transcripción de Tipo Kruppel/fisiología , Neuritas/fisiología , Sinapsis/metabolismo , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Células Cultivadas , Espinas Dendríticas/ultraestructura , Humanos , Factores de Transcripción de Tipo Kruppel/efectos de los fármacos , Neuritas/ultraestructura , Neuronas/metabolismo , Neuronas/fisiología , Neuronas/ultraestructura , Trastornos Psicóticos/genética , ARN Interferente Pequeño/farmacología , Ratas , Sinapsis/ultraestructura
10.
Horm Behav ; 74: 228-42, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-26143621

RESUMEN

This article is part of a Special Issue "Estradiol and Cognition". Over recent years tremendous progress has been made towards understanding the molecular and cellular mechanism by which estrogens exert enhancing effects on cognition, and how they act as a neuroprotective or neurotrophic agent in disease. Currently, much of this work has been carried out in animal models with only a limited number of studies using native human tissue or cells. Recent advances in stem cell technology now make it possible to reprogram somatic cells from humans into induced pluripotent stem cells (iPSCs), which can subsequently be differentiated into neurons of specific lineages. Importantly, the reprogramming of cells allows for the generation of iPSCs that retain the genetic "makeup" of the donor. Therefore, it is possible to generate iPSC-derived neurons from patients diagnosed with specific diseases, that harbor the complex genetic background associated with the disorder. Here, we review the iPSC technology and how it's currently being used to model neural development and neurological diseases. Furthermore, we explore whether this cellular system could be used to understand the role of estrogens in human neurons, and present preliminary data in support of this. We further suggest that the use of iPSC technology offers a novel system to not only further understand estrogens' effects in human cells, but also to investigate the mechanism by which estrogens are beneficial in disease. Developing a greater understanding of these mechanisms in native human cells will also aid in the development of safer and more effective estrogen-based therapeutics.


Asunto(s)
Estrógenos/farmacología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Neuronas/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , Humanos , Células Madre Pluripotentes Inducidas/fisiología , Modelos Biológicos , Neurogénesis/efectos de los fármacos , Neurogénesis/fisiología , Neuronas/fisiología
11.
PLoS One ; 9(3): e91269, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24651281

RESUMEN

TDP-43 is found in cytoplasmic inclusions in 95% of amyotrophic lateral sclerosis (ALS) and 60% of frontotemporal lobar degeneration (FTLD). Approximately 4% of familial ALS is caused by mutations in TDP-43. The majority of these mutations are found in the glycine-rich domain, including the variant M337V, which is one of the most common mutations in TDP-43. In order to investigate the use of allele-specific RNA interference (RNAi) as a potential therapeutic tool, we designed and screened a set of siRNAs that specifically target TDP-43(M337V) mutation. Two siRNA specifically silenced the M337V mutation in HEK293T cells transfected with GFP-TDP-43(wt) or GFP-TDP-43(M337V) or TDP-43 C-terminal fragments counterparts. C-terminal TDP-43 transfected cells show an increase of cytosolic inclusions, which are decreased after allele-specific siRNA in M337V cells. We then investigated the effects of one of these allele-specific siRNAs in induced pluripotent stem cells (iPSCs) derived from an ALS patient carrying the M337V mutation. These lines showed a two-fold increase in cytosolic TDP-43 compared to the control. Following transfection with the allele-specific siRNA, cytosolic TDP-43 was reduced by 30% compared to cells transfected with a scrambled siRNA. We conclude that RNA interference can be used to selectively target the TDP-43(M337V) allele in mammalian and patient cells, thus demonstrating the potential for using RNA interference as a therapeutic tool for ALS.


Asunto(s)
Alelos , Esclerosis Amiotrófica Lateral/genética , Proteínas de Unión al ADN/genética , Técnicas de Silenciamiento del Gen , Células Madre Pluripotentes Inducidas/patología , Mutación/genética , Células-Madre Neurales/metabolismo , Sustitución de Aminoácidos/genética , Secuencia de Bases , Células HEK293 , Humanos , Cuerpos de Inclusión/metabolismo , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo
12.
Proc Natl Acad Sci U S A ; 110(12): 4697-702, 2013 Mar 19.
Artículo en Inglés | MEDLINE | ID: mdl-23401527

RESUMEN

Glial proliferation and activation are associated with disease progression in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia. In this study, we describe a unique platform to address the question of cell autonomy in transactive response DNA-binding protein (TDP-43) proteinopathies. We generated functional astroglia from human induced pluripotent stem cells carrying an ALS-causing TDP-43 mutation and show that mutant astrocytes exhibit increased levels of TDP-43, subcellular mislocalization of TDP-43, and decreased cell survival. We then performed coculture experiments to evaluate the effects of M337V astrocytes on the survival of wild-type and M337V TDP-43 motor neurons, showing that mutant TDP-43 astrocytes do not adversely affect survival of cocultured neurons. These observations reveal a significant and previously unrecognized glial cell-autonomous pathological phenotype associated with a pathogenic mutation in TDP-43 and show that TDP-43 proteinopathies do not display an astrocyte non-cell-autonomous component in cell culture, as previously described for SOD1 ALS. This study highlights the utility of induced pluripotent stem cell-based in vitro disease models to investigate mechanisms of disease in ALS and other TDP-43 proteinopathies.


Asunto(s)
Esclerosis Amiotrófica Lateral , Astrocitos , Células Madre Pluripotentes Inducidas , Neuronas Motoras , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Astrocitos/metabolismo , Astrocitos/patología , Línea Celular , Proliferación Celular , Supervivencia Celular , Técnicas de Cocultivo , Proteínas de Unión al ADN/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Masculino , Persona de Mediana Edad , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Mutación
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