RESUMO
DNA methylation (DNAm), the addition of a methyl group to a cytosine in DNA, plays an important role in the regulation of gene expression. Single-nucleotide polymorphisms (SNPs) associated with schizophrenia (SZ) by genome-wide association studies (GWAS) often influence local DNAm levels. Thus, DNAm alterations, acting through effects on gene expression, represent one potential mechanism by which SZ-associated SNPs confer risk. In this study, we investigated genome-wide DNAm in postmortem superior temporal gyrus from 44 subjects with SZ and 44 non-psychiatric comparison subjects using Illumina Infinium MethylationEPIC BeadChip microarrays, and extracted cell-type-specific methylation signals by applying tensor composition analysis. We identified SZ-associated differential methylation at 242 sites, and 44 regions containing two or more sites (FDR cutoff of q = 0.1) and determined a subset of these were cell-type specific. We found mitotic arrest deficient 1-like 1 (MAD1L1), a gene within an established GWAS risk locus, harbored robust SZ-associated differential methylation. We investigated the potential role of MAD1L1 DNAm in conferring SZ risk by assessing for colocalization among quantitative trait loci for methylation and gene transcripts (mQTLs and tQTLs) in brain tissue and GWAS signal at the locus using multiple-trait-colocalization analysis. We found that mQTLs and tQTLs colocalized with the GWAS signal (posterior probability >0.8). Our findings suggest that alterations in MAD1L1 methylation and transcription may mediate risk for SZ at the MAD1L1-containing locus. Future studies to identify how SZ-associated differential methylation affects MAD1L1 biological function are indicated.
Assuntos
Proteínas de Ciclo Celular , Metilação de DNA , Esquizofrenia , Encéfalo/metabolismo , Proteínas de Ciclo Celular/genética , DNA/metabolismo , Estudo de Associação Genômica Ampla , Humanos , Polimorfismo de Nucleotídeo Único , Esquizofrenia/genética , Esquizofrenia/metabolismoRESUMO
BACKGROUND: Genetic studies have implicated rare and common variations in liability for autism spectrum disorder (ASD). Of the discovered risk variants, those rare in the population invariably have large impact on liability, while common variants have small effects. Yet, collectively, common risk variants account for the majority of population-level variability. How these rare and common risk variants jointly affect liability for individuals requires further study. METHODS: To explore how common and rare variants jointly affect liability, we assessed two cohorts of ASD families characterized for rare and common genetic variations (Simons Simplex Collection and Population-Based Autism Genetics and Environment Study). We analyzed data from 3011 affected subjects, as well as two cohorts of unaffected individuals characterized for common genetic variation: 3011 subjects matched for ancestry to ASD subjects and 11,950 subjects for estimating allele frequencies. We used genetic scores, which assessed the relative burden of common genetic variation affecting risk of ASD (henceforth "burden"), and determined how this burden was distributed among three subpopulations: ASD subjects who carry a potentially damaging variant implicated in risk of ASD ("PDV carriers"); ASD subjects who do not ("non-carriers"); and unaffected subjects who are assumed to be non-carriers. RESULTS: Burden harbored by ASD subjects is stochastically greater than that harbored by control subjects. For PDV carriers, their average burden is intermediate between non-carrier ASD and control subjects. Both carrier and non-carrier ASD subjects have greater burden, on average, than control subjects. The effects of common and rare variants likely combine additively to determine individual-level liability. LIMITATIONS: Only 305 ASD subjects were known PDV carriers. This relatively small subpopulation limits this study to characterizing general patterns of burden, as opposed to effects of specific PDVs or genes. Also, a small fraction of subjects that are categorized as non-carriers could be PDV carriers. CONCLUSIONS: Liability arising from common and rare risk variations likely combines additively to determine risk of any individual diagnosed with ASD. On average, ASD subjects carry a substantial burden of common risk variation, even if they also carry a rare PDV affecting risk.
Assuntos
Transtorno do Espectro Autista , Transtorno Autístico , Transtorno do Espectro Autista/diagnóstico , Transtorno do Espectro Autista/genética , Predisposição Genética para Doença , HumanosRESUMO
BACKGROUND: Evidence supports raised circulating levels of inflammatory mediators, such as interleukin-6 (IL-6) and tumor necrosis factor (TNFα), among clinically depressed adults, although preliminary findings in adolescents are mixed. Independently, meta-analyses identify correlations between childhood trauma and elevated cytokine levels in adulthood. Here, we examine the possible role of individual differences in exposure to childhood trauma in contributing to variability in cytokine levels in depressed adolescents. METHODS: 52 depressed adolescents and 20 healthy adolescents completed measures of childhood trauma and provided blood for the assessment of plasma IL-6 and TNFα. Cross-sectional associations of childhood trauma and cytokine measures were assessed in both depressed and healthy adolescents, along with exploratory analysis of childhood trauma subtypes. Longitudinal relationships between childhood trauma and cytokine measures were also studied in an exploratory fashion within a subset of depressed participants (n = 36). RESULTS: Higher childhood trauma (particularly emotional abuse) was positively associated with TNFα in depressed adolescents. Childhood trauma was not linked to longitudinal changes in cytokine levels. DISCUSSION: In depressed adolescents, childhood trauma may relate to higher levels of the proinflammatory cytokine TNFα and contribute to heterogeneity in cytokine elevation among depressed adolescents. Such findings may ultimately help guide more effective individualized treatments for adolescents with depression.
Assuntos
Experiências Adversas da Infância , Citocinas/sangue , Depressão/sangue , Depressão/complicações , Trauma Psicológico/sangue , Trauma Psicológico/complicações , Adolescente , Estudos de Casos e Controles , Estudos Transversais , Feminino , Humanos , Interleucina-6/sangue , Masculino , Fator de Necrose Tumoral alfa/sangueRESUMO
Psychotic symptoms, defined as the occurrence of delusions or hallucinations, are frequent in Alzheimer disease (AD with psychosis, AD + P). AD + P affects ~50% of individuals with AD, identifies a subgroup with poor outcomes, and is associated with a greater degree of cognitive impairment and depressive symptoms, compared to subjects without psychosis (AD - P). Although the estimated heritability of AD + P is 61%, genetic sources of risk are unknown. We report a genome-wide meta-analysis of 12,317 AD subjects, 5445 AD + P. Results showed common genetic variation accounted for a significant portion of heritability. Two loci, one in ENPP6 (rs9994623, O.R. (95%CI) 1.16 (1.10, 1.22), p = 1.26 × 10-8) and one spanning the 3'-UTR of an alternatively spliced transcript of SUMF1 (rs201109606, O.R. 0.65 (0.56-0.76), p = 3.24 × 10-8), had genome-wide significant associations with AD + P. Gene-based analysis identified a significant association with APOE, due to the APOE risk haplotype ε4. AD + P demonstrated negative genetic correlations with cognitive and educational attainment and positive genetic correlation with depressive symptoms. We previously observed a negative genetic correlation with schizophrenia; instead, we now found a stronger negative correlation with the related phenotype of bipolar disorder. Analysis of polygenic risk scores supported this genetic correlation and documented a positive genetic correlation with risk variation for AD, beyond the effect of ε4. We also document a small set of SNPs likely to affect risk for AD + P and AD or schizophrenia. These findings provide the first unbiased identification of the association of psychosis in AD with common genetic variation and provide insights into its genetic architecture.
Assuntos
Doença de Alzheimer , Transtornos Psicóticos , Esquizofrenia , Doença de Alzheimer/genética , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla , Alucinações , Humanos , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Polimorfismo de Nucleotídeo Único/genética , Transtornos Psicóticos/genética , Esquizofrenia/genéticaRESUMO
Aim: Neonatal necrotizing enterocolitis (NEC) is a deadly and unpredictable gastrointestinal disease, for which no biomarker exists. We aimed to describe the methylation patterns in stool and colon from infants with NEC. Methods: We performed a high-resolution genome-wide epigenomic analysis using solution-phase hybridization and next-generation sequencing of bisulfite-converted DNA. Results: Our data reveal significant genomic hypermethylation in NEC tissues compared with non-NEC controls. These changes were more pronounced in regions outside CpG islands and gene regulatory elements, suggesting that NEC-specific hypermethylation is not a nonspecific global phenomenon. Conclusions: This study provides evidence of a methylomic signature associated with NEC that is detectable noninvasively and provides a new opportunity for the development of a novel diagnostic method for NEC.
Assuntos
Biomarcadores , Metilação de DNA , Suscetibilidade a Doenças , Enterocolite Necrosante/etiologia , Ilhas de CpG , Enterocolite Necrosante/diagnóstico , Enterocolite Necrosante/metabolismo , Epigênese Genética , Epigenômica/métodos , Fezes , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Lactente , Recém-Nascido , Masculino , Análise de Sequência de DNA , Transdução de SinaisRESUMO
Obsessive-compulsive disorder (OCD) is a chronic and severe psychiatric disorder for which effective treatment options are limited. Structural and functional neuroimaging studies have consistently implicated the orbitofrontal cortex (OFC) and striatum in the pathophysiology of the disorder. Recent genetic evidence points to involvement of components of the excitatory synapse in the etiology of OCD. However, the transcriptional alterations that could link genetic risk to known structural and functional abnormalities remain mostly unknown. To assess potential transcriptional changes in the OFC and two striatal regions (caudate nucleus and nucleus accumbens) of OCD subjects relative to unaffected comparison subjects, we sequenced messenger RNA transcripts from these brain regions. In a joint analysis of all three regions, 904 transcripts were differentially expressed between 7 OCD versus 8 unaffected comparison subjects. Region-specific analyses highlighted a smaller number of differences, which concentrated in caudate and nucleus accumbens. Pathway analyses of the 904 differentially expressed transcripts showed enrichment for genes involved in synaptic signaling, with these synapse-associated genes displaying lower expression in OCD subjects relative to unaffected comparison subjects. Finally, we estimated that cell type fractions of medium spiny neurons were lower whereas vascular cells and astrocyte fractions were higher in tissue of OCD subjects. Together, these data provide the first unbiased examination of differentially expressed transcripts in both OFC and striatum of OCD subjects. These transcripts encoded synaptic proteins more often than expected by chance, and thus implicate the synapse as a vulnerable molecular compartment for OCD.
Assuntos
Transtorno Obsessivo-Compulsivo , Transcriptoma , Corpo Estriado , Substância Cinzenta , Humanos , Imageamento por Ressonância Magnética , Transtorno Obsessivo-Compulsivo/genética , SinapsesRESUMO
OBJECTIVE: We explored the potential of genome-wide epigenomic liquid biopsy for the comprehensive analysis of cell-free DNA (cfDNA) methylation signatures in maternal plasma in early gestation. METHOD: We used solution phase hybridization for targeted region capture of bisulfite-converted DNA obtained from plasma of pregnant women in early gestation and nonpregnant female controls. RESULTS: Targeted sequencing of ~80.5 Mb of the plasma methylome generated an average read depth across all 17 plasma samples of ~42x. We used these data to explore the pregnancy-specific characteristics of cfDNA methylation in plasma and found that pregnancy resulted in clearly detectable global alterations in DNA methylation patterns that were influenced by genomic location. We analyzed similar, previously published, data from first-trimester maternal leukocyte populations and gestational age-matched chorionic villus (CV) and confirmed that tissue-specific DNA methylation signatures in these samples had a significant influence on global and gene-specific methylation in the plasma of pregnant women. CONCLUSION: We describe an approach for targeted epigenomic liquid biopsy in pregnancy and discuss our findings in the context of noninvasive prenatal testing with respect to phenotypic pregnancy monitoring and the early detection of complex gestational phenotypes such as preeclampsia and preterm birth.
Assuntos
Ácidos Nucleicos Livres/análise , Metilação de DNA , Epigenômica/métodos , Teste Pré-Natal não Invasivo/métodos , Gravidez/metabolismo , Estudos de Casos e Controles , Ilhas de CpG , Feminino , Humanos , Testes para Triagem do Soro MaternoRESUMO
BACKGROUND: Depression has been associated with low-grade elevation of plasma cytokines (e.g. interleukin-6, IL-6; tumor necrosis factor alpha, TNFα) in both cross-sectional and longitudinal studies in adults. Preclinical and clinical studies also suggest that IL-6 and TNFα elevation are associated with anhedonia. However, few studies have examined longitudinal relationships between cytokines and depression/anhedonia in clinically depressed samples, particularly adolescents. METHODS: Thirty-six adolescents with a depressive disorder receiving standard-of-care community treatment were assessed at a baseline and a follow-up timepoint. Self-report and clinical measures of depression and anhedonia, along with plasma IL-6 and TNFα levels, were obtained at both timepoints. Baseline cytokine measures were examined in association with baseline and follow-up clinical measures. On an exploratory basis, change in clinical measures over time was examined in relation to change in cytokine levels over time. RESULTS: Higher baseline TNFα levels predicted higher follow-up depression severity after approximately four months (controlling for baseline depression). Higher baseline TNFα levels also associated positively with baseline anhedonia and predicted higher anhedonia at follow-up (controlling for baseline anhedonia). No association was found between change in clinical measures and change in cytokine levels over time. CONCLUSIONS: Among adolescents receiving standard-of-care community treatment for depression, higher levels of TNFα predicted greater depressive symptoms at 4-month follow-up, suggesting this cytokine may be used to help identify patients in need of more intensive treatment. Elevated TNFα levels were also associated with concurrent and future anhedonia symptoms, suggesting a specific mechanism in which TNFα affects depression trajectories. Future studies should examine the relationships between cytokine levels and depression/anhedonia symptoms at multiple timepoints in larger cohorts of depressed adolescents.
Assuntos
Anedonia , Citocinas , Depressão , Adolescente , Adulto , Estudos Transversais , Humanos , Interleucina-6 , Estudos Longitudinais , Fator de Necrose Tumoral alfaRESUMO
BACKGROUND: Necrotizing enterocolitis (NEC) remains one of the overall leading causes of death in premature infants, and the pathogenesis is unpredictable and not well characterized. The aim of our study was to determine the molecular phenotype of NEC via transcriptomic and epithelial cell-specific epigenomic analysis, with a specific focus on DNA methylation. METHODS: Using laser capture microdissection, epithelial cell-specific methylation signatures were characterized by whole-genome bisulfite sequencing of ileal and colonic samples at the time of surgery for NEC and after NEC had healed at reanastomosis (n = 40). RNA sequencing was also performed to determine the transcriptomic profile of these samples, and a comparison was made to the methylome data. RESULTS: We found that surgical NEC has a considerable impact on the epigenome by broadly increasing DNA methylation levels, although these effects are less pronounced in genomic regions associated with the regulation of gene expression. Furthermore, NEC-related DNA methylation signatures were influenced by tissue of origin, with significant differences being noted between colon and ileum. We also identified numerous transcriptional changes in NEC and clear associations between gene expression and DNA methylation. CONCLUSIONS: We have defined the intestinal epigenomic and transcriptomic signatures during surgical NEC, which will advance our understanding of disease pathogenesis and may enable the development of novel precision medicine approaches for NEC prediction, diagnosis and phenotyping.
Assuntos
Enterocolite Necrosante/genética , Enterocolite Necrosante/cirurgia , Células Epiteliais/metabolismo , Microdissecção e Captura a Laser/métodos , Animais , Estudos de Casos e Controles , Colo/patologia , Colo/cirurgia , Ilhas de CpG/genética , Metilação de DNA , Enterocolite Necrosante/etiologia , Enterocolite Necrosante/patologia , Epigenômica/métodos , Células Epiteliais/patologia , Estudo de Associação Genômica Ampla/métodos , Humanos , Íleo/patologia , Íleo/cirurgia , Recém-Nascido , Intestinos/patologia , Microdissecção e Captura a Laser/efeitos adversos , Modelos Animais , Análise de Sequência de RNA/métodos , Transcriptoma/genéticaRESUMO
Our goal was to undertake a genome-wide epigenomic liquid biopsy of cerebrospinal fluid (CSF) for the comprehensive analysis of cell-free DNA (cfDNA) methylation signatures in the human central nervous system (CNS). Solution-phase hybridization and massively parallel sequencing of bisulfite converted human DNA was employed to compare methylation signatures of cfDNA obtained from CSF with plasma. Recovery of cfDNA from CSF was relatively low (68-840 pg/mL) compared to plasma (2720-8390 pg/mL) and cfDNA fragments from CSF were approximately 20 bp shorter than their plasma-derived counterparts. Distributions of CpG methylation signatures were significantly altered between CSF and plasma, both globally and at the level of functional elements including exons, introns, CpG islands, and shores. Sliding window analysis was used to identify differentially methylated regions. We found numerous gene/locus-specific differences in CpG methylation between cfDNA from CSF and plasma. These loci were more frequently hypomethylated in CSF compared to plasma. Differentially methylated CpGs in CSF were identified in genes related to branching of neurites and neuronal development. Using the GTEx RNA expression database, we found clear association between tissue-specific gene expression in the CNS and cfDNA methylation patterns in CSF. Ingenuity pathway analysis of differentially methylated regions identified an enrichment of functional pathways related to neurobiology. In conclusion, we present a genome-wide analysis of DNA methylation in human CSF. Our methods and the resulting data demonstrate the potential of epigenomic liquid biopsy of the human CNS for molecular phenotyping of brain-derived DNA methylation signatures.
Assuntos
Epigenômica , Sequenciamento de Nucleotídeos em Larga Escala , Encéfalo , Ilhas de CpG , Metilação de DNA , Humanos , Biópsia LíquidaRESUMO
The synthesis of a lead anti-viral cyclopropyl carboxy acyl hydrazone 4F17 (5) and three sequential arrays of structural analogues along with the initial assessment and optimization of the antiviral pharmacophore against the herpes simplex virus type 1 (HSV-1) are reported.
Assuntos
Antivirais/química , Antivirais/farmacologia , Herpesvirus Humano 1/efeitos dos fármacos , Hidrazonas/química , Hidrazonas/farmacologia , Antivirais/síntese química , Linhagem Celular , Técnicas de Química Sintética , Herpes Simples/tratamento farmacológico , Humanos , Hidrazonas/síntese química , Relação Estrutura-AtividadeRESUMO
Individuals diagnosed with major depressive disorder not responding to at least two adequate treatments are defined as treatment-refractory major depressive disorder (TR-MDD). Some TR-MDD patients have altered metabolic phenotypes that may be pharmacologically reversed. The characterization of these phenotypes and their underlying etiologies is paramount, particularly their genetic components. In this study, TR-MDD patients (n = 124) were recruited and metabolites were quantified in their cerebrospinal fluid (CSF) and peripheral blood. Three sub-categories of deficiencies were examined, namely 5-methyltetrahydrofolte (in CSF; n = 13), tetrahydrobiopterin (in CSF; n = 11), and abnormal acylcarnitine profiles (in peripheral blood; n = 8). Whole exome sequencing was performed on genomic DNA from the entire TR-MDD cohort and exonic variant allele frequencies for cases were compared to a control cohort (1:5 matching on ancestry). Low frequency, damaging alleles were identified and used for in silico pathway analyses. Three association signals for TR-MDD approached genome-wide significance on chromosomes 22, 7, and 3. Three risk-associated variants from a prior depression study were replicated. Relevant biological pathways were identified that contained an enrichment of rare, damaging variants in central nervous system (CNS)-specific pathways, including neurotransmitter receptors, potassium channels, and synapse transmission. Some TR-MDD patients had rare variants in genes that were previously associated with other psychiatric disorders, psychiatric endophenotypes, CNS structural defects, and CNS-related cellular and molecular functions. Exome analysis of metabolically phenotyped TR-MDD patients has identified potentially functional gene pathways and low frequency, deleterious gene variants for further investigation. Further studies in larger cohorts of biochemically phenotyped TR-MDD patients are desirable to extend and confirm these findings.
Assuntos
Biopterinas/análogos & derivados , Carnitina/análogos & derivados , Transtorno Depressivo Resistente a Tratamento/sangue , Tetra-Hidrofolatos/sangue , Adolescente , Adulto , Alelos , Biopterinas/sangue , Carnitina/sangue , Simulação por Computador , Transtorno Depressivo Resistente a Tratamento/genética , Feminino , Predisposição Genética para Doença , Humanos , Masculino , Fenótipo , Polimorfismo de Nucleotídeo Único , Sequenciamento do Exoma , Adulto JovemRESUMO
BACKGROUND: Self-reported consanguinity is associated with risk for schizophrenia (SZ) in several inbred populations, but estimates using DNA-based coefficients of inbreeding are unavailable. Further, it is not known whether recessively inherited risk mutations can be identified through homozygosity by descent (HBD) mapping. METHODS: We studied self-reported and DNA-based estimates of inbreeding among Egyptian patients with SZ (nâ¯=â¯421, DSM IV criteria) and adult controls without psychosis (nâ¯=â¯301), who were evaluated using semi-structured diagnostic interview schedules and genotyped using the Illumina Infinium PsychArray. Following quality control checks, coefficients of inbreeding (F) and regions of homozygosity (ROH) were estimated using PLINK software for HBD analysis. Exome sequencing was conducted in selected cases. RESULTS: Inbreeding was associated with schizophrenia based on self-reported consanguinity (χ2â¯=â¯4.506, 1 df, pâ¯=â¯0.034) and DNA-based estimates for inbreeding (F); the latter with a significant Fâ¯×â¯age interaction (ßâ¯=â¯32.34, pâ¯=â¯0.0047). The association was most notable among patients older than age 40 years. Eleven ROH were over-represented in cases on chromosomes 1, 3, 6, 11, and 14; all but one region is novel for schizophrenia risk. Exome sequencing identified six recessively-acting genes in ROH with loss-of-function variants; one of which causes primary hereditary microcephaly. CONCLUSIONS: We propose consanguinity as an age-dependent risk factor for SZ in Egypt. HBD mapping is feasible for SZ in adequately powered samples.
Assuntos
Endogamia , Esquizofrenia , Adulto , Consanguinidade , Egito/epidemiologia , Homozigoto , Humanos , Polimorfismo de Nucleotídeo Único , Esquizofrenia/epidemiologia , Esquizofrenia/genéticaRESUMO
Major depressive disorder (MDD) affects approximately 15 million Americans. Approximately 2 million of these are classified as being refractory to treatment (TR-MDD). Because of the lack of available therapies for TR-MDD, and the high risk of suicide, there is interest in identifying new treatment modalities and diagnostic methods. Understanding of the impact of genomic copy number variation in the etiology of a variety of neuropsychiatric phenotypes is increasing. Low copy repeat elements at 15q13.3 facilitate non-allelic homologous recombination, resulting in recurrent copy number variants (CNVs). Numerous reports have described association between microdeletions in this region and a variety of neuropsychiatric phenotypes, with CHRNA7 implicated as a candidate gene. However, the pathogenicity of 15q13.3 duplications is less clear. As part of an ongoing study, in which we have identified a number of metabolomic anomalies in spinal fluid from TR-MDD patients, we also evaluated genomic copy number variation in patients (n = 125) and controls (n = 26) via array-based copy number genomic hybridization (CGH); the case frequency was compared with frequencies reported in a prior study as well as a larger population-sized cohort. We identified five TR-MDD patients with microduplications involving CHRNA7. CHRNA7 duplications are the most common CNVs identified by clinical CGH in this cohort. Therefore, this study provides insight into the potential involvement of CHRNA7 duplications in the etiology of TR-MDD and informs those involved with care of affected individuals.
Assuntos
Duplicação Cromossômica , Cromossomos Humanos Par 15/genética , Transtorno Depressivo Maior/genética , Adolescente , Adulto , Antidepressivos/uso terapêutico , Variações do Número de Cópias de DNA , Transtorno Depressivo Maior/tratamento farmacológico , Transtorno Depressivo Maior/metabolismo , Feminino , Humanos , Masculino , Metaboloma , Resultado do Tratamento , Receptor Nicotínico de Acetilcolina alfa7/genéticaRESUMO
Herpes simplex virus 1 (HSV-1) establishes latency in both peripheral nerve ganglia and the central nervous system (CNS). The outcomes of acute and latent infections in these different anatomic sites appear to be distinct. It is becoming clear that many of the existing culture models using animal primary neurons to investigate HSV-1 infection of the CNS are limited and not ideal, and most do not recapitulate features of CNS neurons. Human induced pluripotent stem cells (hiPSCs) and neurons derived from them are documented as tools to study aspects of neuropathogenesis, but few have focused on modeling infections of the CNS. Here, we characterize functional two-dimensional (2D) CNS-like neuron cultures and three-dimensional (3D) brain organoids made from hiPSCs to model HSV-1-human-CNS interactions. Our results show that (i) hiPSC-derived CNS neurons are permissive for HSV-1 infection; (ii) a quiescent state exhibiting key landmarks of HSV-1 latency described in animal models can be established in hiPSC-derived CNS neurons; (iii) the complex laminar structure of the organoids can be efficiently infected with HSV, with virus being transported from the periphery to the central layers of the organoid; and (iv) the organoids support reactivation of HSV-1, albeit less efficiently than 2D cultures. Collectively, our results indicate that hiPSC-derived neuronal platforms, especially 3D organoids, offer an extraordinary opportunity for modeling the interaction of HSV-1 with the complex cellular and architectural structure of the human CNS.IMPORTANCE This study employed human induced pluripotent stem cells (hiPSCs) to model acute and latent HSV-1 infections in two-dimensional (2D) and three-dimensional (3D) CNS neuronal cultures. We successfully established acute HSV-1 infections and infections showing features of latency. HSV-1 infection of the 3D organoids was able to spread from the outer surface of the organoid and was transported to the interior lamina, providing a model to study HSV-1 trafficking through complex neuronal tissue structures. HSV-1 could be reactivated in both culture systems; though, in contrast to 2D cultures, it appeared to be more difficult to reactivate HSV-1 in 3D cultures, potentially paralleling the low efficiency of HSV-1 reactivation in the CNS of animal models. The reactivation events were accompanied by dramatic neuronal morphological changes and cell-cell fusion. Together, our results provide substantive evidence of the suitability of hiPSC-based neuronal platforms to model HSV-1-CNS interactions in a human context.
Assuntos
Sistema Nervoso Central/metabolismo , Herpes Simples/metabolismo , Herpesvirus Humano 1/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios/metabolismo , Animais , Sistema Nervoso Central/patologia , Sistema Nervoso Central/virologia , Chlorocebus aethiops , Herpes Simples/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Células-Tronco Pluripotentes Induzidas/virologia , Neurônios/patologia , Neurônios/virologia , Células VeroRESUMO
Acyclovir (ACV) is an effective antiviral agent for treating lytic Herpes Simplex virus, type 1 (HSV-1) infections, and it has dramatically reduced the mortality rate of herpes simplex encephalitis. However, HSV-1 resistance to ACV and its derivatives is being increasingly documented, particularly among immunocompromised individuals. The burgeoning drug resistance compels the search for a new generation of more efficacious anti-herpetic drugs. We have previously shown that trans-dihydrolycoricidine (R430), a lycorane-type alkaloid derivative, effectively inhibits HSV-1 infections in cultured cells. We now report that R430 also inhibits ACV-resistant HSV-1 strains, accompanied by global inhibition of viral gene transcription and enrichment of H3K27me3 methylation on viral gene promoters. Furthermore, we demonstrate that R430 prevents HSV-1 reactivation from latency in an ex vivo rodent model. Finally, among a panel of DNA viruses and RNA viruses, R430 inhibited Zika virus with high therapeutic index. Its therapeutic index is comparable to standard antiviral drugs, though it has greater toxicity in non-neuronal cells than in neuronal cells. Synthesis of additional derivatives could enable more efficacious antivirals and the identification of active pharmacophores.
Assuntos
Alcaloides de Amaryllidaceae/farmacologia , Antivirais/farmacologia , Infecções por Vírus de DNA/tratamento farmacológico , Vírus de DNA/efeitos dos fármacos , Infecções por Vírus de RNA/tratamento farmacológico , Vírus de RNA/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Células Cultivadas , Chlorocebus aethiops , Infecções por Vírus de DNA/virologia , Humanos , Camundongos , Infecções por Vírus de RNA/virologia , Células VeroRESUMO
BACKGROUND: A variety of neurological disorders including neurodegenerative diseases and infection by neurotropic viruses can cause structural and functional changes in the central nervous system (CNS), resulting in long-term neurological sequelae. An improved understanding of the pathogenesis of these disorders is important for developing efficacious interventions. Human induced pluripotent stem cells (hiPSCs) offer an extraordinary window for modeling pathogen-CNS interactions, and other cellular interactions, in three-dimensional (3D) neuronal cultures that can recapitulate several aspects of in vivo brain tissue. METHODS: Herein, we describe a prototype of scaffold-free hiPSC-based adherent 3D (A-3D) human neuronal cultures in 96-well plates. To test their suitability for drug screening, A-3D neuronal cultures were infected with herpes simplex virus type 1 (HSV-1) with or without acyclovir. RESULTS: The half maximal inhibitory concentration (IC50) of acyclovir was 3.14 µM and 3.12 µM determined using flow cytometry and the CX7 High Content Screening platform, respectively. CONCLUSIONS: Our A-3D neuronal cultures provide an unprecedented opportunity for high-content drug screening programs to treat human CNS infections.
Assuntos
Sistema Nervoso Central/metabolismo , Neurônios/metabolismo , Viroses/genética , Diferenciação Celular , Humanos , Neurônios/citologia , Viroses/metabolismo , Viroses/patologiaRESUMO
The discovery of antiviral activity of 2,3-disubstituted quinazolinones, prepared by a one-pot, three-component condensation of isatoic anhydride with amines and aldehydes, against Herpes Simplex Virus (HSV)-1 is reported. Sequential iterative synthesis/antiviral assessment allowed structure-activity relationship (SAR) generation revealing synergistic structural features required for potent anti-HSV-1 activity. The most potent derivatives show greater efficacy than acyclovir against acute HSV-1 infections in neurons and minimal toxicity to the host.
Assuntos
Herpesvirus Humano 1/efeitos dos fármacos , Quinazolinonas/química , Quinazolinonas/farmacologia , Aciclovir/farmacologia , Animais , Antivirais/química , Antivirais/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Imunoprecipitação da Cromatina , Avaliação Pré-Clínica de Medicamentos , Humanos , Relação Estrutura-Atividade , Células VeroRESUMO
Patients with schizophrenia and their relatives have reduced prevalence of rheumatoid arthritis. Schizophrenia and rheumatoid arthritis genome-wide association studies also indicate negative genetic correlations, suggesting that there may be shared pathogenesis at the DNA level or downstream. A portion of the inverse prevalence could be attributed to pleiotropy, i.e., variants of a single nucleotide polymorphism that could confer differential risk for these disorders. To study the basis for such an interrelationship, we initially compared lists of single nucleotide polymorphisms with significant genetic associations (p < 1e-8) for schizophrenia or rheumatoid arthritis, evaluating patterns of linkage disequilibrium and apparent pleiotropic risk profiles. Single nucleotide polymorphisms that conferred risk for both schizophrenia and rheumatoid arthritis were localized solely to the extended HLA region. Among single nucleotide polymorphisms that conferred differential risk for schizophrenia and rheumatoid arthritis, the majority were localized to HLA-B, TNXB, NOTCH4, HLA-C, HCP5, MICB, PSORS1C1, and C6orf10; published functional data indicate that HLA-B and HLA-C have the most plausible pathogenic roles in both disorders. Interactomes of these eight genes were constructed from protein-protein interaction information using publicly available databases and novel computational predictions. The genes harboring apparently pleiotropic single nucleotide polymorphisms are closely connected to rheumatoid arthritis and schizophrenia associated genes through common interacting partners. A separate and independent analysis of the interactomes of rheumatoid arthritis and schizophrenia genes showed a significant overlap between the two interactomes and that they share several common pathways, motivating functional studies suggesting a relationship in the pathogenesis of schizophrenia/rheumatoid arthritis.