Deciphering novel TCF4-driven mechanisms underlying a common triplet repeat expansion-mediated disease.

Fuchs endothelial corneal dystrophy (FECD) is an age-related cause of vision loss, and the most common repeat expansion-mediated disease in humans characterised to date. Up to 80% of European FECD cases have been attributed to expansion of a non-coding CTG repeat element (termed CTG18.1) located within the ubiquitously expressed transcription factor encoding gene, TCF4. The non-coding nature of the repeat and the transcriptomic complexity of TCF4 have made it extremely challenging to experimentally decipher the molecular mechanisms underlying this disease. Here we comprehensively describe CTG18.1 expansion-driven molecular components of disease within primary patient-derived corneal endothelial cells (CECs), generated from a large cohort of individuals with CTG18.1-expanded (Exp+) and CTG 18.1-independent (Exp-) FECD. We employ long-read, short-read, and spatial transcriptomic techniques to interrogate expansion-specific transcriptomic biomarkers. Interrogation of long-read sequencing and alternative splicing analysis of short-read transcriptomic data together reveals the global extent of altered splicing occurring within Exp+ FECD, and unique transcripts associated with CTG18.1-expansions. Similarly, differential gene expression analysis highlights the total transcriptomic consequences of Exp+ FECD within CECs. Furthermore, differential exon usage, pathway enrichment and spatial transcriptomics reveal TCF4 isoform ratio skewing solely in Exp+ FECD with potential downstream functional consequences. Lastly, exome data from 134 Exp- FECD cases identified rare (minor allele frequency <0.005) and potentially deleterious (CADD>15) TCF4 variants in 7/134 FECD Exp- cases, suggesting that TCF4 variants independent of CTG18.1 may increase FECD risk. In summary, our study supports the hypothesis that at least two distinct pathogenic mechanisms, RNA toxicity and TCF4 isoform-specific dysregulation, both underpin the pathophysiology of FECD. We anticipate these data will inform and guide the development of translational interventions for this common triplet-repeat mediated disease.

Developmentally dynamic changes in DNA methylation in the human pancreas.

Development of the human pancreas requires the precise temporal control of gene expression via epigenetic mechanisms and the binding of key transcription factors. We quantified genome-wide patterns of DNA methylation in human fetal pancreatic samples from donors aged 6 to 21 post-conception weeks. We found dramatic changes in DNA methylation across pancreas development, with > 21% of sites characterized as developmental differentially methylated positions (dDMPs) including many annotated to genes associated with monogenic diabetes. An analysis of DNA methylation in postnatal pancreas tissue showed that the dramatic temporal changes in DNA methylation occurring in the developing pancreas are largely limited to the prenatal period. Significant differences in DNA methylation were observed between males and females at a number of autosomal sites, with a small proportion of sites showing sex-specific DNA methylation trajectories across pancreas development. Pancreas dDMPs were not distributed equally across the genome and were depleted in regulatory domains characterized by open chromatin and the binding of known pancreatic development transcription factors. Finally, we compared our pancreas dDMPs to previous findings from the human brain, identifying evidence for tissue-specific developmental changes in DNA methylation. This study represents the first systematic exploration of DNA methylation patterns during human fetal pancreas development and confirms the prenatal period as a time of major epigenomic plasticity.

Evaluation of nanopore sequencing for epigenetic epidemiology: a comparison with DNA methylation microarrays.

Most epigenetic epidemiology to date has utilized microarrays to identify positions in the genome where variation in DNA methylation is associated with environmental exposures or disease. However, these profile less than 3% of DNA methylation sites in the human genome, potentially missing affected loci and preventing the discovery of disrupted biological pathways. Third generation sequencing technologies, including Nanopore sequencing, have the potential to revolutionize the generation of epigenetic data, not only by providing genuine genome-wide coverage but profiling epigenetic modifications direct from native DNA. Here we assess the viability of using Nanopore sequencing for epidemiology by performing a comparison with DNA methylation quantified using the most comprehensive microarray available, the Illumina EPIC array. We implemented a CRISPR-Cas9 targeted sequencing approach in concert with Nanopore sequencing to profile DNA methylation in three genomic regions to attempt to rediscover genomic positions that existing technologies have shown are differentially methylated in tobacco smokers. Using Nanopore sequencing reads, DNA methylation was quantified at 1779 CpGs across three regions, providing a finer resolution of DNA methylation patterns compared to the EPIC array. The correlation of estimated levels of DNA methylation between platforms was high. Furthermore, we identified 12 CpGs where hypomethylation was significantly associated with smoking status, including 10 within the AHRR gene. In summary, Nanopore sequencing is a valid option for identifying genomic loci where large differences in DNAm are associated with a phenotype and has the potential to advance our understanding of the role differential methylation plays in the etiology of complex disease.

Phenotypic effect of GBA1 variants in individuals with and without Parkinson's disease: The RAPSODI study.

BACKGROUND: Variants in the GBA1 gene cause the lysosomal storage disorder Gaucher disease (GD). They are also risk factors for Parkinson's disease (PD), and modify the expression of the PD phenotype. The penetrance of GBA1 variants in PD is incomplete, and the ability to determine who among GBA1 variant carriers are at higher risk of developing PD, would represent an advantage for prognostic and trial design purposes. OBJECTIVES: To compare the motor and non-motor phenotype of GBA1 carriers and non-carriers. METHODS: We present the cross-sectional results of the baseline assessment from the RAPSODI study, an online assessment tool for PD patients and GBA1 variant carriers. The assessment includes clinically validated questionnaires, a tap-test, the University of Pennsyllvania Smell Identification Test and cognitive tests. Additional, homogeneous data from the PREDICT-PD cohort were included. RESULTS: A total of 379 participants completed all parts of the RAPSODI assessment (89 GBA1-negative controls, 169 GBA1-negative PD, 47 GBA1-positive PD, 47 non-affected GBA1 carriers, 27 GD). Eighty-six participants were recruited through PREDICT-PD (43 non-affected GBA1 carriers and 43 GBA1-negative controls). GBA1-positive PD patients showed worse performance in visual cognitive tasks and olfaction compared to GBA1-negative PD patients. No differences were detected between non-affected GBA1 carriers carriers and GBA1-negative controls. No phenotypic differences were observed between any of the non-PD groups. CONCLUSIONS: Our results support previous evidence that GBA1-positive PD has a specific phenotype with more severe non-motor symptoms. However, we did not reproduce previous findings of more frequent prodromal PD signs in non-affected GBA1 carriers.

Postpandemic rebound of adeno-associated virus type 2 (AAV2) infections temporally associated with an outbreak of unexplained severe acute hepatitis in children in the United Kingdom.

Over 1000 cases of unexplained severe acute hepatitis in children have been reported to date worldwide. An association with adeno-associated virus type 2 (AAV2) infection, a human parvovirus, prompted us to investigate the epidemiology of AAV in the United Kingdom. Three hundred pediatric respiratory samples collected before (April 03, 2009-April 03, 2013) and during (April 03, 2022) the COVID-19 pandemic were obtained. Wastewater samples were collected from 50 locations in London (August 2021-March 2022). Samples were tested for AAV using real-time polymerase chain reaction followed by sequencing. Selected adenovirus (AdV)-positive samples were also sequenced. The detection frequency of AAV2 was a sevenfold higher in 2022 samples compared with 2009-2013 samples (10% vs. 1.4%) and highest in AdV-positive samples compared with negatives (10/37, 27% vs. 5/94, 5.3%, respectively). AAV2-positive samples displayed high genetic diversity. AAV2 sequences were either very low or absent in wastewater collected in 2021 but increased in January 2022 and peaked in March 2022. AAV2 was detected in children in association with AdV of species C, with a highest frequency in 2022. Our findings are consistent with the expansion of the population of children unexposed to AAV2, leading to greater spread of the virus once distancing restrictions were lifted.

Growth disrupting mutations in epigenetic regulatory molecules are associated with abnormalities of epigenetic aging.

Germline mutations in fundamental epigenetic regulatory molecules including DNA methyltransferase 3 alpha (DNMT3A) are commonly associated with growth disorders, whereas somatic mutations are often associated with malignancy. We profiled genome-wide DNA methylation patterns in DNMT3A c.2312G > A; p.(Arg771Gln) carriers in a large Amish sibship with Tatton-Brown-Rahman syndrome (TBRS), their mosaic father, and 15 TBRS patients with distinct pathogenic de novo DNMT3A variants. This defined widespread DNA hypomethylation at specific genomic sites enriched at locations annotated as genes involved in morphogenesis, development, differentiation, and malignancy predisposition pathways. TBRS patients also displayed highly accelerated DNA methylation aging. These findings were most marked in a carrier of the AML-associated driver mutation p.Arg882Cys. Our studies additionally defined phenotype-related accelerated and decelerated epigenetic aging in two histone methyltransferase disorders: NSD1 Sotos syndrome overgrowth disorder and KMT2D Kabuki syndrome growth impairment. Together, our findings provide fundamental new insights into aberrant epigenetic mechanisms, the role of epigenetic machinery maintenance, and determinants of biological aging in these growth disorders.

Long-lasting transcription in hippocampal area CA1 after contextual fear conditioning.

A fundamental question is how memory is stored for several weeks and even longer. A long-lasting increase in gene transcription has been suggested to mediate such long-term memory storage. Here, we used contextual fear conditioning in mice to search for lasting transcription that may contribute to long-term memory storage. Our study focussed on hippocampal area CA1, which has been suggested to have a role for at least one week in contextual fear memory. Using an unbiased microarray analysis followed by confirmatory quantitative real-time PCR, we identified an upregulation of two transcription factors, Fosl2 and Nfil3, which lasted for seven days after conditioning. To our knowledge these are the longest transcriptional changes ever detected in the hippocampus after contextual fear conditioning. Thus, our findings suggest novel transcriptional candidates for long-term memory storage.

Knockdown of the psychosis susceptibility gene ZNF804A alters expression of genes involved in cell adhesion.

Genome-wide association studies have convincingly implicated several novel genes in susceptibility to schizophrenia and bipolar disorder. The first genome-wide significant association with the broad phenotype of psychosis was with a polymorphism in the ZNF804A gene. However, the biological function(s) of ZNF804A have, to date, been entirely unknown. In this study, we manipulated the expression of ZNF804A in neural progenitor cells derived from human cortical neuroepithelium and assessed its effects on the cellular transcriptome. Gene ontology analysis of differentially expressed genes indicated a significant effect of ZNF804A knockdown on the expression of genes involved in cell adhesion, suggesting a role for ZNF804A in processes such as neural migration, neurite outgrowth and synapse formation. Several highly significant gene expression changes were confirmed in repeat cell culture experiments. Most consistent gene expression changes were seen for C2ORF80, a gene of as-yet-unknown function, and STMN3, a gene involved in neurite outgrowth and axonal and dendritic branching. These data, generated in a hypothesis-free manner, provide a basis for more targeted investigations of ZNF804A function.

In field use of water samples for genomic surveillance of infectious spleen and kidney necrosis virus (ISKNV) infecting tilapia fish in Lake Volta, Ghana.

Viral outbreaks are a constant threat to aquaculture, limiting production for better global food security. A lack of diagnostic testing and monitoring in resource-limited areas hinders the capacity to respond rapidly to disease outbreaks and to prevent viral pathogens becoming endemic in fisheries productive waters. Recent developments in diagnostic testing for emerging viruses, however, offers a solution for rapid in situ monitoring of viral outbreaks. Genomic epidemiology has furthermore proven highly effective in detecting viral mutations involved in pathogenesis and assisting in resolving chains of transmission. Here, we demonstrate the application of an in-field epidemiological tool kit to track viral outbreaks in aquaculture on farms with reduced access to diagnostic labs, and with non-destructive sampling. Inspired by the "lab in a suitcase" approach used for genomic surveillance of human viral pathogens and wastewater monitoring of COVID19, we evaluated the feasibility of real-time genome sequencing surveillance of the fish pathogen, Infectious spleen and kidney necrosis virus (ISKNV) in Lake Volta. Viral fractions from water samples collected from cages holding Nile tilapia (Oreochromis niloticus) with suspected ongoing ISKNV infections were concentrated and used as a template for whole genome sequencing, using a previously developed tiled PCR method for ISKNV. Mutations in ISKNV in samples collected from the water surrounding the cages matched those collected from infected caged fish, illustrating that water samples can be used for detecting predominant ISKNV variants in an ongoing outbreak. This approach allows for the detection of ISKNV and tracking of the dynamics of variant frequencies, and may thus assist in guiding control measures for the rapid isolation and quarantine of infected farms and facilities.

Long-read transcript sequencing identifies differential isoform expression in the entorhinal cortex in a transgenic model of tau pathology.

Increasing evidence suggests that alternative splicing plays an important role in Alzheimer's disease (AD) pathology. We used long-read sequencing in combination with a novel bioinformatics tool (FICLE) to profile transcript diversity in the entorhinal cortex of female transgenic (TG) mice harboring a mutant form of human tau. Our analyses revealed hundreds of novel isoforms and identified differentially expressed transcripts - including specific isoforms of Apoe, App, Cd33, Clu, Fyn and Trem2 - associated with the development of tau pathology in TG mice. Subsequent profiling of the human cortex from AD individuals and controls revealed similar patterns of transcript diversity, including the upregulation of the dominant TREM2 isoform in AD paralleling the increased expression of the homologous transcript in TG mice. Our results highlight the importance of differential transcript usage, even in the absence of gene-level expression alterations, as a mechanism underpinning gene regulation in the development of AD neuropathology.

Allelic skewing of DNA methylation is widespread across the genome.

DNA methylation is assumed to be complementary on both alleles across the genome, although there are exceptions, notably in regions subject to genomic imprinting. We present a genome-wide survey of the degree of allelic skewing of DNA methylation with the aim of identifying previously unreported differentially methylated regions (DMRs) associated primarily with genomic imprinting or DNA sequence variation acting in cis. We used SNP microarrays to quantitatively assess allele-specific DNA methylation (ASM) in amplicons covering 7.6% of the human genome following cleavage with a cocktail of methylation-sensitive restriction enzymes (MSREs). Selected findings were verified using bisulfite-mapping and gene-expression analyses, subsequently tested in a second tissue from the same individuals, and replicated in DNA obtained from 30 parent-child trios. Our approach detected clear examples of ASM in the vicinity of known imprinted loci, highlighting the validity of the method. In total, 2,704 (1.5%) of our 183,605 informative and stringently filtered SNPs demonstrate an average relative allele score (RAS) change > or =0.10 following MSRE digestion. In agreement with previous reports, the majority of ASM ( approximately 90%) appears to be cis in nature, and several examples of tissue-specific ASM were identified. Our data show that ASM is a widespread phenomenon, with >35,000 such sites potentially occurring across the genome, and that a spectrum of ASM is likely, with heterogeneity between individuals and across tissues. These findings impact our understanding about the origin of individual phenotypic differences and have implications for genetic studies of complex disease.

Stochastic choice of allelic expression in human neural stem cells.

Monoallelic gene expression, such as genomic imprinting, is well described. Less well-characterized are genes undergoing stochastic monoallelic expression (MA), where specific clones of cells express just one allele at a given locus. We performed genome-wide allelic expression assessment of human clonal neural stem cells derived from cerebral cortex, striatum, and spinal cord, each with differing genotypes. We assayed three separate clonal lines from each donor, distinguishing stochastic MA from genotypic effects. Roughly 2% of genes showed evidence for autosomal MA, and in about half of these, allelic expression was stochastic between different clones. Many of these loci were known neurodevelopmental genes, such as OTX2 and OLIG2. Monoallelic genes also showed increased levels of DNA methylation compared to hypomethylated biallelic loci. Identified monoallelic gene loci showed altered chromatin signatures in fetal brain, suggesting an in vivo correlate of this phenomenon. We conclude that stochastic allelic expression is prevalent in neural stem cells, providing clonal diversity to developing tissues such as the human brain.

Comparison of metagenomic and targeted methods for sequencing human pathogenic viruses from wastewater.

Wastewater-based epidemiology is a powerful tool for monitoring the emergence and spread of viral pathogens at the population scale. Typical polymerase chain reaction (PCR)-based methods of quantitative and genomic monitoring of viruses in wastewater provide high sensitivity and specificity. However, these methods are limited to the surveillance of target viruses in a single assay and require prior knowledge of the target genome(s). Metagenomic sequencing methods may represent a target-agnostic approach to viral wastewater monitoring, allowing for the detection of a broad range of target viruses, including potentially novel and emerging pathogens. In this study, targeted and untargeted metagenomic sequencing methods were compared with tiled-PCR sequencing for the detection and genotyping of viral pathogens in wastewater samples. Deep shotgun metagenomic sequencing was unable to generate sufficient genome coverage of human pathogenic viruses for robust genomic epidemiology, with samples dominated by bacteria. Hybrid-capture enrichment of shotgun libraries for respiratory viruses led to significant increases in genome coverage for a range of targets. Tiled-PCR sequencing led to further improvements in genome coverage compared to hybrid capture for severe acute respiratory syndrome coronavirus 2, enterovirus D68, norovirus GII, and human adenovirus F41 in wastewater samples. In conclusion, untargeted shotgun sequencing was unsuitable for genomic monitoring of the low virus concentrations in wastewater samples analyzed in this study. Hybrid-capture enrichment represented a viable method for simultaneous genomic epidemiology of a range of viral pathogens, while tiled-PCR sequencing provided the optimal genome coverage for individual viruses with the minimum sequencing depth. IMPORTANCE Most public health initiatives that monitor viruses in wastewater have utilized quantitative polymerase chain reaction (PCR) and whole genome PCR sequencing, mirroring techniques used for viral epidemiology in individuals. These techniques require prior knowledge of the target viral genome and are limited to monitoring individual or small groups of viruses. Metagenomic sequencing may offer an alternative strategy for monitoring a broad spectrum of viruses in wastewater, including novel and emerging pathogens. In this study, while amplicon sequencing gave high viral genome coverage, untargeted shotgun sequencing of total nucleic acid samples was unable to detect human pathogenic viruses with enough sensitivity for use in genomic epidemiology. Enrichment of shotgun libraries for respiratory viruses using hybrid-capture technology provided genotypic information on a range of viruses simultaneously, indicating strong potential for wastewater surveillance. This type of targeted metagenomics could be used for monitoring diverse targets, such as pathogens or antimicrobial resistance genes, in environmental samples.

Optimised protocol for monitoring SARS-CoV-2 in wastewater using reverse complement PCR-based whole-genome sequencing.

Monitoring the spread of viral pathogens in the population during epidemics is crucial for mounting an effective public health response. Understanding the viral lineages that constitute the infections in a population can uncover the origins and transmission patterns of outbreaks and detect the emergence of novel variants that may impact the course of an epidemic. Population-level surveillance of viruses through genomic sequencing of wastewater captures unbiased lineage data, including cryptic asymptomatic and undiagnosed infections, and has been shown to detect infection outbreaks and novel variant emergence before detection in clinical samples. Here, we present an optimised protocol for quantification and sequencing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in influent wastewater, used for high-throughput genomic surveillance in England during the COVID-19 pandemic. This protocol utilises reverse compliment PCR for library preparation, enabling tiled amplification across the whole viral genome and sequencing adapter addition in a single step to enhance efficiency. Sequencing of synthetic SARS-CoV-2 RNA provided evidence validating the efficacy of this protocol, while data from high-throughput sequencing of wastewater samples demonstrated the sensitivity of this method. We also provided guidance on the quality control steps required during library preparation and data analysis. Overall, this represents an effective method for high-throughput sequencing of SARS-CoV-2 in wastewater which can be applied to other viruses and pathogens of humans and animals.

Utility of wastewater genomic surveillance compared to clinical surveillance to track the spread of the SARS-CoV-2 Omicron variant across England.

The world has moved into a new stage of managing the SARS-CoV-2 pandemic with minimal restrictions and reduced testing in the population, leading to reduced genomic surveillance of virus variants in individuals. Wastewater-based epidemiology (WBE) can provide an alternative means of tracking virus variants in the population but decision-makers require confidence that it can be applied to a national scale and is comparable to individual testing data. We analysed 19,911 samples from 524 wastewater sites across England at least twice a week between November 2021 and February 2022, capturing sewage from >70% of the English population. We used amplicon-based sequencing and the phylogeny based de-mixing tool Freyja to estimate SARS-CoV-2 variant frequencies and compared these to the variant dynamics observed in individual testing data from clinical and community settings. We show that wastewater data can reconstruct the spread of the Omicron variant across England since November 2021 in close detail and aligns closely with epidemiological estimates from individual testing data. We also show the temporal and spatial spread of Omicron within London. Our wastewater data further reliably track the transition between Omicron subvariants BA1 and BA2 in February 2022 at regional and national levels. Our demonstration that WBE can track the fast-paced dynamics of SARS-CoV-2 variant frequencies at a national scale and closely match individual testing data in time shows that WBE can reliably fill the monitoring gap left by reduced individual testing in a more affordable way.

Effects of cis-regulatory variation differ across regions of the adult human brain.

Cis-regulatory variation is considered to be an important determinant of human phenotypic variability, including susceptibility to complex disease. Recent studies have shown that the effects of cis-regulatory polymorphism on gene expression can differ widely between tissues. In the present study, we tested whether the effects of cis-regulatory variation can also differ between regions of the adult human brain. We used relative allelic expression to measure cis-effects on the RNA expression of five candidate genes for neuropsychiatric illness (ZNF804A, NOS1, RGS4, AKT1 and TCF4) across multiple discrete brain regions within individual subjects. For all five genes, we observed significant differences in allelic expression between brain regions in several individual subjects, suggesting regional differences in the effects of cis-regulatory polymorphism to be a common phenomenon. As well as highlighting an important caveat for studies of regulatory polymorphism in the brain, our findings indicate that it is possible to delineate brain areas in which cis-regulatory variants are active. This may provide important insights into the fundamental biology of neuropsychiatric phenotypes with which such variants are associated.

Attenuated Induction of the Unfolded Protein Response in Adult Human Primary Astrocytes in Response to Recurrent Low Glucose.

Aims/hypothesis: Recurrent hypoglycaemia (RH) is a major side-effect of intensive insulin therapy for people with diabetes. Changes in hypoglycaemia sensing by the brain contribute to the development of impaired counterregulatory responses to and awareness of hypoglycaemia. Little is known about the intrinsic changes in human astrocytes in response to acute and recurrent low glucose (RLG) exposure. Methods: Human primary astrocytes (HPA) were exposed to zero, one, three or four bouts of low glucose (0.1 mmol/l) for three hours per day for four days to mimic RH. On the fourth day, DNA and RNA were collected. Differential gene expression and ontology analyses were performed using DESeq2 and GOseq, respectively. DNA methylation was assessed using the Infinium MethylationEPIC BeadChip platform. Results: 24 differentially expressed genes (DEGs) were detected (after correction for multiple comparisons). One bout of low glucose exposure had the largest effect on gene expression. Pathway analyses revealed that endoplasmic-reticulum (ER) stress-related genes such as HSPA5, XBP1, and MANF, involved in the unfolded protein response (UPR), were all significantly increased following low glucose (LG) exposure, which was diminished following RLG. There was little correlation between differentially methylated positions and changes in gene expression yet the number of bouts of LG exposure produced distinct methylation signatures. Conclusions/interpretation: These data suggest that exposure of human astrocytes to transient LG triggers activation of genes involved in the UPR linked to endoplasmic reticulum (ER) stress. Following RLG, the activation of UPR related genes was diminished, suggesting attenuated ER stress. This may be a consequence of a successful metabolic adaptation, as previously reported, that better preserves intracellular energy levels and a reduced necessity for the UPR.

Full-length transcript sequencing of human and mouse cerebral cortex identifies widespread isoform diversity and alternative splicing.

Alternative splicing is a post-transcriptional regulatory mechanism producing distinct mRNA molecules from a single pre-mRNA with a prominent role in the development and function of the central nervous system. We used long-read isoform sequencing to generate full-length transcript sequences in the human and mouse cortex. We identify novel transcripts not present in existing genome annotations, including transcripts mapping to putative novel (unannotated) genes and fusion transcripts incorporating exons from multiple genes. Global patterns of transcript diversity are similar between human and mouse cortex, although certain genes are characterized by striking differences between species. We also identify developmental changes in alternative splicing, with differential transcript usage between human fetal and adult cortex. Our data confirm the importance of alternative splicing in the cortex, dramatically increasing transcriptional diversity and representing an important mechanism underpinning gene regulation in the brain. We provide transcript-level data for human and mouse cortex as a resource to the scientific community.

Novel epigenetic clock for fetal brain development predicts prenatal age for cellular stem cell models and derived neurons.

Induced pluripotent stem cells (iPSCs) and their differentiated neurons (iPSC-neurons) are a widely used cellular model in the research of the central nervous system. However, it is unknown how well they capture age-associated processes, particularly given that pluripotent cells are only present during the earliest stages of mammalian development. Epigenetic clocks utilize coordinated age-associated changes in DNA methylation to make predictions that correlate strongly with chronological age. It has been shown that the induction of pluripotency rejuvenates predicted epigenetic age. As existing clocks are not optimized for the study of brain development, we developed the fetal brain clock (FBC), a bespoke epigenetic clock trained in human prenatal brain samples in order to investigate more precisely the epigenetic age of iPSCs and iPSC-neurons. The FBC was tested in two independent validation cohorts across a total of 194 samples, confirming that the FBC outperforms other established epigenetic clocks in fetal brain cohorts. We applied the FBC to DNA methylation data from iPSCs and embryonic stem cells and their derived neuronal precursor cells and neurons, finding that these cell types are epigenetically characterized as having an early fetal age. Furthermore, while differentiation from iPSCs to neurons significantly increases epigenetic age, iPSC-neurons are still predicted as being fetal. Together our findings reiterate the need to better understand the limitations of existing epigenetic clocks for answering biological research questions and highlight a limitation of iPSC-neurons as a cellular model of age-related diseases.

The role of omega-3 fatty acids in preventing glucocorticoid-induced reduction in human hippocampal neurogenesis and increase in apoptosis.

Glucocorticoids have been suggested to be involved in several neuropsychiatric disorders, including depression. One of the possible mechanisms through which glucocorticoids contribute to the development of the depressive symptomatology is via regulation of distinct neurogenic mechanisms in the brain. A preventive or protective approach for these patients might be the use of omega-3 polyunsaturated fatty acids (n-3 PUFAs), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are known for they neuroprotective properties. We used the human hippocampal progenitor cell line HPC0A07/03C and pre-treated cells with either EPA or DHA, followed by treatment with the glucocorticoid cortisol either alone, or in co-treatment with the same n-3 PUFA during subsequent 3 days of proliferation and 7 days of differentiation. During proliferation, both EPA and DHA were able to prevent cortisol-induced reduction in proliferation and increase in apoptosis, when used in pre-treatment, and both pre- and co-treatment. During differentiation, EPA was able to prevent cortisol-induced reduction in neurogenesis and increase in apoptosis, when used in pre-treatment, and both pre- and co-treatment only during the proliferation stage; however, DHA required continuous treatment also during the differentiation stage to prevent cortisol-induced reduction in neurogenesis. Using transcriptomic analyses, we showed that both EPA and DHA regulated pathways involved in oxidative stress and immune response [e.g., nuclear factor (erythroid-derived 2)-like 2 (Nrf2), Signal transducer and activator of transcription 3 (STAT3), Interferon (IFN) and Interleukin (IL)-1 signaling], whereas DHA also regulated pathways involved in cell development and neuronal formation [e.g., cAMP-response element binding protein (CREB) signaling]. We provide the first evidence for treatment with both EPA and DHA to prevent cortisol-induced reduction in human hippocampal neurogenesis, and identify novel molecular mechanisms underlying these effects.