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Human epidemiological studies reveal that dietary and environmental alterations influence the health of the offspring and that the effect is not limited to the F1 or F2 generations. Non-Mendelian transgenerational inheritance of traits in response to environmental stimuli has been confirmed in non-mammalian organisms including plants and worms and are shown to be epigenetically mediated. However, transgenerational inheritance beyond the F2 generation remains controversial in mammals. Our lab previously discovered that the treatment of rodents (rats and mice) with folic acid significantly enhances the regeneration of injured axons following spinal cord injury in vivo and in vitro, and the effect is mediated by DNA methylation. The potential heritability of DNA methylation prompted us to investigate the following question: Is the enhanced axonal regeneration phenotype inherited transgenerationally without exposure to folic acid supplementation in the intervening generations? In the present review, we condense our findings showing that a beneficial trait (i.e., enhanced axonal regeneration after spinal cord injury) and accompanying molecular alterations (i.e., DNA methylation), triggered by an environmental exposure (i.e., folic acid supplementation) to F0 animals only, are inherited transgenerationally and beyond the F3 generation.

Introduction The biological basis of daytime sleepiness and excessive sleep duration in patients with unexplained hypersomnolence is unknown. However, a growing body of evidence suggests modulations in the epigenome coincide with objective measures of sleep, suggesting epigenetic mechanisms may be related to hypersomnolence. Previously, we identified significant correlations between saliva DNA methylation abundance at nine CpG sites located in the PAX8/PAX8-AS1 genes and total sleep time measured using polysomnography in unmedicated, clinical patients with unexplained hypersomnolence. Since psychotropic medications frequently affect clinical sleep testing and may also affect DNA methylation, here we attempted to replicate prior findings in a separate, medicated sample. Methods Forty-three medicated clinical patients with unexplained hypersomnolence were drawn from a larger study of a multimodal sleep assessment that included ad libitum overnight polysomnography. Pyrosequencing quantified DNA methylation at the previously identified CpG sites in PAX8/PAX8-AS1. Multivariate linear models, adjusting for age, sex, and body mass index (BMI), assessed relationships between total sleep time and DNA methylation at these prespecified CpG sites. Results Participants were young -to middle-aged (Mean Age = 32.2±10 years) and predominantly female (Percent Female = 95.3%), with a mean BMI of 27.8±5.4kg/m2. Epworth Sleepiness Scale (ESS) score was consistent with complaints of daytime sleepiness (Mean ESS =15.1±-4.3). Total sleep time on ad libitum polysomnography was 9.2±1.7 hours (range 5.6-12.8 hours). No significant associations between DNA methylation at the PAX8/PAX8-AS1 CpG sites and sleep duration were identified in adjusted models. Conclusion We were unable to replicate findings of an association between sleep duration and DNA methylation levels at specific PAX8/PAX8-AS1 CpG sites in medicated patients with unexplained hypersomnolence. Further research to clarify whether this is a result of medications affecting the DNA methylation levels at these CpG sites or previously identified associations between DNA methylation of PAX8/PAX8-AS1 and sleep length are spurious will require replication of prior findings in a novel unmedicated cohort and quantification of within-subjects effects of psychotropic medications on DNA methylation levels in genes of interest. Support (if any) AASM Foundation (229 SR-20 - Diversity Supplement Grant)

While embryonic mammalian central nervous system (CNS) axons readily grow and differentiate, only a minority of fully differentiated mature CNS neurons are able to regenerate injured axons, leading to stunted functional recovery after injury and disease. To delineate DNA methylation changes specifically associated with axon regeneration, we developed a Fluorescent-Activated Cell Sorting (FACS)-based methodology in a rat optic nerve transection model to segregate the injured retinal ganglion cells (RGCs) into regenerating and non-regenerating cell populations. Whole-genome DNA methylation profiling of these purified neurons revealed known and novel genes and pathways linked to mammalian RGC regeneration. Moreover, whole-methylome sequencing of purified uninjured adult and embryonic RGCs identified embryonic molecular profiles reactivated after injury in mature neurons, and others that correlate specifically with embryonic or adult axon growth, but not both. The results highlight the contribution to both embryonic growth and adult axon regeneration of subunits encoding the Na+/K+-ATPase. In turn, both biochemical and genetic inhibition of the Na+/K+-ATPase pump significantly reduced RGC axon regeneration. These data provide critical molecular insights into mammalian CNS axon regeneration, pinpoint the Na+/K+-ATPase as a key regulator of regeneration of injured mature CNS axons, and suggest that successful regeneration requires, in part, reactivation of embryonic signals. Identification of the specific role of DNA methylation in CNS regeneration promises novel therapeutic targets for CNS injury and disease.

We recently reported the COVID-19-induced circulating leukocytes DNA methylation profile. Here, we hypothesized that some of these genes would persist differentially methylated after disease resolution. Fifteen participants previously hospitalized for SARS-CoV-2 infection were epityped one year after discharge. Of the 1505 acute illness-induced differentially methylated regions (DMRs) previously identified, we found 71 regions with persisted differentially methylated, with an average of 7 serial CpG positions per DMR. Sixty-four DMRs persisted hypermethylated, and 7 DMR persisted hypomethylated. These data are the first reported evidence that DNA methylation changes in circulating leukocytes endure long after recovery from acute illness.

Maternal malnutrition remains one of the major adversities affecting brain development and long-term mental health outcomes, increasing the risk to develop anxiety and depressive disorders. We have previously shown that malnutrition-induced anxiety-like behaviours can be rescued by a social and sensory stimulation (enriched environment) in male mice. Here, we expand these findings to adult female mice and profiled genome-wide ventral hippocampal 5hmC levels related to malnutrition-induced anxiety-like behaviours and their rescue by an enriched environment. This approach revealed 508 differentially hydroxymethylated genes associated with protein malnutrition and that several genes (N = 34) exhibited a restored 5hmC abundance to control levels following exposure to an enriched environment, including genes involved in neuronal functions like dendrite outgrowth, axon guidance, and maintenance of neuronal circuits (e.g. Fltr3, Itsn1, Lman1, Lsamp, Nav, and Ror1) and epigenetic mechanisms (e.g. Hdac9 and Dicer1). Sequence motif predictions indicated that 5hmC may be modulating the binding of transcription factors for several of these transcripts, suggesting a regulatory role for 5hmC in response to perinatal malnutrition and exposure to an enriched environment. Together, these findings establish a role for 5hmC in early-life malnutrition and reveal genes linked to malnutrition-induced anxious behaviours that are mitigated by an enriched environment.

Folate supplementation in F0 mating rodents increases regeneration of injured spinal axons in vivo in 4 or more generations of progeny (F1-F4) in the absence of interval folate administration to the progeny. Transmission of the enhanced regeneration phenotype to untreated progeny parallels axonal growth in neuron culture after in vivo folate administration to the F0 ancestors alone, in correlation with differential patterns of genomic DNA methylation and RNA transcription in treated lineages. Enhanced axonal regeneration phenotypes are observed with diverse folate preparations and routes of administration, in outbred and inbred rodent strains, and in two rodent genera comprising rats and mice, and are reversed in F4-F5 progeny by pretreatment with DNA demethylating agents prior to phenotyping. Uniform transmission of the enhanced regeneration phenotype to progeny together with differential patterns of DNA methylation and RNA expression is consistent with a non-Mendelian mechanism. The capacity of an essential nutritional co-factor to induce a beneficial transgenerational phenotype in untreated offspring carries broad implications for the diagnosis, prevention, and treatment of inborn and acquired disorders.

Study Objectives Patients with unexplained hypersomnolence have significant impairment related to daytime sleepiness and excessive sleep duration, the biological bases of which are poorly understood. This investigation sought to examine relationships between objectively measured hypersomnolence phenotypes and epigenetic modification of candidate hypersomnolence genes to advance this line of inquiry. Methods Twenty-eight unmedicated clinical patients with unexplained hypersomnolence were evaluated using overnight ad libitum polysomnography, multiple sleep latency testing, infrared pupillometry, and the psychomotor vigilance task. DNA methylation levels on CpG sites annotated to 11 a priori hypersomnolence candidate genes were assessed for statistical association with hypersomnolence measures using independent regression models with adjusted local index of significance (aLIS) P-value threshold of 0.05. Results Nine CpG sites exhibited significant associations between DNA methylation levels and total sleep time measured using ad libitum polysomnography (aLIS p-value < .05). All nine differentially methylated CpG sites were annotated to the paired box 8 (PAX8) gene and its related antisense gene (PAX8-AS1). Among these nine differentially methylated positions was a cluster of five CpG sites located in the body of the PAX8 gene and promoter of PAX8-AS1. Conclusions This study demonstrates that PAX8/PAX8-AS1 DNA methylation levels are associated with total sleep time in persons with unexplained hypersomnolence. Given prior investigations that have implicated single nucleotide polymorphisms in PAX8/PAX8-AS1 with habitual sleep duration, further research that clarifies the role of DNA methylation levels on these genes in the phenotypic expression of total sleep time is warranted.

Maternal and environmental factors influence brain networks and architecture via both physiological pathways and epigenetic modifications. In particular, prenatal maternal depression and anxiety symptoms appear to impact infant white matter (WM) microstructure, leading us to investigate whether epigenetic modifications (i.e., DNA methylation) contribute to these WM differences. To determine if infants of women with depression and anxiety symptoms exhibit epigenetic modifications linked to neurodevelopmental changes, 52 umbilical cord bloods (CBs) were profiled. We observed 219 differentially methylated genomic positions (DMPs; FDR p p value 0.5), which were annotated to 98 and 81 genes, respectively. Together, these findings suggest that umbilical CB DNA methylation levels at birth are associated with 1-month WM microstructure.

Meningeal lymphatic vessels residing in the dural layer above the sinuses of the brain, meninges at the base of the brain, and near the cribriform plate have all been shown to drain fluid, cells, and antigens. We have previously reported that meningeal lymphatics near the cribriform plate undergo VEGFR3-dependent lymphangiogenesis during experimental autoimmune encephalomyelitis (EAE) to facilitate excess drainage. Using single-cell RNA sequencing (scRNA-seq), we report that neuroinflammation changes the phenotype and function of cribriform plate lymphatic endothelial cells (cpLECs). Upregulation of genes involved in antigen presentation, adhesion to leukocytes, and immunoregulatory molecules were verified by flow cytometry and functional assays. The inflamed cpLECs retain dendritic cells and to lesser extent CD4 T cells, creating an immune-regulatory niche that represents a previously underappreciated interface in the regulation of neuroinflammation. Additionally, the discontinuity of the arachnoid membrane near cpLECs provides unrestricted access to the cerebrospinal fluid (CSF) for immune surveillance. These findings may lead to new therapeutic approaches to neuroinflammatory diseases.

Background There are no prior reports that compare differentially methylated regions of DNA in blood samples from COVID-19 patients to samples collected before the SARS-CoV-2 pandemic using a shared epigenotyping platform. We performed a genome-wide analysis of circulating blood DNA CpG methylation using the Infinium Human MethylationEPIC BeadChip on 124 blood samples from hospitalized COVID-19-positive and COVID-19-negative patients and compared these data with previously reported data from 39 healthy individuals collected before the pandemic. Prospective outcome measures such as COVID-19-GRAM risk-score and mortality were combined with methylation data. Results Global mean methylation levels did not differ between COVID-19 patients and healthy pre-pandemic controls. About 75% of acute illness-associated differentially methylated regions were located near gene promoter regions and were hypo-methylated in comparison with healthy pre-pandemic controls. Gene ontology analyses revealed terms associated with the immune response to viral infections and leukocyte activation; and disease ontology analyses revealed a predominance of autoimmune disorders. Among COVID-19-positive patients, worse outcomes were associated with a prevailing hyper-methylated status. Recursive feature elimination identified 77 differentially methylated positions predictive of COVID-19 severity measured by the GRAM-risk score. Conclusion Our data contribute to the awareness that DNA methylation may influence the expression of genes that regulate COVID-19 progression and represent a targetable process in that setting.

Meningeal lymphatic vessels residing in the dural layer surrounding the dorsal regions of the brain, basal regions, and near the cribriform plate have all been implicated in the management of neuroinflammation and edema. Interestingly, only the lymphatic vessels near the cribriform plate undergo functional lymphangiogenesis in a mouse model of Multiple Sclerosis, suggesting these particular lymphatics uniquely undergo dynamic changes in response to neuroinflammation and may have distinct access to pro-lymphangiogenic factors in the CNS. However, it is unknown if these newly formed lymphangiogenic vessels are functionally similar to steady-state or if they have any other functional changes during neuroinflammation. In this study, we generated a novel protocol to isolate lymphatic endothelial cells from the cribriform plate for single cell analysis. We demonstrate that neuroinflammation-induced lymphangiogenic vessels undergo unique changes, including the capture of CNS-derived antigens, upregulation of adhesion and immune-modulatory molecules to interact with dendritic cells, and display IFN-γ dependent changes in response to the microenvironment. Single-cell trajectory analysis showed that cribriform plate lymphangiogenic vessels are post-proliferative and not generated from trans-differentiation of myeloid cells. Additionally, we show that these lymphangiogenic vessels have access to a CSF reservoir, express the water pore Aquaporin-1, and may have direct access to the CSF due to gaps in the arachnoid epithelial layer separating the dura from the subarachnoid space. These data characterize cribriform plate lymphatics and demonstrate that these vessels are dynamic structures that engage in leukocyte interactions, antigen sampling, and undergo expansion to drain excess fluid during neuroinflammation. Neuroinflammation not only induces efficient drainage of CSF but also alters the functions of lymphatic vessels near the cribriform plate.

Alterations in environmentally sensitive epigenetic mechanisms (e.g., DNA methylation) influence axonal regeneration in the spinal cord following sharp injury. Conventional DNA methylation detection methods using sodium bisulphite treatment do not distinguish between methylated and hydroxymethylated forms of cytosine, meaning that past studies report a composite of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). To identify the distinct contributions of DNA methylation modifications to axonal regeneration, we collected spinal cord tissue after sharp injury from untreated adult F3 male rats with enhanced regeneration of injured spinal axons or controls, derived from folate- or water-treated F0 lineages, respectively. Genomic DNA was profiled for genome-wide 5hmC levels, revealing 658 differentially hydroxymethylated regions (DhMRs). Genomic profiling with whole genome bisulphite sequencing disclosed regeneration-related alterations in composite 5mC + 5hmC DNA methylation levels at 2,260 differentially methylated regions (DMRs). While pathway analyses revealed that differentially hydroxymethylated and methylated genes are linked to biologically relevant axon developmental pathways, only 22 genes harbour both DhMR and DMRs. Since these differential modifications were more than 60 kilobases on average away from each other, the large majority of differential hydroxymethylated and methylated regions are unique with distinct functions in the axonal regeneration phenotype. These data highlight the importance of distinguishing independent contributions of 5mC and 5hmC levels in the central nervous system, and denote discrete roles for DNA methylation modifications in spinal cord injury and regeneration in the context of transgenerational inheritance.

Differentially methylated positions (DMPs) between persons with and without late-onset Alzheimer’s disease (LOAD) were observed at 477 of 769,190 loci in a plurality of genes. Of these, 17 were shared with DMPs identified using clinical LOAD markers analyzed independently as continuous variables comprising Rey Auditory Verbal Learning Test scores, cerebrospinal fluid total tau (t-tau) and phosphorylated tau 181 (p-tau(181)) levels, and t-tau/Aβ1–42 (Aβ(42)), p-tau(181)/Aβ(42), and Aβ(42)/Aβ1–40 (Aβ(40)) ratios. In patients with LOAD, 12 of the shared 17 DMPs were hypomethylated in B3GALT4 (Beta-1,3-galatcosyltransferase 4) (EC 2.4.1.62), and5 were hypomethylated in ZADH2 (Prostaglandin reductase 3) (EC 1.3.1.48).

Environmental stress contributes to the development of psychiatric disorders, including posttraumatic stress disorder and anxiety. While even acute stress alters gene expression, the molecular mechanisms underlying these changes remain largely unknown. 5-hydroxymethylcytosine (5hmC) is a novel environmentally sensitive DNA modification that is highly enriched in the brain and is associated with active transcription of neuronal genes. Here we examined behavioral and molecular alterations in adult mice that experienced an early-life stress before weaning (postnatal day 12 to 18) and found anxiety-like behaviors in adult female mice that were accompanied by correlated disruptions of hypothalamic 5hmC and gene expression in 118 genes, revealing potentially functional 5hmC (i.e., gene regulation). These genes are known and potentially novel stress-related targets, including Nr3c2, Nrxn1, Nfia, and Clip1, that have a significant enrichment for neuronal ontological functions, such as neuronal development and differentiation. Sequence motif predictions indicated that 5hmC may regulate gene expression by mediating transcription factor binding and alternative splicing of many of these transcripts. Together, these findings represent a critical step toward understanding the effects of early environment on the neuromolecular mechanisms that underlie the risk to develop anxiety disorders.

The original version of this article unfortunately contained error in Figure 4a to where some of the text was overlapping.

Environmental stress is among the most important contributors to increased susceptibility to develop psychiatric disorders. While it is well known that acute environmental stress alters gene expression, the molecular mechanisms underlying these changes remain largely unknown. 5-hydroxymethylcytosine (5hmC) is a novel environmentally sensitive epigenetic modification that is highly enriched in neurons and is associated with active neuronal transcription. Recently, we reported a genome-wide disruption of hippocampal 5hmC in male mice following acute stress that was correlated to altered transcript levels of genes in known stress related pathways. Since sex-specific endocrine mechanisms respond to environmental stimulus by altering the neuronal epigenome, we examined the genome-wide profile of hippocampal 5hmC in female mice following exposure to acute stress and identified 363 differentially hydroxymethylated regions (DhMRs) linked to known (e.g., Nr3c1 and Ntrk2) and potentially novel genes associated with stress response and psychiatric disorders. Integration of hippocampal expression data from the same female mice found stress-related hydroxymethylation correlated to altered transcript levels. Finally, characterization of stress-induced sex-specific 5hmC profiles in the hippocampus revealed 778 sex-specific acute stress-induced DhMRs some of which were correlated to altered transcript levels that produce sex-specific isoforms in response to stress. Together, the alterations in 5hmC presented here provide a possible molecular mechanism for the adaptive sex-specific response to stress that may augment the design of novel therapeutic agents that will have optimal effectiveness in each sex.

Meningeal lymphatics in the dural layer surrounding the dorsal regions of the brain, basal regions, and near the cribriform plate have all been implicated in the management of neuroinflammation and edema by facilitating the drainage of fluid, macromolecules, antigens, and cells. We have previously published that during neuroinflammation only the lymphatics near the cribriform plate are able to undergo extensive morphological changes through lymphangiogenesis in response to neuroinflammation during Experimental Autoimmune Encephalomyelitis (EAE), a mouse model of Multiple Sclerosis. In this study, we hypothesize that these cribriform plate lymphatics likely have other phenotypic changes as a consequence of neuroinflammation-induced lymphangiogenesis, and that identifying these phenotypic alterations may shed light on potentially novel roles of meningeal lymphatics during neuroinflammation. Single cell RNA sequencing reveals the upregulation of genes involved in leukocyte crosstalk and regulation such as chemotaxis/adhesion and antigen processing/presentation, suggesting that cribriform plate lymphatics may play a direct role in regulating adaptive immunity in addition to its canonical roles in facilitating drainage. We also demonstrate that cribriform plate lymphatics are able to capture CNS-derived antigens and functionally engage in crosstalk with dendritic cells and CD4 T cells. These data characterize cribriform plate lymphatics and demonstrate that these vessels become dynamic in response to neuroinflammation to facilitate excess drainage as well as directly modulate immunity through leukocyte crosstalk.

Environmental stress is among the most important contributors to increased susceptibility to develop psychiatric disorders, including anxiety and post-traumatic stress disorder. While even acute stress alters gene expression, the molecular mechanisms underlying these changes remain largely unknown. 5-hydroxymethylcytosine (5hmC) is a novel environmentally sensitive DNA modification that is highly enriched in post-mitotic neurons and is associated with active transcription of neuronal genes. Recently, we found a hippocampal increase of 5hmC in the glucocorticoid receptor gene (Nr3c1) following acute stress, warranting a deeper investigation of stress-related 5hmC levels. Here, we used an established chemical labeling and affinity purification method coupled with high-throughput sequencing technology to generate the first genome-wide profile of hippocampal 5hmC following exposure to acute restraint stress and a one-hour recovery. This approach found a genome-wide disruption in 5hmC associated with acute stress response, primarily in genic regions, and identified known and potentially novel stress-related targets that have a significant enrichment for neuronal ontological functions. Integration of these data with hippocampal gene expression data from these same mice found stress-related hydroxymethylation correlated to altered transcript levels and sequence motif predictions indicated that 5hmC may function by mediating transcription factor binding to these transcripts. Together, these data reveal an environmental impact on this newly discovered epigenetic mark in the brain and represent a critical step toward understanding stress-related epigenetic mechanisms that alter gene expression and can lead to the development of psychiatric disorders.

Chronic and severe stress exposure in early childhood is associated with the development of psychiatric disorders. Yet, the molecular mechanisms underlying this relationship remain poorly understood. Here, we profile molecular marks (DNA methylation and gene expression) throughout the human genome to determine the associations between childhood stress exposure and gene regulation. To do so, we collected saliva tissue from prepubertal girls (mean age 10.9 ± 1.26 years) who had experienced different levels of childhood adversity, ranging from mild to severe. We found 122 differentially methylated genes (FDR P-value

Mapping patterns of DNA methylation throughout the epigenome are critical to our understanding of several important biological and regulatory functions, such as transcriptional regulation, genomic imprinting, and embryonic development. The development and rapid advancement of next-generation sequencing (NGS) technologies have provided clinicians and researchers with accurate and reliable read-outs of genomic and epigenomic information at the nucleotide level. Such improvements have significantly lowered the cost required for genome-wide sequencing, facilitating the vast acquisition of data that has led to many improvements in patient care. However, the torrid rate of NGS data generation has left targeted validation approaches behind, including the confirmation of epigenetic marks such as DNA methylation. To overcome these shortcomings, we present a rapid and robust protocol for the parallel examination of multiple methylated sequences that we have termed simultaneous targeted methylation sequencing (sTM-Seq). Key features of this technique include the elimination of the need for large amounts of high-molecular weight DNA and the nucleotide specific distinction of both 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Moreover, sTM-Seq is scalable and can be used to investigate multiple loci in dozens of samples within a single sequencing run. By utilizing freely available web-based software and universal primers for multipurpose barcoding, library preparation, and customized sequencing, sTM-Seq is affordable, efficient, and widely applicable. Together, these features enable sTM-Seq to have wide-reaching clinical applications that will greatly improve turnaround rates for same-day procedures and allow clinicians to collect high-resolution data that can be used in a variety of patient settings. © 2019 by John Wiley & Sons, Inc.

Historically biomedical research has examined genetic influences on mental health but these approaches have been limited, likely due to the broad heritability of brain-related disorders (e.g., 30–90%). Epigenetic modifications, such as DNA methylation, are environmentally sensitive mechanisms that may play a role in the origins and progression of mental illness. Recently, genome-wide disruptions of 5-hydroxymethylcytosine (5hmC) were associated with the development of early and late onset mental illnesses such as autism and Alzheimer’s disease, bringing new hope to the field of psychiatry. Here, we review the recent links of 5hmC to mental illness and discuss several putative functions of 5hmC in the context of its promising clinical relevance.