Defining RNA oligonucleotides that reverse deleterious phase transitions of RNA-binding proteins with prion-like domains.

RNA-binding proteins with prion-like domains, such as FUS and TDP-43, condense into functional liquids, which can transform into pathological fibrils that underpin fatal neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD). Here, we define short RNAs (24-48 nucleotides) that prevent FUS fibrillization by promoting liquid phases, and distinct short RNAs that prevent and, remarkably, reverse FUS condensation and fibrillization. These activities require interactions with multiple RNA-binding domains of FUS and are encoded by RNA sequence, length, and structure. Importantly, we define a short RNA that dissolves aberrant cytoplasmic FUS condensates, restores nuclear FUS, and mitigates FUS proteotoxicity in optogenetic models and human motor neurons. Another short RNA dissolves aberrant cytoplasmic TDP-43 condensates, restores nuclear TDP-43, and mitigates TDP-43 proteotoxicity. Since short RNAs can be effectively delivered to the human brain, these oligonucleotides could have therapeutic utility for ALS/FTD and related disorders.

Liquid-Liquid Phase Separation of TDP-43 and FUS in Physiology and Pathology of Neurodegenerative Diseases.

Liquid-liquid phase separation of RNA-binding proteins mediates the formation of numerous membraneless organelles with essential cellular function. However, aberrant phase transition of these proteins leads to the formation of insoluble protein aggregates, which are pathological hallmarks of neurodegenerative diseases including ALS and FTD. TDP-43 and FUS are two such RNA-binding proteins that mislocalize and aggregate in patients of ALS and FTD. They have similar domain structures that provide multivalent interactions driving their phase separation in vitro and in the cellular environment. In this article, we review the factors that mediate and regulate phase separation of TDP-43 and FUS. We also review evidences that connect the phase separation property of TDP-43 and FUS to their functional roles in cells. Aberrant phase transition of TDP-43 and FUS leads to protein aggregation and disrupts their regular cell function. Therefore, restoration of functional protein phase of TDP-43 and FUS could be beneficial for neuronal cells. We discuss possible mechanisms for TDP-43 and FUS aberrant phase transition and aggregation while reviewing the methods that are currently being explored as potential therapeutic strategies to mitigate aberrant phase transition and aggregation of TDP-43 and FUS.

Cross-species Association Between Telomere Length and Glucocorticoid Exposure.

CONTEXT: Chronic exposure to glucocorticoids (GCs) or stress increases the risk of medical disorders, including cardiovascular and neuropsychiatric disorders. GCs contribute to accelerated aging; however, while the link between chronic GC exposure and disease onset is well established, the underpinning mechanisms are not clear. OBJECTIVE: We explored the potential nexus between GCs or stress exposure and telomere length. METHODS: In addition to rats exposed to 3 weeks of chronic stress, an iatrogenic mouse model of Cushing syndrome (CS), and a mouse neuronal cell line, we studied 32 patients with CS and age-matched controls and another cohort of 75 healthy humans. RESULTS: (1) Exposure to stress in rats was associated with a 54.5% (P = 0.036) reduction in telomere length in T cells. Genomic DNA (gDNA) extracted from the dentate gyrus of stressed and unstressed rats showed 43.2% reduction in telomere length (P = 0.006). (2) Mice exposed to corticosterone had a 61.4% reduction in telomere length in blood gDNA (P = 5.75 × 10-5) and 58.8% reduction in telomere length in the dentate gyrus (P = 0.002). (3) We observed a 40.8% reduction in the telomere length in patients with active CS compared to healthy controls (P = 0.006). There was a 17.8% reduction in telomere length in cured CS patients, which was not different from that of healthy controls (P = 0.08). For both cured and active CS, telomere length correlated significantly with duration of hypercortisolism (R2 = 0.22, P = 0.007). (4) There was a 27.6% reduction in telomere length between low and high tertiles in bedtime cortisol levels of healthy participants (P = 0.019). CONCLUSION: Our findings demonstrate that exposure to stress and/or GCs is associated with shortened telomeres, which may be partially reversible.

DNA methylation and behavioral dysfunction in males with 47,XXY and 49,XXXXY: a pilot study.

BACKGROUND: Equal dosage of X-linked genes between males and females is maintained by the X-inactivation of the second X chromosome in females through epigenetic mechanisms. Boys with aneuploidy of the X chromosome exhibit a host of symptoms such as low fertility, musculoskeletal anomalies, and cognitive and behavioral deficits that are presumed to be caused by the abnormal dosage of these genes. The objective of this pilot study is to assess the relationship between CpG methylation, an epigenetic modification, at several genes on the X chromosome and behavioral dysfunction in boys with supernumerary X chromosomes. RESULTS: Two parental questionnaires, the Behavior Rating Inventory of Executive Function (BRIEF) and Child Behavior Checklist (CBCL), were analyzed, and they showed expected differences in both internal and external behaviors between neurotypical (46,XY) boys and boys with 49,XXXXY. There were several CpGs in AR and MAOA of boys with 49,XXXXY whose methylation levels were skewed from levels predicted from having one active (Xa) and three inactive (Xi) X chromosomes. Further, methylation levels of multiple CpGs in MAOA showed nominally significant association with externalizing behavior on the CBCL, and the methylation level of one CpG in AR showed nominally significant association with the BRIEF Regulation Index. CONCLUSIONS: Boys with 49,XXXXY displayed higher levels of CpG methylation at regulatory intronic regions in X-linked genes encoding the androgen receptor (AR) and monoamine oxidase A (MAOA), compared to that in boys with 47,XXY and neurotypical boys. Our pilot study results suggest a link between CpG methylation levels and behavior in boys with 49,XXXXY.

Characterization of glucocorticoid-induced loss of DNA methylation of the stress-response gene Fkbp5 in neuronal cells.

Exposure to stress or glucocorticoids (GCs) is associated with epigenetic and transcriptional changes in genes that either mediate or are targets of GC signalling. FKBP5 (FK506 binding protein 5) is one such gene that also plays a central role in negative feedback regulation of GC signalling and several stress-related psychiatric disorders. In this study, we sought to examine how the mouse Fkbp5 gene is regulated in a neuronal context and identify requisite factors that can mediate the epigenetic sequelae of excess GC exposure. Mice treated with GCs were used to establish the widespread changes in DNA methylation (DNAm) and expression of Fkbp5 across four brain regions. Then two cell lines were used to test the persistence, decay, and functional significance of GC-induced methylation changes near two GC response elements (GREs) in the fifth intron of Fkbp5. We also tested the involvement of DNMT1, cell proliferation, and MeCP2 in mediating the effect of GCs on DNAm and gene activation. DNAm changes at some CpGs persist while others decay, and reduced methylation states are associated with a more robust transcriptional response. Importantly, the ability to undergo GC-induced DNAm loss is tied to DNMT1 function during cell division. Further, GC-induced DNAm loss is associated with reduced binding of MeCP2 at intron 5 and a physical interaction between the fifth intron and promoter of Fkbp5. Our results highlight several key factors at the Fkbp5 locus that may have important implications for GC- or stress-exposure during early stages of neurodevelopment.

A Rat Methyl-Seq Platform to Identify Epigenetic Changes Associated with Stress Exposure.

As genomes of a wider variety of animals become available, there is an increasing need for tools that can capture dynamic epigenetic changes in these animal models. The rat is one particular model animal where an epigenetic tool can complement many pharmacological and behavioral studies to provide insightful mechanistic information. To this end, we adapted the SureSelect Target Capture System (referred to as Methyl-Seq) for the rat, which can assess DNA methylation levels across the rat genome. The rat design targeted promoters, CpG islands, island shores, and GC-rich regions from all RefSeq genes. To implement the platform on a rat experiment, male Sprague Dawley rats were exposed to chronic variable stress for 3 weeks, after which blood samples were collected for genomic DNA extraction. Methyl-Seq libraries were constructed from the rat DNA samples by shearing, adapter ligation, target enrichment, bisulfite conversion, and multiplexing. Libraries were sequenced on a next-generation sequencing platform and the sequenced reads were analyzed to identify DMRs between DNA of stressed and unstressed rats. Top candidate DMRs were independently validated by bisulfite pyrosequencing to confirm the robustness of the platform. Results demonstrate that the rat Methyl-Seq platform is a useful epigenetic tool that can capture methylation changes induced by exposure to stress.