Engineered LINE-1 retrotransposition in nondividing human neurons

Half the human genome is made of transposable elements (TEs), whose ongoing activity continues to impact our genome. LINE-1 (or L1) is an autonomous non-LTR retrotransposon in the human genome, comprising 17% of its genomic mass and containing an average of 80-100 active L1s per average genome that provide a source of inter-individual variation. New LINE-1 insertions are thought to accumulate mostly during human embryogenesis. Surprisingly, the activity of L1s can further impact the somatic human brain genome. However, it is currently unknown whether L1 can retrotranspose in other somatic healthy tissues or if L1 mobilization is restricted to neuronal precursor cells (NPCs) in the human brain. Here, we took advantage of an engineered L1 retrotransposition assay to analyze L1 mobilization rates in human mesenchymal (MSCs) and hematopoietic (HSCs) somatic stem cells. Notably, we have observed that L1 expression and engineered retrotransposition is much lower in both MSCs and HSCs when compared to NPCs. Remarkably, we have further demonstrated for the first time that engineered L1s can retrotranspose efficiently in mature nondividing neuronal cells. Thus, these findings suggest that the degree of somatic mosaicism and the impact of L1 retrotransposition in the human brain is likely much higher than previously thought.We thank current members of the J.L.G.-P. laboratory for helpful discussions. We also thank Drs. Geoffrey Faulkner (Mater Research, Australia) and John V. Moran (University of Michigan) for sharing unpublished data and for critical input during the project; Ms. Raquel Marrero (Microscopy Unit, Genyo) for technical support; Simon Mendez-Ferrer (CNIC, Spain) for providing total RNA isolated from human mesenspheres; Dr. Oliver Weichenrieder (Max-Planck, Tubingen, Germany) for providing a polyclonal L1-ORF1p antibody; and Dr. Aurelien Doucet (IRCAN, Nice, France) for providing a plasmid containing an UBC-driven EGFP retrotransposition indicator cassette. J.L.G. was funded by the US Department of Defense, Breast Cancer Research Program (award #BC051386), the National Institutes of Health (NIH) National Institute of Neurological Disorders and Stroke (1R03NS087290-01), and the ALS Therapy Alliance (2013-F-067). A.M. has been partially funded by a Marie Curie IRG project (FP7-PEOPLE-2007-4-3-IRG: SOMATIC LINE-1). J.L.G.-P's laboratory is supported by CICE-FEDER-P09-CTS-4980, CICE-FEDER-P12-CTS-2256, Plan Nacional de I+D+I 2008–2011 and 2013–2016 (FIS-FEDER-PI11/01489 and FIS-FEDER-PI14/02152), PCIN-2014-115-ERA-NET NEURON II, the European Research Council (ERC-Consolidator ERC-STG-2012-233764), by an International Early Career Scientist grant from the Howard Hughes Medical Institute (IECS-55007420), and by The Wellcome Trust–University of Edinburgh Institutional Strategic Support Fund (ISFF2).S

Interrogating intervention delivery and participants’ emotional states to improve engagement and implementation: A realist informed multiple case study evaluation of Engager

BACKGROUND: 'Engager' is an innovative 'through-the-gate' complex care intervention for male prison-leavers with common mental health problems. In parallel to the randomised-controlled trial of Engager (Trial registration number: ISRCTN11707331), a set of process evaluation analyses were undertaken. This paper reports on the depth multiple case study analysis part of the process evaluation, exploring how a sub-sample of prison-leavers engaged and responded to the intervention offer of one-to-one support during their re-integration into the community. METHODS: To understand intervention delivery and what response it elicited in individuals, we used a realist-informed qualitative multiple 'case' studies approach. We scrutinised how intervention component delivery lead to outcomes by examining underlying causal pathways or 'mechanisms' that promoted or hindered progress towards personal outcomes. 'Cases' (n = 24) were prison-leavers from the intervention arm of the trial. We collected practitioner activity logs and conducted semi-structured interviews with prison-leavers and Engager/other service practitioners. We mapped data for each case against the intervention logic model and then used Bhaskar's (2016) 'DREIC' analytic process to categorise cases according to extent of intervention delivery, outcomes evidenced, and contributing factors behind engagement or disengagement and progress achieved. RESULTS: There were variations in the dose and session focus of the intervention delivery, and how different participants responded. Participants sustaining long-term engagement and sustained change reached a state of 'crises but coping'. We found evidence that several components of the intervention were key to achieving this: trusting relationships, therapeutic work delivered well and over time; and an in-depth shared understanding of needs, concerns, and goals between the practitioner and participants. Those who disengaged were in one of the following states: 'Crises and chaos', 'Resigned acceptance', 'Honeymoon' or 'Wilful withdrawal'. CONCLUSIONS: We demonstrate that the 'implementability' of an intervention can be explained by examining the delivery of core intervention components in relation to the responses elicited in the participants. Core delivery mechanisms often had to be 'triggered' numerous times to produce sustained change. The improvements achieved, sustained, and valued by participants were not always reflected in the quantitative measures recorded in the RCT. The compatibility between the practitioner, participant and setting were continually at risk of being undermined by implementation failure as well as changing external circumstances and participants' own weaknesses. TRIAL REGISTRATION NUMBER: ISRCTN11707331, Wales Research Ethics Committee, Registered 02-04-2016-Retrospectively registered https://doi.org/10.1186/ISRCTN11707331

Development and evaluation of a collaborative care intervention for male prison leavers with mental health problems: the Engager research programme

BackgroundMany male prison leavers have significant mental health problems. Prison leavers often have a history of trauma, ongoing substance misuse and housing insecurity. Only a minority of prison leavers receive mental health care on release from prison.ObjectivesThe aim of the Engager research programme was to develop and evaluate a theory- and evidence-informed complex intervention designed to support individuals with common mental health problems (e.g. anxiety, depression) and other complex needs, including mental health comorbidity, before and after release from prison.MethodsIn phase 1, the intervention was developed through a set of realist-informed substudies, including a realist review of psychosocial care for individuals with complex needs, case studies within services demonstrating promising intervention features, focus groups with individuals from under-represented groups, a rapid realist review of the intervention implementation literature and a formative process evaluation of the prototype intervention. In a parallel randomised trial, methodological development included selecting outcome measures through reviewing literature, piloting measures and a consensus process, developing ways to quantify intervention receipt, piloting trial procedures and modelling economic outcomes. In phase 2, we conducted an individually randomised superiority trial of the Engager intervention, cost-effectiveness and cost–consequence analyses and an in-depth mixed-methods process evaluation. Patient and public involvement influenced the programme throughout, primarily through a Peer Researcher Group.ResultsIn phase 1, the Engager intervention included multiple components. A practitioner offered participants practical support, emotional help (including mentalisation-based approaches) and liaison with other services in prison on the day of the participant’s release and for 3–5 months post release. An intervention delivery platform (i.e. training, manual, supervision) supported implementation. Outcome measures were selected through testing and stakeholder consensus to represent a broad range of domains, with a general mental health outcome as the primary measure for the trial. Procedures for recruitment and follow-up were tested and included flexible approaches to engagement and retention. In phase 2, the trial was conducted in three prison settings, with 280 participants randomised in a 1 : 1 ratio to receive either Engager plus usual care (n = 140) or usual care only (n = 140). We achieved a follow-up rate of 65% at 6 months post release from prison. We found no difference between the two groups for the Clinical Outcomes in Routine Evaluation – Outcome Measure at 6 months. No differences in secondary measures and sensitivity analyses were found beyond those expected by chance. The cost-effectiveness analysis showed that Engager cost significantly more at £2133 (95% of iterations between £997 and £3374) with no difference in quality-adjusted life-years (–0.017, 95% of iterations between –0.042 and 0.007). The mixed-methods process evaluation demonstrated implementation barriers. These barriers included problems with retention of the intervention team, and the adverse health and criminal justice system context. Seventy-seven per cent (108/140) of individuals had at least one community contact. Significant proportions of participants engaging received day release work and practical support. In contrast, there was evidence that the psychological components, mentalisation and developing a shared understanding were used less consistently. When engagement was positive, these components were associated with positive achievement of goals for individuals. We were also able to identify how to improve the intervention programme theory, including how to support individuals who were unrealistic in their perception of their ability to cope with challenges post release.Strengths and limitationsOur development work provides a worked example of the development of a complex intervention, particularly given little prior evidence or theory specific to male offenders to build on. Our trial methodological development enabled the completion of, to the best of our knowledge, the first fully powered trial of a mental health intervention for prison leavers with common mental health problems. There were potential weaknesses in the trial methodology in terms of follow-up rates and outcome measures, with the latter potentially being insufficiently sensitive to important but highly individual changes in participants who responded to the intervention.ConclusionsDelivering a randomised controlled trial for prison leavers with acceptable levels of follow-up is possible, despite adverse conditions. Full intervention implementation was challenging, but this is to be expected. Some individuals did respond well to the intervention when both practical and psychological support were flexibly deployed as intended, with evidence that most components were experienced as helpful for some individuals. It is recommended that several key components be developed further and tested, along with improved training and supervision, to support delivery of the Engager intervention within existing teams working with prison leavers

LINE-1 Evasion of Epigenetic Repression in Humans

Epigenetic silencing defends against LINE-1 (L1) retrotransposition in mammalian cells. However, the mechanisms that repress young L1 families and how L1 escapes to cause somatic genome mosaicism in the brain remain unclear. Here we report that a conserved Yin Yang 1 (YY1) transcription factor binding site mediates L1 promoter DNA methylation in pluripotent and differentiated cells. By analyzing 24 hippocampal neurons with three distinct single-cell genomic approaches, we characterized and validated a somatic L1 insertion bearing a 3' transduction. The source (donor) L1 for this insertion was slightly 5' truncated, lacked the YY1 binding site, and was highly mobile when tested in\ua0vitro. Locus-specific bisulfite sequencing revealed that the donor L1 and other young L1s with mutated YY1 binding sites were hypomethylated in embryonic stem cells, during neurodifferentiation, and in liver and brain tissue. These results explain how L1 can evade repression and retrotranspose in the human body

The Microprocessor controls the activity of mammalian retrotransposons

More than half of the human genome is made of transposable elements whose ongoing mobilization is a driving force in genetic diversity; however, little is known about how the host regulates their activity. Here, we show that the Microprocessor (Drosha-DGCR8), which is required for microRNA biogenesis, also recognizes and binds RNAs derived from human long interspersed element 1 (LINE-1), Alu and SVA retrotransposons. Expression analyses demonstrate that cells lacking a functional Microprocessor accumulate LINE-1 mRNA and encoded proteins. Furthermore, we show that structured regions of the LINE-1 mRNA can be cleaved in vitro by Drosha. Additionally, we used a cell culture–based assay to show that the Microprocessor negatively regulates LINE-1 and Alu retrotransposition in vivo. Altogether, these data reveal a new role for the Microprocessor as a post-transcriptional repressor of mammalian retrotransposons and a defender of human genome integrity.S.R.H. was supported by a Marie Curie Intra-European Fellowship and a Marie Curie CIG-Grant (PCIG10-GA-2011-303812). M.P. and E.E. were supported by the Spanish Ministry of Science (BIO2011-23920) and by the Sandra Ibarra Foundation (CSD2009-00080). M.P. is supported by the Novo Nordisk Foundation. J.L.G.-P. is supported by FP7-PEOPLE-2007-4-3-IRG, CICE-FEDER-P09-CTS-4980, PeS-FEDER-PI-002, FIS-FEDER-PI11/01489 and the Howard Hughes Medical Institute (IECS-55007420). J.F.C. was supported by Core funding from the Medical Research Council and by the Wellcome Trust (grant 095518/B/11/Z)

Transcriptional profiling of HERV-K(HML-2) in amyotrophic lateral sclerosis and potential implications for expression of HML-2 proteins

Abstract Background Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder. About 90% of ALS cases are without a known genetic cause. The human endogenous retrovirus multi-copy HERV-K(HML-2) group was recently reported to potentially contribute to neurodegeneration and disease pathogenesis in ALS because of transcriptional upregulation and toxic effects of HML-2 Envelope (Env) protein. Env and other proteins are encoded by some transcriptionally active HML-2 loci. However, more detailed information is required regarding which HML-2 loci are transcribed in ALS, which of their proteins are expressed, and differences between the disease and non-disease states. Methods For brain and spinal cord tissue samples from ALS patients and controls, we identified transcribed HML-2 loci by generating and mapping HML-2-specific cDNA sequences. We predicted expression of HML-2 env gene-derived proteins based on the observed cDNA sequences. Furthermore, we determined overall HML-2 transcript levels by RT-qPCR and investigated presence of HML-2 Env protein in ALS and control tissue samples by Western blotting. Results We identified 24 different transcribed HML-2 loci. Some of those loci are transcribed at relatively high levels. However, significant differences in HML-2 loci transcriptional activities were not seen when comparing ALS and controls. Likewise, overall HML-2 transcript levels, as determined by RT-qPCR, were not significantly different between ALS and controls. Indeed, we were unable to detect full-length HML-2 Env protein in ALS and control tissue samples despite reasonable sensitivity. Rather our analyses suggest that a number of HML-2 protein variants other than full-length Env may potentially be expressed in ALS patients. Conclusions Our results expand and refine recent publications on HERV-K(HML-2) and ALS. Some of our results are in conflict with recent findings and call for further specific analyses. Our profiling of HML-2 transcription in ALS opens up the possibility that HML-2 proteins other than canonical full-length Env may have to be considered when studying the role of HML-2 in ALS disease

Additional file 2: of Transcriptional profiling of HERV-K(HML-2) in amyotrophic lateral sclerosis and potential implications for expression of HML-2 proteins

Figure S1. HERV-K(HML-2) locus-specific nucleotide differences in cDNA sequences derived from gag amplicon. Figure S2. HERV-K(HML-2) locus-specific nucleotide differences in cDNA sequences derived from an env amplicon. Figure S3. HERV-K(HML-2) locus-specific nucleotide differences in cDNA sequences derived from a rec amplicon. Figure S4. HERV-K(HML-2) locus-specific nucleotide differences in cDNA sequences derived from an np9 amplicon. Figure S5. Normalized levels of HERV-W transcripts identified in various ALS and control tissue samples. Figure S6. Normalized levels of HERV-W transcripts identified in various ALS and control tissue samples. Figure S7. Expression of HERV-K(HML-2) Env protein detected with the HERM-1811-5 antibody. Figure S8. Agarose gel photos of HML-2 env-specific endpoint RT-PCRs for subsequent HML-2 transcription profiling. Figure S9. RNA qualities and correlations with GAPDH Ct-values. Figure S10. No correlation of relative HERV-K(HML-2) transcript levels with age or gender of donors. (PDF 2700 kb

Properties of LINE-1 proteins and repeat element expression in the context of amyotrophic lateral sclerosis

ABSTRACT Background Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease involving loss of motor neurons and having no known cure and uncertain etiology. Several studies have drawn connections between altered retrotransposon expression and ALS. Certain features of the LINE-1 (L1) retrotransposon-encoded ORF1 protein (ORF1p) are analogous to those of neurodegeneration-associated RNA-binding proteins, including formation of cytoplasmic aggregates. In this study we explore these features and consider possible links between L1 expression and ALS. Results We first considered factors that modulate aggregation and subcellular distribution of LINE-1 ORF1p, including nuclear localization. Changes to some ORF1p amino acid residues alter both retrotransposition efficiency and protein aggregation dynamics, and we found that one such polymorphism is present in endogenous L1s abundant in the human genome. We failed, however, to identify CRM1-mediated nuclear export signals in ORF1p nor strict involvement of cell cycle in endogenous ORF1p nuclear localization in human 2102Ep germline teratocarcinoma cells. Some proteins linked with ALS bind and colocalize with L1 ORF1p ribonucleoprotein particles in cytoplasmic RNA granules. Increased expression of several ALS-associated proteins, including TAR DNA Binding Protein (TDP-43), strongly limits cell culture retrotransposition, while some disease-related mutations modify these effects. Using quantitative reverse transcription PCR (RT-qPCR) of ALS tissues and reanalysis of publicly available RNA-Seq datasets, we asked if changes in expression of retrotransposons are associated with ALS. We found minimal altered expression in sporadic ALS tissues but confirmed a previous report of differential expression of many repeat subfamilies in C9orf72 gene-mutated ALS patients. Conclusions Here we extended understanding of the subcellular localization dynamics of the aggregation-prone LINE-1 ORF1p RNA-binding protein. However, we failed to find compelling evidence for misregulation of LINE-1 retrotransposons in sporadic ALS nor a clear effect of ALS-associated TDP-43 protein on L1 expression. In sum, our study reveals that the interplay of active retrotransposons and the molecular features of ALS are more complex than anticipated. Thus, the potential consequences of altered retrotransposon activity for ALS and other neurodegenerative disorders are worthy of continued investigation

Evaluation of a Complex Intervention for Prisoners with Common Mental Health Problems, Near To and After Release: The Engager Randomised Controlled Trial

BACKGROUND: Many male prisoners have significant mental health problems, including anxiety and depression. High proportions struggle with homelessness and substance misuse. AIMS: This study aims to evaluate whether the Engager intervention improves mental health outcomes following release. METHOD: The design is a parallel randomised superiority trial that was conducted in the North West and South West of England (ISRCTN11707331). Men serving a prison sentence of 2 years or less were individually allocated 1:1 to either the intervention (Engager plus usual care) or usual care alone. Engager included psychological and practical support in prison, on release and for 3-5 months in the community. The primary outcome was the Clinical Outcomes in Routine Evaluation Outcome Measure (CORE-OM), 6 months after release. Primary analysis compared groups based on intention-to-treat (ITT). RESULTS: In total, 280 men were randomised out of the 396 who were potentially eligible and agreed to participate; 105 did not meet the mental health inclusion criteria. There was no mean difference in the ITT complete case analysis between groups (92 in each arm) for change in the CORE-OM score (1.1, 95% CI -1.1 to 3.2, P = 0.325) or secondary analyses. There were no consistent clinically significant between-group differences for secondary outcomes. Full delivery was not achieved, with 77% (108/140) receiving community-based contact. CONCLUSIONS: Engager is the first trial of a collaborative care intervention adapted for prison leavers. The intervention was not shown to be effective using standard outcome measures. Further testing of different support strategies for prison with mental health problems is needed