EpimiRNA
Bicoll © Kai Lamottke
Bicoll © Kai Lamottke

Welcome to our first EpimiRNA newsletter!

 

In this 1st issue of the EpimiRNA newsletter, we would like to update you on the latest news from the project, including publications, presentations upcoming project events and provide some useful information on the EpimiRNA Database by BCPlatforms. Your feedback and suggestions for the newsletter are welcome!

NEWS ON EPIMIRNA & FROM THE PARTNERS

 EpimiRNA GA meeting in Copenhagen!

 

 Thanks to all partners for a successful GA meeting and fruitful discussions!

EC feedback to the 3rd EpimiRNA Periodic Report

The EpimiRNA 3rd report has been approved by the EC. The feedback is very positive and encouraging. The Project Officer has pointed out that the Consortium is under contractual obligation to provide open access to EpimiRNA related research (clause 39 of the GA). Therefore, as much as possible, any past publications funded by EpimiRNA should be made public (e.g. using OpenAire or possibly PubMed Central (PMC). All future publications should also be with an open access.

EpimiRNA publications

In the last 6 months the EpimiRNA partners were very active in publishing new publications! For further information on the publications please see the short summaries below in this newsletter.

EpimiRNA on Twitter

EpimiRNA is on Twitter! For the latest project updates, please follow our page and spread our news! Please provide any news/highlights/etc you would like to share to ARTTIC and they will tweet it!

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EpimiRNA Database

The consortium data is collected into a centralized database system located at Aarhus University High Performance Computing Cluster (GenomeDK).  The system is based on BC Platform’s BC|GENOME application providing database backend and user interface components for storing and accessing the data.  The data is stored into predefined data tables, which can be queried using standard tools and programming languages like SQL and R or accessed from web based user interface. An example of a user interface is shown in the figure below.

 

 

The aim is to use simple data structures and common naming conventions for data submissions making it possible to integrate data produced by different labs easily. As an example tab-delimited submission format for the animal models:

Sample table contains animal and sample level information

  • Subject  - Produced by a scheme illustrated here by an example:  RMK_RH48_050, where RMK: R=RCSI, M=Mouse and K means KA treated. RH48: Means right hippocampus, 48 h time point. 050 is animal number here chosen by RCSI
  • Produced_by:  Abbreviation of lab producing the sample (e.g. RCSI)
  • Species: Species (e.g. Mouse)
  • Mode: Name of the model (e.g. Kainic acid model)
  • Group:  Id of a treatment group (e.g. 1h_KA)
  • Animal id: Animal id provided by lab
  • Task: Id of the study task (e.g. 1.1)
  • Other info: Free form additional info

Measurement tables contain expression values done over the sample and marker (miRNA identifiers) ids. Normalization method and other experimental conditions are stored into the description field and structured later if needed.

 

In addition to the structured data, raw source data files e.g. files used in analysis are stored into file system. Data submission to the system is made easy and the process can start already often from these raw data files, which will be structured by readymade tools based on the agreed data formats. New conversion tools and formats can be developed based on need.

All EpimiRNA partners are encouraged to upload their experimental data in this database and to make sure that all relevant information on experimental conditions is made available for the whole EpimiRNA consortium!

It is important to take care that identifiers, like animal ids, used in different labs can be cross-referenced. More information is available from Morten Venø and Juha Muilu. Please do not hesitate to contact them!

 

Events

EpimiRNA events

5th Steering Committee meeting in Amsterdam 10th March 2017

The EpimiRNA Steering Committee members will meet in Amsterdam in March 2017. The meeting has for objective to report on progress and discuss next steps on WP level.

5th General Assembly & Steering Committee Meeting

The EpimiRNA General Assembly will meet in August/September 2017, venue needs to be confirmed. The meeting will enable partners to share progress made in the previous year and organise upcoming activities.

Upcoming events of interest

6th Status epilepticus colloquium, Salzburg, Austria, 4 – 6 April 2017

D. Henshall will speak at the upcoming 6th Status epilepticus colloquium, Salzburg, Austria, April 4 – 6 2017 on the topic of epigenetics and status epilepticus.

Science workshop during the 2017 AES meeting Washington, DC, USA, 1 – 5 December 2017

A basic science workshop was proposed by Dr Cristina Gross, for the 2017 AES meeting in Washington DC which will include presentations by EpimiRNA members D. Henshall and D. Goldstein and EpiTarget’s E. Aronica.

KNAW colloquium, Amsterdam, Netherlands, 6-7 November 2017

Prof. J. Pasterkamp (UMCU) is organising the KNAW colloquium on the topic ‘Cell biology of the axon: progress made, promises ahead’

 

 

EPIMIRNA TEAM CHANGES

Dr. Andreas Hartlep will be leaving his position at Cerbomed in January 2017. Thank you for your contribution and continuous support to EpimiRNA. The new representative of Cerbomed will be Herbert Frosch. Welcome to the EpimiRNA team!

 

 

NEW GRANTS OBTAINED BY EPIMIRNA PARTNERS

Aarhus University in Denmark

Villum Foundation has funded a project entitled "Uncovering the new world of circular RNAs". The amount granted to AU is of 700.000€.

Innovation Fund Denmark has funded a project titles “Indo-Danish Centre for Musculo-Skeletal Stem Cell Targeting”. The amount granted to AU is of 1M €

 

Royal College of Surgeons in Ireland

Medical Research Charities Group and Epilepsy Ireland have funded a project titled “MicroRNAs in the mechanism of ketogenic diet therapies and as biomarkers in paediatric epilepsy”. The Principal investigator of the project is David Henshall.

 

University Medical Center Utrecht & Aarhus University

UMCU and AU are both partners of the circRTrain (circRNA biology Training Network: from biogenesis to biomarker), a Marie Curie Innovative Training Network (ITN) funded by the European Union within the H2020 Programme.

 

 

EPIMIRNA PARTNERS ARE RECRUITING

Postdoctoral Fellowship in Bioinformatics for Molecular Neurosciences: Campinas, SP, Brazil

The Medical Genetics Department of the Medical Sciences Faculty of Medical Sciences, Unicamp (University of Campinas), Brazil, is seeking postdoctoral candidates with a degree in bioinformatics or a mathematical discipline with a strong interest in neurosciences. The selected candidate will join a recently funded large project of multimodal approach of epilepsy. The main responsibility will be to analyse a large gene expression data set (RNAseq) generated at the university to identify molecular perturbations linked to epilepsy in animal models. Applicants should have strong modelling/applied statistics experience in analysing complex dataset in biology. Skills requirements include programing and knowledge of at least one statistical computing environment. Applicants must have a Ph.D. degree and show good scientific communication skills by having published at least three papers in peer-reviewed scientific journals in the last three years. Language requirements: proficiency in English, Portuguese or Spanish. Funds are available for an initial 2 years period. Please send CV and 2 recommendation letters to undefinedProf. Iscia Lopes-Cendes.

 

2 PhD positions within circRTrain at UMCU

circRTrain (circular RNA biology Training Network) investigators are hiring in total 15 PhD candidates who will start their projects between January and September 2017. 2 positions are available at UMCU. They are looking for highly motivated young researchers to investigate the formation and role of circRNAs. Each position will be appointed for 36 months. Detailed information can be found here.

 

 

GOOD TO KNOW

Development of computer programs/tools and software

Rqc package offers the user efficient strategies for quality assessment of Next-Generation Sequencing data. It processes FASTQ and BAM files without compromising the system performance, allowing the user to evaluate possible issues during the sequencing experiment. Try!

 

GA4GHclient is a R package for accessing GA4GH API data servers. This software is based on official Data Model Schemas created by Global Alliance for Genomics and Health. Try!

 

 

EpimiRNA Publications - Featured Article

New study by EpimiRNA collaborators shows antagomirs targeting miR-134 work in multiple animal models of epilepsy

Targeting microRNA-134 was originally shown to have anti-seizure effects in 2012, one of several findings that helped shape the concepts behind the EpimiRNA consortium. Since then, members of the EPimiRNA consortium have been working hard to understand the mechanism of this effect and begin to drive the research toward preclinical development. This journey took a step forward with exciting findings reported this month in the journal Molecular Therapy – Nucleic Acids. Dr Cristina Reschke and colleagues tested the effects of oligonucleotide inhibitors of microRNA-134 (Ant-134) in four different animal models of epilepsy. The research showed that seizures were reduced in 3 out of 4 tested models which brings to a total of 6 the number of models in which anti-seizure effects of targeting microRNA-134 have been reported. The new study also found that levels of the microRNA were higher in the part of the brain responsible for seizures in many patients with temporal lobe epilepsy. Notably, the targeting approach was effective in a model of acquired temporal lobe epilepsy, preventing the development of epilepsy in 86% of rats in the study.  The research included contributions from several members of EpimiRNA including Dr Braxton Norwood and members of Prof Rosenow’s group, Stephanie Schorge’s team at UCL, Jeroen Pasterkamp and colleagues at UMCU and clinical colleagues at Beaumont hospital in Dublin.

Cristina Reschke, first author of the study commented “I am delighted to see the new study published. The project began outside of EpimiRNA and was only planned to test the inhibitor in one more mouse model. After discussion at an EpimiRNA meeting others joined in and it became a larger, more impactful collaborative effort that took advantage of the many skilled researchers and their models in the consortium. Hopefully this approach will be useful for new miRNAs currently under study by the consortium

Cristina R. Reschke, Luiz F. Almeida Silva, Braxton A. Norwood, Ketharini Senthilkumar, Gareth Morris,Amaya Sanz-Rodriguez, Ronán M. Conroy, Lara Costard, Valentin Neubert, Sebastian Bauer, Michael A. Farrell, Donncha F. O’Brien, Norman Delanty, Stephanie Schorge, R. Jeroen Pasterkamp, Felix Rosenow and David C. Henshall Potent Anti-seizure Effects of Locked Nucleic Acid Antagomirs Targeting miR-134 in Multiple Mouse and Rat Models of Epilepsy. Molecular Therapy: Nucleic Acids in press

 

 

EpimiRNA Publications -Highlights from the Consortium

Henshall D.C., Hamer H., Pasterkamp R.J., Goldstein D.B., Kjems J., Prehn J.H., Schorge S., Lamottke K., Rosenow F. MicroRNAs in epilepsy: pathophysiology and clinical utility. Lancet Neurol 15, 1368-1376 (2016)

Temporal lobe epilepsy is a common and frequently intractable seizure disorder. Its pathogenesis is thought to involve large-scale alterations to the expression of genes controlling neurotransmitter signalling, ion channels, synaptic structure, neuronal death, gliosis, and inflammation. Identification of mechanisms coordinating gene networks in patients with temporal lobe epilepsy will help to identify novel therapeutic targets and biomarkers. MicroRNAs (miRNAs) are a family of small non-coding RNAs that control the expression levels of multiple proteins by decreasing mRNA stability and translation, and could therefore be key regulatory mechanisms and therapeutic targets in epilepsy.

RECENT DEVELOPMENTS: In the past 5 years, studies have found changes in miRNA levels in the hippocampus of patients with temporal lobe epilepsy and in neural tissues from animal models of epilepsy. Early functional studies showed that silencing of brain-specific miR-134 using antisense oligonucleotides (antagomirs) had potent antiseizure effects in animal models, whereas genetic deletion of miR-128 produced fatal epilepsy in mice. Levels of certain miRNAs were also found to be altered in the blood of rodents after seizures. In the past 18 months, functional studies have identified nine novel miRNAs that appear to influence seizures or hippocampal pathology. Their targets include transcription factors, neurotransmitter signalling components, and modulators of neuroinflammation. New approaches to manipulate miRNAs have been tested, including injection of mimics (agomirs) to enhance brain levels of miRNAs. Altered miRNA expression has also been reported in other types of refractory epilepsy and our understanding of how miRNA levels are controlled has grown, with studies on DNA methylation indicating epigenetic regulation. Biofluids (blood) of patients with epilepsy have shown differences in quantity of circulating miRNAs, implying diagnostic biomarker potential. WHERE NEXT?: Recent functional studies need to be replicated to build a robust evidence base. The specific cell types in which miRNAs execute their functions and their primary targets have to be identified, to fully explain the phenotypic effects of modulating miRNAs. Delivery of large molecules such as antisense inhibitors or mimics to the brain poses a challenge, and the multi-targeting effects of miRNAs create additional risks of unanticipated side effects. Potential genetic variation in miRNAs should be explored as the basis for disease susceptibility. The latest findings provide a rich source of new miRNA targets, but substantial challenges remain before their role in the pathogenesis, diagnosis, and treatment of epilepsy can be translated into clinical practice.

 

Brennan G.P., Henshall D.C. microRNAs in the Pathophysiology of Epilepsy. Neurosci Lett. 2017 Jan 16

Temporal lobe epilepsy is a common and often drug-resistant seizure disorder. The underlying pathological processes which give rise to the development of spontaneous seizures include neuroinflammation, cell loss, neurogenesis and dendritic abnormalities and many of these are driven by insult-induced changes in gene expression and gene expression regulation. MicroRNAs are powerful modulators of post-transcriptional gene expression which are dysregulated during epileptogenesis. The advent of locked nucleic acid (LNA) based inhibitory methods and mimic technology has facilitated in vivo functional assessment of these molecules in epilepsy. Here we review recent advances in our understanding of the role of these short non-coding RNAs in the pathophysiology of epilepsy.   

 

Engel T., Brennan G.P., Sanz-Rodriguez A., Alves M., Beamer E., Watters O., Henshall D.C., Jimenez-Mateos EM. A calcium-sensitive feed-forward loop regulating the expression of the ATP-gated purinergic P2X7 receptor via specificity protein 1 and microRNA-22. Biochim Biophys Acta. 1864, 255-266 (2017)

Cells have developed complex transcriptional regulatory mechanisms to maintain intracellular homeostasis and withstand pathophysiological stressors. Feed-forward loops comprising transcription factors that drive expression of both target gene and a microRNA as negative regulator, are gaining increasing recognition as key regulatory elements of cellular homeostasis. The ATP-gated purinergic P2X7 receptor (P2X7R) is an important driver of inflammation and has been implicated in the pathogenesis of numerous brain diseases including epilepsy. Changes in P2X7R expression have been reported in both experimental models and in epilepsy patients but the mechanism(s) controlling P2X7R levels remain incompletely understood. The specificity protein 1 (Sp1) has been shown to induce P2X7R transcription in vitro and recent data has identified microRNA-22 as a post-transcriptional repressor of P2X7R expression after seizures. In the present study we show that Sp1 can induce the transcription of both microRNA-22 and P2X7R in vitro during increased neuronal activity and in vivo in a mouse model of status epilepticus. We further show that Sp1-driven microRNA-22 transcription is calcium-sensitive and Sp1 occupancy of the microRNA-22 promoter region is blocked under conditions of seizure activity sufficient to elicit neuronal death. Taken together, our results suggest a neuronal activity-dependent P2X7R expression which is induced by the transcription factor Sp1 and repressed in a calcium-dependent manner by microRNA-22.

Almeida Silva, L.F., Engel T., Reschke C.R., Conroy R., Langa E., Henshall D.C. Distinct behavioural and epileptic phenotype differences in 129/P mice compared to C57BL/6 mice subject to intraamygdala kainic acid-induced status epilepticus. Epilepsy Behav 64,186-194 (2016)

Animal models of status epilepticus are important tools to understand the pathogenesis of epileptic brain injury and evaluate potential seizure-suppressive, neuroprotective, and antiepileptogenic treatments. Focal elicitation of status epilepticus by intraamygdala kainic acid in mice produces unilateral hippocampal damage and the emergence of spontaneous recurrent seizures after a short latent period. The model has been characterized in C57BL/6, BALB/c, and SJL mice where strain-specific differences were found in the extent of hippocampal damage. 129/P mice are a common background strain for genetic models and may display unique characteristics in this model. We therefore compared responses to intraamygdala kainic acid between 129/P and C57BL/6 mice. Racine scale-scored convulsive behavior during status epilepticus was substantially lower in 129/P mice compared with that in C57BL/6 mice. Analysis of surface-recorded electroencephalogram (EEG) showed differences between strains in several frequency bands; EEG total power was greater during ictal episodes while duration of seizures was slightly shorter in 129/P mice. Histological analysis revealed similar hippocampal injury between strains, with neuronal death mainly confined to the ipsilateral CA3 subfield. Expression of genes associated with gliosis and neuroinflammatory responses was also similar between strains after seizures. Video-EEG telemetry recordings showed that 129/P mice first display spontaneous seizures within a few days of status epilepticus similar to C57BL/6 mice. However, high mortality in 129/P mice prevented a quantitative comparison of the epileptic seizure phenotypes between strains. This study defined behavioral, EEG, and histopathologic features of this mouse strain in a model increasingly useful for the study of the genetic contribution to acquired epilepsy. Intraamygdala kainic acid in 129/P mice could serve as a model of nonconvulsive status epilepticus, but long-term assessments will require model adjustment to mitigate the severity of the emergent epileptic phenotype.

Gross C., Yao X., Engel T., Xing L., Danielson S.W., Thomas K.T., Jimenez-Mateos E.M., Henshall D.C., & Gary J. Bassell. MicroRNA-mediated downregulation of the potassium channel Kv4.2 contributes to seizure onsethttps://www.ncbi.nlm.nih.gov/pubmed/27681419 Cell Rep 17, 37-45 (2016)

Seizures are bursts of excessive synchronized neuronal activity, suggesting that mechanisms controlling brain excitability are compromised. The voltage-gated potassium channel Kv4.2, a major mediator of hyperpolarizing A-type currents in the brain, is a crucial regulator of neuronal excitability. Kv4.2 expression levels are reduced following seizures and in epilepsy, but the underlying mechanisms remain unclear. Here, we report that Kv4.2 mRNA is recruited to the RNA-induced silencing complex shortly after status epilepticus in mice and after kainic acid treatment of hippocampal neurons, coincident with reduction of Kv4.2 protein. We show that the microRNA miR-324-5p inhibits Kv4.2 protein expression and that antagonizing miR-324-5p is neuroprotective and seizure suppressive. MiR-324-5p inhibition also blocks kainic-acid-induced reduction of Kv4.2 protein in vitro and in vivo and delays kainic-acid-induced seizure onset in wild-type but not in Kcnd2 knockout mice. These results reveal an important role for miR-324-5p-mediated silencing of Kv4.2 in seizure onset.

Cattani AA, Allene C, Seifert V, Rosenow F, Henshall DC, Freiman TM Involvement of microRNAs in epileptogenesis. Epilepsia. 2016 Jul;57(7):1015-26.

Patients who have sustained brain injury or had developmental brain lesions present a non-negligible risk for developing delayed epilepsy. Finding therapeutic strategies to prevent development of epilepsy in at-risk patients represents a crucial medical challenge. Noncoding microRNA molecules (miRNAs) are promising candidates in this area. Indeed, deregulation of diverse brain-specific miRNAs has been observed in animal models of epilepsy as well as in patients with epilepsy, mostly in temporal lobe epilepsy (TLE). Herein we review deregulated miRNAs reported in epilepsy with potential roles in key molecular and cellular processes underlying epileptogenesis, namely neuroinflammation, cell proliferation and differentiation, migration, apoptosis, and synaptic remodeling. We provide an up-to-date listing of miRNAs altered in epileptogenesis and assess recent functional studies that have interrogated their role in epilepsy. Last, we discuss potential applications of these findings for the future development of disease-modifying therapeutic strategies for antiepileptogenesis.

Mooney C, Becker BA, Raoof R, Henshall DC (2016).  EpimiRBase: a comprehensive database of microRNA-epilepsy associations. Bioinformatics. 1;32(9):1436-8.

MicroRNAs are short non-coding RNA which function to fine-tune protein levels in all cells. This is achieved mainly by sequence-specific binding to 3' untranslated regions of target mRNA. The result is post-transcriptional interference in gene expression which reduces protein levels either by promoting destabilisation of mRNA or translational repression. Research published since 2010 shows that microRNAs are important regulators of gene expression in epilepsy. A series of microRNA profiling studies in rodent and human tissue has revealed that epilepsy is associated with wide ranging changes to microRNA levels in the brain. These are thought to influence processes including cell death, inflammation and re-wiring of neuronal networks. MicroRNAs have also been identified in the blood after injury to the brain and therefore may serve as biomarkers of epilepsy. EpimiRBase is a manually curated database for researchers interested in the role of microRNAs in epilepsy. The fully searchable database includes information on up- and down-regulated microRNAs in the brain and blood, as well as functional studies, and covers both rodent models and human epilepsy.

Other Pubications from EpimiRNA partners

Quinlan S., Henke C., Brennan G.P., Henshall D.C., Jimenez-Mateos E.M. Detection of microRNAs in brain slices using in situ hybridization. Methods Mol Biol 1509, 85-91 (2017)

MicroRNAs are key posttranscriptional regulators of protein levels in cells. The brain is particularly enriched in microRNAs, and important roles have been demonstrated for these noncoding RNAs in various neurological disorders. To this end, visualization of microRNAs in specific cell types and subcellular compartments within tissue sections provides researchers with essential insights that support understanding of the cell and molecular mechanisms of microRNAs in brain diseases. In this chapter we describe an in situ hybridization protocol for the detection of microRNAs in mouse brain sections, which provides cellular resolution of the expression of microRNAs in the brain.

Hansen, T.B., Venø, M.T., Jensen, T.I., Schaefer, A., Damgaard, C.K. & Kjems, J.: Argonaute associated short introns are a novel class of gene regulators Nature Commun. 7:11538 (2016).

MicroRNAs (miRNAs) are short (22 nucleotides) regulators of gene expression acting by direct base pairing to 3'-UTR target sites in messenger RNAs. Mature miRNAs are produced by two sequential endonucleolytic cleavages facilitated by Drosha in the nucleus and Dicer in the cytoplasm. A subclass of miRNAs, termed mirtrons, derives from short introns and enters the miRNA biogenesis pathway as Dicer substrates. Here we uncover a third biogenesis strategy that, similar to mirtron biogenesis, initiates from short introns but bypasses Dicer cleavage. These short introns (80-100 nucleotides), coined agotrons, are associated with and stabilized by Argonaute (Ago) proteins in the cytoplasm. Some agotrons are completely conserved in mammalian species, suggesting that they are functionally important. Furthermore, we demonstrate that the agotrons are capable of repressing mRNAs with seed-matching target sequences in the 3'-UTR. These data provide evidence for a novel RNA regulator of gene expression, which bypasses the canonical miRNA biogenesis machinery.

Svenningsen K, Venø MT, Henningsen K, Mallien AS, Jensen L, Christensen T, Kjems J, Vollmayr B, Wiborg O. MicroRNA Profiling in the Medial and Lateral Habenula of Rats Exposed to the Learned Helplessness Paradigm: Candidate Biomarkers for Susceptibility and Resilience to Inescapable Shockhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4975463/. PLoS One. 11(8):e0160318 (2016)

Depression is a highly heterogeneous disorder presumably caused by a combination of several factors ultimately causing the pathological condition. The genetic liability model of depression is likely to be of polygenic heterogeneity. miRNAs can regulate multiple genes simultaneously and therefore are candidates that align with this model. The habenula has been linked to depression in both clinical and animal studies, shifting interest towards this region as a neural substrate in depression. The goal of the present study was to search for alterations in miRNA expression levels in the medial and lateral habenula of rats exposed to the learned helplessness (LH) rat model of depression. Ten miRNAs showed significant alterations associating with their response to the LH paradigm. Of these, six and four miRNAs were significantly regulated in the MHb and LHb, respectively. In the MHb we identified miR-490, miR-291a-3p, MiR-467a, miR-216a, miR-18b, and miR-302a. In the LHb miR-543, miR-367, miR-467c, and miR-760-5p were significantly regulated. A target gene analysis showed that several of the target genes are involved in MAPK signaling, neutrophin signaling, and ErbB signaling, indicating that neurotransmission is affected in the habenula as a consequence of exposure to the LH paradigm.

Yan Y.; Wang X.; Venø M.T., Bakholdt V., Sørensen, J.A., Krogdahl, A., Zheng, S., Gao, S., Kjems, J.: Circulating miRNAs as biomarkers for oral squamous cell carcinoma recurrence in operated patients. Oncotarget In Press (2016).

MicroRNAs (miRNAs) are small regulatory non-coding RNAs for which altered expression in cancers can serve as potential biomarkers for diseases. We here investigated whether circulating miRNAs can serve as biomarkers for predicting post-operational recurrence of oral squamous cell carcinoma (OSCC) in patients. Plasma samples from 8 Danish OSCC patients were collected before, and one year after surgical operation, as well as from 3 Danish healthy controls and subjected to miRNA profiling by next generation sequencing. Disease recurrence did not occur in the 8 patients when the post-operative plasma samples were collected. Based on the sequencing data, three up-regulated miRNAs (miR-148a-3p, miR-26a-5p and miR-21-5p) and three down-regulated miRNAs (miR-375, miR-92b-3p and miR-486-5p) in the OSCC samples compared to healthy controls were selected for qRT-PCR validation in a Chinese cohort of 20 plasma samples collected before, and 9-12 months after surgical operation, and 18 healthy controls. Disease recurrence had occurred in 8 out of the 20 Chinese patients at the time their post-operative plasma samples were collected. The results of qRT-PCR showed that down-regulation of miR-486-5p, miR-375 and miR-92b-3p were highly associated with OSCC recurrence. This study indicates that the plasma miRNA profile is altered in OSCC during its progression and can be used to monitor the likelihood of OSCC recurrence in patients after surgery.

Pronker MF, Lemstra S, Snijder J, Heck AJR, Thies-Weesie DME, Pasterkamp RJ, Janssen BJC (2016). Structural basis of myelin-associated glycoprotein adhesion and signalling. Nature Communications, in press.

Myelin-associated glycoprotein (MAG) is a myelin-expressed cell-adhesion and bi-directional signalling molecule. MAG maintains the myelin-axon spacing by interacting with specific neuronal glycolipids (gangliosides), inhibits axon regeneration and controls myelin formation. The mechanisms underlying MAG adhesion and signalling are unresolved. We present crystal structures of the MAG full ectodomain, which reveal an extended conformation of five Ig domains and a homodimeric arrangement involving membrane-proximal domains Ig4 and Ig5. MAG-oligosaccharide complex structures and biophysical assays show how MAG engages axonal gangliosides at domain Ig1. Two post-translational modifications were identified-N-linked glycosylation at the dimerization interface and tryptophan C-mannosylation proximal to the ganglioside binding site-that appear to have regulatory functions. Structure-guided mutations and neurite outgrowth assays demonstrate MAG dimerization and carbohydrate recognition are essential for its regeneration-inhibiting properties. The combination of trans ganglioside binding and cis homodimerization explains how MAG maintains the myelin-axon spacing and provides a mechanism for MAG-mediated bi-directional signalling.

Schellino R, Trova S, Cimino I, Farinetti A, Jongbloets BC, Pasterkamp RJ, Panzica G, Giacobini P, De Marchis S, Peretto P (2016). https://www.ncbi.nlm.nih.gov/pubmed/27782186Opposite-sex attraction in male mice requires testosterone-dependent regulation of adult olfactory bulb neurogenesis. Sci Rep 6, 36063.

Opposite-sex attraction in most mammals depends on the fine-tuned integration of pheromonal stimuli with gonadal hormones in the brain circuits underlying sexual behaviour. Neural activity in these circuits is regulated by sensory processing in the accessory olfactory bulb (AOB), the first central station of the vomeronasal system. Recent evidence indicates adult neurogenesis in the AOB is involved in sex behaviour; however, the mechanisms underlying this function are unknown. By using Semaphorin 7A knockout (Sema7A ko) mice, which show a reduced number of gonadotropin-releasing-hormone neurons, small testicles and subfertility, and wild-type males castrated during adulthood, we demonstrate that the level of circulating testosterone regulates the sex-specific control of AOB neurogenesis and the vomeronasal system activation, which influences opposite-sex cue preference/attraction in mice. Overall, these data highlight adult neurogenesis as a hub for the integration of pheromonal and hormonal cues that control sex-specific responses in brain circuits.

van Rossum D, Verheijen BM, Pasterkamp RJ (2016). Circular RNAs: Novel Regulators of Neuronal Developmenthttps://www.ncbi.nlm.nih.gov/pubmed/27616979. Front Mol Neurosci 9, 74.

Circular RNAs (circRNAs) are highly stable, circularized long non-coding RNAs. circRNAs are conserved across species and appear to be specifically enriched in the nervous system. Recent studies show that many circRNAs are expressed in a tissue- and developmental-stage-specific manner, reveal a striking regulation of circRNAs during neuronal development, and detect their presence at synaptic sites. The exact functions of circRNAs remain poorly understood, but evidence from analysis of some circRNA molecules suggests that they could substantially contribute to the regulation of gene expression, particularly in architecturally complex and polarized cells such as neurons. Emerging evidence also indicates that circRNAs are involved in the development and progression of various neurological disorders. In this review, we summarize the molecular characteristics of circRNAs and discuss their proposed functions and mechanism-of-action in developing neurons.

van Rheenen W, Shatunov A, Dekker AM, Pasterkamp RJ, et al.(2016). Genome-wide association analyses identify new risk variants and the genetic architecture of amyotrophic lateral sclerosis. Nat Genet 48, 1043-8.

To elucidate the genetic architecture of amyotrophic lateral sclerosis (ALS) and find associated loci, we assembled a custom imputation reference panel from whole-genome-sequenced patients with ALS and matched controls (n = 1,861). Through imputation and mixed-model association analysis in 12,577 cases and 23,475 controls, combined with 2,579 cases and 2,767 controls in an independent replication cohort, we fine-mapped a new risk locus on chromosome 21 and identified C21orf2 as a gene associated with ALS risk. In addition, we identified MOBP and SCFD1 as new associated risk loci. We established evidence of ALS being a complex genetic trait with a polygenic architecture. Furthermore, we estimated the SNP-based heritability at 8.5%, with a distinct and important role for low-frequency variants (frequency 1-10%). This study motivates the interrogation of larger samples with full genome coverage to identify rare causal variants that underpin ALS risk.

Kong Y, Janssen BJ, Malinauskas T, Vangoor VR, Coles CH, Kaufmann R, Ni T, Gilbert RJ, Padilla-Parra S, Pasterkamp RJ*, Jones EY* (2016). Structural Basis for Plexin Activation and Regulation. Neuron 91, 548-60. *Shared senior authors.

Class A plexins (PlxnAs) act as semaphorin receptors and control diverse aspects of nervous system development and plasticity, ranging from axon guidance and neuron migration to synaptic organization. PlxnA signaling requires cytoplasmic domain dimerization, but extracellular regulation and activation mechanisms remain unclear. Here we present crystal structures of PlxnA (PlxnA1, PlxnA2, and PlxnA4) full ectodomains. Domains 1-9 form a ring-like conformation from which the C-terminal domain 10 points away. All our PlxnA ectodomain structures show autoinhibitory, intermolecular "head-to-stalk" (domain 1 to domain 4-5) interactions, which are confirmed by biophysical assays, live cell fluorescence microscopy, and cell-based and neuronal growth cone collapse assays. This work reveals a 2-fold role of the PlxnA ectodomains: imposing a pre-signaling autoinhibitory separation for the cytoplasmic domains via intermolecular head-to-stalk interactions and supporting dimerization-based PlxnA activation upon ligand binding. More generally, our data identify a novel molecular mechanism for preventing premature activation of axon guidance receptors.

Siebold C, Yamashita T, Monnier PP, Mueller BK, Pasterkamp RJ (2016). RGMs: Structural insights, molecular regulation and downstream signalinghttps://www.ncbi.nlm.nih.gov/pubmed/28007423. Trends in Cell Biology, in press.

Although originally discovered as neuronal growth cone-collapsing factors, repulsive guidance molecules (RGMs) are now known as key players in many fundamental processes, such as cell migration, differentiation, iron homeostasis, and apoptosis, during the development and homeostasis of many tissues and organs, including the nervous, skeletal, and immune systems. Furthermore, three RGMs (RGMa, RGMb/DRAGON, and RGMc/hemojuvelin) have been linked to the pathogenesis of various disorders ranging from multiple sclerosis (MS) to cancer and juvenile hemochromatosis (JHH). While the molecular details of these (patho)biological effects and signaling modes have long remained unknown, recent studies unveil several exciting and novel aspects of RGM processing, ligand-receptor interactions, and downstream signaling. In this review, we highlight recent advances in the mechanisms-of-action and function of RGM proteins.

Engelen-Lee Y, Blokhuis AM, Spliet WGM, Pasterkamp RJ, Aronica E, Demmers JAA, Broekhuizen R, Nardo G, Bovenschen N, Van den Berg LH (2016). Proteomic profiling of the spinal cord in ALS: decreased ATP5D levels suggest synaptic dysfunction in ALS pathogenesis. ALS Frontotemp Degeneration, in press.

BACKGROUND:We aimed to gain new insights into the pathogenesis of sporadic ALS (sALS) through a comprehensive proteomic analysis. METHODS:Protein profiles of the anterior and posterior horn in post-mortem spinal cord samples of 10 ALS patients and 10 controls were analysed using 2D-differential gel electrophoresis. The identified protein spots with statistically significant level changes and a spot ratio >2.0 were analysed by LC-MS/MS. RESULTS:In the posterior horn only 3 proteins were differentially expressed. In the anterior horn, 16 proteins with increased levels and 2 proteins with decreased levels were identified in ALS compared to controls. The identified proteins were involved in mitochondrial metabolism, calcium homeostasis, protein metabolism, glutathione homeostasis, protein transport and snRNP assembly. The two proteins with decreased levels, ATP5D and calmodulin, were validated by Western blot and immunostaining. Immunohistochemical and immunofluorescent double staining of ATP5D and synaptophysin showed that the reduction of ATP5D was most pronounced at synapses. CONCLUSIONS:We speculate that mitochondrial dysfunction in synaptic clefts could play an important role in sALS pathogenesis. A similar approach revealed decreased calmodulin expression mainly in the neuronal body and dendrites of ALS patients. These findings contribute to a deeper understanding of the disease process underlying ALS.

Zuko A, Aguro-Ando A, Post H, Taggenbrock RLRE, Van Dijk RE, Altelaar M, Heck AJR, Petrenko AG, Van der Zwaag B, Shimoda Y, Pasterkamp RJ, Burbach JPH (2016) Association of cell adhesion molecules contactin-6 and latrophilin-1 regulates neuronal apoptosishttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5156884/. Front Mol Neuroscience, in press.

In view of important neurobiological functions of the cell adhesion molecule contactin-6 (Cntn6) that have emerged from studies on null-mutant mice and autism spectrum disorders patients, we set out to examine pathways underlying functions of Cntn6 using a proteomics approach. We identified the cell adhesion GPCR latrophilin-1 (Lphn1, a.k.a. CIRL1/CL, ADGRL1) as a binding partner for Cntn6 forming together a heteromeric cis-complex. Lphn1 expression in cultured neurons caused reduction in neurite outgrowth and increase in apoptosis, which was rescued by coexpression of Cntn6. In cultured neurons derived from Cntn6-/- mice, Lphn1 knockdown reduced apoptosis, suggesting that the observed apoptosis was Lphn1-dependent. In line with these data, the number of apoptotic cells was increased in the cortex of Cntn6-/- mice compared to wild-type littermate controls. These results show that Cntn6 can modulate the activity of Lphn1 by direct binding and suggests that Cntn6 may prevent apoptosis thereby impinging on neurodevelopment.

Vieira AS, de Matos AH, do Canto AM, Rocha CS, Carvalho BS, Pascoal VD, Norwood B, Bauer S, Rosenow F, Gilioli R, Cendes F, Lopes-Cendes I (2016) RNA sequencing reveals region-specific molecular mechanisms associated with epileptogenesis in a model of classical hippocampal sclerosis. Sci Rep. 2016 Mar 3;6:22416

We report here the first complete transcriptome analysis of the dorsal (dDG) and ventral dentate gyrus (vDG) of a rat epilepsy model presenting a hippocampal lesion with a strict resemblance to classical hippocampal sclerosis (HS). We collected the dDG and vDG by laser microdissection 15 days after electrical stimulation and performed high-throughput RNA-sequencing. There were many differentially regulated genes, some of which were specific to either of the two sub-regions in stimulated animals. Gene ontology analysis indicated an enrichment of inflammation-related processes in both sub-regions and of axonal guidance and calcium signaling processes exclusively in the vDG. There was also a differential regulation of genes encoding molecules involved in synaptic function, neural electrical activity and neuropeptides in stimulated rats. The data presented here suggests, in the time point analyzed, a remarkable interaction among several molecular components which takes place in the damaged hippocampi. Furthermore, even though similar mechanisms may function in different regions of the DG, the molecular components involved seem to be region specific.

de Andrade HM, de Albuquerque M, Avansini SH, de S Rocha C, Dogini DB, Nucci A, Carvalho B, Lopes-Cendes I, França MC Jr. (2016) MicroRNAs-424 and 206 are potential prognostic markers in spinal onset amyotrophic lateral sclerosis.https://www.ncbi.nlm.nih.gov/pubmed/27538595 J Neurol Sci. 2016 Sep 15;368:19-24.

INTRODUCTION: Skeletal muscle microRNAs (miRNAs) are potential candidate biomarkers for amyotrophic lateral sclerosis (ALS) that deserve further investigation. OBJECTIVES: To identify miRNAs abnormally expressed in the skeletal muscle and plasma of patients with ALS, and to correlate them with parameters of disease progression. METHODS: Expression profile of miRNAs in muscle was evaluated using an array platform. Subsequently we assessed the plasmatic expression of candidate miRNAs in a set of 39 patients/39 controls. We employed generalized estimating equations to investigate correlations with clinical data. RESULTS: We identified 11 miRNAs differentially expressed in the muscle of ALS patients; of these, miR424, miR-214 and miR-206 were validated by qPCR in muscle samples. In plasma, we found only miR-424 and miR 206 to be overexpressed. Baseline expression of miR-424 and 206 correlated with clinical deterioration over time. CONCLUSION: MiR-424 and miR-206 are potential prognostic markers for ALS.

Upcoming Publications

Bielefield P., Mooney C., Henshall D.C., Fitzsimons C.P. MicroRNAs in the control of neurogenesis in neurological diseases. Brain Plasticity in press

Henshall D.C. Focally applied chemoconvulsants. in “Models of seizures and epilepsy”, Second Edition (Elsevier).

Henshall D.C. Post-status epilepticus models: Focal kainic acid in “Models of seizures and epilepsy”,Second Edition (Elsevier).

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