From microRNA target validation to therapy : lessons learned from studies on BDNF

During the past decade, the identification of microRNA (miR) targets has become common laboratory practice, and various strategies are now used to detect interactions between miRs and their mRNA targets. However, the current lack of a standardized identification process often leads to incomplete and/or conflicting results. Here, we review the problems most commonly encountered when verifying miR-mRNA interactions, and we propose a workflow for future studies. To illustrate the challenges faced when validating a miR target, we discuss studies in which the regulation of brain-derived neurotrophic factor by miRs was investigated, and we highlight several controversies that emerged from these studies. Finally, we discuss the therapeutic use of miR inhibitors, and we discuss several questions that should be addressed before proceeding to preclinical testing.Peer reviewe

Myenteric Neurons Do Not Replicate in Small Intestine Under Normal Physiological Conditions in Adult Mouse

BACKGROUND & AIMS: The enteric nervous system (ENS) is the largest part of the peripheral nervous system; moreover, abnormal ENS development and function are associated with multiple human pathologies. Data from several groups suggest that under normal physiological conditions in adult animals, enteric nerve cells do not replicate. A study by Kulkarni et al in 2017 challenged this view and proposed that nearly 70% of enteric neurons in the myenteric ganglia are born in 1 week The authors of this study suggested that differences in DNA labelling times and DNA denaturation conditions might explain discrepancies with previous reports. Previous studies were carried out using different conditions and labelling techniques in various regions of the gastrointestinal tract; thus, conclusions have remained elusive. METHODS: Here, we have eliminated those variables by analyzing the whole small intestine using the reagents and conditions that Kulkarni et al used. To exclude variables related to immunohistochemistry, we carried out parallel experiments with "click chemistry"-based detection of DNA replication. RESULTS: Although proliferation was readily detected in the epithelium, we found no evidence of neuronal replication in the myenteric ganglia. CONCLUSIONS: We conclude that within 1 week under normal physiological conditions, myenteric neurons in the small intestine do not replicate.Peer reviewe

Biology of GDNF and its receptors — Relevance for disorders of the central nervous system

a b s t r a c t a r t i c l e i n f o Article history: Received 7 December 2015 Revised 14 January 2016 Accepted 25 January 2016 Available online xxxx A targeted effort to identify novel neurotrophic factors for midbrain dopaminergic neurons resulted in the isolation of GDNF (glial cell line-derived neurotrophic factor) from the supernatant of a rat glial cell line in 1993. Over two decades and 1200 papers later, the GDNF ligand family and their different receptor systems are now recognized as one of the major neurotrophic networks in the nervous system, important for the development, maintenance and function of a variety of neurons and glial cells. The many ways in which the four members of the GDNF ligand family can signal and function allow these factors to take part in the control of multiple types of processes, from neuronal survival to axon guidance and synapse formation in the developing nervous system, to synaptic function and regenerative responses in the adult. In this review, we will briefly summarize basic aspects of GDNF signaling mechanisms and receptor systems and then review our current knowledge of the physiology of GDNF activities in the central nervous system, with an eye to its relevance for neurodegenerative and neuropsychiatric diseases

Two-fold elevation of endogenous GDNF levels in mice improves motor coordination without causing side-effects

Glial cell line-derived neurotrophic factor (GDNF) promotes the survival of dopaminergic neurons in vitro and in vivo. For this reason, GDNF is currently in clinical trials for the treatment of Parkinson’s disease (PD). However, how endogenous GDNF influences dopamine system function and animal behavior is not fully understood. We recently generated GDNF hypermorphic mice that express increased levels of endogenous GDNF from the native locus, resulting in augmented function of the nigrostriatal dopamine system. Specifically, Gdnf wt/hyper mice have a mild increase in striatal and midbrain dopamine levels, increased dopamine transporter activity, and 15% increased numbers of midbrain dopamine neurons and striatal dopaminergic varicosities. Since changes in the dopamine system are implicated in several neuropsychiatric diseases, including schizophrenia, attention deficit hyperactivity disorder (ADHD) and depression, and ectopic GDNF delivery associates with side-effects in PD models and clinical trials, we further investigated Gdnf wt/hyper mice using 20 behavioral tests. Despite increased dopamine levels, dopamine release and dopamine transporter activity, there were no differences in psychiatric disease related phenotypes. However, compared to controls, male Gdnf wt/hyper mice performed better in tests measuring motor function. Therefore, a modest elevation of endogenous GDNF levels improves motor function but does not induce adverse behavioral outcomes.Peer reviewe

DNA-korjauksen kahdet kasvot : genomin ylläpito ja geenien muokkaus

Vertaisarvioitu.DNA:n korjausmekanismit ovat kehittyneet säilyttämään DNA-molekyylit riittävän virheettöminä solun ja eliön elinkyvyn säilyttämiseksi sukupolvelta toiselle. Periytyvät muutokset DNA:ssa voivat johtaa perinnöllisiin sairauksiin, mutta toisaalta ne sallivat perimän vaihtelun ja sitä kautta evoluution. Somaattisten kudosten solujen DNA:han kerääntyvät virheet liittyvät lääketieteen näkökulmasta ennen kaikkea syöpien kehitykseen sekä ikääntymiseen, ja DNA:n korjausmekanismien tutkimus onkin aiemmin liittynyt pääasiallisesti näihin ilmiöihin. Viime aikoina valtavasti kehittyneet geenien muokkausmenetelmät, kuten CRISPR-Cas, luovat uusia mahdollisuuksia hoitaa ihmisten sairauksia genomia muokkaamalla. Solunsisäiset DNA:n korjausmekanismit ovat keskeisiä ja luovat reunaehdot genomin muokkaukselle.Peer reviewe

Improving CRISPR/Cas9 mutagenesis efficiency by delaying the early development of zebrafish embryos

CRISPR/Cas9 driven mutagenesis in zygotes is a popular tool for introducing targeted mutations in model organisms. Compared to mouse, mutagenesis in zebrafish is relatively inefficient and results in somatic mosaicism most likely due to a short single-cell stage of about 40 min. Here we explored two options to improve CRISPR/Cas9 mutagenesis in zebrafish-extending the single-cell stage and defining conditions for carrying out mutagenesis in oocytes prior to in vitro fertilization. Previous work has shown that ovarian fluid from North American salmon species (coho and chinook salmon) prolong oocyte survival ex vivo so that they are viable for hours instead of dying within minutes if left untreated. We found that commonly farmed rainbow trout (Oncorhynchus mykiss) ovarian fluid (RTOF) has similar effect on zebrafish oocyte viability. In order to prolong single-cell stage, we incubated zebrafish zygotes in hydrogen sulfide (H2S) and RTOF but failed to see any effect. However, the reduction of temperature from standard 28 to 12 degrees C postponed the first cell division by about an hour. In addition, the reduction in temperature was associated with increased CRISPR/Cas9 mutagenesis rate. These results suggest that the easily applicable reduction in temperature facilitates CRISPR/Cas9 mutagenesis in zebrafish.Peer reviewe

Female C57BL/6J Mice Show Alcohol-Seeking Behaviour after Withdrawal from Prolonged Alcohol Consumption in the Social Environment.

Aims Recently we developed a model to study alcohol-seeking behaviour after withdrawal in a social context in female mice. The model raised several questions that we were eager to address to improve methodology. Methods In our model, female mice were group-housed in automated cages with three conditioned (CS+) corners and water in both sides of one separate non-conditioned corner. Water was available with opened doors at all the time of training. We established conditioning by pairing alcohol drinking with light cues. Here, we introduced prolonged access to increasing concentrations of alcohol instead of intermittent access. To study motivation to drink alcohol, we carried out the extinction tests on withdrawal days 1 (WD1) and 10 (WD10). During tests, the light cues were present in conditioned corners, but there was no liquid in the bottles. Results We found that the number of visits and nosepokes in the CS+ corner in the alcohol group was much higher than in the water group. Also, during training, the consumption of alcohol was increasing. In the extinction tests, we found that the number of nosepokes in the CS+ corner increased in the alcohol group on both WD1 and WD10. Conclusions Our study supports that alcohol-seeking behaviour after withdrawal can be modelled and studied in group-housed animals and environments without social isolation. Short Summary: We developed a model to study alcohol drinking behaviour in an enriched and social environment. Long-term conditioning coupling with alcohol reward results in cue-induced alcohol-seeking behaviour in group-housed female C57BL/6J mice. Moreover, a high number of nosepokes on the last day of alcohol drinking conditioning might potentiate alcohol-seeking after withdrawal response.Peer reviewe

Towards developing a model to study alcohol drinking and craving in female mice housed in automated cages

It is about half a century ago when the so-called "Wise model" to study alcohol drinking behavior in rats was established. The model was based on voluntary intermittent access to increasing concentrations of alcohol. We aimed to establish a model of alcohol craving and used an extinction test on withdrawal days 1 and 10 to study motivation for alcohol. For this purpose, the alcohol drinking training was paired with light cues to establish conditioning. The extinction test was carried out without alcohol but in the presence of light cues and empty bottles. The outcome measures were number of visits, nosepokes, and licks in the conditioned corner where the number of nosepokes represents how much mice "want" alcohol and number of licks shows how much mice "like" alcohol. The number of nosepokes during withdrawal is a measure of craving. Late withdrawal craving was found when intermittent alcohol access was carried out in the automated cages. In this case, we observed a significant increase in the number of nosepokes on both withdrawal days 1 and 10 as compared to water control. The number of nosepokes in the withdrawal days did not correlate with alcohol dose, but number of nosepokes on withdrawal day 1 correlated with the number of nosepokes on the last training day. Although we did not observe incubation of alcohol craving after withdrawal, the craving was increased at the late time point. We conclude that we have established a new tool to study alcohol drinking behavior and craving in female mice.Peer reviewe

A Coalgebraic View of Infinite Trees and Iteration

AbstractThe algebra of infinite trees is, as proved by C. Elgot, completely iterative, i.e., all ideal recursive equations are uniquely solvable. This is proved here to be a general coalgebraic phenomenon: let H be an endofunctor such that for every object X a final coalgebra, TX, of H(_) + X exists. Then TX is an object-part of a monad which is completely iterative. Moreover, a similar contruction of a “completely iterative monoid” is possible in every monoidal category satisfying mild side conditions