L1 Antisense Promoter Drives Tissue-Specific Transcription of Human Genes

Transcription of transposable elements interspersed in the genome is controlled by complex interactions between their regulatory elements and host factors. However, the same regulatory elements may be occasionally used for the transcription of host genes. One such example is the human L1 retrotransposon, which contains an antisense promoter (ASP) driving transcription into adjacent genes yielding chimeric transcripts. We have characterized 49 chimeric mRNAs corresponding to sense and antisense strands of human genes. Here we show that L1 ASP is capable of functioning as an alternative promoter, giving rise to a chimeric transcript whose coding region is identical to the ORF of mRNA of the following genes: KIAA1797, CLCN5, and SLCO1A2. Furthermore, in these cases the activity of L1 ASP is tissue-specific and may expand the expression pattern of the respective gene. The activity of L1 ASP is tissue-specific also in cases where L1 ASP produces antisense RNAs complementary to COL11A1 and BOLL mRNAs. Simultaneous assessment of the activity of L1 ASPs in multiple loci revealed the presence of L1 ASP-derived transcripts in all human tissues examined. We also demonstrate that L1 ASP can act as a promoter in vivo and predict that it has a heterogeneous transcription initiation site. Our data suggest that L1 ASP-driven transcription may increase the transcriptional flexibility of several human genes

Combination of native and denaturing PAGE for the detection of protein binding regions in long fragments of genomic DNA

<p>Abstract</p> <p>Background</p> <p>In a traditional electrophoresis mobility shift assay (EMSA) a ³²P-labeled double-stranded DNA oligonucleotide or a restriction fragment bound to a protein is separated from the unbound DNA by polyacrylamide gel electrophoresis (PAGE) in nondenaturing conditions. An extension of this method uses the large population of fragments derived from long genomic regions (approximately 600 kb) for the identification of fragments containing protein binding regions. With this method, genomic DNA is fragmented by restriction enzymes, fragments are amplified by PCR, radiolabeled, incubated with nuclear proteins and the resulting DNA-protein complexes are separated by two-dimensional PAGE. Shifted DNA fragments containing protein binding sites are identified by using additional procedures, i. e. gel elution, PCR amplification, cloning and sequencing. Although the method allows simultaneous analysis of a large population of fragments, it is relatively laborious and can be used to detect only high affinity protein binding sites. Here we propose an alternative and straightforward strategy which is based on a combination of native and denaturing PAGE. This strategy allows the identification of DNA fragments containing low as well as high affinity protein binding regions, derived from genomic DNA (<10 kb) of known sequence.</p> <p>Results</p> <p>We have combined an EMSA-based selection step with subsequent denaturing PAGE for the localization of protein binding regions in long (up to10 kb) fragments of genomic DNA. Our strategy consists of the following steps: digestion of genomic DNA with a 4-cutter restriction enzyme (<it>Alu</it>I, <it>Bsu</it>RI, <it>Tru</it>I, etc), separation of low and high molecular weight fractions of resultant DNA fragments, ³²P-labeling with Klenow polymerase, traditional EMSA, gel elution and identification of the shifted bands (or smear) by denaturing PAGE. The identification of DNA fragments containing protein binding sites is carried out by running the gel-eluted fragments alongside with the full "spectrum" of initial restriction fragments of known size. Here the strategy is used for the identification of protein-binding regions in the 5' region of the rat p75 neurotrophin receptor (<it>p75NTR</it>) gene.</p> <p>Conclusion</p> <p>The developed strategy is based on a combination of traditional EMSA and denaturing PAGE for the identification of protein binding regions in long fragments of genomic DNA. The identification is straightforward and can be applied to shifted bands corresponding to stable DNA-protein complexes as well as unstable complexes, which undergo dissociation during electrophoresis.</p

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

Cerebral Dopamine Neurotrophic Factor Diffuses Around the Brainstem and Does Not Undergo Anterograde Transport After Injection to the Substantia Nigra

Cerebral dopamine neurotrophic factor (CDNF) has shown therapeutic potential in rodent and non-human primate models of Parkinson's disease by protecting the dopamine neurons from degeneration and even restoring their phenotype and function. Previously, neurorestorative efficacy of CDNF in the 6-hydroxydopamine (6-OHDA) model of Parkinson's disease as well as diffusion of the protein in the striatum (STR) has been demonstrated and studied. Here, experiments were performed to characterize the diffusion and transport of supra-nigral CDNF in non-lesioned rats. We injected recombinant human CDNF to the substantia nigra (SN) of naive male Wistar rats and analyzed the brains 2, 6, and 24 h after injections. We performed immunohistochemical stainings using an antibody specific to human CDNF and radioactivity measurements after injecting iodinated CDNF. Unlike the previously reported striatonigral retrograde transport seen after striatal injection, active anterograde transport of CDNF to the STR could not be detected after nigral injection. There was, however, clear diffusion of CDNF to the brain areas surrounding the SN, and CDNF colocalized with tyrosine hydroxylase (TH)-positive neurons. Overall, our results provide insight on how CDNF injected to the SN may act in this region of the brain.Peer reviewe

Molecular profile of the rat peri-infarct region four days after stroke: Study with MANF

The peri-infarct region after ischemic stroke is the anatomical location for many of the endogenous recovery processes, and the molecular events in the peri-infarct region remain poorly characterized. In this study, we examine the molecular profile of the peri-infarct region on post-stroke day four, time when reparative processes are ongoing. We used a multiomics approach, involving RNA sequencing, and mass spectrometry-based proteomics and metabolomics to characterize molecular changes in the peri-infarct region. We also took advantage of our previously developed method to express transgenes in the peri-infarct region where self-complementary adeno-associated virus (AAV) vectors were injected into the brain parenchyma on post-stroke day 2. We have previously used this method to show that mesencephalic astrocyte-derived neurotrophic factor (MANF) enhances functional recovery from stroke and recruits phagocytic cells to the peri-infarct region. Here, we first analyzed the effects of stroke to the peri-infarct region on post-stroke day 4 in comparison to sham-operated animals, finding that stroke induced changes in 3345 transcripts, 341 proteins, and 88 metabolites. We found that after stroke genes related to inflammation, proliferation, apoptosis, and regeneration were upregulated, whereas genes encoding neuroactive ligand receptors and calcium-binding proteins were downregulated. In proteomics, we detected upregulation of proteins related to protein synthesis and downregulation of neuronal proteins. Metabolomic studies indicated that in after stroke tissue there is increase in saccharides, sugar phosphates, ceramides and free fatty acids and decrease of adenine, hypoxantine, adenosine and guanosine. We then compared the effects of post-stroke delivery AAV1-MANF delivery to AAV1-eGFP (enhanced green fluorescent protein). MANF administration increased the expression of 77 genes, most of which were related to immune response. In proteomics, MANF administration reduced S100A8 and S100A9 protein levels. In metabolomics, no significant differences between MANF and eGFP treatment were detected, but relative to sham surgery group, most of the changes in lipids were significant in the AAV-eGFP group only. This work describes the molecular profile of the peri-infarct region during recovery from ischemic stroke, and establishes a resource for further stroke studies. These results provide further support for parenchymal MANF as a modulator of phagocytic function.Peer reviewe

Antagonism of peripheral opioid receptors by methylnaltrexone does not prevent morphine tolerance in rats

Opioids are effective analgesics in the management of severe pain. However, tolerance, leading to dose escalation and adverse effects are significant limiting factors in their use. The role of peripheral opioid receptors in analgesia has been discussed especially under inflammatory conditions. The results from pharmacological and conditional knockout studies together do not provide a clear picture of the contribution of peripheral opioid receptors on antinociceptive tolerance and this needs to be evaluated. Therefore, we studied whether the peripherally restricted opioid receptor antagonist, methylnaltrexone (MNTX), could prevent morphine tolerance without attenuating the antinociceptive effect of morphine. Male Sprague-Dawley rats were treated for 7 days with increasing subcutaneous doses of morphine (5-30 mg/kg) and were coadministered saline, MNTX (0.5 or 2 mg/kg), or naltrexone (NTX; 2 mg/kg). Nociception was assessed with tail-flick, hotplate, and von Frey tests. Morphine, MNTX, and NTX concentrations in the plasma, brain, and spinal cord were measured by liquid chromatography-tandem mass spectrometry. In acute coadministration, NTX, but not MNTX, abolished the acute antinociceptive effects of morphine in all nociceptive tests. The antinociceptive tolerance after repeated morphine administration was also prevented by NTX but not by MNTX. MNTX penetrated to the spinal cord and the brain to some extent after repeated administration. The results do not support the use of MNTX for preventing opioid tolerance and also suggest that morphine tolerance is mediated by central rather than peripheral opioid receptors in the rat.Peer reviewe

Post-stroke Intranasal (+)-Naloxone Delivery Reduces Microglial Activation and Improves Behavioral Recovery from Ischemic Injury

Peer reviewe

Synthesis of 7β-hydroxy-8-ketone opioid derivatives with antagonist activity at mu- and delta-opioid receptors

Despite extensive years of research, the direct oxidation of the 7,8-double bond of opioids has so far received little attention and knowledge about the effects of this modification on activity at the different opioid receptors is scarce. We herein report that potassium permanganate supported on iron(II) sulfate heptahydrate can be used as a convenient oxidant in the one-step, heterogeneous conversion of Delta(7.8)-opioids to the corresponding 7 beta-hydroxy-8-ketones. Details of the reaction mechanism are given and the effects of the substituent at position 6 of several opioids on the reaction outcome is discussed. The opioid hydroxy ketones prepared are antagonists at the mu- and delta-opioid receptors. Docking simulations and detailed structure-activity analysis revealed that the presence of the 7 beta-hydroxy-8-ketone functionality in the prepared compounds can be used to gain activity towards the delta opioid receptor. The 7 beta-hydroxy-8-ketones prepared with this method can also be regarded as versatile intermediates for the synthesis of other opioids of interest. (C) 2018 Elsevier Masson SAS. All rights reserved.Peer reviewe

Mitoxantrone, pixantrone, and mitoxantrone (2-hydroxyethyl)piperazine are toll-like receptor 4 antagonists, inhibit NF-κB activation, and decrease TNF-alpha secretion in primary microglia

Toll-like receptor 4 (TLR4) recognizes various endogenous and microbial ligands and is an essential part in the innate immune system. TLR4 signaling initiates transcription factor NF-κB and production of proinflammatory cytokines. TLR4 contributes to the development or progression of various diseases including stroke, neuropathic pain, multiple sclerosis, rheumatoid arthritis and cancer, and better therapeutics are currently sought for these conditions. In this study, a library of 140 000 compounds was virtually screened and a resulting hit-list of 1000 compounds was tested using a cellular reporter system. The topoisomerase II inhibitor mitoxantrone and its analogues pixantrone and mitoxantrone (2-hydroxyethyl)piperazine were identified as inhibitors of TLR4 and NF-κB activation. Mitoxantrone was shown to bind directly to the TLR4, and pixantrone and mitoxantrone (2- hydroxyethyl)piperazine were shown to inhibit the production of proinflammatory cytokines such as tumor necrosis factor alpha (TNFα) in primary microglia. The inhibitory effect on NF-κB activation or on TNFα pro-duction was not mediated through cytotoxity at ≤ 1 μM concentration for pixantrone and mitoxantrone (2- hydroxyethyl)piperazine treated cells, as assessed by ATP counts. This study thus identifies a new mechanism of action for mitoxantrone, pixantrone, and mitoxantrone (2-hydroxyethyl)piperazine through the TLR4.Peer reviewe

MANF and CDNF in the rat model of cortical ischemia

Acute ischemic stroke is among the leading causes of long-term disability in developed countries. Among the contributing factors are our inability to limit brain damage in the acute-phase of stroke and the infrequency of complete spontaneous functional recovery. Effective pharmacological therapies that would protect neurons from ischemia-induced death are not available. Moreover, as of now, there are no proven pharmacological treatments to enhance the limited neurological recovery process in stroke patients. Neurotrophic factors MANF (mesencephalic astrocyte-derived neurotrophic factor) and CDNF (cerebral dopamine neurotrophic factor) are currently among the most promising molecules for the treatment for Parkinson s disease (PD). Namely, both have shown great therapeutic potential in rodent models that mimic the most conspicuous and debilitating symptoms of PD, caused by the progressive degeneration of dopaminergic (DA) neurons in the substantia nigra (SN). However, in these preclinical studies the fate of CDNF and MANF after their therapeutic intracerebral administration has been poorly characterized. While MANF has been shown to protect cortical neurons from death in a rat model of ischemic brain injury, it is unknown whether CDNF has a similar effect. Moreover, the neurorestorative effects CDNF and MANF have been shown to have in animal models of PD raise the question of whether these factors could act similarly after cerebral ischemic damage and enhance functional recovery. In order to have a better understanding of what happens to recombinant human CDNF (rhCDNF) in brain tissue, we studied its distribution, intracellular localization and clearance after infusion into rat brain. We present the pharmacokinetic properties of striatally infused rhCDNF and describe the main intracellular localization patterns after its neuronal uptake. We also present data that after intrastriatal infusion, the rhCDNF found in the SN is almost exclusively localized to the DA neurons, thus showing that it is retrogradely transported. To shed light on the possible neuroprotective potential of CDNF for ischemic stroke, we studied the effect of CDNF in the rat model of ischemic brain injury achieved by temporary unilateral occlusion of the middle cerebral artery. We show that CDNF is protective against cortical ischemic injury when administered as a recombinant protein, but unlike MANF, not when delivered via a CDNF-expressing adeno-associated virus vector (AAV-CDNF). Our findings thus unmask an important difference in MANF and CDNF s capacity for therapeutic action. Using the same rat model of ischemic stroke, we studied the effect of delayed AAV-MANF administration on post-stroke behavioural recovery, taking advantage of the delivery method we have developed in order to target the expression of AAV vector-delivered genes to the peri-infarct area. We report that AAV-MANF, delivered to the peri-infarct area two days after transient ischemia, accelerates the reversal of ischemia-induced behavioral deficits without affecting lesion size. While histological analyses of brain tissue could not point out which cellular process mediates MANF s effect, an unbiased transcriptomics approach implies modulation of the activity of innate immune cells as a possible mechanism. In summary, MANF should be considered as a possible therapeutic agent or a drug target for promoting functional recovery after stroke. In addition, we show that in cultured peripheral neurons MANF needs to be in the endoplasmic reticulum in order to exert its intracellular survival-promoting activity. We also present data on the importance of two sequence motifs of MANF for its intracellular survival-promoting activity and the neuroprotective efficacy that extracellularly applied recombinant human MANF has in the rat model of cortical ischemic injury. In summary, this work has extended the knowledge on MANF and CDNF s capacity for therapeutic action, and the pharmacokinetic and structural properties important for their therapeutic use in further studies.Aivohalvaus on teollisuusmaissa yksi yleisimmistä kuolinsyistä ja yleisin syy heikentyneeseen elämänlaatuun. Valtaosassa aivohalvaustapauksista on häiriön syynä aivovaltimoa tukkiva veritulppa joka aiheuttaa veren ja hapen puutteen, ja lopulta aivokudoksen vaurioitumisen. Nykyiset hoitokeinot verenkierron palauttamiseen ovat rajalliset ja farmakologisia menetelmiä hermosolujen kuoleman estämiseksi ei ole. Lisäksi, potilaiden toimintakyvyn palautuminen aivoinfarktin jälkeen on vain osittaista ja palautumista nopeuttavilla kuntoutusmuodoilla vaikutus on rajallinen. Näistä syistä johtuen aivoinfarktista aiheutuva vammautuminen on hyvin yleistä ja valitettavasti myös pysyvää. MANF- ja CDNFhermokasvutekijöillä on havaittu olevan vahva hermosoluja suojaava ja niiden toimintaa palauttava vaikutus Parkinsonin taudin eläinmalleissa. MANF:lla on lisäksi osoitettu olevan vahva hermosoluja suojaava vaikutus aivoinfarktin rottamallissa, mutta CDNF:n hermosoluja aivoiskemialta suojaavaa vaikutusta ei ole tähän asti tutkittu. Aivokudokseen annosteltujen CDNF ja MANF proteiinien vaikutuksista on hyvin vähän tietoa, mutta pre-kliinisten ja kliinisten jatkotutkimusten suunnittelun kannalta tämä tieto olisi olennaista. Tässä työssä on selvitetty mihin soluihin aivokudokseen annosteltu CDNF pääsee, miten se niissä sijoittuu ja miten nopeasti se aivokudoksesta häviää. Lisäksi olemme osoittaneet, että aivokudokseen annosteltu CDNF kuljetetaan myös keskiaivojen dopaminergisiin hermosoluihin, joiden rappeutuminen aiheuttaa Parkinsonin taudin motoriset oireet. Näytämme aivoinfarktin rottamallia käyttäen, että aivokudokseen annostellulla CDNF proteiinilla on kyky suojata aivokuoren soluja iskemialta. Tämän tuloksen mukaan on aivohalvauksen mahdollisen hoitokeinon löytämiseksi syytä jatkaa CDNF:n suojaavan vaikutuksen tutkimista. Lisäksi olemme havainneet geeniterapiaa käyttäen, että MANF, annosteltuna kaksi päivää aivoiskemian jälkeen, vähentää aivoinfarktin rottamallissa eläinten motorista toimintahäiriötä aivoinfarktia seuraavien viikkojen aikana. Mahdollista MANF-pohjaista hoitokeinoa voisi tämän havainnon perusteella kehittää aivoinfarktipotilaiden toiminnallisen palautumisen nopeuttamiseksi. Lopulta olemme tutkineet MANF proteiinin rakenteellisten aiheiden tärkeyttä hermosoluja suojaavan ominaisuuden kannalta, verraten niiden tarpeellisuutta viljellyissä hermosoluissa ja aivoinfarktin rottamallissa. Tämä työ on lisännyt tietojamme MANF:n ja CDNF:n terapeuttisesta potentiaalista ja näiden proteiinien farmakokineettisistä ja rakenteellisista ominaisuuksista