Glial cell line-derived neurotrophic factor receptor REarranged during transfection agonist supports dopamine neurons in Vitro and enhances dopamine release In Vivo

Background Motor symptoms of Parkinson's disease (PD) are caused by degeneration and progressive loss of nigrostriatal dopamine neurons. Currently, no cure for this disease is available. Existing drugs alleviate PD symptoms but fail to halt neurodegeneration. Glial cell line-derived neurotrophic factor (GDNF) is able to protect and repair dopamine neurons in vitro and in animal models of PD, but the clinical use of GDNF is complicated by its pharmacokinetic properties. The present study aimed to evaluate the neuronal effects of a blood-brain-barrier penetrating small molecule GDNF receptor Rearranged in Transfection agonist, BT13, in the dopamine system. Methods We characterized the ability of BT13 to activate RET in immortalized cells, to support the survival of cultured dopamine neurons, to protect cultured dopamine neurons against neurotoxin-induced cell death, to activate intracellular signaling pathways both in vitro and in vivo, and to regulate dopamine release in the mouse striatum as well as BT13's distribution in the brain. Results BT13 potently activates RET and downstream signaling cascades such as Extracellular Signal Regulated Kinase and AKT in immortalized cells. It supports the survival of cultured dopamine neurons from wild-type but not from RET-knockout mice. BT13 protects cultured dopamine neurons from 6-Hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridinium (MPP+)-induced cell death only if they express RET. In addition, BT13 is absorbed in the brain, activates intracellular signaling cascades in dopamine neurons both in vitro and in vivo, and also stimulates the release of dopamine in the mouse striatum. Conclusion The GDNF receptor RET agonist BT13 demonstrates the potential for further development of novel disease-modifying treatments against PD. (c) 2019 International Parkinson and Movement Disorder SocietyPeer reviewe

Evaluation of health and environmental risks for juvenile tilapia (Oreochromis niloticus) exposed to florfenicol.

Abstract: Intensive fish cultivation has a high incidence of infection, which is often controlled by administering antibiotics. Florfenicol (FF) is one of the two antimicrobial drugs permitted for aquaculture in Brazil. Due to their intensive use, potentially harmful effects on aquatic organisms are of great concern. In this sense, we investigated whether the presence of FF in cultivation water could change the health parameters of Nile tilapia. For this, we evaluated hemoglobin, hematocrit, mean corpuscular hemoglobin (MCHC) concentration, mean corpuscular volume (MCV), total plasma protein (TPP), number of circulating red blood cells and leukocytes, as lipid peroxidation levels, catalase activity and glutathione S-transferase activity of fish exposed to 11.72 mg L-1 of FF in water for 48 h. The fish were divided into two groups: Nile tilapia in water with FF or without FF (control). Exposure to FF in cultivation water for a short period didn\'t change the hematological variables analyzed, but caused changes in liver ROS (Reactive oxygen species) markers of the Nile tilapia, which was revealed by lipid peroxidation levels, catalase activity, and glutathione S-transferase. The 48h exposure period was enough to induce oxidative stress in hepatocytes, causing cellular oxidative damage. Therefore, the antibiotic florfenicol may cause toxicity to organisms and aquatic ecosystems, even at a sublethal concentrations near 1/100 LC50-48h for fish species

GDNF Overexpression from the Native Locus Reveals its Role in the Nigrostriatal Dopaminergic System Function

Peer reviewe

Endogeeninen GDNF dopamiinijärjestelmän säätelijänä : tutkimusvälineenä amfetamiini

Amphetamine and its derivatives are widely used as medicines but also abused as psychostimulant drugs. The most important action of amphetamine in the central nervous system is to release dopamine to the extracellular space which leads to enhanced dopaminergic neurotransmission. Amphetamine also releases serotonin and norepinephrine by similar mechanisms and it affects indirectly other neurotransmitter systems too. It still remains partly unsolved how amphetamine exactly releases monoamines but it is known to have multiple sites of action. Amphetamine is a substrate for dopamine transporter (DAT) and it acts as a competitive inhibitor of the transporter reducing uptake of dopamine. Amphetamine enters the cell mainly through DAT and partly by diffusing through the cell membrane. The drug induces changes in DAT leading to reverse transport of dopamine from the cytoplasm into the synaptic cleft through DAT. Amphetamine is also substrate for vesicular monoamine transporter 2 (VMAT2) preventing the uptake of dopamine into storage vesicles and promoting its release from the vesicles to cytoplasm. Additionally, amphetamine inhibits monoamine oxidase (MAO), enzyme which degrades monoamines. It also enhances dopamine synthesis and according to recent studies amphetamine augments exocytotic dopamine release. Drug addiction is a chronic disorder related to structural and functional adaptive changes of neurons, called neuronal plasticity. GDNF (glial cell line-derived neurotrophic factor) is one of the many molecules regulating plasticity. It is especially important to the dopaminergic system and some investigations have suggested that it has potential as a protective agent against addiction. The aim of this study was to investigate how the overexpression of endogenic GDNF affects dopaminergic system and how it changes drug responses. A hypermorphic mouse strain (GDNFh), which is overexpressing physiological GDNF, was used. Their wild-type littermates were used as controls. Using brain microdialysis it was measured how the extracellular dopamine concentration changes in striatum and nucleus accumbens (NAcc) after amphetamine stimulation. Amphetamine was administered straight to the brain through the microdialysis probe. Microdialysis was performed on days 1 and 4, and on days 2 and 3 the mice were given amphetamine intraperitoneally. This was done to find out if the response to amphetamine changed after repeated dosing. In addition to these experiments, the biological activity of three small-molecule GDNF mimetics in intact brains was tested by microdialysis. On the first day amphetamine increased striatal dopamine output more in the heterozygous GDNFh mouse than in the wild-type mice. This stronger reaction to amphetamine may be explained by the enhanced activity of DAT in the GDNFh-het mice leading to higher intracellular amphetamine concentration. Also the striatal dopamine levels are increased in the GDNFh-het. On the fourth day no differences were detected between the genotypes. In the NAcc no significant difference was found between the genotypes. Instead in NAcc amphetamine caused a smaller increase in the dopamine output on day 4 than on day 1 in both genotypes suggesting that tolerance was developed. These results confirm that endogenic GDNF has a remarkable role in the regulation of the dopamine system and hence addiction but further investigations are needed to clarify its versatile actions. The small-molecule GDNF mimetics increased striatal dopamine output thus showing biological activity and encouraging to further investigations.Amfetamiinia ja sen monia johdannaisia käytetään sekä lääkkeinä että stimuloivina huumausaineina. Amfetamiinin merkittävin vaikutus keskushermostossa on voimakas dopamiinin vapautuminen ulos soluista, mikä johtaa dopaminergisen hermovälityksen tehostumiseen. Amfetamiini vaikuttaa samoin myös noradrenaliinin ja serotoniin vapautumiseen, ja epäsuorasti muihinkin välittäjäainejärjestelmiin. Amfetamiinin tarkka vaikutusmekanismi ei ole edelleenkään täysin selvä, mutta sen tiedetään vaikuttavan moniin solunosiin ja hermovälityksen säätelymekanismeihin. mfetamiini on dopamiinitransportterin (DAT) substraatti, ja toimii sen kilpailevana inhibiittorina vähentäen näin dopamiinin takaisinottoa soluihin. Kulkeuduttuaan soluun amfetamiini saa aikaan muutoksia DAT:n toiminnassa, jolloin dopamiinia alkaakin kulkeutua DAT:n kautta ulos solusta. Amfetamiini on myös vesikulaarisen monoamiinitransportteri-2:n (VMAT2) substraatti ja kykenee vapauttamaan dopamiinia solunsisäisistä varastorakkuloista lisäten vapaan dopamiinin pitoisuutta solussa. Lisäksi amfetamiini estää dopamiinin metaboliaa monoamiinioksidaasin (MAO) kautta, lisää dopamiinin synteesiä sekä uusimpien tutkimusten mukaan lisää dopamiinihermosolujen aktiivisuutta ja dopamiinin eksosytoottista vapautumista. Huumausaineriippuvuus on krooninen sairaus, johon liittyy hermoston plastisia muutoksia. GDNF eli gliasolulinjaperäinen hermokasvutekijä on yksi, erityisesti dopamiinijärjestelmän kannalta tärkeä, plastisuutta säätelevä molekyyli, jolla on toivottu olevan jopa riippuvuudelta suojaavia ominaisuuksia. Tässä erikoistyössä tutkittiin, miten endogeenisen GDNF:n ylituotanto vaikuttaa dopamiinijärjestelmään ja erityisesti, millaisina muutoksina tämä näkyy reagoinnissa huumausaineelle. Tutkimuksessa käytettiin endogeenistä GDNF:ää normaalia enemmän ilmentävää hypermorfista hiirikantaa (GDNFh), ja verrokkeina samojen poikueiden villityypin hiiriä. Kokeissa mitattiin mikrodialyysin avulla, miten striatumin ja accumbens-tumakkeen solunulkoinen dopamiinipitoisuus muuttuu amfetamiinistimulaation seurauksena. Amfetamiini annosteltiin suoraan aivoihin mikrodialyysikoettimen kautta. Mikrodialyysi tehtiin kullekin eläimelle koepäivinä 1 ja 4. Välipäivinä hiirille annosteltiin amfetamiinia intraperitoneaalisesti. Tarkoituksena oli selvittää, muuttuuko aivojen dopamiinivaste toistetun amfetamiinistimulaation seurauksena. Lisäksi erikoistyössä tutkittiin mikrodialyysin avulla kolmen pienmolekyylisen GDNF:ää matkivan yhdisteen biologista aktiivisuutta intakteissa aivoissa. GDNFh-heterotsygooteilla solunulkoinen dopamiinipitoisuus nousi striatumissa ensimmäisenä koepäivänä enemmän kuin villityypin hiirillä, eli ne reagoivat amfetamiinille villityypin hiiriä voimakkaammin. DAT-aktiivisuuden on todettu olevan näillä hypermorfisilla hiirillä normaalia suurempi, jolloin amfetamiini kulkeutuu soluihin tehokkaammin. Lisäksi dopamiinin kudospitoisuus striatumissa on korkeampi kuin villityypin hiirillä, joten suuremmat dopamiinivarastot mahdollistavat runsaamman vapautumisen. Neljäntenä koepäivänä genotyyppien välistä eroa ei kuitenkaan enää havaittu. Accumbens-tumakkeen mikrodialyysissä ei havaittu tilastollisesti merkitsevää eroa genotyyppien välillä. Tällä aivoalueella sen sijaan vaikutti kehittyvän toleranssia amfetamiinille, sillä neljäntenä koepäivänä dopamiinia vapautui molemmilla genotyypeillä vähemmän kuin ensimmäisenä. Kokeiden tulokset vahvistavat GDNF:llä olevan merkittävän roolin dopamiinijärjestelmän ja riippuvuuden säätelyssä. Riippuvuudelta suojaavaa vaikutusta ei kuitenkaan havaittu. Tutkitut pienmolekyyliset GDNF-mimeetit osoittivat biologista aktiivisuutta antaen jatkotutkimuksille aihetta

Correction: GDNF Overexpression from the Native Locus Reveals its Role in the Nigrostriatal Dopaminergic System Function.

[This corrects the article DOI: 10.1371/journal.pgen.1005710.]

Identification of <i>Gdnf</i>-regulating miRNAs.

<p><b>(A)</b> Putative miR binding sites cluster within the conserved areas of the <i>Gdnf</i> 3’UTR. The miRNAs underlined with red bars were co-immunoprecipitated with <i>Gdnf</i> mRNA in a genome-wide screen of a mouse brain tissue [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005710#pgen.1005710.ref034" target="_blank">34</a>]. Predicted putative RNA-binding protein (RBP) sites are indicated with triangles. Source: Blast, TragetScan, AREsite, and <a href="http://servers.binf.ku.dk/antar/" target="_blank">http://servers.binf.ku.dk/antar/</a>; Hs, human; Mm, mouse. <b>(B)</b> Luciferase expression from a Ren-<i>Gdnf</i> 3’UTR construct after co-transfection with the indicated pre-miRNAs in HEK293 cells. scr1 and scr2 are scrambled pre-miRNA controls; N = 3 experiments/miRNA with 3–5 biological repeats/miRNA/experiment. <b>(C)</b> Predicted binding sites (ddG<-3) for miR-9 (green), miR-96 (blue), miR-133a/b (orange), and miR-146a (red) based on “predict microRNA targets” analysis (<a href="http://genie.weizmann.ac.il/" target="_blank">http://genie.weizmann.ac.il/</a>). Boxes indicate the mutated miRNA-binding sites in each mutant. Note that miR-9/96/133m contains overlapping sites for miR-9, miR-96, and miR-133, all of which were mutated in this construct. <b>(D)</b> Luciferase assay of miR-9, miR-96, miR-133a, and miR-146a mutants (mutated sequences indicated with boxes in panel C; scr1 is a scrambled pre-miRNA control; N = 2 experiments/miRNA with 3 biological repeats/miRNA/experiment. <b>(E-F)</b> Expression of endogenous <i>GDNF</i> mRNA (E) and GDNF protein (F) is inhibited in U87 cells by co-transfection with the indicated pre-miRNAs; N = 3–4 experiments with 2–3 biological replicates/experiment. <b>(G)</b> Adenoviral transduction of Cre—but not GFP—in homozygous “floxed” Dicer-1^F/F primary cortical neurons increases endogenous GDNF protein levels; N = 2 experiments with 3–4 mice/group. <b>(H)</b> AAV-based constructs encoding shRNAs against miR-9, miR-96, and miR-146a, as well as siRNA against Dicer, increase endogenous <i>GDNF</i> expression in HEK293 cells; N = 5 experiments/construct (except for the shRNA against miR-9, where N = 2 experiments/construct) with 2 biological repeats per experiment. Ad, adenovirus; m, mutant.</p

<i>Gdnf</i> expression is increased in cells that normally express <i>Gdnf</i>.

<p><b>(A)</b><i>In situ</i> hybridization showing <i>Gdnf</i> mRNA expression in thalamic nuclei at P7.5 (white arrowheads). <b>(B)</b><i>In situ</i> hybridization showing <i>Gdnf</i> mRNA expression in Clarke’s column in the thoracic part of the spinal cord at P12.5 (white arrowheads). <b>(C)</b><i>Gdnf</i> mRNA-positive cells (black dots) in the whole striatum (upper panel); a magnified view of <i>Gdnf</i> mRNA-positive cells (arrowheads) is shown in the lower panel. <b>(D)</b> Representative images of <i>Pvalb (PV</i>, blue) and <i>Gdnf</i> (red) mRNA in the striatum of 3-month-old mice detected using RNAscope. <b>(E)</b> Summary of <i>PV</i>-positive only, <i>Gdnf</i>-positive only, and double-positive cells in striatal slices obtained from <i>Gdnf</i>^<i>wt/wt</i> and <i>Gdnf</i>^{<i>wt/hyper</i>} mice; N = 5 animals/group. <b>(F)</b> QPCR analysis of <i>Gdnf</i> mRNA levels in the indicated brain regions in P7.5 mice; N = 6–8 mice/group. <b>(G)</b> QPCR analysis of <i>Gdnf</i> mRNA levels in the indicated brain regions of adult mice; N = 4–8 mice/group. <b>(H)</b> ELISA analysis of GDNF protein levels in the dorsal striatum of adult mice; N = 6–8 mice/group. Scale bars: A, 150 μm; B, 200 μm; C (upper panels), 500 μm; C (lower panels), 50 μm; D, 10 μm. Abbreviations: dSTR, dorsal striatum; OB, olfactory bulb; Hyp, hypothalamus; PFC, prefrontal cortex; vSTR, ventral striatum; Hip, hippocampus; vMB, ventral midbrain; CB, cerebellum; RN, dorsal raphe nucleus; SC, spinal cord; SN, substantia nigra; VTA, ventral tegmental area; PV, parvalbumin; m, months; P, postnatal day.</p

Increased endogenous GDNF expression affects the development and function of the nigrostriatal dopaminergic system.

<p><b>(A)</b> Levels of phosphorylated ERK2 at P7.5 in the striatum of <i>Gdnf</i>^<i>wt/wt</i>, <i>Gdnf</i>^{<i>wt/hyper</i>} and <i>Gdnf</i>^{<i>hyper/hyper</i>} mice. N = 5 mice/group; ERK was used for normalization. <b>(B)</b> HPLC analysis of DA levels in the rostral brain; N = 5–8 mice/group (F = 7.44, P = 0.016). <b>(C)</b> Quantification of tyrosine hydroxylase (TH)-positive (a marker of DA neurons) cells in the SNpc; N = 6–8 mice/group (F = 7.44, P = 0.0048). <b>(D)</b> HPLC analysis of DA levels in the dSTR; N = 11 for <i>Gdnf</i>^<i>wt/wt</i>, 8 for <i>Gdnf</i>^{<i>wt/hyper</i>} mice/group (P = 0.000164). HPLC analysis of DA levels in the dorsal striatum of <i>Gdnf 3’UTR</i>^<i>wt/wt</i> and <i>Gdnf</i>^<i>wt/KO</i> mice; N = 6 mice/group. <b>(E-F)</b> The number of TH-positive (E; N = 8 <i>Gdnf</i>^wt/wt, N = 7 <i>Gdnf</i>^wt/hyper; P = 0.025) and VMAT2-positive neurons (F; N = 7 <i>Gdnf</i>^wt/wt, N = 7 <i>Gdnf</i>^wt/hyper; P = 0.016) in the SNpc. <b>(G)</b> The number of DAT+ varicosities (N = 9 <i>Gdnf</i>^wt/wt, N = 7 <i>Gdnf</i>^wt/hyper; P = 0.042) in the dSTR. <b>(H-K)</b> Cyclic voltammetry analysis of acute striatal slices (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005710#pgen.1005710.s008" target="_blank">S3J Fig</a>); N = 5–7 mice/group with 1–3 slices per mouse. <b>(H)</b> DA release in response to electrical stimulation [two-way repeated measures ANOVA, F (1,29) = 5.866]; <b>(I)</b> Averaged traces of DA events. <b>(J)</b> Short-term depression of striatal DA release after prior DA exocytosis, shown as percent of the first DA release. <b>(K)</b> The ratio of DA release after a single stimulus and after a 5 pulse burst at 20Hz. <b>(L)</b><i>In vivo</i> amperometry following intrastriatal DA injection reveals that dopamine transporter (DAT) activity in <i>Gdnf</i>^{<i>wt/hyper</i>} mice is dependent on the concentration of DA; N = 4 mice/group (F = 47.931). <b>(M)</b> Locomotor activity after an injection of amphetamine (1 mg/kg, i.p.); N = 9–10 mice/group (F = 4.386, P = 0.04). <b>(N)</b><i>In vivo</i> microdialysis analysis of extracellular striatal DA levels; amphetamine was applied as indicated by the horizontal bar; N = 9 mice/group. <b>(O)</b> Cyclic voltammetry analysis shows that amphetamine (5 μM) depletes stimulated DA release faster in the striata of <i>Gdnf</i>^{<i>wt/hyper</i>} mice compared to <i>Gdnf</i>^<i>wt/wt</i> mice; two-way repeated-measures ANOVA reveals an effect of time (P<0.0001) and genotype (P = 0.031), as well as an interaction between time and genotype (P = 0.049); N = 6 mice/group with 1–3 slices per mouse. <b>(P-Q)</b> Analysis of a 6-OHDA induced PD model. <b>(P)</b> Quantification of DA in the dSTR 2 weeks after striatal 6-OHDA injection, relative to the intact side (N = 12 <i>Gdnf</i>^wt/wt; N = 10 <i>Gdnf</i>^wt/hyper), (F = 40.62, P = 0.00549, Students t-test). The intact and lesioned side differed significantly (P = 2.71×10⁻¹⁵). <b>(Q)</b> Quantification of TH-positive neurons in the SNpc 2 weeks after striatal 6-OHDA injection, relative to the intact side, (F = 7.04, P = 0.0143, Students t-test). The intact and lesioned side differed significantly (P = 3.00×10⁻¹¹). <b>(R-T)</b> Analysis of a lactacystin-induced PD model. <b>(R)</b> The percentage of sugar pellet retrievals from the contralateral side in the corridor test; N = 5–7 mice/group (F = 6.087, P = 0.033). <b>(S)</b> Quantification of DA, DOPAC, and HVA in the dSTR 5 weeks after supranigral lactacystin injection, relative to the intact side; N = 5 <i>Gdnf</i>^<i>wt/wt</i>, N = 7 <i>Gdnf</i>^{<i>wt/hyper</i>}; P = 0.046 for DA, P = 0.015 for DOPAC, P = 0.011 for HVA. The intact and lesioned side differed significantly; P = 0.00016 for DA, P = 0.015 for DOPAC, P = 0.010 for HVA. <b>(T)</b> Quantification of TH-positive neurons in the SNpc 5 weeks after lactacystin injection, relative to the intact side; N = 4 <i>Gdnf</i>^<i>wt/wt</i>, N = 7 <i>Gdnf</i>^{<i>wt/hyper</i>}; P = 0.236. The intact and lesioned side differed significantly (P = 0.00029). <b>(U-W)</b> Evaluation of side effects associated with intracranial ectopic GDNF expression. <b>(U)</b> Spontaneous locomotor activity in an open field; N = 31–34 mice/group. <b>(V)</b> Food intake by adult mice during a 72-hour period; N = 10 mice/group. <b>(W)</b> Body weight of adult mice; N = 9–34 mice/group. Abbreviations: DA, dopamine; DOPAC, 3,4-dihydroxyphenylacetic acid; HVA, homovanillic acid; dSTR, dorsal striatum; SNpc, substantia nigra pars compacta.</p