GDNF sen reseptori GFRa1 hermoston kehittymisessä ja toiminnassa

Neurotrophic factor glial cell line-derived neurotrophic factor (GDNF), its co-receptor GDNF family receptor alpha 1 (GFRa1), and signaling receptor RET tyrosine kinase are essential to enteric nervous system (ENS) development; mice knockout for Gdnf, Gfra1 or Ret lack the whole ENS distal to the stomach. These Gdnf/Gfra1/Ret knockout mice die at birth because of lack of ENS and kidneys hindering analysis of postnatal function of those proteins. Transgenic overexpression in animal models on the other hand relates to loss of physiological spatiotemporal regulation of gene expression. These two bottlenecks have hindered the understanding of the role and therapeutic potential of GDNF/GFRa1/RET signaling in congenital diseases, such as Hirschsprung’s disease, and degenerative neurological diseases, such as Parkinson’sdisease. To tackle at least some of these problems, we have generated and characterized new mouse models with either increased or decreased gene expression dose - from the gene’s endogenous locus and limited to naturally expressing cells. Novel mouse models with increased expression were generated by editing 3’ untranslated region (3’UTR) of the Gdnf gene in such a way that the edited 3’UTR lacks binding sites for negative regulators such as microRNAs. By preventing the posttranscriptional downregulation via the 3’UTR we were able to achieve Gdnf overexpression from the endogenous locus limited to the naturally Gdnf expressing cells. We showed that 3’UTR replacement or 3’UTR editing results in increased GDNF levels in the brain and kidneys, maintaining the spatiotemporal expression pattern with positive effects on the dopaminergic system and negative effects on the kidney size and urogenital tract development. We also found that 3’UTR regulates GDNF levels in the gastrointestinal tract and that 3’UTR controlled GDNF levels determine proportions of neuronal subtypes in the ENS. More specifically, inactivation of negative Gdnf 3’UTR regulation enhances nitrergic and cholinergic neuron numbers, and leads to increased gastrointestinal transit time, increased stool pellet size, and increased stool water content. In congenital Hirschsprung’s disease (HSCR) patients, on the other hand, lack of ENS ganglia in the distal gut leads to constipation and megacolon. Even though RET mutations are the most common cause of Hirschsprung’s disease, no causative mutations in GFRa1 are known. However, one study reported low GFRa1 mRNA levels in some HSCR patients, suggesting that perhaps instead of being caused by mutations some HSCR cases could be triggered by reduced GFRa1 levels. Complicating the establishment of disease etiology in GDNF/GFRa1/RET related HSCR, postnatal viable HSCR mouse models with a defect in GDNF/GFRa1/RET signaling are not available. Here, we generated GFRa1 hypomorphic mice by insertion of a selectable marker gene in opposite transcriptional direction after the Gfra1 exon 6. Insertion of an expression cassette in the opposite transcriptional direction often leads to under-expression from the other strand, resulting in hypomorph allele. We showed that a 70-80 % reduction in GFRa1 levels in mice resulted in congenital Hirschsprung’s disease and associated enterocolitis phenotype with 100 % penetrance. We were also able to shed light in the chronology of events in the pathogenesis of Hirschsprung’s disease associated enterocolitis: first goblet cell dysplasia accompanied by an abnormal mucin phenotype is proceeding into epithelial damage, later followed by microbial enterocyte adherence and bacterial tissue invasion which likely leads to death by sepsis. Previously all those features had been described in patients but the sequence of events had remained unclear. Our results suggest that dysregulation of GDNF or GFRa1 levels by epigenetic mechanisms may play a role in normal and pathogenic development of the enteric nervous system.Gliasolulinjaperäinen hermokasvutekijä (GDNF), sen ligandia sitova reseptori, GDNF perheen reseptori alfa1 (GFRa1), ja signaalin välittävä reseptori RET-tyrosiinikinaasi ovat kaikki välttämättömiäruuansulatuskanavan enteerisen hermoston kehittymiselle. Gdnf-, Gfra1- tai Ret-poistogeenisilta hiiriltä puuttuu koko enteerinen hermosto mahalaukusta nähden distaalisesti. Näiden poistogeenisten hiirten enteeristä hermostoa ei siis pystytä tutkimaan syntymän jälkeen, koska poikaset eivät ole elinkykyisiä puuttuvien munuaisten ja enteerisen hermoston takia. Lisäksi transgeeniseen yliekspressioon liittyyongelmia, jotka johtuvat spatiotemporaalisen geenin ilmentymisen säätelyn puuttumisesta. Edellä mainitut kaksi eläinmallien yleistä ongelmaa ovat haitanneet GDNF/GFRa1/RET viestinvälityksen tutkimista synnynnäisten sairauksien, kuten Hirschsprungin taudin, ja hermorappeumasairauksien, kuten Parkinsonin taudin, tutkimuksessa. Aiempiin eläinmalleihin liittyvien ongelmien ratkaisemiseksi olemme kehittäneet ja karakterisoineet uusia hiirimalleja, jotka joko yli- tai ali-ilmentävät tutkittavaa geeniä endogeenisestä lokuksesta, jolloin geenin ilmentyminen rajoittuu sitä luontaisesti tuottaviin soluihin. Geeniä yli-ilmentävät uudet hiirimallit tuotettiin muokkaamalla Gdnf geenin 3′-ei-transloitua aluetta (3’UTR) siten, että geenin ilmentymistä vähentävät tekijät, kuten mikroRNA:t, eivät voi enää siihen sitoutua. Kun transkription jälkeistä negatiivista säätelyä estettiin, pystyttiin GDNF:n yli-ilmentyminen rajoittamaan sitä luontaisesti tuottaviin soluihin. Osoitimme 3’UTR:n korvaamisen tai sen muokkaamisen johtavan lisääntyneeseen GDNF-tasoon aivoissa ja munuaisissa siten, että spatiotemporaalinen ilmentyminen säilyy vastaavana kuin villin tyypin hiirillä. Suuremmalla GDNF-tasolla on positiivisia vaikutuksia aivojen dopaminergiseen järjestelmään ja negatiivisia vaikutuksia munuaisten ja lisääntymiselinten kehittymiseen. Lisäksi havaitsimme, että 3’UTR säätelee GDNF-tasoa ruuansulatuskanavassa ja vaikuttaa enteerisen hermoston hermosolutyyppien suhteisiin. Tarkemmin sanottuna 3’UTR:n kautta tapahtuvan negatiivisen säätelyn estäminen lisää etenkin typpioksidia mutta myös asetyylikoliinia välittäjäaineena käyttävän hermotuksen määrää ja vaikuttaa luultavasti sitä kautta ruuansulatuskanavan toimintaan hidastaen suolen läpikulkuaikaa, suurentaen ulostepellettien kokoa ja lisäten ulosteen vesipitoisuutta. Geeniä ali-ilmentävää eläinmallia käytettiin mallintamaan synnynnäistä Hirschsprungin tautia. Hirschsprungin tautia sairastavilta potilailta puuttuvat suoliston loppuosan enteerisen hermoston gangliot. Tämä aiheuttaa ummetusta ja johtaa paksusuolen laajentumiseen, megakooloniin. Vaikka RET-mutaatiot ovat yleisimpiä Hirschsprungin taudin aiheuttajia, ei tällaisia tautia aiheuttavia mutaatioita ole löydetty GFRa1-geenistä, vaikkakin yhdessä tutkimuksessa on raportoitu Hirscsprungin taudin potilailla vähentyneestä GFRa1-mRNA-tasosta. Tämä viittaa siihen, että mutaatioiden sijaan vähentynyt GFRa1:n määrä voi osalla potilaista osaltaan vaikuttaa Hirschsprungin taudin patogeneesiin. GDNF/GFRa1/RETsignaloinnin roolin tutkimista Hirschsprungin taudissa on vaikeuttanut elinkykyisten eläinmallien puuttuminen. Tässä tutkimuksessa kehitettiin GFRa1-hypomorfinen hiirimalli siten, että Gfra1 geenin 6. eksonin jälkeen sijoitettiin selektiivinen markkerigeeni vastakkaiseen suuntaan transkriptioon nähden. Ekspressiokasetin lisääminen tällä tavalla johtaa usein vähentyneeseen ilmentymiseen toisesta juosteesta eli hypomorfiseen alleeliin. Osoitimme, että 70-80 % vähennys GFRa1-tasossa aiheuttaa hiirille 100 % penetranssilla fenotyypin, joka vastaa synnynnäistä Hirschsprungin tautia ja siihen liittyvää enterokoliittia. Pystyimme myös selvittämään Hirschsprungin tautiin liittyvän enterokoliitin patogeneesin aikajärjestystä: ensin pikarisolujen dysplasia, johon liittyvät epänormaalit musiinit, sitten etenevä epiteelivaurio joiden jälkeen mikrobit voivat päästä kiinnittymään enterosyytteihin ja etenemään kudokseen, joka taas voiaiheuttaa sepsiksen ja kuoleman. Nämä kaikki on kuvattu potilailla, mutta tähän asti järjestys on ollut epäselvä. Tuloksiemme perusteella GDNF tai GFRa1 geenien ilmentymisen epigeneettinen säätely voi liittyä sekä enteerisen hermoston normaaliin että tauteihin liittyvään kehittymiseen

Gfra1 Underexpression Causes Hirschsprung's Disease and Associated Enterocolitis in Mice

BACKGROUND & AIMS: RET, the receptor for the glial cell line-derived neurotrophic factor (GDNF) family ligands, is the most frequently mutated gene in congenital aganglionic megacolon or Hirschsprung's disease (HSCR). The leading cause of mortality in HSCR is HSCR-associated enterocolitis (HAEC), which is characterized by altered mucin composition, mucin retention, bacterial adhesion to enterocytes, and epithelial damage, although the order of these events is obscure. In mice, loss of GDNF signaling leads to a severely underdeveloped enteric nervous system and neonatally fatal kidney agenesis, thereby precluding the use of these mice for modeling postnatal HSCR and HAEC. Our aim was to generate a postnatally viable mouse model for HSCR/HAEC and analyze HAEC etiology. METHODS: GDNF family receptor alpha-1 (GFRa1) hypomorphic mice were generated by placing a selectable marker gene in the sixth intron of the Gfra1 locus using gene targeting in mouse embryonic stem cells. RESULTS: We report that 70%-80% reduction in GDNF co-receptor GFRa1 expression levels in mice results in HSCR and HAEC, leading to death within the first 25 postnatal days. These mice mirror the disease progression and histopathologic findings in children with untreated HSCR/HAEC. CONCLUSIONS: In GFRa1 hypomorphic mice, HAEC proceeds from goblet cell dysplasia, with abnormal mucin production and retention, to epithelial damage. Microbial enterocyte adherence and tissue invasion are late events and therefore unlikely to be the primary cause of HAEC. These results suggest that goblet cells may be a potential target for preventative treatment and that reduced expression of GFRa1 may contribute to HSCR susceptibility.Peer reviewe

Elevated endogenous GDNF induces altered dopamine signalling in mice and correlates with clinical severity in schizophrenia

Presynaptic increase in striatal dopamine is the primary dopaminergic abnormality in schizophrenia, but the underlying mechanisms are not understood. Here, we hypothesized that increased expression of endogenous GDNF could induce dopaminergic abnormalities that resemble those seen in schizophrenia. To test the impact of GDNF elevation, without inducing adverse effects caused by ectopic overexpression, we developed a novel in vivo approach to conditionally increase endogenous GDNF expression. We found that a 2-3-fold increase in endogenous GDNF in the brain was sufficient to induce molecular, cellular, and functional changes in dopamine signalling in the striatum and prefrontal cortex, including increased striatal presynaptic dopamine levels and reduction of dopamine in prefrontal cortex. Mechanistically, we identified adenosine A2a receptor (A(2A)R), a G-protein coupled receptor that modulates dopaminergic signalling, as a possible mediator of GDNF-driven dopaminergic abnormalities. We further showed that pharmacological inhibition of A(2A)R with istradefylline partially normalised striatal GDNF and striatal and cortical dopamine levels in mice. Lastly, we found that GDNF levels are increased in the cerebrospinal fluid of first episode psychosis patients, and in post-mortem striatum of schizophrenia patients. Our results reveal a possible contributor for increased striatal dopamine signalling in a subgroup of schizophrenia patients and suggest that GDNF-A(2A)R crosstalk may regulate dopamine function in a therapeutically targetable manner.</p

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.]

Elevated endogenous GDNF induces altered dopamine signalling in mice and correlates with clinical severity in schizophrenia.

Presynaptic increase in striatal dopamine is the primary dopaminergic abnormality in schizophrenia, but the underlying mechanisms are not understood. Here, we hypothesized that increased expression of endogenous GDNF could induce dopaminergic abnormalities that resemble those seen in schizophrenia. To test the impact of GDNF elevation, without inducing adverse effects caused by ectopic overexpression, we developed a novel in vivo approach to conditionally increase endogenous GDNF expression. We found that a 2-3-fold increase in endogenous GDNF in the brain was sufficient to induce molecular, cellular, and functional changes in dopamine signalling in the striatum and prefrontal cortex, including increased striatal presynaptic dopamine levels and reduction of dopamine in prefrontal cortex. Mechanistically, we identified adenosine A2a receptor (A2AR), a G-protein coupled receptor that modulates dopaminergic signalling, as a possible mediator of GDNF-driven dopaminergic abnormalities. We further showed that pharmacological inhibition of A2AR with istradefylline partially normalised striatal GDNF and striatal and cortical dopamine levels in mice. Lastly, we found that GDNF levels are increased in the cerebrospinal fluid of first episode psychosis patients, and in post-mortem striatum of schizophrenia patients. Our results reveal a possible contributor for increased striatal dopamine signalling in a subgroup of schizophrenia patients and suggest that GDNF-A2AR crosstalk may regulate dopamine function in a therapeutically targetable manner

<i>Gdnf</i> levels are a critical determinant of embryonic renal growth and morphogenesis.

<p><b>(A)</b> Schematic representation of <i>Gdnf</i> expression (blue) in the mouse kidney at E11.5. <b>(B)</b> Representative image of <i>in situ</i> hybridization of <i>Gdnf</i> mRNA (blue) in the urogenital tract of E11.5 mice. N = 4 mice/group. <b>(C-E)</b><i>Gdnf</i> mRNA [E14.5 (C) and E18.5 (D)] and protein [E18.5; (E)] levels in the kidney measured using QPCR (C and D) and ELISA (E). N = 2–10 mice/group. <b>(F)</b> Representative image of kidneys obtained from P7.5 mice. <b>(G)</b> Representative image of hematoxylin-and-eosin‒stained sections from E18.5 kidneys. The renal cortex is indicated with a yellow bar, the medulla is indicated with a green bar, and collecting duct cysts are indicated with yellow arrowheads. <b>(H)</b> At the time of renal differentiation initiation (E11.75), a wild-type kidney (left) contains a typical UB branching pattern with an interim stalk (red arrowhead), elongated ureter stalk (white arrowhead), and locally enlarged UB tips (arrows). In contrast, a kidney from a <i>Gdnf</i>^{<i>hyper/hyper</i>} embryo (right) contains one large UB that appears bumpy (arrows), lacks an interim stalk, and lacks normal elongation of the UB (white arrowhead). <b>(I)</b> Images of a wild-type (left) and <i>Gdnf</i>^{<i>hyper/hyper</i>} (right) kidney at E13.5; the kidney from the <i>Gdnf</i>^{<i>hyper/hyper</i>} embryo is smaller in size, has enlarged ureteric buds (arrows), and shortened stalks (arrow head). For F-I, N = 3–20 mice/group. Scale bars: B, 10 μm; F, 1 mm; G, 300 μm H, 50 μm; I, 100 μm. Abbreviations: E, embryonic day; MM, metanephric mesenchyme; P, postnatal day; UB, ureteric bud. In this and subsequent figures, all summary data are presented as the mean ± SEM; *P<0.05, **P<0.01, and ***P<0.001; Student’s <i>t</i>-test, unless noted otherwise.</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

<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

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