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A MicroRNA Feedback Circuit in Midbrain Dopamine Neurons

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Science  31 Aug 2007:
Vol. 317, Issue 5842, pp. 1220-1224
DOI: 10.1126/science.1140481

Abstract

MicroRNAs (miRNAs) are evolutionarily conserved, 18- to 25-nucleotide, non–protein coding transcripts that posttranscriptionally regulate gene expression during development. miRNAs also occur in postmitotic cells, such as neurons in the mammalian central nervous system, but their function is less well characterized. We investigated the role of miRNAs in mammalian midbrain dopaminergic neurons (DNs). We identified a miRNA, miR-133b, that is specifically expressed in midbrain DNs and is deficient in midbrain tissue from patients with Parkinson's disease. miR-133b regulates the maturation and function of midbrain DNs within a negative feedback circuit that includes the paired-like homeodomain transcription factor Pitx3. We propose a role for this feedback circuit in the fine-tuning of dopaminergic behaviors such as locomotion.

MicroRNAs (miRNAs) are derived from long primary transcripts through sequential processing by the Drosha ribonuclease and the Dicer enzyme (1). In the context of an RNA-induced silencing complex, miRNAs guide the cleavage of target mRNAs and/or inhibit their translation. miRNAs regulate developmental cell fate decisions in the nervous system and elsewhere (2).

Midbrain dopaminergic neurons (DNs) play a central role in complex behaviors such as reward and addiction, and these cells are lost in Parkinson's disease. A number of transcription factors have been identified that regulate midbrain DN development, function, and survival (3). However, the role of posttranscriptional mechanisms is unknown. To establish a function for miRNAs, we first used an in vitro model system: the differentiation of murine embryonic stem (ES) cells into DNs (4, 5). An ES cell line was obtained that expresses Dicer enzyme conditionally [containing LoxP recombinase sites that flank both chromosomal copies of the Dicer gene, herein termed floxed Dicer (6)]. Introduction of Cre recombinase into these cells by lentiviral transduction leads to the deletion of Dicer in nearly 100% of cells (fig. S1A).

ES cultures were differentiated to a midbrain DN phenotype using the embryoid body (EB) protocol (fig. S1B) (5, 7). Cre-mediated deletion of Dicer at a stage when postmitotic DNs first arise led to a nearly complete loss of DN accumulation, as quantified by the expression of markers including tyrosine hydroxylase (TH) (Fig. 1A). Other mature neuronal classes, including GABAergic neurons, were reduced in these cultures to a lesser extent (by approximately 50%), as were cells expressing TujI, an early general neuronal marker that first appears at the neural precursor stage of EB differentiation. The Dicer deletion phenotype is partially rescued by transfection of RNA species with low molecular weight (but not high molecular weight) derived from embryonic mouse midbrain, consistent with a model in which miRNAs play a role in midbrain DN terminal differentiation and survival (Fig. 1B and fig. S1D).

Fig. 1.

Dicer is essential for the midbrain DN phenotype. (A) Floxed Dicer conditional knockout ES cultures (flx/flx) were differentiated by the EB method, transduced with Cre or control green fluorescent protein (GFP) lentivirus, and analyzed by immunostaining with antibodies specific for TH (red), TujI (green), and GABA (blue). Cultures transduced with a lentiviral Cre vector (vCre) but not control GFP lentivirus (vGFP) were essentially devoid of TH+ neurons, whereas TujI+ and GABA+ cells were reduced by approximately 40 to 60%. (n = 3 independent samples per group). Scale bar, 100 μm. Data represent mean ± SEM; analysis of variance (ANOVA) test, *P < 0.05. (B) The Dicer deletion phenotype, as in (A), can be “rescued” by transfection of midbrain-derived small RNAs (<200 base pairs) but not large RNAs (>200 base pairs). Two independent experiments of three sets each were performed, with 10 visual fields per set; data represent mean ± SEM; ANOVA test, **P < 0.01. (C) Immunostaining of brain sections from 8-week-old DATCRE/+:Dicer flox/flox mice for TH demonstrates loss of 90% of midbrain DNs in the substantia nigra (SN) and ventral tegmental area (VTA) and their axonal projections to the striatum relative to control littermates (DATCRE/+:Dicer flox/+)(n = 3 for each genotype). Scale bars, 200 μm. (D) Locomotor activity of DATCRE/+:Dicerflox/flox mice in the open field. The total distance traveled was significantly decreased in DATCRE/+:Dicerflox/flox mice (n = 4 for each genotype). Data represent mean ± SEM; Student's t test, *P < 0.05,**P < 0.01.

To extend these findings to the intact rodent central nervous system, we generated mice that were homozygous for the conditional floxed Dicer allele and expressed Cre recombinase under the regulation of dopamine transporter regulatory sequences [DATCRE/+:Dicer flox/flox (6)], leading to the specific deletion of Dicer in postmitotic midbrain DNs (8). These mice display a progressive loss of midbrain DNs, as quantified by TH and dopamine transporter (DAT) immunostaining (Fig. 1C and fig. S1E), due to apoptosis (fig. S2A).

Behavioral studies of mice that harbor a midbrain DN-specific deletion of Dicer revealed markedly reduced locomotion in an open-field assay (Fig. 1D and fig. S2B). This is mostly a consequence of long periods of immobility, which is reminiscent of the phenotype of human patients with Parkinson's disease. These results suggest that miRNAs are essential for the terminal differentiation and/or maintenance of multiple neuron types, including midbrain DNs.

Because no specific miRNAs had been implicated in midbrain DNs, we sought to identify some. We took a subtractive approach and compared miRNA expression profiles of the normal adult midbrain with the profiles of a midbrain depleted of DNs. Expression analyses were performed by quantitative real-time reverse transcription polymerase chain reaction (qPCR) for a panel of 224 miRNA precursors in midbrain, cerebellum, and cerebral cortex samples from Parkinson's disease patients and normal controls (fig. S3 and table S1). Expression of one of these precursor miRNAs, miR-133b, was specifically enriched in the midbrain and deficient in the context of Parkinson's disease patient samples, as determined by ribonuclease (RNase) protection assays, qPCR, and Northern blotting for mature miR-133b (Fig. 2A and fig. S4A).

Fig. 2.

miR-133b is enriched in the midbrain and is deficient in the tissue of Parkinson's disease patients. (A) Expression analysis of miR-133b in cerebral cortex (CX), midbrain (MB), or cerebellum (CB) of unaffected controls and Parkinson's disease (PD) brain. RNA protection assays were performed for miR133a1 and miR133b expression, showing specific expression in control midbrain but not PD midbrain (n = 3 per group). Data represent mean ± SEM; ANOVA test, *P < 0.05. (B) Expression analysis for miR-133b in cerebral cortex (CX), midbrain (MB), or cerebellum (CB) of control (WT) and Aphakia mutant mice. RNA protection assays were performed for miR133a1 and miR133b expression in control and Aphakia mutant mouse brain (three independent experiments). Data represent mean ± SEM;ANOVAtest,*P < 0.05. (C) qPCR analysis of murine ES cultures differentiated by the EB method and transduced with lentiviral vectors for pitx3, the transcription factor nurr1 (as a negative control), both, or GFP vector control. Pitx3 transduction leads to the specific induction of miR-133b precursor expression; miR-133a1 and miR-133a2 precursors are not induced by Pitx3 overexpression (three independent experiments were performed). Data represent mean ± SEM; ANOVA test,*P < 0.05.

We investigated expression of miR-133b in two additional DN deficiency models: adult Aphakia mice deficient in the transcription factor Pitx3 (911) and mice treated with the DN-specific toxin 6-hydroxydopamine (5). miR-133b was specifically expressed in the midbrain of normal mice, as in humans, and expression was markedly reduced in both rodent dopamine-deficiency models as demonstrated by RNase protection assays, qPCR, and Northern blotting (Fig. 2B and fig. S4, B and C).

The relative deficiency of miR-133b expression in Aphakia mouse strain midbrain was surprising, given that adult Aphakia mice do maintain a population of midbrain DNs within the ventral tegmental area (10); this suggested the possibility that miR-133b is a direct target of Pitx3 transcription activation (as pitx3 is mutated in Aphakia mice). Consistent with this model, overexpression of Pitx3 in differentiating ES cultures led to up-regulation of miR-133b precursor expression (Fig. 2C). Furthermore, expression of a luciferase reporter vector that harbors 350 base pairs of proximal miR-133b promoter sequences was specifically induced by overexpression of Pitx3 in COS cells (fig. S4D).

Next, we investigated the consequences of increased miR-133b expression in either ES cell–derived cultures or in primary embryonic day 14.5 (E14.5) midbrain cultures. miR-133b precursor overexpression (using a lentiviral vector; fig. S3A) led to a relative reduction in transcription of the late midbrain DN maturation marker DAT, although transcription of early midbrain DN markers, such as Pitx3 and the transcription factor Nurr1, appeared unaltered or increased (Fig. 3, A and B). Consistent with this, dopamine release in the context of potassium-induced depolarization was markedly reduced with miR-133b overexpression. Overexpression of miR-133b at the neural precursor stage of EB-differentiated ES cultures led to a significant reduction in the number of TH-positive cells (but not TujI-positive cells; Fig. 3C).

Fig. 3.

miR-133b suppresses DN maturation and function. (A) Overexpression of miR-133b precursor in primary embryonic rat midbrain cultures led to decreased expression of the mature DN marker, DAT. A lentivirus vector was used to overexpress either miR-133b precursor or control (GFP) sequences. Expression of TH showed a trend toward reduced expression that is not statistically significant, whereas Nurr1 and Pitx3 mRNA expression appeared unaffected. Data represent mean ± SEM; three independent experiments were performed; Student's t test, *P < 0.05. (B) Depolarization-induced dopamine release was quantified in ES-derived, EB differentiated cultures transduced with miR-133b precursor lentiviral vector or GFP control. miR-133b precursor overexpression reduced dopamine release in murine ES culture-derived DNs. Data represent mean ± SEM; five independent experiments were performed; ANOVA test, *P < 0.05. (C) Overexpression of miR-133b during EB differentiation resulted in a significant decrease in the accumulation of TH-positive cells. Data represent mean ± SEM; three independent experiments were performed; ANOVA test, *P < 0.05. (D) Reduction of miR-133b by penetratin-conjugated antisense miR133b 2′-O-methyl–modified oligonucleotide in primary embryonic rat midbrain culture leads to increased expression of DN mRNAs including TH and DAT, whereas Nurr1 and Pitx3 mRNAs are not significantly altered. Data represent mean ± SEM; five independent experiments were performed; Student's t test, *P < 0.05. (E) Depolarization-induced dopamine release was quantified in murine ES-derived, EB differentiated cultures transduced with miR-133b reduction– (or control-) modified oligonucleotide. miR-133b reduction induced dopamine release in these cultures. Data represent mean ± SEM; three independent experiments were performed; Student's t test, *P < 0.05.

The activity of miR-133b can be inhibited using a 2′-O-methyl–modified RNA oligonucleotide homologous to the miR-133b sequence and linked to a short peptide derived from the Drosophila Antennapedia protein that mediates cell transduction (12) (fig. S5A). Suppression of miR-133b in ES cell EB differentiation cultures induced expression of DN markers including DAT and TH (quantified by qPCR analyses; Fig. 3, D and E). In ES-derived cultures, transduction of the miR-133b inhibitory oligonucleotide potentiated potassium-stimulated dopamine release. Taken together, these data implicate miR-133b in the regulation of midbrain DN maturation and function.

Individual miRNAs appear to regulate the expression of numerous targets posttranslationally (13). To identify potential physiological targets for miR-133b activity, we used available miRNA target prediction programs based on 3′ untranslated sequence homology to miR-133b (14, 15). The Pitx3 3′-untranslated region (3′ UTR) was identified as a potential target of miR-133b activity, and consistent with this, Pitx3 3′ UTR sequences were subject to suppression by miR-133b when placed downstream of a luciferase reporter gene (fig. S6A). Thus, a hypothetical model for the observed phenotypes associated with altered miR-133b expression is that miR-133b functions within a negative feedback circuit that normally suppresses Pitx3 expression posttranscriptionally, and, in turn, Pitx3 activates midbrain DN gene expression (5, 16) and induces transcription of miR-133b. Fluorescence-activated cell sorter (FACS) analysis of permeablized primary rat midbrain cells with an antibody that recognizes Pitx3 protein revealed that miR-133b overexpression induced a reduction in Pitx3 protein levels in TH+ cells (Fig. 4A), whereas miR-133b reduction led to an increase in Pitx3 protein in TH+ cells (Fig. 4B).

Fig. 4.

Pitx3 is a target of miR-133b activity. (A) Primary midbrain cultures (at day 1 in vitro) transduced with miR-133b precursor or control lentiviral vectors were analyzed (after 7 days) by FACS using antibodies for Pitx3 and TH. Expression of TH and Pitx3 protein is reduced in cells transduced with miR-133b precursor relative to control vector (GFP) or miR-18 precursor. Data represent mean ± SEM; three independent experiments were performed; ANOVA test, *P < 0.05. (B) Reduction of miR-133b in primary midbrain cultures (at day 7 in vitro) with the use of 2′-O-methyl–modified oligonucleotide but not control oligonucleotide leads to a significant induction in Pitx3 and TH protein as quantified by FACS analysis (after 7 days). Data represent mean ± SEM; three independent experiments were performed; Student's t test, *P < 0.05. (C) miR-133b inhibition by 2′-O-methyl–modified oligonucleotide in Pitx3-deficient Aphakia primary neuron cultures fails to induce TH or DAT transcription. Data represent mean ± SEM; three independent experiments were performed; Student's t test, *P < 0.05. (D) Pitx3 protein expression was significantly increased in TH-positive cells from 10-day-old miRNA-deficient DAT CRE/+:Dicerflox/flox mice relative to control DATCRE/+:Dicerflox/+ mice. FACS analyses were performed on acutely dissociated, permeablized midbrain cells with the use of TH- and Pitx3-specific antibodies. Data represent mean ± SEM; three independent experiments were performed; Student's t test, *P < 0.05.

If Pitx3 is a direct target of miR-133b, one prediction is that miR-133b inhibition by modified oligonucleotide transduction would fail to induce TH and DATexpression in Pitx3-deficient, Aphakia primary neuron cultures, and this was observed (Fig.4C). Finally, FACS analysis on acutely dissociated midbrain DNs from young Dicer mutant mice (10 days old, derived from DATCRE/+:Dicer flox/flox mice) revealed that Pitx3 protein expression is up-regulated in TH-positive neurons (relative to control DATCRE/+:Dicer flox/+ cells; Fig. 4D and fig. S6C), consistent with a role for miRNA in Pitx3 regulation.

Our data support a model in which miR-133b functions within a feedback loop, as Pitx3 specifically induces transcription of miR-133b, and Pitx3 activity is down-regulated by miR-133b posttranscriptionally (fig. S6D). Midbrain DN function is dynamic, and such feedback circuitry has been shown to increase the robustness and speed response time and stability in the context of dynamic changes (17). Furthermore, we present evidence that Dicer deletion leads to the progressive loss of midbrain DNs, suggesting that miRNAs in addition to miR-133b function in these cells.

Supporting Online Material

www.sciencemag.org/cgi/content/full/317/5842/1220/DC1

Materials and Methods

Figs. S1 to S6

Tables S1 to S3

References

References and Notes

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