A MicroRNA feedback circuit in midbrain dopamine neurons

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.

Figure 1

Dicer is essential for terminal differentiation of ES cells to a DN phenotype. (A) Floxed Dicer conditional knockout cells were transduced with CRE lentivirus at EB stage 4 and analyzed by immunostaining at day 7 of stage 5 with antibodies specific for TH (Red), TujI (Green) and GABA (blue). Cultures transduced with a lentiviral Cre vector (vCre) are essentially devoid of TH+ neurons, whereas TujI+ and GABA+ cells are reduced by approximately 40%-60%. (Scale bars, 100μm). (n=3 independent samples per group; 2 additional independent sets of samples showed consistent results). Data represent means ± S.E.M. ANOVA-test, *p < 0.05. (C) The Dicer deletion phenotype, as in (A) can be ‘rescued’ by transfection of midbrain-derived small RNAs (<200bp) not large RNAs (>200bp) (Two independent experiments of 3 sets each with 10 visual fields per set), Data represent means ± S.E.M. ANOVA-test, **p < 0.01. (D) Immunostaining of brain sections from 8 week old DAT^CRE/+ :Dicer^flox/flox mice for tyrosine hydroxylase (TH) demonstrates loss of approximately 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 (DAT^CRE/+ : Dicer^flox/+; N=3 for each genotype). (Scale bars,200μm) (F) Locomotor activity of DAT^CRE/+ :Dicer^flox/flox mice in the open field. The total distance traveled during the test was significantly decreased in DAT^CRE/+ :Dicer^flox/flox mice. (N=4 for each genotype), Data represent means ± S.E.M. Student's t test, *p < 0.05, **p < 0.01.

Figure 2

miR-133b is enriched in midbrain and is deficient in PD samples. (A) Expression analysis for precursor miR-133b in cerebral cortex (CX), midbrain (MB), or cerebellum (CB) of healthy human and Parkinson's disease (PD) patient brain. RNA protection assay for miR133a1 and miR133b expression from normal or PD human brain and quantification of RNA protection assay. n=3 independent experiments; data represent means ± S.E.M. ANOVA test, *p < 0.05. (B) Expression analysis for precursor miR-133b in cerebral cortex (CX), midbrain (MB), or cerebellum (CB) of control mice and Aphakia mutant. RNA protection assay for miR133a1 and miR133b expression from control mice and Aphakia mutant mice brain and quantification of RNA protection assay. n=3 independent experiments; data represent means ± S.E.M. ANOVA test, *p < 0.05. (C) qPCR analysis of murine ES cultures differentiated by EB and transduced at stage 4 with viral vectors for Nurr1, Pitx3, both, or GFP vector control. Pitx3 transduction (but not Nurr1 transduction) leads to the specific induction of miR-133b precursor expression assayed at stage 5; miR-133a1 and miR-133a2 precursors are not induced by Pitx3 overexpression. n=3 independent experiments; data represent means ± S.E.M. ANOVA test, *p < 0.05.

Figure 3

miR-133b suppresses dopamine neuron terminal differentiation and function. (A) Overexpression of miR-133b precursor in primary embryonic rat midbrain cultures leads to decreased expression of dopamine neuron mRNAs including TH and DAT, whereas Nurr1 and Pitx3 mRNAs are not altered. Data represent means ± S.E.M. n=3 independent experiments; Student's t test, *p < 0.05. (B) Depolarization-induced dopamine release was quantified in cultures transduced with miR-133b precursor vector or GFP control. miR-133b overexpression reduced dopamine release in murine ES cultures derived dopamine neurons. Data represent means ± S.E.M. n=5; ANOVA test, *p < 0.05. (C) Overexpression of miR-133b during EB stage 4 of mouse ES cell differentiation resulted in a significant decrease in TH-positive cells. Data represent means ± S.E.M. n=3 independent experiments; ANOVA test, *p < 0.05. (D) Knockdown of miR-133b by penetratin conjugated antisense miR133b 2OM oligo in primary embryonic rat midbrain cultures leads to increased expression of dopamine neuron mRNAs including TH and DAT, whereas Nurr1 and Pitx3 mRNAs are not significantly altered. Data represent means ± S.E.M. n=5 independent experiments; Student's t test, *p < 0.05. (E) Depolarization-induced dopamine release was quantified in cultures with miR-133b knockdown or oligonucleotide control. miR-133b knockdown induced dopamine release in murine ES cultures. Data represent means ± S.E.M. n=3 independent experiments; Student's t test, *p < 0.05.

Figure 4

Pitx3 is a target of miR-133b activity. (A) Upper Panel: Schematic of a luciferase assay vector, pGL3-Pitx3 and pGL3-Mut-Pitx3 that harbors 300bp Pitx3 3′-UTR sequences predicted to be subject to miR-133b regulation. Lower panel: Overexpression of miR-133b (but not miR-18) in 293 cells decreases luciferase expression from pGL3-Pitx3 (but not control vector and pGL3-mut Pitx3). Data represent means ± S.E.M. n=3 independent experiments; Student's t test, *p < 0.05. (B) miR-133b and Pitx3 define a negative feedback loop in midbrain DN function and differentiation. It is likely that miR-133b has additional significant targets. (C) Upper panels: FACS analysis of primary midbrain cultures transduced with miR-133b or control vectors using antibodies for Pitx3 and TH reveals that miR-133b overexpression decrease in both Pitx3 and TH protein expression. Pitx3 is reduced in TH-positive cells transduced with miR-133b relative to control vector or miR-18. Data represent means ± S.E.M. n=3 independent experiments; ANOVA test, *p < 0.05. Lower panels: Knockdown of miR-133b using modified oligonucleotides leads to a significant induction in Pitx3 and TH protein levels relative to control oligonucleotides. Data represent means ± S.E.M. n=3 independent experiments; Student's t test, *p < 0.05. (E) miR-133b inhibition by modified oligonucleotides in Pitx3-deficient Aphakia primary neuron cultures fails to induce TH or DAT transcription. Data represent means ± S.E.M. n=3 independent experiments; Student's t test, *p < 0.05.