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A Microrna Feedback Circuit in Midbrain Dopamine Neurons

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A Microrna Feedback Circuit in Midbrain Dopamine Neurons REPORTS rins, is not sufficient to colocalize these proteins at tacts, potentially increasing the local level of 22. Z. Wang et al., Science 304, 1164 (2004). the cell junctions. The PTPs generally have little phosphatase activity. Because of the high affinity 23. J. A. Besco, R. Hooft van Huijsduijnen, A. Frostholm, A. Rotter, Brain Res. 1116, 50 (2006). substrate specificity, and they rely on noncatalytic of the trans interaction, the balance between cell 24. M. Fuchs, T. Muller, M. M. Lerch, A. Ullrich, J. Biol. Chem. domains to control their subcellular distribution adhesion versus mobility can only be shifted by 271, 16712 (1996). and therefore indirectly regulate their activity by the action of the ADAM 10 protease (14). In both 25. G. C. M. Zondag, A. B. Reynolds, W. H. Moolenaar, J. Biol. restricting access to particular substrates at de- CD45 and RPTPm, however, ectodomain size and Chem. 275, 11264 (2000). 6 32 26. S. M. Brady-Kalnay et al., J. Cell Biol. 141, 287 (1998). fined locations ( , ). RPTPs are known to be rigidity appear to provide a mechanism to allow 27. X. F. Sui et al., Am. J. Pathol. 166, 1247 (2005). constitutively active, and ligand-induced inactiva- cell-cell spacings to regulate intercellular multi- 28. K. Miyaguchi, J. Struct. Biol. 132, 169 (2000). tion has been reported for type I and IV subfam- molecular assemblies. 29. T. J. Boggon et al., Science 296, 1308 (2002). ilies. Such a mechanism is unlikely to apply to type 30. L. A. Staehelin, Int. Rev. Cytol. 39, 191 (1974). IIB RPTPs, where an active enzyme would be References and Notes 31. W. He, P. Cowin, D. L. Stokes, Science 302, 109 (2003). 32. I. A. Yudushkin et al., Science 315, 115 (2007). required to maintain cadherin-catenin complexes 1. M. Perez-Moreno, C. Jamora, E. Fuchs, Cell 112, 535 (2003). 33. S. J. Davis, P. A. van der Merwe, Nat. Immunol. 7, 803 in a dephosphorylated state and thus contribute to 2. B. M. Gumbiner, Nat. Rev. Mol. Cell Biol. 6, 622 (2005). (2006). the stability of cell contacts (2). In this context, for 3. J. L. Sallee, E. S. Wittchen, K. Burridge, J. Biol. Chem. 34. K. Choudhuri, D. Wiseman, M. H. Brown, K. Gould, type IIB RPTPs, the ectodomain-mediated trans 281, 16189 (2006). P. A. van der Merwe, Nature 436, 578 (2005). 35. Single-letter abbreviations for the amino acid residues are homophilic interactions appear to represent the 4. R. L. Del Vecchio, N. K. Tonks, J. Biol. Chem. 280, 1603 (2005). as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; driving force for correct localization and function. 5. M. F. Gebbink et al., J. Cell Biol. 131, 251 (1995). H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; Our results on RPTPm suggest how the type 6. N. K. Tonks, Nat. Rev. Mol. Cell Biol. 7, 833 (2006). R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. IIB RPTPs modulate the stability of adherens 7. J. Besco, M. C. Popesco, R. V. Davuluri, A. Frostholm, 36. T. Maretzky et al., Proc. Natl. Acad. Sci. U.S.A. 102, 9182 A. Rotter, BMC Genomics 5, 14 (2004). (2005). junctions (Fig. 4). The ectodomain trans interac- m 8. A. R. Aricescu et al., EMBO J. 25, 701 (2006). 37. Coordinates and structure factors of eRPTP have been tionisswitchedoffatacidpH(8, 18) (i.e., until deposited in the PDB (www.rcsb.org) with the accession m 9. A. R. Aricescu, W. Lu, E. Y. Jones, Acta Crystallogr. D 62, RPTP reaches the cell surface). The rigid, ruler- 1243 (2006). number 2V5Y. We thank the staff of the ID 29 beamline like ectodomain then acts as a sensor of inter- 10. V. T. Chang et al., Structure 15, 267 (2007). at the European Synchrotron Radiation Facility for m cellular distances, matching cadherin-mediated 11. Materials and methods are available as supporting assistance with data collection; M. Gebbink for the RPTP material on Science Online. cDNA; P. Reeves and H. G. Khorana for the human cell contacts, at which point the trans interaction – – 12. V. Soroka et al., Structure 11, 1291 (2003). embryonic kidney 293S GnTI cell line; M. Shaw for serves as a spacer clamp, locking the phosphatase 13. M. Campan et al., Biochemistry 35, 3797 (1996). assistance with EM imaging; and J. Brown, M. Crispin, activity into proximity with the target substrates. 14. L. Anders et al., Mol. Cell. Biol. 26, 3917 (2006). W.-C. Hon, and D. Stuart for discussions. The work was The spacer-clamp action of RPTPm represents the 15. V. B. Cismasiu, S. A. Denes, H. Reilander, H. Michel, funded by Cancer Research UK (CR-UK). E.Y.J. is a CR-UK Principal Research Fellow. inverse strategy to the size-exclusion mechanism S. E. Szedlacsek, J. Biol. Chem. 279, 26922 (2004). 16. S. M. Brady-Kalnay, A. J. Flint, N. K. Tonks, J. Cell Biol. proposed to regulate the cell surface location of 122, 961 (1993). Supporting Online Material www.sciencemag.org/cgi/content/full/317/5842/1217/DC1 another RPTP, CD45; in that case, the mismatch 17. J. Cheng et al., J. Biol. Chem. 272, 7264 (1997). Materials and Methods between the RPTP ectodomain and the inter- 18. M. F. Gebbink et al., J. Biol. Chem. 268, 16101 (1993). 19. G. C. M. Zondag et al., J. Biol. Chem. 270, 14247 (1995). Figs. S1 to S6 cellular spacing is thought to contribute to T cell Table S1 20. J. Sap, Y. P. Jiang, D. Friedlander, M. Grumet, J. Schlessinger, References signaling by expelling the phosphatase activity Mol. Cell. Biol. 14, 1 (1994). from local zones of cell-cell contact (33, 34). 21. M. Fuchs, H. Wang, T. Ciossek, Z. Chen, A. Ullrich, Mech. 4 May 2007; accepted 17 July 2007 Unlike CD45, RPTPm is maintained at cell con- Dev. 70, 91 (1998). 10.1126/science.1144646 the differentiation of murine embryonic stem A MicroRNA Feedback Circuit (ES) cells into DNs (4, 5). An ES cell line was obtained that expresses Dicer enzyme condition- in Midbrain Dopamine Neurons ally [containing LoxP recombinase sites that flank both chromosomal copies of the Dicer Jongpil Kim,1 Keiichi Inoue,1 Jennifer Ishii,1 William B. Vanti,1 Sergey V. Voronov,1 gene, herein termed floxed Dicer (6)]. Introduc- Elizabeth Murchison,2 Gregory Hannon,2 Asa Abeliovich1* tion of Cre recombinase into these cells by lentiviral transduction leads to the deletion of MicroRNAs (miRNAs) are evolutionarily conserved, 18- to 25-nucleotide, non–protein coding Dicer in nearly 100% of cells (fig. S1A). transcripts that posttranscriptionally regulate gene expression during development. miRNAs also ES cultures were differentiated to a midbrain occur in postmitotic cells, such as neurons in the mammalian central nervous system, but their DN phenotype using the embryoid body (EB) function is less well characterized. We investigated the role of miRNAs in mammalian midbrain protocol (fig. S1B) (5, 7). Cre-mediated deletion dopaminergic neurons (DNs). We identified a miRNA, miR-133b, that is specifically expressed in of Dicer at a stage when postmitotic DNs first midbrain DNs and is deficient in midbrain tissue from patients with Parkinson’s disease. miR-133b arise led to a nearly complete loss of DN accu- regulates the maturation and function of midbrain DNs within a negative feedback circuit that mulation, as quantified by the expression of includes the paired-like homeodomain transcription factor Pitx3. We propose a role for this markers including tyrosine hydroxylase (TH) feedback circuit in the fine-tuning of dopaminergic behaviors such as locomotion. (Fig. 1A). Other mature neuronal classes, includ- ing GABAergic neurons, were reduced in these icroRNAs (miRNAs) are derived from Midbrain dopaminergic neurons (DNs) play a long primary transcripts through se- central role in complex behaviors such as reward 1Departments of Pathology and Neurology, Center for Mquential processing by the Drosha and addiction, and these cells are lost in Par- Neurobiology and Behavior, and Taub Institute, Columbia ribonuclease and the Dicer enzyme (1). In the kinson’s disease. A number of transcription fac- University, College of Physicians and Surgeons 15-403, 630 West 168th Street, New York, NY 10032, USA. context of an RNA-induced silencing complex, tors have been identified that regulate midbrain 2 3 Watson School of Biological Sciences, Cold Spring Harbor miRNAs guide the cleavage of target mRNAs DN development, function, and survival ( ). Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY and/or inhibit their translation. miRNAs regulate However, the role of posttranscriptional mecha- 11724, USA. developmental cell fate decisions in the nervous nisms is unknown. To establish a function for *To whom correspondence should be addressed. E-mail: system and elsewhere (2). miRNAs, we first used an in vitro model system: [email protected] 1220 31 AUGUST 2007 VOL 317 SCIENCE www.sciencemag.org REPORTS 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 re- duced by approximately 40 to 60%. (n =3independent samples per group). Scale bar, 100 mm. Data represent mean ± SEM; analysis loss of 90% of midbrain DNs in the substantia nigra (SN) and ventral of variance (ANOVA) test, *P <0.05.(B) The Dicer deletion phenotype, as in tegmental area (VTA) and their axonal projections to the striatum relative to (A), can be “rescued” by transfection of midbrain-derived small RNAs (<200 control littermates (DATCRE/+:Dicer flox/+)(n = 3 for each genotype).
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