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A functional screen implicates microRNA-138-dependent regulation of the depalmitoylation enzyme APT1 in dendritic spine morphogenesis

Abstract

The microRNA pathway has been implicated in the regulation of synaptic protein synthesis and ultimately in dendritic spine morphogenesis, a phenomenon associated with long-lasting forms of memory. However, the particular microRNAs (miRNAs) involved are largely unknown. Here we identify specific miRNAs that function at synapses to control dendritic spine structure by performing a functional screen. One of the identified miRNAs, miR-138, is highly enriched in the brain, localized within dendrites and negatively regulates the size of dendritic spines in rat hippocampal neurons. miR-138 controls the expression of acyl protein thioesterase 1 (APT1), an enzyme regulating the palmitoylation status of proteins that are known to function at the synapse, including the α13 subunits of G proteins (Gα13). RNA-interference-mediated knockdown of APT1 and the expression of membrane-localized Gα13 both suppress spine enlargement caused by inhibition of miR-138, suggesting that APT1-regulated depalmitoylation of Gα13 might be an important downstream event of miR-138 function. Our results uncover a previously unknown miRNA-dependent mechanism in neurons and demonstrate a previously unrecognized complexity of miRNA-dependent control of dendritic spine morphogenesis.

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Figure 1: Expression profiling of miRNAs in synaptososomes.
Figure 2: A subset of neuronal miRNAs is localized in the synaptodendritic compartment.
Figure 3: Synaptically enriched miR-138 regulates dendritic spine size.
Figure 4: miR-138 negatively regulates miniature synaptic transmission at excitatory synapses.
Figure 5: APT1 is expressed in neuronal dendrites during synapse development.
Figure 6: APT1 mRNA is a miR-138 target in neurons.
Figure 7: APT1 activity is required for the control of dendritic spine size.
Figure 8: miR-138 regulated membrane-association of Gα13 is required for the control of dendritic spine size.

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Acknowledgements

We thank O. Rocks and P. Wedegaertner for providing reagents; R. Heinen for suggestions on the luciferase reporter assays; T. Wüst for technical assistance; and M. Alenius and A. Keene for critically reading the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (SFB488; to G.M.S.), the Human Frontier Science Program (Career Development Award; to G.M.S.), the National Institute on Drug Abuse (1R21DA025102-01; to G.M.S.), the National Institutes of Health (NS045500; to M.E.G.) the Bioinformatics Initiative (to C.D. and M.R.), the Austrian Academy of Sciences (to J.M. and S.B. (DOC-fFORTE-fellowship)) and the Austrian Government, GEN-AU initiative (to G.O.). C.H. acknowledges the Alexander von Humboldt foundation for a postdoctoral fellowship. F.D. is a recipient of a TALENT-stipendium from the Netherlands Organization for Scientific Research (NWO) and a Marie-Curie fellowship from the European Union.

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Authors and Affiliations

Authors

Contributions

G.M.S. performed microRNA profiling, M.C. analysed the microarray data, G.O., R.F. and C.J.B. performed in situ experiments, G.S., S.B., G.M.S. and C.K. performed and analysed spine measurements, M.O. and A.D. performed and analysed electrophysiology experiments, G.S. and G.M.S. performed and analysed immunostaining, G.S., G.O. and P.J.L. designed and performed the target validation experiments, G.O., C.D. and M.R. carried out bioinformatics, G.S. performed biochemistry, F.D., C.H., B.R. and H.W. were involved in the design and synthesis of specific APT1 inhibitors, R.F. performed sensor assays, G.S, S.B., M.C. and S.Kh. performed RNA expression analysis, K.H. and S.Ka provided reagents, M.E.G. participated in the initial stages of project planning, J.M. and G.M.S. directed the research and G.M.S. coordinated the project and wrote the manuscript.

Corresponding authors

Correspondence to Javier Martinez or Gerhard M. Schratt.

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Siegel, G., Obernosterer, G., Fiore, R. et al. A functional screen implicates microRNA-138-dependent regulation of the depalmitoylation enzyme APT1 in dendritic spine morphogenesis. Nat Cell Biol 11, 705–716 (2009). https://doi.org/10.1038/ncb1876

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