Beruflich Dokumente
Kultur Dokumente
Authors
Bin Wang, Lin Pan, Manyi Wei, ...,
Xing-Yu Jiang, Xu Zhang, Lan Bao
Correspondence
baolan@sibcb.ac.cn
In Brief
Wang et al. find that axon-enriched miR181d regulates axon elongation by
targeting axonal Map1b and Calm1 in
primary sensory neurons. FMRP
mediates delivery of miR-181d and its
targets to the axon. NGF triggers Map1b
and Calm1 release from FMRP and miR181d-repressing granules in axons,
controlling mRNA translation locally to
induce axon elongation.
Highlights
d
Please cite this article in press as: Wang et al., FMRP-Mediated Axonal Delivery of miR-181d Regulates Axon Elongation by Locally Targeting Map1b
and Calm1, Cell Reports (2015), http://dx.doi.org/10.1016/j.celrep.2015.11.057
Cell Reports
Article
FMRP-Mediated Axonal Delivery
of miR-181d Regulates Axon Elongation
by Locally Targeting Map1b and Calm1
Bin Wang,1,5 Lin Pan,1,5 Manyi Wei,1 Qiong Wang,1 Wen-Wen Liu,2 Nuoxin Wang,2 Xing-Yu Jiang,2 Xu Zhang,3,4
and Lan Bao1,4,*
1State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology,
Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
2National Center of Nanoscience and Technology, Beijing 100190, China
3Institute of Neuroscience and State Key Laboratory of Neuroscience, CAS Center for Excellence in Brain Science, Shanghai Institutes for
Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
4School of Life Science and Technology, ShanghaiTech University, Shanghai 200031, China
5Co-first author
*Correspondence: baolan@sibcb.ac.cn
http://dx.doi.org/10.1016/j.celrep.2015.11.057
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
SUMMARY
Please cite this article in press as: Wang et al., FMRP-Mediated Axonal Delivery of miR-181d Regulates Axon Elongation by Locally Targeting Map1b
and Calm1, Cell Reports (2015), http://dx.doi.org/10.1016/j.celrep.2015.11.057
Please cite this article in press as: Wang et al., FMRP-Mediated Axonal Delivery of miR-181d Regulates Axon Elongation by Locally Targeting Map1b
and Calm1, Cell Reports (2015), http://dx.doi.org/10.1016/j.celrep.2015.11.057
Figure 1. Identification of Axon-Enriched miR-181d and Its Targets, Map1b and Calm1, in Embryonic DRG Neurons
(A) miR-181d was enriched in the axons of embryonic DRG neurons. Immunostaining for Tuj-1 demonstrated the successful establishment of microfluidic
cultured E13.5 DRG neurons at 7 DIV (upper panel). A qPCR analysis of miR-181d showed a 5.4-fold increase in its axonal abundance compared to the cell
bodies. The results are presented as mean SEM. *p < 0.05 versus the level of miR-181d in the cell body (n = 6). Scale bar, 300 mm.
(B) Localization of miR-181d (red) in the axons and cell bodies of E13.5 DRG neurons at 4 DIV used LNA-modified probes. A scramble probe was used as the
negative control. Immunostaining for Tuj-1 (green) indicated the neuronal profile. Scale bar, 5 mm.
(C) Luciferase reporter assays showed that Map1b and Calm1 are potential downstream targets of miR-181d. The luciferase activity of the WT 30 UTR, but not the
mutated Map1b and Calm1, was downregulated by miR-181d mimics in HEK293 cells. The results are presented as mean SEM. *p < 0.05 and **p < 0.01 versus
the negative control (n = 3).
(D) RT-PCR showed that Map1b and Calm1 were distributed in the cell bodies and axons of cells in the microfluidic cultures of E13.5 DRG neurons at 7 DIV.
b-actin and g-actin were the positive and negative controls, respectively, for axonal distribution.
(E) In situ hybridization showed that Map1b and Calm1 (red) were localized in the cell bodies and axons of cultured E13.5 DRG neurons at 4 DIV. Immunostaining
for tubulin (green) was used to indicate the neuronal profile. Scale bar, 5 mm.
(F and G) Live-cell imaging demonstrated the anterograde movement of Map1b-30 UTR (F) and Calm1-30 UTR (G) in the axons of cultured P14 DRG neurons at
4 DIV (arrowheads). Scale bar, 5 mm.
(H and I) Immunoblotting showed that applying a miR-181d inhibitor (H) and its mimics (I) increased and decreased, respectively, the protein levels of MAP1B and
calmodulin in cultured E13.5 DRG neurons at 4 DIV. Tubulin was used as a loading control. The results are presented as mean SEM. *p < 0.05 and **p < 0.01
versus the negative control (n = 3).
See also Figures S1 and S2.
Cell Reports 13, 114, December 29, 2015 2015 The Authors 3
Please cite this article in press as: Wang et al., FMRP-Mediated Axonal Delivery of miR-181d Regulates Axon Elongation by Locally Targeting Map1b
and Calm1, Cell Reports (2015), http://dx.doi.org/10.1016/j.celrep.2015.11.057
Figure 2. miR-181d Negatively Regulates Axon Elongation by Locally Targeting Axonal Map1b and Calm1
(A and B) Representative images of microfluidic cultured E13.5 DRG axons at 4 DIV (A) and quantitative results of axon growth rates (B) showed that the axonal
application of a miR-181d inhibitor and its mimics increased and decreased axon elongation, respectively, in the microfluidic chamber. The results are presented
as mean SEM. *p < 0.05 versus the negative control (n = 3). Scale bar, 300 mm.
4 Cell Reports 13, 114, December 29, 2015 2015 The Authors
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and Calm1, Cell Reports (2015), http://dx.doi.org/10.1016/j.celrep.2015.11.057
(C and D) Representative immunoblots and quantitative data showed that si-Map1b and si-Calm1 decreased the protein levels of MAP1B (C) and calmodulin (D) in
cultured E13.5 DRG neurons at 4 DIV. Tubulin was used as a loading control. The results are presented as mean SEM and normalized to the levels in the siRNA
control. **p < 0.01 versus the siRNA control (n = 3).
(E and F) Representative images of microfluidic cultured E13.5 DRG axons at 4 DIV (E) and quantitative results of axon growth rates (F) showed that the axonal
application of si-Map1b or si-Calm1 reduced axon elongation. The results are presented as mean SEM. *p < 0.05 versus the siRNA control (n = 3). Scale bar,
300 mm.
(G) Representative immunoblots and quantitative data showed that the axonal application of Map1b-TP or Calm1-TP blocked the interaction of miR-181d with the
30 UTR of Map1b or Calm1, respectively, and increased the protein levels of MAP1B and calmodulin in the axon, but not in the cell body, in microfluidic cultures of
E13.5 DRG neurons at 4 DIV. Tubulin was used as a loading control. The results are presented as mean SEM and normalized to the mock control. *p < 0.05 and
**p < 0.01 versus the negative control (n = 3).
(H and I) Representative images of microfluidic cultured E13.5 DRG axons at 4 DIV (H) and quantitative results of axon growth rates (I) demonstrated that the
axonal application of either Map1b-TP or Calm1-TP increased axon elongation in the microfluidic chamber. The results are presented as mean SEM. *p < 0.05
versus the negative control (n = 3). Scale bar, 300 mm.
See also Figures S2 and S3.
Cell Reports 13, 114, December 29, 2015 2015 The Authors 5
Please cite this article in press as: Wang et al., FMRP-Mediated Axonal Delivery of miR-181d Regulates Axon Elongation by Locally Targeting Map1b
and Calm1, Cell Reports (2015), http://dx.doi.org/10.1016/j.celrep.2015.11.057
(Figures 3I and 3J). Taken together, our data suggest that FMRP
associates with miR-181d, Map1b, and Calm1 in DRG neurons.
FMRP Deficiency Causes Defects in the Axonal Delivery
of miR-181d, Map1b, and Calm1
We assessed the function of FMRP in the axonal distribution of
miR-181d, Map1b, and Calm1 in vivo. The qPCR showed that
6 Cell Reports 13, 114, December 29, 2015 2015 The Authors
Please cite this article in press as: Wang et al., FMRP-Mediated Axonal Delivery of miR-181d Regulates Axon Elongation by Locally Targeting Map1b
and Calm1, Cell Reports (2015), http://dx.doi.org/10.1016/j.celrep.2015.11.057
report (Gumy et al., 2011). The qPCR showed that the expression
levels of miR-181d, Map1b, and Calm1 in the axons of DRG neurons were decreased, whereas their expression in the cell bodies
remained unchanged (Figure 4E). Furthermore, we transfected
MS2-mCherry with Map1b-30 UTR or Calm1-30 UTR into cultured
P14 DRG neurons expressing either FMRP short hairpin RNA
(shRNA) or a control shRNA. Knockdown of FMRP reduced the
numbers of Map1b-30 UTR and Calm1-30 UTR granules in axons
showing either anterograde or retrograde movement, in addition
to increasing the number of granules showing non-significant
movementwhether stationary, Brownian, or otherwise not
showing displacement over long distances (Figures 4F4H).
However, the average anterograde and retrograde velocities of
Map1b-30 UTR and Calm1-30 UTR granules were not changed
(Figure 4I). Co-expression of shRNA-resistant FMRP (FMRPR)
rescued the axonal transport of Map1b-30 UTR and Calm1-30
UTR granules by silencing FMRP (Figures 4F4H), indicating
that the defect caused by FMRP shRNA was not due to an offtarget effect. In addition, phase contrast images showed that
the DRG neurons affected by knockdown of FMRP by either
AAV infection or electroporation displayed morphologies similar
to those of neurons that were transfected with control shRNA
(Figures S5A and S5B), which excluded the effect of significant
stress in neurons affected by FMRP shRNA. As a control,
b-actin-30 UTR was not localized in the FMRP-GFP-positive
granules, and the axonal transport of b-actin-30 UTR was unaffected by knockdown of FMRP (Figures S5CS5F), suggesting
the specificity of FMRP-mediated mRNA transport. Thus,
FMRP is required for the axonal delivery of miR-181d, Map1b,
and Calm1.
FMRP Deficiency Decreases the Protein Level of MAP1B
and Calmodulin
We further assessed the protein levels of MAP1B and calmodulin in the FMRP-deficient axons of DRG neurons. Consistent
with a previous report (Zang et al., 2009), we found that the
protein level of FMRP was dramatically reduced in the DRGs
and sciatic nerves of the Fmr1I304N mice (Figures 5A and 5B).
We then showed that Fmr1I304N mice had decreased levels of
MAP1B and calmodulin in sciatic nerves, whereas the levels
of these proteins in DRGs were unchanged (Figures 5A and
5B). In microfluidic cultures of E13.5 DRG neurons infected
with AAV-shFMRP-GFP-FLAG, the amounts of MAP1B and
calmodulin in the axon compartment were significantly reduced
(Figures 5C and 5D). Co-expression of AAV-FMRPR-mCherryFLAG rescued the decrease in the protein level of FMRP in
the cell bodies and axons of cultured E13.5 DRG neurons
induced by silencing FMRP (Figures 5C and 5D). The protein
levels of MAP1B and calmodulin, which were reduced by
silencing FMRP in axons, were markedly recovered by expression of FMRPR (Figures 5C and 5D). However, the protein levels
of MAP1B and calmodulin in the cell bodies of cultured E13.5
DRG neurons were not affected by the knockdown or rescue
of FMRP (Figure 5D). Furthermore, the stability and protein
transport of MAP1B and calmodulin were not affected by
silencing FMRP because the protein levels of MAP1B and
calmodulin in cell bodies remained stable (Figure 5), and the
fluorescence recovery after photobleaching (FRAP) of
Please cite this article in press as: Wang et al., FMRP-Mediated Axonal Delivery of miR-181d Regulates Axon Elongation by Locally Targeting Map1b
and Calm1, Cell Reports (2015), http://dx.doi.org/10.1016/j.celrep.2015.11.057
Figure 4. FMRP Deficiency Impairs Axonal Targeting of miR-181d, Map1b, and Calm1
(A and B) A qPCR analysis showed that miR-181d (A), as well as Map1b and Calm1 (B), was decreased in the sciatic nerves, but not in the DRGs, of P14 Fmr1I304N
mice. The results are presented as mean SEM. *p < 0.05 and **p < 0.01 versus the WT (n = 5).
(C) Schematic of the AAV-shFMRP-GFP-FLAG construct used for FMRP knockdown.
(D) A representative image showed highly efficient infection of AAV-shFMRP-GFP-FLAG in a microfluidic culture of E13.5 DRG neurons at 7 DIV (upper panel).
Immunoblotting revealed significant reductions in FMRP in the cell bodies and axons (lower panel). Scale bar, 300 mm.
(E) A qPCR analysis showed that miR-181d, Map1b, and Calm1 were decreased in the axons, but not in the cell bodies, of microfluidic cultured E13.5 DRG
neurons expressing AAV-shFMRP-GFP-FLAG at 7 DIV. The results are presented as mean SEM. *p < 0.05 and **p < 0.01 versus the control (Ctrl) shRNA (n = 4).
8 Cell Reports 13, 114, December 29, 2015 2015 The Authors
Please cite this article in press as: Wang et al., FMRP-Mediated Axonal Delivery of miR-181d Regulates Axon Elongation by Locally Targeting Map1b
and Calm1, Cell Reports (2015), http://dx.doi.org/10.1016/j.celrep.2015.11.057
(FI) Representative live-cell images (F and G) and quantitative data (H) showed that knockdown of FMRP by FMRP shRNA (green) reduced the numbers of
Map1b-30 UTR and Calm1-30 UTR granules (red) moving in both anterograde and retrograde directions, as well as increasing the number of granules displaying
non-significant movement in the axons of cultured P14 DRG neurons at 4 DIV. These phenotypes were rescued by co-expression of FMRPR-BFP-FLAG (blue).
Mean velocity of anterograde and retrograde movements by Map1b-30 UTR and Calm1-30 UTR granules was not significantly changed (I). A 300 s video was
collected, and a kymograph was created to demonstrate the movement of Map1b-30 UTR and Calm1-30 UTR granules. The results are presented as mean SEM.
*p < 0.05 and **p < 0.01 versus the Ctrl shRNA, and #p < 0.05 and ##p < 0.01 versus the indicated treatments (n = 3). Scale bar, 10 mm.
See also Figure S5.
Cell Reports 13, 114, December 29, 2015 2015 The Authors 9
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and Calm1, Cell Reports (2015), http://dx.doi.org/10.1016/j.celrep.2015.11.057
Figure 6. NGF Signaling Regulates the Association of Map1b and Calm1 with FMRP
(A and B) Representative immunoblots (A) and
quantitative data (B) showed that axonal MAP1B
and calmodulin were increased after 2 hr of NGF
treatment in the axon compartment of microfluidic
cultured E13.5 DRG neurons at 7 DIV. These
phenotypes were abolished in the presence of
CHX. Tubulin was used as a loading control. The
results are presented as mean SEM. *p < 0.05
versus vehicle, and #p < 0.05 and ##p < 0.01 versus
the indicated treatments (n = 3).
(C and D) A qPCR analysis showed that miR-181d
(C), as well as Map1b and Calm1 (D), levels were
not changed in either cell bodies or axons after 2 hr
of NGF treatment in the axon compartment of
microfluidic cultured E13.5 DRG neurons at 7 DIV.
The results are presented as mean SEM (n = 3).
(E and F) RT-PCR (E) and qPCR analysis (F)
showed that the association of Map1b and Calm1
with FMRP was decreased in the FLAG immunoprecipitates after NGF treatment in PC12 cells
expressing FMRP-GFP-FLAG. The results are
presented as mean SEM. *p < 0.05 versus
vehicle (n = 4).
(G) A qPCR analysis showed that the association of
miR-181d with FMRP was not affected, as analyzed
in FLAG immunoprecipitates, by NGF treatment in
PC12 cells expressing FMRP-GFP-FLAG. The results are presented as mean SEM (n = 4).
(H and I) Representative immunoblots (H) and
quantitative data (I) showed that the association of
FMRP with Ago2 was not affected by NGF treatment in PC12 cells expressing FMRP-GFP-FLAG.
The results are presented as mean SEM (n = 3).
See also Figure S6.
Please cite this article in press as: Wang et al., FMRP-Mediated Axonal Delivery of miR-181d Regulates Axon Elongation by Locally Targeting Map1b
and Calm1, Cell Reports (2015), http://dx.doi.org/10.1016/j.celrep.2015.11.057
of microtubules (Montenegro-Venegas et al., 2010; VillarroelCampos and Gonzalez-Billault, 2014). The local role of calmodulin-mediated calcium signaling has been investigated in axon
development (Nakamuta et al., 2011); however, our data
demonstrating the axonal distribution of Calm1 in fluorescence
in situ hybridization and the transport of Calm1-30 UTR in livecell imaging are the evidence to support the presence of
Calm1 in axons. The selective knockdown of miR-181d or the
blockade of the interacting domain of Map1b and Calm1 with
miR-181d in the axon compartment increased local levels of
MAP1B and calmodulin and promoted axon elongation, suggesting a physiological role for miR-181d in the axons of primary sensory neurons.
A recent study has also reported that miR-132 is abundant in
the axons of sensory neurons and that it promotes axon
extension by targeting the mRNA of the Ras guanosine triphosphatase activator Rasa1 (Hancock et al., 2014). Considering that miR-181d negatively regulates axon elongation by
affecting Map1b and Calm1 translation, our data demonstrating that different miRNAs cooperatively affect axon elongation by targeting different downstream molecules are
interesting.
Please cite this article in press as: Wang et al., FMRP-Mediated Axonal Delivery of miR-181d Regulates Axon Elongation by Locally Targeting Map1b
and Calm1, Cell Reports (2015), http://dx.doi.org/10.1016/j.celrep.2015.11.057
EXPERIMENTAL PROCEDURES
Animals, Plasmids, miRNA Mimics and Inhibitors, siRNAs, and TPs
All animal experiments were approved by the Committee of Use of Laboratory
Animals in the Institute of Biochemistry and Cell Biology, Chinese Academy of
Sciences. Descriptions of animal information and the synthesis of miRNA
12 Cell Reports 13, 114, December 29, 2015 2015 The Authors
mimics and inhibitors, siRNAs, and TPs are provided in the Supplemental
Experimental Procedures. The construction of plasmids Luc-MS2-24XbsMap1b-30 UTR, Luc-MS2-24Xbs-Calm1-30 UTR, and other plasmids used
for luciferase reporter or shRNA assays and for FMRP and scrambles is also
provided in the Supplemental Experimental Procedures.
In Situ Hybridization
A detailed description of the procedure is provided in the Supplemental Experimental Procedures. miR-181d and its targeting mRNAs were detected as reported, with some modifications (Bicker et al., 2013; Li et al., 2012).
Live-Cell Imaging for mRNA Granules
Electroporated P14 DRG neurons at 4 days in vitro (DIV) were cultured on
glass-bottom dishes on a temperature-controlled workstation (37 C) and
imaged using a UltraView Vox system (PerkinElmer). Regions of axon segments 100150 mm from the cell body were selected, and 150 frames were
collected during a 5 min period. A kymograph was generated using ImageJ
1.4 software (NIH). The number and average velocity of RNA granules in a
kymograph were analyzed using Image-Pro Plus 5.1 software (Media
Cybernetics).
Axon Elongation Assay
A detailed description of the procedure is provided in the Supplemental Experimental Procedures. We detected the axon growth rate as described previously, with some modifications (Ye et al., 2003). Briefly, in microfluidic cultures
of E13.5 DRG neurons at 7 DIV, the clear and longest distal axons in the axon
compartment were selected before and 48 hr after transfection with synthetic
siRNA, miRNA mimics, or an inhibitor. The increase in axon length over 48 hr
was analyzed using Neurolucida software (MBF Bioscience). For NGF-induced
axon growth assays, axons from microfluidic chambers were monitored after
the indicated treatment or after AAV-mediated infection using 10 min intervals
for a total of 60 min using a live-cell imaging microscope, as previously reported (Hengst et al., 2009). Axon length was analyzed using an ImageJ
plug-in (NIH).
RNA Immunoprecipitation
A detailed description of the procedure is provided in the Supplemental Experimental Procedures. Briefly, the supernatants from DRG tissues and cultured
PC12 cells were pre-cleared using protein G sepharose and then incubated
with 5 mg FMRP antibody (Abcam) conjugated to immobilized beads, as previously reported (Zalfa et al., 2003). Total RNA was extracted from the beads
using Trizol and prepared for the subsequent detection of FMRP-associated
mRNAs and miRNAs.
Cell Culture, Transfection, and Immunostaining; RNA Extraction,
RT-PCR, and qPCR; Luciferase Reporter Assay; Immunoblotting;
Drug Treatment; and FRAP
Detailed descriptions of these procedures are provided in the Supplemental
Experimental Procedures.
Statistical Analysis
All data are presented as mean SEM. Statistical analyses were performed
using two-tailed paired Students t tests in Prism (GraphPad). Differences
were considered significant at p < 0.05.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
seven figures, and four tables and can be found with this article online at
http://dx.doi.org/10.1016/j.celrep.2015.11.057.
AUTHOR CONTRIBUTIONS
L.B., B.W., L.P., and X.Z. designed the research. B.W. and L.P. performed the
majority of the experiments with the help of Q.W. and M.W. and analyzed the
Please cite this article in press as: Wang et al., FMRP-Mediated Axonal Delivery of miR-181d Regulates Axon Elongation by Locally Targeting Map1b
and Calm1, Cell Reports (2015), http://dx.doi.org/10.1016/j.celrep.2015.11.057
data. W.-W.L. and N.W. made the microfluidic devices as instructed by X.-Y.J.
L.B., B.W., and L.P. wrote the manuscript.
ACKNOWLEDGMENTS
Hancock, M.L., Preitner, N., Quan, J., and Flanagan, J.G. (2014). MicroRNA132 is enriched in developing axons, locally regulates Rasa1 mRNA, and promotes axon extension. J. Neurosci. 34, 6678.
We thank Dr. Mofang Liu, Dr. Hong Cheng, and Minghua Lu for technical
help to set up the experiments and for comments on the project, and we
thank Dr. Zilong Qiu for the Fmr1I304N mice. This work was supported by
grants from the National Natural Science Foundation of China (31271141
and 31330046), National Basic Research Program of China
(2014CB942802), China Postdoctoral Science Foundation (2012M510096
and 2013T60469), Postdoctoral Foundation from Shanghai Institutes for Biological Sciences (2012KIP505), and Shanghai Postdoctoral Science Foundation (12R21417300).
Kaplan, B.B., Kar, A.N., Gioio, A.E., and Aschrafi, A. (2013). MicroRNAs in the
axon and presynaptic nerve terminal. Front. Cell. Neurosci. 7, 126.
Klemmer, P., Meredith, R.M., Holmgren, C.D., Klychnikov, O.I., Stahl-Zeng, J.,
Loos, M., van der Schors, R.C., Wortel, J., de Wit, H., Spijker, S., et al. (2011).
Proteomics, ultrastructure, and physiology of hippocampal synapses in a fragile X syndrome mouse model reveal presynaptic phenotype. J. Biol. Chem.
286, 2549525504.
REFERENCES
Andreassi, C., Zimmermann, C., Mitter, R., Fusco, S., De Vita, S., Saiardi, A.,
and Riccio, A. (2010). An NGF-responsive element targets myo-inositol monophosphatase-1 mRNA to sympathetic neuron axons. Nat. Neurosci. 13,
291301.
Antar, L.N., Li, C., Zhang, H., Carroll, R.C., and Bassell, G.J. (2006). Local functions for FMRP in axon growth cone motility and activity-dependent regulation
of filopodia and spine synapses. Mol. Cell. Neurosci. 32, 3748.
Bassell, G.J., and Warren, S.T. (2008). Fragile X syndrome: loss of local mRNA
regulation alters synaptic development and function. Neuron 60, 201214.
Bicker, S., Khudayberdiev, S., Wei, K., Zocher, K., Baumeister, S., and
Schratt, G. (2013). The DEAH-box helicase DHX36 mediates dendritic localization of the neuronal precursor-microRNA-134. Genes Dev. 27, 991996.
Cheever, A., and Ceman, S. (2009). Translation regulation of mRNAs by the
fragile X family of proteins through the microRNA pathway. RNA Biol. 6,
175178.
Chen, X.Q., Wang, B., Wu, C., Pan, J., Yuan, B., Su, Y.Y., Jiang, X.Y., Zhang,
X., and Bao, L. (2012). Endosome-mediated retrograde axonal transport of
P2X3 receptor signals in primary sensory neurons. Cell Res. 22, 677696.
Hengst, U., Deglincerti, A., Kim, H.J., Jeon, N.L., and Jaffrey, S.R. (2009).
Axonal elongation triggered by stimulus-induced local translation of a polarity
complex protein. Nat. Cell Biol. 11, 10241030.
Jung, H., Yoon, B.C., and Holt, C.E. (2012). Axonal mRNA localization and local
protein synthesis in nervous system assembly, maintenance and repair. Nat.
Rev. Neurosci. 13, 308324.
Li, L., Wei, D., Wang, Q., Pan, J., Liu, R., Zhang, X., and Bao, L. (2012). MEC-17
deficiency leads to reduced a-tubulin acetylation and impaired migration of
cortical neurons. J. Neurosci. 32, 1267312683.
Lin, A.C., and Holt, C.E. (2008). Function and regulation of local axonal translation. Curr. Opin. Neurobiol. 18, 6068.
McCue, H.V., Haynes, L.P., and Burgoyne, R.D. (2010). The diversity of calcium sensor proteins in the regulation of neuronal function. Cold Spring
Harb. Perspect. Biol. 2, a004085.
McNeill, E., and Van Vactor, D. (2012). MicroRNAs shape the neuronal landscape. Neuron 75, 363379.
Montenegro-Venegas, C., Tortosa, E., Rosso, S., Peretti, D., Bollati, F., Bisbal,
M., Jausoro, I., Avila, J., Caceres, A., and Gonzalez-Billault, C. (2010). MAP1B
regulates axonal development by modulating Rho-GTPase Rac1 activity. Mol.
Biol. Cell 21, 35183528.
Muddashetty, R.S., Nalavadi, V.C., Gross, C., Yao, X., Xing, L., Laur, O., Warren, S.T., and Bassell, G.J. (2011). Reversible inhibition of PSD-95 mRNA
translation by miR-125a, FMRP phosphorylation, and mGluR signaling. Mol.
Cell 42, 673688.
Chi, S.W., Zang, J.B., Mele, A., and Darnell, R.B. (2009). Argonaute HITS-CLIP
decodes microRNA-mRNA interaction maps. Nature 460, 479486.
Nakamuta, S., Funahashi, Y., Namba, T., Arimura, N., Picciotto, M.R., Tokumitsu, H., Soderling, T.R., Sakakibara, A., Miyata, T., Kamiguchi, H., and Kaibuchi, K. (2011). Local application of neurotrophins specifies axons through
inositol 1,4,5-trisphosphate, calcium, and Ca2+/calmodulin-dependent protein
kinases. Sci. Signal. 4, ra76.
Dajas-Bailador, F., Bonev, B., Garcez, P., Stanley, P., Guillemot, F., and Papalopulu, N. (2012). MicroRNA-9 regulates axon extension and branching by targeting Map1b in mouse cortical neurons. Nat. Neurosci. 15, 697699.
Ouyang, Y.B., Lu, Y., Yue, S., Xu, L.J., Xiong, X.X., White, R.E., Sun, X., and
Giffard, R.G. (2012). miR-181 regulates GRP78 and influences outcome from
cerebral ischemia in vitro and in vivo. Neurobiol. Dis. 45, 555563.
Darnell, J.C., Van Driesche, S.J., Zhang, C., Hung, K.Y., Mele, A., Fraser, C.E.,
Stone, E.F., Chen, C., Fak, J.J., Chi, S.W., et al. (2011). FMRP stalls ribosomal
translocation on mRNAs linked to synaptic function and autism. Cell 146,
247261.
Patel, T.D., Jackman, A., Rice, F.L., Kucera, J., and Snider, W.D. (2000). Development of sensory neurons in the absence of NGF/TrkA signaling in vivo.
Neuron 25, 345357.
Dictenberg, J.B., Swanger, S.A., Antar, L.N., Singer, R.H., and Bassell, G.J.
(2008). A direct role for FMRP in activity-dependent dendritic mRNA transport
links filopodial-spine morphogenesis to fragile X syndrome. Dev. Cell 14,
926939.
Saba, R., Storchel, P.H., Aksoy-Aksel, A., Kepura, F., Lippi, G., Plant, T.D., and
Schratt, G.M. (2012). Dopamine-regulated microRNA miR-181a controls
GluA2 surface expression in hippocampal neurons. Mol. Cell. Biol. 32,
619632.
Fineberg, S.K., Kosik, K.S., and Davidson, B.L. (2009). MicroRNAs potentiate
neural development. Neuron 64, 303309.
Sasaki, Y., Gross, C., Xing, L., Goshima, Y., and Bassell, G.J. (2014). Identification of axon-enriched microRNAs localized to growth cones of cortical neurons. Dev. Neurobiol. 74, 397406.
Fusco, D., Accornero, N., Lavoie, B., Shenoy, S.M., Blanchard, J.M., Singer,
R.H., and Bertrand, E. (2003). Single mRNA molecules demonstrate probabilistic movement in living mammalian cells. Curr. Biol. 13, 161167.
Staton, A.A., and Giraldez, A.J. (2011). Use of target protector morpholinos to
analyze the physiological roles of specific miRNA-mRNA pairs in vivo. Nat.
Protoc. 6, 20352049.
Goldie, B.J., Dun, M.D., Lin, M., Smith, N.D., Verrills, N.M., Dayas, C.V., and
Cairns, M.J. (2014). Activity-associated miRNA are packaged in Map1b-enriched exosomes released from depolarized neurons. Nucleic Acids Res. 42,
91959208.
Gumy, L.F., Yeo, G.S., Tung, Y.C., Zivraj, K.H., Willis, D., Coppola, G., Lam,
B.Y., Twiss, J.L., Holt, C.E., and Fawcett, J.W. (2011). Transcriptome analysis
Wang, H., Dictenberg, J.B., Ku, L., Li, W., Bassell, G.J., and Feng, Y. (2008).
Dynamic association of the fragile X mental retardation protein as a messenger
ribonucleoprotein between microtubules and polyribosomes. Mol. Biol. Cell
19, 105114.
Cell Reports 13, 114, December 29, 2015 2015 The Authors 13
Please cite this article in press as: Wang et al., FMRP-Mediated Axonal Delivery of miR-181d Regulates Axon Elongation by Locally Targeting Map1b
and Calm1, Cell Reports (2015), http://dx.doi.org/10.1016/j.celrep.2015.11.057
White, F.A., Silos-Santiago, I., Molliver, D.C., Nishimura, M., Phillips, H., Barbacid, M., and Snider, W.D. (1996). Synchronous onset of NGF and TrkA survival dependence in developing dorsal root ganglia. J. Neurosci. 16, 4662
4672.
Zang, J.B., Nosyreva, E.D., Spencer, C.M., Volk, L.J., Musunuru, K., Zhong, R.,
Stone, E.F., Yuva-Paylor, L.A., Huber, K.M., Paylor, R., et al. (2009). A mouse
model of the human fragile X syndrome I304N mutation. PLoS Genet. 5,
e1000758.
Ye, H., Kuruvilla, R., Zweifel, L.S., and Ginty, D.D. (2003). Evidence in support
of signaling endosome-based retrograde survival of sympathetic neurons.
Neuron 39, 5768.
Zhao, J., Lee, M.C., Momin, A., Cendan, C.M., Shepherd, S.T., Baker, M.D.,
Asante, C., Bee, L., Bethry, A., Perkins, J.R., et al. (2010). Small RNAs control
sodium channel expression, nociceptor excitability, and pain thresholds.
J. Neurosci. 30, 1086010871.
Zalfa, F., Giorgi, M., Primerano, B., Moro, A., Di Penta, A., Reis, S., Oostra, B.,
and Bagni, C. (2003). The fragile X syndrome protein FMRP associates with
BC1 RNA and regulates the translation of specific mRNAs at synapses. Cell
112, 317327.
Zhou, F.Q., Zhou, J., Dedhar, S., Wu, Y.H., and Snider, W.D. (2004). NGFinduced axon growth is mediated by localized inactivation of GSK-3beta
and functions of the microtubule plus end binding protein APC. Neuron 42,
897912.
14 Cell Reports 13, 114, December 29, 2015 2015 The Authors