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Identification of a Vav2-dependent mechanism for GDNF/Ret control of mesolimbic DAT trafficking

Nature Neuroscience volume 18, pages 1084–1093 (2015)Cite this article

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Abstract

Dopamine (DA) homeostasis is essential for a variety of brain activities. Dopamine transporter (DAT)-mediated DA reuptake is one of the most critical mechanisms for normal DA homeostasis. However, the molecular mechanisms underlying the regulation of DAT activity in the brain remain poorly understood. Here we show that the Rho-family guanine nucleotide exchange factor protein Vav2 is required for DAT cell surface expression and transporter activity modulated by glial cell line–derived neurotrophic factor (GDNF) and its cognate receptor Ret. Mice deficient in either Vav2 or Ret displayed elevated DAT activity, which was accompanied by an increase in intracellular DA selectively in the nucleus accumbens. Vav2−/− mice exposed to cocaine showed reduced DAT activity and diminished behavioral cocaine response. Our data demonstrate that Vav2 is a determinant of DAT trafficking in vivo and contributes to the maintenance of DA homeostasis in limbic DA neuron terminals.

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Figure 1: Vav2 is a VTA-enriched protein required for DA homeostasis in mesolimbic neuron terminals.
Figure 2: Enhanced surface expression and DAT activity in Vav2−/− mice.
Figure 3: Vav2 is a substrate of Ret receptor tyrosine kinase contributing to the regulation of DAT trafficking and function.
Figure 4: GDNF/Ret/Vav2 signaling modulates DAT activity.
Figure 5: Ret interacts with DAT.
Figure 6: Vav2 is required for DAT trafficking in the context of cocaine.
Figure 7: Altered cocaine-induced behavioral sensitization in Vav2−/− mice.

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Acknowledgements

We thank X.C. Zhen (Soochow University) for providing the DAT cDNA, C. He (Second Military Medical University, China) for human RET cDNA, H.L. Hu and Z.L. Qiu (Institute of Neuroscience, Chinese Academy of Sciences) for the AAV virus constructs, L.P. Cheng (Institute of Neuroscience, Chinese Academy of Sciences) for the Ret mutant mice and the Riken BioResource Center for the TH-GFP transgenic mice. We also thank the Optical Imaging Center of the Institute of Neuroscience, Chinese Academy of Sciences, for technical support in confocal microscopy and the Behavioral Core of ION for providing assistance. This work was supported by grants from the National Key Basic Research Program of China (2015CB553500, 2011CB504102), Natural Science Foundation of China (31123002, 31321091 and 30621130075), Beijing Institute for Brain Disorders (BIBD-PXM2013_014226_07_000084) and Strategic Priority Research Program (B) of the Chinese Academy of Sciences (XDB01020300). X.R.B.'s work has been funded by the Spanish Ministry of Economy and Competitiveness (SAF2009-07172, SAF2012-3171, RD06/0020/0001 and RD12/0036/0002), the Castilla-León Autonomous Government (CSI101U13) and Spanish Association of Science Communication.

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Author notes

  1. Shuyong Zhu

    Present address: Present address: Institute of Virology, Hannover Medical School, Hannover, Germany.,

  2. Shuyong Zhu, Chengjiang Zhao and Yingying Wu: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Neuroscience, State Key Laboratory of Neuroscience, Chinese Academy of Sciences Center for Excellence in Brain Science, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

    Shuyong Zhu, Chengjiang Zhao, Yingying Wu, Qiaoqiao Yang, Aiyun Shao, Tiepeng Wang, Jianfu Wu, Yanqing Yin, Yandong Li, Jincan Hou, Xinhua Zhang & Jiawei Zhou

  2. University of the Chinese Academy of Sciences, Shanghai, China

    Chengjiang Zhao & Yingying Wu

  3. Department of Anatomy, Histology and Embryology, Shanghai Medical School, Fudan University, Shanghai, China

    Qiaoqiao Yang & Guomin Zhou

  4. Co-innovation Center of Neuroregeneration, School of Medicine, Nantong University, Nantong, China

    Xinhua Zhang & Xiaosong Gu

  5. Center of Parkinson's Disease, Beijing Institute for Brain Disorders, Beijing, China

    Xiaomin Wang

  6. Centro de Investigación del Cáncer, Consejo Superior de Investigaciones Científicas, University of Salamanca, Salamanca, Spain

    Xosé R Bustelo

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Contributions

S.Z. conducted most of the biochemical experiments and the data analysis, and C.Z. conducted most of the in vivo experiments and the data analysis. Y.W. conducted some of the in vivo and in vitro experiments. Q.Y. contributed to the in vitro experiments. A.S. and J.W. contributed to mutant and protein interaction analysis. T.W. contributed to the CPP assay. Y.Y. conducted in situ hybridization; Y.Y., Y.L. and J.H. conducted genotyping; X.Z. contributed to in vitro experiments. X.G., G.Z. and X.W. provided advice; X.R.B. provided Vav2−/− and Vav3−/− mice and Vav2 and 3 constructs; and J.Z. supervised the project and wrote the manuscript.

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Correspondence to Jiawei Zhou.

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Integrated supplementary information

Supplementary Figure 1 Vav2, but not Vav3, is preferentially expressed in the VTA of adult mice and is required for the maintenance of dopamine homeostasis.

a, Immunoblotting reveals the Vav2 expression levels in the ventral tagmental area (VTA) is significantly higher than in the substantia nigra (SN) of adult brain. b, Quantitative data of a. (n = 4); t(6)= 6.317, *P = 0.0007. c. Western analysis of brain tissue lysates from adult mouse brain shows that Vav3 is ubiquitously expressed in major brain regions. The experiments were repeated at least twice. d-f, Measurement of DA, 5-HT and their metabolites levels were performed in the olfactory bulb (OB) of Vav2 mutants (d), nucleus accumbens (NAc) (e) and dorsal striatum (dSTR) (f) of Vav3 mutant and wild-type mice. d, n = 3 per group; unpaired two-tailed t-test, t(4) = 0.8303, P = 0.4530 for DA. e, f, n = 10 for WT, n = 8 for Vav3 mutants. unpaired two-tailed t-test. e, t(16) = 1.196, P = 0.2490 for DA. f, t(16) = 0.02254, P = 0.9823 for DA. g-h, Tyrosine hydroxylase immunosignal density was measured in the NAc (g) and dSTR (h) of Vav2 mutant and WT mice. (g, h, n = 4). unpaired two-tailed t-test. g, t(46) = 1.913, P = 0.0620. h, t(60) = 1.108, P = 0.2721. i, Western blotting shows that AAV-mediated overexpression of Vav2 following injection in the VTA or SN, validating the transfection efficiency in vivo. j, [3H]DA uptake assays were performed in the NAc or dSTR synapotosome preparation from Vav3-null and wild-type. (n = 3 mice per group). unpaired two-tailed t-test. t(4) = 0.8844, P = 0.4264 for NAc, t(4) = 0.2033, P = 0.8488 for dSTR. k, Immunoblotting reveals that the levels of Vav2 expression were significantly down-regulated 48, 72 h following transfection with shVav2 #2 in N2a cells. Error bars represent s.e.m. i, k are cropped blot images from their corresponding full-length blots presented in Supplementary Figure 12.

Supplementary Figure 2 DA biosynthesis and degradation in the NAc of Vav2 mutant mice are not significantly altered.

a, Western analysis of VTA tissue lysates from adult Vav2-null mouse brain, in which rabbit monoclonal anti-Vav2 antibody was used, shows the absence of Vav2 protein. The experiments were repeated at least twice. b, Quantitative data of a. There is no significant difference in the levels of TH and VMAT2 protein expression between genotypes. unpaired two-tailed t-test. (n = 3 per group); t(4) = 1.460, P = 0.2182. *P = 0.2182. c-e, Western analysis of phosphor-TH (ser 40) expression in the NAc (c, d) or VTA (c, e) of Vav2 mutant and WT mice. d, e, Quantitative data of (c). unpaired two-tailed t-test. (d, n = 6 mice per group, t(10) = 0.8557, P = 0.4122, e, n = 5 mice per group, t(8) = 0.3065, P = 0.7670). f, The accumulation of l-dopa in the NAc was measured in the animals exposed to NSD-1015, an aromatic acid decarboxylase (AADC) inhibitor, using HPLC. Lower levels of L-dopa were seen in the NAc of Vav2 mutant mice, suggesting that TH enzymatic activity was markedly inhibited. unpaired two-tailed t-test. (n = 5 mice per group); t(8) = 2.400, *P = 0.0432. g, h, qPCR analysis of mRNA expression of MAO-A (g) and MAO-B (h), which are responsible for enzymatic degradation of 5-HT, NE and DA in the VTA and NAc of Vav2 mutant and WT mice. unpaired two-tailed t-test. (n = 3 mice). (g, t(4) = 0.3998, P = 0.7097 for VTA, t(4) = 0.06329, P = 0.9526 for NAc; h, t(4) = 2.946, P = 0.0421 for VTA, t(4) = 2.382, P = 0.0758 for NAc). i, qPCR analysis of MAO-A and Vav2 mRNA expression in MN9D dopaminergic cell line transfected with shVav2 #2. unpaired two-tailed t-test. (n = 3 independent experiments). t(4) = 35.55, *P < 0.0001 for Vav2, t(4) = 0.0, *P > 0.9999 for MAO-A. j, Measurement of enzymatic activities of MAO-A were performed in the VTA and NAc of Vav2 mutant and WT mice. unpaired two-tailed t-test. (n = 3 mice per group). t(4) = 0.1290, P = 0.9036 for VTA, t(4) = 1.202, P =0.2958 for NAc. k, Immunoblotting reveals that the levels of VMAT2 expression were significantly altered in the striatum of Vav2 mutant mice compared with wild-type counterparts. l, Quantitative data shown in k. unpaired two-tailed t-test. (n = 6 mice per group). t(10) = 0.02854, P = 0.9778. m, Full-length gel showing immunoblotting analysis of phosphor-Thr53-DAT expression. Their expression levels were not significantly changed between genotypes in various brain regions. n, Quantitative data shown in l. unpaired two-tailed t-test. (n = 5 mice). t(8) = 1.290, P = 0.2331 for NAc, t(8) = 1.849, P = 0.1016 for dSTR, t(8) = 1.146, P = 0.2847 for VTA. Error bars represent s.e.m. n.s., not significant. k shows cropped blot images from their corresponding full-length blot presented in Supplementary Figure 12.

Supplementary Figure 3 Quantification of total DAT expression in synaptosomal preparations derived from Vav2–/– mice and WT control under various experimental conditions.

a-c, correspond to Fig. 2f-i. (a, n = 4). Two-way ANOVA with Bonferroni's multiple comparisons test. F(1, 12) = 1.067, P > 0.9999 for Nac, P = 0.6297 for dSTR. (b, c, n = 3). One-way ANOVA with Bonferroni's multiple comparisons test. b, F(2, 6) = 0.2230, P = 0.7976 between control and shVav2 #1, P = 0.8972 between control and rescue. c, F(2, 6) = 0.1670, P > 0.9999 between control and RFP-Vav2, P > 0.9999 between control and Y172F. d, corresponds to Fig. 3b. (n = 3). Two-way ANOVA with Bonferroni's multiple comparisons test. F(1, 8) = 0.1616. P > 0.9999 between NAc WT and NAc Ret+/−, P > 0.9999 between dSTR WT and dSTR Ret+/−. e, f, correspond to Fig. 4e, g. (n = 3). e, unpaired two-tailed t-test. (f, n = 2). Two-way ANOVA with Bonferroni's multiple comparisons test. F(1, 4) = 0.2292, P > 0.9999 between scrambled control and scrambled GDNF, P > 0.9999 between shVav2 control and shVav2 GDNF. g, h, correspond to Fig. 6b, c. (g, n = 2). Two-way ANOVA with Bonferroni's multiple comparisons test. F(1, 4) = 0.5856, P > 0.9999 between WT saline and WT cocaine, P> 0.9999 between Vav2 mutant saline and Vav2 mutant cocaine. (h, n = 4). Two-way ANOVA with Sidak's multiple comparisons test. F(1, 12) = 0.3031. P = 0.9998 between WT saline and WT cocaine, P = 0.9983 between Vav2 mutant saline and Vav2 mutant cocaine. n.s. no significance. The data are expressed as ratios of total DAT to β-actin. Error bars represent s.e.m.

Supplementary Figure 4 Ret+/– mutant mice exhibit no significant alterations in the levels of DA, 5-HT or their metabolites in the olfactory bulb and expression levels of DAT and phospho-Thr53-DAT in the NAc and VTA.

a, The levels of DA, 5-HT and their metabolites were measured in the olfactory bulb of Ret+/- and wild-type mice. The olfactory bulb (OB) is one of brain regions enriched in dopaminergic neurons. unpaired two-tailed t-test. (n=5 mice per group). t(8) = 0.8544. P = 0.4177 for DA. b, Western blotting reveals that the levels of phosphor-Thr53-DAT expression are not significantly altered in the NAc and VTA between genotypes. b shows cropped blot images from their corresponding full-length blots presented in Supplementary Figure 12. c, Quantitative data of b. unpaired two-tailed t-test. (n = 3 mice per group). t(4) = 1.092, P = 0.3364 for NAc, t(4) = 1.672, P = 0.1698 for VTA. Error bars represent s.e.m.

Supplementary Figure 5 Vav2, but not Vav3, strongly binds to Ret.

a, Y2H assays reveal that Vav2 strongly binds to Ret intracellular domain (ICD). In contrast, Vav3 weakly interacts with Ret ICD. b, Co-IP assays show that Ret-flag is immunoprecipitated with Myc-Vav2. c, [3H]DA uptake assays reveal that overexpression of TrkA in HEK293 cells stably expressing YFP-DAT does not significantly alter DAT activity compared to control. (n = 4). One-way ANOVA with Bonferroni's multiple comparisons test: F(2, 9) = 40.37, P < 0.0001; post hoc: t(9) = 6.610, *P = 0.0002. Error bars represent s.e.m. d, Western blotting reveals that treatment of N2a cells with 100 ng/ml GDNF increases the phosphorylation of Ret at Tyr905 site. e, Western blotting reveals that overexpression of Flag-tagged Ret elevates the levels of phosphor-Ret Tyr905 in HEK293T cells. These experiments were repeated at least three times. b, d, e are cropped blot images from their corresponding full-length blots presented in Supplementary Figure 12.

Supplementary Figure 6 Involvement of PKC in the Vav2-mediated DAT activity.

a, shVav2 #2-induced Vav2 knockdown attenuates PMA-induced reduction of DAT activity in N2a cells stably expressing RFP-DAT and GFRa1. Two-way ANOVA with Bonferroni‘s post-hoc test was performed. (n = 10). Main effect of genotype (F(1, 36) = 38.06, P <0.0001), treatment (F(1, 36) = 58.23, P <0.0001) and interaction (F(1, 36) = 4.376, P = 0.0436); post hoc: t(36) = 6.875, P < 0.0001 for Scrambled group, t(36) = 3.917, P = 0.0008 for shVav2 group. *P < 0.05. b, Treatment of N2a cells stably expressing RFP-DAT and GFRa1 with either PMA (1 mm, 30 min) or GDNF (100 ng/ml, 30 min) significantly down-regulate DAT-mediated DA uptake. These effects could not be significantly attenuated by pretreatment with PKC inhibitor GF-109203X (1 mM, 30 min). One way ANOVA with Tukey's multiple comparisons test was performed. (n = 11). F(3, 40) = 28.13, P<0.0001; q(40)=12.58, P < 0.0001 between control and PMA, q(40) = 8.185, P < 0.0001 between Control and GDNF, q(40) = 3.219, P = 0.1208 between GDNF and GDNF+GF. *P <0.05. Error bars represent s.e.m.

Supplementary Figure 7 Characterization of interaction among Vav2, DAT and Ret.

a, Colony growth assay on the SD-His medium showed the DAT-Cub-LexA-VP16 and Ret-NubG were expressed with no self-activation of the histidine reporter gene expression. b, Co-IP assays show that treatment with PP2 does not alter their interaction between Ret and DAT in HEK293 cells. c, Immunoblotting reveals that activation of Ret signaling by overexpression of Ret-Flag in HEK293 cells does not cause significant alteration in the levels of phosphor-Thr53-DAT. These experiments were repeated at least twice. c shows cropped blot images from their corresponding full-length blots presented in Supplementary Figure 12.

Supplementary Figure 8 Altered cocaine-induced behavioral response in Vav2–/– mice.

a, Measurement of extracellular DA concentrations in the NAc of WT and Vav2 mutant mice using in vivo microdialysis combined with HPLC analysis. The mice received single injection of cocaine (20 mg/kg, i.p.) as indicated by an arrow. The effects of cocaine on the dopamine output were presented as relative to the baseline (the average concentration of four consecutive stable samples defined as 100%). (n = 8 for WT, n = 11 for Vav2 mutants). Two-way ANOVA test. F(7, 136) = 0.5922, P = 0.7614. b, The spontaneous activities are recorded during a 24-h period. There is no alteration between genotypes (n = 5 mice for WT, n = 7 mice for mutants). Two-way ANOVA test. (no effect of genotype (F(1, 170) = 0.02575, P = 0.8727) and interaction (F(16, 170) = 1.017, P = 0.4410)). c, d, The locomotor activity of Vav2 mutants administrated with nomifensine (10 mg/kg, i.p) is altered compared with WT mice (n = 13 mice for WT, n = 14 mice for mutants). c, Two-way ANOVA with Bonferroni’s post test. main effect of genotype (F(1, 375) = 9.091, P = 0.0027) and interaction (F(14, 375) = 3.072, P = 0.0002); d, F(1, 50) = 8.137, *P = 0.0003. e, The performance in sucrose preference test is not altered in Vav2 mutants. (n = 9 mice per group). unpaired two-tailed t-test, t(16) = 0.1830, P = 0.8571. f, Recent memory of Vav2 mutant and WT mice is expressed as the percentage of time spent in freezing during contextual fear conditioning test. (n = 16 mice per group). unpaired two-tailed t-test, t(30) = 1.114, P = 0.2739. g, The performance in CPP is not significantly altered in Vav3 mutants (n = 8 mice per group), t(14) = 1.304, P = 0.2132. h, i. The response to acoustic startle stimulation (h) and prepulse inhibition (i) is not significantly altered in Vav2 mutant mice compared with control. (n = 17 mice for WT, n = 19 mice for Vav2 mutant). h, unpaired two-tailed t-test, t(34) = 1.530, P = 0.1354. i, Two-way ANOVA test. (no effect of genotype (F(1, 102) = 0.4790, P = 0.4904) and interaction (F(2, 102) = 0.06487, P = 0.9372)). Error bars represent s.e.m.

Supplementary Figure 9 A proposed model for how Vav2 regulates GDNF/Ret signaling, thereby modulating DAT internalization and DA homeostasis, in the mesolimbic DA neuron terminals of adult mice under physiological conditions.

Our data also suggest that cocaine could influence the trafficking of DAT in a way that intersects with RET/Vav2, given that the conformation of DAT when bound to cocaine disables the DAT/RET interaction, with no effect if Vav2 is absent. Moreover, elevated extracellular DA produced by cocaine acting normally to produce RET dephosphorylation and reduced Vav2 associations, and which is of no significance in the context of a Vav2 knockout.

Supplementary Figure 10 Full-length images of cropped immunoblots presented in Figures 1a,c,e, 2c–f,h,i and 3b,g,h,j.

Supplementary Figure 11 Full-length images of cropped immunoblots presented in Figures 4b,c,e,f, 5b,c,e and 6a,f.

Supplementary Figure 12 Full-length images of cropped immunoblots presented in Supplementary Figures 1i,k, 2k, 4b, 5b,d,e and 7c.

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Zhu, S., Zhao, C., Wu, Y. et al. Identification of a Vav2-dependent mechanism for GDNF/Ret control of mesolimbic DAT trafficking. Nat Neurosci 18, 1084–1093 (2015). https://doi.org/10.1038/nn.4060

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