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. 2010 Jul 21;29(14):2421-32.
doi: 10.1038/emboj.2010.120. Epub 2010 Jun 15.

CIN85 regulates dopamine receptor endocytosis and governs behaviour in mice

Noriaki Shimokawa  1 , Kaisa Haglund, Sabine M Hölter, Caroline Grabbe, Vladimir Kirkin, Noriyuki Koibuchi, Christian Schultz, Jan Rozman, Daniela Hoeller, Chun-Hong Qiu, Marina B Londoño, Jun Ikezawa, Peter Jedlicka, Birgit Stein, Stephan W Schwarzacher, David P Wolfer, Nicole Ehrhardt, Rainer Heuchel, Ioannis Nezis, Andreas Brech, Mirko H H Schmidt, Helmut Fuchs, Valerie Gailus-Durner, Martin Klingenspor, Oliver Bogler, Wolfgang Wurst, Thomas Deller, Martin Hrabé de Angelis, Ivan Dikic
Affiliations

CIN85 regulates dopamine receptor endocytosis and governs behaviour in mice

Noriaki Shimokawa et al. EMBO J. .

Abstract

Despite extensive investigations of Cbl-interacting protein of 85 kDa (CIN85) in receptor trafficking and cytoskeletal dynamics, little is known about its functions in vivo. Here, we report the study of a mouse deficient of the two CIN85 isoforms expressed in the central nervous system, exposing a function of CIN85 in dopamine receptor endocytosis. Mice lacking CIN85 exon 2 (CIN85(Deltaex2)) show hyperactivity phenotypes, characterized by increased physical activity and exploratory behaviour. Interestingly, CIN85(Deltaex2) animals display abnormally high levels of dopamine and D2 dopamine receptors (D2DRs) in the striatum, an important centre for the coordination of animal behaviour. Importantly, CIN85 localizes to the post-synaptic compartment of striatal neurons in which it co-clusters with D2DRs. Moreover, it interacts with endocytic regulators such as dynamin and endophilins in the striatum. Absence of striatal CIN85 causes insufficient complex formation of endophilins with D2DRs in the striatum and ultimately decreased D2DR endocytosis in striatal neurons in response to dopamine stimulation. These findings indicate an important function of CIN85 in the regulation of dopamine receptor functions and provide a molecular explanation for the hyperactive behaviour of CIN85(Deltaex2) mice.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
CIN85 is highly expressed in neurons in which it localizes to post-synaptic sites. (A) Western blot showing CIN85 expression in different mouse brain regions. Lysates (15 μg protein/lane) of the indicated brain regions from wild-type (+/+) and CIN85Δex2 knockout (−/−) mice were separated by 7% SDS–PAGE and immunoblotted with antibodies against CIN85 (CT). Equal protein loading was confirmed by re-blotting with anti-β-actin antibodies. (B) In primary hippocampal neurons derived from wild-type rat, CIN85 (SETA antibody, green) is localized to dendritic spines, in which it is accumulated in post-synaptic compartments, co-localizing with F-actin (red, upper panel) and PSD-95 (red, middle panel), closely juxtaposing the pre-synaptic synaptophysin protein (red, lower panel). Scale bars: 10 μm. (C) CIN85 (green) localizes to actin-positive (red), spine-like structures in dendrites of primary rat striatal neurons. Cells were fixed and stained with antibodies against CIN85 (SETA antibody, green) and with rhodamine phalloidin (red). Scale bars: 10 μm. (D) CIN85 is present in post-synaptic compartments in mouse brains. Synaptosome (SNS) fractions were isolated using the Percoll-step-gradient method from whole brain lysates as described in the Supplementary data. The western blot shows that CIN85, PSD-95 and synaptophysin are present in SNS fractions and that extraction with Triton X-100 solubilizes pre-synaptic synaptophysin into supernatant 1 (S1), whereas CIN85 and PSD-95 are present in post-synaptic fractions (pellet 2, P2, after a second Triton X-100 extraction). Supernatant 2 (S2) is the supernatant after the second Triton X-100 extraction. (E) CIN85 localizes to post-synaptic compartments in the striatum. Synaptosomes from mouse striata were prepared using a sucrose density-gradient method as described in the Supplementary data and immunoblotted with antibodies against CIN85 and PSD-95. WSL, whole striatal lysates; SNS, synaptosomes; PSD-s, supernatant of PSD fraction; PSD-p, pellet of PSD fraction. Each lane contains 15 μg of total protein.
Figure 2
Figure 2
Generation of CIN85Δex2 knockout mice. (A) Gene targeting of the CIN85 locus by removal of exon 2 by homologous recombination. The targeting vector consisted of a 6.0 kb 5′ homology region, the pGNA backbone containing a LacZ/neomycin cassette followed by a 3.0 kb 3′ homology region. The wild-type and targeted loci with their respective restriction sites are indicated. The arrows (1, 2, 3, 4) represent the primers used for genotyping of ES cells and CIN85Δex2 knockout mice (see Supplementary Figure S3 for primer sequences). The 1.2 kb probe used for Southern blot detects an 8 kb product in wild-type mice and a 13 kb product in CIN85Δex2 knockout mice after HindIII digestion. (B) Confirmation of recombinant targeting events in ES cell clones by Southern blot. Genomic DNA from wild-type ES cells and targeted ES cell clones (D3 and D8) was digested with HindIII and analysed by Southern blotting using the probe indicated in (A). The two targeted ES cell clones showed a 13 kb mutant band, indicating successful integration of the targeting vector by homologous recombination, whereas the wild-type ES cells, as expected, showed the 8 kb band of the wild-type locus (NC). An extended form of a linearized targeting vector was used as a positive control (PC). (C) Confirmation of the targeting event in CIN85Δex2 knockout mice by PCR, using a mixture of primers 1, 2 and 3. CIN85Δex2 knockout mice (−/−) give rise to the expected 1.5 kb product (primers 2 and 3), wild-type mice (+/+) the expected 0.5 kb product (primers 1 and 3) and heterozygous mice (+/−) to both products (primers 1, 2 and 3). Primer sequences are found in Supplementary Figure S3A. (D) Whole tissue lysates from mouse brain, thymus and spleen (200 μg/lane) from wild-type (+/+) and CIN85Δex2 knockout mice (−/−) were subjected to immunoprecipitation with anti-CIN85 (CT) antibodies. Subsequent western blot analysis with antibodies against CIN85 confirms the removal of CIN85-xl and CIN85-l in the brain and of CIN85-l in thymus and spleen in CIN85Δex2 knockouts. CIN85-ΔA remains in thymus and spleen. Some smaller isoforms present in thymus and spleen are also removed in CIN85Δex2 knockouts.
Figure 3
Figure 3
CIN85Δex2 knockout mice display metabolic and behavioural phenotypes. (A) CIN85Δex2 mutant mice display abnormalities in several metabolic parameters. Both male (M) and female (F) CIN85Δex2 knockout mice (−/−) showed a leaner body mass, as well as a decrease in total and subcutaneous fat content as compared with their wild-type littermates (+/+). Female CIN85Δex2 mice also showed a significantly increased energy uptake compared with their wild-type littermates (+/+). The graphs are based on the values in the table presented in Supplementary Figure S4B. *P<0.05. (B) CIN85Δex2 knockout mice are hyperactive. Behavioural analysis of 8–10-week-old CIN85Δex2 (−/−) mice and wild-type (+/+) littermate controls (n=22–26 per genotype) by the modified hole-board test (described in detail in the Supplementary data). The CIN85Δex2 knockout mice showed significantly increased forward locomotor activity, speed, turning frequency, board entry frequency and hole exploration frequency, as compared with wild-type controls; *P<0.05; **P<0.01, ***P<0.001, +/+ versus −/−.
Figure 4
Figure 4
Impaired endocytic internalization of D2 dopamine receptors in mice lacking CIN85 expression in the CNS. (A) The levels of dopamine (DA) and its metabolites (3,4-dihydroxyphenylacetic acid, DOPAC and homovanillic acid, HVA) are increased in the striatum of CIN85Δex2 knockout mice. The contents of total striatal dopamine, DOPAC and HVA were determined by high performance liquid chromatography using an electro-chemical (HPLC-EC) detector as described in the Supplementary data. Differences between wild-type (+/+) and CIN85Δex2 knockout mice (−/−) were analysed using a Student's t-test, with the level of significance set at *P<0.05. +/+: n=4; −/−: n=4. (B) D2 dopamine receptor endocytosis is impaired in striatal neurons deficient of CIN85. Representative immunoblots (left panel) and quantification (right panel) showing the amount of surface-associated D2 dopamine receptors (D2DR) in cultured striatal neurons after dopamine stimulation. Striatal neurons were treated with dopamine hydrochloride (3-hydroxytyramine, 20 μM) for the indicated times before biotinylation. The remaining cell surface proteins were subsequently isolated with avidin and analysed by western blotting. The quantification of surface-associated D2DRs after dopamine stimulation is normalized to and depicted as the reduction of band intensity as compared with non-stimulated cells at the indicated times. Differences between wild type (+/+) and CIN85Δex2 knockout (−/−) were analysed by ANOVA and Duncan multiple range test for post hoc between group comparisons, with the level of significance set at *P<0.05. Glutamate receptor-2 (GluR-2) was used as an internal control. +/+: n=3; −/−: n=3. (C) Endocytosis of D1 dopamine receptors (D1DR) is not affected by loss of CIN85. Representative immunoblots and quantification showing the amount of surface D1DR in cultured striatal neurons before and after dopamine stimulation. Striatal neurons were treated with dopamine hydrochloride (20 μM) for 1 h before biotinylation. The remaining cell surface proteins were subsequently isolated with avidin and analysed by western blotting. D1DRs of cultured striatal neurons from wild-type (+/+) and CIN85Δex2 knockout mice (−/−) were internalized 69±5.7% and 63±8.8%, respectively. Glutamate receptor-2 (GluR-2) was used as an internal control. +/+: n=3; −/−: n=3. (D) CIN85 co-clusters with D2 dopamine receptors (D2DRs) at punctuate synapse-like structures in primary rat striatal neurons. Cells were fixed and stained with antibodies against CIN85 (SETA antibody, green) and D2DRs (red). Scale bars: 5 μm.
Figure 5
Figure 5
Defective complex formation between endophilins and D2 dopamine receptors in CIN85Δex2 knockouts. (A) Locomotor responses (left panel) and [3H]spiperone binding (right panel) of quinpirole-treated mice. Mice of the indicated genotypes were repeatedly injected with quinpirole (0.5 mg/kg, s.c.) or saline in 48 h intervals until a total of eight injections were completed as described in the Supplementary data. Immediately after each injection, the locomotor activity was measured for 30 min using an actimeter. The left panel shows the distance travelled (m) during the last 5 min of the 30-min testing period. Data are presented as the mean±s.e.m. (n=3). The difference between saline-injected wild types and the other genotypes/treatments were analysed by ANOVA and Student's t-test, with the level of significance set at *P<0.05. Right panel: After the final measurement of locomotor activity, the membrane fraction of the striatum (12.5 μg/reaction) was prepared and incubated with [3H]spiperone. Concentrations of [3H]spiperone ranging from 10 to 500 pM were used, and non-specific binding was determined in the presence of D-butaclamol. Data are presented as the mean±s.e.m. (n=3). The differences between saline-injected wild types and the other genotypes/treatments were analysed by ANOVA and Duncan multiple range test for post hoc between group comparisons, with the level of significance set at *P<0.05. (B) Total distance travelled after a threshold dose of quinpirole; 19–26-week-old CIN85Δex2 (−/−) and wild-type (+/+) littermate control mice (n=10–15 per genotype) were subjected to the modified hole-board test 30 min after quinpirole injection (0 or 0.01 mg/kg i.p.) as described in the Supplementary data. (C) Total distance travelled after a low dose of haloperidol; 14–36-week-old CIN85Δex2 (−/−) and wild-type (+/+) littermate control mice (n=10–13 per genotype) were subjected to the modified hole-board test 30 min after haloperidol injection (0 or 0.05 mg/kg i.p.) as described in the Supplementary data. *P<0.05, vehicle versus haloperidol, Bonferroni's post-tests. (D) Total distance travelled after a low dose SCH23390; 10–11-week-old CIN85Δex2 (−/−) and wild-type (+/+) littermate control mice (n=10–12 per genotype) were subjected to the modified hole-board test 30 min after SCH23390 injection (0 or 0.03 mg/kg i.p.) as described in the Supplementary data. *P<0.05, **P<0.01, vehicle versus SCH23390, Bonferroni's post-tests. (E) Synaptosome fractions were prepared from wild-type and CIN85Δex2 knockout mouse striata by sucrose density-gradient centrifugation. In total, 25 μg of fractionated protein was analysed by immunoprecipitation (IP) followed by western blotting (WB) using the indicated antibodies. CIN85 co-immunoprecipitates with D2DRs, the endocytic proteins endophilin and dynamin, as well as the post-synaptic density protein PSD-95, in wild type, but not in CIN85Δex2 knockout samples. The upper band evident in the dynamin IP may be CIN85-xl. (F) The level of endophilin in complex with the D2DR is decreased in the striatal synaptosome fractions in CIN85Δex2 knockout mice. Synaptosomes of wild-type (+/+) and CIN85Δex2 knockout (−/−) mouse striata were prepared as in (E) and subjected to IP with anti-D2DR antibodies and subsequent immunoblot analysis with antibodies against endophilin, dynamin, PSD-95 and the D2DR (left and middle panels). Western blots from four independent experiments are shown in the middle panel. Relative intensities of the endophilin levels in CIN85Δex2 knock outs compared with wild type for each of the four experiments are indicated. A graph depicting the mean value of the relative endophilin intensities in CIN85Δex2 knockout (−/−) compared with wild-type (+/+) mice from the four experiments is shown in the right panel. The value is presented as mean±s.e. Differences between wild-type (+/+) and CIN85Δex2 knockout mice (−/−) were analysed using a Student's t-test, with the level of significance set at *P<0.05. +/+: n=4; −/−: n=4.
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