RGS 9‐2 rescues dopamine D2 receptor levels and signaling in DYT 1 dystonia mouse models
2018; Springer Nature; Volume: 11; Issue: 1 Linguagem: Inglês
10.15252/emmm.201809283
ISSN1757-4684
AutoresPaola Bonsi, Giulia Ponterio, Valentina Vanni, Annalisa Tassone, Giuseppe Sciamanna, Sara Migliarini, Giuseppina Martella, Maria Meringolo, Benjamin Dehay, Évelyne Doudnikoff, Venetia Zachariou, Rose E Goodchild, Nicola Biagio Mercuri, Marcello D’Amelio, Massimo Pasqualetti, Erwan Bézard, Antonio Pisani,
Tópico(s)Neurotransmitter Receptor Influence on Behavior
ResumoResearch Article14 December 2018Open Access Source DataTransparent process RGS9-2 rescues dopamine D2 receptor levels and signaling in DYT1 dystonia mouse models Paola Bonsi Corresponding Author Paola Bonsi [email protected] orcid.org/0000-0001-5940-9028 Laboratory of Neurophysiology and Plasticity, IRCCS Fondazione Santa Lucia, Rome, Italy Search for more papers by this author Giulia Ponterio Giulia Ponterio Laboratory of Neurophysiology and Plasticity, IRCCS Fondazione Santa Lucia, Rome, Italy Department of Systems Medicine, University Tor Vergata, Rome, Italy Search for more papers by this author Valentina Vanni Valentina Vanni Laboratory of Neurophysiology and Plasticity, IRCCS Fondazione Santa Lucia, Rome, Italy Department of Systems Medicine, University Tor Vergata, Rome, Italy Search for more papers by this author Annalisa Tassone Annalisa Tassone Laboratory of Neurophysiology and Plasticity, IRCCS Fondazione Santa Lucia, Rome, Italy Department of Systems Medicine, University Tor Vergata, Rome, Italy Search for more papers by this author Giuseppe Sciamanna Giuseppe Sciamanna Laboratory of Neurophysiology and Plasticity, IRCCS Fondazione Santa Lucia, Rome, Italy Department of Systems Medicine, University Tor Vergata, Rome, Italy Search for more papers by this author Sara Migliarini Sara Migliarini Unit of Cell and Developmental Biology, Department of Biology, University of Pisa, Pisa, Italy Search for more papers by this author Giuseppina Martella Giuseppina Martella Laboratory of Neurophysiology and Plasticity, IRCCS Fondazione Santa Lucia, Rome, Italy Department of Systems Medicine, University Tor Vergata, Rome, Italy Search for more papers by this author Maria Meringolo Maria Meringolo Laboratory of Neurophysiology and Plasticity, IRCCS Fondazione Santa Lucia, Rome, Italy Department of Systems Medicine, University Tor Vergata, Rome, Italy Search for more papers by this author Benjamin Dehay Benjamin Dehay orcid.org/0000-0003-1723-9045 Université de Bordeaux, Institut des Maladies Neurodégénératives, UMR 5293, Bordeaux, France CNRS, Institut des Maladies Neurodégénératives, UMR 5293, Bordeaux, France Search for more papers by this author Evelyne Doudnikoff Evelyne Doudnikoff Université de Bordeaux, Institut des Maladies Neurodégénératives, UMR 5293, Bordeaux, France CNRS, Institut des Maladies Neurodégénératives, UMR 5293, Bordeaux, France Search for more papers by this author Venetia Zachariou Venetia Zachariou Department of Neuroscience, Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA Search for more papers by this author Rose E Goodchild Rose E Goodchild orcid.org/0000-0002-4524-5728 Department of Neurosciences, VIB-KU Leuven Center for Brain and Disease Research, KU Leuven, Leuven, Belgium Search for more papers by this author Nicola B Mercuri Nicola B Mercuri Laboratory of Neurophysiology and Plasticity, IRCCS Fondazione Santa Lucia, Rome, Italy Department of Systems Medicine, University Tor Vergata, Rome, Italy Search for more papers by this author Marcello D'Amelio Marcello D'Amelio Laboratory Molecular Neurosciences, IRCCS Fondazione Santa Lucia, Rome, Italy Unit of Molecular Neurosciences, Department of Medicine, University Campus-Biomedico, Rome, Italy Search for more papers by this author Massimo Pasqualetti Massimo Pasqualetti Unit of Cell and Developmental Biology, Department of Biology, University of Pisa, Pisa, Italy Center for Neuroscience and Cognitive Systems @UniTn, Istituto Italiano di Tecnologia, Rovereto, Italy Search for more papers by this author Erwan Bezard Erwan Bezard orcid.org/0000-0002-0410-4638 Université de Bordeaux, Institut des Maladies Neurodégénératives, UMR 5293, Bordeaux, France CNRS, Institut des Maladies Neurodégénératives, UMR 5293, Bordeaux, France Search for more papers by this author Antonio Pisani Corresponding Author Antonio Pisani [email protected] orcid.org/0000-0002-8432-594X Laboratory of Neurophysiology and Plasticity, IRCCS Fondazione Santa Lucia, Rome, Italy Department of Systems Medicine, University Tor Vergata, Rome, Italy Search for more papers by this author Paola Bonsi Corresponding Author Paola Bonsi [email protected] orcid.org/0000-0001-5940-9028 Laboratory of Neurophysiology and Plasticity, IRCCS Fondazione Santa Lucia, Rome, Italy Search for more papers by this author Giulia Ponterio Giulia Ponterio Laboratory of Neurophysiology and Plasticity, IRCCS Fondazione Santa Lucia, Rome, Italy Department of Systems Medicine, University Tor Vergata, Rome, Italy Search for more papers by this author Valentina Vanni Valentina Vanni Laboratory of Neurophysiology and Plasticity, IRCCS Fondazione Santa Lucia, Rome, Italy Department of Systems Medicine, University Tor Vergata, Rome, Italy Search for more papers by this author Annalisa Tassone Annalisa Tassone Laboratory of Neurophysiology and Plasticity, IRCCS Fondazione Santa Lucia, Rome, Italy Department of Systems Medicine, University Tor Vergata, Rome, Italy Search for more papers by this author Giuseppe Sciamanna Giuseppe Sciamanna Laboratory of Neurophysiology and Plasticity, IRCCS Fondazione Santa Lucia, Rome, Italy Department of Systems Medicine, University Tor Vergata, Rome, Italy Search for more papers by this author Sara Migliarini Sara Migliarini Unit of Cell and Developmental Biology, Department of Biology, University of Pisa, Pisa, Italy Search for more papers by this author Giuseppina Martella Giuseppina Martella Laboratory of Neurophysiology and Plasticity, IRCCS Fondazione Santa Lucia, Rome, Italy Department of Systems Medicine, University Tor Vergata, Rome, Italy Search for more papers by this author Maria Meringolo Maria Meringolo Laboratory of Neurophysiology and Plasticity, IRCCS Fondazione Santa Lucia, Rome, Italy Department of Systems Medicine, University Tor Vergata, Rome, Italy Search for more papers by this author Benjamin Dehay Benjamin Dehay orcid.org/0000-0003-1723-9045 Université de Bordeaux, Institut des Maladies Neurodégénératives, UMR 5293, Bordeaux, France CNRS, Institut des Maladies Neurodégénératives, UMR 5293, Bordeaux, France Search for more papers by this author Evelyne Doudnikoff Evelyne Doudnikoff Université de Bordeaux, Institut des Maladies Neurodégénératives, UMR 5293, Bordeaux, France CNRS, Institut des Maladies Neurodégénératives, UMR 5293, Bordeaux, France Search for more papers by this author Venetia Zachariou Venetia Zachariou Department of Neuroscience, Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA Search for more papers by this author Rose E Goodchild Rose E Goodchild orcid.org/0000-0002-4524-5728 Department of Neurosciences, VIB-KU Leuven Center for Brain and Disease Research, KU Leuven, Leuven, Belgium Search for more papers by this author Nicola B Mercuri Nicola B Mercuri Laboratory of Neurophysiology and Plasticity, IRCCS Fondazione Santa Lucia, Rome, Italy Department of Systems Medicine, University Tor Vergata, Rome, Italy Search for more papers by this author Marcello D'Amelio Marcello D'Amelio Laboratory Molecular Neurosciences, IRCCS Fondazione Santa Lucia, Rome, Italy Unit of Molecular Neurosciences, Department of Medicine, University Campus-Biomedico, Rome, Italy Search for more papers by this author Massimo Pasqualetti Massimo Pasqualetti Unit of Cell and Developmental Biology, Department of Biology, University of Pisa, Pisa, Italy Center for Neuroscience and Cognitive Systems @UniTn, Istituto Italiano di Tecnologia, Rovereto, Italy Search for more papers by this author Erwan Bezard Erwan Bezard orcid.org/0000-0002-0410-4638 Université de Bordeaux, Institut des Maladies Neurodégénératives, UMR 5293, Bordeaux, France CNRS, Institut des Maladies Neurodégénératives, UMR 5293, Bordeaux, France Search for more papers by this author Antonio Pisani Corresponding Author Antonio Pisani [email protected] orcid.org/0000-0002-8432-594X Laboratory of Neurophysiology and Plasticity, IRCCS Fondazione Santa Lucia, Rome, Italy Department of Systems Medicine, University Tor Vergata, Rome, Italy Search for more papers by this author Author Information Paola Bonsi *,1, Giulia Ponterio1,2, Valentina Vanni1,2, Annalisa Tassone1,2, Giuseppe Sciamanna1,2, Sara Migliarini3, Giuseppina Martella1,2, Maria Meringolo1,2, Benjamin Dehay4,5, Evelyne Doudnikoff4,5, Venetia Zachariou6, Rose E Goodchild7, Nicola B Mercuri1,2, Marcello D'Amelio8,9, Massimo Pasqualetti3,10, Erwan Bezard4,5 and Antonio Pisani *,1,2 1Laboratory of Neurophysiology and Plasticity, IRCCS Fondazione Santa Lucia, Rome, Italy 2Department of Systems Medicine, University Tor Vergata, Rome, Italy 3Unit of Cell and Developmental Biology, Department of Biology, University of Pisa, Pisa, Italy 4Université de Bordeaux, Institut des Maladies Neurodégénératives, UMR 5293, Bordeaux, France 5CNRS, Institut des Maladies Neurodégénératives, UMR 5293, Bordeaux, France 6Department of Neuroscience, Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA 7Department of Neurosciences, VIB-KU Leuven Center for Brain and Disease Research, KU Leuven, Leuven, Belgium 8Laboratory Molecular Neurosciences, IRCCS Fondazione Santa Lucia, Rome, Italy 9Unit of Molecular Neurosciences, Department of Medicine, University Campus-Biomedico, Rome, Italy 10Center for Neuroscience and Cognitive Systems @UniTn, Istituto Italiano di Tecnologia, Rovereto, Italy *Corresponding author. Tel: +39-06501703211; E-mail: [email protected] *Corresponding author. Tel: +39-06501703153; E-mail: [email protected] EMBO Mol Med (2019)11:e9283https://doi.org/10.15252/emmm.201809283 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Dopamine D2 receptor signaling is central for striatal function and movement, while abnormal activity is associated with neurological disorders including the severe early-onset DYT1 dystonia. Nevertheless, the mechanisms that regulate D2 receptor signaling in health and disease remain poorly understood. Here, we identify a reduced D2 receptor binding, paralleled by an abrupt reduction in receptor protein level, in the striatum of juvenile Dyt1 mice. This occurs through increased lysosomal degradation, controlled by competition between β-arrestin 2 and D2 receptor binding proteins. Accordingly, we found lower levels of striatal RGS9-2 and spinophilin. Further, we show that genetic depletion of RGS9-2 mimics the D2 receptor loss of DYT1 dystonia striatum, whereas RGS9-2 overexpression rescues both receptor levels and electrophysiological responses in Dyt1 striatal neurons. This work uncovers the molecular mechanism underlying D2 receptor downregulation in Dyt1 mice and in turn explains why dopaminergic drugs lack efficacy in DYT1 patients despite significant evidence for striatal D2 receptor dysfunction. Our data also open up novel avenues for disease-modifying therapeutics to this incurable neurological disorder. Synopsis Striatal dopamine D2 receptor (DRD2) dysfunction in DYT1 dystonia mouse models was uncovered and shown to be mediated by an abnormal and selective trafficking to lysosomal degradation. The regulatory protein RGS9-2 over-expression can restore both the striatal level and the function of DRD2. Correlated developmental changes in the expression level of DRD2 and its regulatory protein RGS9-2 are observed in wild-type mouse striatum. A simultaneous reduction in striatal DRD2 and RGS9-2 protein expression levels is observed in adult Dyt1 mouse models. DRD2 downregulation is mediated by its selective trafficking to lysosomal degradation. Viral-induced expression of RGS9-2 is able to restore both the expression level and function of striatal DRD2. Introduction Striatal dopaminergic transmission is central to movement control and several disease conditions (Redgrave et al, 2010; Gittis & Kreitzer, 2012). Dopaminergic dysfunction has been implicated in early-onset generalized DYT1-TOR1A dystonia, a highly disabling and incurable neurological disease typically manifesting in childhood, which generalizes within a few years causing involuntary movements and abnormal postures (Balint et al, 2018). This disorder is most frequently caused by an autosomal dominant ∆gag mutation in the TOR1A gene, causing loss of function of the gene product torsinA, a member of the AAA+ (ATPases associated with cellular activities) family of proteins (Ozelius et al, 1997). Mutant ∆E-torsinA is mislocalized from the endoplasmic reticulum to the nuclear envelope, causing abnormalities in folding, assembly, and trafficking of proteins targeted for secretion or to membranes (Torres et al, 2004; Burdette et al, 2010; Granata et al, 2011). Clinical neuroimaging studies have revealed decreased caudate–putamen dopamine D2 receptor (DRD2) availability in DYT1 patients compared to controls (Asanuma et al, 2005; Carbon et al, 2009). Reduced striatal DRD2 binding and protein level have also been reported in several different DYT1 experimental models (Napolitano et al, 2010; Yokoi et al, 2011; Dang et al, 2012). Notably, multiple lines of evidence demonstrated reduced coupling between the DRD2 and its cognate G proteins and severely altered receptor function (Pisani et al, 2006; Napolitano et al, 2010; Sciamanna et al, 2011, 2012a; Martella et al, 2014; Scarduzio et al, 2017). DRD2 signaling via G proteins inhibits cAMP production and in turn PKA activity. However, there is also accumulating evidence for G protein-independent DRD2 signaling functions (Beaulieu & Gainetdinov, 2011), as well as G protein-independent regulation of the GPCR activity of DRD2. The reciprocal interactions of DRD2 with spinophilin or arrestin represent a regulatory mechanism for fine-tuning receptor-mediated signaling. Indeed, β-arrestin 2 (β-Arr2) is involved in internalization and G protein-independent signaling of DRD2 (Beaulieu et al, 2011; Del'guidice et al, 2011), while spinophilin antagonizes arrestin actions (Wang et al, 2004). In addition, the striatal-enriched regulator of G protein signaling 9-2 (RGS9-2) regulates the amplitude of the behavioral responses to DRD2 activation (Rahman et al, 2003; Gold et al, 2007; Traynor et al, 2009), inhibits DRD2 internalization (Celver et al, 2010), and specifically modulates DRD2 signaling in striatal neuronal subtypes (Cabrera-Vera et al, 2004). On the other hand, the receptor can target the RGS protein to the plasma membrane (Kovoor et al, 2005; Celver et al, 2012), and exposure to DRD2 ligands can alter RGS9-2 level in wild-type animals (Seeman et al, 2007) indicating a reciprocal modulation. In the present work, we investigated the molecular mechanisms underlying DRD2 reduced levels and altered signaling in the striatum of DYT1 dystonia models, Tor1a+/−-knock-out and Tor1a∆gag/+-knock-in mice (Goodchild et al, 2005). Our findings shed new light on DRD2 dysfunction in DYT1 striatum and show that in vivo delivery of RGS9-2 is able to rescue DRD2 expression levels and to recover striatal D2DR signaling. These findings might explain the paradox of the lack of efficacy of dopaminergic drugs in DYT1-TOR1A dystonia patients, despite strong evidence that abnormal dopamine signaling is central to disease pathophysiology. Further, they also define a potential therapeutic target that restores dopaminergic responses. Results DRD2 and RGS9-2 protein levels are simultaneously downregulated in DYT1 striatum In order to analyze the molecular mechanisms of DRD2 dysfunction, we utilized the Tor1a+/− mouse model that mimics the loss of function effect of the DYT1 dystonia TOR1A mutation. First, we measured receptor expression levels in the striatum of adult (P60–P90) Tor1a+/+ (Fig EV1) and Tor1a+/− male littermates. Tor1a+/− samples showed, as expected, lower amounts of torsinA protein (Fig 1A1; Tor1a+/+ 1.000 ± 0.053 N = 7, Tor1a+/− 0.602 ± 0.055 N = 6, t-test P = 0.0003). In lysates of Tor1a+/− striatum, we also observed significantly reduced levels of DRD2 protein compared to Tor1a+/+ control samples (Fig 1A1; Tor1a+/+ 1.000 ± 0.027 N = 18, Tor1a+/− 0.839 ± 0.044 N = 16, t-test P = 0.0029), despite similar mRNA levels (Fig EV2; 2−dCt: Tor1a+/+ 0.649 ± 0.081 N = 3, Tor1a+/− 0.587 ± 0.175 N = 3; Mann–Whitney test P = 1.0000). Based on the well-characterized reciprocal regulatory relationship between GPCRs and regulator of G protein signaling (RGS) proteins, we next examined whether RGS protein levels were affected and might give further insight on the nature of the impairment of D2R-mediated transmission in DYT1 mutant mice. We focused this analysis on striatal levels of RGS9-2, an R7 RGS family member specifically regulating DRD2 function, and the closely related RGS7. Western blotting (WB) analysis revealed significantly reduced RGS9-2 levels in Tor1a+/− mice (Fig 1A1; Tor1a+/+ 1.000 ± 0.036 N = 32, Tor1a+/− 0.844 ± 0.049 N = 33, t-test P = 0.0115). Conversely, RGS7 levels were comparable between genotypes (Fig 1A1; Tor1a+/+ 1.000 ± 0.077 N = 7; Tor1a+/− 0.980 ±0.097 N = 7, t-test P = 0.8721), ruling out a generalized protein downregulation and pointing to a specific deficit of DRD2 and its signaling pathway. Click here to expand this figure. Figure EV1. Representative DRD2 immunoblotting of striatal tissuePattern of DRD2 immunolabeling in the striatum of adult wild-type Tor1a+/+ mice. 30 μg of striatal (Str) lysate was loaded. DRD2 antibody recognized a prominent band at ~ 63 kDa. Download figure Download PowerPoint Figure 1. Striatal levels of DRD2 and RGS9-2 are reduced in the Tor1a+/− and Tor1a∆gag/+ DYT1 dystonia mouse models A. (A1) Representative WBs showing DRD2 and RGS9-2 downregulation in adult (P60–P90) Tor1a+/− (+/−) striata, characterized by a reduced torsinA protein level with respect to Tor1a+/+ (+/+) littermates. The striatal level of RGS7 is unchanged. The dot plot shows Tor1a+/− data, normalized to Tor1a+/+ controls of the same experiment. TorsinA: Tor1a+/+ N = 7, Tor1a+/− N = 6, t-test ***P = 0.0003; DRD2: Tor1a+/+ N = 18, Tor1a+/− N = 16, t-test **P = 0.0029; RGS9-2: Tor1a+/+ N = 32, Tor1a+/− N = 33, t-test *P = 0.0115; RGS7: Tor1a+/+ N = 7, Tor1a+/− N = 7, t-test P = 0.8721. WB quantification data, expressed as the ratio of protein vs. loading control intensity level, are normalized to the wild-type samples of the same experiment. (A2–A4) Time-course of changes in torsinA, DRD2, and RGS9-2 striatal levels along postnatal development. Summary plot data are normalized to the Tor1a+/+ P60 sample of each independent experiment. (A2) TorsinA level is significantly reduced in mutants throughout the developmental period considered (P7 N = 3; P14–P60 N = 4; one-way ANOVA P < 0.0001 and Bonferroni's multiple comparison test **P < 0.01 at P7, P14, and P60). (A3, A4) DRD2 and RGS9-2 levels show parallel courses, with a similar increase from P7 to P21 in Tor1a+/+ as well as Tor1a+/− striatal lysates (P7: DRD2 N = 3, RGS9-2 N = 4; P14–P21 N = 4; one-way ANOVA with Bonferroni's multiple comparison test P > 0.05). At P60, a simultaneous reduction of DRD2 and RGS9-2 levels is observed in Tor1a+/− striatal lysates (N = 4; Tor1a+/− one-sample t-test DRD2: *P = 0.0250, RGS9-2: *P = 0.0175). WB quantification data, expressed as the ratio of protein vs. loading control intensity level, are normalized to the wild-type P60 sample of the same experiment. Mean ± SEM is represented in the graphs. B. Representative WB images and the dot plot show downregulation of torsinA, DRD2, and RGS9-2 striatal levels also in adult P60–P90 Tor1a∆gag/+ (∆gag/+) mice. TorsinA: Tor1a+/+ N = 10, Tor1aΔgag/+ N = 12, t-test ***P = 0.0006; DRD2: Tor1a+/+ N = 7, Tor1aΔgag/+ N = 9, t-test *P = 0.0183; RGS9-2: Tor1a+/+ N = 10, Tor1aΔgag/+ N = 9, t-test *P = 0.0192. WB quantification data, expressed as the ratio of protein vs. loading control intensity level, are normalized to the wild-type samples of the same experiment. Mean ± SEM is represented in the graph. C. Left: Representative image of coronal striatal slices of fresh-frozen brains of wild-type and Tor1a+/− mice probed with the DRD2 radioligand 3H-spiperone. Scale bar 1.5 mm. Right: Graph showing DRD2 binding density in Tor1a+/− striatal sections, obtained from the densitometric quantification analysis, expressed as percentage variation compared to control animals. Tor1a+/+ N = 5, Tor1a+/− N = 6, t-test **P = 0.0042. Mean ± SEM is represented. Source data are available online for this figure. Source Data for Figure 1 [emmm201809283-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Similar DRD2 mRNA expression in Tor1a+/+ and Tor1a+/− striatumDRD2 mRNA expression is unaltered in the striatum of Tor1a+/− mice (N = 3) with respect to wild-types (N = 3), as determined by qRT–PCR (Mann–Whitney test P > 0.05). Data are represented as mean ± SEM. Download figure Download PowerPoint To assess the reciprocal relationship among these dysregulated proteins, we then analyzed the time-course of striatal expression levels of torsinA, DRD2, and RGS9-2 during postnatal development (P7, P14, P21, P60). TorsinA levels (Fig 1A2) were significantly reduced in Tor1a+/− with respect to control mice at P7, P14 and P60 (one-way ANOVA P < 0.0001 and Bonferroni's multiple comparison test P < 0.01). In contrast, DRD2 levels increased similarly between P7 and P21 in both genotypes (Fig 1A3; Tor1a+/+: P7 0.404 ± 0.169, P21 0.991 ± 0.072; Tor1a+/−: P7 0.595 ±0.140, P21 0.980 ± 0.111; one-way ANOVA P = 0.0023 and Bonferroni's multiple comparison test: Tor1a+/+ vs. Tor1a+/− P > 0.05), consistent with the previously reported developmental progression of mRNA levels in the murine striatum (Araki et al, 2007). However, while the receptor remained at steady level at P60 in Tor1a+/+ striatum, it showed a significant reduction in P60 Tor1a+/− samples (DRD2 P60 Tor1a+/− 0.768 ± 0.055, N = 4, one-sample t-test P = 0.0250). Interestingly, in Tor1a+/+ mice the developmental profile of RGS9-2 protein expression (Anderson et al, 2007a) was superimposable to that of DRD2 (Pearson's r correlation test: R2 0.97, P = 0.0152). Similarly, in Tor1a+/− striatum, RGS9-2 expression levels paralleled the course of DRD2 protein and showed a significant decrease at P60 (Fig 1A4; RGS9-2 P60 Tor1a+/− 0.861 ± 0.029, N = 4, one-sample t-test P = 0.0175). To confirm the involvement of DRD2 dysregulation in DYT1 dystonia, we measured striatal receptor levels in a different model, the Tor1aΔgag/+ mouse. TorsinA levels were reduced in adult (P60–P90) male mutant mice with respect to wild-type littermates (Fig 1B; Tor1a+/+ 1.000 ± 0.075 N = 10, Tor1aΔgag/+ 0.649 ± 0.048 N = 12; t-test P = 0.0006), providing further evidence that the Δgag is a loss of function mutation that alters torsinA protein expression level (Goodchild et al, 2005; Gordon & Gonzalez-Alegre, 2008). Of note, DRD2 and RGS9-2 protein expression levels were also reduced in Tor1aΔgag/+ striatum (Fig 1B; DRD2: Tor1a+/+ 1.000 ± 0.123 N = 7, Tor1aΔgag/+ 0.648 ± 0.067 N = 9, t-test P = 0.0183; RGS9-2: Tor1a+/+ 1.000 ± 0.123 N = 10, Tor1aΔgag/+ 0.556 ± 0.119 N = 9, t-test P = 0.0192), similarly to what we observed in Tor1a+/− mice, further strengthening the relevance of dopaminergic dysfunction in DYT1 dystonia. Finally, we performed an in situ DRD2 binding study with 3H-spiperone (Zeng et al, 2004). As reported in Fig 1C, a significant 15% decrease in DRD2 binding density was found in Tor1a+/− as compared to wild-type striatum, in accordance with Western blot analysis (Fig 1C; Tor1a+/+ 100.00 ± 1.83, N = 5; Tor1a+/− 84.94 ± 3.83, N = 6, t-test P = 0.0042). DRD2 downregulation in Tor1a+/− striatum is mediated by lysosomal degradation RGS9-2 possesses specific determinants which target the protein for constitutive degradation by lysosomal proteases, unless it is shielded by its membrane anchor R7 binding protein (R7BP; Anderson et al, 2007b). Additionally, RGS9-2 interacts with another binding partner, the atypical G protein subunit type 5 G protein beta (Gβ5) subunit (Masuho et al, 2011). We therefore investigated whether RGS9-2 downregulation may be determined by changes in striatal levels of its binding partners. Unexpectedly, we found that striatal levels of R7BP were significantly increased, whereas Gβ5 protein amount was unaltered (Fig 2A1–A3; Gβ5 Tor1a+/+: 1.000 ± 0.049 N = 11, Tor1a+/−: 1.002 ± 0.036 N = 10, Mann–Whitney test P = 0.2453; R7BP Tor1a+/+: 1.000 ± 0.057 N = 12, Tor1a+/−: 1.279 ± 0.051 N = 12, t-test P = 0.0014). Therefore, we evaluated the stability of RGS9-2 protein, by measuring its degradation by lysosomal proteases. Tor1a+/+ and Tor1a+/− dorsal striatum contralateral slices were incubated with or without the lysosomal protease inhibitor leupeptin (Anderson et al, 2007a; leu, 100 μM; Fig 2B1 and B2). In the absence of lysosomal proteolysis inhibition, we observed a similar extent of RGS9-2 degradation in Tor1a+/− and wild-type mice (Fig 2B2; Tor1a+/+: with leu 1.000 ± 0.115 N = 5, without leu 0.712 ± 0.041 N = 5, t-test P = 0.0464; Tor1a+/−: with leu 0.917 ± 0.095 N = 5, without leu 0.610 ± 0.043 N = 5, t-test P = 0.0190; Tor1a+/+ 74.00 ± 7.58% reduction; Tor1a+/− 70.58 ± 10.26% reduction, Tor1a+/+ vs. Tor1a+/− t-test P = 0.7956), indicating that RGS9-2 protein turnover is unaltered in mutant mice. Since also the long-lived DRD2 protein can be targeted to lysosomal degradation (Li et al, 2012), we wondered whether the lower DRD2 levels in the Tor1a+/− striatum could be caused by increased lysosomal degradation. Thus, we quantified DRD2 protein expression level in the previously described experimental conditions, and found similar levels in lysates of Tor1a+/+ striatal slices incubated with or without leu, indicating a long protein half-life (Fig 2B2; Tor1a+/+: with leu 1.000 ± 0.049 N = 4, without leu 0.925 ± 0.076 N = 4, t-test P = 0.4376). Surprisingly, however, in Tor1a+/− striatal lysates incubated without leu we observed a reduced level of the DRD2 protein (Fig 2B2; Tor1a+/−: with leu 0.904 ± 0.088 N = 4, without leu 0.664 ± 0.030 N = 4, t-test P = 0.0427), suggesting that loss of torsinA may disrupt DRD2 protein stability. Figure 2. DRD2 downregulation is mediated by lysosomal degradation A. (A1) Representative WB of RGS9-2, R7BP, and Gβ5 proteins on four Tor1a+/+ and four Tor1a+/− striatal lysates. The bar histogram on the right shows the quantification of the three proteins in each sample. (A2) Dot plot showing that Gβ5 striatal levels are unchanged in Tor1a+/− mice (Tor1a+/+ N = 11, Tor1a+/− N = 10, Mann–Whitney test P = 0.2453). (A3) Conversely, the level of the R7 binding protein R7BP is increased in Tor1a+/− striatum (Tor1a+/+ N = 12, Tor1a+/− N = 12, t-test **P = 0.0014). Values are represented as ratio of protein vs. loading control intensity level, normalized to the mean of Tor1a+/+ control values of the same experiment. Mean ± SEM is represented. B. (B1) Representative WB of lysates of Tor1a+/+ and Tor1a+/− dorsal striatum slices incubated for 5 h in the presence (w/ leu) or absence (w/o leu; contralateral striatum) of the protease inhibitor leupeptin. Samples showing enhanced degradation of DRD2 and/or RGS9-2 proteins show additional bands at lower molecular weight. (B2) Summary plot reporting mean ± SEM of RGS9-2 and DRD2 protein level values, expressed as the ratio of protein vs. loading control intensity level, normalized to the value of the Tor1a+/+ w/leu sample measured in the same experiment. DRD2: Tor1a+/+ N = 4, t-test P = 0.4376; Tor1a+/− N = 4, t-test *P = 0.0427; RGS9-2: Tor1a+/+ N = 5, t-test *P = 0.0464; Tor1a+/− N = 5, t-test *P = 0.0190. C. Dot plots showing DRD2, RGS9-2, R7BP, and Gβ5 protein levels measured in striatal detergent-resistant-membrane (DRM) preparations from Tor1a+/− (+/−) and WT (+/+) mice (DRD2: Tor1a+/+ N = 16, Tor1a+/− N = 13, t-test *P = 0.0129; RGS9-2: Tor1a+/+ N = 7, Tor1a+/− N = 6, t-test *P = 0.0396; R7BP: Tor1a+/+ N = 6, Tor1a+/− N = 6, t-test ***P = 0.0002; Gβ5: Tor1a+/+ N = 9, Tor1a+/− N = 9, t-test P = 0.8461). Values are reported as ratio of protein vs. PSD-95 intensity level, normalized to the mean value of the Tor1a+/+ samples measured in the same experiment. Mean ± SEM is represented. Source data are available online for this figure. Source Data for Figure 2 [emmm201809283-sup-0004-SDataFig2.pdf] Download figure Download PowerPoint Detergent-resistant membranes (DRM) are thought to contain structures such as lipid rafts and to be involved in membrane compartmentalization. Notably, torsinA levels affect cellular lipid metabolism (Grillet et al, 2016) and therefore may alter the portion of DRD2 and RGS9-2 that is expressed in the DRM fraction. We therefore measured the level of DRD2 and RGS9-2 expressed in the DRM of control and Tor1a+/− striatum (Celver et al, 2012). We observed a reduction of DRD2 protein expression level in the DRM of Tor1a+/− striatum (Fig 2C1; Tor1a+/+ 1.000 ± 0.070 N = 16; Tor1a+/− 0.714 ± 0.083 N = 13; t-test P = 0.0129), further supporting a downregulation of the receptor. Notably, we found instead an increase in RGS9-2 in the DRM of Tor1a+/− striatum (Fig 2C2; Tor1a+/+ 1.000 ± 0.108 N = 7; Tor1a+/− 1.572 ± 0.234 N = 6; t-test P = 0.0396). This observation may explain the reduced RGS9-2 level in striatal lysates in the absence of changes in protein stability, suggesting an increased compartmentalization of the protein in the DRM. Indeed, the level of the RGS9-2 binding
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