The association of spinophilin with disks large-associated protein 3 (SAPAP3) is regulated by metabotropic glutamate receptor (mGluR) 5

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Highlights

  • SAPAP3 and mGluR5 co-immunoprecipitate with spinophilin.

  • Spinophilin association with SAPAP3 is enhanced by mGluR5.

  • Spinophilin association with SAPAP3 is enhanced by PKC activity.

  • Spinophilin association with mGluR5 is enhanced by SAPAP3.

  • Loss of spinophilin attenuates amphetamine-induced locomotor sensitization.

Abstract

Spinophilin is the most abundant protein phosphatase 1 targeting protein in the postsynaptic density of dendritic spines. Spinophilin associates with myriad synaptic proteins to regulate normal synaptic communication; however, the full complement of spinophilin interacting proteins and mechanisms regulating spinophilin interactions are unclear. Here we validate an association between spinophilin and the scaffolding protein, disks large-associated protein 3 (SAP90/PSD-95 associated protein 3; SAPAP3). Loss of SAPAP3 leads to obsessive-compulsive disorder (OCD)-like behaviors due to alterations in metabotropic glutamate receptor (mGluR) signaling. Here we report that spinophilin associates with SAPAP3 in the brain and in a heterologous cell system. Moreover, we have found that expression or activation of group I mGluRs along with activation of the mGluR-dependent kinase, protein kinase C β, enhances this interaction. Functionally, global loss of spinophilin attenuates amphetamine-induced hyperlocomotion, a striatal behavior associated with dopamine dysregulation and OCD. Together, these data delineate a novel link between mGluR signaling, spinophilin, and SAPAP3 in striatal pathophysiology.

Introduction

Spinophilin knockout mice have deficits in long-term depression (LTD) in the striatum and hippocampus (Allen et al., 2006; Di Sebastiano et al., 2016) and behavioral pathologies, such as enhanced cocaine-induced conditioned place preference (Allen et al., 2006) and increased sedation in response to α2 adrenergic agonists (Lu et al., 2010). As spinophilin is the major protein phosphatase 1 (PP11) targeting protein in the Postsynaptic density (Colbran et al., 1997), many of spinophilin's functions are thought to be due to alterations in spinophilin-dependent PP1 targeting. Using a global proteomics analysis of spinophilin immunoprecipitates, we have previously reported that spinophilin binding to multiple classes of proteins are diminished following dopamine depletion in the striatum (Hiday et al., 2017), an animal model of Parkinson disease (PD). One protein that was detected in this screen to potentially have a decreased association with spinophilin was SAP90/PSD-95 associated protein 3 (SAPAP3).

SAPAP3 is implicated in obsessive-compulsive disorder (OCD) pathologies as knockout of this protein leads to excessive grooming in mice, causing them to develop facial lesions (Welch et al., 2007). These alterations are due to increases in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) internalization and alterations in synaptic plasticity (Chen et al., 2011; Wan et al., 2011). Furthermore, this internalization is caused by excessive metabotropic glutamate receptor (mGluR) 5-dependent activity and plasticity (Chen et al., 2011; Wan et al., 2011). Interestingly, others have found that in addition to SAPAP3, spinophilin associates with mGluR5 and loss of spinophilin enhances mGluR endocytosis (Di Sebastiano et al., 2016). Moreover, treatment with the group I mGluR agonist, dihydroxyphenylglycine (DHPG), fails to elicit LTD in hippocampus of spinophilin KO animals. However, the mechanisms regulating the spinophilin/SAPAP3 complex are unknown. Here we identify SAPAP3 as a spinophilin interacting protein whose association with spinophilin is regulated by mGluR signaling. Specifically, mGluR expression and downstream activation of protein kinase C appear to play a role. Moreover, we find that SAPAP3 also regulates spinophilin binding to mGluR5. Functionally, we observed that a loss of spinophilin attenuates amphetamine-dependent locomotor sensitization, a model of dopamine-dependent striatal dysfunction that overlaps with compulsive behaviors. Together these data shed mechanistic light on the association of spinophilin with SAPAP3 and mGluR5 and delineate a novel potential role for spinophilin in striatal pathophysiology.

Section snippets

Animals

Dissected striatum for immunoprecipitations from WT and spinophilin KO animals were performed from female 2-month old mice. Dissected forebrains for immunoprecipitations from WT and SAPAP3 KO animals were performed from male (N = 2, 1 WT and 1 KO) and female (N = 2, 1 WT and 1 KO) 1-month old mice. Whole brains from these mice were a kind gift of Dr. Nicole Calakos (Duke University School of Medicine, Durham, NC). Striatal slices for DHPG studies were prepared from female, 1.5–3.5-month old

Validation of the spinophilin and SAPAP3 protein interaction in mouse brain

Mouse striata were homogenized and spinophilin and SAPAP3 immunoprecipitations were performed from the lysates. These isolated immunoprecipitates were separated by SDS-PAGE and immune complexes were analyzed via western blot. Spinophilin immunoprecipitates contained SAPAP3 whereas SAPAP3 immunoprecipitates contained spinophilin (Fig. 1A). Less co-immunoprecipitation was observed in IgG controls (Fig. 1A). To further validate this association, we utilized spinophilin KO animals as a control. As

Spinophilin-SAPAP3-mGluR5 association

Previous studies have identified group I mGluRs in spinophilin immunoprecipitates (Di Sebastiano et al., 2016). Moreover, our previous studies have detected the mGluR-interacting protein, homer, in spinophilin immunoprecipitates isolated from WT, but not spinophilin KO mice (Baucum 2nd et al., 2010). In a recent study using proteomics approaches, we found that spinophilin associates with SAPAP3, a regulator of mGluRs (Ade et al., 2016; Chen et al., 2011), and that this association may be

Conclusion

Taken together, our data are the first to describe mechanisms modulating the interaction between spinophilin and SAPAP3 and have potential implications in normal striatal MSN physiology and biochemical pathologies in striatal MSNs associated with PD and OCD. Future studies will need to delineate how modulation of the spinophilin/mGluR5/SAPAP3 complex regulates MSN function and striatal disease pathology.

Acknowledgements and funding

Support for these studies was from NIH (K01NS073700, R21/R33DA041876 to AJB), Department of Biology Start-up funds, Bridge Funding, and a research support funds grant (RSFG) (to AJB), and a summer Undergraduate Research Opportunity Program (UROP) fellowship (to CWM). We thank the Calakos lab (Duke University School of Medicine) for providing SAPAP3 KO and WT brains and the Mastracci lab (Indiana Biosciences Research Institute) for use of the Zeiss Elyra Microscope. The authors have no competing

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