Browsing by Author "Baucum, Anthony J., II"
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Item The association of spinophilin with disks large-associated protein 3 (SAPAP3) is regulated by metabotropic glutamate receptor (mGluR) 5(Elsevier, 2018) Morris, Cameron W.; Watkins, Darryl S.; Salek, Asma B.; Edler, Michael C.; Baucum, Anthony J., II; Biology, School of ScienceSpinophilin 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.Item Correction: Baucum II, Anthony J. et al. Proteomic Analysis of the Spinophilin Interactome in Rodent Striatum Following Psychostimulant Sensitization. Proteomes 2018, 6, 53(MDPI, 2019-02-13) Watkins, Darryl S.; True, Jason D.; Mosley, Amber L.; Baucum, Anthony J., II; Biochemistry and Molecular Biology, School of MedicineThe author wishes to make the following corrections to the methods section of their paper [...]. Erratum for Proteomic Analysis of the Spinophilin Interactome in Rodent Striatum Following Psychostimulant Sensitization. [Proteomes. 2018]Item Maternal deprivation induces alterations in cognitive and cortical function in adulthood(Nature Publishing Group, 2018-03-27) Janetsian-Fritz, Sarine S.; Timme, Nicholas M.; Timm, Maureen M.; McCane, Aqilah M.; Baucum, Anthony J., II; O’ Donnell, Brian F.; Lapish, Christopher C.; Psychology, School of ScienceEarly life trauma is a risk factor for a number of neuropsychiatric disorders, including schizophrenia (SZ). The current study assessed how an early life traumatic event, maternal deprivation (MD), alters cognition and brain function in rodents. Rats were maternally deprived in the early postnatal period and then recognition memory (RM) was tested in adulthood using the novel object recognition task. The expression of catechol-o-methyl transferase (COMT) and glutamic acid decarboxylase (GAD67) were quantified in the medial prefrontal cortex (mPFC), ventral striatum, and temporal cortex (TC). In addition, depth EEG recordings were obtained from the mPFC, vertex, and TC during a paired-click paradigm to assess the effects of MD on sensory gating. MD animals exhibited impaired RM, lower expression of COMT in the mPFC and TC, and lower expression of GAD67 in the TC. Increased bioelectric noise was observed at each recording site of MD animals. MD animals also exhibited altered information theoretic measures of stimulus encoding. These data indicate that a neurodevelopmental perturbation yields persistent alterations in cognition and brain function, and are consistent with human studies that identified relationships between allelic differences in COMT and GAD67 and bioelectric noise. These changes evoked by MD also lead to alterations in shared information between cognitive and primary sensory processing areas, which provides insight into how early life trauma confers a risk for neurodevelopmental disorders, such as SZ, later in life.Item Mechanisms and Consequences of Dopamine Depletion-Induced Attenuation of the Spinophilin/Neurofilament Medium Interaction(Hindawi, 2017) Hiday, Andrew C.; Edler, Michael C.; Salek, Asma B.; Morris, Cameron W.; Thang, Morrent; Rentz, Tyler J.; Rose, Kristie L.; Jones, Lisa M.; Baucum, Anthony J., II; Biology, School of ScienceSignaling changes that occur in the striatum following the loss of dopamine neurons in the Parkinson disease (PD) are poorly understood. While increases in the activity of kinases and decreases in the activity of phosphatases have been observed, the specific consequences of these changes are less well understood. Phosphatases, such as protein phosphatase 1 (PP1), are highly promiscuous and obtain substrate selectivity via targeting proteins. Spinophilin is the major PP1-targeting protein enriched in the postsynaptic density of striatal dendritic spines. Spinophilin association with PP1 is increased concurrent with decreases in PP1 activity in an animal model of PD. Using proteomic-based approaches, we observed dopamine depletion-induced decreases in spinophilin binding to multiple protein classes in the striatum. Specifically, there was a decrease in the association of spinophilin with neurofilament medium (NF-M) in dopamine-depleted striatum. Using a heterologous cell line, we determined that spinophilin binding to NF-M required overexpression of the catalytic subunit of protein kinase A and was decreased by cyclin-dependent protein kinase 5. Functionally, we demonstrate that spinophilin can decrease NF-M phosphorylation. Our data determine mechanisms that regulate, and putative consequences of, pathological changes in the association of spinophilin with NF-M that are observed in animal models of PD.Item Mechanisms and consequences of regulating the spinophilin/NMDA receptor interaction(2016-07-12) Beiraghi Salek, Asma; Baucum, Anthony J., II; Belecky-Adams, Teri; Watson, John C.; Cummins, Theodore R.Parkinson disease (PD) is the second most common neurodegenerative disease. It is characterized by loss of dopaminergic cells in the substantia nigra, which causes loss of dopaminergic synapses onto striatal medium spiny neurons (MSNs). Dendritic spines that are localized to these striatal MSNs receive synaptic inputs from both the nigral dopamine neurons and cortical glutamate neurons. Signaling downstream of excitatory, glutamatergic drive is modulated by dopamine. This tripartite connection: glutamate, dopamine, and MSN dendritic spine, is important for normal motor function. Glutamate released from presynaptic terminals binds to and activates two classes of inotropic glutamate receptors that are localized to dendritic spines on striatal MSNs: the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and the N-methyl-D-aspartate receptor (NMDAR). Once these receptors are activated, they allow for Ca2+ influx, which in turn activates Ca2+-dependent processes that underlie neural plasticity, including long-term potentiation (LTP) and long-term depression (LTD). Proper machinery in the pre- and post-synaptic neurons is required for normal signal transduction. Moreover, this signal transduction requires proper organization of synaptic proteins, which is achieved by specific protein-protein interactions. These protein-protein interactions are dynamic and can be modulated under various conditions, including pathological changes in the phosphorylation status of a specific protein. Catalytically active proteins called phosphatases and kinases specifically regulate the phosphorylation status of synaptic proteins. Pathologically, in PD there is increased autophosphorylation and activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII). This increased phosphorylation may be due to changes in the activity of the serine/threonine protein phosphatase 1 (PP1), a highly conserved protein serine/threonine phosphatase that has a diverse set of functions in eukaryotes. Serine/threonine phosphatase substrate specificity is obtained via interactions with targeting and regulatory proteins. One such protein, spinophilin, is a scaffolding protein that targets PP1 to various synaptic substrates to regulate their phosphorylation. Interestingly, the association of PP1 with spinophilin is enhanced in a rat model of PD. The NMDAR is another protein that has altered phosphorylation in animal models of PD. We have found that there is a decrease in the NMDAR-spinophilin interaction in an animal model of PD. Here, we have found that spinophilin and the NMDAR interact in brain tissue and when overexpressed in a mammalian cell system. Moreover, we have identified novel mechanisms that regulate this interaction and have identified putative consequences of altering this association. These studies give us novel insight into mechanisms and consequences underlying pathological changes observed in an animal model of PD. Understanding these changes will inform novel therapeutic targets that may be useful in modulating striatal function.Item Mechanisms of spinophilin-dependent pancreas dysregulation underlying diabesity(Cold Spring Harbor Laboratory, 2023-02-08) Stickel, Kaitlyn C.; Mosley, Amber L.; Doud, Emma H.; Belecky-Adams, Teri L.; Baucum, Anthony J., II; Biology, School of ScienceObjective: Spinophilin is an F-actin binding and protein phosphatase 1 (PP1) targeting protein that acts as a scaffold of PP1 to its substrates. Spinophilin knockout (Spino-/-) mice have decreased fat mass, increased lean mass, and improved glucose tolerance, with no difference in feeding behaviors. While spinophilin is enriched in neurons, its roles in non-neuronal tissues, such as beta cells of the pancreatic islets, are unclear. Methods & results: We have corroborated and expanded upon previous studies to determine that Spino-/- mice have decreased weight gain and improved glucose tolerance in two different models of obesity. Using proteomics and immunoblotting-based approaches we identified multiple putative spinophilin interacting proteins isolated from intact pancreas and observed increased interactions of spinophilin with exocrine, ribosomal, and cytoskeletal protein classes that mediate peptide hormone production, processing, and/or release in Leprdb/db and/or high fat-fed (HFF) models of obesity. Moreover, loss of spinophilin specifically in pancreatic beta cells improved glucose tolerance without impacting body weight. Conclusion: Our data further support a role for spinophilin in mediating pathophysiological changes in body weight and whole-body metabolism associated with obesity and provide the first evidence that spinophilin mediates obesity-dependent pancreatic dysfunction that leads to deficits in glucose homeostasis or diabesity.Item Mechanisms Regulating the Association of Protein Phosphatase 1 with Spinophilin and Neurabin(American Chemical Society, 2018-11-21) Edler, Michael C.; Salek, Asma B.; Watkins, Darryl S.; Kaur, Harjot; Morris, Cameron W.; Yamamoto, Bryan K.; Baucum, Anthony J., II; Biology, School of ScienceProtein phosphorylation is a key mediator of signal transduction, allowing for dynamic regulation of substrate activity. Whereas protein kinases obtain substrate specificity by targeting specific amino acid sequences, serine/threonine phosphatase catalytic subunits are much more promiscuous in their ability to dephosphorylate substrates. To obtain substrate specificity, serine/threonine phosphatases utilize targeting proteins to regulate phosphatase subcellular localization and catalytic activity. Spinophilin and its homologue neurabin are two of the most abundant dendritic spine-localized protein phosphatase 1 (PP1) targeting proteins. The association between spinophilin and PP1 is increased in the striatum of animal models of Parkinson's disease (PD). However, mechanisms that regulate the association of spinophilin and neurabin with PP1 are unclear. Here, we report that the association between spinophilin and PP1α or PP1γ1 was increased by CDK5 expression and activation in a heterologous cell system. This increased association is at least partially due to phosphorylation of PP1. Conversely, CDK5 expression and activation decreased the association of PP1 with neurabin. As with dopamine depletion, methamphetamine (METH) abuse causes persistent alterations in dopamine signaling which influence striatal medium spiny neuron function and biochemistry. Moreover, both METH toxicity and dopamine depletion are associated with deficits in motor control and motor learning. Pathologically, we observed a decreased association of spinophilin with PP1 in rat striatum evaluated one month following a binge METH paradigm. Behaviorally, we found that loss of spinophilin recapitulates rotarod pathology previously observed in dopamine-depleted and METH-treated animals. Together, these data have implications in multiple disease states associated with altered dopamine signaling such as PD and psychostimulant drug abuse and delineate a novel mechanism by which PP1 interactions with spinophilin and neurabin may be differentially regulated.Item Proteomic Analysis of Postsynaptic Protein Complexes Underlying Neuronal Plasticity(American Chemical Society, 2017-04-19) Baucum, Anthony J., II; Biology, School of ScienceNormal neuronal communication and synaptic plasticity at glutamatergic synapses requires dynamic regulation of postsynaptic molecules. Protein expression and protein post-translational modifications regulate protein interactions that underlie this organization. In this Review, we highlight data obtained over the last 20 years that have used qualitative and quantitative proteomics-based approaches to identify postsynaptic protein complexes. Herein, we describe how these proteomics studies have helped lay the foundation for understanding synaptic physiology and perturbations in synaptic signaling observed in different pathologies. We also describe emerging technologies that can be useful in these analyses. We focus on protein complexes associated with the highly abundant and functionally critical proteins: calcium/calmodulin-dependent protein kinase II, the N-methyl-d-aspartate, and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptors, and postsynaptic density protein of 95 kDa.Item Proteomic Analysis of the Spinophilin Interactome in Rodent Striatum Following Psychostimulant Sensitization(MDPI, 2018-12-17) Watkins, Darryl S.; True, Jason D.; Mosley, Amber L.; Baucum, Anthony J., II; Biochemistry and Molecular Biology, School of MedicineGlutamatergic projections from the cortex and dopaminergic projections from the substantia nigra or ventral tegmental area synapse on dendritic spines of specific GABAergic medium spiny neurons (MSNs) in the striatum. Direct pathway MSNs (dMSNs) are positively coupled to protein kinase A (PKA) signaling and activation of these neurons enhance specific motor programs whereas indirect pathway MSNs (iMSNs) are negatively coupled to PKA and inhibit competing motor programs. An imbalance in the activity of these two programs is observed following increased dopamine signaling associated with exposure to psychostimulant drugs of abuse. Alterations in MSN signaling are mediated by changes in MSN protein post-translational modifications, including phosphorylation. Whereas direct changes in specific kinases, such as PKA, regulate different effects observed in the two MSN populations, alterations in the specific activity of serine/threonine phosphatases, such as protein phosphatase 1 (PP1) are less well known. This lack of knowledge is due, in part, to unknown, cell-specific changes in PP1 targeting proteins. Spinophilin is the major PP1-targeting protein in striatal postsynaptic densities. Using proteomics and immunoblotting approaches along with a novel transgenic mouse expressing hemagglutainin (HA)-tagged spinophilin in dMSNs and iMSNs, we have uncovered cell-specific regulation of the spinophilin interactome following a sensitizing regimen of amphetamine. These data suggest regulation of spinophilin interactions in specific MSN cell types and may give novel insight into putative cell-specific, phosphatase-dependent signaling pathways associated with psychostimulants.Item Retinal inflammation in murine models of type 1 and type 2 diabetes with diabetic retinopathy(Springer, 2023) Dharmarajan, Subramanian; Carrillo, Casandra; Qi, Zhonghua; Wilson, Jonathan M.; Baucum, Anthony J., II; Sorenson, Christine M.; Sheibani, Nader; Belecky‑Adams, Teri L.; Biology, School of ScienceAims/hypothesis: The loss of pericytes surrounding the retinal vasculature in early diabetic retinopathy underlies changes to the neurovascular unit that lead to more destructive forms of the disease. However, it is unclear which changes lead to loss of retinal pericytes. This study investigated the hypothesis that chronic increases in one or more inflammatory factors mitigate the signalling pathways needed for pericyte survival. Methods: Loss of pericytes and levels of inflammatory markers at the mRNA and protein levels were investigated in two genetic models of diabetes, Ins2Akita/+ (a model of type 1 diabetes) and Leprdb/db (a model of type 2 diabetes), at early stages of diabetic retinopathy. In addition, changes that accompany gliosis and the retinal vasculature were determined. Finally, changes in retinal pericytes chronically incubated with vehicle or increasing amounts of IFNγ were investigated to determine the effects on pericyte survival. The numbers of pericytes, microglia, astrocytes and endothelial cells in retinal flatmounts were determined by immunofluorescence. Protein and mRNA levels of inflammatory factors were determined using multiplex ELISAs and quantitative reverse transcription PCR (qRT-PCR). The effects of IFNγ on the murine retinal pericyte survival-related platelet-derived growth factor receptor β (PDGFRβ) signalling pathway were investigated by western blot analysis. Finally, the levels of cell death-associated protein kinase C isoform delta (PKCδ) and cleaved caspase 3 (CC3) in pericytes were determined by western blot analysis and immunocytochemistry. Results: The essential findings of this study were that both type 1 and 2 diabetes were accompanied by a similar progression of retinal pericyte loss, as well as gliosis. However, inflammatory factor expression was dissimilar in the two models of diabetes, with peak expression occurring at different ages for each model. Retinal vascular changes were more severe in the type 2 diabetes model. Chronic incubation of murine retinal pericytes with IFNγ decreased PDGFRβ signalling and increased the levels of active PKCδ and CC3. Conclusions/interpretation: We conclude that retinal inflammation is involved in and sustains pericyte loss as diabetic retinopathy progresses. Moreover, IFNγ plays a critical role in reducing pericyte survival in the retina by reducing activation of the PDGFRβ signalling pathway and increasing PKCδ levels and pericyte apoptosis.