Indiana University-Purdue University Indianapolis (IUPUI) / Glioblastoma is the most aggressive type of brain cancer with very poor prognosis. Due to the lack of understanding of underlying mechanisms, there are no anti-invasive clinical therapeutics available. SRC terminal kinase (SRC) is a tumorigenic protein that is highly expressed in glioblastoma samples. SRC inhibitor kinase 1 (SRCIN1), also known as p140Cap is a negative regulator of SRC. Silencing SRCIN1 results in increased tumor invasion. Our lab has discovered two novel scaffolding proteins Spinophilin (Spn) and neurabin (Nrb) that bind to SRCIN1. They may play a role in regulating SRCIN1 activity, as well as its downstream effects that ultimately decrease SRC’s tumorigenic activity. Spn and Nrb are two scaffolding proteins that are heavily expressed in the central nervous system. Spn knockout mice develop more tumors, indicating that Spn acts as a tumor suppressor protein, although the mechanisms of Spn’s anti-tumor properties are not well understood. Spn and Nrb are PP1 targeting proteins that target PP1 to other substrates, resulting in dephosphorylation and alteration of function. We found that PP1 increases Spn association with SRCIN1, but decreases Nrb association with SRCIN1, indicating that the two proteins might have opposite effects to balance the activity of p140Cap. We also found that cyclin-dependent kinase 5 (CDK5) phosphorylates and regulates the association of these scaffolding proteins with the tumor suppressor protein, p140Cap. Understanding these mechanisms provides insight into new therapeutic targets that may ultimately decrease SRC activity and its tumorigenic and invasive properties.

Indiana University-Purdue University Indianapolis (IUPUI) / A histological marker of Parkinson’s disease (PD) is the loss of synapses located on striatal medium spiny neurons (MSNs) as a result of dopaminergic nigral cell depletion. The dendritic spines that give MSNs their name have a well-characterized structure and are the main regions of post-synaptic input. It has been shown that spines have altered functionality and morphology in many neurodegenerative diseases. Spine morphology, and potentially function, is dictated by an array of structural proteins and their associations with other proteins in a region dubbed the post-synaptic density (PSD). Spinophilin and neurofilament medium (NF-M) are two proteins that are enriched in the PSD and have potential implications in PD. Interestingly, preliminary data show that there is a decrease in the NF-M-spinophilin interaction in animal models of PD. Here it is shown that these two proteins interact in brain tissue and when overexpressed in a mammalian cell system. Moreover, we have begun to determine mechanisms that regulate this interaction. It is known that there is a misregulation of protein phosphatases and kinases in many neurodegenerative diseases. Moreover, the phosphorylation state of a protein can regulate its association with other proteins. Therefore, we hypothesize that the phosphorylation state of either protein affects the interaction between spinophilin and NF-M. Furthermore, we have conducted experiments utilizing protein phosphatases and kinases that are known to modulate the phosphorylation state of NF-M and/or spinophilin. Data show that both kinase and phosphatase activity and/or expression modulates the NF-M-spinophilin interaction in heterologous cell lines. Through the use of MS/MS analysis, we have begun to map specific phosphorylation sites that may play a role in regulating this interaction. Currently, we are elucidating the specific effects of these post-translational modifications on regulating the spinophilin-NF-M interaction. These data will enhance our knowledge of spinophilin’s interactions and how these interactions are altered in neurological disorders such as PD.

Indiana University-Purdue University Indianapolis (IUPUI) / Striatal-mediated pathological disease-states such as Obsessive-Compulsive Disorder (OCD), Parkinson’s Disease (PD), and psychostimulant drug addiction/abuse are coupled with distinct motor movement abnormalities. In addition, these disorders are associated with perturbed synaptic transmission. Proper synaptic transmission is critical for maintaining neuronal communication. Furthermore, in many striatal-dependent disease-states, the principle striatal neurons, medium spiny neurons (MSNs), exhibit differential perturbations in downstream signaling. Signal transduction pathways that are localized to the glutamatergic post-synaptic density (PSD) of GABAergic MSNs regulate protein phosphorylation in a tightly controlled manner. Alterations in the control of this phosphorylation in striatal MSNs are observed in myriad striatal pathological diseasestates and can give rise to perturbations in synaptic transmission. While serine/threonine kinases obtain substrate specificity, in part, by phosphorylating specific consensus sites, serine/threonine phosphatases such as protein phosphatase 1 (PP1) are much more promiscuous. To obtain substrate selectivity, PP1 associates with targeting proteins. The major targeting protein for PP1 in the PSD of striatal dendritic spines is spinophilin. Spinophilin not only binds PP1, but also concurrently interacts with myriad synaptic proteins. Interestingly, dopamine depletion, an animal model of PD, modulates spinophilin protein-protein interactions in the striatum. However, spinophilin function on basal striatal-mediated motor behaviors such as the rotarod or under hyperdopaminergic states such as those observed following psychostimulant-induced behavioral sensitization are less well characterized. To elucidate spinophilin function more specifically, we have generated multiple transgenic animals that allow for cell type-specific loss of spinophilin as well as cell-specific interrogation of spinophilin protein interactions. Here, I report the functional role of spinophilin in regulating striatal mediated motor behaviors and functional changes associated with amphetamine-induced locomotor sensitization. In addition, we define changes in spinophilin protein-protein interactions that may mediate these behavioral changes. Furthermore, global loss of spinophilin abrogates amphetamine-induced sensitization and plays a critical role in striatal motor learning and performance. The data suggest that the striatal spinophilin protein interactome is upregulated in MSNs following psychostimulant administration. In addition, loss of spinophilin changes protein expression in myriad psychostimulant-mediated striatal adaptations. Taken together the data suggests that spinophilin’s protein-protein interactions in the striatum are obligate for appropriate striatal mediated motor function.

Indiana University-Purdue University Indianapolis (IUPUI) / The striatum is a key brain region involved in regulating motor output and integration. The dorsal and ventral subdivisions of the striatum work in concert to mediate the reinforcing and motor behavioral outputs of the striatum. Moreover, dysfunction of these striatal regions is involved in various diseases including Parkinson’s disease and drug addiction. Therefore, understanding and characterizing biochemical and molecular changes within the striatum associated with these diseases is key in devolving novel therapeutics to treat these disease states. The main output neurons of the striatum are GABAergic, medium-spiny neurons (MSNs), and striatal functionality is mediated by neuroplastic changes in MSN activity. Within MSNs, dopaminergic receptor activation triggers a cascade of reversable phosphorylation, which is facilitated by the activation of specific protein kinases and inhibition of specific protein phosphatases. In comparison to the 350 serine/threonine protein kinases expressed within the striatum, there are only 40 major serine/threonine protein phosphatases. However, serine/threonine protein phosphatases, such as protein phosphatase 1 (PP1), gain their target specificity by interacting with phosphatase-targeting proteins. Within the striatum, the neurabins, termed neurabin and spinophilin, are the most abundant PP1 targeting proteins in dendritic spines. Spinophilin’s expression in the striatum has been strongly characterized, and spinophilin has been shown to regulate striatal-dependent motor-skill learning and amphetamine-induced locomotor sensitization. In contrast to spinophilin, neurabin’s expression within the striatum and its involvement in these striatal-dependent behaviors has not been fully probed. I found that neurabin expression in the striatum is not sex-dependent but is age-dependent. In addition to these data, I also present validation of new global, constitutive and conditional neurabin knock-out mouse lines. Finally, I present data that, unlike previous studies in spinophilin knockout mice, neurabin knockout mice have enhanced striatal-dependent motor-skill learning, but do not impact amphetamine-induced locomotor sensitization. Further characterization of neurabin’s expression in the striatum, and its role in these key striatal behaviors could provide a druggable target for therapeutics designed to address striatal dysfunction.

It is estimated that one in 59 children in the US are affected by autism spectrum disorder (ASD). ASD is distinguished by social and communication deficits that can be displayed throughout a wide range of severity. This resulting spectrum of behaviors observed in ASD suggests that a complex etiology is involved. Previous studies have shown a genetic susceptibility to autism including paternal age, twin and sibling concordance. Genetic sequencing of those affected as well as first order relatives have identified alterations in genes associated with neuronal synaptic communication. However, very little information is available regarding the pathophysiology of synapses in ASD. Neuronal communication between anterior cingulate cortical neurons via synapses with other brain regions is vital in the execution of social behaviors in individuals. The aim of this study was to evaluate the protein expression of the synaptic marker spinophilin and post-synaptic density protein-95 (PSD-95) in postmortem ASD gray matter brain tissue from the anterior cingulate and frontal cortex to compare to typically developing (TD) control brain tissue. Postmortem brain tissue of ASD and TD subjects was acquired from nationally funded brain repositories previously matched by brain area, age and gender. Immunoblotting for spinophilin and PSD-95 was performed using anterior cingulate and frontal cortical gray matter brain tissue from matched ASD and TD brain tissue. Spinophilin and PSD-95 protein amounts for all donors were normalized using GAPDH. Frontal cortical tissue demonstrated no significant differences in spinophilin protein expression between TD and ASD groups (N=6). Anterior cingulate tissue demonstrated no significant differences in spinophilin protein expression between TD and ASD groups (N=5). PSD-95 protein expression levels did not result in any significant differences between ASD donors and their control pairs for either brain tissue region. Although no changes were detected in the frontal cortex or anterior cingulate cortex, more brain areas and subjects must be evaluated to determine if spinophilin or PSD-95 can be reliable markers for synaptic alterations in ASD. These data are critical in determining synaptic pathology in ASD which may lead to future treatments.

Indiana University-Purdue University Indianapolis (IUPUI) / 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.

Indiana University-Purdue University Indianapolis (IUPUI) / Excitotoxicity, a major hallmark of neurodegeneration associated with cerebral ischemia, is a result of accumulation of extracellular glutamate. This excess glutamate leads to hyperactivation of glutamate receptors such as the N-methyl-D-asparate (NMDA) receptors (NMDARs) following the activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPARs). Excessive activation of NMDARs causes an influx of calcium, which can eventually activate apoptotic pathways and lead to death of neurons. Regulation of NMDAR subunit composition, localization, surface expression, and activity can balance cell survival via activation of either pro-death or pro-survival pathways after a course of an ischemic insult. Specifically, phosphorylation of different NMDAR subunits defines their activity and downstream signaling pathways. NMDARs are phosphorylated by multiple kinases and dephosphorylated by different phosphatases. Besides phosphatases and kinases, per se, phosphorylation of synaptic proteins that regulate kinase or phosphatase targeting and activity also mediate NMDAR phosphorylation. Spinophilin, a major synaptic scaffolding and protein phosphatase 1 (PP1) targeting protein, mediates substrate phosphorylation via its ability to bind PP1. Our studies focus on delineating the role of spinophilin in the regulation of phosphorylation and function of the GluN2B subunit of the NMDA receptor as well as the role of spinophilin in modulating glutamate-induced neurotoxicity. Interestingly, our data demonstrate that spinophilin sequesters PP1 away from GluN2B thereby enhancing phosphorylation of GluN2B at Ser-1284. These changes impact GluN2B protein interactions, subcellular localization, and surface expression, leading to alterations in the amount of calcium entering the neuron via GluN2B-containing NMDARs. Our data show that spinophilin biphasically regulates GluN2B function. Specifically, Ser-1284 phosphorylation enhances calcium influx through GluN2B containing NMDA receptors, but spinophilin leads to dramatic decreases in the surface expression of the receptor independent of Ser-1284 phosphorylation. Moreover, in spinophilin knockout mice, we observe less PP1 binding to GluN2B and less phosphorylation of Ser-1284, but more surface expression of GluN2B and greater levels of caspase activity. Together, these observations suggest a potential neuroprotective role for spinophilin by decreasing GluN2B-containing NMDA receptor-dependent surface expression and thereby decreasing intracellular calcium and neuronal cell death.

No description available.

<p> The striatum is a key brain region involved in regulating motor output and integration. The dorsal and ventral subdivisions of the striatum work in concert to mediate the reinforcing and motor behavioral outputs of the striatum. Moreover, dysfunction of these striatal regions is involved in various diseases including Parkinson’s disease and drug addiction. Therefore, understanding and characterizing biochemical and molecular changes within the striatum associated with these diseases is key in devolving novel therapeutics to treat these disease states. The main output neurons of the striatum are GABAergic, medium-spiny neurons (MSNs), and striatal functionality is mediated by neuroplastic changes in MSN activity. Within MSNs, dopaminergic receptor activation triggers a cascade of reversable phosphorylation, which is facilitated by the activation of specific protein kinases and inhibition of specific protein phosphatases. In comparison to the 350 serine/threonine protein kinases expressed within the striatum, there are only 40 major serine/threonine protein phosphatases. However, serine/threonine protein phosphatases, such as protein phosphatase 1 (PP1), gain their target specificity by interacting with phosphatase-targeting proteins. Within the striatum, the neurabins, termed neurabin and spinophilin, are the most abundant PP1 targeting proteins in dendritic spines. Spinophilin’s expression in the striatum has been strongly characterized, and spinophilin has been shown to regulate striatal-dependent motor-skill learning and amphetamine-induced locomotor sensitization. In contrast to spinophilin, neurabin’s expression within the striatum and its involvement in these striatal-dependent behaviors has not been fully probed. I found that neurabin expression in the striatum is not sex-dependent but is age-dependent. In addition to these data, I also present validation of new global, constitutive and conditional neurabin knock-out mouse lines. Finally, I present data that, unlike previous studies in spinophilin knockout mice, neurabin knockout mice have enhanced striatal-dependent motor-skill learning, but do not impact amphetamine-induced locomotor sensitization. Further characterization of neurabin’s expression in the striatum, and its role in these key striatal behaviors could provide a druggable target for therapeutics designed to address striatal dysfunction.   </p>

Excitotoxicity, a major hallmark of neurodegeneration associated with cerebral ischemia, is a result of accumulation of extracellular glutamate. This excess glutamate leads to hyperactivation of glutamate receptors such as the N-methyl-D-asparate (NMDA) receptors (NMDARs) following the activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPARs). Excessive activation of NMDARs causes an influx of calcium, which can eventually activate apoptotic pathways and lead to death of neurons. Regulation of NMDAR subunit composition, localization, surface expression, and activity can balance cell survival via activation of either pro-death or pro-survival pathways after a course of an ischemic insult. Specifically, phosphorylation of different NMDAR subunits defines their activity and downstream signaling pathways. NMDARs are phosphorylated by multiple kinases and dephosphorylated by different phosphatases. Besides phosphatases and kinases, per se, phosphorylation of synaptic proteins that regulate kinase or phosphatase targeting and activity also mediate NMDAR phosphorylation. Spinophilin, a major synaptic scaffolding and protein phosphatase 1 (PP1) targeting protein, mediates substrate phosphorylation via its ability to bind PP1. Our studies focus on delineating the role of spinophilin in the regulation of phosphorylation and function of the GluN2B subunit of the NMDA receptor as well as the role of spinophilin in modulating glutamate-induced neurotoxicity. Interestingly, our data demonstrate that spinophilin sequesters PP1 away from GluN2B thereby enhancing phosphorylation of GluN2B at Ser-1284. These changes impact GluN2B protein interactions, subcellular localization, and surface expression, leading to alterations in the amount of calcium entering the neuron via GluN2B-containing NMDARs. Our data show that spinophilin biphasically regulates GluN2B function. Specifically, Ser-1284 phosphorylation enhances calcium influx through GluN2B containing NMDA receptors, but spinophilin leads to dramatic decreases in the surface expression of the receptor independent of Ser-1284 phosphorylation. Moreover, in spinophilin knockout mice, we observe less PP1 binding to GluN2B and less phosphorylation of Ser-1284, but more surface expression of GluN2B and greater levels of caspase activity. Together, these observations suggest a potential neuroprotective role for spinophilin by decreasing GluN2B-containing NMDA receptor-dependent surface expression and thereby decreasing intracellular calcium and neuronal cell death.