Browsing by Author "Baucum, Anthony J., II."

Now showing 1 - 6 of 6
  • Thumbnail Image
    Item
    CaMKII enhances voltage-gated sodium channel Nav1.6 activity and neuronal excitability
    (American Society for Biochemistry and Molecular Biology, 2020-08-14) Zybura, Agnes S.; Baucum, Anthony J., II.; Rush, Anthony M.; Cummins, Theodore R.; Hudmon, Andy; Biology, School of Science
    Nav1.6 is the primary voltage-gated sodium channel isoform expressed in mature axon initial segments and nodes, making it critical for initiation and propagation of neuronal impulses. Thus, Nav1.6 modulation and dysfunction may have profound effects on input-output properties of neurons in normal and pathological conditions. Phosphorylation is a powerful and reversible mechanism regulating ion channel function. Because Nav1.6 and the multifunctional Ca2+/CaM-dependent protein kinase II (CaMKII) are independently linked to excitability disorders, we sought to investigate modulation of Nav1.6 function by CaMKII signaling. We show that inhibition of CaMKII, a Ser/Thr protein kinase associated with excitability, synaptic plasticity, and excitability disorders, with the CaMKII-specific peptide inhibitor CN21 reduces transient and persistent currents in Nav1.6-expressing Purkinje neurons by 87%. Using whole-cell voltage clamp of Nav1.6, we show that CaMKII inhibition in ND7/23 and HEK293 cells significantly reduces transient and persistent currents by 72% and produces a 5.8-mV depolarizing shift in the voltage dependence of activation. Immobilized peptide arrays and nanoflow LC-electrospray ionization/MS of Nav1.6 reveal potential sites of CaMKII phosphorylation, specifically Ser-561 and Ser-641/Thr-642 within the first intracellular loop of the channel. Using site-directed mutagenesis to test multiple potential sites of phosphorylation, we show that Ala substitutions of Ser-561 and Ser-641/Thr-642 recapitulate the depolarizing shift in activation and reduction in current density. Computational simulations to model effects of CaMKII inhibition on Nav1.6 function demonstrate dramatic reductions in spontaneous and evoked action potentials in a Purkinje cell model, suggesting that CaMKII modulation of Nav1.6 may be a powerful mechanism to regulate neuronal excitability.
  • Thumbnail Image
    Item
    A Multi-Omic Analysis of the Dorsal Striatum in an Animal Model of Divergent Genetic Risk for Alcohol Use Disorder
    (Wiley, 2021) Grecco, Gregory G.; Haggerty, David L.; Doud, Emma H.; Fritz, Brandon M.; Yin, Fuqin; Hoffman, Hunter; Mosley, Amber L.; Simpson, Edward; Liu, Yunlong; Baucum, Anthony J., II.; Atwood, Brady K.; Pharmacology and Toxicology, School of Medicine
    The development of selectively bred high and low alcohol-preferring mice (HAP and LAP, respectively) has allowed for an assessment of the polygenetic risk for pathological alcohol consumption and phenotypes associated with alcohol use disorder (AUD). Accumulating evidence indicates that the dorsal striatum (DS) is a central node in the neurocircuitry underlying addictive processes. Therefore, knowledge of differential gene, protein, and phosphorylated protein expression in the DS of HAP and LAP mice may foster new insights into how aberrant DS functioning may contribute to AUD-related phenotypes. To begin to elucidate these basal differences, a complementary and integrated analysis of DS tissue from alcohol-naïve male and female HAP and LAP mice was performed using RNA sequencing, quantitative proteomics, and phosphoproteomics. These datasets were subjected to a thorough analysis of gene ontology, pathway enrichment, and hub gene assessment. Analyses identified 2,108, 390, and 521 significant differentially expressed genes, proteins, and phosphopeptides, respectively between the two lines. Network analyses revealed an enrichment in the differential expression of genes, proteins, and phosphorylated proteins connected to cellular organization, cytoskeletal protein binding, and pathways involved in synaptic transmission and functioning. These findings suggest that the selective breeding to generate HAP and LAP mice may lead to a rearrangement of synaptic architecture which could alter DS neurotransmission and plasticity differentially between mouse lines. These rich datasets will serve as an excellent resource to inform future studies on how inherited differences in gene, protein, and phosphorylated protein expression contribute to AUD-related phenotypes.
  • Thumbnail Image
    Item
    Neurabin's Influence on Striatal Dependent Behaviors
    (2022-08) Corey, Wesley; Cummins, Theodore; Berbari, Nicolas; Baucum, Anthony J., II.
    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.
  • Thumbnail Image
    Item
    Spinophilin limits GluN2B-containing NMDAR activity and sequelae associated with excessive hippocampal NMDAR function
    (Cold Spring Harbor Laboratory, 2021-01-01) Salek, Asma B.; Bansal, Ruchi; Berbari, Nicolas F.; Baucum, Anthony J., II.; Biology, School of Science
    N-methyl-D-Aspartate receptors (NMDARs) are calcium-permeable ion channels that are ubiquitously expressed within the glutamatergic postsynaptic density. Phosphorylation of NMDAR subunits defines receptor activity and surface localization. Modulation of NMDAR phosphorylation by kinases and phosphatases regulates calcium entering the cell and subsequent activation of calcium-dependent processes. Spinophilin is the major synaptic protein phosphatase 1 (PP1) targeting protein that controls phosphorylation of myriad substrates via targeting or inhibition of PP1. Spinophilin limits NMDAR function in a PP1-dependent manner and we have previously shown that spinophilin sequesters PP1 away from the GluN2B subunit of the NMDAR, which results in increased phosphorylation of Ser-1284. However, how spinophilin modifies NMDAR function is unclear. Herein, we detail that while Ser-1284 phosphorylation increases calcium influx via GluN2B-containing NMDARs, overexpression of spinophilin decreases GluN2B-containing NMDAR activity by decreasing its surface expression. In hippocampal neurons isolated from spinophilin knockout animals there is an increase in cleaved caspase-3 levels compared to wildtype mice; however, this effect is not exclusively due to NMDAR activation; suggesting multiple putative mechanisms by which spinophilin may modulate caspase cleavage. Behaviorally, our data suggest that spinophilin knockout mice have deficits in spatial cognitive flexibility, a behavior associated GluN2B function within the hippocampus. Taken together, our data demonstrate a unique mechanism by which spinophilin modulates GluN2B containing NMDAR phosphorylation, channel function, and trafficking and that loss of spinophilin promotes pathological sequelae associated with GluN2B dysfunction.
  • Thumbnail Image
    Item
    Spinophilin-dependent regulation of GluN2B-containing NMDAR-dependent calcium influx, GluN2B surface expression, and cleaved caspase expression
    (Wiley, 2023) Salek, Asma B.; Claeboe, Emily T.; Bansal, Ruchi; Berbari, Nicolas F.; Baucum, Anthony J., II.; Biology, School of Science
    N-methyl-d-aspartate receptors (NMDARs) are calcium-permeable ion channels that are ubiquitously expressed within the glutamatergic postsynaptic density. Phosphorylation of NMDAR subunits defines receptor conductance and surface localization, two alterations that can modulate overall channel activity. Modulation of NMDAR phosphorylation by kinases and phosphatases regulates the amount of calcium entering the cell and subsequent activation of calcium-dependent processes. The dendritic spine enriched protein, spinophilin, is the major synaptic protein phosphatase 1 (PP1) targeting protein. Depending on the substrate, spinophilin can act as either a PP1 targeting protein, to permit substrate dephosphorylation, or a PP1 inhibitory protein, to enhance substrate phosphorylation. Spinophilin limits NMDAR function in a PP1-dependent manner. Specifically, we have previously shown that spinophilin sequesters PP1 away from the GluN2B subunit of the NMDAR, which results in increased phosphorylation of Ser-1284 on GluN2B. However, how spinophilin modifies NMDAR function is unclear. Herein, we utilize a Neuro2A cell line to detail that Ser-1284 phosphorylation increases calcium influx via GluN2B-containing NMDARs. Moreover, overexpression of spinophilin decreases GluN2B-containing NMDAR activity by decreasing its surface expression, an effect that is independent of Ser-1284 phosphorylation. In hippocampal neurons isolated from spinophilin knockout animals, there is an increase in cleaved caspase-3 levels, a marker of calcium-associated apoptosis, compared with wildtype mice. Taken together, our data demonstrate that spinophilin regulates GluN2B containing NMDAR phosphorylation, channel function, and trafficking and that loss of spinophilin enhances neuronal cleaved caspase-3 expression.
  • Thumbnail Image
    Item
    Zebrafish Asd Discovery Models for Epileptic Mutations of Scn2a and Scn8a
    (2022-12) Milder, Patrick; Marrs, James A.; Baucum, Anthony J., II.; Overholser, Brian R.; Cummins, Theodore R.; Mastracci, Teresa L.
    Approximately 30% of patients with epilepsy do not achieve adequate seizure control through current anti-seizure drugs (ASD) and treatment methods. Therefore, a critical need exists to efficiently screen ASDs to enhance our ability to tailor treatment protocols and improve patient outcomes. The zebrafish pentylenetetrazol (PTZ) seizure model has become an increasingly popular screening paradigm for novel ASDs. Here, we present an optimized PTZ assay to improve reliability and reproducibility based on work in our laboratory. This optimized assay improves robustness in our screening of anti-seizure drugs (topiramate, lamotrigine, carbamazepine and GS967). These findings show that electroencephalogram (EEG) and calcium sensitive GFP from fusion protein (GCaMP) assays largely correlate with the behavioral findings, helping us connect physiological and behavioral responses to ASDs. Genetic epilepsy syndromes, like voltage gated sodium channel SCN2A and SCN8A pathogenic variants, are often poorly controlled by current medications. Our optimized assay relied on a fast and precise zebrafish seizure model using mRNA overexpression of hSCN2A and hSCN8A variants including: hSCN2A R1882Q and R853Q and hSCN8A R1872Q. All three pathogenic variants increased seizure activity, and the ASDs significantly decreased this seizure activity. This mRNA overexpression assay can be used to quickly evaluate seizure activity induced by pathogenic variants in voltage gated sodium channel genes and test ASDs to determine efficacy. In a separate study, we tested if the addition of the human SCN2A sodium channel could potentially rescue the loss of the zebrafish scn1Lab gene. Our GCaMP assay data indicates that this loss was successfully rescued. Cumulatively, these findings can be used to improve the screening of novel ASDs and treatments for patients with refractory epilepsy.