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Sealing of Transected Neurites of Rat B104 Cells Requires a Diacylglycerol PKC-Dependent Pathway and a PKA-Dependent Pathway

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Abstract

To survive, neurons and other eukaryotic cells must rapidly repair (seal) plasmalemmal damage. Such repair occurs by an accumulation of intracellular vesicles at or near the plasmalemmal disruption. Diacylglycerol (DAG)-dependent and cAMP-dependent proteins are involved in many vesicle trafficking pathways. Although recent studies have implicated the signaling molecule cAMP in sealing, no study has investigated how DAG and DAG-dependent proteins affect sealing. By means of dye exclusion to assess Ca2+-dependent vesicle-mediated sealing of transected neurites of individually identifiable rat hippocampal B104 cells, we now report that, compared to non-treated controls, sealing probabilities and rates are increased by DAG and cAMP analogs that activate PKC and Munc13-1 and PKA. Sealing is decreased by inhibiting DAG-activated novel protein kinase C isozymes η (nPKCη) and θ (nPKCθ) and Munc13-1, the PKC effector myristoylated alanine rich PKC substrate (MARCKS) or phospholipase C (PLC). DAG-increased sealing is prevented by inhibiting MARCKS or protein kinase A (PKA). Sealing probability is further decreased by simultaneously inhibiting nPKCη, nPKCθ, and PKA. Extracellular Ca2+, DAG, or cAMP analogs do not affect this decrease in sealing. These and other data suggest that DAG increases sealing through MARCKS and that nPKCη, nPKCθ, and PKA are all required to seal plasmalemmal damage in B104 and likely all eukaryotic cells.

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Acknowledgments

The research was supported by grants from the Lone Star Paralysis Foundation (www.lonestarparalysis.org). The authors would like to thank Dr. Van A. Herd and Robert “Rip” Wilcott for their input and commentary, and Deborah Surman for her maintenance of B104 cell cultures.

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  1. Christopher S. Spaeth

    Present address: Basic Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA

Authors and Affiliations

  1. Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, 78712, USA

    Aleksej Zuzek, Christopher S. Spaeth & George D. Bittner

  2. Section of Neurobiology, University of Texas at Austin, Austin, TX, 78712, USA

    Jerry D. Fan & George D. Bittner

  3. Institute for Neuroscience, University of Texas at Austin, Austin, TX, 78712, USA

    George D. Bittner

  4. Division of Pharmacology and Toxicology, University of Texas at Austin, Austin, TX, 78712, USA

    George D. Bittner

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  1. Aleksej Zuzek
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Correspondence to Aleksej Zuzek.

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Zuzek, A., Fan, J.D., Spaeth, C.S. et al. Sealing of Transected Neurites of Rat B104 Cells Requires a Diacylglycerol PKC-Dependent Pathway and a PKA-Dependent Pathway. Cell Mol Neurobiol 33, 31–46 (2013). https://doi.org/10.1007/s10571-012-9868-5

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