Papers by Christopher Spaeth
The Journal of Neuroscience, Nov 24, 2010
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Developmental Neurobiology, Jul 13, 2012
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Cellular and Molecular Neurobiology, Aug 3, 2012
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Nature Cell Biology, Nov 10, 2013
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Journal of Neurophysiology, Aug 1, 2010
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Journal of Neuroscience Research, 2012
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Nature Cell Biology, 2013
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Journal of Neuroscience Research, 2011
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Journal of Neuroscience Research, 2012
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Journal of Neurophysiology, 2010
The inability to rapidly (within minutes to hours) improve behavioral function after severance of... more The inability to rapidly (within minutes to hours) improve behavioral function after severance of peripheral nervous system axons is an ongoing clinical problem. We have previously reported that polyethylene glycol (PEG) can rapidly restore axonal integrity (PEG-fusion) between proximal and distal segments of cut- and crush-severed rat axons in vitro and in vivo. We now report that PEG-fusion not only reestablishes the integrity of crush-severed rat sciatic axons as measured by the restored conduction of compound action potentials (CAPs) and the intraaxonal diffusion of fluorescent dye across the lesion site, but also produces more rapid recovery of appropriate hindlimb motor behaviors. Improvement in recovery occurred during the first few postoperative weeks for the foot fault (FF) asymmetry test and between week 2 and week 3 for the Sciatic Functional Index (SFI) based on analysis of footprints. That is, the FF test was the more sensitive indicator of early behavioral recovery, sh...
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Developmental Neurobiology, 2012
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Cellular and Molecular Neurobiology, 2012
To survive, neurons and other eukaryotic cells must rapidly repair (seal) plasmalemmal damage. Su... more 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 Ca(2+)-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 Ca(2+), 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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Journal of Neuroscience, 2010
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Neural regeneration research, 2016
The repair (sealing) of plasmalemmal damage, consisting of small holes to complete transections, ... more The repair (sealing) of plasmalemmal damage, consisting of small holes to complete transections, is critical for cell survival, especially for neurons that rarely regenerate cell bodies. We first describe and evaluate different measures of cell sealing. Some measures, including morphological/ultra-structural observations, membrane potential, and input resistance, provide very ambiguous assessments of plasmalemmal sealing. In contrast, measures of ionic current flow and dye barriers can, if appropriately used, provide more accurate assessments. We describe the effects of various substances (calcium, calpains, cytoskeletal proteins, ESCRT proteins, mUNC-13, NSF, PEG) and biochemical pathways (PKA, PKC, PLC, Epac, cytosolic oxidation) on plasmalemmal sealing probability, and suggest that substances, pathways, and cellular events associated with plasmalemmal sealing have undergone a very conservative evolution. During sealing, calcium ion influx mobilizes vesicles and other membranous s...
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Journal of Neuroscience Research, 2012
To survive, cells must rapidly repair (seal) plasmalemmal damage. Cytosolic oxidation has been sh... more To survive, cells must rapidly repair (seal) plasmalemmal damage. Cytosolic oxidation has been shown to increase cell survival in some cases and produce cell death in other protocols. An antioxidant (melatonin; Mel) has been reported to decrease the probability of sealing plasmalemmal damage. Here we report that plasmalemmal damage produces cytosolic oxidation, as assayed by methylene blue (MB) color change in rat B104 hippocampal cells. Plasmalemmal sealing is affected by duration of Ca²⁺ deprivation and length of exposure to, and concentration of, oxidizing agents such as H₂O₂ and thimerosal (TH). Cytosolic oxidation by 10 μM to 50 mM H₂O₂ or 100 μM to 2 mM TH increases the probability of Ca²⁺-dependent plasmalemmal sealing, whereas higher concentrations of H₂O₂ decrease sealing probability and also damage uninjured cells. We also show that antioxidants (Mel, MB) or reducing agents (dithiothreitol) decrease sealing. Proteins, such as protein kinase A, SNAP-25, synaptobrevin, and N-ethylmaleimide-sensitive factor (previously reported to enhance sealing in other pathways), also enhance sealing in this oxidation pathway. In brief, our data show that plasmalemmal damage produces cytosolic oxidation that increases the probability of plasmalemmal sealing, which is strongly correlated with cell survival in other studies. Our results may provide new insights into the etiology and treatment of oxidation-dependent neurodegenerative disorders, such as Parkinson's, Huntington's, and Alzheimer's diseases.
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Objective The objective was immediate restoration of axonal membrane continuity and distal motor ... more Objective The objective was immediate restoration of axonal membrane continuity and distal motor and sensory innervation after peripheral nerve transection, using both the lipid membrane fusogen polyethylene glycol (PEG) and the antioxidant methylene blue (MB), enhancing functional recovery. Methods Animals were subjected to sciatic nerve transection (with the exception of sham) followed by: no repair; PEG alone; suture alone; suture+PEG; and suture+PEG/MB. Compound action potentials (CAPs) were measured by directly stimulating/recording from the Sprague Dawley rat sciatic nerve: before transection; after transection; and after repair as a physiological measure of axonal continuity. Ex vivo nerve dye diffusion studies were performed. Histological parameters of nerve regeneration including: nerve fiber counts; axon/fiber diameter; g ratio, and myelin thickness were measured 5 mm proximal and distal to the repair at 1,2 and 12 weeks. Weekly hindlimb behavioral assessments were underta...
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Journal of Neuroscience, 2010
Plasmalemmal repair is necessary for survival of damaged eukaryotic cells. Ca(2+) influx through ... more Plasmalemmal repair is necessary for survival of damaged eukaryotic cells. Ca(2+) influx through plasmalemmal disruptions activates calpain, vesicle accumulation at lesion sites, and membrane fusion proteins; Ca(2+) influx also initiates competing apoptotic pathways. Using the formation of a dye barrier (seal) to assess plasmalemmal repair, we now report that B104 hippocampal cells with neurites transected nearer (<50 μm) to the soma seal at a lower frequency and slower rate compared to cells with neurites transected farther (>50 μm) from the soma. Analogs of cAMP, including protein kinase A (PKA)-specific and Epac-specific cAMP, each increase the frequency and rate of sealing and can even initiate sealing in the absence of Ca(2+) influx at both transection distances. Furthermore, Epac activates a cAMP-dependent, PKA-independent, pathway involved in plasmalemmal sealing. The frequency and rate of plasmalemmal sealing are decreased by a small molecule inhibitor of PKA targeted to its catalytic subunit (KT5720), a peptide inhibitor targeted to its regulatory subunits (PKI), an inhibitor of a novel PKC (an nPKCη pseudosubstrate fragment), and an antioxidant (melatonin). Given these and other data, we propose a model for redundant parallel pathways of Ca(2+)-dependent plasmalemmal sealing of injured neurons mediated in part by nPKCs, cytosolic oxidation, and cAMP activation of PKA and Epac. We also propose that the evolutionary origin of these pathways and substances was to repair plasmalemmal damage in eukaryotic cells. Greater understanding of vesicle interactions, proteins, and pathways involved in plasmalemmal sealing should suggest novel neuroprotective treatments for traumatic nerve injuries and neurodegenerative disorders.
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Journal of Neuroscience Research, 2012
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Papers by Christopher Spaeth