Pathways for plasmalemmal repair mediated by PKA, Epac, and cytosolic oxidation in rat B104 cells in vitro and rat sciatic axons ex vivo

Abstract

Plasmalemmal repair (sealing) is necessary for survival of damaged eukaryotic cells. Ca(2+) influx through plasmalemmal disruptions activates pathways that initiate sealing, which is commonly assessed by exclusion of extracellular dye. These sealing pathways include PKA, Epac, and cytosolic oxidation. In this article, we investigate whether PKA, Epac, and/or cytosolic oxidation, activate specific proteins required to produce a plasmalemmal seal. We report that toxin cleavage of proteins required for neurotransmitter release (SNAP-25), inhibition of Golgi trafficking (with Brefeldin A: Bref A) or inhibition of N-ethylmaleimide sensitive factor (NSF) all decrease sealing of rat B104 hippocampal cells with transected neuritis in vitro. Epac, but not PKA or cytosolic oxidation, partly overcomes the decrease in sealing produced by cleavage of SNAP-25. PKA and increased cytosolic oxidation, but not Epac, can partly overcome the decrease in sealing due to Bref A. PKA, Epac, and/or cytosolic oxidation cannot overcome NSF inhibition. Substances that affect plasmalemmal sealing of B104 neurites in vitro have similar effects on plasmalemmal sealing in rat sciatic axons ex vivo. From these and other data, we propose a model of plasmalemmal sealing having three redundant, evolutionarily conserved, parallel pathways that all converge on NSF.

Intracellular cAMP Sensor EPAC: Physiology, Pathophysiology, and Therapeutics Development

“…Considering the actions of EPAC on neuronal differentiation, the functions of specific activators and inhibitors of EPACs are becoming an attractive idea for therapies targeting neuronal injuries. For example, plasmalemmal repair is essential for neurons to maintain circuit connections postinjury (764,971). This repair process is accomplished by merging undamaged membranes (263, 970) through myelin delamination (52) and calcium-dependent accumulation of vesicles and undamaged membrane (262,558,858,1031).…”

The curious ability of polyethylene glycol fusion technologies to restore lost behaviors after nerve severance

“…Epac, exchange protein activated by cAMP; DAG, diacylglycerol; PKA, phosphokinase A; PKC, phosphokinase C; PKI, phosphokinase in hibitor. Modified and used with permission from Spaeth et al ().…”

“…Two decades of research has subsequently revealed that, in more than 20 preparations studied to date, all eukaryotic cells (including neurons) seal plasmalemmal damage by Ca 2+ ‐dependent production of vesicles that form a plug, often at a partially constricted cut end (Krause et al, ; Steinhardt et al, ; Bi et al, ; Eddleman et al, ; Bittner and Fishman, ; Detrait et al, ; Yoo et al, ; Nguyen et al, ; McNeil, ; Spaeth et al, ; Zuzek et al, ; Jimenez et al, ). Vesicles from nearby intact membrane (Eddleman et al, ), lysosomes (Reddy et al, ), and/or myelin delaminations (Ballinger et al, ) migrate, accumulate, and pack tightly at the damage site to seal small holes within seconds to minutes and to seal complete axonal transections within 5–20 min (Fig.…”

“…Since these initial studies, we and others have described in some detail four or five parallel pathways for endogenous plasmalemmal sealing that have various proteins (Fig. ), many activated by Ca 2+ , and that have isomers involved in vesicle trafficking or fusion at synapses or the Golgi apparatus (McNeil, ; Spaeth et al, ; Zuzek et al, ; Jimenez et al, ). Examples of such proteins include isoforms of SNARES, SNAPS, VAMPS, NSF, synaptobrevin, syntaxin, synaptotagmin, calpains, TRIM proteins, phosphokinase A (PKA), and phosphokinase C (PKC).…”

“…), such as Ca 2+ , cAMP, PKA, EPAC, synaptobrevin, syntaxin, H 2 O 2 , and brilliant blue, that promote vesicle formation and membrane repair and MB, MEL, and phosphokinase inhibitor (PKI). Various substances that impede membrane repair have also been identified, such as (η, θ) pseudosubstrate fragments (PSFs; Spaeth et al, ; Zuzek et al, ). Especially relevant to developing the bioengineered series of PEG‐fusion solutions described below were data showing that membrane sealing was decreased by antioxidants such as MB, MEL, and PKI and enhanced by oxidants such as H 2 O 2 or thimerosal (Figs.…”