A model for sealing plasmalemmal damage in neurons and other eukaryotic cells.

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.

Polyethylene glycol rapidly restores axonal integrity and improves the rate of motor behavior recovery after sciatic nerve crush injury.

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, showing significant postoperative improvement of motor behavior in PEG-treated animals at 24-48 h. In contrast, the SFI more sensitively measured longer-term postoperative behavioral recovery and deficits at 4-8 wk, perhaps reflecting the development of fine (distal) motor control. These and other data show that PEG-fusion not only rapidly restores physiological and morphological axonal continuity, but also more quickly improves behavioral recovery.