Plasmalemmal Damage Research Articles

Repair of traumatic lesions to the plasmalemma of neurons and other cells: Commonalities, conflicts, and controversies.

Neuroscientists and Cell Biologists have known for many decades that eukaryotic cells, including neurons, are surrounded by a plasmalemma/axolemma consisting of a phospholipid bilayer that regulates trans-membrane diffusion of ions (including calcium) and other substances. Cells often incur plasmalemmal damage via traumatic injury and various diseases. If the damaged plasmalemma is not rapidly repaired within minutes, activation of apoptotic pathways by calcium influx often results in cell death. We review publications reporting what is less-well known (and not yet covered in neuroscience or cell biology textbooks): that calcium influx at the lesion sites ranging from small nm-sized holes to complete axonal transection activates parallel biochemical pathways that induce vesicles/membrane-bound structures to migrate and interact to restore original barrier properties and eventual reestablishment of the plasmalemma. We assess the reliability of, and problems with, various measures (e.g., membrane voltage, input resistance, current flow, tracer dyes, confocal microscopy, transmission and scanning electron microscopy) used individually and in combination to assess plasmalemmal sealing in various cell types (e.g., invertebrate giant axons, oocytes, hippocampal and other mammalian neurons). We identify controversies such as plug versus patch hypotheses that attempt to account for currently available data on the subcellular mechanisms of plasmalemmal repair/sealing. We describe current research gaps and potential future developments, such as much more extensive correlations of biochemical/biophysical measures with sub-cellular micromorphology. We compare and contrast naturally occurring sealing with recently-discovered artificially-induced plasmalemmal sealing by polyethylene glycol (PEG) that bypasses all natural pathways for membrane repair. We assess other recent developments such as adaptive membrane responses in neighboring cells following injury to an adjacent cell. Finally, we speculate how a better understanding of the mechanisms involved in natural and artificial plasmalemmal sealing is needed to develop better clinical treatments for muscular dystrophies, stroke and other ischemic conditions, and various cancers.

Natural mechanisms and artificial PEG-induced mechanism that repair traumatic damage to the plasmalemma in eukaryotes.

Eukaryotic tissues are composed of individual cells surrounded by a plasmalemma that consists of a phospholipid bilayer with hydrophobic heads that bind cell water. Bound-water creates a thermodynamic barrier that impedes the fusion of a plasmalemma with other membrane-bound intracellular structures or with the plasmalemma of adjacent cells. Plasmalemmal damage consisting of small or large holes or complete transections of a cell or axon results in calcium influx at the lesion site. Calcium activates fusogenic pathways that have been phylogenetically conserved and that lower thermodynamic barriers for fusion of membrane-bound structures. Calcium influx also activates phylogenetically conserved sealing mechanisms that mobilize the gradual accumulation and fusion of vesicles/membrane-bound structures that seal the damaged membrane. These naturally occurring sealing mechanisms for different cells vary based on the type of lesion, the type of cell, the proximity of intracellular membranous structures to the lesion and the relation to adjacent cells. The reliability of different measures to assess plasmalemmal sealing need be carefully considered for each cell type. Polyethylene glycol (PEG) bypasses calcium and naturally occurring fusogenic pathways to artificially fuse adjacent cells (PEG-fusion) or artificially seal transected axons (PEG-sealing). PEG-fusion techniques can also be used to rapidly rejoin the closely apposed, open ends of severed axons. PEG-fused axons do not (Wallerian) degenerate and PEG-fused nerve allografts are not immune-rejected, and enable behavioral recoveries not observed for any other clinical treatment. A better understanding of natural and artificial mechanisms that induce membrane fusion should provide better clinical treatment for many disorders involving plasmalemmal damage.

Repair of traumatic plasmalemmal damage to neurons and other eukaryotic cells.

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 structures (lysosomes, mitochondria, etc.) in a continuous fashion to form a vesicular plug that gradually restricts diffusion of increasingly smaller molecules and ions over a period of seconds to minutes. Furthermore, we find no direct evidence that sealing occurs through the collapse and fusion of severed plasmalemmal leaflets, or in a single step involving the fusion of one large wound vesicle with the nearby, undamaged plasmalemma. We describe how increases in perikaryal calcium levels following axonal transection account for observations that cell body survival decreases the closer an axon is transected to the perikaryon. Finally, we speculate on relationships between plasmalemmal sealing, Wallerian degeneration, and the ability of polyethylene glycol (PEG) to seal cell membranes and rejoin severed axonal ends – an important consideration for the future treatment of trauma to peripheral nerves. A better knowledge of biochemical pathways and cytoplasmic structures involved in plasmalemmal sealing might provide insights to develop treatments for traumatic nerve injuries, stroke, muscular dystrophy, and other pathologies.

Microvesicle Shedding and Lysosomal Repair Fulfill Divergent Cellular Needs during the Repair of Streptolysin O-Induced Plasmalemmal Damage

Pathogenic bacteria secrete pore-forming toxins that permeabilize the plasma membrane of host cells. Nucleated cells possess protective mechanisms that repair toxin-damaged plasmalemma. Currently two putative repair scenarios are debated: either the isolation of the damaged membrane regions and their subsequent expulsion as microvesicles (shedding) or lysosome-dependent repair might allow the cell to rid itself of its toxic cargo and prevent lysis. Here we provide evidence that both mechanisms operate in tandem but fulfill diverse cellular needs. The prevalence of the repair strategy varies between cell types and is guided by the severity and the localization of the initial toxin-induced damage, by the morphology of a cell and, most important, by the incidence of the secondary mechanical damage. The surgically precise action of microvesicle shedding is best suited for the instant elimination of individual toxin pores, whereas lysosomal repair is indispensable for mending of self-inflicted mechanical injuries following initial plasmalemmal permeabilization by bacterial toxins. Our study provides new insights into the functioning of non-immune cellular defenses against bacterial pathogens.

P2X7 receptors mediate resistance to toxin-induced cell lysis

In the majority of cells, the integrity of the plasmalemma is recurrently compromised by mechanical or chemical stress. Serum complement or bacterial pore-forming toxins can perforate the plasma membrane provoking uncontrolled Ca2+ influx, loss of cytoplasmic constituents and cell lysis. Plasmalemmal blebbing has previously been shown to protect cells against bacterial pore-forming toxins. The activation of the P2X7 receptor (P2X7R), an ATP-gated trimeric membrane cation channel, triggers Ca2+ influx and induces blebbing. We have investigated the role of the P2X7R as a regulator of plasmalemmal protection after toxin-induced membrane perforation caused by bacterial streptolysin O (SLO).Our results show that the expression and activation of the P2X7R furnishes cells with an increased chance of surviving attacks by SLO. This protective effect can be demonstrated not only in human embryonic kidney 293 (HEK) cells transfected with the P2X7R, but also in human mast cells (HMC-1), which express the receptor endogenously. In addition, this effect is abolished by treatment with blebbistatin or A-438079, a selective P2X7R antagonist. Thus blebbing, which is elicited by the ATP-mediated, paracrine activation of the P2X7R, is part of a cellular non-immune defense mechanism. It pre-empts plasmalemmal damage and promotes cellular survival. This mechanism is of considerable importance for cells of the immune system which carry the P2X7R and which are specifically exposed to toxin attacks.

Sealing of transected neurites of rat B104 cells requires a diacylglycerol PKC-dependent pathway and a PKA-dependent pathway.

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.

Rapid, effective, and long‐lasting behavioral recovery produced by microsutures, methylene blue, and polyethylene glycol after completely cutting rat sciatic nerves

Behavioral function lost in mammals (including humans) after peripheral nerve severance is slowly (weeks to years) and often poorly restored by 1-2-mm/day, nonspecifically directed outgrowths from proximal axonal stumps. To survive, proximal stumps must quickly repair (seal) plasmalemmal damage. We report that, after complete cut- or crush-severance of rat sciatic nerves, morphological continuity, action potential conduction, and behavioral functions can be consistently (>98% of trials), rapidly (minutes to days), dramatically (70-85% recovery), and chronically restored and some Wallerian degeneration prevented. We assess axoplasmic and axolemmal continuity by intra-axonal dye diffusion and action potential conduction across the lesion site and amount of behavioral recovery by Sciatic Functional Index and Foot Fault tests. We apply well-specified sequences of solutions containing FDA-approved chemicals. First, severed axonal ends are opened and resealing is prevented by hypotonic Ca²⁺-free saline containing antioxidants (especially methylene blue) that inhibit plasmalemmal sealing in sciatic nerves in vivo, ex vivo, and in rat B104 hippocampal cells in vitro. Second, a hypotonic solution of polyethylene glycol (PEG) is applied to open closely apposed (by microsutures, if cut) axonal ends to induce their membranes to flow rapidly into each other (PEG-fusion), consistent with data showing that PEG rapidly seals (PEG-seals) transected neurites of B104 cells, independently of any known endogenous sealing mechanism. Third, Ca²⁺-containing isotonic saline is applied to induce sealing of any remaining plasmalemmal holes by Ca²⁺-induced accumulation and fusion of vesicles. These and other data suggest that PEG-sealing is neuroprotective, and our PEG-fusion protocols that repair cut- and crush-severed rat nerves might rapidly translate to clinical procedures.

Neurite transection produces cytosolic oxidation, which enhances plasmalemmal repair

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.

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.

Plasmalemmal sealing of transected mammalian neurites is a gradual process mediated by Ca2+‐regulated proteins

Cultured mammalian PC12 or B104 cells do not instantaneously restore a plasmalemmal barrier (seal) after neurite transection, as measured using fluorescent dye probes of various sizes and saline solutions with different [Ca(2+)](o). Rather, transected cells gradually (from 15 to 60 min postseverance) exclude probes (dye molecules) of progressively smaller size. Furthermore, an inhibitor (calpeptin) of a Ca(2+)-activated cysteine protease (calpain) and antibodies or toxins to a Ca(2+)-regulated protein (synaptotagmin) and other membrane fusion proteins (syntaxin and synaptobrevin) inhibit plasmalemmal sealing. These data obtained using molecular probes on mammalian cell lines are consistent with previous data on invertebrate giant axons indicating that Ca(2+) plays many roles in the formation, accumulation, and fusion/interaction of vesicles gradually forming a seal at a site of plasmalemmal damage.

Vesicle-mediated restoration of a plasmalemmal barrier in severed axons.

Ca(2+)-induced endocytotic vesicles undergo protein-mediated interactions to restore a selectively permeable barrier and propagated action potentials in severed invertebrate giant axons. Similar barrier-restoration phenomena observed in cultured mammalian cells with transected neurites suggest that cellular/molecular mechanisms that repair plasmalemmal damage are phylogenetically conserved.

Delaminating myelin membranes help seal the cut ends of severed earthworm giant axons.

Transected axons are often assumed to seal by collapse and fusion of the axolemmal leaflets at their cut ends. Using photomicroscopy and electronmicroscopy of fixed tissues and differential interference contrast and confocal fluorescence imaging of living tissues, we examined the proximal and distal cut ends of the pseudomyelinated medial giant axon of the earthworm, Lumbricus terrestris, at 5-60 min post-transection in physiological salines and Ca2+-free salines. In physiological salines, the axolemmal leaflets at the cut ends do not completely collapse, much less fuse, for at least 60 min post-transection. In fact, the axolemma is disrupted for 20-100 microm from the cut end at 5-60 min post-transection. However, a barrier to dye diffusion is observed when hydrophilic or styryl dyes are placed in the bath at 15-30 min post-transection. At 30-60 min post-transection, this barrier to dye diffusion near the cut end is formed amid an accumulation of some single-layered and many multilayered vesicles and other membranous material, much of which resembles delaminated pseudomyelin of the glial sheath. In Ca2+-free salines, this single and multilayered membranous material does not accumulate, and a dye diffusion barrier is not observed. These and other data are consistent with the hypothesis that plasmalemmal damage in eukaryotic cells is repaired by Ca2+-induced vesicles arising from invaginations or evaginations of membranes of various origin which form junctional contacts or fuse with each other and/or the plasmalemma.

Repair of plasmalemmal lesions by vesicles.

Crayfish medial giant axons (MGAs) transected in physiological saline form vesicles which interact with each other, pre-existing vesicles, and/or with the plasmalemma to form an electrical and a physical barrier that seals a cut axonal end within 60 min. The formation of this barrier (seal) was assessed by measuring the decay of injury current at the cut end; its location at the cut end was determined by the exclusion of fluorescent hydrophilic dye at the cut end. When a membrane-incorporating styryl dye was placed in the bath prior to axonal transection and a hydrophilic dye was placed in the bath just after axonal transection, many vesicles near the barrier at the cut axonal end had their limiting membrane labeled with the styryl dye and their contents labeled with the hydrophilic dye, indicating that these vesicles originated from the axolemma by endocytosis. This barrier does not form in Ca2+-free salines. Similar collections of vesicles have been observed at regions of plasmalemmal damage in many cell types. From these and other data, we propose that plasmalemmal lesions in most eukaryotic cells (including axons) are repaired by vesicles, at least some of which arise by endocytosis induced by Ca2+ inflow resulting from the plasmalemmal damage. We describe several models by which vesicles could interact with each other and/or with intact or damaged regions of the plasmalemma to repair small (1-30 microm) plasmalemmal holes or a complete transection of the plasmalemma.