Repair of traumatic plasmalemmal damage to neurons and other eukaryotic cells

Abstract

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.

Keywords: Ca2+; axon regeneration; membrane damage; neuron; plasmalemmal sealing; vesicle mediated repair.

Conflict of interest statement

Conflicts of Interest: None declared.

Figures

Figure 1

Sealing pathway schematic for multiple proteins and pathways involved in vesicle mediated repair of plasmalemmal damage See text for references. Ca2+ influx at a site of plasmalemmal damage activates Ca2+ binding proteins, many of which increase vesicular traffic. The P2X7 protein, activated by ATP, also increases intracellular Ca2+. To date, three parallel sealing pathways have been identified that involve 1) oxidation, 2) calpains, and 3) cAMP (whose levels are mediated by Ca2+ activated adenylate cyclases). (1) The oxidation pathway activates vesicle formation, synaptic membrane fusion proteins (labeled in yellow), and Tri-partite motif proteins (TRIM proteins). (2) Calpains cleave cytoskeletal elements to free vesicles bound to the cytoskeleton, phospholipase C (PLC) and regulatory subunits of PKC isomers. PLC activation, in turn, cleaves the membrane lipid PIP2 into second messengers IP3 and diacylglycerol (DAG). DAG binding to regulatory subunits activates some PKC isomers. Ca2+ and DAG activate additional PKC isomers and the membrane fusion protein MUNC-13. (Note: not all PKC isomers are implicated in plasmalemmal sealing). PKC phosphorylates various isomers of synaptic membrane fusion proteins, and the membrane bound protein MARCKS. MARCKS increases DAG production, thereby enhancing PKC activity. (3) cAMP activates PKA and Epac. PKA directly phosphorylates SNARE proteins, such as SNAP-25 (inhibited by Botulinum toxin (BonT) A/E), synaptotagmin (inhibited by BonT B/TeNT), and syntaxin (inhibited by BonT E). PKA likely does not activate isomers of SNARE proteins required for Golgi membrane fusion (labeled in green) and ESCRT proteins that are inhibited by the fungal toxin Brefeldin A. In contrast, Epac (the guanine exchange factor) activates SNARE proteins in both the Golgi and synaptic membrane fusion pathways that converge at N-ethylmaleimide sensitive factor (NSF: labeled in blue). Finally, both lipid rafts and PEG produce membrane fusion, bypassing NSF.

Figure 2

Schematic of plasmalemmal sealing probabilities (A) and mechanisms (B–D). (A) Probability (sealing frequency) of plasmalemmal sealing of proximal stump decreases exponentially with decreasing distance of transection site from axon hillock. (B–D) Sealing of transected proximal and distal stumps by (B) plasmalemmal collapse and fusion, (C) formation of a new plasmalemmal partition, or (D) accumulation of vesicles or other membrane-bound structures that arise by plasmalemmal endocytosis, breakup or budding of the Golgi apparatus, smooth endoplasmic reticulum (SER), adaxonal (non-myelinating) glial membrane, or myelin sheath delaminatyion. Lysosomes and mitochondria can also contribute to the accumulation of membrane bound structures at a hole or complete transection of a cytoplasmic process (axon, muscle fiber, dendrite, etc.). These vesicular structures form a vesicular plug and pack tighter and tighter and/or fuse with each other and the plasmalemma (D1) or “wound vesicle” (D2). The vesicles are eventually resorbed and/or integrated to form a continuous membrane with the morphological and barrier characteristics of an undamaged plasmalemma (D3).