A new way of understanding bioenergetics
Abstract
Extra-mitochondrial oxidative phosphorylation that leads to the production of energy,
in the form of ATP, has been shown to occur in the rod outer segments in the retina. This
supplies energy for the process whereby light is converted to an electrical impulse and
carried by the optic nerve to the brain. Professor Alessandro Maria Morelli and Professor
Isabella Panfoli, from the University of Genoa, Italy, show that extra-mitochondrial
oxidative phosphorylation also occurs in the myelin sheath surrounding nerves, and
suggest that it is a vital energy supply for the nerve.
... Extensive research over the past 15 years by the Dr. Morelli group has revealed the existence of aerobic ATP synthesis in the myelin membrane, a subcellular structure devoid of mitochondria (Ravera et al. 2009(Ravera et al. , 2016a(Ravera et al. , b, 2021Morelli et al. 2011a; Morelli and Scholkmann 2024; Bartolucci et al. 2015). Oxidative phosphorylation (OxPhos) machinery that serves to synthesize extra-mitochondrial aerobic ATP needed to support efficient transmission of electrical impulses through the axon membrane (Ravera et al 2016a, b;Morelli 2022) could be delivered to the myelin membrane from mitochondria via the endoplasmic reticulum Marchi et al. 2014;Lee and Min 2018) or exported to the myelin sheath from mitochondrial components of oligodendrocytes (Morelli et al. 2011b). ...
... While the insulating properties of the myelin sheath are traditionally attributed to its multilayered structure, the sheath's role in facilitating rapid transmission through saltatory conduction-where action potentials jump from one node of Ranvier to the next-suggests a simpler and direct process in the myelin membranes that enables faster transmission of electrical signals along the nerve fiber. A new concept for the role of the myelin sheath, which has been developing over the past 15 years, suggests that ATP is synthesized in myelin membranes to provide energy for the rapid transmembrane influxes of sodium ions in the nodes of Ranvier, thereby generating efficient transmission of electrical impulses through the axons' membrane (Fields 2014;Ravera et al. 2009;Morelli 2022;Morelli et al. 2011a,b,c;Ravera et al. 2016aRavera et al. , b, 2021Bartolucci et al. 2015;Morelli and Scholkmann 2024;Panfoli et al. 2011;Marchi et al. 2014;Lee and Min 2018). It has been proposed that ATP synthesized extra-mitochondrially in myelin membranes is delivered through spaces between the myelin membranes to the axon through the gap junctions in the myelin membrane (Ravera et al 2016a, b;Morelli 2022). ...
... A new concept for the role of the myelin sheath, which has been developing over the past 15 years, suggests that ATP is synthesized in myelin membranes to provide energy for the rapid transmembrane influxes of sodium ions in the nodes of Ranvier, thereby generating efficient transmission of electrical impulses through the axons' membrane (Fields 2014;Ravera et al. 2009;Morelli 2022;Morelli et al. 2011a,b,c;Ravera et al. 2016aRavera et al. , b, 2021Bartolucci et al. 2015;Morelli and Scholkmann 2024;Panfoli et al. 2011;Marchi et al. 2014;Lee and Min 2018). It has been proposed that ATP synthesized extra-mitochondrially in myelin membranes is delivered through spaces between the myelin membranes to the axon through the gap junctions in the myelin membrane (Ravera et al 2016a, b;Morelli 2022). Additionally, it has been suggested that the myelin sheath serves as a proton capacitor by inducing the accumulation and ensued storage of protons on the myelin membrane surface (Morelli 2022;Morelli et al. 2011c), a concept supported by experimental evidence from our research group (Chaudary et al. 2024). ...
In this work, the heterodimeric phospholipase A2, HDP-2, from viper venom was investigated for its hydrolytic activity in model myelin membranes as well as for its effects on intermembrane exchange of phospholipids (studied by phosphorescence quenching) and on phospholipid polymorphism (studied by ¹H-NMR spectroscopy) to understand the role of sphingomyelin (SM) in the demyelination of nerve fibers. By using well-validated in vitro approaches, we show that the presence of SM in model myelin membranes leads to a significant inhibition of the hydrolytic activity of HDP-2, decreased intermembrane phospholipid exchange, and reduced phospholipid polymorphism. Using AutoDock software, we show that the NHδ+ group of the sphingosine backbone of SM binds to Tyr22(C=Opbδ–) of HDP-2 via a hydrogen bond which keeps only the polar head of SM inside the HDP-2’s active center and positions the sn-2 acyl ester bond away from the active center, thus making it unlikely to hydrolyze the alkyl chains at the sn-2 position. This observation strongly suggests that SM inhibits the catalytic activity of HDP-2 by blocking access to other phospholipids to the active center of the enzyme. Should this observation be verified in further studies, it would offer a tantalizing opportunity for developing effective pharmaceuticals to stop the demyelination of nerve fibers by aberrant PLA2s with overt activity – as observed in brain degenerative diseases – by inhibiting SM hydrolysis and/or facilitating SM synthesis in the myelin sheath membrane.
Graphical Abstract
Binding of sphingomyelin (SM) to catalytic subunits of HDP-2 predicted by Autodock. SM is given in sticks representation and HDP-2P is displayed in molecular surface (A) and line stick representation (B). For the stick diagrams, emerald represents carbon, blue–nitrogen, orange–phosphorus, red–oxygen, white–hydrogen. For molecular surface and lines, green represents carbon, blue–nitrogen, red–oxygen, white–hydrogen, yellow–sulfur. Yellow broken lines identify intermolecular bonds. Arrows point to sn-2 bonds. Hydrogen bond between NHδ+ group and Tyr22(C=Opbδ–) and ionic bond between PO4– group and His48 drive the positioning of the sn-2 acyl ester bond away from the active center that suggests that SM binds non-productively in the active center of HDP-2 enzyme.
... In some neurons, the presence of F1 subunits of ATP synthase in myelin sheaths was determined by oligomycin titration experiments [88]. Also, the considerable presence of mitochondrial respiratory components in lipid rafts in myelin was discovered [91,92], suggesting the existence of a route for delivering mitochondrial respiratory complexes to the myelin sheath [93,94]. Morelli et al. put forward the idea that mitochondria can provide the necessary apparatus of complex proteins required for synthesizing ATP in extra-mitochondrial regions of the cell [94]. ...
... Also, the considerable presence of mitochondrial respiratory components in lipid rafts in myelin was discovered [91,92], suggesting the existence of a route for delivering mitochondrial respiratory complexes to the myelin sheath [93,94]. Morelli et al. put forward the idea that mitochondria can provide the necessary apparatus of complex proteins required for synthesizing ATP in extra-mitochondrial regions of the cell [94]. Whereas the majority of mitochondrial proteins are synthesized in the nucleus, the mitochondria have their own DNA, termed mtDNA, which transcribes and synthesizes a limited number of proteins including subunits of respiratory complexes and ATP synthase to drive oxidative phosphorylation (OxPhos). ...
... Whereas the majority of mitochondrial proteins are synthesized in the nucleus, the mitochondria have their own DNA, termed mtDNA, which transcribes and synthesizes a limited number of proteins including subunits of respiratory complexes and ATP synthase to drive oxidative phosphorylation (OxPhos). Imaging experiments have demonstrated that mitochondria and ER are closely associated, which suggests that the two function in a coordinated manner to maintain homeostasis and when this association is lost, the bioenergetic capacity is impaired [94]. The close interaction of mitochondria with the ER has been also well documented in other studies [95,96]. ...
In this paper, we provide an overview of mitochondrial bioenergetics and specific conditions that lead to the formation of non-bilayer structures in mitochondria. Secondly, we provide a brief overview on the structure/function of cytotoxins and how snake venom cytotoxins have contributed to increasing our understanding of ATP synthesis via oxidative phosphorylation in mito-chondria, to reconcile some controversial aspects of the chemiosmotic theory. Specifically, we provide an emphasis on the biochemical contribution of delocalized and localized proton movement, involving direct transport of protons though the Fo unit of ATP synthase or via the hydrophobic environment at the center of the inner mitochondrial membrane (proton circuit) on oxidative phos-phorylation, and how this influences the rate of ATP synthesis. Importantly, we provide new insights on the molecular mechanisms through which cobra venom cytotoxins affect mitochondrial ATP synthesis, mitochondrial structure, and dynamics. Finally, we provide a perspective for the use of cytotoxins as novel pharmacological tools to study membrane bioenergetics and mitochondrial biology, how they can be used in translational research, and their potential therapeutic applications. Key Contribution: Cardiotoxin-induced inverted micelles in model inner mitochondrial membranes incited discovery of cardiolipin-made inverted micelles serving as proton nanocarriers for transporting protons from the Fo unit of ATP synthase to respiratory complexes at the inner mito-chondrial membrane.
... Professor Alessandro Morelli of Genoa University proposed that mitochondria, which have their own DNA for respiratory complexes and ATP synthase to drive oxidative phosphorylation, deliver all the protein complexes necessary for ATP synthesis to the myelin sheath with the help of the endoplasmic reticulum (ER) [64], with which mitochondria are closely associated in cytoplasmic space and function [65,66]. It has been determined that mitochondria produce necessary vesicles providing the ER with the necessary oxidative phosphorylation complexes [67,68]. ...
... From the respiratory complexes to the F1 subunit of ATP synthase, and in the middle of the F1 subunit, protons turn to the hydrophobic center of the membrane through which protons move back to the respiratory complexes [23]. Thus, the proton circuit, taking place entirely inside a single membrane, couples respiration with ATP synthesis [23,64,80]. In our view, the experimental evidence described above supports localized proton coupling, which is based on the membrane's ability to absorb protons on its surface. ...
... One of the mechanisms for enhancing ATP synthase activity facilitated by the non-bilayer phase was recently proposed by us, in which inverted micelles transfer protons across the hydrophobic environment of the crista membrane in the inner aqueous volume of inverted micelles [35,86]. The non-bilayer phase observed in our study in model myelin membranes likely exists in the form of inverted micelles, which transport protons in the myelin membrane's hydrophobic environment, not across the membrane but through the membrane center from the F1 subunit to the respiratory complexes to complete the proton circuit inside a single membrane, as proposed by Professor Morelli [64,80]. ...
This study describes the isolation of novel acidic and basic proteins from the venom of the Latrodectus pallidus (white widow spider), which exhibit high amino acid sequence homology to the 18.5 kDa isoforms of myelin basic proteins. This study explores the ability of model myelin membranes, composed of phospholipids and the acidic and basic proteins from spider venom, to absorb protons and form a non-bilayer lipid phase. The results of this study support a previously suggested concept by A.M. Morelli, which proposes that the myelin membrane may accumulate protons on its surface to store energy. The energy stored on membrane surface could then be used to drive proton circuits, potentially coupling hypothetical redox processes and ATP synthesis within the myelin membrane.
The existence of different conductive patterns in unmyelinated and myelinated axons is uncertain. It seems that considering exclusively physical electrical phenomena may be an oversimplification. A novel interpretation of the mechanism of nerve conduction in myelinated nerves is proposed, to explain how the basic mechanism of nerve conduction has been adapted to myelinated conditions.
The neurilemma would bear the voltage-gated channels and Na⁺/K⁺-ATPase in both unmyelinated and myelinated conditions, the only difference being the sheath wrapping it. The dramatic increase in conduction speed of the myelinated axons would essentially depend on an increment in ATP availability within the internode: myelin would be an aerobic ATP supplier to the axoplasm, through connexons. In fact, neurons rely on aerobic metabolism and on trophic support from oligodendrocytes, that do not normally duplicate after infancy in humans.
Such comprehensive framework of nerve impulse propagation in axons may shed new light on the pathophysiology of nervous system disease in humans, seemingly strictly dependent on the viability of the pre-existing oligodendrocyte.
The disks of the vertebrate retinal rod Outer Segment (OS), devoid of mitochondria, are the site of visual transduction, a very energy demanding process. In a previous proteomic study we reported the expression of the respiratory chain complexes I-IV and the oxidative phosphorylation Complex V (F(1)F(0)-ATP synthase) in disks. In the present study, the functional localization of these proteins in disks was investigated by biochemical analyses, oxymetry, membrane potential measurements, and confocal laser scanning microscopy. Disk preparations, isolated by Ficoll flotation, were characterized for purity. An oxygen consumption, stimulated by NADH and Succinate and reverted by rotenone, antimycin A and KCN was measured in disks, either in coupled or uncoupled conditions. Rhodamine-123 fluorescence quenching kinetics showed the existence of a proton potential difference across the disk membranes. Citrate synthase activity was assayed and found enriched in disks with respect to ROS. ATP synthesis by disks (0.7 micromol ATP/min/mg), sensitive to the common mitochondrial ATP synthase inhibitors, would largely account for the rod ATP need in the light. Overall, data indicate that an oxidative phosphorylation occurs in rod OS, which do not contain mitochondria, thank to the presence of ectopically located mitochondrial proteins. These findings may provide important new insight into energy production in outer segments via aerobic metabolism and additional information about protein components in OS disk membranes.