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THEORETICAL B I O LO G Y F O RU M 104 · 2/2011

PISA · ROMA FABRIZIO SERRA EDITORE MMXI

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CONTENTS editorial Giuseppe Damiani, Lamarckian evolution: the dark side of the biology

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letters to the editors Mesut Tez, Complex system perspective in colorectal carcinogenesis

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Dewu Ding, Of Fish and Men: A comparative study for genome-scale metabolic networks

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articles Federico Morganti, Intelligence as the plasticity of instinct: George J. Romanes and Darwin’s earthworms

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Sergio Pennazio, The dawn of photosynthesis

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perspectives Alessandro Morelli, Silvia Ravera, Daniela Calzia, Isabella Panfoli, Exportability of the mitochondrial oxidative phosphorylation machinery into myelin sheath.

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Silvano Traverso, Mechanical signalling in tissues and its possible role in nociception

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EXPORTABILITY OF THE MITOCHONDRIAL OXIDATIVE PHOSPHORYLATION MACHINERY INTO MYELIN SHEATH Alessandro Morelli · Silvia Raver a Daniela Calzia · Isabella Panfoli DIPTERIS_Biochemistry Lab, University of Genova, Faculty of Sciences Viale Benedetto XV, 3, 16132 Genova, Italy, Tel/Fax: +39 010 353 8153 Corresponding Author: Alessandro Morelli E-mail: [email protected] Contents: 1. Introduction. 2. Objectives. 2. 1. A novel function for myelin sheath 3. Conclusions. 3. 1. Discussion. Keywords: myelin, ATP, bioenergetics, gap junctions, oligodendrocyte. Abstract: White matter comprises over half of the brain, and its role in axonal survival is being reconsidered, consistently with the observation that axonal degeneration follows demyelination. The recent evidence of an extra-mitochondrial aerobic ATP production in isolated myelin vesicles, thanks to the expression therein of the mitochondrial Oxydative Phosphorylation (OXPHOS) machinery, stands in for myelin playing a functional bioenergetic role in ATP supply for the axon. The observation that subunits of the OXPHOS encoded by the mitochondrial genome are expressed in myelin, suggests that they can be the

same as those of the inner mitochondrial membrane. This would mean that the OXPHOS is exportable. Here the hypothesis is exposed that the mitochondrion is the unique site of the assembly of the OXPHOS, so that this is exported to those sub cellular districts displaying high energy demand, such as myelin sheath. There the OXPHOS would display a higher efficiency in oxidative ATP production than inside the mitochondrion itself. In this respect, the role of the glia in the nervous conduction is shed new light and the oligodendrocyte mitochondrial OXPHOS are hypothesized to be delivered to nascent myelin.

1. Introduction

T

he human brain is the organ displaying the highest energy expenditure of the body, as well as the highest oxygen (O2) consumption. However, it does not seem likely that mitochondria are the only site of combustion in brain, because of its scarcity in mitochondria [1]. Another district besides mitochondria seems necessary for combustion in brain. Moreover, O2 is intensely absorbed by brain and some device must be operative for oxygen trapping, similarly to myoglobin, the common oxygen trap, that is however absent in brain. It may be asked whether a structure meeting both requirements does exist. Aiming to identify possible structures that can vicariate mitochondria in the Nervous System (NS), attention is being posed on the role exerted by glia, particularly on its interaction with the axon. It has been shown that myelin can influence

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a. morelli · s. ravera · d. calzia · i. panfoli

conduction velocity depending on the thickness of the sheath and axon diameter, which is reduced in myelin-deficient mutants [2]. Generally larger-caliber fibers conduct impulses at higher speeds, however differences are seen between fibers [3] even along the same fiber [4]. The number of myelin wraps increases as animals grow and their axons lengthen. Even the g-ratio (i.e.: the axon diameter divided by the total fiber diameter i.e.: including the myelin sheath) can change with respect to the theoretical optimal value of 0.65 [3]. In addition to controlling conduction velocity, myelin proteins directly controls synapse formation by inhibiting axon sprouting. It was shown that electrical activity promotes proliferation of oligodendrocyte progenitor cells in optic nerve [5]. Such induction of myelination by axonal electric activity appears as a central topic in teleological growth of myelin [6]. It appears that myelin grows in response to an energy demand by the axon: this is the core of our present speculation. Moreover, survival of axons and neurons is dependent on trophic support and signaling from myelinating glia. For example, in multiple sclerosis, chronically demyelinated axons are lost. The long period of myelination in humans, that coincides with the period of synaptic remodeling in response to environmental experience, would imply that myelin plays a role in optimizing information processing. In fact the speed of impulse conduction through axons is controlled by myelin [7]. Interestingly, it was reported that the oligodendroglial differentiation is dependent on an amplification of mitochondrial transcripts and mtDNA and that oligodendrocytes treated with rotenone (an electron transport chain Complex I inhibitor) display decreased levels of the transcripts of specific myelin proteins (2’,3’-Cyclic Nucleotide-3’-Phosphodiesterase and Myelin Basic Protein (MBP)) [8]. Cordeau-Lossouarn et al. [9] found that an increase in mitochondrial proteins accompanies the steps of oligodendrocyte differentiation [9]. Recently we have formulated a supported hypothesis on the existence of extramitochondrial subcellular sites for oxidative phosphorylation (OXPHOS) [10]. Accordingly, myelin would be functional in glucose combustion for axonal supply, thanks to mitochondrial components therein expressed [11-13] [14] [15, 16] [17] and catalytically active [18] [19]. Moreover, the neutral lipids of which myelin is rich would absorb the O2 coming from blood [20]. Neutral lipids are good candidates for oxygen trapping. So the myelin sheath appears as an integrated device for optimized glucose combustion to realize an energetic supply of the axon, by colocalization of an ‘O2 sponge’ represented by the abundant neutral lips, and OXPHOS machinery. 2. Objectives The primary goal of this study was to theoretically investigate: – the possibility that ATP is supplied to the axons with the contribution of both mitochondria and myelin, via a mechanism requiring the expression therein of mitochondrial OXPHOS;

extramitochondrial oxidative phosphorylation in myelin

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– the source and the route followed by mitochondrial protein complexes found expressed in myelin in the oligodendrocyte membranes that form myelin sheath in CNS; – whether the transfer of mitochondrial components from mitochondria to myelin, the central intriguing question of present speculation, may involve endoplasmic reticulum (ER). 2. 1. A novel function for myelin sheath Recently, an extramitochondrial aerobic ATP production was reported in Isolated Myelin Vesicles (IMV) [18] and retinal rod outer segments [21] [19]. An hypothesis on myelin functioning has also been proposed [22]. Moreover, it was shown that IMV membranes express IF1, a protein inhibitor of the F1 moiety of F0F1-ATP synthase acting in low pH [23]. IF1 plays a pivotal role in the prevention of the reversal of ATP synthase for example during central NS ischemic events [23] [24]. In many proteomic analyses of myelin [13] a considerable amount of mitochondrial proteins were found, as we have discussed [17]. We proposed that gap junctions, transmembrane channels formed by docking of two connexons, each of which is composed of one out of six connexin (Cx) proteins, may allow ATP synthesized into the sheath to reach the axoplasm, supporting the bioenergetics of the axonal firing [22] together with axonal mitochondria. In this view a novel interaction between oligodendrocyte and axons is depicted in which the connexons would cross the major dense line and let ATP synthesized by ATP synthase thanks to the accumulating H+, to flow to the periplasmic face, and eventually into the axoplasm. The insert of schematic in Figure 1 depicts a vision of myelin sheath as a respiring wrap where O2 is reduced to H2O (Figure 1). An ectopic sidedness of ATP synthase in the oligodendrocyte plasma membrane is hypothesized, so that F0 would face the external milieu. ATP would be aerobically synthesized in periplasmic myelin where the respiratory complexes I-IV would be expressed [11] [12] with a sidedness similar to that in inner mitochondrial membranes. H+ transferred by a putative electron transport chain would accumulate in the major dense line formed by the two cytoplasmic faces of the oligodendrocyte, thanks to an H+ buffering by myelin basic protein (MBP), particularly abundant (25-30 % of total protein) in myelin, whose function is as yet unclear. So this buffering function appears very functional for energy accumulation and supply, in a organ, the brain, substantially devoid of classical energy stores (triglycerides and glycogen). The possible role of this chemiosmotic buffer in sleep mechanism has been pointed out [10]. H+ would flow back to the periplasmic face through ATP synthase and the synthesized ATP would be radially transferred into the axoplasm, through gap-junctions. Contacts among the major dense line and axolemma of regenerating axons have been identified [25]. Functional gap junctions are expressed in myelin [26] [27] [28] (Alvarez-Maubecin et al., 2000; Suadicani et al., 2009) [29]. In CNS, gap junctions have been hypothesized to form a glial “sponge” that siphons K+ away from the firing axons [30]. However, ATP was shown to flow 300-fold better than any other tested compound through Cx 43 [31].

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Fig. 1. Exportation of mitochondrial components from oligodendrocyte to myelin sheath. The insert magnifies the probable assembly of the F0F1-ATP synthase at the opposite sides of major dense line.

An ectocellular ATP synthase has been reported in the cellular membranes of many cell types, however a number of reports ascribe to it functions other than ATP synthesis, such as of a cell-surface receptor (reviewed in [32] [33]). For example, recently Kim et al. [34] challenged the conclusion that ATP synthase is always required for extracellular ATP synthesis. If myelin sheath is active in aerobic ATP synthesis, and all the components of OXPHOS are expressed in myelin, how are these mitochondrial proteins transferred to the oligodendrocyte membranes? Whatever the real pathway is, it is clear that mitochondrial protein are abundant in myelin and that OXPHOS is fully functional there. The molecular components of myelin can only originate from mitochondria, at least as far as the proteins codified by mitochondrial DNA are concerned. The pathway followed remains a matter for investigation. Nevertheless, the possibility of a fusion among mitochondria, nuclear membrane, ER, and plasma membrane, has been depicted [35]. Close dynamic contacts between sub-domains of the endoplasmic and mitochondrial reticulum, have been suggested to exists [reviewed in [36]]. A recent proteomic study showed that about 30% of brain ER proteins are mitochondrial [37]. The ER seems to express TIM and TOM [37]. ER stress in myelinating cells has been involved in the pathogenesis of various myelin disorders [38].


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3. Conclusions – An extramitocondrial energy elaboration is an absolute necessity for the nervous system and a new fascinating energetic role for myelin is depicted. – Myelinating glia can influence the axon connectivity and neuronal survival under both physiological and pathological conditions, raising the possibility that survival of individual axons could be influenced by glia under normal conditions. – Mitochondrial OXPHOS found expressed in myelin would be assembled in mitochondria and delivered to myelin sheath by a fusion pathway that would operate from oligodendrocyte to mature myelin processes, with possible participation of the ER. 3. 1. Discussion Processing of energy is the most peculiar and essential feature of all living systems. This is particularly true in the NS where aerobic metabolism is higher (~ 10 folds) than – for example – in liver that consumes 10-fold less oxygen but possesses many more mitochondria than neurons. Retinal rod outer segment disks and bovine brain IMV, which are devoid of mitochondria, display functional OXPHOS for ectopic aerobic ATP synthesis [19] [18]. Recently we have shown that the enzymes of Tricarboxylic acid Cycle (TCA) are functional in rod OS (Panfoli et al. [2011]). Many facts on myelin that were puzzling from the older perspective of its being a simple insulating sheath are shed new light by the present hypothesis. The putative presence of respiring extramitochondrial membranes in NS offers a new vision in the pathogenic role of mitochondrial disorders, a group of human diseases characterized by defects in OXPHOS whose most frequent clinical presentations are neurological syndromes [39], and of many inherited and acquired retinopathies, hereditary optic neuropathies [40], as well as demyelinating/dysmyelinating diseases of NS. Some of these have oxidative stress or energy impairment as a common denominator [41]. So, these demyelinating diseases may be observed under a new perspective, considering the energetic factor, which may allow to better understand their etiopathogenesis and to improve the treatments and the preventive interventions. The identification of the sites involved in oxygen absorption and energetic production in SN is fundamental for the comprehension of many neuronal pathologies. In fact, myelin is attracting new interest as an unexpected contributor to a wide range of psychiatric disorders, including depression and schizophrenia. Myelin disorders from damage or disease result in many psychiatric and neurological disorders. Conduction failure consequent to demyelination results in sensorymotor dysfunction, cognitive impairment, to cite only a few. White matter changes are associated with learning in people and cognitive impairment is associated with demyelination consequent to therapy with 5 fluorouracyl [42]. The central question on the pathway followed by the OXPHOS machinery to oligodendrocyte processes forming the nascent myelin is a matter of future research. We had supposed that the assembly of the OXPHOS takes place in the mitochondrion itself, being the process complex and plausibly needing a dedicate site.

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Probably this is the most important role of mitochondria. On the way of delivery, it is possible a intermediation of the ER, in consideration of its impressively high content in mitochondrial proteins [37]. Moreover this assessment opens a new biological scenario for other non-neural cells so that the exportability of mitochondrial machinery would be a general process for optimisation of energy conversion in the subcellular sites. Myelin turnover is lower than that of other neuronal proteins. The expression of the OXPHOS in myelin gets them more long-lived, which stands in for a greater efficiency but also for a higher vulnerability to dysfunction: myelin can reveal the mitochondrial dysfunctions, which is in fact the case. Mutations in the OXPHOS proteins cause diseases that affect almost exclusively CNS, PNS and visual system. In conclusion, the present hypothesis gives to the glia a central role in the energetic support of synaptic transmission, which can overturn the long-lived idea that myelin is only a mechanical support, a ‘glue’, which has been recently challenged [7]. In fact, axonal degeneration and not a simple slowing down of conduction follows demyelization. May we be more optimist about our understanding of myelin function than it has been stated [43]? References 1. Veltri KL, Espiritu M, Singh G. Distinct genomic copy number in mitochondria of different mammalian organs. J Cell Physiol. 1990 Apr;143(1): 160-4. 2. Fields RD, Stevens-Graham B. New insights into neuron-glia communication. Science (New York, NY. 2002 Oct 18;298(5593): 556-62. 3. Berthold CH, Nilsson I, Rydmark M. Axon diameter and myelin sheath thickness in nerve fibres of the ventral spinal root of the seventh lumbar nerve of the adult and developing cat. Journal of anatomy. 1983 May;136(Pt 3): 483-508. 4. Baker GE, Stryker MP. Retinofugal fibres change conduction velocity and diameter between the optic nerve and tract in ferrets. Nature. 1990 Mar 22;344(6264): 342-5. 5. Barres BA, Raff MC. Proliferation of oligodendrocyte precursor cells depends on electrical activity in axons. Nature. 1993 Jan 21;361(6409): 258-60. 6. Demerens C, Stankoff B, Logak M, Anglade P, Allinquant B, Couraud F, et al. Induction of myelination in the central nervous system by electrical activity. Proceedings of the National Academy of Sciences of the United States of America. 1996 Sep 3;93(18): 9887-92. 7. Fields RD. White matter in learning, cognition and psychiatric disorders. Trends in neurosciences. 2008 Jul;31(7): 361-70. 8. Schoenfeld R, Wong A, Silva J, Li M, Itoh A, Horiuchi M, et al. Oligodendroglial differentiation induces mitochondrial genes and inhibition of mitochondrial function represses oligodendroglial differentiation. Mitochondrion. 2010 Mar;10(2): 143-50. 9. Cordeau-Lossouarn L, Vayssiere JL, Larcher JC, Gros F, Croizat B. Mitochondrial maturation during neuronal differentiation in vivo and in vitro. Biol Cell. 1991;71(1-2): 57-65. 10. Morelli A, Ravera S, Panfoli I. Myelin sheath: a new possible role in sleep mechanism. Sleep medicine. 2011 Feb;12(2): 199. 11. Taylor CM, Marta CB, Claycomb RJ, Han DK, Rasband MN, Coetzee T, et al. Proteomic mapping provides powerful insights into functional myelin biology. Proceedings of the National Academy of Sciences of the United States of America. 2004 Mar 30;101(13): 4643-8.


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