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Title: Impulse Conduction Increases Mitochondrial Transport in Adult Mammalian Peripheral Nerves In Vivo}
Article:
Abstract:
Matching energy supply and demand is critical in the bioenergetic homeostasis of all cells. This is a special problem in neurons where high levels of energy expenditure may occur at sites remote from the cell body, given the remarkable length of axons and enormous variability of impulse activity over time. Positioning mitochondria at areas with high energy requirements is an essential solution to this problem, but it is not known how this is related to impulse conduction in vivo. Therefore, to study mitochondrial trafficking along resting and electrically active adult axons in vivo, confocal imaging of saphenous nerves in anaesthetised mice was combined with electrical and pharmacological stimulation of myelinated and unmyelinated axons, respectively. We show that low frequency activity induced by electrical stimulation significantly increases anterograde and retrograde mitochondrial traffic in comparison with silent axons. Higher frequency conduction within a physiological range( 50 Hz) dramatically further increased anterograde, but not retrograde, mitochondrial traffic, by rapidly increasing the number of mobile mitochondria and gradually increasing their velocity. Similarly, topical application of capsaicin to skin innervated by the saphenous nerve increased mitochondrial traffic in both myelinated and unmyelinated axons. In addition, stationary mitochondria in axons conducting at higher frequency become shorter, thus supplying additional mitochondria to the trafficking population, presumably through enhanced fission. Mitochondria recruited to the mobile population do not accumulate near Nodes of Ranvier, but continue to travel anterogradely. This pattern of mitochondrial redistribution suggests that the peripheral terminals of sensory axons represent sites of particularly high metabolic demand during physiological high frequency conduction. As the majority of mitochondrial biogenesis occurs at the cell body, increased anterograde mitochondrial traffic may represent a mechanism that ensures a uniform increase in mitochondrial density along the length of axons during high impulse load, supporting the increased metabolic demand imposed by sustained conduction.
Introduction:
Axons can be a metre, or more, in length and mitochondria traffic along them to become correctly distributed to fulfil local metabolic requirements[1]. Indeed, defects in mitochondrial transport are associated with a number of diseases affecting long neuronal tracts[2]. The devastating consequence of impaired mitochondrial trafficking is illustrated in the axonal form of Charcot-Marie-Tooth disease, which is caused by selective impairment of mitochondrial transport due to mutations of the mitofusin2 gene[3],[4]. However, studies of dynamic processes such as mitochondrial transport are not feasible in humans and are still very challenging in animal models. Thus, the study of axonal mitochondrial traffic has largely been limited to in vitro methods, e. g., employing cultured dorsal root ganglia( DRG), organotypic slice cultures[5], or excised myelinated nerves[6]. However, observations in vitro are inevitably limited by inherent constraints. For example, neurites in DRG cultures are not only typically unmyelinated, but they also continue to develop and grow while being examined, so mitochondrial trafficking reflects changes in growth and development as well as the steady state energy requirements of axons. Organotypic slices and excised nerves may contain myelinated axons, but they have no perfused vasculature providing oxygen and nutrients, thus lacking physiological regulatory mechanisms crucial for metabolic adaptation in response to increased demand. Furthermore, excised nerves lack cell bodies and are therefore incipiently undergoing degeneration. To avoid these problems, zebrafish embryos have been used to image mitochondrial trafficking in vivo[7], but it is not known whether developing axons in fish reflect the properties of adult mammalian axons. Axonal mitochondria provide the ATP required to support impulse activity, yet almost no information is available about the modulation of axonal mitochondrial traffic by neuronal activity. To overcome the limitations imposed by in vitro conditions we have studied mitochondrial traffic in vivo, along resting and electrically active adult mammalian axons. Thus, confocal time lapse imaging of mitochondrial movements, distribution, and morphology along saphenous nerve axons in anaesthetised mice was combined with electrical and pharmacological stimulation of myelinated and unmyelinated axons.
Discussion:
Our findings demonstrate a physiological relationship between impulse activity and mitochondrial trafficking along normal, adult, myelinated, and unmyelinated mammalian peripheral axons in vivo. Earlier studies conducted in vitro have reported different observations in excised frog nerves[6] and cultured mammalian Purkinje cells in tissue slices[5], where induced activity was associated with a decrease in mitochondrial transport, and an accumulation of mitochondria at, or near, nodes of Ranvier. The explanation for the different results is not clear, although there are obvious differences in methodological detail. Zheng et al. applied tetanic stimulation( 200 Hz) to excised frog nerves in vitro, which is presumably a supra-physiological frequency for this cold-blooded species. Ohno et al. described pharmacologically induced activity in central nervous system Purkinje cells in vitro, measured in numbers of calcium transients. The stimulation resulted in an increase from one, to three calcium transients in 200 s, which appears to be an increase in frequency from 0. 005 Hz to 0. 015 Hz. We have studied impulse activity in mammalian peripheral nervous system( PNS) axons in vivo within the physiological range at 0, 1, and 50 Hz( impulse activity at 50 Hz is well within the physiological range for many mammalian axons[12]), suggesting that the relatively high number and speed( average ∼0. 7 µm/s and anterograde ∼0. 85 µm/s) of moving mitochondria detected with 50 Hz activity may indicate the steady state mitochondrial traffic in sensory axons in awake, active animals. Misgeld and colleagues reported an average speed of mitochondria of 1–1. 5 µm/s in intercostal nerve in the triangularis sterni muscle explant[13], but whether the axons were active was not reported. It is possible that the higher speed in the intercostal nerve is due to the presence of motor axons rather than solely sensory axons as in the saphenous nerve, although there is a range of speeds in the saphenous nerve, perhaps reflecting different sensory modalities and their inherently different average levels of impulse activity. To our knowledge, the link between molecular motors( e. g., kinesin and dynein), which carry mitochondria along the microtubules, and impulse conduction is not known. The previous studies into mechanisms of regulation of mitochondrial traffic studied growing, or damaged, axons, and therefore tended to detect and examine docking, rather than the mechanisms of increases in number of moving mitochondria or their speed. In contrast to descriptions of activity-dependent nodal accumulation of mitochondria in vitro[5],[6], we found no such evidence in vivo. Although it seems reasonable to expect a greater increase in energy demand at nodes of Ranvier during sustained impulse activity, sodium entry to the axon during impulse activity is a passive process and the energy-demanding act of sodium extrusion via the Na+/K+ ATPase appears to be accomplished along the internode[14]. The absence of nodal recruitment of mitochondria in response to physiological levels of impulse activity is therefore unsurprising. Also, one might expect that the mitochondria in the mouse nerves are already optimally positioned along the axons by physiological impulse activity because of prior life experience, and so re-positioning in response to the applied impulse load is unnecessary. The nodal accumulation observed in the frog nerves may have been related to the unusually high stimulus rate of 200 Hz applied to this amphibian species, or to the fact that amphibian nodes can have different biophysical properties to mammalian nodes, e. g., with regard to the distribution of potassium channels[15], and perhaps to the distribution of the Na+/K+ ATPase. The large number of mitochondria normally found at sensory axon terminals[16],[17] indicates that the terminals have a high metabolic demand, and the surge of anterograde transport we have observed will help to satisfy this demand. Indeed, we demonstrated increased labelling for the mitochondrial marker, VDAC1, in the dermal terminals of saphenous nerves conducting at 50 Hz, suggesting that the sensory endings are one of the destinations of the anterogradely transported mitochondria. A recent study found that just one sensory neuron can branch to make contact with around 200 hair follicles or Merkel cells[18], and individual neuronal arbors in hairy skin may have around 1, 000 branch points, and a total cutaneous axon length of up to one metre. In this case, providing even a small increase in mitochondrial density to all the terminals of one axon would require a substantial increase in anterograde mitochondrial trafficking in the parent axon. Indeed, failure to satisfy such potential demand for mitochondria in sensory terminals in disease states may explain the dying-back neuropathies, such as Charcot-Marie-Tooth( CMT) or diabetic neuropathy: indeed, CMT is associated with an impairment of mitochondrial trafficking due to mutation of the mitofusin2 gene. The destiny of mitochondria accumulated within peripheral terminals, following cessation of high frequency impulse conduction, is still unclear. If the purpose of transport is to deliver functional mitochondria to the target destination( e. g., sensory terminal), then it seems counterproductive for the neuron to degrade these, presumably healthy, mitochondria in the terminals once the period of increased need is ceased. Previous studies suggested that mitochondrial degradation and autophagy is indeed initiated at the axonal terminal of cultured DRG neurons, and that the autophagosomes are then transported retrogradely towards the cell body, becoming increasingly acidified along the way[19]. In our study, the increase in the mobile cohort of mitochondria appears to be composed of( a) static mitochondria that become mobile, particularly from regions densely populated by small mitochondria, and( b) portions of mitochondria that are pinched off the static population by fission. A possibly comparable phenomenon of depolarisation-induced fission has been observed in the dendritic spines of cultured hippocampal neurons[20]. The mechanism( s) by which fission contributes to neuroprotection are not established. Our study suggests a hitherto unsuspected role of fission in providing a rapid supply of mobile mitochondria to axons that are required promptly to conduct impulses at high frequencies over relatively long periods. Our observations indicate a mechanism by which axons solve the problem of how to match mitochondrial density and distribution to the changing energy demands imposed by a variable impulse load. It would be wasteful to provide more mitochondria than necessary, and yet dangerous to provide too few, so a mechanism to maintain dynamic balance is necessary. A sustained increase in impulse load( e. g., resulting from an injury that causes a shift in body stance) requires an increase in mitochondrial mass, but this has to be achieved promptly and to be evenly distributed along an axon that may be a metre long, even in a moderately sized body such as a human. The conundrum appears to be solved by a mechanism where the longest mitochondria stay anchored( which ensures that no regions are deprived of mitochondria), but “pinch off” daughter mitochondria by fission, adding them to the anterograde traffic so that they increase mitochondrial density evenly along the axon, building an increased mitochondrial mass from the terminals backwards as the demand is satisfied. New mitochondria are presumably added to the proximal axon at the cell body to maintain the proximal population, and to sustain a raised mitochondrial mass suitable for the new impulse load. Aside from responding to changes in impulse load, the effect of impulse activity on mitochondrial trafficking also provides an effective mechanism to ensure that mitochondria are replenished more rapidly along axons that have a higher impulse activity. It seems likely that mitochondria with high rates of oxidative phosphorylation in very active axons will require earlier replacement, and in this case the mechanism would ensure that mitochondria cycle rapidly through very active axons, but linger longer in more quiescent axons.
As mitochondria are the main power source for most cells , their correct localization is vital for cellular homeostasis . The morphological and functional complexity of neurons , and the unpredictability of energy demand resulting from sustained impulse activity , means that correct mitochondrial function and location is particularly important for these cells . However , it is unclear how mitochondrial transport responds to the wide range of physiological conduction frequencies experienced by an intact nervous system . Here we image mitochondria moving along mouse saphenous nerve axons in vivo and observe the effects of sustained trains of impulses , evoked either by electrical stimulation of myelinated axons at physiological frequencies ( 1 and 50 Hz ) , or chemical stimulation ( cutaneous capsaicin ) in unmyelinated axons . We find that the number and speed of mitochondria moving towards the peripheral sensory terminals dramatically increases in active axons . The increased numbers of mobile mitochondria seem to originate from “pinched off” segments of stationary mitochondria ( mitochondrial fission ) , and from recruitment of small stationary mitochondria . The transported mitochondria appear to accumulate in sensory nerve terminals in the skin . This study suggests that mitochondrial fission is involved in rapidly supplying mobile mitochondria to aid their re-distribution to regions of increased metabolic demand along axons .