Molecular evolution of cytochrome C oxidase underlies high-altitude adaptation in the bar-headed goose.

Bar-headed geese (Anser indicus) fly at up to 9,000 m elevation during their migration over the Himalayas, sustaining high metabolic rates in the severe hypoxia at these altitudes. We investigated the evolution of cardiac energy metabolism and O(2) transport in this species to better understand the molecular and physiological mechanisms of high-altitude adaptation. Compared with low-altitude geese (pink-footed geese and barnacle geese), bar-headed geese had larger lungs and higher capillary densities in the left ventricle of the heart, both of which should improve O(2) diffusion during hypoxia. Although myoglobin abundance and the activities of many metabolic enzymes (carnitine palmitoyltransferase, citrate synthase, 3-hydroxyacyl-coA dehydrogenase, lactate dehydrogenase, and pyruvate kinase) showed only minor variation between species, bar-headed geese had a striking alteration in the kinetics of cytochrome c oxidase (COX), the heteromeric enzyme that catalyzes O(2) reduction in oxidative phosphorylation. This was reflected by a lower maximum catalytic activity and a higher affinity for reduced cytochrome c. There were small differences between species in messenger RNA and protein expression of COX subunits 3 and 4, but these were inconsistent with the divergence in enzyme kinetics. However, the COX3 gene of bar-headed geese contained a nonsynonymous substitution at a site that is otherwise conserved across vertebrates and resulted in a major functional change of amino acid class (Trp-116 → Arg). This mutation was predicted by structural modeling to alter the interaction between COX3 and COX1. Adaptations in mitochondrial enzyme kinetics and O(2) transport capacity may therefore contribute to the exceptional ability of bar-headed geese to fly high.

A new organellar complex in rat sympathetic neurons.

Membranous compartments of neurons such as axons, dendrites and modified primary cilia are defining features of neuronal phenotype. This is unlike organelles deep to the plasma membrane, which are for the most part generic and not related directly to morphological, neurochemical or functional specializations. However, here we use multi-label immunohistochemistry combined with confocal and electron microscopy to identify a very large (approximately 6 microns in diameter), entirely intracellular neuronal organelle which occurs singly in a ubiquitous but neurochemically distinct and morphologically simple subset of sympathetic ganglion neurons. Although usually toroidal, it also occurs as twists or rods depending on its intracellular position: tori are most often perinuclear whereas rods are often found in axons. These 'loukoumasomes' (doughnut-like bodies) bind a monoclonal antibody raised against beta-III-tubulin (SDL.3D10), although their inability to bind other beta-III-tubulin monoclonal antibodies indicate that the responsible antigen is not known. Position-morphology relationships within neurons and their expression of non-muscle heavy chain myosin suggest a dynamic structure. They associate with nematosomes, enigmatic nucleolus-like organelles present in many neural and non-neural tissues, which we now show to be composed of filamentous actin. Loukoumasomes also separately interact with mother centrioles forming the basal body of primary cilia. They express gamma tubulin, a microtubule nucleator which localizes to non-neuronal centrosomes, and cenexin, a mother centriole-associated protein required for ciliogenesis. These data reveal a hitherto undescribed organelle, and depict it as an intracellular transport machine, shuttling material between the primary cilium, the nematosome, and the axon.

Differential regulation of dendritic plasticity by neurotrophins following deafferentation of the adult spinal cord is independent of p75(NTR).

Spontaneous and/or treatment-evoked re-modeling of the CNS following spinal cord injury is a prerequisite for functional recovery. While there has been considerable interest in the role of endogenous neurotrophins in spontaneous plasticity of several populations of spinal axons, the same cannot be said for morphological changes to dendrites. Here, we examined the responses of dendrites in the mouse lateral spinal nucleus (LSN, a site of sensory integration in the dorsolateral white matter) to exogenous and endogenous neurotrophins. We performed a septuple dorsal rhizotomy, which permanently eliminates sensory input to the spinal cord, and stimulates sprouting of spinal axons. While dendrites showed no change in density following injury alone, they sprouted vigorously (a two-fold increase in density) upon addition of exogenous brain-derived neurotrophic factor (BDNF). On the other hand, endogenous nerve growth factor (NGF) severely restricted dendritic sprouting, as TrkA-Fc treatment also roughly doubled the density of dendritic processes in the LSN. Spontaneous, BDNF- and TrkA-Fc mediated sprouting was unaffected by the absence of p75(NTR). Importantly, TrkA-Fc treatment markedly reduced expression of the truncated BDNF receptor TrkBT1 in both p75(+/+) and p75(-/-) mice, which was robustly-upregulated by deafferentation in both genotypes. We propose that the upregulation of TrkBT1 by NGF results in a reduced availability of endogenous BDNF to dendrites. Accordingly, sprouting of serotonergic axons, a BDNF-dependent consequence of dorsal root injury, was significantly enhanced in TrkA-Fc-treated animals. These results suggest that NGF and BDNF signaling differentially regulates dendritic plasticity in the deafferented spinal cord.

Schwann cell p75NTR prevents spontaneous sensory reinnervation of the adult spinal cord.

Schwann cells are attractive candidates for repair of the injured spinal cord. Transplanted Schwann cells are permissive to regeneration, but their ability to promote regeneration into distal spinal cord remains weak despite their production of growth-promoting neurotrophins. Schwann cell activation such as that which accompanies peripheral nerve injury results in massive upregulation of the p75(NTR) pan-neurotrophin-receptor. Here we test the hypothesis that this p75(NTR) upregulation following dorsal root injury limits availability of endogenous neurotrophin to axons and restricts regeneration of injured axons into the spinal cord. We injured dorsal roots (fourth cervical to second thoracic) in mice lacking the neurotrophin-binding domain of p75(NTR) and in wild-type littermates. Axonal regeneration was assessed by selective tracing of neurotrophin-responsive and non-responsive dorsal root ganglion neurons. Functional reinnervation of the spinal cord was assessed in behavioural experiments and via Fos immunohistochemistry following formalin injection into the forepaw. We also measured levels of nerve growth factor and neurotrophin-3 following nerve injury in knockout and wild-type mice, and used Trk-Fc receptor chimeras to block nerve growth factor and neurotrophin-3 signalling in dorsal root ganglion/Schwann cell co-cultures and following dorsal root injury in vivo. The roles of neuronal and glial p75(NTR) were assessed in transplant experiments in vivo and in co-cultures. We found that nerve growth factor and neurotrophin-3-responsive axons regenerated into the spinal cord of p75(NTR) knockout mice where they made functional connections with dorsal horn neurons. Despite equivalent levels of nerve growth factor and neurotrophin-3 in wild-type and knockout mice, successful regeneration in knockouts was neurotrophin-dependent. Transplantation of p75(-/-) neurons into a wild-type environment, p75(-/-) peripheral nerve grafts into the injured p75(+/+) spinal cord, and dissociated sensory neuron/Schwann cell co-cultures showed that the absence of p75(NTR) from glia, not from neurons, promotes regeneration. These findings indicate that Schwann cell p75(NTR) restricts neurotrophin availability to the extent that it prevents spontaneous sensory axon regeneration into the spinal cord. The implication is that inactivating p75(NTR) in Schwann (or olfactory ensheathing) cells may enable axons to grow beyond transplants, improving the outcome of spinal cord injury.

Targeting myelin to optimize plasticity of spared spinal axons.

Functional re-innervation of target neurons following neurological damage such as spinal cord injury is an essential requirement of potential therapies. There are at least two avenues by which this can be achieved: (a) through the regeneration of injured axons and (b) through promoting plasticity of those spared by the initial insult. There are several reasons why the latter approach may be more feasible, not the least of which are the inhibitory character of the glial scar, the often long distances over which injured axons must regrow, and the fact that spared axons are often already in the vicinity of denervated targets. The challenge is to unveil the well-recognized intrinsic plasticity of spared axons in a way that avoids complications, such as pain or autonomic dysfunction. One approach that we as well as others have taken is to target growth-suppressing signaling pathways initiated in spared axons by myelin-derived proteins. This article reviews models used for the study of spinal axon plasticity and describes the anatomical and behavioral effects of interfering with myelinderived proteins, their receptors, and components of their intracellular signaling cascades.

Deafferentation and neurotrophin-mediated intraspinal sprouting: a central role for the p75 neurotrophin receptor.

Axonal plasticity in the adult spinal cord is governed by intrinsic neuronal growth potential and by extracellular cues. The p75 receptor (p75(NTR)) binds growth-promoting neurotrophins (NTs) as well as the common receptor for growth-inhibiting myelin-derived proteins (the Nogo receptor) and so is well situated to gauge the balance of positive and negative influences on axonal plasticity. Using transgenic mice lacking the extracellular NT-binding domain of p75(NTR) (p75-/- mice), we have examined the influence of p75(NTR) on changes in the density of primary afferent (calcitonin gene-related peptide-expressing) and descending monoaminergic (serotonin- and tyrosine hydroxylase-expressing) projections to the dorsal horn after dorsal rhizotomy, with and without concomitant application of exogenous nerve growth factor and NT-3. We found that, in intact p75-/- mice, the axon density of all populations was equal to or less than that in wild-type mice but that rhizotomy-induced intraspinal sprouting was significantly augmented. Monoaminergic axon sprouting was enhanced in both nerve growth factor- and NT-3-treated p75-/- mice compared with similarly treated wild-type mice. Primary afferent sprouting was particularly robust in NT-3-treated p75-/- mice. These in vivo results illustrate the interactions of p75(NTR) with NTs, with their respective tropomyosin-related kinase receptors and with inhibitory myelin-derived molecules. Our findings illustrate the pivotal role of p75(NTR) in spinal axonal plasticity and identify it as a potential therapeutic target for spinal cord injury.