How does the eye breathe? Evidence for neuroglobin-mediated oxygen supply in the mammalian retina

Visual performance of the vertebrate eye requires large amounts of oxygen, and thus the retina is one of the highest oxygen-consuming tissues of the body. Here we show that neuroglobin, a neuron-specific respiratory protein distantly related to hemoglobin and myoglobin, is present at high amounts in the mouse retina (approximately 100 microm). The estimated concentration of neuroglobin in the retina is thus about 100-fold higher than in the brain and is in the same range as that of myoglobin in the muscle. Neuroglobin is expressed in all neurons of the retina but not in the retinal pigment epithelium. Neuroglobin mRNA was detected in the perikarya of the nuclear and ganglion layers of the neuronal retina, whereas the protein was present mainly in the plexiform layers and in the ellipsoid region of photoreceptor inner segment. The distribution of neuroglobin correlates with the subcellular localization of mitochondria and with the relative oxygen demands, as the plexiform layers and the inner segment consume most of the retinal oxygen. These findings suggest that neuroglobin supplies oxygen to the retina, similar to myoglobin in the myocardium and the skeletal muscle.     

Divergent distribution in vascular and avascular mammalian retinae links neuroglobin to cellular respiration

The visual function of the vertebrate retina relies on sufficient supply with oxygen. Neuroglobin is a respiratory protein thought to play an essential role in oxygen homeostasis of neuronal cells. For further understanding of its function, we compared the distribution of neuroglobin and mitochondria in both vascular and avascular mammalian retinae. In the vascular retinae of mouse and rat, oxygen is supplied by the outer choroidal, deep retinal, and inner capillaries. We show that in this type of retina, mitochondria are concentrated in the inner segments of photoreceptor cells, the outer and the inner plexiform layers, and the ganglion cell layer. These are the same regions in which oxygen consumption takes place and in which neuroglobin is present at high levels. In the avascular retina of guinea pig the deep retinal and inner capillaries are absent. Therefore, only the inner segments of the photoreceptors adjacent to choroidal capillaries display an oxidative metabolism. We demonstrate that in the retina of guinea pigs both neuroglobin and mitochondria are restricted to this layer. Our results clearly demonstrate an association of neuroglobin and mitochondria, thus supporting the hypothesis that neuroglobin is a respiratory protein that supplies oxygen to the respiratory chain.     

缺血缺氧疾病中脑红蛋白的表达

脑红蛋白是继血红蛋白和肌红蛋白后由Burmester等[1]于2000年发现的体内第3类携氧蛋白,脑红蛋白在视网膜组织中的含量高达100μmol/L,占视网膜总蛋白量的2%-4%,其浓度约为脑中浓度的100倍,而体内不同组织脑红蛋白表达量的增加均与组织的缺血缺氧密切相关。视网膜作为中枢神经系统的重要组成部分之一,对氧的需求远远大于其他神经组织,临床实践中存在着与视网膜相关的大量的缺血缺氧性疾病,视网膜的这种特征为我们深入研究脑红蛋白与视网膜细胞之间的关系提供了临床基础。本文就脑红蛋白在缺血缺氧疾病中的表达展开论述,为视网膜缺血缺氧损伤中脑红蛋白的表达提供相关依据。     

Rat optic nerve oligodendrocytes develop in the absence of viable retinal ganglion cell axons.

Retinal ganglion cell axons and axonal electrical activity have been considered essential for migration, proliferation, and survival of oligodendrocyte lineage cells in the optic nerve. To define axonal requirements during oligodendrogenesis, the developmental appearance of oligodendrocyte progenitors and oligodendrocytes were compared between normal and transected optic nerves. In the absence of viable axons, oligodendrocyte precursors migrated along the length of the nerve and subsequently multiplied and differentiated into myelin basic protein-positive oligodendrocytes at similar densities and with similar temporal and spatial patterns as in control nerves. Since transected optic nerves failed to grow radially, the number of oligodendrocyte lineage cells was reduced compared with control nerves. However, the mitotic indices of progenitors and the percentage of oligodendrocytes undergoing programmed cell death were similar in control and transected optic nerves. Oligodendrocytes lacked their normal longitudinal orientation, developed fewer, shorter processes, and failed to form myelin in the transected nerves. These data indicate that normal densities of oligodendrocytes can develop in the absence of viable retinal ganglion axons, and support the possibility that axons assure their own myelination by regulating the number of myelin internodes formed by individual oligodendrocytes.     

Neuroglobin Promotes Neurite Outgrowth via Differential Binding to PTEN and Akt

Neuroglobin, the third mammalian globin with a hexa-coordinated heme, exists predominantly in neurons of the brain. Neuroglobin plays an important role in neuronal death upon ischemia and oxidative stress. The physiological function of neuroglobin remains unclear. Here, we report a novel function of neuroglobin in neurite development. Knocking-down neuroglobin exhibited a prominent neurite-deficient phenotype in mouse neuroblastoma N2a cells. Silencing neuroglobin prevented neurite outgrowth, while ectopic expression of neuroglobin but not homologous cytoglobin promoted neurite outgrowth of N2a cells upon serum withdrawal. In primary cultured rat cerebral cortical neurons, neuroglobin was upregulated and preferentially distributed in neurites during neuronal development. Overexpression of neuroglobin but not cytoglobin in cultured cortical neurons promoted axonal outgrowth, while knocking-down of neuroglobin retarded axonal outgrowth. Neuroglobin overexpression suppressed phosphatase and tensin homolog (PTEN) but increased Akt phosphorylation during neurite induction. Bimolecular fluorescence complementation and glutathione S-transferase pull-down assays revealed that neuroglobin and various mutants (E53Q, E118Q, K119N, H64A, H64L, and Y44D) bound with Akt and PTEN differentially. Neuroglobin E53Q showed a prominent reduced PTEN binding but increased Akt binding, resulting in decreased p-PTEN, increased p-Akt, and increased neurite length. Taken together, we demonstrate a critical role of neuroglobin in neuritogenesis or development via interacting with PTEN and Akt differentially to activate phosphatidylinositol 3-kinase/Akt pathway, providing potential therapeutic applications of neuroglobin for axonopathy in neurological diseases.     

Neuroglobin,an oxygen-binding protein in the mammalian nervous system (localization and putative functions)

The review summarizes current data on neuroglobin, the heme-containing protein discovered in mammalian nerve cells in 2000. It presents general characteristics of neuroglobin as well as data on its evolutionary changes and expression across different taxa. Neuroglobin distribution in specific brain structures and outside the brain is described. The issue of the occurrence of neuroglobin not only in neurons but also in astroglial cells is discussed. Subcellular localization of neuroglobin is characterized with a special focus on its detection in the nucleus of nerve cells, suggesting its involvement in nuclear functions. Current ideas on the probable functional significance of neuroglobin are reported. Neuroglobin is presumed to be involved in metabolism of reactive nitrogen and oxygen species as well as in intracellular signaling pathways. Besides, neuroglobin has neuroprotective and antiapoptotic functions. Since its expression changes during ontogenesis, its neuroprotective role in ageing is specifically highlighted. Changes in expression and localization of neuroglobin are suggested to influence the adaptive potential of an organism.     

A rare natural lipid induces neuroglobin expression to prevent amyloid oligomers toxicity and retinal neurodegeneration

Most neurodegenerative diseases such as Alzheimer''s disease are proteinopathies linked to the toxicity of amyloid oligomers. Treatments to delay or cure these diseases are lacking. Using budding yeast, we report that the natural lipid tripentadecanoin induces expression of the nitric oxide oxidoreductase Yhb1 to prevent the formation of protein aggregates during aging and extends replicative lifespan. In mammals, tripentadecanoin induces expression of the Yhb1 orthologue, neuroglobin, to protect neurons against amyloid toxicity. Tripentadecanoin also rescues photoreceptors in a mouse model of retinal degeneration and retinal ganglion cells in a Rhesus monkey model of optic atrophy. Together, we propose that tripentadecanoin affects p‐bodies to induce neuroglobin expression and offers a potential treatment for proteinopathies and retinal neurodegeneration.     

Retinal Ganglion Cell Dendritic Atrophy in DBA/2J Glaucoma

Glaucoma is a complex disease affecting an estimated 70 million people worldwide, characterised by the progressive degeneration of retinal ganglion cells and accompanying visual field loss. The common site of damage to retinal ganglion cells is thought to be at the optic nerve head, however evidence from other optic neuropathies and neurodegenerative disorders suggests that dendritic structures undergo a prolonged period of atrophy that may accompany or even precede soma loss and neuronal cell death. Using the DBA/2J mouse model of glaucoma this investigation aims to elucidate the impact of increasing intraocular pressure on retinal ganglion cell dendrites using DBA/2J mice that express YFP throughout the retinal ganglion cells driven by Thy1 (DBA/2J.Thy1(YFP)) and DiOlistically labelled retinal ganglion cells in DBA/2J mice. Here we show retinal ganglion cell dendritic degeneration in DiOlistically labelled DBA/2J retinal ganglion cells but not in the DBA/2J.Thy1(YFP) retinal ganglion cells suggesting that a potential downregulation of Thy1 allows only ‘healthy’ retinal ganglion cells to express YFP. These data may highlight alternative pathways to retinal ganglion cell loss in DBA/2J glaucoma.     

Functional properties of neuroglobin and cytoglobin. Insights into the ancestral physiological roles of globins

Neuroglobin and cytoglobin are two recently discovered vertebrate globins, which are expressed at low levels in neuronal tissues and in all tissues investigated so far, respectively. Based on their amino acid sequences, these globins appear to be phylogenetically ancient and to have mutated less during evolution in comparison to the other vertebrate globins, myoglobin and hemoglobin. As with some plant and bacterial globins, neuroglobin and cytoglobin hemes are hexacoordinate in the absence of external ligands, in that the heme iron atom coordinates both a proximal and a distal His residue. While the physiological role of hexacoordinate globins is still largely unclear, neuroglobin appears to participate in the cellular defence against hypoxia. We present the current knowledge on the functional properties of neuroglobin and cytoglobin, and describe a mathematical model to evaluate the role of mammalian retinal neuroglobin in supplying O2 supply to the mitochondria. As shown, the model argues against a significant such role for neuroglobin, that more likely plays a role to scavenge reactive oxygen and nitrogen species that are generated following brain hypoxia. The O2 binding properties of cytoglobin, which is upregulated upon hypoxia, are consistent with a role for this protein in O2-requiring reactions, such as those catalysed by hydroxylases.     

In Vitro Protein Synthesis in the Goldfish Retinotectal Pathway During Regeneration: Evidence for Specific Axonal Proteins of Retinal Origin in the Optic Nerve

Four proteins with molecular weights of 58,000 can be separated as a linear array by two-dimensional gel electrophoresis. They are highly concentrated in the goldfish optic nerve and are designated as ON1, ON2, ON3, and ON4. Proteins ON1 and ON2 are undetectable in the optic nerve after disconnection and their concentration is gradually restored during regeneration. In vitro incubations of retinas, optic nerves, or tecta in the presence of [35S]methionine indicate that proteins ON1 and ON2 are of retinal origin. The labeling rate of these proteins in the retina increases fourfold after optic nerve crush whereas the overall labeling rate in the retina remains largely constant. Their synthesis cannot be detected in tissues devoid of retinal ganglion cells. This is consistent with the view that ON1 and ON2 are synthesized by retinal ganglion cells and are consequently of neuronal origin in the optic nerve. In contrast, similar experiments indicate that ON3 and ON4 are of nonneuronal origin. They are synthesized in the optic nerve in the absence of retinal ganglion cells.     

Neuroglobin and cytoglobin in search of their role in the vertebrate globin family

Neuroglobin and cytoglobin are two recent additions to the family of heme-containing respiratory proteins of man and other vertebrates. Here, we review the present state of knowledge of the structures, ligand binding kinetics, evolution and expression patterns of these two proteins. These data provide a first glimpse into the possible physiological roles of these globins in the animal's metabolism. Both, neuroglobin and cytoglobin are structurally similar to myoglobin, although they contain distinct cavities that may be instrumental in ligand binding. Kinetic and structural studies show that neuroglobin and cytoglobin belong to the class of hexa-coordinated globins with a biphasic ligand-binding kinetics. Nevertheless, their oxygen affinities resemble that of myoglobin. While neuroglobin is evolutionarily related to the invertebrate nerve-globins, cytoglobin shares a more recent common ancestry with myoglobin. Neuroglobin expression is confined mainly to brain and a few other tissues, with the highest expression observed in the retina. Present evidence points to an important role of neuroglobin in neuronal oxygen homeostasis and hypoxia protection, though other functions are still conceivable. Cytoglobin is predominantly expressed in fibroblasts and related cell types, but also in distinct nerve cell populations. Much less is known about its function, although in fibroblasts it might be involved in collagen synthesis.     

Zebrafish reveals different and conserved features of vertebrate neuroglobin gene structure, expression pattern, and ligand binding

Neuroglobin has been identified as a respiratory protein that is primarily expressed in the mammalian nervous system. Here we present the first detailed analysis of neuroglobin from a non-mammalian vertebrate, the zebrafish Danio rerio. The zebrafish neuroglobin gene reveals a mammalian-type exon-intron pattern in the coding region (B12.2, E11.0, and G7.0), plus an additional 5'-non-coding exon. Similar to the mammalian neuroglobin, the zebrafish protein displays a hexacoordinate deoxy-binding scheme. Flash photolysis kinetics show the competitive binding on the millisecond timescale of external ligands and the distal histidine, resulting in an oxygen affinity of 1 torr. Western blotting, immune staining, and mRNA in situ hybridization demonstrate neuroglobin expression in the fish central nervous system and the retina but also in the gills. Neurons containing neuroglobin have a widespread distribution in the brain but are also present in the olfactory system. In the fish retina, neuroglobin is mainly present in the inner segments of the photoreceptor cells. In the gills, the chloride cells were identified to express neuroglobin. Neuroglobin appears to be associated with mitochondria-rich cell types and thus oxygen consumption rates, suggesting a myoglobin-like function of this protein in facilitated oxygen diffusion.     

损伤视神经后斑马鱼视网膜神经节细胞数量和分布的变化

中枢神经系统的再生是神经科学领域的一个重要课题。鱼类和两栖类的视神经作为中枢神经系统的一部分,具有再生的能力。已知在损伤视神经后,对与视神经纤维直接相连的视网膜神经节细胞的形态结构,数量和分布等产生一系列的影响。视神经再生过程中细胞学研究在很大程度上依赖于示踪方法和其它技术的发展,结合光镜和电镜,它们仅对神经细胞末梢的精细结构和神经细胞间突触连接构筑等研究较准确详实,但对视网膜神经节细     

RaxL regulates chick ganglion cell development

RaxL is a paired-like homeobox gene involved in vertebrate eye morphogenesis. We examined RaxL protein expression patterns during chick retinal development in combination with ganglion cell markers including the RA4 antigen, cBrn-3, Islet-1 and neuronal type III beta-tubulin. Double-immunostaining demonstrated that downregulation of RaxL protein correlates with upregulation of ganglion cell markers in the ganglion cell layer (GCL). To explore this correlation in vivo, we performed gain- and loss-of-function experiments by electroporating retroviral vectors encoding wild-type and dominant-negative-RaxL into the optic vesicles of stage 10 chick embryos. Infection with virus expressing RaxL led to a 35% decrease in Islet-1-positive ganglion cells at E5.0 and a complete loss of ganglion cells at E15, with no effect on displaced amacrine cells in the GCL. When dominant-negative RaxL was expressed, the total number of cells in the GCL increased by approximately 40% at E5.0 but was reduced to 40% at E15, due to ectopic apoptosis in the GCL from E9 to E15. These results suggest that RaxL gives an inhibitory effect on ganglion cell development and that the loss of RaxL expression is required for maintenance of ganglion cells.     

Functional characterization of fish neuroglobin: Zebrafish neuroglobin is highly expressed in amacrine cells after optic nerve injury and can translocate into ZF4 cells

Neuroglobin (Ngb) is a recently discovered vertebrate heme protein that is expressed in the brain and can reversibly bind oxygen. Mammalian Ngb is involved in neuroprotection under conditions of oxidative stress, such as ischemia and reperfusion. We previously found that zebrafish Ngb can penetrate the mammalian cell membrane. In the present study, we investigated the functional characteristics of fish Ngb by using the zebrafish cell line ZF4 and zebrafish retina. We found that zebrafish Ngb translocates into ZF4 cells, but cannot protect ZF4 cells against cell death induced by hydrogen peroxide. Furthermore, we demonstrated that a chimeric ZHHH Ngb protein, in which module M1 of human Ngb is replaced by that of zebrafish, is a cell-membrane-penetrating protein that can protect ZF4 cells against hydrogen peroxide exposure. Moreover, we investigated the localization of Ngb mRNA and protein in zebrafish retina and found that Ngb mRNA is expressed in amacrine cells in the inner nuclear layer and is significantly increased in amacrine cells 3 days after optic nerve injury. Immunohistochemical studies clarified that Ngb protein levels were increased in both amacrine cells and presynaptic regions in the inner plexiform layer after nerve injury. Taken together, we hypothesize that fish Ngb, whose expression is upregulated in amacrine cells after optic nerve injury, might be released from amacrine cells, translocate into neighboring ganglion cells, and function in the early stage of optic nerve regeneration. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

Target-Dependent Regulation of Retinal Nicotinic Acetylcholine Receptor and Tubulin RNAs During Optic Nerve Regeneration in Goldfish

A fundamental issue in central nervous system development regards the effect of target tissue on the differentiation of innervating neurons. We address this issue by characterizing the role the retinal ganglion cell target, i.e., the optic tectum, plays in regulating expression of tubulin and nicotinic acetylcholine receptor genes in regenerating retinal ganglion cells. Tubulins are involved in axonal growth, whereas nicotinic acetylcholine receptors mediate communication across synapses. Retinal ganglion cell axons were induced to regenerate by crushing the optic nerve. Following crush, there was a rapid increase in alpha-tubulin RNAs (3 days), which preceded the increase in nicotinic acetylcholine receptor RNAs (10-15 days). Both classes of RNAs approached control levels by the time retinotectal synapses and functional recovery were restored (4-6 weeks). If the optic nerve was repeatedly crushed or its target ablated, tubulin RNAs remained elevated, and the increase in receptor RNAs that would otherwise be seen 2 weeks after a single nerve crush did not occur. The interaction of retinal ganglion cell axons with their targets in the optic tectum appears, then, to exert a suppressive effect on the RNA encoding a cytoskeletal protein, tubulin, and an inductive effect on RNAs encoding nicotinic acetylcholine receptors involved in synaptic communication.     

The A5 antigen, a candidate for the neuronal recognition molecule, has homologies to complement components and coagulation factors

The A5 antigen is a neuronal cell surface protein of Xenopus presumed to be involved in the neuronal recognition between the optic nerve fibers and the visual centers. Analyses of cDNA clones revealed that the A5 antigen is a class I membrane protein containing two different internal repeats in the extracellular segment. The first repeat bears homology to domain III of complement components C1r and C1s, and the second repeat is homologous to the C1 and C2 domains of coagulation factors V and VIII. The mRNA for the A5 antigen was present in retinal ganglion cells and visual center neurons. Nonneuronal cells in the peripheral and central nervous systems did not express the mRNA for the A5 antigen.     

Susceptibility to neurodegeneration in a glaucoma is modified by Bax gene dosage

In glaucoma, harmful intraocular pressure often contributes to retinal ganglion cell death. It is not clear, however, if intraocular pressure directly insults the retinal ganglion cell axon, the soma, or both. The pathways that mediate pressure-induced retinal ganglion cell death are poorly defined, and no molecules are known to be required. DBA/2J mice deficient in the proapoptotic molecule BCL2-associated X protein (BAX) were used to investigate the roles of BAX-mediated cell death pathways in glaucoma. Both Bax+/- and Bax-/- mice were protected from retinal ganglion cell death. In contrast, axonal degeneration was not prevented in either Bax+/- or Bax-/- mice. While BAX deficiency did not prevent axonal degeneration, it did slow axonal loss. Additionally, we compared the effects of BAX deficiency on the glaucoma to its effects on retinal ganglion cell death due to two insults that are proposed to participate in glaucoma. As in the glaucoma, BAX deficiency protected retinal ganglion cells after axon injury by optic nerve crush. However, it did not protect retinal ganglion cells from N-methyl-D-aspartate (NMDA)-induced excitotoxicity. BAX is required for retinal ganglion cell death in an inherited glaucoma; however, it is not required for retinal ganglion cell axon degeneration. This indicates that distinct somal and axonal degeneration pathways are active in this glaucoma. Finally, our data support a role for optic nerve injury but not for NMDA receptor-mediated excitotoxicity in this glaucoma. These findings indicate a need to understand axon-specific degeneration pathways in glaucoma, and they suggest that distinct somal and axonal degeneration pathways may need to be targeted to save vision.     

Apoptosis of Central and Peripheral Neurons Can Be Prevented with Cyclin-Dependent Kinase/Mitogen-Activated Protein Kinase Inhibitors

Abstract: Previous studies have indicated that certain members of the cyclin-dependent kinase/mitogen-activated protein kinase superfamily are involved in apoptosis of neuronal cells. Here, we have examined programmed cell death induced by withdrawal of neurotrophic support from CNS (rat retinal) and PNS (chick sympathetic, sensory, and ciliary) neurons. All four neuron types were equally rescued by the purine analogues olomoucine and roscovitine. Olomoucine inhibits multiple cyclin-dependent and mitogen-activated protein kinases with similar potency. Roscovitine is a more selective cyclin-dependent kinase inhibitor; but, so is butyrolactone I, which did not prevent retinal ganglion cell death. The specific p38^MAPK inhibitor SB-203580 did not prevent apoptosis in retinal ganglion cells. Death of these cells in the absence of neurotrophic factors was accompanied by morphological changes indicative of apoptosis, including nuclear condensation and fragmentation. Treatment with olomoucine or roscovitine not only prevented these apoptotic changes in retinal ganglion cells but also blocked neurite outgrowth. The survival-promoting activity of olomoucine correlated with its in vitro IC₅₀ for c-Jun N-terminal kinase-1 and its potency to repress c- jun induction in live PC12 cells. Roscovitine was more potent in rescuing neurons than in inhibiting Jun kinase. Thus, the antiapoptotic action of roscovitine might be due to inhibition of additional kinases.