HNK-1 Carrier Glycoproteins Are Decreased in the Alzheimer’s Disease Brain

The human natural killer-1 (HNK-1), 3-sulfonated glucuronic acid, is a glycoepitope marker of cell adhesion that participates in cell-cell and cell-extracellular matrix interactions and in neurite growth. Very little is known about the regulation of the HNK-1 glycan in neurodegenerative disease, particularly in Alzheimer’s disease (AD). In this study, we investigate changes in the levels of HNK-1 carrier glycoproteins in AD. We demonstrate an overall decrease in HNK-1 immunoreactivity in glycoproteins extracted from the frontal cortex of AD subjects, compared with levels from non-demented controls (NDC). Immunoblotting of ventricular post-mortem and lumbar ante-mortem cerebrospinal fluid with HNK-1 antibodies indicate similar levels of carrier glycoproteins in AD and NDC samples. Decrease in HNK-1 carrier glycoproteins were not paralleled by changes in messenger RNA (mRNA) levels of the enzymes involved in the synthesis of the glycoepitope, β-1,4-galactosyltransferase (β4GalT), glucuronyltransferases GlcAT-P and GlcAT-S, or sulfotransferase HNK-1ST. Over-expression of amyloid precursor protein in Tg2576 transgenic mice and in vitro treatment of SH-SY5Y neuroblastoma cells with the amyloidogenic Aβ42 peptide resulted in a decrease in HNK-1 immunoreactivity levels in brain and cellular extracts, whereas the levels of soluble HNK-1 glycoproteins detected in culture media were not affected by Aβ treatment. HNK-1 levels remain unaffected in the brain extracts of Tg-VLW mice, a model of mutant hyperphosphorylated tau, and in SH-SY5Y cells over-expressing hyperphosphorylated wild-type tau. These results provide evidence that cellular levels of HNK-1 carrier glycoforms are decreased in the brain of AD subjects, probably influenced by the β-amyloid protein.     

Perturbation of cranial neural crest migration by the HNK-1 antibody

The HNK-1 antibody recognizes a carbohydrate moiety that is shared by a family of cell adhesion molecules and is also present on the surface of migrating neural crest cells. Here, the effects of the HNK-1 antibody on neural crest cells were examined in vitro and in vivo. When the HNK-1 antibody was added to neural tube explants in tissue culture, neural crest cells detached from laminin substrates but were unaffected on fibronectin substrates. In order to examine the effects of the HNK-1 antibody in vivo, antibody was injected lateral to the mesencephalic neural tube at the onset of cranial neural crest migration. The injected antibody persisted for approximately 16 hr on the injected side of the embryo and appeared to be most prevalent on the surface of neural crest cells. Embryos fixed within the first 24 hr after injection of HNK-1 antibodies (either whole IgMs or small IgM fragments) showed one or more of the following abnormalities: (1) ectopic neural crest cells external to the neural tube, (2) an accumulation of neural crest cell volume on the lumen of the neural tube, (3) some neural tube anomalies, or (4) a reduction in the neural crest cell volume on the injected side. The ectopic cells and neural tube anomalies persisted in embryos fixed 2 days postinjection. Only embryos having 10 or less somites at the time of injection were affected, suggesting a limited period of sensitivity to the HNK-1 antibody. Control embryos injected with a nonspecific antibody or with a nonblocking antibody against the neural cell adhesion molecule (N-CAM) were unaffected. Previous experiments from this laboratory have demonstrated than an antibody against integrin, a fibronectin and laminin receptor caused defects qualitatively similar to those resulting from HNK-1 antibody injection (M. Bronner-Fraser, J. Cell Biol., 101, 610, 1985). Coinjection of the HNK-1 and integrin antibodies resulted in a greater percentage of affected embryos than with either antibody alone. The additive nature of the effects of the two antibodies suggests that they act at different sites. These results demonstrate that the HNK-1 antibody causes abnormalities in cranial neural crest migration, perhaps by perturbing interactions between neural crest cells and laminin substrates.     

Expression of zebrafish fkd6 in neural crest-derived glia

The zebrafish fkd6 gene is a marker for premigratory neural crest. In this study, we analyze later expression in putative glia of the peripheral nervous system. Prior to neural crest migration, fkd6 expression is downregulated in crest cells. Subsequently, expression appears initially in loose clusters of cells in positions corresponding to cranial ganglia. Double labelling with a neuronal marker shows that fkd6-expressing cells are not differentiated neurones and generally lie peripheral to neurones in ganglia. Later, expression appears associated with the posterior lateral line and other cranial nerves. For the posterior lateral line nerve, we show that fkd6-labeling extends caudally along this nerve in tight correlation with lateral line primordium migration and axon elongation. Expression in colourless mutant embryos is consistent with these cells being satellite glia and Schwann cells.     

Mutations affecting the formation of posterior lateral line system in Medaka, Oryzias latipes

We performed a systematic screen for mutations affecting the trajectory of axons visualized by immunohistochemical staining of Medaka embryos with anti-acetylated tubulin antibody. Among the mutations identified, yanagi (yan) and kazura (kaz) mutations caused specific defects in projection of the posterior lateral line (PLL) nerve. In yan and kaz mutant embryos, the PLL nerve main bundle was misrouted ventrally and dorsally or anteriorly. Medaka semaphorin3A, sdf1, and cxcr4 cDNA fragments were cloned to allow analysis of these mutants. There were no changes in semaphorin3A or sdf1 expression in mutant embryos, suggesting that the tissues expressing semaphorin3A or sdf1 that are involved in PLL nerve guidance are present in these mutant embryos. Double staining revealed that the mislocated PLL primordium and growth cone of the ectopically projected PLL nerve were always colocalized in both yan and kaz mutant embryos, suggesting that migration of PLL primordia and PLL nerve growth cones are not uncoupled in these mutants. Although homozygous yan larvae showed incomplete migration of the PLL primordium along the anteroposterior axis, ventral proneuromast migration was complete, suggesting that ventral migration of the proneuromast does not require the signaling affected in yan mutants. In addition to the PLL system, the distribution of primordial germ cells (PGCs) was also affected in both yan and kaz mutant embryos, indicating that yan and kaz genes are required for the migration of both PLL primordia and PGCs. Genetic linkage analysis indicated that kaz is linked to cxcr4, but yan is not linked to sdf1 or cxcr4. These mutations will provide genetic clues to investigate the molecular mechanism underlying formation of the PLL system.     

斑马鱼胚胎发育过程中TGF-1基因的表达特征分析

为研究转化生长因子 (Transforming growth factor , TGF)1对斑马鱼胚胎发育的调控作用, 通过NCBI获得TGF-1基因序列, TGF-1 cDNA全长1571 bp, 编码377个氨基酸。系统进化树分析发现, TGF-蛋白按照不同的类型严格聚类, 斑马鱼TGF-1与其他鱼类的TGF-1聚集到一个分支, 在进化中非常保守。对斑马鱼胚胎进行RT-PCR和Real-Time PCR检测显示, TGF-1基因为母源表达基因, 在分节期之前的表达水平比较低, 而从咽囊期开始持续高水平的表达。胚胎整体原位杂交发现, TGF-1基因在斑马鱼24 hpf 胚胎中开始有特异信号出现, TGF-1基因的表达主要分布在腮弓、侧线原基、耳囊、嗅觉基板、心脏和前肾等处, 表明TGF-1基因可能参与斑马鱼胚胎免疫调节、循环系统发育和侧线形成。用低氧处理斑马鱼胚胎, 发现低氧处理24h后斑马鱼胚胎发育延迟。利用Real-Time PCR和胚胎整体原位杂交检测发现, 低氧处理后发育延迟的斑马鱼胚胎中TGF-1 mRNA表达量较常氧组显著降低。以上结果表明, TGF-1基因参与斑马鱼胚胎发育调控, 并且可能与低氧处理后斑马鱼胚胎发育延迟有关。研究结果将为深入研究斑马鱼TGF-1基因的功能奠定基础。       

Pathfinding by zebrafish motoneurons in the absence of normal pioneer axons.

Individually identified primary motoneurons of the zebrafish embryo pioneer cell-specific peripheral motor nerves. Later, the growth cones of secondary motoneurons extend along pathways pioneered by primary motor axons. To learn whether primary motor axons are required for pathway navigation by secondary motoneurons, we ablated primary motoneurons and examined subsequent pathfinding by the growth cones of secondary motoneurons. We found that ablation of the primary motoneuron that pioneers the ventral nerve delayed ventral nerve formation, but a normal-appearing nerve eventually formed. Therefore, the secondary motoneurons that extend axons in the ventral nerve were able to pioneer that pathway in the absence of the pathway-specific primary motoneuron. In contrast, in the absence of the primary motoneuron that normally pioneers the dorsal nerve, secondary motoneurons did not pioneer a nerve in the normal location, instead they formed dorsal nerves in an atypical position. This difference in the ability of these two groups of motoneurons to pioneer their normal pathways suggests that the guidance rules followed by their growth cones may be very different. Furthermore, the observation that the atypical dorsal nerves formed in a consistent incorrect location suggests that the growth cones of the secondary motoneurons that extend dorsally make hierarchical pathway choices.     

Functional anatomy of the canine mediastinal cardiac nerves located at the base of the heart

The major canine cardiopulmonary nerves which arise from the middle cervical and stellate ganglia and the vagi course toward the heart in the dorsal mediastinum where they form, at the base of the heart dorsal to the pulmonary artery and aorta, the dorsal mediastinal cardiac nerves. In addition, the left caudal pole and interganglionic nerves project onto the left lateral side of the heart as the left lateral cardiac nerve. These nerves contain afferent and (or) efferent axons which, upon stimulation, modify specific cardiac regions and (or) systemic pressure. In addition, with the exception of the left lateral cardiac nerve, stimulation of each of these nerves produces compound action potentials in the cranial ends of the majority of the major cardiopulmonary nerves demonstrating that axons in each dorsal mediastinal cardiac nerve interconnect with axons in the majority of the cardiopulmonary nerves. Axons in the left lateral cardiac nerve connect primarily with axons in the left caudal pole and left interganglionic nerves. The dorsal mediastinal nerves project distally onto the heart as coronary nerves accompanying the right or left coronary arteries. These innervated the ventricular myocardium which is supplied by their respective vessels. The left lateral cardiac nerve projects directly onto the lateral epicardium of the left ventricle. The dorsal mediastinal and left lateral cardiac nerves are the major sympathetic cardiac nerves. Thus, the cardiac nerves located in the mediastinum at the base of the heart are not simple extensions of cardiopulmonary nerves, but rather have a unique anatomy and function of their own.     

Myelin Structure and Composition in Zebrafish

To establish a standard for genotype/phenotype studies on the myelin of zebrafish (Danio rerio), an organism increasingly popular as a model system for vertebrates, we have initiated a detailed characterization of the structure and biochemical composition of its myelinated central and peripheral nervous system (CNS; PNS) tissues. Myelin periods, determined by X-ray diffraction from whole, unfixed optic and lateral line nerves, were approximately 153 and approximately 162 Angstrom, respectively. In contrast with the lability of PNS myelin in higher vertebrates, zebrafish lateral line nerve myelin exhibited structural stability when exposed to substantial changes in pH and ionic strength. Neither optic nor lateral line nerves showed swelling at the cytoplasmic apposition in CaCl(2)-containing Ringer's solution, in contrast with nerves from other teleost and elasmobranch fishes. Zebrafish optic nerve showed greater stability against changes in NaCl and CaCl(2) than lateral line nerve. The nerves from zebrafish having mutations in the gene for myelin basic protein (mbpAla2Thr and mbpAsp25Val) showed similar myelin periods as the wildtype (WT), but gave approximately 20% less compact myelin. Analysis of proteins by SDS-PAGE and Western blotting identified in both CNS and PNS of WT zebrafish two orthologues of myelin P0 glycoprotein that have been characterized extensively in trout--intermediate protein 1 (24 kDa) and intermediate protein 2 (28 kDa). Treatment with endoglycosidase-F demonstrated a carbohydrate moiety of approximately 7 kDa, which is nearly threefold larger than for higher vertebrates. Thin-layer chromatography for lipids revealed a similar composition as for other teleosts. Taken together, these data will serve as a baseline for detecting changes in the structure and/or amount of myelin resulting from mutations in myelin-related genes or from exogenous, potentially cytotoxic compounds that could affect myelin formation or stability.     

alphaII-spectrin is essential for assembly of the nodes of Ranvier in myelinated axons

Saltatory conduction in myelinated axons requires organization of the nodes of Ranvier, where voltage-gated sodium channels are prominently localized [1]. Previous results indicate that alphaII-spectrin, a component of the cortical cytoskeleton [2], is enriched at the paranodes [3, 4], which flank the node of Ranvier, but alphaII-spectrin's function has not been investigated. Starting with a genetic screen in zebrafish, we discovered in alphaII-spectrin (alphaII-spn) a mutation that disrupts nodal sodium-channel clusters in myelinated axons of the PNS and CNS. In alphaII-spn mutants, the nodal sodium-channel clusters are reduced in number and disrupted at early stages. Analysis of chimeric animals indicated that alphaII-spn functions autonomously in neurons. Ultrastructural studies show that myelin forms in the posterior lateral line nerve and in the ventral spinal cord in alphaII-spn mutants and that the node is abnormally long; these findings indicate that alphaII-spn is required for the assembly of a mature node of the correct length. We find that alphaII-spectrin is enriched in nodes and paranodes at early stages and that the nodal expression diminishes as nodes mature. Our results provide functional evidence that alphaII-spectrin in the axonal cytoskeleton is essential for stabilizing nascent sodium-channel clusters and assembling the mature node of Ranvier.     

Zebrafish semaphorin Z1b inhibits growing motor axons in vivo.

Zebrafish semaphorin 1b (sema Z1b) is a new member of the semaphorin family, related to mammalian sema D/III. It is expressed in rhombomeres three and five, and in the posterior half of newly formed somites which is avoided by ventrally extending motor axons. Embryos injected at the 1-2 cell stage with synthetic sema Z1b mRNA developed normally but many (63%) showed missing or severely stunted ventral motor nerves. Other axons, somites, and hindbrain rhombomeres were not affected. No abnormalities were seen in control embryos injected with lacZ mRNA. Sema Z1b might normally influence the midsegmental pathway choice of the ventrally extending motor axons by contributing to a repulsive domain in the posterior somite.     

Peripheral nervous system of the ocean sunfish Mola mola (Tetraodontiformes: Molidae)

Dissection of peripheral nerves in the ocean sunfish Mola mola showed the lateral line system to comprise 6 cephalic and 1 trunk lateral lines, all neuromasts being superficial. The trunk line was restricted to the anterior half of the body, the number of neuromasts (27) being fewer than those previously recorded in other tetraodontiforms. The lateral ramus of the posterior lateral line nerve did not form a “serial collector nerve” along the body. The number of foramina in the neurocranium, serving as passages for the cranial nerves, was fewer than in primitive tetraodontiforms, the reduction being related to modifications in the posterior cranium. Some muscle homologies were reinterpreted based on nerve innervation patterns. The cutaneous branch innervation pattern in the claval fin rays was clearly identical with that in the dorsal and anal fin rays, but differed significantly from that in the caudal fin rays, providing strong support for the hypothesis that the clavus comprises highly modified components of the dorsal and anal fins.     

Developmental changes in the ultrastructure of the lamprey lateral line nerve during metamorphosis

The ultrastructure of the trunk lateral line nerve of larval and adult lampreys was studied with transmission electron microscopy. We confirmed that lampreys' lateral line nerve lacks myelin. Nevertheless, all axons were wrapped by Schwann cell processes. In the larval nerve, gaps between Schwann cells were observed, where the axolemma was covered only by a basal lamina, indicating an earlier developmental stage. In the adult nerve, glial (Schwann cell) ensheathment was mostly complete. Additionally, we observed variable ratios of axons to Schwann cells in larval and adult preparations. In the larval nerve, smaller axons were wrapped by one Schwann cell. Occasionally, a single Schwann cell surrounded two axons. Larger axons were associated with two to five Schwann cells. In the adult nerve, smaller axons were surrounded by one, but larger axons by three to eight Schwann cells. The larval epineurium contained large adipose cells, separated from each other by single fibroblast processes. This layer of adipose tissue was reduced in adult preparation. The larval perineurium was thin, and the fibroblasts, containing large amounts of glycogen granules, were arranged loosely. The adult perineurium was thicker, consisting of at least three layers of fibroblasts separated by collagen fibrils. The larval and adult endoneurium contained collagen fibrils oriented orthogonally to each other. Both larval and adult lateral line nerves possessed a number of putative fascicles weakly defined by a thin layer of perineurial fibroblasts. These results indicate that after a prolonged larval stage, the lamprey lateral line nerve is subjected to additional maturation processes during metamorphosis. J. Morphol. 2009. © 2009 Wiley‐Liss, Inc.     

The development of a simple scaffold of axon tracts in the brain of the embryonic zebrafish, Brachydanio rerio

We have examined neuronal differentiation and the formation of axon tracts in the embryonic forebrain and midbrain of the zebrafish, between 1 and 2 days postfertilisation. Axons were visualised with three techniques; immunocytochemistry (using HNK-1 and antiacetylated tubulin antibodies) and horseradish peroxidase (HRP) labelling in whole-mounted brains, and transmission electron microscopy. Differentiation was monitored by histochemical staining for acetylcholinesterase (AChE). These independent methods demonstrated that a simple grid of tracts and commissures forms the initial axon scaffold of the brain. At 1 day, the olfactory nerve, four commissures, their associated tracts and three other non-commissural tracts are present. By 2 days, these tracts and commissures have all greatly enlarged and, in addition, the optic nerve and tract, and three new commissures and their associated tracts have been added. Small applications of HRP at various sites revealed the origins and projections of some of these earliest axons. Retrogradely labelled cell bodies originated from regions that were also positive for AChE activity. At 1 day, HRP-labelled axons were traced: (1) from the olfactory placode through the olfactory nerve to the dorsal telencephalon; (2) from the telencephalon into the tract of the anterior commissure and also to the postoptic region of the diencephalon; (3) from the hindbrain through the ventral midbrain and diencephalon to the postoptic commissure; (4) from the dorsal diencephalon (in or near the epiphysis) to the tract of the postoptic commissure; (5) from ventral and rostral midbrain through the posterior commissure. Three new projections were demonstrated at 2 days: (1) from the retina through the tract of the postoptic commissure to the tectum; (2) from the telencephalon to the contralateral diencephalon; and (3) from the telencephalon to the ventral flexure. These results show that at 1 day, the zebrafish brain is impressively simple, with a few small, well-separated tracts but by 2 days the brain is already considerably more complex. Most of the additional axons added onto pre-existent tracts rather than pioneered new ones supporting the notion that other axons play a crucial role in the guidance of early central nervous system (CNS) axons.     

On the topographical differences in the localization of alkaline and acid phosphatases in nerves of teleosts Saccobranchus fossilis and Mystus seenghala

Summary The present study describes the distribution of alkaline and acid phosphatases in the lateral line nerve of Saccobranchus fossilis and optic nerve of Mystus seenghala. The distribution of the phosphatases is quite different in these nerves, i.e., in the lateral line nerve the axons are intensely positive for alkaline and acid phosphatases whereas the myelin sheaths are negative for the enzymes. In the optic nerve, however, the axons are negative and the activity of the alkaline and acid phosphatases appear in the form of bands in the myelin sheaths. The significance of these differences is discussed with special reference to the controversy about the neurokeratin network and the physiological activities of nerves.The glial processes intervening between the myelinated axons of the optic nerve are also stained by the reactions for alkaline and acid phosphatases. The metabolic significance of phosphatases at these sites is discussed.     

Early axonal contacts during development of an identified dendrite in the brain of the zebrafish

We have identified the initial synaptic contacts made onto the Mauthner (M) cell, an identified neuron that arises during early development of the zebrafish hindbrain. The contacts are made by a small bundle of pioneering trigeminal sensory axons onto the M cell soma before it forms dendrites. The sensory bundle is then partially enveloped by the M cell. The lateral dendrite appears at about the site of the contact, and eventually the trigeminal inputs are shifted to its trunk. As the dendrite elongates, other sensory contacts are made on its distal regions, sequentially from the acoustico-vestibular nerve and the lateral line nerves. To learn whether the earliest inputs induce the initial outgrowth of the M cell dendrite, we ablated the trigeminal neurons by laser irradiation before they contacted the M cell. Morphogenesis of the M cell, including its dendrite, appeared normal.