Rapid neural circuit switching mediated by synaptic plasticity during neural morphallactic regeneration

The aquatic oligochaete, Lumbriculus variegatus (Lumbriculidae), undergoes a rapid regenerative transformation of its neural circuits following body fragmentation. This type of nervous system plasticity, called neural morphallaxis, involves the remodeling of the giant fiber pathways that mediate rapid head and tail withdrawal behaviors. Extra- and intracellular electrophysiological recordings demonstrated that changes in cellular properties and synaptic connections underlie neurobehavioral plasticity during morphallaxis. Sensory-to-giant interneuron connections, undetectable prior to body injury, emerged within hours of segment amputation. The appearance of functional synaptic transmission was followed by interneuron activation, coupling of giant fiber spiking to motor outputs and overt segmental shortening. The onset of morphallactic plasticity varied along the body axis and emerged more rapidly in segments closer to regions of sensory field overlap between the two giant fiber pathways. The medial and lateral giant fibers were simultaneously activated during a transient phase of network remodeling. Thus, synaptic plasticity at sensory-to-giant interneuron connections mediates escape circuit morphallaxis in this regenerating annelid worm.     

Asexual Reproduction and Segmental Regeneration, but not Morphallaxis, are Inhibited by Boric Acid in Lumbriculus variegatus (Annelida: Clitellata: Lumbriculidae)

Body fragmentation, in some animal groups, is a mechanism for survival and asexual reproduction. Lumbriculus variegatus (Müller, 1774), an aquatic oligochaete worm, is capable of regenerating into complete individuals from small body fragments following injury and reproduces primarily by asexual reproduction. Few studies have determined the cellular mechanisms that underlie fragmentation, either regenerative or asexual. We utilized boric acid treatment, which blocks regeneration of segments in amputated fragments and blocks architomic fission during asexual reproduction, to investigate mechanistic relationships and differences between these two modes of development. Neural morphallaxis, involving changes in sensory fields and giant fiber conduction, was detected in amputated fragments in the absence of segmental regeneration. Furthermore, neural morphallactic changes occurred as a result of developmental mechanisms of asexual reproduction, even when architomy was prevented. These results show that fragmentation in L. variegatus, during injury or asexual reproduction, employs developmental and morphallactic processes that can be mechanistically dissociated by boric acid exposure. In regeneration following injury, compensatory morphallaxis occurred in response to fragmentation. In contrast, anticipatory morphallaxis was induced in preparation for fragmentation during asexual reproduction. Thus, various forms of regeneration in this lumbriculid worm can be activated independently and in different developmental contexts.     

Effects of nerve injury and segmental regeneration on the cellular correlates of neural morphallaxis

Functional recovery of neural networks after injury requires a series of signaling events similar to the embryonic processes that governed initial network construction. Neural morphallaxis, a form of nervous system regeneration, involves reorganization of adult neural connectivity patterns. Neural morphallaxis in the worm, Lumbriculus variegatus, occurs during asexual reproduction and segmental regeneration, as body fragments acquire new positional identities along the anterior-posterior axis. Ectopic head (EH) formation, induced by ventral nerve cord lesion, generated morphallactic plasticity including the reorganization of interneuronal sensory fields and the induction of a molecular marker of neural morphallaxis. Morphallactic changes occurred only in segments posterior to an EH. Neither EH formation, nor neural morphallaxis was observed after dorsal body lesions, indicating a role for nerve cord injury in morphallaxis induction. Furthermore, a hierarchical system of neurobehavioral control was observed, where anterior heads were dominant and an EH controlled body movements only in the absence of the anterior head. Both suppression of segmental regeneration and blockade of asexual fission, after treatment with boric acid, disrupted the maintenance of neural morphallaxis, but did not block its induction. Therefore, segmental regeneration (i.e., epimorphosis) may not be required for the induction of morphallactic remodeling of neural networks. However, on-going epimorphosis appears necessary for the long-term consolidation of cellular and molecular mechanisms underlying the morphallaxis of neural circuitry.     

Phylogenetic conservation of the cell-type-specific Lan3-2 glycoepitope in Caenorhabditis elegans

The biological function of a cell-type-specific glycosylation of an adhesion molecule belonging to the L1CAM immunoglobulin superfamily was previously determined in the nervous system of the embryonic leech, Hirudo medicinalis. The Lan3-2 glycoepitope is a surface marker of sensory afferent neurons and is required for their appropriate developmental collateral branching and synaptogenesis in the CNS. The chemical structure of the Lan3-2 glycoepitope consists of β-(1,4)-linked mannopyranose. Here, we show the conservation of the cell-type-specific expression of this mannose polymer in Caenorhabditis elegans. The Lan3-2 glycoepitope is expressed on the cell surface of a subset of dissociated embryonic neurons and, in the adult worm, by the pharyngeal motor neuron, M5, and the chemosensory afferents, the amphids. Additionally, the vulval epithelium expresses the Lan3-2 glycoepitope in late L4 larvae and in adult hermaphrodites. To investigate proteins carrying this restrictively expressed glycoepitope, worm extract was immunoaffinity purified with Lan3-2 monoclonal antibody and Western blotted. A polyclonal antibody reactive with the cytoplasmic tail of LAD-1/SAX-7, a C. elegans member of the L1CAM family, recognizes a 270 kDa protein band while Lan3-2 antibody also recognizes a 190 kDa glycoform, its putative Lan3-2 ectodomain. Thus, in C. elegans, as in leech, the Lan3-2 epitope is located on a L1CAM homologue. The cell-type-specific expression of the Lan3-2 glycoepitope shared by leech and C. elegans will be useful for understanding how cell-type-specific glycoepitopes mediate cell–cell interactions during development.     

The Lan3‐2 glycoepitope of Hirudo medicinalis consists of β‐(1,4)‐linked mannopyranose

While glycosyltransferases are restrictively expressed in invertebrate model organisms, little is known of their glycan end products. One such restrictively expressed glycoepitope was localized to sensory and epithelial cells of leech and Caenorhabditis elegans using the Lan3‐2 monoclonal antibody. A biological function for the neural Lan3‐2 epitope was previously determined in the leech. Here we report on the chemical structure of this mannosidic epitope harvested from whole Hirudo medicinalis. Crude glycans were liberated from glycoproteins by hydrazinolysis. Re‐N‐acetylated glycans were subjected to immunoaffinity purification. The affinity‐purified glycans were fractioned by size chromatography into oligosaccharides and polysaccharides. Lan3‐2 oligosaccharide structure was characterized by gas chromatography of alditol acetates, methylation analysis, 500 MHz ¹H NMR spectroscopy, matrix‐assisted laser desorption/ionization mass spectrometry, and electrospray ionization tandem MS‐MS of permethylated derivatives. The predominant components of the Lan3‐2 oligosaccharide fraction were a series of linear β‐(1,4)‐linked mannose polymers. The homologous expression of the Lan3‐2 epitope in C. elegans will facilitate the exploration of its glycosylation pathway. Other invertebrates expressing the Lan3‐2 epitope are Planaria dugesia, Capitella sp. I and Lumbriculus variegatus. The glycoepitope was not detected in the diploblastic animals Hydra littoralis and Aptaisia sp. or in deuterostomes.     

Differential Glycosylation of Tractin and LeechCAM, Two Novel Ig Superfamily Members, Regulates Neurite Extension and Fascicle Formation

By immunoaffinity purification with the mAb Lan3-2, we have identified two novel Ig superfamily members, Tractin and LeechCAM. LeechCAM is an NCAM/FasII/ApCAM homologue, whereas Tractin is a cleaved protein with several unique features that include a PG/YG repeat domain that may be part of or interact with the extracellular matrix. Tractin and LeechCAM are widely expressed neural proteins that are differentially glycosylated in sets and subsets of peripheral sensory neurons that form specific fascicles in the central nervous system. In vivo antibody perturbation of the Lan3-2 glycoepitope demonstrates that it can selectively regulate extension of neurites and filopodia. Thus, these experiments provide evidence that differential glycosylation can confer functional diversity and specificity to widely expressed neural proteins.     

Morphallaxis in an aquatic oligochaete, Lumbriculus variegatus: reorganization of escape reflexes in regenerating body fragments

We describe functional and anatomical correlates of the reorganization of giant nerve fiber-mediated escape reflexes in body fragments of an aquatic oligochaete, Lumbriculus variegatus, a species that reproduces asexually by fragmentation. Since fragments from any axial position always regenerate short heads (seven or eight segments long) and much longer tail sections, segments originating from posterior fragments become transposed along the longitudinal axis and acquire, by morphallaxis, features of escape reflex organization that conform to their new anterior position. Using noninvasive electrophysiological recordings we have quantified, on a day-to-day and a segment-by-segment basis, the reorganization that occurs in sensory field arrangements of the medial (MGF) and lateral (LGF) giant nerve fibers, as well as changes in giant fiber conduction velocity and morphometry. Our results show that (1) posterior fragments, originally subserved by the LGF sensory field gradually become subserved by the MGF sensory field; (2) appropriate increases in the ratio of MGF:LGF cross-sectional area, perimeter, and conduction velocity accompany the reorganization in giant fiber sensory fields; and (3) sensory field reorganization can be repeatedly reversed by additional amputations. These results demonstrate that the functional organization of escape reflexes is highly plastic and that morphallaxis may result from the counterbalance of morphogenic influences localized within the anterior and posterior ends of regenerating body fragments.     

The role of human natural killer-1 (HNK-1) carbohydrate in neuronal plasticity and disease

BackgroundThe human natural killer-1 (HNK-1) carbohydrate, a unique trisaccharide possessing sulfated glucuronic acid in a non-reducing terminus (HSO₃-3GlcAß1-3Galß1-4GlcNAc-), is highly expressed in the nervous system and its spatiotemporal expression is strictly regulated. Mice deficient in the gene encoding a key enzyme, GlcAT-P, of the HNK-1 biosynthetic pathway exhibit almost complete disappearance of the HNK-1 epitope in the brain, significant reduction of long-term potentiation, and aberration of spatial learning and memory formation. In addition to its physiological roles in higher brain function, the HNK-1 carbohydrate has attracted considerable attention as an autoantigen associated with peripheral demyelinative neuropathy, which relates to IgM paraproteinemia, because of high immunogenicity. It has been suggested, however, that serum autoantibodies in IgM anti-myelin-associated glycoprotein (MAG) antibody-associated neuropathy patients show heterogeneous reactivity to the HNK-1 epitope.Scope of reviewWe have found that structurally distinct HNK-1 epitopes are expressed in specific proteins in the nervous system. Here, we overview the current knowledge of the involvement of these HNK-1 epitopes in the regulation of neural plasticity and discuss the impact of different HNK-1 antigens of anti-MAG neuropathy patients.Major conclusionsWe identified the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptor subunit GluA2 and aggrecan as HNK-1 carrier proteins. The HNK-1 epitope on GluA2 and aggrecan regulates neural plasticity in different ways. Furthermore, we found the clinical relationship between reactivity of autoantibodies to the different HNK-1 epitopes and progression of anti-MAG neuropathy.General significanceThe HNK-1 epitope is indispensable for the acquisition of normal neuronal function and can be a good target for the establishment of diagnostic criteria for anti-MAG neuropathy.     

Nervous system dynamics during fragmentation and regeneration in Enchytraeus japonensis (Oligochaeta, Annelida)

Enchytraeus japonensis is a small terrestrial oligochaete which primarily reproduces asexually by fragmentation and regeneration. In order to introduce a molecular approach to the study of regeneration we developed a whole-mount immunostaining procedure for the worm. Using an antibody directed against acetylated tubulin in conjunction with confocal laser-scanning microscopy, we succeeded in clarifying the three- dimensional structure of the entire nervous system in the full-grown worm and its dynamics during the fragmentation and regeneration process. In addition, we examined the expression of neurotransmitters and neuropeptides in the worm using a fluorescently-labeled antagonist and various antibodies. In particular, we found two circumferential structures in the body wall muscle of each segment that react strongly with α-bungarotoxin, an antagonist of nicotinic acetylcholine receptors, and detected nerve fibers just underneath these structures. During the fragmentation process, the circular body wall muscles contract near one of these circumferential structures in the middle of the segment, which causes constriction and results in fission of the body. This α-bungarotoxin-positive structure was designated the neuromuscular junction of the circular muscle. During the regeneration process nerve fibers grow from the remaining ventral nerve cord and gradually form networks in both the anterior and posterior regeneration buds. The growing fibers extend to the prostomium (a sensory organ) at the anterior end prior to connecting to the presumptive brain rudiment. A neural network appears around the pygidium, and this is followed by growth of the body at the posterior end. The nervous system appears to play an important role in both anterior and posterior regeneration. Received: 9 June 1999 / Accepted: 30 December 1999     

Tract formation and axon fasciculation of molecularly distinct peripheral neuron subpopulations during leech embryogenesis.

In leech, the central projections of peripheral sensory neurons segregate into specific axonal tracts, which are distinguished by differential expression of surface antigens recognized by the monoclonal antibodies Lan3-2 and Lan4-2. Lan3-2 recognizes an epitope expressed on axons that segregate into three distinct axon fascicles. In contrast, the Lan4-2-positive axons selectively project into only one of the Lan3-2-positive axon tracts. These observations provide evidence for a hierarchy of guidance cues mediating specific pathway formation in this system. Since the Lan3-2 antibody has been shown to perturb this process and since, as shown here, the Lan3-2 and Lan4-2 antigens are closely molecularly interrelated, these antibodies may help define molecules and epitopes mediating neuronal recognition and axonal guidance.     

Brain alpha-dystroglycan displays unique glycoepitopes and preferential binding to laminin-10/11

alpha-Dystroglycan was quantitatively enriched from mammalian brain based on its uniform reactivity with Vicia villosa agglutinin and resolved into sub-populations possessing or lacking the sulfated glucuronic acid epitope recognized by monoclonal antibody HNK-1. We generated a new monoclonal antibody specific for a glycoepitope on brain alpha-dystroglycan but absent from alpha-dystroglycan expressed in all other tissues examined. Finally, we found that laminin-10/11 preferentially bound to brain alpha-dystroglycan compared to skeletal muscle alpha-dystroglycan. Our results suggest that tissue-specific glycosylation modifies the laminin binding specificity of alpha-dystroglycan.     

A Sulfated Glycosaminoglycan Linkage Region Is a Novel Type of Human Natural Killer-1 (HNK-1) Epitope Expressed on Aggrecan in Perineuronal Nets

Human natural killer-1 (HNK-1) carbohydrate (HSO₃-3GlcAβ1-3Galβ1-4GlcNAc-R) is highly expressed in the brain and required for learning and neural plasticity. We previously demonstrated that expression of the HNK-1 epitope is mostly abolished in knockout mice for GlcAT-P (B3gat1), a major glucuronyltransferase required for HNK-1 biosynthesis, but remained in specific regions such as perineuronal nets (PNNs) in these mutant mice. Considering PNNs are mainly composed of chondroitin sulfate proteoglycans (CSPGs) and regulate neural plasticity, GlcAT-P-independent expression of HNK-1 in PNNs is suggested to play a role in neural plasticity. However, the function, structure, carrier glycoprotein and biosynthetic pathway for GlcAT-P-irrelevant HNK-1 epitope remain unclear. In this study, we identified a unique HNK-1 structure on aggrecan in PNNs. To determine the biosynthetic pathway for the novel HNK-1, we generated knockout mice for GlcAT-S (B3gat2), the other glucuronyltransferase required for HNK-1 biosynthesis. However, GlcAT-P and GlcAT-S double-knockout mice did not exhibit reduced HNK-1 expression compared with single GlcAT-P-knockout mice, indicating an unusual biosynthetic pathway for the HNK-1 epitope in PNNs. Aggrecan was purified from cultured cells in which GlcAT-P and -S are not expressed and we determined the structure of the novel HNK-1 epitope using liquid chromatography/mass spectrometry (LC/MS) as a sulfated linkage region of glycosaminoglycans (GAGs), HSO₃-GlcA-Gal-Gal-Xyl-R. Taken together, we propose a hypothetical model where GlcAT-I, the sole glucuronyltransferase required for synthesis of the GAG linkage, is also responsible for biosynthesis of the novel HNK-1 on aggrecan. These results could lead to discovery of new roles of the HNK-1 epitope in neural plasticity.     

Microscopical evaluation of neural connectivity between paired stages of Eudiplozoon nipponicum (Monogenea: Diplozoidae)

Diplozoidae monogeneans am fish-gill ectoparasites comprising 2 individuals fused in so-called permanent copula. This unique situation occurs when 2 larvae (diporpee) make contact on the host gin, such that their union triggers maturation into an individual adult worm. The present study examined paired stages of Eudiplozoon nipponioun microscopicaily to ascertain whether somatic fusion involves neural connectivity between these 2 heterogenic larvae. Neuronal pathways were demonstrated in whole-mount preparations of the worm, using indirect immunocytochemical techniques interfaced with confocal scanning laser microscopy for peptidergic and serotoninergic innervations and enzyme cytochemical methodology and light microscopy for cholinergic component. Elements of the central nervous systems of paired worms are connected by commissures in the region of fusion so that the 2 systems are in structural continuity. Interindividual connections were mast apparent between corresponding ventral nerve cords. All 3 classes of neuronal mediators were identified throughout both central and peripheral connections of the 2 nervous systems. The anatomical complexity and apparent plasticity of the diplozoon nervous system suggest that it has a pivotal role not only in motility, feeding, and reproductive behavious but also in the events of larval pairing and somatic fusion.     

6-Sulphated chondroitins have a positive influence on axonal regeneration

Chondroitin sulphate proteoglycans (CSPGs) upregulated in the glial scar inhibit axon regeneration via their sulphated glycosaminoglycans (GAGs). Chondroitin 6-sulphotransferase-1 (C6ST-1) is upregulated after injury leading to an increase in 6-sulphated GAG. In this study, we ask if this increase in 6-sulphated GAG is responsible for the increased inhibition within the glial scar, or whether it represents a partial reversion to the permissive embryonic state dominated by 6-sulphated glycosaminoglycans (GAGs). Using C6ST-1 knockout mice (KO), we studied post-injury changes in chondroitin sulphotransferase (CSST) expression and the effect of chondroitin 6-sulphates on both central and peripheral axon regeneration. After CNS injury, wild-type animals (WT) showed an increase in mRNA for C6ST-1, C6ST-2 and C4ST-1, but KO did not upregulate any CSSTs. After PNS injury, while WT upregulated C6ST-1, KO showed an upregulation of C6ST-2. We examined regeneration of nigrostriatal axons, which demonstrate mild spontaneous axon regeneration in the WT. KO showed many fewer regenerating axons and more axonal retraction than WT. However, in the PNS, repair of the median and ulnar nerves led to similar and normal levels of axon regeneration in both WT and KO. Functional tests on plasticity after the repair also showed no evidence of enhanced plasticity in the KO. Our results suggest that the upregulation of 6-sulphated GAG after injury makes the extracellular matrix more permissive for axon regeneration, and that the balance of different CSs in the microenvironment around the lesion site is an important factor in determining the outcome of nervous system injury.     

Mechanisms of axon regeneration: The significance of proteoglycans

BackgroundTherapeutics specific to neural injury have long been anticipated but remain unavailable. Axons in the central nervous system do not readily regenerate after injury, leading to dysfunction of the nervous system. This failure of regeneration is due to both the low intrinsic capacity of axons for regeneration and the various inhibitors emerging upon injury. After many years of concerted efforts, however, these hurdles to axon regeneration have been partially overcome.Scope of reviewThis review summarizes the mechanisms regulating axon regeneration. We highlight proteoglycans, particularly because it has become increasingly clear that these proteins serve as critical regulators for axon regeneration.Major conclusionsStudies on proteoglycans have revealed that glycans not only assist in the modulation of protein functions but also act as main players—e.g., as functional ligands mediating intracellular signaling through specific receptors on the cell surface. By regulating clustering of the receptors, glycans in the proteoglycan moiety, i.e., glycosaminoglycans, promote or inhibit axon regeneration. In addition, proteoglycans are involved in various types of neural plasticity, ranging from synaptic plasticity to experience-dependent plasticity.General significanceAlthough studies on proteins have progressively facilitated our understanding of the nervous system, glycans constitute a new frontier for further research and development in this field. This article is part of a Special Issue entitled Neuro-glycoscience, edited by Kenji Kadomatsu and Hiroshi Kitagawa.     

The human natural killer-1 (HNK-1) glycan mimetic ursolic acid promotes functional recovery after spinal cord injury in mouse

Human natural killer-1 (HNK-1) cell antigen is a glycan epitope involved in several neural events, such as neuritogenesis, myelination, synaptic plasticity and regeneration of the nervous system after injury. We have recently identified the small organic compound ursolic acid (UA) as a HNK-1 mimetic with the aim to test its therapeutic potential in the central nervous system. UA, a plant-derived pentacyclic triterpenoid, is well known for its multiple biological functions, including neuroprotective, antioxidant and anti-inflammatory activities. In the present study, we evaluated its functions in a mouse model of spinal cord injury (SCI) and explored the molecular mechanisms underlying its positive effects. Oral administration of UA to mice 1 h after SCI and thereafter once daily for 6 weeks enhanced the regaining of motor functions and axonal regrowth, and decreased astrogliosis. UA administration decreased levels of proinflammatory markers, including interleukin-6 and tumor necrosis factor-α, in the injured spinal cord at the acute phase of inflammation and activated the mitogen-activated protein kinase and phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin pathways in the injured spinal cord. Taken together, these results suggest that UA may be a candidate for treatment of nervous system injuries.     

Defasciculation as a neuronal pathfinding strategy: involvement of a specific glycoprotein

Leech sensory afferents change their growth behavior as they enter the CNS. Arriving from the periphery in fasciculated tracts, they abruptly defasciculate and expand into diffuse trees before reassembling into four distinct central tracts. In the organ-cultured germinal plate, growing sensory afferents were incubated with monovalent Fab fragments of the Lan3-2 antibody, which recognizes a 130 kd sensory neuron protein by its mannose epitope. Very low concentrations of Lan3-2 (6 and 12 nM) specifically inhibited the central defasciculation of sensory afferents, which then continued growing as a single tract. In contrast, monoclonal antibody Lan3-6, which binds to an internal sensory antigen, failed to yield the same effect. These observations suggest that this sensory neuron 130 kd surface glycoprotein participates in a developmentally significant heterophilic interaction specific for the CNS.     

The Specificity of 130-kDa Leech Sensory Afferent Proteins Is Encoded by Their Carbohydrate Epitopes

From early development through adulthood in the leech, sensory afferents, glial cells, and connective tissue express different epitopes located on a group of 130-kDa glycoproteins. The sensory epitope [reactive with monoclonal antibody (mAb) Lan3-2] is shared by the peripheral sensory afferents of different sensory modalities. In contrast, three other immunocytochemically distinct epitopes (reactive with mAbs Laz2-369, Laz7-79, and Laz6-212) differentiate these sensory afferents according to their sensory modalities. The glial epitope (mAb Laz6-297) is expressed on all macroglial processes, and the connective tissue epitope (mAb Laz9-84) is located on connective tissue surrounding the CNS, as well as in the peripheral tissues. The hydrophilic-hydrophobic nature of the 130-kDa sensory afferent and glial proteins was determined by phase separation with Triton X-114 and hypoosmotic extraction. They behave as peripheral membrane proteins. Deglycosylation of 130-kDa glycoproteins with N-Glycanase or preincubation of their respective mAbs with alpha-methylmannoside showed that the sensory epitope contains mannose, whereas the modality epitopes are of an undefined carbohydrate character. Immunoprecipitation and a peptide mapping experiment confirmed the existence of four distinct sensory afferent epitopes. Previous studies provided evidence that the mannose-containing Lan3-2 epitope mediates normal sensory afferent growth in the synaptic neuropile. We, therefore, postulate that the carbohydrate epitopes on sensory afferent glycoproteins participate in synapse formation.     

Chondroitin sulfate: a key molecule in the brain matrix

Chondroitin sulfate is a glycosaminoglycan composed of N-acetylgalactosamine and glucuronic acid. It attaches to a core protein to form chondroitin sulfate proteoglycan (CSPG). Being a major component of the brain extracellular matrix, CSPGs are involved in neural development, axon pathfinding and guidance, plasticity and also regeneration after injury in the nervous system. In this review, we shall discuss the structure, the biosynthetic pathway, its functions in the nervous system and how we can improve regeneration in the nervous system by modulating its structure and binding properties.