Mesial temporal lobe epilepsy (TLE) is associated with pronounced anatomical and biochemical changes in the hippocampal formation including extensive neurodegeneration, reorganization of mossy fibres and sprouting of interneurons. Although the anatomical features and some of the physiological consequences of hippocampal remodeling have been well documented, the molecular mechanisms underlying the profound and orientated outgrowth of hippocampal neurons in TLE are not yet understood. The reticulon protein Nogo-A has been associated with an inhibitory action on axon growth and plasticity. Using immunohistochemistry and in situ hybridization, we investigated the expression of Nogo-A in specimens obtained at surgery from patients with TLE compared with those obtained from autopsy controls. In control specimens, Nogo-A immunoreactivity and mRNA were mainly confined to oligodendrocytes. Only approximate to 40% of the specimens revealed low expression of Nogo-A mRNA in neurons. In contrast, in TLE patients with and without Ammon's horn sclerosis, Nogo-A mRNA and immunoreactivity were markedly up-regulated in most neurons (3.6- and 4.4-fold increases in Nogo-A mRNA in granule cells of sclerotic and nonsclerotic specimens) and their processes throughout the hippocampal formation. Similar elevations in Nogo-A mRNA and protein levels were determined by quantitative RT-PCR and Western blotting. Since Nogo-A expression was also up-regulated in specimens without hippocampal sclerosis, it may be induced by seizures prior to progressing neurodegeneration.

Nogo-A is a potent neurite growth inhibitor in vitro and plays a role both in the restriction of axonal regeneration after injury and in structural plasticity in the CNS of higher vertebrates. The regions that mediate inhibition and the topology of the molecule in the plasma membrane have to be defined. Here we demonstrate the presence of three different active sites: (1) an N-terminal region involved in the inhibition of fibroblast spreading, (2) a stretch encoded by the Nogo-A-specific exon that restricts neurite outgrowth and cell spreading and induces growth cone collapse, and (3) a C-terminal region (Nogo-66) with growth cone collapsing function. We show that Nogo-A-specific active fragments bind to the cell surface of responsive cells and to rat brain cortical membranes, suggesting the existence of specific binding partners or receptors. Several antibodies against different epitopes on the Nogo-A-specific part of the protein as well as antisera against the 66 aa loop in the C-terminus stain the cell surface of living cultured oligodendrocytes. Nogo-A is also labeled by nonmembrane-permeable biotin derivatives applied to living oligodendrocyte cultures. Immunofluorescent staining of intracellular, endoplasmic reticulum-associated Nogo-A in cells after selective permeabilization of the plasma membrane reveals that the epitopes of Nogo-A, shown to be accessible at the cell surface, are exposed to the cytoplasm. This suggests that Nogo-A could have a second membrane topology. The two proposed topological variants may have different intracellular as well as extracellular functions.

Nogo-A is a protein associated with central nervous system (CNS) myelin thought to impair regenerative responses and to suppress sprouting and plastic changes of synaptic terminals. In this study, we report that serum IgM autoantibodies to the recombinant large N-terminal inhibitory domain of Nogo-A are a frequent finding in multiple sclerosis (MS) and acute inflammatory (IND) and non-inflammatory neurological diseases (OND), but not in neurodegenerative diseases (ND), systemic inflammatory disease and healthy controls. Furthermore, we demonstrate intrathecal production of anti-Nogo-A antibodies measured by increased IgG indices. Intrathecal anti-Nogo antibodies were significantly more frequent in patients with relapsing-remitting as compared to chronic progressive (CP) MS. We also found a highly significant negative correlation of these antibody responses with age indicating that they are more frequent in younger patients. We finally demonstrate that human anti-Nogo-A antibodies recognize native Nogo-A in brain extracts, oligodendrocytes and cells expressing human Nogo-A.

The adult mammalian CNS has a limited capacity for nerve regeneration and structural plasticity. The presence of glia-derived inhibitory factors myelin-associated glycoprotein (MAG) and Nogo-A have been suggested to provide a nonpermissive environment for elongating nerve fibers. In particular, Nogo-A, an integral membrane protein predominantly expressed by oligodendrocytes, has been demonstrated to impair neurite growth in vitro and in vivo. Structure function analysis revealed that Nogo-A protein contains at least two active domains, NiG and Nogo-66, with diverse effects on neurite outgrowth and cell spreading. We now provide evidence that these inhibitory domains mediate their effects via an antagonistic regulation of the small GTPases RhoA and Rac1, resulting in activation of RhoA and suppression of Rac1. By inactivating RhoA with C3 transferase or the downstream effector Rho-kinase ROCK with Y27632, the inhibitory effects of both Nogo-A fragments and MAG on neurite outgrowth and oligodendrocyte-mediated growth cone collapse were abolished. Furthermore, we show that the recently cloned receptor for Nogo-66 and MAG, NgR, is not necessary for either NiG- or MAG-induced RhoA activation.

Proteoglycans are a heterogeneous class of proteins bearing sulfated glycosaminoglycans. Some of the proteoglycans have distinct core protein structures, and others display similarities and thus may be grouped into families such as the syndecans, the glypicans, or the hyalectans (or lecticans). Proteoglycans can be found in almost all tissues being present in the extracellular matrix, on cellular surfaces, or in intracellular granules. In recent years, brain proteoglycans have attracted growing interest due to their highly regulated spatiotemporal expression during nervous system development and maturation. There is increasing evidence that different proteoglycans act as regulators of cell migration, axonal pathfinding, synaptogenesis, and structural plasticity. This review summarizes the most recent data on structures and functions of brain proteoglycans and focuses on new physiological concepts for their potential roles in the developing central nervous system.

In this paper, we identify the chondroitin sulfate proteoglycan versican V2 as a major inhibitor of axonal growth in the extracellular matrix of the mature central nervous system. In immunohistochemical and in situ hybridization experiments we show that this tissue-specific splice variant of versican is predominantly present in myelinated fiber tracts of the brain and in the optic nerve, most likely being expressed by oligodendrocytes. We demonstrate that isolated versican V2 strongly inhibits neurite outgrowth of central and peripheral neurons in stripe-choice assays using laminin-1 as permissive substrate. The inhibitory character of versican V2 is maintained after removal of chondroitin sulfate and N- and O-linked oligosaccharide side chains, but it is abolished after core protein digestion with proteinase-K. Our data support the notion, that intact versican V2 prevents excessive axonal growth during late phases of development and hereby participates in the structural stabilization of the mature central nervous system.

After the transection of the Schaffer collateral pathway in hippocampal slice cultures, reactive sprouting is induced in the CA3 area, and eventually synaptic transmission between areas CA1 and CA3 is restored. Using this model, we have studied the role of ionotropic glutamate receptors in the initiation of axonal sprouting and the regeneration of functional synapses. We show that neither reactive sprouting nor functional recovery of synaptic transmission occur in the presence of the non-N-methyl-D-aspartate (NMDA) receptor antagonist 6-nitro-7-sulfamoylbenzoquinoxaline-2,3-dione (CNQX). In contrast, the NMDA receptor antagonists methyl-10, 11-dihydro-5-H-dibenzocyclohepten-5,10-imine (MK-801) or 3-(RS)-2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid (CPP) did not interfere with these processes. Moreover, we observed that the application of NMDA receptor antagonists induced massive axonal sprouting and an increase in the frequency of miniature excitatory postsynaptic currents in unlesioned cultures. Our results thus indicate that NMDA and non-NMDA receptors exert a differential effect on reactive sprouting and the recovery of synaptic transmission after injury in the hippocampus. Activation of non-NMDA receptors appears necessary for these processes to occur, whereas activation of NMDA receptors suppresses growth-associated protein -43 expression and axonal outgrowth.

The absence of fiber regrowth in the injured mammalian CNS is influenced by several different factors and mechanisms. Besides the nonconducive properties of the glial scar tissue that forms around the lesion site, individual molecules present in CNS myelin and expressed by oligodendrocytes, such as NI-35/NI-250, bNI-220, and myelin-associated glycoprotein (MAG), have been isolated and shown to inhibit axonal growth. Here, we report an additional neurite growth-inhibitory activity purified from bovine spinal cord myelin that is not related to bNI-220 or MAG. This activity can be ascribed to the presence of two chondroitin sulfate proteoglycans (CSPGs), brevican and the brain-specific versican V2 splice variant. Neurite outgrowth of neonatal cerebellar granule cells and of dorsal root ganglion neurons in vitro was strongly inhibited by this myelin fraction enriched in CSPGs. Immunohistochemical staining revealed that brevican and versican V2 are present on the surfaces of differentiated oligodendrocytes. We provide evidence that treatment of oligodendrocytes with the proteoglycan synthesis inhibitors beta-xylosides can strongly influence the growth permissiveness of oligodendrocytes. beta-Xylosides abolished cell surface presentation of brevican and versican V2 and reversed growth cone collapse in encounters with oligodendrocytes as demonstrated by time-lapse video microscopy. Instead, growth cones were able to grow along or even into the processes of oligodendrocytes. Our results strongly suggest that brevican and versican V2 are additional components of CNS myelin that contribute to its nonpermissive substrate properties for axonal growth. Expression of these CSPGs on oligodendrocytes may indicate that they participate in the restriction of structural plasticity and regeneration in the adult CNS.

Growth cones are known as the site of action of many factors that influence neurite growth behavior. To assess how different collapsing agents influence the growth cone cytoskeleton, we used recombinant human Semaphorin III (hSema III) and the serine protease thrombin. Embryonic chick dorsal root ganglion neurons showed a dramatic depolymerization of actin filaments within 5 min upon hSema III exposure and virtually no influence on microtubules (MT). Only at later time points (20–30 min) was the polymerization/depolymerization rate of MT significantly affected. Thrombin induced a morphologically and kinetically similar growth cone collapse. Moreover, thrombin induced an early and selective depolymerization of dynamic MT, accompanied by the formation of loops of stable MT bundles. Selective changes in the phosphorylation pattern of tau were associated with microtubule assembly in thrombin-induced responses. Our data provide evidence that different signal transduction pathways lead to distinct changes of the growth cone cytoskeleton.

The poor axonal regeneration that follows lesions of the central nervous system (CNS) is crucially influenced by the local CNS tissue environment through which neurites have to grow. In addition to an inhibitory role of the glial scar, inhibitory substrate effects of CNS myelin and oligodendrocytes have been demonstrated. Several proteins including NI-35/250, myelin-associated glycoprotein, tenascin-R, and NG-2 have been described to have neurite outgrowth inhibitory or repulsive properties in vitro. Antibodies raised against NI-35/250 (monoclonal antibody IN-1) were shown to partially neutralize the growth inhibitory effect of CNS myelin and oligodendrocytes, and to result in long distance fiber regeneration in the lesioned adult mammalian CNS in vivo. We report here the purification of a myelin protein to apparent homogeneity from bovine spinal cord which exerts a potent neurite outgrowth inhibitory effect on PC12 cells and chick dorsal root ganglion cells, induces collapse of growth cones of chick dorsal root ganglion cells, and also inhibits the spreading of 3T3 fibroblasts. These activities could be neutralized by the monoclonal antibody IN-1. The purification procedure includes detergent solubilization, anion exchange chromatography, gel filtration, and elution from high resolution SDS-polyacrylamide gel electrophoresis. The active protein has a molecular mass of 220 kDa and an isoelectric point between 5.9 and 6.2. Its inhibitory activity is sensitive to protease treatment and resists harsh treatments like 9 M urea or short heating. Glycosylation is, if present at all, not detectable. Microsequencing resulted in six peptides and strongly suggests that this proteins is novel.

The extent of fibre regeneration in the adult injured vertebrate nervous system appears to be primarily determined by the local environment. Thus, the failure of axon regrowth in the central nervous system (CNS) is crucially influenced by the presence of the myelin-associated neurite growth inhibitor NI-35/250 and possibly also by molecules such as the myelin-associated glycoprotein and the proteoglycans. Developmental time course studies have shown that the capacity for regeneration declines sharply with the appearance of mature oligodendrocytes and myelin, which indicates a role of NI-35/250 in restricting CNS regeneration and plasticity. However, recent in vitro and in vivo studies showed that embryonic neurons are capable of extending fibres on and in adult CNS tissue apparently unaffected by myelinated areas. A possible explanation is that very immature neurons have yet to express the appropriate receptors and response mechanisms for factors that normally induce growth inhibition at a later stage of development. Here we report that embryonic rat dorsal root ganglion and chick retinal ganglion cells display different sensitivity to bovine NI-35/250 compared with mature neurons. In older neurons NI-35/250 could evoke long-lasting collapse responses accompanied by a large increase in the intracellular calcium level, persisting for several minutes. In contrast, their embryonic counterparts collapsed only transiently when exposed to NI-35/250, and increases in intracellular calcium concentration were small and transient. Calcium influx induced experimentally by the calcium ionophore A23187 revealed that it was not the maximal size of the calcium increase but rather the duration of elevated calcium concentration that was the most important determinant for subsequent morphological alterations of the growth cone. Our data further suggest that developing neurons acquire their complete sensitivity for NI-35/250 around the time of myelination.

Numerous studies have shown that the developing tip of a neurite, the growth cone, can respond to environmental cues with behaviors such as guidance or collapse. To assess whether a given cell type can use more than one second-messenger pathway for a single behavior, we compared the influence of two well-characterized guidance cues on growth cones of chick temporal retinal ganglion cells. The first cue was the repulsive activity derived from the posterior optic tectum (p-membranes), and the second was the collapse-inducing activity derived from oligodendrocytes known as NI35/NI250. p-Membranes caused permanent growth cone collapse with no recovery after several hours, while NI35 caused transient collapse followed by recovery after about 10 min. The p-membrane-induced collapse was found to be Ca2+ independent, as shown using the Ca2+-sensitive dye Fura-2 and by the persistence of collapse in Ca2+-free medium. Dantrolene, a blocker of the ryanodine receptor, had only a minor effect on the collapse frequency caused by p-membranes. In contrast, the NI35-induced collapse was clearly Ca2+ dependent. [Ca2+]i increased sevenfold preceding collapse, and both dantrolene and antibodies against NI35 significantly reduced both the Ca2+ increase and the collapse frequency. Thus, even in a single cell type, growth cone collapse induced by two different signals can be mediated by two different second-messenger systems.

The MAG-deficient mouse was used to test whether MAG acts as a significant inhibitor of axonal regeneration in the adult mammalian CNS, as suggested by cell culture experiments. Cell spreading, neurite elongation, or growth cone collapse of different cell types in vitro was not significantly different when myelin preparations or optic nerve cryosections from either MAG-deficient or wild-type mice were used as a substrate. More importantly, the extent of axonal regrowth in lesioned optic nerve and corticospinal tract in vivo was similarly poor in MAG-deficient and wild-type mice. However, axonal regrowth increased significantly and to a similar extent in both genotypes after application of the IN-1 antibody directed against the neurite growth inhibitors NI-35 and NI-250. These observations do not support the view that MAG is a significant inhibitor of axonal regeneration in the adult CNS.

To determine whether optic nerve myelin of goldfish carries mammalian-like neurite growth inhibitory proteins which can be neutralized by the antibody IN-1, myelin fractions of fish optic nerves were used as substrates for fish retinal ganglion cell axons and rat dorsal root ganglia (DRG). Axonal growth was monitored and compared with that of IN-1 treated preparations. Growth of fish retinal axons and rat DRG neurites was substantial on goldfish optic nerve myelin and no improvement was observed with IN-1. In contrast, rat CNS myelin allowed only poor growth, and number of axons and length of DRG neurites increased significantly with IN-1. In addition, proteins of fish optic nerve myelin and bovine CNS myelin were extracted, reconstituted in liposomes and applied to growth cones. When goldfish myelin proteins in liposomes were seeded onto growth cones, 77% of fish and 89% of rat DRG growth cones continued to elongate, and the proportion of elongating fish growth cones (80%) did not significantly change when liposomes were pretreated with IN-1. But 73% of fish and 93% of rat growth cones collapsed with liposomes containing proteins from bovine CNS myelin. Upon IN-1 treatment, only 24% of fish growth cones collapsed. Thus, axon growth in vitro indicates that goldfish optic nerves, which permit successful axon regeneration in vivo, lack mammalian-like neurite growth inhibitors which are neutralized by IN-1.

Adhesion and neurite outgrowth of PC12 cells, as well as the spreading of 3T3 fibroblasts, were inhibited in a dose dependent manner by detergent solubilized mouse central nervous system myelin proteins as a tissue culture substrate. These inhibitory effects could be neutralized by the monoclonal antibody IN-1 directed against the neurite growth inhibiting proteins NI-35 and NI-250. Separation of the detergent soluble proteins of bovine spinal cord by an anion exchange column showed that the peaks of inhibitory activity for the two cell lines overlapped, such that the PC12 cells were inhibited by a larger number of fractions comprising those inhibitory for 3T3 cells. Neurite outgrowth of PC12 cells was not influenced by the myelin associated glycoprotein, MAG.

A comparison was made of neurite growth across spinal cord lesions in the isolated central nervous system (CMS) of newborn opossums ( Monodelphis domestica ) at various stages of development. The aim was to define the critical period at which growth after injury ceases to occur, with emphasis on growth‐inhibitory proteins, myelin and glial cells. In postnatal opossums 3–6 days old (P3–6), repair was observed 5 days after lesions were made in culture at the cervical level (C7) by crushing with forceps. Through‐conduction of action potentials was re‐established and axons stained by Oil grew into and beyond the crush. In a series of 66 animals 29 showed repair. In 28 animals at P11–12 with comparable lesions repair was observed in five preparations. At P13–14, the CMS was still viable in culture, but none of the 25 preparations examined showed any axonal growth into the crush or conduction through it. The rostra‐caudal gradient of development permitted lesions to be made in mature cervical and immature lumbar regions of P11–12 spinal cord. Growth across crushes occurred in lumbar but not in cervical segments of the same preparation. The development of glial cells and myelin was assessed by electron microscopy and by staining with specific antibodies (Rip‐1 and myelin‐associated glycoprotein) in cervical segments of neonatal P6–14 opossums. At P8, oligodendrocytes and thin myelin sheaths started to appear followed at P9 by astrocytes stained with antibody against glial fibrillary acidic protein. By P14, astrocytes, oligodendrocytes and well‐developed myelin sheaths were abundant. The cervical crush sites of P12 cords contained occasional astrocytes but no oligodendrocytes. Specific antibodies (IN‐1) to neurite growth‐inhibiting proteins (NI‐35/250) associated with oligodendrocytes and myelin in the rat CNS cross‐reacted with opossum proteins. Assays using the spreading of 3T3 fibroblasts and IN‐1 showed that by P7 inhibitory proteins became apparent, particularly in the hindbrain and cervical spinal cord. The concentrations of NI‐35/250 thereafter increased and became abundant in the adult opossum. Our finding of a well‐defined critical period, encompassing only 5 days, in CNS preparations that can be maintained in culture offers advantages for analysing mechanisms that promote or prevent CNS repair.

The brain's default mode network consists of discrete, bilateral and symmetrical cortical areas, in the medial and lateral parietal, medial prefrontal, and medial and lateral temporal cortices of the human, nonhuman primate, cat, and rodent brains. Its ...Read More