Christine E. Bandtlow

Lack of evidence that myelin-associated glycoprotein is a major inhibitor of axonal regeneration in the CNS

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.

Identification and characterization of a bovine neurite growth inhibitor (bNI-220).

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 9m 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.

Versican V2 and the central inhibitory domain of Nogo-A inhibit neurite growth via p75NTR/NgR-independent pathways that converge at RhoA.

Myelin is a major obstacle for regenerating nerve fibers of the adult mammalian central nervous system (CNS). Several proteins including Nogo-A, myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgp) and the chondroitin-sulfate proteoglycan (CSPG) Versican V2 have been identified as inhibitory components present in CNS myelin. MAG, OMgp as well as the Nogo specific domain Nogo-66 exert their inhibitory activity by binding to a neuronal receptor complex containing the Nogo-66 receptor NgR and the neurotrophin receptor p75(NTR). While this suggests a converging role of the p75(NTR)/NgR receptor complex for myelin-derived neurite growth inhibitors, we show here that NgR/p75(NTR) is not required for mediating the inhibitory activity of the two myelin components NiG, unlike Nogo-66 a distinct domain of Nogo-A, and Versican V2. Primary neurons derived from a complete null mutant of p75(NTR) are still sensitive to NiG and Versican V2. In line with this result, neurite growth of p75(NTR) deficient neurons is still significantly blocked on total bovine CNS myelin. Furthermore, modulation of RhoA and Rac1 in p75(NTR)-/- neurons persists with NiG and Versican V2. Finally, we demonstrate that neither NiG nor Versican V2 interact with the p75(NTR)/NgR receptor complex and provide evidence that the binding sites of NiG and Nogo-66 are physically distinct from each other on neural tissue. These results indicate not only the existence of neuronal receptors for myelin inhibitors independent from the p75(NTR)/NgR receptor complex but also establish Rho GTPases as a common point of signal convergence of diverse myelin-induced regeneration inhibitory pathways.

Nogo-A Stabilizes the Architecture of Hippocampal Neurons

Although the role of myelin-derived Nogo-A as an inhibitor of axonal regeneration after CNS injury has been thoroughly described, its physiological function in the adult, uninjured CNS is less well known. We address this question in the hippocampus, where Nogo-A is expressed by neurons as well as oligodendrocytes. We used 21 d in vitro slice cultures of neonatal hippocampus where we applied different approaches to interfere with Nogo-A signaling and expression and analyze their effects on the dendritic and axonal architecture of pyramidal cells. Neutralization of Nogo-A by function-blocking antibodies induced a major alteration in the dendrite structure of hippocampal pyramidal neurons. Although spine density was not influenced by Nogo-A neutralization, spine type distribution was shifted toward a more immature phenotype. Axonal complexity and length were greatly increased. Nogo-A KO mice revealed a weak dendritic phenotype resembling the effect of the antibody treatment. To discriminate a possible cell-autonomous role of Nogo-A from an environmental, receptor-mediated function, we studied the effects of short hairpin RNA-induced knockdown of Nogo-A or NgR1, a prominent Nogo-A receptor, within individual neurons. Knockdown of Nogo-A reproduced part of the dendritic and none of the spine or axon alterations. However, downregulation of NgR1 replicated the dendritic, the axonal, and the spine alterations observed after Nogo-A neutralization. Together, our results demonstrate that Nogo-A plays a major role in stabilizing and maintaining the architecture of hippocampal pyramidal neurons. Mechanistically, although the majority of the activity of Nogo-A relies on a receptor-mediated mechanism involving NgR1, its cell-autonomous function plays a minor role.

The Critical Period for Repair of CNS of Neonatal Opossum (Monodelphis domestica) in Culture: Correlation with Development of Glial Cells, Myelin and Growth-inhibitory Molecules

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.

Developmental Changes in Neuronal Responsiveness to the CNS Myelin‐associated Neurite Growth Inhibitor N I‐35/250

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 Nl‐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 Nl‐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 Nl‐35/250 compared with mature neurons. In older neurons Nl‐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 Nl‐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 Nl‐35/250 around the time of myelination.

Differential cytoskeletal changes during growth cone collapse in response to hSema III and thrombin.

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.

Understanding myelination through studying its evolution.

Publisher Summary This chapter discusses many studies that provide insights into the origins of central (CNS) and peripheral nervous system (PNS) myelination and into the reasons that CNS regeneration in adult vertebrates is restricted to fish and amphibians. As in other fields, studying biological processes through their evolutionary history, in their own right, provides important and novel insights into human diseases in which myelin sheaths develop abnormally and/or following normal development they disassemble and are degraded. The chapter studies mechanisms for the adaptation of sophisticated regulation that underlies myelination for animals living under conditions far different from example, without thermoregulation or different osmotic environments. Among the animal models with a rapidly growing following is the zebra, which is being used more frequently by ‘‘myelin ’’ scientists. As more and more sequences from genome-sequencing projects relevant to vertebrate and invertebrate evolution become available, appreciation of evolutionary adaptations made by the plethora of molecules associated with differing facets of the myelination process is realized.

Increased expression of Nogo-A in hippocampal neurons of patients with temporal lobe epilepsy

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 ≈ 40% of the specimens revealed low expression of Nogo‐A mRNA in neurons. In contrast, in TLE patients with and without Ammons 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.

From Cell Death to Neuronal Regeneration, Effects of the p75 Neurotrophin Receptor Depend on Interactions with Partner Subunits

In the adult mammalian central nervous system (CNS), growth of neuronal fibers is actively inhibited by myelin. The proteins myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgP), and Nogo-66 have been identified as inhibitory components present in CNS myelin. All three proteins exert their inhibitory activity by binding to a neuronal receptor complex containing the Nogo-66 receptor (NgR) and the neurotrophin (NT) receptor p75NTR. In their recent publication, Mi et al. identify the novel protein Lingo-1 as an interactor of p75NTR and NgR. The Lingo-1-NgR-p75NTR complex is shown to confer the inhibitory effects on nerve cell regeneration of Nogo-66, OMgP, and MAG by activating the small guanosine triphosphatase (GTPase) RhoA. Together with the recent finding that p75NTR interacts with the transmembrane protein sortilin to form a different receptor complex with cell death-promoting activity, the results of Mi et al. indicate that p75NTR exerts its diverse cellular functions by associating with function-specific co-receptors.