Martin E. Schwab

Nogo-A is a myelin-associated neurite outgrowth inhibitor and an antigen for monoclonal antibody IN-1.

The capacity of the adult brain and spinal cord to repair lesions by axonal regeneration or compensatory fibre growth is extremely limited. A monoclonal antibody (IN-1) raised against NI-220/250, a myelin protein that is a potent inhibitor of neurite growth, promoted axonal regeneration and compensatory plasticity following lesions of the central nervous system (CNS) in adult rats. Here we report the cloning of nogo A, the rat complementary DNA encoding NI-220/250. The nogo gene encodes at least three major protein products (Nogo-A, -B and -C). Recombinant Nogo-A is recognized by monoclonal antibody IN-1, and it inhibits neurite outgrowth from dorsal root ganglia and spreading of 3T3 fibroblasts in an IN-1-sensitive manner. Antibodies against Nogo-A stain CNS myelin and oligodendrocytes and allow dorsal root ganglion neurites to grow on CNS myelin and into optic nerve explants. These data show that Nogo-A is a potent inhibitor of neurite growth and an IN-1 antigen produced by oligodendrocytes, and may allow the generation of new reagents to enhance CNS regeneration and plasticity.

Antibody against myelin-associated inhibitor of neurite growth neutralizes nonpermissive substrate properties of CNS white matter.

CNS white matter from higher vertebrates and cultured differentiated oligodendrocytes are nonpermissive substrates for neurite growth and fibroblast spreading. Membrane proteins of 35 kd and 250 kd with highly nonpermissive substrate properties could be extracted from CNS myelin fractions. Monoclonal antibodies were raised against these proteins: IN-1 and IN-2 bound both to the 35 kd and 250 kd inhibitors and to the surface to differentiated cultured oligodendrocytes. Adsorption of nonpermissive CNS myelin or nonpermissive oligodendrocytes with either antibody markedly improved their substrate properties. Optic nerve explants injected with IN-1 or IN-2 allowed axon ingrowth of cocultured sensory and sympathetic neurons. We conclude that the nonpermissive substrate properties of CNS white matter are due to these membrane proteins on the surface of differentiated oligodendrocytes and to their in vivo product, myelin.

The injured spinal cord spontaneously forms a new intraspinal circuit in adult rats

In contrast to peripheral nerves, central axons do not regenerate. Partial injuries to the spinal cord, however, are followed by functional recovery. We investigated the anatomical basis of this recovery and found that after incomplete spinal cord injury in rats, transected hindlimb corticospinal tract (CST) axons sprouted into the cervical gray matter to contact short and long propriospinal neurons (PSNs). Over 12 weeks, contacts with long PSNs that bridged the lesion were maintained, whereas contacts with short PSNs that did not bridge the lesion were lost. In turn, long PSNs arborize on lumbar motor neurons, creating a new intraspinal circuit relaying cortical input to its original spinal targets. We confirmed the functionality of this circuit by electrophysiological and behavioral testing before and after CST re-lesion. Retrograde transynaptic tracing confirmed its integrity, and revealed changes of cortical representation. Hence, after incomplete spinal cord injury, spontaneous extensive remodeling occurs, based on axonal sprout formation and removal. Such remodeling may be crucial for rehabilitation in humans.

NGF-Mediated increase of choline acetyltransferase (ChAT) in the neonatal rat forebrain: Evidence for a physiological role of NGF in the brain?

Abstract Neonatal rats received intraventricular injections of mouse submandibular gland nerve growth factor (NGF) on days 1, 3, 5 and 7 postpartum. After killing the animals at day 8, activities of choline acetyltransferase (ChAT), acetylcholine esterase (AChE) and tyrosine hydroxylase (TH) were measured in different brain areas. NGF treatment increased ChAT activity in the septal area by 78%, in the hippocampus by 30%, and in the cortex by 73% relative to control animals. No increase was observed in other brain areas. The elevation of ChAT activity was not accompanied by an increased activity of AChE, and the concomitant 30–40% increase of TH activity observed in the cortex and brainstem was abolished after immunosympathectomy, reflecting the ingrowth of peripheral sympathetic peripheral fibers into the central nervous system (CNS) in response to centrally administered NGF23. In adult rats, repeated injections of NGF over 4 weeks caused a small but statistically significant increase of ChAT activity (15%) in the forebrain. In contrast, repeated intraventricular or intracortical injections into neonatal rats of large amounts of purified antibodies against mouse NGF (anit-NGF) failed to reduce ChAT activity in the same forebrain areas. Moreover, the offspring of rats autoimmunized against mouse NGF showed no reduction of ChAT activity in the brain, even though the TH activity was reduced by 76% in the superior cervical ganglia (SCG) of these animals. Antibodies against mouse NGF were also without effect on ChAT activity in cultures of dissociated septal neurons, though these cells also responded to NGF with an increase in ChAT activity. Anti-NGF blocked the effect of exogenous NGF but failed to reduce basal ChAT activity in these cultures. It is concluded that exogenous NGF can affect forebrain cholinergic neurons during their development. NGF does not seem to be identical with an endogenous neurotrophic factor produced by hippocampus or neocortex acting on cholinergic neurons of the forebrain.

Plasticity of motor systems after incomplete spinal cord injury

Although spontaneous regeneration of lesioned fibres is limited in the adult central nervous system, many people that suffer from incomplete spinal cord injuries show significant functional recovery. This recovery process can go on for several years after the injury and probably depends on the reorganization of circuits that have been spared by the lesion. Synaptic plasticity in pre-existing pathways and the formation of new circuits through collateral sprouting of lesioned and unlesioned fibres are important components of this recovery process. These reorganization processes might occur in cortical and subcortical motor centres, in the spinal cord below the lesion, and in the spared fibre tracts that connect these centres. Functional and anatomical evidence exists that spontaneous plasticity can be potentiated by activity, as well as by specific experimental manipulations. These studies prepare the way to a better understanding of rehabilitation treatments and to the development of new approaches to treat spinal cord injury.

Specific retrograde transport of nerve growth factor (NGF) from neocortex to nucleus basalis in the rat

[125I]labeled NGF injected in very small quantities into the frontal or dorsal anterior occipital cortex of adult rats, was specifically taken up and transported retrogradely to large, presumably cholinergic neurons in the nucleus basalis region (lateral preoptic nucleus, anterior lateral hypothalamic nucleus, substantia innominata, ventral globus pallidus and internal capsule), as revealed by light microscopic autoradiography. Cells projecting to the injection site in the frontal cortex were localized ipsilaterally in the more caudal parts of the nucleus basalis region, whereas cells projecting to the dorsal anterior occipital cortex could be found throughout the entire extent of the nucleus basalis and also in the vertical and horizontal limb of the nucleus of the diagonal band of Broca. Other nuclei known to project to the cortex (locus coeruleus, substantia nigra, nucleus raphe, thalamus) were consistently found to be unlabeled. In contrast to [125I]NGF, injection of [125I]cytochrome C failed to label any cell bodies in the basal forebrain nuclei by retrograde transport. This high selectivity for uptake and retrograde transport of NGF indicates the presence of membrane receptors for NGF or a closely related molecule on these cholinergic neurons of the basal forebrain innervating the cerebral cortex.

Nogo and axon regeneration

Nogo-A is one of several neurite growth inhibitory components present in oligodendrocytes and CNS myelin membranes. Nogo has a crucial role in restricting axonal regeneration and compensatory fibre growth in the injured adult mammalian CNS. Recent studies have shown that in vivo applications of Nogo neutralizing antibodies, peptides blocking the Nogo receptor subunit NgR, or blockers of the postreceptor components Rho-A and ROCK induce long-distance axonal regeneration and compensatory sprouting, accompanied by an impressive enhancement of functional recovery, in the rat and mouse spinal cord.

Secondary Cell Death and the Inflammatory Reaction After Dorsal Hemisection of the Rat Spinal Cord

Local spinal cord lesions are often greatly enlarged by secondary damage, a process which leads to massive additional cell death. This process is poorly understood. In order to investigate which types of cells could play a role in increasing the size of the lesion, we have analysed the events occurring at rat spinal cord lesion sites from 1 h to 3 months after partial transection using cell type‐specific markers. One hour after transection, the lesion site was small and corresponded to the zone of primary mechanical damage. Extravasation of blood and an opening of the blood – brain barrier occurred. Rapidly thereafter, at 3 and 6 h, an area of secondary cell death developed around the zone of the primary lesion. This secondary cell death, which was probably largely of the necrotic type, affected neurons, macroglia and microglial cells indiscriminately. It was virtually complete at 12 h. Recruitment of inflammatory cells followed a time course which lagged behind that of secondary cell death. Adhesion of neutrophils to the inside of blood vessels was observed at 3 h. They appeared in large numbers at 6 h at the site of the primary lesion, but not yet in the area of secondary cell death. They were numerous throughout the lesion site at 24 h and then disappeared rapidly. Proliferation and recruitment of macrophages and microglial cells became predominant 2 days after injury. Their density was highest within the lesion site between 4 and 8 days. Very few astrocytes were present in the lesion site during the first week. In contrast, the surrounding area contained numerous activated astrocytes, which began to delineate the lesion site. After 2 weeks, the microglial cells and macrophages progressively disappeared from the lesion site, and a cavity formed. A glial scar surrounded this cavity and consisted of reactive astrocytes and activated microglial cells. The time course of the cellular reactions observed here suggests that secondary damage is not primarily due to destructive effects of neutrophils and macrophages. The inflammatory process after spinal cord transection is qualitatively similar to that observed outside the CNS. Inflammatory cells, which can release cytokines and growth factors, could play important roles in protective reactions of the tissue and glial scar formation.

Brain-derived neurotrophic factor supports the survival of cultured rat retinal ganglion cells

Brain-derived neurotrophic factor (BDNF) is a small, basic protein purified from the mammalian brain that has been shown previously to support the survival of cultured spinal sensory neurons (Barde et al., 1982). In current studies, BDNF was tested for its ability to support the survival of cultured CNS cells isolated from the perinatal rat retina. Both immunofluorescent labeling of Thy-1 and prior retrograde labeling with HRP were used as retinal ganglion cell markers in vitro. With embryonic day (E) 17 retinas, it was found that BDNF allowed the survival of a small subpopulation of neurons (about 7% of the cells plated at this age) identified by the immunofluorescent labeling of Thy- 1. No detectable effects were seen when either the total number of cells or the number of tetanus toxin-positive neurons was measured. BDNF also had an effect on cultured neurons retrogradely labeled after HRP injections in the superior colliculi of neonatal rats. The BDNF- responsive population was therefore detected only in retinal cultures with specific markers and identified as consisting of retinal ganglion cells. These cells could be enriched about 80-fold by density gradient centrifugation, and purified ganglion cell cultures were shown to be responsive to BDNF. Whereas with E17 retinas, the number of surviving Thy-1 positive neurons could be kept constant for at least 4 d, the survival of postnatal neurons was only transiently increased by BDNF. We conclude that in the retina, BDNF affects only the survival of ganglion cells in vitro by a direct action on these cells. The results are discussed in terms of target-derived neurotrophic support during development.

Nerve growth factor increases choline acetyl-transferase but not survival or fiber outgrowth of cultured fetal septal cholinergic neurons

Neurons dissociated from the septal area of fetal rat brains were grown in culture. Cholinergic neurons were identified by immunocytochemical visualization of choline acetyltransferase and cytochemical demonstration of acetyl cholinesterase. Choline acetyltransferase immunocytochemistry stained cell bodies and proximal processes while acetylcholinesterase cytochemistry visualized the entire neuron. Choline acetyltransferase-positive neurons could only be identified in cultures grown under conditions that produced the maximal choline acetyltransferase activity, measured biochemically. All of the choline acetyltransferase-positive neurons were double stained for acetylcholinesterase while only 6% of the acetylcholinesterase-positive cells were choline acetyltransferase negative in these cultures. These results indicate that acetylcholinesterase is a reliable marker for cholinergic cells in cultures of dissociated septal neurons. Being the more sensitive method, acetylcholinesterase staining was therefore used to identify cholinergic cells in cultures with choline acetyltransferase levels insufficient for immunocytochemical visualization of this enzyme. Addition of nerve growth factor or antibodies to nerve growth factor to the medium did not affect the number of cholinergic neurons surviving in culture. Furthermore, nerve growth factor and anti-nerve growth factor failed to influence the general morphological appearance and the number of processes of these neurons. However, nerve growth factor elevated the biochemically measured activity of choline acetyltransferase up to two-fold. The nerve growth factor-mediated increase in choline acetyltransferase activity was dose dependent with an ED50 of 10 ng/ml (4 X 10(-10) M). The increase was highly specific for nerve growth factor. It was blocked by anti-nerve growth factor, and epidermal growth factor, insulin and other control proteins failed to exert a similar effect. Nerve growth factor had to be present for at least 3 days in the culture medium to increase choline acetyltransferase activity, suggesting that the increase was due to an elevated choline acetyltransferase synthesis rather than to an activation of the enzyme.