Ted Ebendal

Chronic implants of chromaffin tissue into the dopamine-denervated striatum. Effects of NGF on graft survival, fiber growth and rotational behavior

SummaryAdult rat chromaffin tissue was transplanted into striatum of adult rat recipients whose nigrostriatal dopamine pathway had been lesioned on the grafted side by 6-hydroxydopamine. Long-term survival of the intrastriatal chromaffin grafts and the effects of treatment with nerve growth factor (NGF) was studied histochemically using Falck-Hillarp fluorescence histochemistry and functionally using rotational behavior induced by apomorphine. Small, cortex-free adrenal chromaffin tissue grafts survived permanently in striatum. The number of surviving cells was significantly increased by NGF. NGF treatment also caused transformation of many cells towards a more neuronal phenotype and greatly enhanced the adrenergic nerve fiber outgrowth into host brain tissue. NGF was either injected stereotaxically into the site of transplantation or infused continuously using implantable osmotic minipumps and a stereotaxically placed chronic indwelling dialysis fiber through striatum. The latter arrangement permitted continuous infusion of NGF for 14–28 days and caused a vigorous adrenergic nerve growth response by the grafts directed towards the source of NGF in the brain. There was a clearcut correlation between morphological signs of taking and rotational behavior. Grafts, and in particular grafts treated with NGF, were able to significantly and permanently counteract the rotational behavior induced by apomorphine. There seemed to be a dose relationship between NGF treatments and amount of reduction of asymmetric behavior. NGF treatment probably decreased the relative importance of diffuse release of catecholamines from chromaffin cells in the graft and increased the importance of adrenergic innervation of host striatum by cells in the graft. Immunofluorescence using antibodies against glial fibrillary acidic protein did not reveal any marked gliosis around the grafts nor were there any marked gliotic reactions around chronic indwelling dialysis fibers. We conclude that implantation of chromaffin tissue into striatum in conjunction with NGF treatments is an effective means of counteracting some of the symptoms of experimentally induced unilateral parkinsonism in rats.

Chimeric molecules with multiple neurotrophic activities reveal structural elements determining the specificities of NGF and BDNF.

Nerve growth factor (NGF) and brain‐derived neurotrophic factor (BDNF) are two members of a family of neurotrophic factors which show both overlapping and distinct neurotrophic activities. Using site‐directed mutagenesis, chimeric molecules were constructed where different combinations of sequences from BDNF replaced the corresponding sequences in NGF. The resulting molecules were transiently expressed in COS cells and conditioned media containing the chimeric proteins were assayed for biological activity in explanted chick sympathetic, spinal and nodose ganglia. Our results show that the biological specificities of the two proteins are obtained by specific combinations of a set of sequences that differ between the two molecules. Some of these combinations allowed us to engineer molecules which display multiple neurotrophic activities recruited from both the NGF and BDNF proteins.

Structure-function studies of nerve growth factor : functional importance of highly conserved amino acid residues.

Selected amino acid residues in chicken nerve growth factor (NGF) were replaced by site‐directed mutagenesis. Mutated NGF sequences were transiently expressed in COS cells and the yield of NGF protein in conditioned medium was quantified by Western blotting. Binding of each mutant to NGF receptors on PC12 cells was evaluated in a competition assay. The biological activity was determined by measuring stimulation of neurite outgrowth from chick sympathetic ganglia. The residues homologous to the proposed receptor binding site of insulin (Ser18, Met19, Val21, Asp23) were substituted by Ala. Replacement of Ser18, Met19 and Asp23 did not affect NGF activity. Modification of Val21 notably reduced both receptor binding and biological activity, suggesting that this residue is important to retain a fully active NGF. The highly conserved Tyr51 and Arg99 were converted into Phe and Lys respectively, without changing the biological properties of the molecule. However, binding and biological activity were greatly impaired after the simultaneous replacement of both Arg99 and Arg102 by Gly. The three conserved Trp residues at positions 20, 75 and 98 were substituted by Phe. The Trp mutated proteins retained 15‐60% of receptor binding and 40‐80% of biological activity, indicating that the Trp residues are not essential for NGF activity. However, replacement of Trp20 significantly reduced the amount of NGF in the medium, suggesting that this residue may be important for protein stability.

NGF and Anti-NGF: Evidence against Effects on Fiber Growth in Locus coeruleus from Cultures of Perinatal CNS Tissues

The present study examines whether the developing noradrenergic neurons of locus coeruleus depend on endogenous nerve growth factor (NGF) for nerve fiber production and if exogenous NGF stimulates fiber growth in this nucleus, using a collagen gel tissue culture technique. Lucus coeruleus from perinatal rat brain was used in three culture experiments: (1) lucus coeruleus, parietal cerebral cortex, and the superior cervical ganglion, prepared from newborn rats and cultured in different sectors of the same dishes; (2) locus coeruleus and parietal cerebral cortex from 17-day-old rat fetuses cultured in the same manner, and (3) locus coeruleus from 17-day-old rat fetuses co-cultured with spinal, sympathetic and ciliary ganglia from 8-day chick embryos. Experiments 1 and 2 were run with and without addition of NGF and anti-NGF, experiment 3 with and without anti-NGF. Total fiber production in all cultured tissues was evaluated daily by dark field and phase contrast microscopy during 4 days. Adrenergic nerve fiber production was then studied in the same locus coeruleus and superior cervical ganglia from the rats by Falck-Hillarp fluorescence histochemistry. Locus coeruleus and cortex cerebri from fetal rats produced dense fiber halos in culture. Locus coeruleus from newborn rats produced considerably less fibers, newborn cortex only few fibers. Superior cervical ganglia from the same newborn animals produced no or almost no fibers. Addition of NGF was not able to stimulate fiber growth in locus coeruleus nor in cortex cerebri as observed both in the living cultures and by fluorescence microscopy. Likewise, addition of anti-NGF did not affect fiber production in the CNS areas. The negative results with NGF on newborn locus coeruleus and cortex cerebri were in sharp contrast to the strong, highly significant fiber growth response demonstrated by the superior cervical ganglion from the same animals cultured in the same dishes. The third experiment tested whether locus coeruleus in tissue culture contained or produced nerve growth factor or any one of the three chick embryo ganglia. No response whatsoever in these three ganglia was observed. It is concluded that the developing locus coeruleus area does not contain or produce NGF, does not depend on NGF for fiber production, and is not stimulated by exogenous NGF.

Role of growth factors in degeneration and regeneration in the central nervous system; clinical experiences with NGF in Parkinson's and Alzheimer's diseases

Neurotrophin-mediated mechanisms are integral to development and maintenance of the adult central nervous system. Neurotrophin expression has been shown to change rapidly in response to many different types of neuronal stress such as excitotoxic injury, mechanical lesions, epileptogenesis and ischemia. It therefore appears as if they are not only to be regarded as target-derived trophic factors in the classical sense, but also as providers of local trophic support and neuronal protection. These discoveries suggest that neurotrophins or compounds with neurotrophin-like actions might become useful in developing new treatment strategies, not only for neurodegenerative diseases, but also for other diseases and injuries to the nervous system including stroke.

Nerve fiber production by intraocular adrenal medullary grafts: Stimulation by nerve growth factor or sympathetic denervation of the host iris

SummaryThis study evaluates the production of adrenergic nerve fibers by adrenal medullary tissue of the adult rat grafted to the anterior chamber of the eye of adult recipients. The chromaffin grafts attach to and become vascularized by the host iris. They decrease in size intraocularly during the first 3 weeks. This decrease is somewhat counteracted by sympathetic denervation of the host iris, and better counteracted by sympathetic denervation and addition of nerve growth factor (NGF, given at grafting and 1 and 2 weeks after grafting). Outgrowth of adrenergic nerve fibers from the grafts into the host iris was studied in wholemount preparations by use of the Falck-Hillarp technique 3 weeks after grafting. The innervated area of the host iris was approximately doubled in the chronically sympathectomized group and doubled again in the chronically sympathectomized NGF-supplemented group. Chronic sympathetic denervation had no effect on density of outgrowing nerves, whereas addition of NGF more than doubled nerve density. Since sympathetic denervation causes a slight elevation of NGF activity in the iris, the present experiments are taken as evidence that the level of NGF in the iris regulates formation of nerve fibers by adrenal medullary tissue grafts from adult rats.

Neurotrophins and their Receptors

Development and maintenance of the mammalian nervous system require neurotrophic factors. The most well-characterizied neurotrophic factor, nerve growth factor (NGF), has recently been shown to be a member of a family of structurally related proteins that also includes brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4). An evolutionary study of these factors (neurotrophins) in representative groups of vertebrates showed that the four factors are highly conserved from fishes to mammals. Chimeric molecules were contructed where different combinations of sequences from BDNF replaced the corresponding sequences in NGF. Assays for biological activity of such molecules showed that the biological specificities of the two proteins are obtained by specific combinations of a set of sequences that differ between the two molecules. In the brain, the highest levels of NGF, BDNF and NT-3 mRNAs are found within the hippocampus, where they are expressed in a unique set of neurons. Both BDNF and NT-3 mRNAs are transiently expressed in some brain regions during postnatal brain development, suggesting that trophic interactions during brain development are a dynamic phenomenon which may alter with various stages of ontogeny. Expression of NGF and BDNF mRNAs in the brain appears to be regulated by neuronal activity, and the levels of these mRNAs show a marked and transient increase following epileptic seizures, cerebral ischemia and hypoglycemic coma. The increases of NGF and BDNF mRNAs appear to be mediated via a release of glutamate activating AMPA receptors within the hippocampus, as well as a rise in intracellular calcium levels. The protooncogene trkB, an essential component of the high-affinity BDNF receptor, is also expressed in the hippocampus, where it is spatially and temporally regulated in a manner similar to BDNF. These data suggest that BDNF and its receptor could, in a paracrine/autocrine fashion, play a role in kindling-associated neural plasticity and in neuronal protection following ischemic and hypoglycemic insults. Genetically modified cells that express high levels of the four neurotrophins have been established and used for implantation into the rat brain to identify cells that respond to these factors in vivo. Using this approach, NGF has been shown to expert a trophic stimulation for cholinergic neurons in the brain.

Substance P-like immunoreactivity in cultured spinal ganglia from chick embryos.

SummaryThe localization of substance P (SP) or a SP-like peptide in cultured spinal ganglia from chick embryos was studied by the indirect immunofluorescence technique. Ganglia from 8–16 days old chick embryos and from newly hatched chickens were cultured in a control medium or in the presence of nerve growth factor (NGF). Addition of colchicine and exposure to different explanted peripheral tissues were also tried. Ganglia from the younger embryos (8–12 days) cultured for 24 h with added NGF showed a weak SP-like immunoreactivity (SPLI) in some cell bodies and strong specific immunofluorescence in nerve fibres growing out from the ganglia. In spinal ganglia of the older embryos (14 and 16 days) and newly hatched chickens cultured with and without NGF the concentration of SPLI in the cell bodies was considerably higher. Addition of colchicine to spinal ganglia cultured 12 h in NGF-medium, resulted in retraction of nerve fibres and strongly fluorescent, expanded nerve fibres were observed in peripheral parts of the ganglia. Explants of skin placed near the spinal ganglia stimulated the outgrowth of fibres, some of them containing SPLI. A few fluorescent fibres were also seen within the skin explants. Also heart tissue explants stimulated outgrowth of nerve fibres, but innervation of these explants with SPLI-containing nerves could not be observed. Nerve fibre-extension from the spinal ganglia was not stimulated by spinal cord explants. The present results support the existence of SP-containing primary sensory neurons in chickens.

Expression of β-Nerve Growth Factor and its Receptor in the Mammalian Central Nervous System

β-nerve growth factor (NGF) is a target derived protein that in the peripheral nervous system (PNS) is required for the development and maintenance of sympathetic and sensory neurons (LeviMontalcini and Angeletti, 1968; Thoenen and Barde, 1980). NGF mediates its neurotrophic effects by interacting with specific NGF receptors (NGF-R), which in the PNS have been found on the cell surface of sympathetic and sensory neurons (Banerjee et al., 1973: Herrup and Shooter, 1973). The NGF bound to its receptor is internalized by receptor-mediated endocytosis and is transported from nerve terminals to the neuronal cell body (Hendry et al., 1974; Stockel et al., 1974; 1976), where NGF induces several metabolic alterations in the responsive neuron (Schwab et al., 1981; Layer and Shooter, 1983).

Activity and Immunological Properties of Recombinant Nerve Growth Factor (β-NGF)

Nerve growth factor (βNGF) is a basic 118-amino acid protein acting as a trophic factor for many sensory and sympathetic neurons in the peripheral nervous system (Levi-Montalcini and Angeletti, 1968; Thoenen and Barde, 1980; Levi-Montalcini, 1987). The pioneering studies on NGF by Rita Levi-Montalcini and Victor Hamburger in the early 1950’s showed a marked stimulation by NGF on nerve fibre formation, neuron survival and neuron maturation in the sympathetic and some sensory ganglia of the developing peripheral nervous system (Levi-Montalcini and Hamburger, 1953). Nerve growth factor has recently also been found in the brain (Korsching et al., 1985; Whittemore et al., 1986; Shelton & Reichardt, 1986; Goedert et al., 1986) where it serves a trophic function for cholinergic neurons situated in the basal forebrain (Korsching et al.,1985; Large et al., 1986; Richardson et al., 1986; Larkfors et al., 1987b; review by Ebendal, 1989a).