Neuronotrophic Activity Research Articles

Gangliosides -- A new therapeutic agent against stroke and Alzheimer's disease

Gangliosides are glycosphingolipids localized to the outer leaflet of the plasma membrane of vertebrate cells. The highest ganglioside concentration of any organ is found in the mammalian brain, where the gangliosides are enriched in the neuronal membrane, particularly in the synapses. There are four major brain gangliosides with the same neutral tetrasaccharide core to which one to three sialic acids are linked - the simplest being the GM1-ganglioside. These gangliosides have been shown to have neuritogenic and neuronotrophic activity and to facilitate repair of neuronal tissue after mechanical, biochemical or toxic injuries. Mixtures of native bovine brain gangliosides were adopted for pharmacological use in the treatment of peripheral nerve damage, and GM1-ganglioside has been applied for the treatment of CNS injuries and diseases. Beneficial effects of GM1 have been documented in the treatment of stroke and spinal cord injuries, particularly when the treatment has been initiated within a few hours of the acute event. Continuous intraventricular infusion of GM1 has recently been shown to have a significant beneficial effect in Alzheimer disease of early onset (AD Type I).

Dopamine neuronotrophic activity in dopamine-depleted striatum in the rat

Dopamine neuronotrophic activity in dopamine-depleted striatum in the rat

Purification and characterization of fab fragments from anti‐mouse NGF polyclonal antibodies

A functional role for Nerve Growth Factor (NGF) in the peripheral nervous system is well-documented, but a similar case for NGF in the central nervous system remains to be established. One approach to answering this question would be the availability of high-affinity monospecific Fab fragments obtained against NGF. In the present studies we describe the preparation and characterization of such Fab fragments from anti-mouse NGF polyclonal antibodies. Following their purification by the use of a NGF Sepharose-coupled affinity column, the Fab fragments were examined for biological competence in several ways. In vitro, the anti-Fab fragments blocked the neuronotrophic activity of NGF, as measured by the survival of chicken embryonic day 8 dorsal root ganglion neurons. In vivo, these Fab fragments, when administered systemically to neonatal rats, produced a decrease of noradrenaline levels in two sympathetically innervated organs, the heart and the spleen. These findings suggest that affinity purified Fab fragments of anti-NGF antibodies can be a useful tool for studying the physiological function of NGF in the nervous system.

Phosphoglucoisomerase (neuroleukin), fibroblast growth factor and catalase: lack of neuronotrophic activity for cultured rat spinal neurones

Phosphoglucoisomerase (neuroleukin), fibroblast growth factor and catalase: lack of neuronotrophic activity for cultured rat spinal neurones

Chicken NGF and non-NGF trophic factor synthesis and release by sciatic nerves in vitro.

The time course of production and release of nerve growth factor (NGF) and non-NGF neuronotrophic factors for sympathetic neurons by chicken and rat sciatic nerves in culture was examined. These tissues actively synthesize and release neuronotrophic activity as metabolically poisoning nerves with azide dramatically reduced the amount of trophic activity released into the culture medium. The sustained release of this activity also was shown to be dependent on the presence of low-molecular-weight dialysable molecules present in foetal calf serum and amniotic fluid from day 11 chicken embryos. Affinity-purified antimouse NGF antibodies were used to show that sciatic nerves in culture release both NGF and non-NGF trophic factors. These antibodies inhibited all bioactivity of both mouse NGF and of a partially purified preparation of chicken NGF. Immunoblot studies confirm that the antibodies recognize both rodent and avian NGF. Excess antibody inhibited only about 50% of the trophic activity in media conditioned over rat or chicken nerves for the first 24 hr. Relatively similar amounts of this non-NGF trophic activity were released throughout 6 days in culture, and this trophic activity kept sympathetic neurons alive in culture in the absence of NGF for more than 4 days. NGF levels were quantified with a two-site enzyme-linked immunoassay and found to parallel changes in NGF bioactivity. Rat nerves released increasing amounts of NGF with time in culture. Whole chicken sciatic nerves, however, released decreasing amounts of NGF with time in culture, but when these nerves were desheathed by removal of the epineurium and attached tissue, the pattern of NGF release was similar to that observed in the smaller rat sciatic nerves. These studies therefore characterize antibodies recognizing chicken NGF, demonstrate that peripheral nerve tissue synthesize trophic factors other than NGF, and identify factors that influence NGF synthesis.

Reduction in sympathetic neuronotrophic activity in the pulp of the cat canine tooth after denervation

Most of the nerve fibres supplying the mandibular canine on one side were interrupted by sectioning the inferior alveolar nerve (IAN) and, after 1 week, the trophic activity in each mandibular canine pulp was assessed in an in vitro assay using sympathetic neurones from 11-day chick embryos as test cells. In eight of nine animals tested, neuronotrophic activity in the denervated pulp was markedly lower than in the contralateral control pulp. Antiserum to mouse nerve growth factor had no effect on the trophic activity in either control or denervated pulps. Thus, the pulp differs from other peripheral tissues, which undergo increases in neuronotrophic activity after denervation. The basis of this difference may be the high innervation density of the pulp. The IAN distal to the site of nerve transection also had reduced survival-promoting activity.

Development and survival of neurons in dissociated fetal mesencephalic serum-free cell cultures: I. Effects of cell density and of an adult mammalian striatal-derived neuronotrophic factor (SDNF)

The use of CNS cultures for detection and quantification of neuronotrophic activity in the CNS has been analyzed. In particular the development, i.e., neurotransmitter uptake characteristics, and survival of dopaminergic and GABAergic neurons in fetal mouse (E13)-dissociated mesencephalic cells cultured in serum-free, hormone-supplemented medium have been assessed as a function of culture time and cell density. At all times, more than 98% of the cells were classified as neurons on the basis of immunocytochemical criteria. Results indicate that the increase of cell density in vitro significantly enhances specific high-affinity dopamine uptake per dopaminergic cell and cell survival. This effect is not limited to the dopaminergic cells and suggests that the development of neurotransmitter-related traits and cell survival are influenced by cell density-derived trophic signals. The above-mentioned cultures and parameters have also been used to detect neuronotrophic activity in adult mammalian brain extracts or more purified preparations. In particular, bovine striatal extracts contain activity capable of increasing high-affinity neurotransmitter uptake parameters and cell survival of at least the dopaminergic and GABAergic neurons present in the culture system. The neuronotrophic activity from bovine striatum has been partially purified and is associated with a fraction whose main component is a basic protein of approximately 14 kDa.

Alzheimer's disease brain extract stimulates the survival of cerebral cortical neurons from neonatal rats

Cell cultures of neonatal rat cerebral cortex in a serum-free medium were used to investigate a lack of neuronotrophic factors in Alzheimer's disease (AD) brain. A few neurons survived in the absence of brain extract. The addition of normal brain extract resulted in a 2.5-fold increase in neuronal survival. AD brain extract contained 4-fold neuronotrophic activity of normal brain extract. These findings are in contrary to the previous hypothesis of a lacking neuronotrophic factors in AD brain. These new results may change the concept of mechanism of neuronal death in AD.

Neuronotrophic activity of adult human skeletal muscle extract on cultured rat spinal neurons

Neuronotrophic activity of adult human skeletal muscle extract on cultured rat spinal neurons

Potassium-induced release of neuronotoxic activity by astrocytes

Medium conditioned by newborn rat cerebral cortex microexplants contains neuronotoxic activity for cerebellar granule cells and hippocampal neurons. The neuronotoxic activity is associated with low-molecular weight molecule(s) (<1000 Da) and resists to heating and to freezing and thawing. Using nearly homogenous cultures of neurons or astrocytes, we show that the neuronotoxic activity is released by the latter cell type. This release is enhanced by increasing extracellular K +-concentration. Astrocytes also secrete neuronotrophic activity whose release is not affected by external K +. Neurons can be desensitized against the neuronotoxic activity.

Neuronotrophic Activity in Adrenal Glands: Effect of Denervation

Neuronotrophic Activity in Adrenal Glands: Effect of Denervation

Sympathetic neuronotrophic activity in the pulp of the cat canine tooth

Extracts of these dental pulps from adult cats contained a non-dialysable agent or agents which could support neurone survival and neurite development for at least three days in neurone-enriched cultures of sympathetic ganglion cells from 11-day chick embryos. The neurone survival-promoting activity differed from nerve growth factor (NGF) in that: (1) anti-mouse NGF serum did not inhibit it; (2) nearly all ganglionic neurones survived in optimum concentrations of pulp extract, whereas only about 35 per cent were supported by NGF; and (3) cell bodies of NGF-supported neurones were markedly larger than in neurones supported by pulp extracts. The neuronotrophic activity in individual dental pulps was highly variable among different cats, but similar between mandibular canines from the same animal. Smaller pulps had higher concentrations of trophic activity than larger ones. Gingival tissue and the anterior belly of the disgastric muscle contained little neuronotrophic activity.

Nerve growth factor (NGF) in serum: evaluation of serum NGF levels with a sensitive bioassay employing embryonic sensory neurons.

Considerable controversy surrounds the question of whether or not nerve growth factor (NGF) or a related nerve growth-promoting factor is present in serum. Recently, supporting its role as a local neuronotrophic factor, the presence of NGF in glial cells and its production in target tissues of NGF-responsive neurons were demonstrated [Rush: Nature 312:364-367, 1984; Heumann, Korsching, Scott, Thoenen: EMBOJ 3:3183-3189, 1984; Shelton and Reichardt: Proc Natl Acad Sci USA 81:7952-7955, 1984]. At the same time, the concept that NGF may play a role as a humoral factor has been questioned, since careful analyses of serum with specific and sensitive radioimmunoassays [Suda, Barde, Thoenen: Proc Natl Acad Sci USA 75:4042-4046, 1978; Korsching and Thoenen; Proc Natl Acad Sci USA 80:3513-3516, 1983; Furukawa, Kamo, Furukawa, Akazawa, Satoyoshi, Itoh, Hayashi: J Neurochem 40:734-744, 1983] as well as bioassays [Skaper and Varon: Exp Neurol 76:655-665, 1982] have not confirmed earlier reports [Levi-Montalcini and Booker; Proc Natl Acad Sci USA 46:373-391, 1960; Banks, Banthorpe, Charlwood, Pearce, Vernon: Nature 246:503-504, 1973; Hendry: Biochem J 128:1265-1272, 1972] on NGF's representation in serum. In this study serum from mouse, rat, and man was analyzed with an in vitro bioassay system which employs sensory neurons from chicken embyro dorsal root ganglia and which allows the measurement of NGF concentrations as low as 0.8 pM. It was found that sera from all these species contained neuronotrophic activity (S-NGF). The target cell spectrum as well as characteristic parameters of the neuronal growth response of S-NGF and of NGF were identical. S-NGF of mouse serum was completely inhibitable by polyclonal and monoclonal antibodies to mouse submandibular gland beta NGF. On polyacrylamide isoelectric focussing gels mouse and human S-NGF could be recovered from the same position as NGF as well as from the region where alpha 2-macroglobulin and serum albumin focused. In newborn and adult male and female mice basal S-NGF levels were equivalent to 10-50 pM NGF. A fraction of the serum samples of male mice showed elevated S-NGF levels. The incidence of high S-NGF levels was more frequent in NMRI and C57BL/6 males than in BALB/c males. Following sialectomy of male mice only basal S-NGF levels were observed up to 5 weeks after the operation. This indicates that although the submandibular gland may contribute to S-NGF levels in serum under certain conditions that appeared to be stress related, it cannot be the only source of S-NGF.

Purification of a human red blood cell protein supporting the survival of cultured CNS neurons, and its identification as catalase

We have previously reported that red blood cells contain high levels of a protein that supports the survival of a variety of CNS neurons in vitro for 24 hr. Here we report the isolation of this trophic activity from human red blood cells. The active material, purified over 1000-fold by ion-exchange chromatography and isoelectric point, subunit molecular weight, and ability to degrade hydrogen peroxide. Commercially produced bovine liver catalase, lactoperoxidase, HRP, and vitamin E all mimic the ability of the purified human protein to support neuronal survival in vitro. Pharmacological inhibitors of peroxidase activity inhibit the trophic effects of both commercial catalase and the purified blood-derived protein. These results suggest that peroxidase activity mediates the neuronotrophic activity of these agents. The bioassay culture medium itself generates peroxides in the absence of cells. Removal of this toxic material may be the basis for the trophic effects of catalase.

Reduced neuronotrophic activity of fibroblasts from individuals with dysautonomia in cultures of newborn mouse sensory ganglion cells

The neuronotrophic activity of human skin fibroblast cell lines from two normal individuals and 3 individuals with Familial Dysautonomia (FD) was compared in terms of their ability to support neuron survival and neurite regeneration in cultures of newborn mouse sensory neurons. Neuronotrophic activity was assayed by culturing dissociated sensory neurons from newborn mice: (1) on fibroblast ‘cell beds’ consisting of monolayer cultures of living fibroblasts, (2) with medium that had been conditioned by monolayer fibroblast cultures and (3) with extracts of the cultured fibroblasts. Neurite regeneration was compared by determining the percentage of neurons that had regenerated neurites after 24 or 48 h in culture. Neuron survival was determined by counts at 48 h, 7 days and 14 days after culture initiation. Neurite regeneration and neuron survival was found to be significantly less on FD cell beds or with FD-conditioned medium than in replicate cultures with normal fibroblasts or their conditioned medium. The reduced neuronotrophic activity of FD fibroblasts or conditioned medium was still observed in the presence of antibody sufficient to block the neuronotrophic effects of purified nerve growth factor (NGF). Thus, fibroblasts from individuals with FD exhibit reduced neuronotrophic activity for newborn mouse sensory neurons that appears to be due to factor(s) other than NGF.

Increase in neuronotrophic activity during the period of smooth muscle innervation

The expansor secundariorum is a unique smooth muscle of the avian wing that receives a dense sympathetic innervation and contains high concentrations of survival factors for sympathetic neurons. In the present study it has been possible to simultaneously examine the appearance of the neuronotrophic activity and the arrival of nerve fibres during the period of innervation. The results show that catecholamine containing nerve fibres can first be detected within the muscle on the fourteenth day of incubation (stage 40) followed by a rapid increase in the density of fibres during the next few days until the adult pattern is reached shortly before hatch. Biochemical estimation of the innervation process by measurement of dopamine β-hydroxylase activity was supported by the histochemical findings. Estimation of neuronotrophic activity revealed that muscle from stage 40 embryos contains only low levels of activity which increases rapidly as innervation proceeds and further, that this increase in neuronotrophic activity was directly correlated with the dopamine β-hydroxylase activities. Possible mechanisms regulating this dramatic increase in the specific activity of trophic factors are discussed.

Human skeletal muscle cells synthesise a neuronotrophic factor reactive with spinal neurons.

Retrograde trophic influences originating in the skeletal musculature have been postulated to be involved in regulating survival and differentiation of embryonic motor neurons and reactive terminal sprouting of mature motor fibres. We have previously described the use of a quantitative immunoassay for neurofilament protein to bioassay in vitro the cell-type-specific neuronotrophic activity of nerve growth factor (NGF) on sensory ganglion neurons. In the present study, the effect of media conditioned by adult human muscle cells (MCM) on the in vitro development of chicken spinal neurons has been studied using a similar approach. Significant increases in neurofilament protein levels in 7-day chicken embryonic spinal cord cultures were found with doses of MCM protein as low as 0.4 microgram/ml, with a dose-response relationship yielding maximal and half-maximal effects at 4 and 1 microgram/ml, respectively. Maximal increases in neurofilament protein levels were associated with an approximate two-fold increase in neuronal cell survival. MCM also induced increases in choline acetyltransferase activity in chick spinal cord cultures. In both the absence and presence of NGF, MCM did not increase neurofilament protein expression in primary cultures of sensory neurons.

Neuronotrophic factors for mammalian brain neurons: injury induction in neonatal, adult and aged rat brain.

Tissue from neonatal, adult and aged Sprague-Dawley rat brain contained low levels of survival promoting activity for embryonic neurons from various rat brain regions. This basal neuronotrophic activity was 2-fold higher in adult and 4-fold higher in aged rat brain, with respect to that in neonatal brain. Tissue extracts also contained 4-fold higher levels of neuronotoxic activity in adult and aged than in neonatal brain. Ablation of the occipital/entorhinal cortex caused a 3-fold increase in neuronotrophic activity in the tissue surrounding the wound in neonates and 4-fold in adult and aged brain, with respect to the basal levels. Maximal activities occurred at 3 days, 6 days and 15 days postlesion in neonatal, adult and aged tissue respectively, and subsequently returned to basal levels. Neuronotoxic activity was not induced following injury. The slower neuronotrophic response to injury in older animals may be one of the determinant factors of the slower recovery from brain damage observed in aging.

Central nervous system-directed neuronotrophic activity present in red blood cells

A new neuronotrophic factor has been identified in extracts of vertebrate red blood cells. The factor supports the survival in culture of neurons from vertebrate central nervous systems, and does not support the survival of several peripheral ganglionic neurons. The active molecule appears to be a slightly acidic protein of 30,000–100,000 daltons.

Neuronotrophic activity for ciliary ganglion neurons. Induction following injury to the brain of neonatal, adult, and aged rats

The induction of neuronotrophic activity following injury to the brain of neonatal, adult, and aged Sprague-Dawley rats was compared using an improved in vitro assay. The maximal levels of activity in tissue surrounding the wound were reached at 3, 10–15, and about 15 days postlesion in neonatal, adult, and aged animals, respectively. Tissue neuronotrophic levels were always much lower in neonatal animals relative to the older animals. Accumulation of neuronotrophic activity in the gelfoam placed into the wound cavities in neonatal and adult animals lagged behind the levels in tissue by 4–5 days, suggesting that either the neuronotrophic factor itself or the cells which produce it are transferred from the tissue into the gelfoam. Relatively little activity accumulated in the gelfoam taken from aged Sprague-Dawley rats, and this observation was confirmed in aged Fischer rats. Aged animals seem to be unable to produce or release one of a number of neuronotrophic factors in response to injury.